spirulina platensis cl
TRANSCRIPT
Molecular cloning and characterization of
the allophycocyanin gene from Spirulina platensis Cl
MissDauenpen-NIeesapyodsuk B.Sc. (Nursing and Midwifery)
A Thesis Submitted in Partial Fulfillment of the Requirements
for the Degree of Master of Science
Biotechnology Program
School of Bioresources and Technology
King Mongkut’s Institute of Technology Thonburi
1996
Thesis Committee
-5 &L?i,diL LUL 77iLz. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chairman
(Asst. Prof. Dr. Supapon Cheevadhanarak)
(&t&J+ c/lzh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Co-Chairman
(Asst. Prof. Suchada Chaisawadi)
h&J f&h ,,a, oy &/-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Member
(Assoc. Prof. Dr. Morakot Tanticharoen)
. . . . . . . . . . . . . . . . . . . p &t?n, h++L..:................................................................ Member
(Dr. Patcharaporn Deshnium)
ISBN 9741621-904-g
Copyright reserved
ii
Thesis Title Molecular cloning and characterization of the
allophycocyanin gene from Spirulina platensis Cl
Thesis Credits 12
Candidate .Mi-ssDauenpen Meesapyodsuk
Supervisors Asst. Prof. Dr. Supapon Cheevadhanarak
Asst. Prof. Suchada Chaisawadi
Degree of Study Master of Science
Department Biotechnology
Academic Year 1996
Abstract
The apcABC genes encoding a, p subunits of allophycocyanin
and partial part of core linker protein were isolated from S. platensis Cl.
Based on sequence comparisons of apt genes from Synechococcus
PCC7002, Synechococcus PCC6301 and Cyanophora paradoxa, two
highly conserved regions were selected for the synthesis of 2
oligonucleotides, primers for amplifying apt gene probe from genomic
DNA of S. platensis Ci by polymerase chain reaction technique. The
amplified probe was used for screening S. platensis Ci genomic DNA
library. By homologous hybridization the upstream sequence of apcA
and partial sequence of the coding region of apcA gene was obtained. In
order to clone the entire apt genes, primer down in the apcC was
designed from the conserved sequence of the corresponding genes from
five cyanobacteria (Synechococcus PCC6301, Synechocystis PCC6803,
Synechococcus elongatus, FremyeZZa diplosiphon and Synechococcus’v
PCC7002) and primer up from a part of the upstream sequence of apcA
. . .1 1 1
isolated from genomic DNA library. By using these two primers, an
amplified DNA fragment of 1.8 kb was obtained, and its nucleotide
sequence was determined and analysed. Two complete open reading
frames of-qcA and-+zB (16 1 amino acids-for -each gene), and a partial
one of apcC (36 amino acids) were found. The identity of the deduced
amino acid sequence of the products of apcA and apcB to those of
Synechococcus elongatus, Cyanophora paradoxa and Synechocystis
PCC6714 were 85,82 and 80% for apcA, and 93, 84 and 88% for apcB,
respectively. For apcC, the identity of amino acid sequences between S.
platensis Ci and Synechococcus elongatus, Synechocystis PCC67 14 and
Fremyella diplosiphon were 92, 92 and 92%, respectively. Expected
chromophore attachment sites were found at position 81 in both amino
acid sequences of apcA and apcB. The sequence analysis also
demonstrated that the obtained genes form an operon, apcABC, with a
single transcription start site and one possible termination site
downstream of apcB. In addition, by sequence comparison of the
upstream region of apcA of S. platensis Ci with those from other
cyanobacteria, it was found that the apcABC promoters lied in the
conserved sequences centered at -50 and -10 with respect to the start of
transcription.
These results suggest that the obtained genes are putative
apcABC operon of S. platensis Cl.
Keywords: Spirulina platensis Ci/ allophycocyanin gene/ apcABC
operon
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illfil~ElJ (keywords): ~?‘l@El~$7~ULLflU!,~~al Spiruha platensis/ $pd allophycocyanin/
apcABC operon
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Acknowledgements
The au thor i s g ra te fu l to Ass t . Prof . Dr . Supapon
Cheevadhanarak, S.choul~ of Bioresources and Technology, King
Mongkut’s Institute of Technology Thonburi (KMITT), for advice on
all aspects of the work, reading and kindly suggesting improvements of
the manuscript and Asst. Prof. Suchada Chaisawadi, Pilot Plant
Development and Training Institute (PDTI), for her excellent technical
assistance, reading and discussion the manuscript.
I also wish to thank Assoc. Prof. Dr. Morakot Tanticharoen and
Dr. Patcharaporn Deshnium, National Center for Genetic Engineering
and Biotechnology (BIOTEC), for guidance, discussion and reading the
manuscript.
I would like to thank Dr. Christophe Anjard for his valuable
discussion.
Contents
Page
English abstract - - ~._ - ~-
Thai abstract
Acknowledgements
Contents
List of Tables
List of Figures
Abbreviations
Chapter 1 Introduction
1.1 Background
1.2 Objectives
1.3 Scopes
Chapter 2 Literature Review
2.1 Cyanobacteria
2.2 Spirulina
2.3 Phycobilisome and Phycobiliprotein Structure
2.3.1 Phycobilisome Components
2.3.2 Phycobiliproteins
2.3.2.1 PBPs constituing the PBsome core
2.3.2.2 PBPs constituing the rod
elements of PBsome
2.3.3 Linker polypeptides
2.3.4 Pigments
2.3.5 Organization of the genes encoding
the PBsome
ii _
iv
vi
vii
X
xi
xiv
1
1
2
3
4
4
7
1 5
1 5
21
2 6
3 1
38
41
44:r,
. . .Vlll
2.3.6 Energy Transfer in PBsomes
2.4 The molecular biology of cyanobacteria
2.4.1 Genome size of cyanobacteria
-_ - 2.42hlalecularcloning ofphycobilisome
in Cyanobacteria
Chapter 3 Materials and Methods
3.1 Organisms and plasmids
3.1.1 Cyanobacterium strain
3.1.2 Bacterial strains
3.1.3 Plasmids
3.2 Chemicals
3.3 Enzymes
3.4 Media and culture conditions
3.4.1 Spirulina platensis
3.4.2 Bacteria
3.5 Buffers and Solutions
3 .6 Primers/Oligonucleotide synthesis
3.6.1 Primers for PCR
3.6.2 Primers for sequencing
3.7 Molecular biology techniques
3.7.1 Plasmid preparation
3.7.2 Bacterial transformation
3.7.3 Subcloning of DNA fragments
3.7.4 Plating bacteriophages
3.7.5 Preparation of DNA from bacteriophages
3.7.6 DNA sequencing
3.7.7 Southern blotting
4 7
4 8
4 8
48 -
54
54
54
54
55
55
55
55
56
58
59
6 0
6 0
6 1
6 1
6 1
62
63
6 4
65
67k
68
ix
3.7.8 DNA labelling with 32P-dCTP
3.7.9 Dot blotting
3.8 Cloning of allophycocyanin gene from
-_ S. p&ends Cl strain - ~-
3.8.1 Isolation of genomic DNA
3.8.2 Amplification of partial apcAB from genomic
DNA of S. platensis
3.8.3 Screening of genomic library of S. platensis
3.8.4 Southern blot analysis
3.8.5 Characterization of positive clones
3.8.6 Amplification of complete apcAB and partial
apcC from genomic DNA of 5’. platensis
Chapter 4 Results and Discussions
4.1 Construction of apcAB probe from 5’. platensis
4.2 Isolation of allophycocyanin gene from genomic
library of S. platensis
4.3 Cloning of apcAB and partial apcC of 5’. platensis
4.4 Nucleotide sequence analysis
Chapter 5 Conclusion and Suggestion
5.1 Conclusion
5.2 Suggestion
References
Appendix
6 9
6 9
7 0
_
7 0
7 1
73
73
7 4
7 4
7 7
7 7
7 8
86
87
100
100
101
102
116
X
List of Tables
Table
2.1
2.2
2.3
2 .4
2.5
Comparison of-protein content in Spirdina _
and other foods (%)
Chemical analysis of Spirulina from spray dried
Phycobilisome polypeptides and their genes
Abbreviations for the biliprotein subunits and for linker
polypeptides
Genes of 5’. platensis that have been cloned and characterized
as documented in Genbank
Page
10 _
11
2 2
25
5 1
xi
List of Figures
Figure Page
2.1 Schematic diagram&a thin-section ofa cyanobacterial cell 5 _
2 .2 Artist’s representation of the overall three dimensional 6
architecture of Agmenellum quaduplicatum
2.3 Morphology of Spirulinaplatensis 8
2.4 Different morphological types in Spirulina 9
2.5 Life cycle of Spirulina 14
2 .6 Schematic structure of phycobilisomes structure attaching 16
to the photosynthetic membranes or thylakoid membranes
2 .7 Phycobilisomes with two respective three core 1 8
cylinders in cross-section
2 .8 Schematic representation of the hemidiscoidal 1 9
phycobilisomes structure
2 .9 Stereoscopic plot of (@)-monomer
2.10 Schematic side view of a phycocyanin hexamer
2.11 General features of allophycocyanin subunits
2.12 General structure, common features and chromophore
variability of phycocyanins
2.13 General structure, common features and chromophore
variability of phycoerythrins
2.14 A model for rod biogenesis based on
in vitro rod assembly experiments
2.15 Structures of various types of phycobilins
found in cyanobacteria and red algae, linked
by thioether-linkages to PBPs
23
23
2 7
33
37
4 0
43
xii
2.16 Comparison of the organization and transcription of genes
encoding phycobilisome components for the
cyanobacteria Synechococcus sp. PCC7002,
Synechococc~ KC6301 and Anahaena sp. PCC7120
2.17 Energy flow in PBsome of cyanobacteria and red algae
3.1
3.2
3.3
4.1
4 .2
4.3
4 .4
4.5
4 .6
4 .7
Schematic setup of downward capillary transfer of DNA
Comparison of the AP a and p subunits amino acid sequences
of Synechococcus PCC7002, Synechococcus PCC630 1 and
Cyanophora paradoxa
Comparison of the amino acid sequences of
apcC gene products
The amplified PCR products from
genomic DNA of S. platensis
The nucleotide and deduced amino acid sequences
of partial upcAB gene from S. platensis
The deduced amino acid sequence of the amplified
DNA product from S. platensis compared with that of
Synechococcus elongatus, Cyanophora paradoxa and
Synechocystis 67 14
Southern blot analysis of two recombinants hDNAs
isolated from two positive clones, VA1 and VA2 respectively
Southern blot analysis of one positive clone (VAl)
Restriction map 1.6 kb EcoRI-EcoRV fragment of VA1
Lambda phage
The amplified PCR products from
genomic DNA of S. platensis
4 6
4 7
6 8
72
7 6
79
80
8 1
83
84
85
88
. . .x111
4 .8 The nucleotide and deduced amino acid sequences of
the complete apcAB and partial apcC gene of S. platensis Cl
4 .9 The alignment of the amino acid sequence
from-VcAB g - p r o d u c t ~- - . ~
4.10 The alignment of the partial amino acid sequence
from apcC gene product
9 0
9 1
s
92
4.11 Hybridization of the 1.1 kb EC&I-EcoRV fragment
carrying part of the apcAB gene to total genomic
S. platensis DNA
95
4.12 Hybridization of the 1.8 kb EcoRZ fragment of
PCR product carrying apcABC gene to total
genomic S. platensis DNA
96
4.13 Restriction map of a 2 1 kbp EcoRI fragment
of S. platensis composed of the apt gene region
4.14 Alignment of the putative of transcription
start sites and promoters
97
98
4.15 The putative hairpin loop and stem part at the
3’ end of the P-subunit of APC of S. platensis Cl
using GENETYX-MAC program
99
xiv
Abbreviations
DNA
w
*g
k b
O.D.
PCC
PCR
RNase
DNase
rP*
w
=
=
Deoxyribonucleic acid
nanogram ~- ._ - ~-
milligram
kilobase (1 kb = 6.7x lo5 daltons = 1000 base pairs)
optical density
Pasteur Culture Collection
Polymerase Chain Reaction
Ribonuclease
Deoxyribonuclease
revolution per minute
microgram
ul = microlitre
PBsome = phycobilisome
PBPs = phycobiliproteins
APC = allophycocyanin
P C = phycocyanin
P E = phycoerythrin
PEC = phycoerythrocyanin
Chapter 1
Introduction
-
1.1 Background
Spirulina p l a t e n s i s , a phototrophic filamentous
cyanobacterium, has long been used as a staple food in human diet [I].
It is of commercial importance due to its perception as health food
containing high protein content (60% to 70% of dry weight), low fat,
minerals, high vitamin content (particularly Bi2) and essential fatty
acids like gamma-linolenic acid (GLA) Cl 8:3 [2]. Moreover, there are
various kinds of high value substances in Spirulina, e.g. phycocyanin,
allophycocyanin, p-carotene and chlorophylla [3, 41. These substances
including allophycocyanin, a bluish green pigment, are of high value in
their purified forms [5,6].
Allophycocyanin (APC), an accessory pigment in
cyanobacteria, harvests and transfers the solar energy (which has been
transferred from phycocyanin (PC)) to the reaction center of
photosynthesis [7]. In addition, the apcE encoding the core-membrane
linker polypeptide, serves as the terminal transmitter of light energy in
the phycobilisomes core to the reaction center in the thylakoid
membranes [8]. APC is a minor component of the water soluble proteins
constituted macromolecule structures, so called phycobilisomes [9].
Apart from a light-harvesting pigment, allophycocyanin is well suited as;
fluorescent reagent for flow cytometric analysis, since it has a broad
2
excitation spectrum and fluoresces with a high quantum yield.
Moreover, APC has been conjugated to monoclonal and polyclonal
antibodies for use in multicolor FACS (fluorescence-activated cell
sorter) analysis. The-activity of this conjugated antibody remains stable
for at least two years [lo].
Spirulina is the only species of cyanobacteria cultivated
outdoor in large industrial scale as human food or animal feed. Its
growth is affected by outdoor environment extensively. Hence, light
intensity plays a major role in the productivity of cell mass, as well as in
the quantity and quality of the pigments in the cells [ 111. The
knowledge of Spirulina C1 mass cultivation has been developed and
documented widely. However, there is little information concerning the
genetics and mechanisms of gene recombination in this cyanobacterium.
The study of gene(s) involved in light-harvesting protein
pigment(s) of Spirulina may lead to a better understanding in the role of
these pigments in photosynthesis, cell growth and cultivation. As such,
it is the aim of this study to clone and characterize allophycocyanin
operon of S. platensis C1, as the first step to employ molecular biology
as a tool to study allophycocyanin pigment at a molecular level.
1.2 Objectives
1. To clone the allophycocyanin operon from S. platensis Cl
strain.
2. To characterize the obtained operon.
3
1 . 3 Scopes
1. Amplification of allophycocyanin homologous probe by
PCR from -5’. pZateu.sis CL using- the conserved sequence of apt gene
from other organisms for primer designing.
2. Screening of genomic library of 5’. platensis Cr using the
probe from (1).
Cl.
3. Cloning of allophycocyanin gene operon from 5’. platensis
4. Characterization of the obtained gene by restriction
mapping, DNA sequencing and comparison of the gene products with
those of other organisms, as well as analysing the sequence using DNA
analysis program.
Chapter 2
Literature Review
-
2.1 Cyanobacteria
Cyanobacteria or blue-green algae are photoautotrophic
microorganisms [ 121. They are capable of performing oxygenic
photosynthesis similar to that found in eukaryotic algae and higher
plants [ 131. Cyanobacteria have the simplest structural organization
since they are unicellular rods or cocci existing in single or in
aggregated form. Some cyanobacteria exist in a filament of cells or
trichome. The trichomes of cyanobacteria may consist entirely of
vegetative cells, or also contain structurally and functionally
differentiated cells so called heterocyst and akinetes (spores) [ 141. In
cyanobacteria, the outer membrane, plasma membrane and thylakoid
membrane, shown in Fig.2.1, represent three structurally and
functionally distinct membranes [ 151.
The internal organization of the cyanobacterial cell is
prokaryotic, yet it is considerably more complicated structurally than
most bacteria. The structural features of the cell with the light
microscope include a central region (centroplasm or nucleoplasm) rich
in nucleic acid, a peripheral region (chromoplasm) containing the
photosynthetic thylakoid membranes and various inclusions, and several
enveloping layers consisting of plasmalemma, a pellicular wall, and/,
often, a layer of mucilage (Fig.2.1, Fig.2.2) [ 16, 171.
Figure 2.1
G G/
5
Schematic diagram of a thin section of a cyanobacterial cell.
CM, Cell membrane; TH, thylakoid; PBl and PB2, face and
side views of phycobilisomes attached to adjacent
thylakoids; GG, glycogen granules; CY, cyanophycin
granule; P, polyphosphate granule; C, carboxysome,
surrounded by nucleoplasm; R, ribosomes; G, gas vesicles.
(Insert A) Enlarged view of the cell envelope showing the
outer membrane and peptidoglycan wall layers, and the
cytoplasmic membrane. (Insert B) Enlarged view of part of
a thylakoid showing the paired unit membrane with attached;’
phycobilisomes in side view [ 181.
.
Figure 2.2 Artist’s %presentation of the overall three dimensional
architecture of Agmenellum quadruplicatum. 0
carboxysome, (L) lipid body, (P) polyphosphate body, (M)
photosynthetic thylakoid membrane system, and (‘IT)
contacts between thylakoids and cytoplasm membrane.
Thylakoids-cytoplasmic membranes and contact points are
theoretical, as this was not determined precisely (illustration
does not include cell wall, ribosomes, and nuclear material).
(From Nierzwicki-Baur, et al., 1983) [ 171
7
2.2 Spirulina
Spirulina is a multicellular-filamentous cyanobacterium. It
belongs to -phylumLyanophyta, -family Oscillatoriaceae [ 191. Spirulina
can be found in widely differing environments such as soils, marshes,
fresh water, brackish water and sea water. [20]
Under the microscope, Spirulina appears as a blue-green
filament composed of cylindrical cells arranged in unbranched,
helicoidal trichomes (Fig. 2.3) [21]. The filaments are motile, gliding
along their axis, without heterocysts. The helical shape of the trichome
is characteristic of the genus, but the helical parameters (i.e., pith, length
and helix dimensions) vary with the species and even within the same
species (Fig. 2.4). Cell diameter ranges from 1 to 3 pm in the smaller
species and from 3 to 12 urn in the larger ones such as S. platensis and
S. maxima. [20]
Spirulina contains protein as high as 60% of its dry weight and
much higher than that of other agriculture products such as rice, corn,
wheat and soybeans (as shown in Table 2.1) [22]. Besides, Spirulina
contains a rich source of vitamins, especially vitamin B12 and pro-
vitamin A (p-carotene), minerals (especially iron), and pigments (e.g.
phycocyanin and allophycocyanin). Table 2.2 shows the amount of
major compounds found in Spirulina [3].
8
Figure 2.3 - Morphology of Spirulina platensis. (A) Optical microscopy
(x400) of axenic S. platensis. (B) Scanning electroti\’
micrograph of a trichome of axenic S’. platensis [20].
t-,_ \’ ,_
Figure 2.4 Different morphological types in Spirulina (from theFigure 2.4 Different morphological types in Spirulina (from the
collection of the laboratory of Micro-algal Biotechnology atcollection of the laboratory of Micro-algal Biotechnology at
the Jacob Blaustein Institute for Desert Reserch at Sede-the Jacob Blaustein Institute for Desert Reserch at Sede-
Boker, Israel) (a) isolated from the local oxidation pond; (b)Boker, Israel) (a) isolated from the local oxidation pond; (b)
morphological similar trichome as in a developing frommorphological similar trichome as in a developing from
Spirulina platensis typical trichome; (c) Spiruiina pkutensis,Spirulina platensis typical trichome; (c) Spiruiina pkutensis,
nonvacuolated from Lake Chad; (d) straight nonvacuolatednonvacuolated from Lake Chad; (d) straight nonvacuolated
trichomes, isolated from pure culture c, from which theytrichomes, isolated from pure culture c, from which they
have been apparently transformed; (e) S’irulina platensis,have been apparently transformed; (e) S’irulina platensis,
vacuolated; (f) straight vacuolated trichomes isolated fromvacuolated; (f) straight vacuolated trichomes isolated from
pure culture b, (g) Spirulina sp. apparently platensis,pure culture b, (g) Spirulina sp. apparently platensis,
isolated from Lake Bogoria in Kenya, (h) Spirulinaisolated from Lake Bogoria in Kenya, (h) Spirulina
(unidentified), gas vacuolated, appearing during the winter;’(unidentified), gas vacuolated, appearing during the winter;’
in a Spirulina platensis [I2 11.in a Spirulina platensis [I2 11.
10
Table 2.1 Comparison of protein content in Spirulina and other foods
W) PA
-. sours ~.~ - Protein (“A dry-weight)
Beef 1 S-20
Egg 2 8
Fish 16-18
Soybean 41
Wheat 6-10
Rice 7
Chlorella 40-65
Spirulina 60-80
11
Table 2.2 Chemical analysis of Spirulina from spray dried (%dry
weight) [ 31.
Raw protein - - =- .-
Essential amino acids
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
Non essential amino acids
Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Histidine
Proline
Serine
Available lysine
Lipids
Fatty acids
Laurie
Myristic
- 70%
4.13%
5.80%
4.00%
2.17%
3.95%
4.17%
1.13%
6.00%
5.82%
5.98%
6.43%
0.67%
8.94%
1.08%
2.97%
3.18%
8 5 %
7.00%
5.70%
229 mgkg-’
644 mg.kg-’
:i’
1 2
Palmitic
Palmitoleic
Heptadecanoic
Stearic .- z. .~
Oleic
Linoleic
y-linolenic
Others
Ash
Calcium
Phosphorus
Iron
Sodium
Chloride
Magnesium
Manganese
Zinc
Potassium
Others
Carotenoids
p-carotene
Xanthophylls
Carbohydrates
Ramnose
Glucane
Cyclitols
Glucosamine and muramic acid
21,141 mg.kg-’
2,035 mg.kg-’
142 mg.kg-’
353 mg.kg-’
3,009 mg.kg-’
13,784 mg.kg-1
11,970 mg.kg-’
699 mg.kg-’
9.00%
1,3 15 mg.kg-’
8,942 mg.kg-’
580 mg.kg-’
412 mg.kg-’
4,400 mg.kg-’
1,9 15 mg.kg-’
25 mg.kg-’
39 mg.kg-’
15,400 mg.kg-’
57,000 mg.kg-’
4,000 mg.kg“
1,700 mg.kg-’
1,600 mg.kg-’
16.50 mg.kg-’
9.00 mg.kg-’
1.50 mg.kg-’
2.5 mg.kg-’:I ’
2.00%
1 3
Glycogen 0.50%
Sialic acid and others 0.50%
Nucleic acids 4.50%
Ribonucltiid _ ~. - 3 . 5 0 %
Deoxyribonucleic acid
Vitamins
Biotin
Cyanocobalamin
D-Ca-pantothenate
Folic acid
Inositol
Nicotinic acid
Pyridoxine
Riboflavin
Thiamine
Tocopherol
1 .OO%
0.40 mg.kg-’
2.00 mg.kg-’
11 .OO mg.kg-’
0.50 mg.kg-’
350.00 mg.kg-’
118.00 mg.kg-’
3 .OO mg.kg-’
40.00 mg.kg-’
55.00 mg.kg-’
190.00 mg.kg-’
Spirulina has been produced for commercial purpose in many
countries, according to its special characteristics mentioned above and
its safety in not possessing toxicity. The main production sites are in
USA, Japan, Thailand, Taiwan and Israel [ 1, 221. The pigments
(phycocyanin, p-carotene) of Spirulina also make this algae useful in
aquaculture, particularly as feed for tropical fish. [ 19,20,24].
1 4
The life cycle of Spirulina in laboratory culture is rather simple
[20] (Fig. 2.5). A mature trichome is broken in several pieces through
the formation of specialized cells, necridia, that undergo lysis, giving
rise to biconcave sqxtration disks. The fragmentation of the trichome at
the necridia produces gliding, short chain (two to four cells), the
hormogonia, which give rise to a new trichome. The cells in a
hormogonium lost the attached portions of the necridial cells, becoming
rounded at the distal ends with little or no thickening of the walls.
During this process, the cytoplasm appears less granulated and the cells
assume a pale blue-green color. The number of cells in hormogonia
increase by cell fission while the cytoplasm become granulated and the
cells assume a brilliant blue-green color. By this process trichomes
increase in length and assume the typical helicoidal shape. Random but
spontaneous breakage of trichomes together with the formulation of
necridia assure growth and dispersal of the organism.
Figure 2.5 Life cycle of Spirulina [20].
1 5
2.3 Phycobilisome and Phycobiliprotein Structures
2.3.1 Phycobilisome Components
-. The-accessory ~- light-harvesting apparatus of the
cyanobacteria and red algae is a water soluble multiprotein complex
known as the phycobilisome (PBsome). The PBsome is composed of
both phycobiliproteins (85% of the PBsome by mass) and non-
pigmented linker polypeptides (15% of the PBsome by mass) [25]. The
PBsome is attached to the exterior of the thylakoid membranes or
photosynthetic membranes, as shown in Fig.2.6 [26]. Light energy
harvested by the PBsome is transferred to the reaction center of
photosynthesis [27]. The basic building block of any antenna system is a
complex protein involving pigment molecules, chromophore covalently
or non-covalently associated with a protein component. Each type of
pigment-protein complex exhibits a specific spectral characteristic
depending upon the type of pigment molecule and its interaction with
protein component of the complex. Complexes with varying spectral
quality interact with each other to form the complete antenna. In
general, complexes within an antenna distal to the reaction centre
absorb at shorter wavelengths than those complexes proximal to the
reaction centre. The PBsome is assembled from two types of
polypeptide: (I) phycobiliproteins (PBPs) binding pigment molecules,
the chromophores, those serve to harvest light energy, (II) linkers, those
are involved in arranging the PBPs into a functional PBsome and may
also bind chromophores.
16
17
The pigment-proteins of the PBsome are arranged into
two structures, a core attached to the thylakoid membrane and several
rod fanning out from the core (see Fig.2.7). Glazer (1984) [29] has
described-that the corernay have-two or three cylinders depending upon
the species, and the number of rods attached to the core varies between
four and eight. In the case of bicylindrical cores, the two cylinders lie
side by side, and in the tricylindrical cores the third cylinder lies on top
of the two basal cylinders (Fig.2.8). The core structure of Synechocystis
sp. strain PCC 6701 (Fig.2.7) is one of the studied that the core was
composed of three cylinders lying parallel to the membrane consisting
primarily of allophycocyanin (APC). Glazer et al., (1983) [30] has
reported that some Synechococcus species (strain PCC6301 and
PCC7942) had a core made up of only two cylinders. Most of the
cyanobacterial PBsome have a three-cylinder core but in a few strains
the core may contain only two cylinders [31] (Fig.2.8). The major light-
harvesting polypeptide in the core is APC and in the rods is
phycocyanin (PC), phycoerythrins (PE) and phycoerythrocyanin (PEC).
The rods were radiated from the core in a fan-like array as shown in
Fig.2.8 [32].
The composition of PBsome in cyanobacteria and red algae
change in response to environmental changes such as light intensity,
light quality (only cyanobacteria) and nutrient availability. Grossman et
al., (1993) [5] has reported that cyanobacteria generally increase their
cellular contents of antenna proteins and pigments in response to low
light intensity. One of the most spectacular properties of cyanobacteria
is their ability to modulate the pigment composition of the PBsome ini
1 9
1 : PC1 andLRc
2,3 : PC2 or PE and LR
4 : AP, c?~,-~~*.~,
Lc and LcM
5:APandLc
LCM : 94 kDa
A : Calothrix PCC 7601
1 : PC and LRc
2,3:PCandLR
4 : AP, aAPB, /318.3,
Lc and LcM
LCM : 72 kDa
B : Synechococcus PCC 6301 and PCC 7942
Figure2.8 Schematic representa t ion of the hemidiscoidal
phycobilisomes structure [Tandeau de Marsac, N., 1994,
unpublished] A: Calothrix PCC 7601 B: Synechococcus
PCC 6301 and PCC 7942 For abbreviations, see Table 2.4 i’
20
response to light quality in order to maximise the absorption of
available light; a process described as “chromatic adaptation” [33].
Conley et al., (1988) [32] has found that there are at least two sets of
phycocyanin genes-involved in this process, one transcribes two light-
induced transcrips and the other encodes a single transcript present in
both red and green light in the chromatically adapting species Frenzyella
diplosiphon. Tandeau de Marsac (1977) [34] has demonstrated that
Synechocystis PCC670 1, PE-containing cyanobacterium, grown under
green light results in a stimulation of green light-absorbing PE and the
synthesis of the PE associated rod-linkers.
PBsome size and shape vary with species, and are often
dependent on the light conditions in which cells are grown [ 151. It is
known that in photosynthetic organisms, antenna size is inversely
proportional to the light intensity received by the cultures during
growth. Similarly, cyanobacteria respond to light intensity by increasing
(under low light intensity) or decreasing (under high light intensity)
[W-The polypeptide composition of PBsome varies widely
among strains of cyanobacteria. Adaptation mechanisms occurring
within the peripheral rods of PBsome are presently better understood
than those that occur in the core. [36].
Different morphological types of PBsome, they can be
divided into four classes: (1) hemidiscoidal (2) hemiellipsoidal (3)
bundle-shaped and (4) block-shaped [36]. Hemidiscoidal PBsome are
the most common and best described PBsome structures from various
cyanobacteria [5] (as shown in Fig.2.8). It can be described ad,
organelles, about 70 nm along the base, 30-50 nm in height and 14- 17
21
nm in width, attached to the stromal side of the thylakoid membrane
[37]. These PBsome have a mass of 4.5 to 1.5~10~ Da and contain 300-
800 covalently bound phycobilin chromophore [38].
-_ EssenGalJyall genes encoding structural components of
the PBsome have been isolated and characterized from many diffferent
organisms [S]. Table 2.3 [39] enumerates the different polypeptide
constituents of the PBsome, provides their gene designations and offers
a brief description of the function of each.
2.3.2 Phycobiliproteins
The phycobiliproteins (PBPs) are a family of brilliantly
pigmented water-soluble proteins that may constitute 50% of the soluble
proteins of the cyanobacterial cell [39]. The pigment-protein complexes
consist of linear tetrapyrole chromophores covalently attached to the
cysteine residues of the polypeptide via thioether linkage [27]. PBPs
from cyanobacteria and red algae are hetero-monomers consisting of
two nonidentical subunits, denoted a and p, which are present in
equimolar stoichiometry in the (c@) monomer [26] (as shown in
Fig.2.9). Each subunit differs in molecular mass, amino acid sequence
and chromophore content [36]. The fundamental assembly unit for all
PBsome is a stable phycobiliprotein trimer (c@)j. The PBPs (ap)
monomer of C-phycocyanin looks like a boomerang (see Fig. 2.9) [36].
Three monomers are arranged around a 3-fold symmetry axis in the
trimer (ap)3, while two trimers are assembled face-to-face into the
hexamer (@)6 to form a structure like a daugnut [9]. (see Fig.2.10) :i’
2 2
Table 2.3 Phycobilisome polypeptides and their genes [39]
Protein designation Gene designation Position and function
Rods cl” cpcA-_ .- ~--
PP C cpcB
a P E cpeA
PP E cpeB
LK C cpcG
LR (for PC hexamers) cpcCD
cpcHI
LK (for PE hexamers) cpeCDE
Phycobiliproteins that form the hexameric
buildin; blocks of the - peripheral rod
substructure*
Linker polypeptide that serves as an interface
between the peripheral rods a n d the
phycobilisome core
Different linker polypeptides associated with
PC hexamers of the peripheral rods
Linker polypeptides associated with PE
hexamers in the peripheral rods
Core RAP
PA P
0. A P B
P18 5
apcA
apcB
apcD
apcF
Lc apcC
LCM apcE
Phycobiliproteins that form the building blocks
of the phycobilisome core
Terminal energy acceptor in the core
Associate with LCM in the core as part of the
terminal energy acceptor
Small linker polypeptide of the core
Terminal energy acceptor in the core. May
help stabilize the core/or nucleate its
formation. May also establish a physical
association between the PBS and thylakoid
membranes.
* Not all organisms have phycobilisomes containing PE or PE linker polypeptides.
2 3
Figure 2.9 Stereoscopic plot of @$)-monomer [36]
‘?
. .
x
-1-.
Figure 2.10 Schematic side viewof a phycocyanin hexamer.
The1
molecules are labelled 1 to 6 with respect to the symmetrYi’
operation [13 61.
24
There are four major categories of PBPs, based upon
their spectral characteristecs. These are : (1) phycoerythrocyanin (PEC,
hA.max ~575 nm),(2) phycoerythrins (PE, hA,,,=565-575 nm), (3)
phycocyanins (PCj-3tA;,%=61 5-640 nm) arid(4) allophycocyanin (APC,
hA,,,,=650-655 nm). Glazer (1985) [40] has proposed the following
abbreviations for the biliprotein subunits and linker polypeptides of the
PBsomes (as shown in Table 2.4).
PEC and PE are found at the core-distal ends of the
peripheral rods. PC constitutes the portion of the peripheral rods
adjacent to the core and APC forms the major component of the
PBsome core substructure [36]. The intensely colored PBPs (AP, PC
and PE) have two dissimilar polypeptide chain that each subunit have
molecular weights in the range of 14,000 to 23,000. [ 121
Gantt et al., (1976) [41] proposed the first model of a
PBsome of Porphyridium cruentum, the core was composed of APC
surrounded by rods of PC and PE. However, it has been found in the
cyanophyte Mastigocladus laminosus, that the core of APC was similar
to the previous models, but the rods were composed of PEC as the
terminal hexamers in place of the PE [42]. PBsome from a
nonchromatic adapting cyanobacterium, eg. Spirulina platensis, is
composed of a central core containing allophycocyanin and rods with
phycocyanin and linker polypeptides in a regular array [43].
25
Table 2.4 Abbreviations for the biliprotein subunits and for linker
polypeptides [40].
- -Type oC+mlypeptide~~ A b b r e v i a t i o n
Phycoerythrin subunits aPE, pPE,yPE
Phycoerythrocyanin subunits aPEC, PPEC
R-Phycocyanin subunits a R P C, PWC
C-Phycocyanin subunits cxpc, ppc
Allophycocyanin subunits a? P”
Allophycocyanin B a subunit a A P B
P-type core biliprotein subunit PM W
Rod linker polypeptides LRM W
Linker attaching rod elements to core LRCM W
Core linker polypeptides LcM W
Linker attaching core to membrane LCMM W
Abbreviations: PE, phycoerythrin; PEC, phycoerythrocyanin; RPC, R-
phycocyanin; PC, C-phycocyanin; AP, allophycocyanin; APB,
allophycocyanin-B; L, linker polypeptide; MW, apparent molecular
weight; R, rod; C, core; M, thylakoid membrane.
2 6
Comparisons of partial or complete amino acid
sequences of phycobiliproteins have shown that each subunit is highly
conserved among different cyanobacterial species (about 80% identity)
and that-each phycobiliprotein subunit shares homology with all the
others, especially in the region of the conserved chromophore
attachment site [35]. Houmard et al., (1986) [44] reported that in
Synechococcus PCC6301, the sequences around the cystenyl residue
(Cys81) involved in the linkage of the chromophore (region 70-120)
show 100% identity. Glazer (1984) [45] has described that this portion
of the sequence is the most conserved among all the phycobiliprotein
sequences.
2.3.2.1 PBPs constituing the PBsome core
APC is a light-harvesting pigment-protein complex
found in the phytosynthetic apparatus of red algae and cyanobacteria. It
assembles the PBsome core with the assistance of three types of linker
polypeptides (L CM, LRc, and Lc). APC consists of a and p subunit
polypeptides with covalently attached bile pigment chromophores. Each
subunit contains one phycocyanobilin chromophore (PCB) (as shown in
Fig.2.11). From amino acid sequences, the PCBs are shown to be singly
bound to a- and p-Cys 82 [36]. These subunits are the longest
wavelength absorbing and fluorescing molecule in the PBsome. APC
occurs mainly in the trimeric form (oMcpApc)3. The genes for the a and
p subunits of APC are named apcA and apcB [46].
27
Allophycocyanin
common features
a-APCPCB 82
____________________ I____________________ 160 amino acid residues
P-APCPCB 82
____________________ I_________________--- 158-167 amino acid residues
Figure 2.11 General features of allophycocyanin subunits (a and p
subunits). The broken lines represent the linear polypeptide
chain of allophycocyanin and the PCB-binding sites are
indicated by bars. (one per subunit at homologous positions)
28
The apcA and apcB genes were isolated from an
Anabaena variabilis ATCC 29413 by Johnson et al., (1988) [ 121 by
using the allophycocyanin (apt) genes of Cyanophora paradoxa as
heterologous probe. They-found--that the genes appear to be present in~-
only one copy per genome and the apcA is in the upstream of the apcB.
Northern blot analysis showed that the apt genes gave rise to two
transcripts, a 1.4-kb predominant RNA and a minor 1.75-kb form, one
coding for both the alpha and beta subunits as a dicistronic messenger
and the other coding for the both phycobiliproteins (alpha and beta
subunits) and assosiated linker polypeptides. In addition, DiMagno and
Haselkorn (1993) [7] h ave isolated and characterized genes encoding
the phycobilisome core subunits, allophycocyanin a and p, and two
linker proteins from Synechocystis sp. strain PCC6714. They found that
the apcA, apcB, and gene for a small core linker protein (up&,) form an
operon, apcABC, with a single transcription start site and two possible
termination sites, one following apcB and the other following apcC. The
genes are in the order apcA, apcB and apcC. Northern hybridization
experiments identified two mRNAs of 1.5 kb and 1.8 kb. However, it
has found that there is only one transcription start site. The 1.5 kb
transcript is more abundant than the 1.8 kb transcript. This arrangement
is reasonable because the core requires many more copies of the APC
subunits than it does of the Lc linker protein. For the apcE gene,
encoding the protein that links the phycobilisome core to the thylakoid
membrane (LCM) in this organism, is not linked with apcABC operon
which is similar to that of Synechococcus PCC7002 [47]. However, in
C a l o t h r i x PCC7601 [48], C . paradoxa [47] a n d Synechococcus’~~
PCC6301 [49] the apcE gene is found upstream and close to the apcA
29
gene. Nucleotide sequences of Calothrix PCC7601 were determined by
Houmard et al., (1988), [3 l] the five apt genes, namely apcA1 (ahpl),
apcA2 (aAp2), apcB1 (PAP’), apcC (Lc7.*), and apcE (LcMg2) was
identified.- Four oftkese genes -are adjacent on the chromosome and
form the apcElAlBlC gene cluster and no genes have been found close
to the apcA2 gene. Only a single set of genes encoding the aAPC and
P AP’ subunits was found in Mastigocladus Zaminosus. [38] In addition, a
second gene (apcA2), encoding an aAPC subunit (denoted oApc2) with
59% sequence identity to aAPC1 was found in Calothrix PCC7601 [S],
but the function and location of this subunit is not known.
The core-membrane linker phycobiliprotein, also called
the anchor protein, may play a role in the association of PBsome with
thylakoid membranes. It is important in transferring energy from the
PBsome to the photosynthetic reaction centres [5]. It is the largest
chromoprotein in the PBsome with a molecular mass that varies from
70-128 kDa, depending on the organism. It is present in two copies per
PBsome. Lundell et al., (198 1) [8] suggested the LcM to be a new type
of biliprotein and to be one of the terminal energy emitters of the
PBsome. Bryant (199 1) [26] described that the apcE gene of
Synechococcus PCC7002 has been insertionally inactivated or
completely deletion, and the phenotype of the resulting mutants has
been determined. The mutant grows much slower than the wild type.
Immunological studies, as well as amino-terminal amino acid sequence
analyses, had shown that the L CM linker is highly conserved among both
cyanobacteria and red algae. The LcM proteins were shown to be :i’
30
multifunctional, hybrid polypeptides, that can be divided into 3 to 5
domains, each consisting of approximately 220 amino acids. The
amino-terminal portions of these proteins contain phycobiliprotein
domains- with a cyst&e binding site for a single PCB chromophore
WI.Offner and Troxler (1983) [50] had found that the a
subunit and p subunit of allophycocyanin from the unicellular
rhodophyte, Cyanidium caldarium, contain 160 amino acids and 16 1
amino acids, respectively. The a subunit contains one phycocyanobilin
chromophore attached at residue 80 by a cysteinyl-thioether linkage,
and the molecular weight calculated from the sequence is 18,160. The /3
subunit contains one phycocyanobilin chromophore attached at residue
81 by a cysteinyl-thioether linkage, and the molecular weight calculated
from the sequence is 18,125.
The PBsome core receives the excitation energy from the
peripheral rods and transfers it to the reaction centre in the thylakoid
membrane. A portion of the APC-containing complexes is composed of
different APC subunits, either the aAPB subunit, the p16.5 subunit (in
Mastigocladus Zaminosus) or the phycobiliprotein-domain of the LCM
pro te in . The aAPB subuni t i s encoded by the apcD gene.
Allophycocyanin-B was f i rs t purif ied from the unicel lular
cyanobacterium Synechococcus PCC6301 as a trimeric complex with
the composition (aMBpMC) [51]. AP-B was originally proposed to
function as a terminal energy emitter from PBsome and to play a role in
energy transfer from the PBsome to the chlorophyll-proteins of the,
thylakoids [5 13. Bryant (1991) [26] found that the apcD gene in
3 1
Synechococcus PCC7002 contains a polypeptide of 16 1 amino acids,
which shares considerable sequence homology (45% identity, 64%
similarity) with the aAPC subunit.
-. PMyks-tdmit f 11 -ho a op ycocyanin is encoded by the apcF
gene. The apcF gene encoding the AP-P-like polypeptide denoted p” of
Synechococcus PCC7002 reveals a polypeptide of 160 amino acids,
which is similar in amino acid sequence to the pmc subunit (48%
identity, 69% similarity) [26]. The complete amino acid sequence of
p’6.5 subunit of Mastigocladus Zaminosus was found to have 169 amino
acid residues [36].
Both of these genes (apcD and up@ encode minor
components of the PBsome core which have been believed to play
important roles in energy transfer and in the structural asymmetry which
is required to assemble the core on the thylakoid surface. The
conclusion from the analyses of these two mutants is that the apcD and
apcF gene products are not obligately required for PBsome assembly
and function [26].
2.3.2.2 PBPs constituting the rod elements of PBsome
The peripheral rods of PBsome contain phycocyanin
(PC), phycoerythrin (PE) or phycoerythrocyanin (PEC). The
chromophoric proteins in the rod have their light absorption maxima at
a shorter wavelength than the chromophoric proteins in the core thereby
focusing the excitation energy efficiently down to the chlorophyll
molecules in the reaction centre [52]. :i’
32
The Phycocyanins (PC)
The phycocyanin is a major component in the six rods
attached to the core. They are proximal to the core [53]. The PC
monomer- consists-&two dissimilar subunits, a and p, presented in-
equimolar amounts and assembled into trimers (a& or hexamers (@&
(as shown in Fig.2.10). Both subunits have linear tetrapyrrole
chromophores, designated as phycocyanobilin (PCB), covalently
attached to specific cysteine residues in the protein via thioether linkage
[54]. PCB is the chromophore typically found associated with the
subunits of PC. The blue-colored, deeply red-fluorescent C-phycocyanin
(C-PC) is the predominant PC form and contains three PCB
chromophores per (ap) monomer. All of them are located at Cys a-84,
p-84 and p-155 (see Fig.2.12) [36]. William et al., (1978) [55] found
that the a subunit of PC from Synechococcus PCC6301 has a single
PCB chromophore attached to cysteine residue 84, the p subunit of PC
has two chromophores attached to cysteine residues 84 and 155,
respectively. One, or in some cases two, of the peripheral, sensitizing
chromophores (a-84 PCB and/or p-155 PCB) have been replaced by
PXB (phycobi l iv io l in) , PEB (phycoerythrobi l in) or PUB
(phycourobilin) chromophores in each of the variants in order to adapt
to conditions more enriched on blue and green wavelengths of light.
However, the fluorescing p-84 PCB was conserved in all variants.
Glazer and Hixson (1975) [56] isolated R-Phycocyanin (R-PC or R-PC-
I) (R-, Rhodophytan) from the red alga Porphyridium cruentum as an
(a/3) trimer. The molecular mass of the complex was 103 kDa. The a:i’
subunit contains a single PCB while one of the p subunit chromophores,
33
a-PC
WC
- Phycocyania
common features
1 (84)---------_-_----- I-___________________---- 162 amino acid residues
PCB 84 2 (155)----------------- I----------------_ 1----------172 amino acid residues
Figure 2.12 General structure, common features and chromophore
variability of phycocyanins. The broken lines represent the
linear polypeptide chain and the 3 bilin-binding sites are
indicated by bars. All PCs contain a PCB at position p-84
:1,2=PCB;l=PXB,2=PCB;l=PCB,2=PEB;1,2=
PEB; 1 = PUB, 2 = PCB found in C-PC, PEC, R-PC, P-PC11
and WH8501 Phycocyanin (R-PC-III), respectively. (PCB =
Phycocyanobi l in , PXB = Phycobi l iviol in , PEB =
Phycoerythrobilin, PUB = Phycourobilin) [36].
3 4
that at position Cys 155 normally occupied by a PCB in C-PC, is
replaced by a PEB [56]. Ong and Glazer (1987) [57] isolated R-
Phycocyanin-II (R-PC-II) from the marine Synechococcus sp. strains
WH 8 103, WH 802Qand JVII 7803, as thefirst PEB-containing PC of-
cyanobacterial origin. Ong et al., (1984) [58] studied that the position of
the single PCB was assigned to Cys p-84 position and the two PEBs to
Cys a-84 and Cys p-155 by analysis chromopeptides. The PC isolated
from marine Synechococcus sp. strain WI 8501 was the first PUB-
containing PC. A PUB occurs on the a subunit at position Cys 84 and
the /3 subunit of this protein carries two PCB chromophores at position
Cys 84 and Cys 155 [59].
The presence of PC at the rod-core linkage position is
apparently essential for excitation energy transfer from the rods to the
core. At the rod-core linkage positions in the PBsome, PC and special
rod-core linker polypeptides (LRc) form stable rod-core complexes
together with APC complexes: (aPCpPC)6 . LRc .(aAPCpMC)3 .Lc. Conley
et al., (1986) [60] found that Calothrix PCC7601 has three copies of the
genes encoding the a and p subunits of PC.
In both prokaryotes and eukaryotes the p subunit gene
for PC is followed by the a subunit gene [61]. Certain cyanobacteria are
able to modulate the PC and PE composition of the PBsome under
different light qualities, maximizing absorption of prevalent
wavelengths of light. This phenomenon is called complementary
chromatic adaptation. In organisms that exhibit complementary
chromatic adaptation, there are at least two different sets of PC subunits;,
one set accumulates during growth of cyanobacteria in both red and
green light, while the other accumulates to high levels only in PBsome
from cells grown in red light [32].
Phycocyanins involved in rod-core subcomplexes (C-PC,
R-PC-I, R-PC-II orALPGIII) are constitutive’ PCs and are expressed
under all growth conditions under which PBsome are formed [36].
The Phycoervthrocyanin (PEC)
Phycoerythrocyanin is the shortest wavelength absorbing
rod element of those cyanobacteria which neither contain PE nor
perform complementary chromatic adaptation. The function of PEC is
the extension of the light-harvesting capacity of PBsome into the green
portion of the spectrum under medium or low-light conditions. The PEC
content of PBsome is subjected to regulation by light intensity: under
low light PEC-expression is strongly induced [36]. Bryant et al., (1976)
[62] found that the aPEC subunit contains a purple chromophore
(phycobiliviolin, PXB) whereas the two PCB chromophores of the p-
subunit are identical to those found in PC (see Fig.2.12).
The Phvcoervthrins (PE)
The phycobiliproteins of the red-colored phycoerythrin
(PE) family exhibit a great diversity in spectral properties as well as in
their chromophore and subunit composition. PEs carry only one or two
chromophore types, PEB and/or PUB, instead of the four chromophore
types found among members of the PC family (PCB, PXB, PEB, and
PUB) (as shown in Fig.2.13). Bryant (1982) [63] founded that
cyanobacteria contain either PEC or PE in the PBsome but never both, ”
and many cyanobacteria such as in Synechococcus PCC 7002 posses no
3 6
red-colored phycobiliproteins (i.e., neither PE nor PEC) but only the
blue-colored APC and C-PC. PEC does not occur in red algae. PE is the
only phycobiliprotein present in cyanobacteria, red algae as well as in
cryptomonads, and-is thus most suitablefor comparative studies to-
reveal structural, functional and phylogenetic relationships among these
organisms. In the peripheral rods of the PBsome, PE is located at the
core-distal periphery of the structure.
PE chromophore contents differ among cyanobacteria
living in freshwater and soil, and/or marine environments. The y
subunits found in PE of red algae and in PEII of cyanobacteria are the
second type of bihmctional polypeptide combined phycobiliprotein and
linker polypeptide in addition to the LcM [36].
3 7
Phycoerythrin common features
l(84) 2 (143a)a-PE ________-______ I_---------------------- I---------
3 (50/61) PEB 84 PEB (155)-. p-pE--- =-.-z. ------- ) :I 1 1------------- --z----;--; -_----- _______ _--
-30LR -_--_----_---______-____________________---------------------
l(94) 2 (133) 3 (209) 4 (297)y-PE (red -------------- I---------- I------------- I-----------__- I____algae)PE-II
3 (75) PEB (84) 2 (143a)a-PE _-_____ I_______ I_-___------------------ I---------
3 (50/61) PEB 84 PEB (155)P-PE ---------- l-l------------- I------------------- I-----------
PUB (94)y-PE -___-_-------- I--------------------------------------------
Chroomonas PC-645 (crvptomonad)(19) mesohiliverdin
al-PC-645 ------- ----------_-____-____________
a2-PC-645 ------- I-----------------------------
3 (50/61) PEB 8415,16-dihydrobiliverdin 2PEB (155)
p-PC-645 ---------- l-l------------- I------------------- I----------_
Figure 2.13 General structure, common features and chromophore
variability of phycoerythrins. The broken lines represent the
linear polypeptide chain and the bilin binding sites are
indicated by bars. All PEs contain a PEB at position p-84
and p-1 55. The other three bilin binding sites a-75, a-84, a-
143a and p-50-61 may be occupied by a PUB. PE-I
complexes from soil and marine cyanobacteria contain
linker polypeptides without bound bilins (Lam3’). PE-II from
marine cyanobacteria have one PUB and PE-I from red
algae four PUB or PEB bound to the linker polypeptide
(ysubunits). Cryptomonad PEs are modified with PCB, PEB;1
PUB and other novel chromophores [36].J’
38
2.3.3 Linker polypeptides, the skeleton of the PBsome
The non-chromophoric polypeptides (-15%), termed
linker proteins also make up a group of related proteins [5]. They induce
a face-to-fxe aggrzgationof PE,- PEC and PC trimers, and additionally-
cause the tail-to-tail joining of hexameric assemblies to form larger
aggregates such as peripheral rods and core-cylinders. Lundell and
coworkers (1981) [64] have shown that the ratio of PC to linker is
important in determining rod-length. Based upon experimental data
derived from rod assembly in vitro, they have proposed a model for rod
biogenesis, suggesting that PC interacts with another PC trimer in
preference to a PC hexamer (Fig.2.14). Finally, the hexamers assembly
into rods. These proteins also serve to connect the rods to the core, and
last but not least direct the assembly of the PBsome core and its
attachment to the thylakoid surface [36].
Linker polypeptides are the colorless polypeptides. They
are believed to be located mainly in the central cavity of the torus-
shaped phycobiliprotein hexamers or trimers. They can be divided into
four groups according to their function in the PBsome.
1. Lc, the small core linker polypeptides (a@Y)
In Calothrix PCC7601, the apcC gene (204 bp) located
240 nucleotides downstream from apcB1 encoding the small linker
polypeptide Lc7.* associated with AP in the core of the PBsome [35].
Molecular masses of 8-l 3 kDa have been indicated for other organisms
[36]. The core components from Synechococcus PCC7002, the Lc linker
polypeptide is suggested to be associated with trimeric APC at the
peripheries of the core cylinders. This polypeptide seems not to be”’
absolutely required for PBsome assembly, but its presence considerably
3 9
improves both the stability and energy transfer properties of PBsome
P61.2. LcM, the core-membrane linker polypeptide
-. Redlinger and Gantt (1982) [55] described that LCM-
(apcE gene product) is a high molecular weight. chromophoric
polypeptide, anchor protein attaches to the photosynthetic membranes
and may function in transfer of energy from the PBsome to
photosynthetic reaction centres.
3. LRMW, the rod linker polypeptide
They are involved in the assembly of the peripheral rod
substructure. They can be divided into two groups differing in their
molecular masses:
group I: the small rod linker polypeptide consists of an 8
to 10 kDa polypeptide
group II: the rod linker polypeptide consists of
polypeptides with masses of about 30 kDa.
4. LRC, rod-core linker polypeptides
A key position in the PBsome structure and energy
transfer pathway is the rod-core junction. A special class of linker
polypeptide with a molecular mass of about 30 kDa, the C-PC-
associated rod-core linker polypeptide (LRc), attaches the peripheral
rods to the PBsome core and form different types of C-PC-to-APC
interactions. The rod-core linker polypeptides are found to play two
main roles. Firstly, they associate the peripheral rods with the PBsome
core. Secondly, they impart a strong red-shift in the wavelength of
maximum absorbance and fluorescence emission of the central /3-84”’
40
phycocyanobilin chromophores, to enable an optimal rod-to-core energy
transfer [36].
41
2.3.4 Pigments
PBPs are all relatively small with protomers consisting
of an alpha and a beta subunits that each subunit contains one or more
covalently -bound -chromophore.~- [ 121 The brilliant colors of the PBPs
originate from chromophores, linear tetrapyrrole prosthetic groups,
known as phycobilins [36].
Four main types of phycobilins are present in
cyanobacteria and red algae: the blue-colored phycocyanobilin (PCB);
the red-colored phycoerythrobilin (PEB); the yellow-colored
phycourobolin (PUB); and the purple-colored phycobiliviolin (PXB;
also named cryptoviolin) (as shown in Fig.2.9) [36].
Phycobilin chromophores are generally bound to the
polypeptide chain at conserved positions either by one cysteinyl
thioether linkage on the pyrrole ringA of the tetrapyrrole or occasionally
by two cysteinyl thioether linkages on the A and the D pyrrole rings
S&ram et al., (1971) [ 131 has demonstrated that
phycocyanobilin is the prosthetic group of C-phycocyanin (C-
cyanobacterial) and allophycocyanin. Both of PBPs (C-PC and APC)
belong to the important class of photosynthetically active proteins of
blue-green algae. The structure of phycocyanobilin has been
investigated by means of mass spectral. The result of the mass spectrum
of the acid form of phycocyanobilin is clearly that it is a compound of
molecular weight 588 which contains 40 hydrogen atoms.
In a study of R-phycocyanin-I (R-Rhodophytan) a and p
subunits from the red alga Porphyridium cruentum, Ducret et al., (1994):
[66] showed that the a chain carries a phycocyanobilin pigment”
4 2
covalently linked at Cys84 and the p chain carries a phycocyanobilin
pigment covalently linked at Cys82 and a phycoerythrobilin pigment at
cys153.
4 3
Peptide-linked PHYCOCYANOBILIN Peptide-linked PHYCOBILIVIOLIN
C D
Peptide-6inked PHYCOERYTHROBILINS
E F
&w-HV-C)%-c3-- - -l-IN-Crr-CO- M ~-nN-c;I-C~-rP.-’
” /‘. -H n H H
Peptide-linked PHYCOUROBILINS-~
Figure 2.15 Structures of various types of phycobilins found in cyano-
bacteria and red algae, linked by thioether-linkages to PBPs
(A) phycocyanobilin; (B) phycobiliviolin; (C) and (D)
phycoerythrobilin singly or doubly linked, respectively; (E)
and (F) phycourobilin singly or doubly linked to the poly-‘,,
peptide backbone, respectively [36].
4 4
2.3.5 Organization and transcription of genes encoding
PBsome components
The organization patterns for genes encoding PBsome
components of s-al cyanobacteria were summarized by Bryant
(1991) [26]. In Synechococcus PCC7002, it was found that there were
two transcriptional types of genes encoding PBsome components, one
was polycistronic mRNA and the other was monocistronic mRNA. The
first transcriptional unit in the peripheral rods of the PBsome in this
organism is the cpcBACDEF operon, encodes all components of
peripheral rods except for the 29-kDa rod-core linker polypeptide
(LRc29). The second transcriptional unit is the PC-associated, rod-core
linker polypeptide of apparent mass 29 kDa, the product of the cpcG
gene. Pilot and Fox (1984) [67] found that the cpcB gene, which
encodes the ppc subunit of phycocyanin, lies 105 bp 5’ from cpcA
encoding the cc” subunit. These characterictic was found in
phycocyanin of other cyanobacteria (eg. Synechococcus PCC630 1,
Anabaena PCC7120) (see Fig.2.16) and in cyanelle, Cyanophora
paradoxa. The cpcC and cpcD genes, encoding a PC-associated linker
polypeptide were next identified from the region 3’ to the cpcA gene.
The cpcE and cpcF genes lie 5’ from cpcD, do not encode structural
components of the PBsome but are required for attachment of
phycocyanobilin to the cc” subunit.
The six genes (see Fig.2.16) encoding components of the
PBsome cores of Synechococcus sp. PCC7002 are also arranged in two
transcriptional types. The first of these transcriptional types, those{i’
encoding apcA, apcB, and apcC appear to produce polycistronic
4 5
mRNAs. The apcABC operon encodes the a and p subunits of AP and
the AP-associated core linker polypeptide. It is interesting to note that
the order of genes, with the apcA gene occuring 5’ to the apcB gene , is
opposite-that observed-for alll cpcBA operons and cpeBA operons
(operons of phycoerythrin) [68,69]. The second type appears to produce
monocistronic mRNAs, encoding apcD, apcE, and apcF. The apcD,
apcE, and apcF genes were encoded the oAPSB, the p” subunit, and the
LcM linker, respectively. In Calothrix sp. PCC7601 was found that the
organization of the genes encoding core component similars to that of
Synechococcus 7002
46
Synechococcus PCC7002
__ --
.a
(..........--..... -b
Synechococcus PCC6301
atp~ 1 Igene H 0rfW*...-.....-....
apcE
cpcB2
b b
Anabaem PCC7120-- -
cpcE cpcF cpcG1 cpcG2 cpcG3 cpcG4 .~
bb t-.-.
b b*
Figure 2.16 Comparison of the organization of genes encoding phycobilisome
components for the cyanobacteria Synechococcus PCC7002,
Synechococcus PCC6301, and Anabaena PCC7120. The width of the
arrows is roughly proportional to the steady-state abundance of the
transcripts, and dotted arrows indicate possible transcripts that have
not been detected experimentally. The hatched portion of
Synechococcus PCC7002 orfW indicates a region of this putative gene
that has not yet been sequenced. The box indicated “X” for the cpc
operon of Synechococcus PCC7002 indicates the position of a 38j.
codon open reading frame whose function is not known, [26]
4 7
2.3.6 Energy Transfer in PBsomes
PBsomes transfer absorbed light energy primarily to
reaction centre. The efficiency of energy transfer from the PBsome to
photosynthetic reaction centres approaches 100% [ 121. This implies that
the energy transfer mechanism must proceed rapidly in order to avoid
energy losses by competing radiative or non-radiative decay processes
[36]. Spectroscopic studies on PBsome indicate that the following
scheme of energy transfer is unidirectional from PE/PEC discs at the
periphery of the rods through PC discs to APC and the terminal emitters
in the core (LcM or/and aAPB ), terminating at the photosynthetic reaction
centres (as shown diagramatically in Fig.2.17) [SO].
H20'Phy
\erythrin
:(670 nm) ( (670 ILL) PSI1
chlae-
Figure 2.17 Energy flow in PBsome of Cyanobacteria and red algae.
Radiationless excitation energy transfer from short
wavelength (phycoerythrins) to long wavelength absorbing,
pigment-protein complexes (allophycocyanins). Energy is
finally transferred to and distributed between PSI1 and PSIi’
4 8
2.4 The molecular biology of cyanobacteria
2.4.1 Genome size of cyanobacteria
-. The-genome sizes of 12% strains of cyanobacteria,-
representative of all major taxonomic groups, lie in the range of 1.6~10’
to 8.6x 10’ daltons. They were described by Herdman et al., (1979) [70]
which were measured from the kinetics of renaturation of DNA. The
majority of unicellular cyanobacteria, which reproduce either by binary
fission or by budding, contain genomes of 1.6x 10’ to 2.7x 10’
daltons,comparable in size to those of other bacteria. Most
pleurocapsalean and filamentous strains possess larger genomes than
unicellular cyanobacteria. The genome sizes are discontinuously
distributed into four distinct groups which have means of 2.2x log, 3.6
x log, 5.0x 10’ and 7.4x 10’ daltons. The single strain of the genus
Spirulina (filamentous cyanobacteria that produce motile, helical
trichomes) has a small genome size of 2.53x 10gdaltons. The data
suggested that genome evolution in cyanobacteria occurred by a series
of duplications of a small ancestral genome, and that the complex
morphological organization characteristic of many cyanobacteria may
have arisen as a result of this process.
2.4.2 Molecular cloning of phycobilisome in Cyanobacteria
There were different methods employed to clone various
kinds of genes involving in phycobiliproteins and linker polypeptides
synthesis of cyanobacteria and eukaryotic algae. These cloning:i’
49
strategies can be grouped into three categories: cloning by sequence
similarity, polymerase chain reaction and gene expression.
Cloning by sequence similarity
-. - This--procedure for cloning-specific genes of interest is-
by using sequence similarity of previously cloned genes from other
organisms. This approach simply involves obtaining cloned DNA
fragments of published sequences and using them as hybridization
probes, usually at low stringency. Numerous cyanobacterial genes have
been cloned by this approach and due to its rapidity and simplicity, it is
one that should be explored before undertaking more complicated
cloning procedures. For example, Johnson, et al., (1988) [ 121 isolated
the genes encoding phycocyanin and allophycocyanin of Anabaena
variabilis ATCC294 13 using the phycocyanin (cpc) genes of
Agmenellum quadruplicatum and the allophycocyanin (apt) genes of
Cyanophora paradoxa as heterologous probes.
Cloning by polymerase chain reaction (PCR)
With the powerful method of polymerase chain reaction
technique in molecular biology, various kinds of genes have been
cloned by PCR. This cloning strategy employed oligonucleotide derived
from the conserved sequences of known genes as primers. A fragment
of the gene of interest can be amplified from genomic DNA using
primers mentioned above and used as probe for screening library. For
example, DiMagno and Haselkorn (1993) [7] isolated the genes
encoding allophycocyanin subunits and one linker protein from
Synechocystis 6714 using conserved regions at the N-terminal end of
apcA and C-terminal end of apcB for PCR reactions. The PCR produ&
was used as a probe to screen the genomic library.
50
Cloning by gene expression
Cloning by gene expression is rather complicated than
the other two strategies. A cDNA gene library is constructed in an
expression -vector @Z-&i plasmid or phage) and screened for protein-
production by using specific antibodies. Lemaux and Grossman (1984)
[71] identified the gene encoding the p subunit of phycocyanin of C.
paradoxa. Antibodies to mixtures of phycobiliproteins were used to
screen E. coli colonies containing cDNA of plastid DNA.
In Spirulina, various kinds of genes have been cloned
and their sequences are documented in Genbank (see Table 2.5).
Mostly, they are genes involving in photosynthesis and protein
synthesis. Two of them are genes responsible for fatty acid desaturation.
However, until now, there was no genes or sequences of light harvesting
complex of Spirulina published yet. Up to date, there are 26 genes from
S. platensis isolated and characterized which can be retrieved from
Genbank via electronic mail ([email protected]) as shown in
Table 2.5.
51
Table 2.5 Genes of S. platensis that have been cloned and
characterized as documented in Genbank (retrieve
@ncbi.nlm.nih.gov)
Gene name -ReferenceGene product
ribosomal protein S 12
ribosomal protein S7
Translation elongation
factor EF-G
Translation elongation
factor EF-Tu
organism
72rpsL S. platensis
S. platensis
S. platensis
72rpsG
fUS 72
72S. platensis
7316S rRNA 16s ribosomal RNA Spirulina
PCC63 13
Spirulina
PCC63 13
Spirulina
PCC63 13
S. platensis C 1
S. platensis C 1
73tRNA-IIe transfer RNA-IIe
23s rRNA 23s ribosomal RNA 73
serine esterase 74
75
an esterase
ATPase gamma ATPase gamma
subunit gene subunit
rpsI0 ribosomal protein S 10 S. platensis
ribosomal protein S2 S. platens is
76
77
77
rpsB
w- EF-Ts elongation
factor
S. platensis
:i
5 2
Table 2.5 Genes of S. platensis that have been cloned and
characterized. (continued)
Gene name
recA
ilvX
ilvY
fabZ
desA
1euB
desD
glnA
rbcL
rbcS
---Gene product _ organism Reference
recombination protein S. platensis *
strain IAM-
Ml35
Acetohydroxy acid S. platensis C1 7 8
synthase
Acetohydroxy acid S. platensis CI 7 8
synthase
(3R)-hydroxy- S. platensis **
myristoyl acyl carrier strain Italian
protein dehydrase
delta1 2 desaturase S. platensis ***
P-isopropylmalate S. platensis CI 7 9
dehydrogenase
delta6 desaturase S. platensis ****
Glutamine synthetase S. platensis 80
Ribulose-bisphos- S. platensis CI 1 8
phate carboxylase
large subunit
Ribulose-bisphos- S. platensis Cl 1 8
phate carboxylase
small subunit
53
* recA gene (accession U33924)was found by Vachhani, AK.
and Vonshak, A., 1995, unpublished data.
** fab2 gene (accession U4182 1) was found by Los, D.A.and
Murata, N+ 1995, unpublished data. _ _
*** desA gene (accession X86736) was found by Murata, N.,
Deshnium, P. and Tasaka, Y., 1995, unpublished data.
** ** desD gene (accession X87094) was found by Tasaka, Y,
1995, unpublished data.
Chapter 3
Materials and Methods
-. - - :.- -
3.1 Organisms and plasmids
3.1.1 Cyanobacterial strain
S. platensis C1 strain was kindly provided by Prof. Dr. A.
Vonshak, Algal Biotechnology, Ben-Gurian University of the Negev,
Israel. This strain was used as a source of DNA in cloning apt gene by
polymerase chain reaction technique.
3.1.2 Bacterial strains/S. platensis Cl library
The bacterial strain used as recipients for propagation
and subcloning of the different plasmids during this project was
Escherichia coli DH5a (supE4, hsdR17, recA1, endAl, gyrA96, thi-1,
reZA 1).
The host bacterial strain used for transfection and
propagation of recombinant lambda bacteriophages was E. coli LE392
(supE44, supF58, hsdR5 14, galK2, gall22, metB1, trpR55,lacYl).
The library of S. platensis Cl was constructed in
hDASHI1 by Deshnium, 1992 [Sl] and used for screening for the
allophycocyanin gene.
55
3.1.3 Plasmids
pRL498 [82], conferring kanamycin resistance, was
kindly provided by Prof. C. P. Wolk, Michigan State University, USA
and used- for subclonmgthe PCR-fragment-
pGEM4 [83], conferring ampicillin resistance, was used
for cloning and sequencing of apcABC gene.
3.2 Chemicals
All chemicals were of reagent grade and molecular biology
grade.
3.3 Enzymes
Restriction enzymes used in cloning were purchased from
either Boehringer or BRL company (USA). The lysozyme, RNaseA and
DNaseI were obtained from Sigma Co.
3.4 Media and culture conditions
All the media were sterilized for 15 min at 15 PSI, 12 1’C. For
the solid medium, 0.8% (top agar medium) or 1.5% of bacto-agar (agar
medium) was added before sterilization.
56
3.4.1 Spirulina platensis
3.4.1.1 Culture medium, Zarrouk’s medium [84]
-. -- z.- -
Component Concentration (g/l)
NaCl 1 .ooo
MgS04.7H20 0.200
CaClz 0.040
FeS04.7HzO 0.010
EDTA 0.080
K2m04 0.500
NaN03 2.500
K2S04 1 .ooo
NaHC03 16.800
A-5 solution 1 ml
A-6 solution 1 ml
57
A-5 Solution
Component Concentration (g/l)
-. H3B&---- ~- - -~ 2.860-
MnC12.4H20 1.810
ZnS0+7H20 0.222
CuS04.5H20 0.074
Moo3 0.015
B-6 Solution
Component Concentration (g/l)
NH4N03 229.6x 1O-4
K&r2(S0&.24H20 960.0x 1O-4
NiS04.7HzO 478.5x10-’
Na2S04.2Hz0 179.4x1o-4
Ti(SO& 400.0x 1 o-4
CO(NO~)~.~H~O 439.8x 1O-4
Zarrouk’s medium was sterilized for 15 min at 15 PSI. then
cooled slowly in the autoclave to room temperature to prevent
precipitation.
3.4.1.2 Stock and culture conditions
S. platensis C 1 was cultured at 3 5OC in Zarrouk’ si’
medium under illumination with white light of fluorescent lamp (Osram
58
40 watt) at 80 uE.mm2.sec-‘, agitated on a rotary shaker at 150 rpm. Cells
were transferred every two weeks for stock culture. This culture stock
(10 ml) was used to start culture in 250 ml flask containing 100 ml of
Zarrouk’s- medium&Us-in the- mid-log phasem(OD560 =- 0.45 or 4-5
days) were used for isolation of chromosomal DNA.
3.4.2 Bacteria
3.4.2.1 Culture medium, LB-medium (Luria-Bertani
Medium) [ 831
Component Concentration (g/l)
Tryptone 1 0
Yeast extract 5
NaCl 5
adjusted pH to 7.0
3.4.2.2 Culture conditions and storage
Bacteria were stored by adding 15% of steriled glycerol
to overnight culture (grown in liquid medium containing the appropriate
antibiotic) in eppendorf tube. After mixing, the stock culture was kept at
-7O’C until use. This culture stock (20 ~1) was used to start overnight
culture in bottles containing 5 ml of LB (supplemented with antibiotic
when necessary) at 37OC on a rotary shaker, 150 r-pm.
5 9
3.5 Buffers and Solutions (Sambrook, et. al., 1989) [83]
3.5.1 Lysozyme solution (Sigma#L-6876)
-. Lysozyme was dissolved in 10 n-&I Tris-HCl (pH 8.0) at
a concentration of 10 mg/ml.
3.5.2 Ribonuclease A (RNase A) (Sigma#R-5000)
RNase A was dissolved in 10 mM Tris-HCl (pH 7.5), 15
mM NaCl at a concentration of 10 mg/ml. The solution was heated at
1 OO’C for 15 minutes then cooled slowly to room temperature, aliquoted
and kept at -2O’C.
3.5.3 DNase I (Sigma #D-4263)
DNase I was dissolved in TM buffer at a concentration
of 1 mg/ml and kept it at -2O’C.
3.5.4 TM buffer
50 mM Tris-HCl pH7.4,10 mM MgS04
Sterile by autoclave
3.5.5 STET
0.1 M NaCl, 10 mM Tris-HCl (pH S.O), 1 mM EDTA
(pH S.O), 5% Triton X- 100
3.5.6
3.5.7
8 .0
Sterile by autoclave
TE buffer pH8.0
10 n&I Tris-HCl (pH S.O), 1 mM EDTA (pH 8.0)
Sterile by autoclave
TBE buffer
0.089 M Tris base, 0.089 M Boric acid, 1mM EDTA pH:i’
6 0
3.5.8 Denaturing solution
1.5 M NaCl, 0.5 M NaOH
3.5.9 Neutralizing solution
-. 1.5.J&F&Cl, 0.5 M Tris-HCLpH72, 0.001 M EDTA
3.5.102OxSSC
3 M NaCl, 0.3 M Trisodium citrate
3.5.1120xSSPE
pH7.7
3.6 M NaCl, 0.2 M Sodium phosphate, 0.02 M EDTA
3.6 Primers/Oligonucleotide synthesis
Primers for PCR and for sequencing were synthesized by Bio
Service Unit, National Center for Genetic Engineering and
Biotechnology, Bangkok, Thailand.
3.6.1 Primers for PCR
Sequence number : 786: 5’-(GA)TT(GA)TA(ACTG)GT
(TC)TC(TC)TT(ACTG)A(GA)(ACTG)CC(GA)TT-3’
Sequence number : 787: 5’-AA(CT)GC(ACTG)GA(CT)
GC(ACTG)GA(AG)GC(ACTG)CG(ACTG)TA-3’
Sequence number : 1843: 5’-GCCGAATTCAAGTTTTT
CCCCATGAAATG-3’
Sequence number : 1845: 5’-GCCGAATTCC(TG)(TC)
TG(TC)TG(TC)TC(ACTG)C(TG)(AG)AACCA-3’ :i’
6 1
G-3’
GG-3’
3.6.2 Primer for sequencing
Sequence number : 1690: 5’-CTGGCGATGTTACCC-3’
Sequence number : 169 1: 5’-ATCAGGACGTTTTTG
-. -__ I _
Sequence number : 1692: 5’-TGCATCCGTGACCTG-3’
Sequence number : 1693: 5’-AGACTTAGCAACTGC-3’
Sequence number : 1799: 5’-TCATTTTCTACCACAG
Sequence number : 1844: 5’-GCCGAATTCGATTACG
GAAGTGATTGCG-3’
Sequence number : 197 1: 5’-GCTACTGGTGAACTG-3’
3.7 Molecular biology techniques
Most of molecular biology techniques used in this work are as
described by Sambrook et al., (1989) [83] and Davis et al., (1986) [85],
unless otherwise stated.
3.7.1 Plasmid preparation
3.7.1.1 Small-scale preparation
Minipreparation of plasmid DNA was done by the
boiling method (Holmes and Quigley, 1981).
A 5 ml of overnight culture of recombinant bacteria was
centrifuged for 10 min at 6000 rpm. After removing the medium, the
bacterial pellet was resuspended in 450 yl of STET (0.1 M NaCl, 10:i’
mM Tris.Cl pH 8.0, 1 mM EDTA pH 8.0, 5% Triton X- 100). Then
62
suspension was added with 50 ~1 of a freshly prepared lysozyme
solution. The mixture was transferred to an appendorf tube, vortexed for
3 seconds then boiled for 40 seconds. The plasmid solution was clarified
by centrifugation for&l-On&~ at room temperature, The pellet of bacterial
debris was removed by using a sterile toothpick. After adding 50 ~1 of
2.5 M sodium acetate (pH 5.2) and 500 ~1 of isopropanol to the
supernatant, the mixture was incubated for 10 min at room temperature.
The pellet of nucleic acids were recovered by centrifugation for 10 min
at 4’C, 12,000 r-pm. After the pellet of DNA was dried, it was
resuspended in 50 u.1 of TDW.
The DNA solution (0.5 ug/yl) was directly used for
restriction analysis (3 ul per reaction).
3.7.1.2 Large scale preparation
An overnight culture of the recombinant bacteria (0.5
ml) or culture from glycerol stock was inoculated in 100 ml of LB +
ampicillin (100yglml) in 250 ml flask, agitated on a rotary shaker at
37”C, 200 r-pm for 12 to 16 hrs.
After harvesting of cells, the high yield plasmid was
obtained using QIAGEN KIT (Germany), following the method
recommended by the company.
3.7.2 Bacterial transformation
The calcium chloride procedure was used for
transformation of E. coli [ 831. :i’
63
The 1 ml of overnight culture was inoculated to 100 ml
of LB in 250 ml flask, agitated on a rotary shaker at 37’C, 200 rpm.
After 2.5-3 h of incubation (0.D at 560=0.3-0.4), the cells were
incubated- in ice for30 min. The- cells were harvested by centrifugation
at 3,000 rpm for 10 min at 4’C. The cell pellet was resuspended with 40
ml of ice-cold sterile 100 r&l MgC12 and centrifuged again as
previously described. After the pellet was dissolved with 15 ml of ice-
cold sterile 100 mM CaC12 and centrifuged, the pellet was resuspended
in 3 ml of ice-cold 100 mM CaC12. The competent cells were
maintained at 4’C for at least 2 h before use.
The plasmid DNA (10 ~1 containing about 50 ng of
DNA) was added to 200 ~1 of competent cells in a 1.5 ml eppendorf
tube. After 30 min on ice the cells were heat-shocked for 2 min at 42OC.
One millilitre of LB broth was then added and cells were incubated for
45 min at 37OC. The transformed bacteria (200 ~1) were spreaded on LB
agar plates containing the appropriate antibiotic (50 ug ml’ of
kanamycin and 100 ug ml-’ of ampicillin for all plasmids used in this
work). After 12-16 h the transformed bacteria could be picked up and
grown overnight in liquid medium for further screening.
3.7.3 Subcloning of DNA fragments
Plasmids, either pRL498 or pGEM4, were used for
subcloning the fragments of DNA. The fragments of DNA were
separated on a 0.7% agarose gel in 0.5xTBE buffer. The piece of gel
containing the fragment of interest was cut using a scalpel blade, andi,
the fragment was eluted by QIAEX KIT (Germany), following the
64
method recommended by the company. The DNA fragment (0.2 ug)
was then ligated to 0.1 ug of vector DNA using 1 unit of T4 DNA ligase
(BRL company) in a total volume of 10 ~1. All of the ligation mixture
was used to transm -?FcoZi DHSa. Therecombinant colonies were
characterized by small-scale plasmid isolation (section 3.7.1.1) and
restriction analysis.
3.7.4 Plating bacteriophages
3.7.4.1 Preparation of plating bacteria
The plating bacteria were prepared as recommended by
Davis et al., 1986 [85] and Sambrook et al., 1989 [83].
E.coZi LE392 was grown overnight in LB broth
containing 0.2% maltose; the sugar induces the maltose operon, which
contains the gene (ZamB) coded for the bacteriophage h receptor. One
millilitre of the overnight culture was used to inoculate 50 ml of LB
broth containing 0.2% maltose. The culture was grown overnight at
37OC with moderate agitation (250 rpm) until O.D. at 600 nm was
approximately 1.0. The cells were collected by centrimgation at 2,500
xg for 10 min at room temperature. The cell pellet was resuspended in
12.5 ml of sterile 10 mM MgSO4. This yields approximately 1-2x lo9
cells per milliliter. The cell suspension was transferred to a 250 ml
sterile flask and incubate for 1 hour at 37’C, 200 r-pm. The cells could be
stored at 4’C for up to 2 weeks. However, the highest plating
efficiencies were obtained when freshly prepared cells was used.
f
65
3.7.4.2 Plating lambda bacteriophage
The diluted phage suspension was added to lOOu1 of
plating cells. The mixture was incubated at 37’C for 20 min to allow the
bacteriophage particles to-adsorb to E. cati. Then 4 ml molten (45’C)
soft LB agar containing 10 .mM MgS04 was added to the mixture, and
overlaid onto prepoured plates (90 mm) containing 20 ml of LB agar.
The plates were closed and let stand for 5 min at room temperature to
allow the top agar to harden. Plaques appeared after the plates were
inverted at 37’C after about 6 hours of incubation. Plates were stored at
4’C for further screening.
3.7.5 Preparation of DNA from bacteriophage
The method used followed Davis et al., (1986) both for
the growth and preparation of bacteriophage [84].
3.7.5.1 Preparation of clear lysate
With a sterile Pasteur pipette, an agar plaque containing
a single plaque was transferred (one plaque contains approximately lo7
phages) to 50 ml of LB medium supplemented with MgS04 (10 mM
final concentration) and 200 ~1 of plating cells in 250 ml flask. The
culture was shaken vigorously with good aeration (approximately 250
rpm) at 37’C for lo- 12 hrs. The culture then became cloudy and
subsequently clear with lysis. After lysis 500 1-11 of chloroform was
added to the flask, and the flask was shaken further for 5 min at 37’C.
Bacterial debris was removed by centrifugation at 3,000xg for 10 min at
room temperature. The supernatant was transferred to a new tube and\\,
6 6
100 ~1 of 1 M MgS04 was added. The lysate could be stored at 4’C for
several months.
3.7.5.2 DNA preparation from bacteriophage
-. - Ten-mi-lli=kitre of TM buffer@0 -n-&I Tris ~87.4, 10 mM
MgS04) containing 320 ~1 of fresh DNase I solution (80 Kunitz
units/ml in TM) were added to 10 ml lysate. After incubation at room
temperature for 15 min, 2 ml of 5 M NaCl and 2.2 g of solid PEG-6000
were added. The PEG was left to completely dissolve in the lysate
before incubating on ice for 15 min. The phages were centrifuged at
12,000xg for 10 min, 4’C. The pellet was resuspended in 300 ~1 of TM
buffer and transferred into an eppendorf tube. The suspension was
added with 300 ~1 of chloroform, mixed well and centrifuged for 5 min.
The aqueous phase was transferred to a new tube and extracted once
with chloroform. After the addition of 15 ~1 of 0.5M EDTA and 30 ~1 of
5M NaCl, the aqueous phase was extracted with 350 ~1 of phenol,
followed by two more chloroform extractions. The DNA was
precipitated by adding 875 ~1 of ethanol and incubating on ice for 10
min. After centrifugation at 12,000 r-pm for 10 min at 4OC, the pellet was
rinsed with 70% ethanol 2 times. The pellet was dried and resuspended
in 50 ~1 of TDW (triple distilled water).
The DNA obtained still contained a large amount of
bacterial RNA. It was treated with RNaseA (50 ug/ml of final
concentration) for 30 min at 37’C followed by phenol extraction and
ethanol precipitation.
6 7
3.7.6 DNA sequencing
The sequencing of DNA was performed by dideoxy
chain termination method of Sanger et al., [86] using the Sequenase
Version -2;O Kit --(United States Biochemical Corporation, USA).
Double-stranded DNA was used for sequencing, following the protocols
essentially from Sequenase Version 2.0.
The DNA (3-5 pg) (preferably from plasmid miniprep)
was first denatured in an alkaline solution (0.2 M NaOH, 2 mM EDTA)
at 37OC for 30 min. The mixture was then neutralized by 0.1 volume of
3M sodium acetate (pH 4.5-5.5), precipitated with 2-4 volumes of
ethanol at -70°C for 15 min. The DNA pellet was redissolved in 7 pl of
TDW, followed by the addition of 2 pl of sequenase reaction buffer and
1 pl of primer (1 pmollyl). The annealing step was carried out by
warming the mixture to 37’C for 30 min. The primer was extended by
sequenase T7 DNA polymerase with the deoxynucleotide triphosphates
(dGTP, dCTP, dTTP 3.0 pM each of labeling mix). Then the labeling
mixture was added to each termination tube, containing the
deoxynucleotide triphosphate and a dideoxynucleotide triphosphate
(ddGTP, ddATP, ddTTP and ddCTP). The reactions were terminated by
the addition of stop solution (EDTA and formamide). They were
denatured by heating at SO’C for 2 min before loading on sequencing
gels.
6 8
3.7.7 Southern blotting
In Southern blotting, enzyme digested DNA was
transferred onto nylon membrane (HybondTM-N; Amersham, UK) by
downward capilluethod [87]. If the DNA to be transferred
contained fragments larger than 4 kb, the agarose gel was first soaked in
depurination buffer (0.25 M HCl) to just cover the surface of the gel.
After 30 min, the depurination buffer was replaced with denaturation
buffer for 40 min and then by neutralizing buffer for 30 min. The
transfer was set up in a pyramid form as shown in figure 3.1 and carried
out the transfer with 1OxSSC buffer. The membrane transfer was
completed after 1.5 hrs. The buffer was then removed from the
apparatus. Before lifting the gel, the wells of the gel were marked on the
membrane using pencil. Following the transfer, the membrane was
rinsed with 2xSSC and vacuum dried at SO’C for 2 hrs. The DNA was
then ready for hybridization with an appropriate probe.
/-Blotting Membrane
7 p~la&c~;ndxl;
Blotting paper
Paper towels
Figure 3.1 Schematic setup of downward capillary transfer of DNA
6 9
3.7.8 DNA labelling with 32P-dCTP
The labelling of DNA was performed by using Prime-a-
Gene Labelling System (Promega), a random hexanucleotide primers
synthesis, according-to themanufacturer’s instructions.
The DNA template (25 ng/30 ~1) was first denatured by
heating to 95OC for 2 minutes then chilled on ice. After the addition of
10 ~1 of Sxlabelling buffer, 2yl of unlabelled dNTPs, 2 ~1 of BSA and 5
~1 of a-32PdCTP, the mixture was polymerized by using 1 pl of Klenow
enzyme (5 units/pi)/. The reaction tube was mixed gently and incubated
at room temperature for 60 minutes. After the reaction was terminated
by heating at 95-100°C for 2 minutes and subsequently chilling in an ice
bath, the 5 ~1 of 0.2 M EDTA was added into the mixture. Prior to use
the probe was denatured by heating to 95’C for 2 minutes and placed on
ice.
3.7.9 Dot blotting
In dot blotting, DNA was heated to 95’C then chilled on
ice. After adding 1 volume of 2OxSSC, the DNA samples were spotted
onto the nylon membrane which was prewetted with 1OxSSC. Each spot
contained approximately 2 pl (about 0.5 l.tg) of DNA solution. After
allowing of each spot to dry, the membrane was wetted in denaturing
solution for 5 minutes and then in neutralizing buffer for 1 minute.
Following drying with filter paper, the membrane was vacuum dried at
80°C for 2 hrs. The DNA was then ready for hybridization with an
appropriate probe. :i’
7 0
3.8 Cloning of allophycocyanin gene from 5’. platensis Clstrain- -
3.8.1 Isolation of genomic DNA
-. - Gennmic- DNA -was extracted from the cells of S.-
platensis C1, grown to mid-exponential phase, according to the method
of Glatron & Rapoport (1972) [88] with slightly modification. The
harvested cells from 100 ml of the culture of S. platensis Cl (ODsbO =
0.45 or 4-5 days) were resuspended in 650 u.1 of 10 mM sodium acetate
(pH 4.5), 200 n&l Sucrose and 55 mM EDTA. Cell aliquots were
tranferred on to ice cold 15 ml corex centrifuge tube containing : 70 ul
of 20% SDS , 1.5 ml of cold phenol and 1 g of sterile glass beads . Cells
were broken by vortexing 4 to 8 pulses of 15 set at high speed, with an
interval of 30 to 60 set on ice between each pulse to prevent heating.
Then the suspension was centrifuged at 7,500 rpm for 15 min, 4’C. The
aqueous phase was collected and treated with the same volume of
phenol/chloroform/isoamyl alcohol (25:24: 1, by vol.) in order to
remove protein. After centrifugation at 12,000 rpm, 10 min, 4’C,
aqueous phase was extracted with chloroform/isoamyl alcohol (24: 1,
v/v) to remove the remaining phenol. Sodium acetate (pH 5.2) was
added to a final concentration of 0.3 M and DNA was precipitated by
adding 2 volumes of ice-cold ethanol and exposed to -2O’C for at least 2
hrs. DNA was recovered by centrifugation at 12,000 r-pm for 10 min at
4°C. The pellet was washed with ice-cold 70% (v/v) ethanol,
recentrifuged briefly and then dried at room temperature. The dried
DNA was dissolved in 50~1 of TE buffer (pH 8.0) or distilled water and!
kept at 4’C. The obtained DNA concentration was about 0.3 ug ~1~‘.
71
3.8.2 Amplification of partial apt AB from genomic DNA of
5’. platensis Cr by polymerase chain reaction
By comparing the apt AB gene sequences of
Synechowccus PUXOU [26],-- Synechomcus PCC6301 [44] a n d
Cyanophora paradoxa [87], conserved regions at the N-terminal end of
apt A and C - terminal end of apt B could be chosen for designing
oligonucleotide primers for PCR reactions. Two conserved regions at
positions lo- 16 and 110-l 17 (as shown in Fig.3.2), counted from the
amino terminus of the apt A and of the apt B of Synechococcus
PCC7002, respectively were selected. A DNA fragment of 0.9 kb was
amplified by a DNA thermal cycler apparatus (Perkin-Elmer cetus) The
sequences of the designed degenerated primers are as follows :
SAA(CT)GC(ACTG)GA(CT)GC(ACTG)GA(AG)GC(ACTG)CG
(ACTG)TA 3’ contained 2,048 types of primer with 23 bases, and
5’(GA)TT(GA)TA(ACTG)GT(TC)TC(TC)TT(ACTG)A(GA)(ACTG)
CC(GA)TT 3’ contained 4,096 types of primer with 24 bases.
The conditions used for the PCR reactions were: 29
cycles of 1.5 min at 92OC for denaturation, 1 min at 45’C for annealing,
and 1 min at 72’C for polymerization; 1 cycle of 1.5 min at 92’C for
denaturation, 1 min at 45’C for annealing, and 10 min at 72’C for
polymerization. The genomic DNA of 5’. platensis Cl was used as
template. The PCR mixture was then treated with Klenow fragment
enzyme 5 units and incubated at 37’C about 30 minutes for blunt ending
the PCR product . The amplified products were subcloned into pRL 498
and their nucleotide sequences were determined. :i’
72
AP a subunitsSyn 7002 MSIVTKSIW ADAEARYLSP GELDRIKAFV TSGESRLRIA ENLTGSRERI IKSAGDALFQ 6 0Syn 6301 S E T V VG DR QTIAES VKQ NQCyan0 T D S A AS ER QILTDN VRE Q Q
Syn 7002 KRPDWSPGG NAYGEEMTAT CLRDMDYYLR LITYGWAGD VTPIEEIGLV GVREMYKSLG 120Syn 6301 _- V D L V s I I R K-I_ I_Cyan0 I E L V An- L K - N
Syn 7002 TPVDAVAQAV REMKAVATGM MSGDDAAEAG AYFDYVIGAM ESyn 6301 IE EG EL SA AL LT E D A G L SCyan0 VA EG SA SV GL LS D A S ALQ
AP p subunitsSyn 7002 MQDAITSVIN SADVQGKYLD GSAMDKLKAY FTTGALRVRA ASTISANAAA IVKEAVAKSLSyn 6301 A A AS SSALDR S Qs E A S SAL Vcyan0 P A AA TASVEK S QT E A A SSA I
6 0
Syn 7002 LYSDVTRPGG NMYTTRRYAA CIRDLDYYLR YATYAMLAGD PSILDERVLN GLKETYNSLG-~ 120Syn 6301 I E L TCyan0 I D V T
Syn 7002 VPVGSTVQAI QAMKEVTAGL VGADAGREMG VYFDYICSGL SSyn 6301 I A V I S P VL s SCyan0 VA1 A G P IY s G
Figure 3.2 Comparison of the AP a and p subunits amino acid
sequences of Synechococcus PCC7002 [26], Synechococcus
PCC6301 [44] and Cyanophora paradoxa [87]. Selected
amino acid sequences are highly homologous and they are
indicated by underlines and bold. Blank positions indicate
identity with the Synechococcus PCC 7002 sequences at
that position.
73
3.8.3 Screening of genomic library of 5’. platensis Cl
The screening of a genomic DNA library of 5’. platensis
C1 was performed according to the standard method for plaque
hybridization [82],About 1,000 plaques oftherecombinant clones (200
plaques/selection plate) were tranferred to 5 nylon membranes
(HybondTM-N; Amersham UK). The membrane containing hDNAs were
denatured in denaturing solution for 7 minutes, and soaked 2 times in
neutralizing solution for 3 minutes. Then the membranes were washed
once with 2xSSC. The membrane were transferred to dry filter paper
and air dry, plaque side up. The membranes were baked for 2 hours at
8O’C between sheets of filter paper. The baked membranes were pre-
hybridized with SxSSPE, 5xDenhard’s solution, 0.5 %(w/v) SDS and
50% formamide at 42’C for 1 hour. The membranes were hybridized for
at least 12 hours at 42’C with a 32P- labelled probe (amplified partial apt
AB of S. platensis) prepared by using random primer labelling kit
(Promega) as described in section 3.7.8. After hybridization, the
membranes were washed with 2xSSPE, 0.1% SDS at room temperature
for 10 minutes, 2 times and 1 xSSPE, 0.1% SDS at 65’C for 10 minutes
and exposed to X-ray film (Kodak), at -7O’C.
3.8.4 Southern blot analysis
For Southern blot analysis, l-5 pg of each DNA (h or
genomic DNA) was digested with appropriated restriction enzymes. The
digested DNA fragments were fractionated on an 0.7% agarose gel and
transferred to a nylon membrane by downward method and theI ’
membrane was baked for 2 hours at SO’C. The pre-hybridization,
74
hybridization and washing of the membrane were performed as
described in section 3.8.3 and the membrane was exposed to X-ray film
at -70°C.
3.8.5 Characterization of positive clones
The DNA fragment which hybridized with the
appropriate probe was isolated from the positive clones using QIAEX
KIT (Germany), following the method recommended by the company
and subcloning into pGEM 4. The nucleotide sequence of subcloned
DNA fragment was determined by the dideoxy chain termination
method [90] using a DNA sequencing kit as described in section 3.7.6
on single-strand DNA templates according to the protocol of
manufacturer.
3.8.6 Amplification of complete apt AB and partial apt C from
genomic DNA of 5’. platensis Cl by PCR
Two degenerated oligonucleotide primers were designed
from upstream sequences of apt A of 5’. platensis and conserved region
of apcC. The conserved region of apcC was obtained by comparison
and alignment of apcC with sequence of Synechococcus PCC7002 [26],
Synechococcus PCC6301 [44], Synechococcus elongatus [Soga, M.,
1994, unpublished], Calothrix sp. Strain PCC 7601 [3 11, Fischerella sp.
Strain PCC 7603 [89]. The conserved region of apt C at the position
from 37 to 42 (as shown in Fig.3.3), counted from the amino terminus
of apcC of Synechococcus PCC7002. The two oligonucleotides
corresponding to these regions including EcoRI restriction site at 5’ end”
75
(indicated by underlines) were synthesized. The sequences of the
designed primers are as follows :
5’ GCCGAATTCAAGTTTTTCCCCATGAAATG 3’
contained- f type of+imerwith 2-9 bases, and - -
5’ GCCGAATTCC(TG)(TC)TG(TC)TG(TC)TC(ATCG)C(TG)(AG)AA
CCA 3’ contained 256 types of primers with 29 bases.
The oligonucleotides were used as primers (-250 ng) for
amplifying apt AB containing partial apt C by PCR using a DNA
thermal cycler apparatus (Perkin-Elmer cetus).The conditions used for
the PCR reactions were: 29 cycles of 1.5 min at 92’C for denaturation, 1
min at 5O’C for annealing, and 2 min at 72’C for polymerization; 1 cycle
of 1.5 min at 92’C for denaturation, 1 min at 5O’C for annealing, and 10
min at 72’C for polymerization. The genomic DNA of S. platensis Cl (at
various concentration) was used as a template. The amplified products
were subcloned into pGEM4. The nucleotide sequences of the cloned
PCR fragment were determined by using a DNA sequencing kit as
described in section 3.7.6.
76
LC7.8
Syn 7002 MRMEKITACV PSQSRIRTQR ELQNTYFTKL VPYDNWFREO aIMKMGGK1 VKVQLATGKP 60syn 6301 M M R I L PSK F YDA QL I E A R Psyn elan M M K I V QTR Y YEN QM V EF KPCal 7601 --ALKV +--QTS- - ~- Y FEN-m . - M M V EA KQFis 7603 G L K I V QTR Y YDA QM V - E A K Q
Syn 7002 GTNTGLT>
Syn 6301 N T T L>
Syn elan G V T >
Cal 7601 G T T >Fis 7603 GI T A>
Figure 3.3 The comparison of the amino acid sequences of apcC gene
products of Synechococcus PCC 7002[26], Synechococcus
PCC 6301 [44], Synechococcus elongatus [Soga, M. un-
published 19941, Calothrix sp.Strain PCC760 1[3 l] and
Fischerella sp.Strain PCC7603 [89]. Selected amino acid
sequences are highly homologous and they are indicated by
underlines and bold letters. Blank positions indicate identity
with the Synechococcus PCC7002 sequences at that
position.
Chapter 4
Results and Discussions
4.1 Construction of apcAB probe from S. platensis C1 by Polymerase
Chain Reaction
For construction of apcAB probe from S. platensis Cl by PCR,
two amino acid conserved sequences of apcAB gene found in
Synechococcus PCC7002 [26], Synechococcus PCC6301 [44] and
C y a n o p h o r a paradoxa [88] w e r e u s e d f o r d e s i g n i n g t w o
oligonucleotide primers. The sequence NADAEAR (residues lo- 16 in
a-subunits) was chosen for synthesizing the 5’ primer, while
NGLKETYN (residues 110-l 17 in P-subunits) was chosen for the 3’
primer. A 900 bp fragment (as shown in Fig.4.1) was specifically
amplified from S. platensis Cl genomic DNA as template by PCR using
the following primers 5’-AA(CT)GC(ACTG)GA(CT)GC(ACTG)GA
(AG)GC(ACTG)CG(ACTG)TA-3’ (primer up) and 5’-(GA)TT(GA)TA
(ACTG)GT(TC)TC(TC)TT(ACTG)A(GA)(ACTG)CC(GA)TT-3’
(primer down). PCR amplification was performed as described in
materials and methods (chapter 3, section 3.8.2). The amplified DNA
fragment was subcloned into pRL498, (resulting in pMG003), and its
nucleotide sequence was determined by dideoxy nucleotide termination
method as described in materials and methods. The deduced amino acid
sequences were analyzed with the DNA analysis software DNAsis (as’\,
shown in Fig.4.2). A homology search was performed using the BLAST
78
program (National Center for Biotechnology Information (NCBI),
National Library of Medicine, NIH, Bethesda, MD, USA). The analysis
result demonstrated that the amplified fragment contained genes
encoding -parts of thPcxand /3 subunits ofthe allophycocyanin genes.
The deduced amino acid sequence of the a subunit shows 91% (98%),
85% (95%) and 84% (100%) identity (similarity) and the p subunit
shows 88% (97%), 76% (94%) and 82% (92%) identity (similarity)
when aligned with those from Synechococcus elongatus, Cyanophora
paradoxa, Synechocystis PCC6714, respectively (as shown in Fig.4.3).
This result suggests that pMG003 contains a part of apcAB gene of
S. platensis C 1.
4 .2 Isolation of the allophycocyanin gene from a genomic DNA library
of S. platensis C1
The genomic DNA library of S’. platensis Ci constructed in
hDASHI1 by Deshnium, 1992 [8 I] was screened by plaque
hybridization using a 32P-labelled apcAB probe. Two hybridizable phage
clones, namely VAl, VA2 were obtained from 1,000 recombinant
plaques. hDNAs of VA1 and VA2 were isolated and subjected to
Southern blot analysis. The results showed that the two clones were
identical in the restriction and hybridization pattern, and their nucleotide
sequences revealed the sequence of allophycocyanin gene (as shown in
Fig.4.4). As such, VA1 clone was selected for further characterization in
details by Southern blot analysis and nucleotide sequencing. From:i’
7 9
-900 bps
Figure 4.1 The amplified PCR products from genomic DNA of
S. platensis Ci (180 ng) electrophoresed on 1% agarose gel.
A : h-Hind11 (molecular size marker), B : Negative control
(no template), C, D, E, F and G : amplified products from
primer concentrations 300, 250, 200, 100 and 50 ng,
respectively (MgC12 concentration used was 1mM)
7 9
A B C D E F G
-. -
9416 bps-6557 bps-4361 bps-
2322,2027 bps=
-901 D bps
Figure 4.1 The amplified PCR products from genomic DNA of
5’. platensis Ci (180 ng) electrophoresed on 1% agarose gel.
A : h-HindIII (molecular size marker), B : Negative control
(no template), C, D, E, F and G : amplified products from
primer concentrations 300, 250, 200, 100 and 50 ng,
respectively (MgCl, concentration used was 1 mM)
80
a subunit
ATCATCAAGGAAGCAGGJUiACCAACTTTTCC!AAAA?i CGTCCTGATGTAGTCTCTCCCI I K E A G N Q L F Q K R P D V V S P
GGTGGAAATGCCTACGGTGAG-~~TGA~TGCCACCTGCCTGCGGGATCTAGACTACGGNAYGEEMTAT~CL-RD L - D Y
TACCTGCGTCTGATCACCTACGGAATTGTTGTTGCTGGCGATGTTACCCCCATTG~G~Y L R L I T Y G I V A G D V T P I E E
ATCGGGGTTGTAGGTGTTCGCGAAATGTACAAATCTCTCTTGGTI G V V G V R E M Y K S L G
f3 subunit
TCCGTAATCAACTCCTCTGACGTTCAAGGTAAATACCTGGS V I N S S D V Q G K Y L D R S A I Q
AllACTGAAAGCCTATTTCGCTACTGGTGAACTGCGCGTTCGTGCAGCAACCACCATCK L K A Y F A T G E L R V R A A T T I
AGCGCTAATGCAGCTAACATCGTTAAGGAAGCAGTTGCTAGTCTCTGCTGTACTCCS A N A A N I V K E A V A K S L L Y S
GATATCACCCGTCCCGGTGGTAATATGTATGTACACCACTD I T R P G G N M Y T T
Figure 4.2 The nucleotide and deduced amino acid sequences of partial
apcAB gene from S. platensis Cl.
8 1
a subunits
MSVVTKSI YLSPGELDRIKNFVSTGERRLRIAQTLTENRERIVKQAGDQLFQMSIVTKSI YLSPGELDRIKSFAASGERRLRIAQILTDNRERIVREAGQQLFQMSIVTKSI YLSPGELDRIKAFVTGGAARLRIAETLTGSRETIVKQAGDRLFQ
* ** ***IIKEAGNQLFQ
._ - - =--KRPDWSPGGNAYGEEMTATCLRDLDWLRLVTYGIVAGDVTSLGKRPDIVSPGGNAYGEEMTATCLRDLDWLRLVTYGWAGDATSLGKRPDIVSPGGNAYGEEMTATCLRDMDWLRLVTYGWSGDVTRSLG**** ******************* ****** *** * ** l ****** *** *** ***
KRPDWSPGGNAYGEEMTATCLRDLDWLRLITYGIVAGDVTPIEEIGWGVREMYKSLG
Syn elonCyan06714
Sp.Cl
Syn elonCyan06714
Sp.Cl
Syn elon TPIPAVAEGIRAMKNVACSLLSAEDAAEAGSYFDFVIGAMQCyan0 TPVAAVAEGVRSAKSVATGLLSGDDAAEAGSYFDYVIAALQ6714 TPIEAVAQSVREMKEVASGLMSSDDAAEASAYFDFVIGAMSSp.Cl
p subunits
Syn elon MQDAITAVINASDVQGKYLDTAAMEKLKAYFATGELRVRASVISANAANIVKEAVAKSLCyan0 M Q D P I T A V I N A A D V Q G K Y L D T A S V E K L K S Y F Q T G E L R V R A S L6714 MQDAITAVINSADVQGKYLDGAAMDKLKNYFASGELRVRASL
*** ******** *** ** ******** l ** * ********
Sp.Cl SVINSSDVQGKYLDRSAIQKLKAYFATTISASL
Syn elon LYSDITRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDPSILDERV S L GCyan0 LYSDITRPGGNMYTTRRYAACIRDLDYYVRYATYAMLAGDTSILDERV S L G6714 LYSDVTRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDASILDERV S L G
**** *****s-e***
Sp.Cl LYSDITRPGGNMYTT
Syn elon VPIAATVQAIQAMKEVTASLVGADAGKEMGIYFDYICSGLSCyan0 VPVGATIQAIQAAKEVTAGLVGPDAGREMGIYYDYISSGLG6714 VPISSTVQAIQAIKEVTASLVGADAGKEMGWLDYICSGLSSp.Cl
Figure 4.3 The deduced amino acid sequence of the amplified DNA
product from S. platensis Cl (Sp. Cl) compared with
allophycocynin sequences of Synechococcus elongatus (Syn
elon) [Shimazu, T., Soga, M., Hirano, M. and Katoh, S.
Unpublished, 19941, Cyanophora paradoxa (Cyano) [87]
and Synechocystis 6714 (6714) [31]. The identical amino
acids are indicated by asterisks using blast program
([email protected]). Two conserved amino acid
residues are indicated by boxes which were chosen fori’
designing 5’ and 3’ primers in PCR.
82
Southern blot analysis, a restriction map of VA1 clone could be
determined as shown in Fig.4.5 and Fig.4.6. It was found that the apt
gene of 5’. platensis Cl resided on the 1.6 kb EC&I-EcoRV fragment.
However,- the resultfromnucleotide sequencing and DNA analysis (data
not shown) indicated that only the upstream region along with the upper
part of apcA (252 bp) were found on the 1.6 kb EcoRI-EcoRV fragment.
The physical map of the 1.6 kb fragment is shown in Fig.4.6. By
comparing the protein sequence of the Synechococcus PCC7002 [26],
Synechococcus PCC6301 [44] and Cyanophora paradoxa [88] it was
apparent that the 3’ end of the complete apcAB genes were not
contained on the 1.6 EcoRI-EcoRV fragment. Reprobing the same
library with the either apcA probe (1.6 kb EcoRI-EcoRV fragment) or
apcB probe (EcoRV-BamHI fragment of pMG003), it was found that
there was neither plaques hybridizable with apcB probe nor with both
apcA and apcB probe. Only 2 plaques hybridized with apcA probe was
found. As such, it can be concluded that there was no complete apt
genes in this library, resulting in employing cloning of apt gene from S.
platensis C r by using conserved sequences of apt gene in other
organisms as primers by polymerase chain reaction technique.
83
VA1
12 3
0-- - -
0
0
M V-w)- 21226
- 3530
- 1375
VA2
M @P)
-21226
-
-3530
- 1375
Figure 4.4 Southern blot analysis of two recombinants hDNAs isolated
from two positive clones, VA1 and VA2 respectively.
hDNA from each clone digested with restriction enzymes,
EC&I, lane 1; EcoRV, lane 2; EcoRI and EcoRV, lane 3,
were electrophoresed on 0.7% agarose gel and transferred to
a nylon membrane. The blots of DNA were hybridized with
a 32P-labelled apcAB probe (amplified partial apcAB of
5’. platensis). Molecular marker (M) is hDNA digested
with Hi&III and EcoRI, shown on the right of each picture.
-21226
cl48,4973 .--4268
84
1 2 3 4 5 M @P)
-2027-1904
-1584-1375
Figure 4.5 Southern blot analysis of one positive clone (VAl) which
was digested with EcoRI, lane l;EcoRV, lane 2;EcoRI and
EcoRV, lane 3;BgZII, lane 4;EcoRI and BgZII, lane 5. The
blot of DNA was hybridized with a 32P-labelled apcAB
probe (amplified partial apcAB of S. platensis). Molecular
marker (M) is hDNA digested with Hi&III and EcoFCI,
shown on the right site.
85
ECORI EcoRV
I--9kb-1
Rightarmlambda
Figure 4.6 Restriction map of 1.6 kb EC&I-EcoRV fragment of VA1
lambda phage containing upstream region of apcA region
along with the upper part of apcA of 5’. platensis Cl. The
arrow indicates the direction of the apcA gene. The broken
lines indicate left and right arm of h DASHII.
86
4.3 Cloning of apcAB and par t ia l apcC of 5’. pZatensis Cl by
Polymerase Chain Reaction
After geticDNA library of S”p&tensis Ci was screened
three times by using apcB probe, there was no positive clone. The
genomic DNA library was probably obtained from several
amplification, so the wanted gene may be eliminated. In this strategy,
cloning of allophycocyanin gene operon from S. platensis Cl by PCR
was chosen as an alternative, based on the knowledge of the
organization of the allophycocyanin gene.
Two oligonucleotides were designed for amplification of the
complete apcAB gene by PCR. The upstrand primer sequence was
obtained from upstream region apcA of S. platensis C1 (see Fig.4.6) and
the downstrand primer was designed from conserved regions of the
apcC genes of Synechococcus PCC7002, Synechococcus PCC6301,
Synechococcus elongatus, Calothrix PCC760 1 and Fischerella
PCC7603. The sequence FREQQR (residues 37-42 in Lc, see Fig.3.3)
was chosen for the 3’ primer, resulting in the degenerated primer as, 5’-
C(TG)(TC)TG(TC)TG(TC)TC(ATCG)C(TG)(AG)AACCA-3’. The
sequence of 5’ primer was 5’-AAGTTTTTCCCCATGAAATG-3’. The
facilitate subcloning of the amplified product into the plasmid, and
EcoRI restriction site proceeded by GCC (GCCGAATTC was added to
both primers). The optimal conditions used for the PCR reactions were
perfomed as described in materials and methods (chapter 3, section
3.8.6). After amplification, DNA products were checked on 1% agarose
gel (as shown in Fig.4.7), the band showed the equivalent size (about:’
87
1.8 kb) that corresponded to the expected size of apcABC gene of
Synechococcus PCC7002. These amplified DNA fragments were
subcloned into pGEM4, and sequenced by the dideoxy nucleotide
termination method-theobtained fragment-yas named pMG004.
4.4 Nucleotide sequence analysis of the apcABC operon
The nucleotide sequences of 1.8 kb fragment of the amplified
product (present in Fig.4.8) were determined. The 1.8 kb fragment
contains putative apcA, apcB and partial apcC of S. platensis Cl. The
nucleotide sequences of these genes can be seen in Genbank with an
accession number X95898. The deduced amino acid sequences derived
by translation of the apcA, apcB and apcC genes with the DNA analysis
software DNAsis. They are composed with those of the corresponding
phycobilisome component already known using the BLAST program.
The organization of the genes is in the order apcA , apcB and apcC with
ORFs (open reading frames) of 483 bps, 483 bps and 108 bps,
respectively. Bryant (1988) [47] h as described that the apcA gene
encoding the APCa is found 5’ to the apcB gene that encodes APCp in
Synechococcus PCC7002. Generally, the order of the apt genes in all
cyanobacteria, with the apcA gene occurring 5’ to the apcB gene, is
opposite to that observed for all cpcBA operons and cpeBA operons
[26]. In addition, the apcC gene, encoding the small linker polypeptide
Lc, lies downstream from the apcB gene [36]. However, apcC gene has
not been found in Cyanophora paradoxa [36]. Both apcA and apcB
consist of 162 codons. The apcB and apcC genes locate 84 bp&
I
88
A B C D E
-.1800 bp
21226 bp5 148,4268,35302027, 19041584, 1375
947,83 1
564
Figure 4.7 The amplified PCR products from genomic DNA of
S. platensis C, were electrophoresed on 1% agarose gel. A,
B and C : amplified products from the genomic DNA
template at the concentration of 28,00, 280 and 28 ng
respectively, (1 mM MgC12 and 250 ng primer), D :
Negative control (no template), E : h marker (Hi&III and
EcoRI)
89
downstream from apcA and 251 bps downstream from apcB,
respectively (Fig.4.8). The deduced amino acid sequence of the a
subunit shows 85%, 82% and 80% identity, and the p subunit shows
93%, 84’Sand 88%--&&y when they were-aligned with those from
Synechococcus elongatus, Cyanophora paradoxa and S’ynechocystis
PCC67 14, respectively (Fig.4.9). In addition, Lc polypeptide shows
92% (100%) ident i ty (s imilar i ty) with three organisms of
Synechococcus elongatus, Synechocystis PCC67 14 and Frenzyella
diplosiphon (as shown in Fig.4.10). Closer examination of these
sequences showed that stretches of 100% identity exist in around the
cysteinyl residue (Cys 81) involved in the linkage of the chromophore
(region 70- 120) of a and p subunits. This portion of the sequence is the
most conserved among all the phycobiliprotein sequences.
The position of the chromophore of the a and p subunit of
allophycocyanin in S. platensis Ct were determined by comparison of
the amino acid sequence of the chromopeptides with the putative apcA
and apcB. This located the chromophore at residue 81 of both th a and
p subunits. Offner and Troxler (1983) has determined the complete
amino acid sequence of the a and p subunits of allophycocyanin from
the unicellular rhodophyte, Cyanidium caldarium by automated Edman
degradation of the proteins and peptides derived from them by chemical
and enzymatic cleavages. They found that the a subunit contains 160
amino acids, one phycocyanobilin chromophore attached at residue 80
and j3 subunit contains 161 amino acids, one phycocyanobilin
chromophore attached at residue 81 [50]. :i,
90
-60 -401 CCTCGTCCCTAATTATTAAGTTTTTCCCCATGAAATGTTA CTATTACAAATATACTAATATGTGA
-20 +111 ACATAATGCCTCAAPLATACATTTCGAGGTAGTCATGTCATGAGGTTTCATTTGGGGGACC~TAGGGAC
141 ACCCGAAACTCGTGGCGGCGTATAATCAAATACGCCCGCCCGATCGCGATCGAT~TGACTCGGC~TCTTGG211 TAATAGCCAAAAGTTGCCTGCTCAGGAGAAGTTGCCTGCCTGCT~CCGCCACCTGTGGCAGGTT~TGGTAC280 TTCCCAAAGCTGAGGAGCCACGACACCGGGCTGACCGAAACACATTAGCT351 AAACCCTGTGGTAGAAAATGAGTATCGTTACCAAATCCATGCGCGTTATCT
MetSerIleValThrLysSerIleValAsnAlaAspAlaAspAlaGl~l~rgTyrLem ;-421 GAGC~~TGGTG~TTAGATCGGATCAAATCCTTTGTTACCTCTGGC~~~-GC~GGGTTCGGAT~CTG~
uSerProGlyGluLeuAspArgIleLysSerPheValThrSerGlyGl~rgArgValArgIleAlaGlu
491 ACCATGACAGGTGCTCGTGAGCGCATCATCAAGGAAGCAGCTTTTCC UCGTCCTGATGThrMetThrGlyAlaArgGluArgIleIleLysGluAlaGlyAsnGlnLeuPheGlnLysArgProAspV
561 TAGTCTCTCCCGGTGGAAATGCCTACGGTGAGGAAATGACTCTAGACTACTAalValSerProGlyGlyAs~laTyrGlyGluGl~etThrAlaThrCysLe~rgAspLe~spTyrTy
631 CCTGCGTCTGATCACCTACGGAATTGTTGTTGTTCGGGGTTGTAGGTrLeuArgLeuIleThrTyrGlyIleValAlaGlyAspValThrProIleGluGluIleGlyValValGly
701 GTTCGCGAAATGTACAAATCTCTTGGTACTCCCATCGAAGValArgGluMetTyrLysSerLeuGlyThrProIleGl~laValAlaGluGlyValArgAlaMetLysS
771 GTGTAGCCACTTCCCTGCTGTCTGGAGAAGACGCAGCCGACGCAGCCG~GCAGGTGCTTACTTCGACTACCT~TTGGerValAlaThrSerLeuLeuSerGlyGluAspAlaAlaGl~laGlyAlaTyrPheAspTyrLeuIleGl
841 TGCAATGTCATAAGCACTGGCGATTATCTCTTATTAATCGACCAAGATTTCCTAGATCyAlaMetSer***
911 AAGCGACCATTAGCAAACGAAACCATCATCATGCAACGTTCMetGlnAspAlaIleThrSerValIleAsnSerSerAspValG
981 AAGGTAAATACCTGGATCGTAGCGCTATCCAAAAA CTGAAAGCCTATTTCGCTACTGGTGAACTGCGCGTlnGlyLysTyrLeuAspArgSerAlaIleGlnLysLeuLysAlaTyrPheAlaThrGlyGluLeuArgVa
1051 TCGTGCAGCAACCACCATCAGCGCTAATGCAGCTAACATCGTCTCTGCTGlArgAlaAlaThrThrIleSerAlaAsnAlaAlaAsnIleValLysGluAlaValAlaLysSerLeuLeu
1121 TACTCCGATATCACCCGTCCCGGTGGTAATATGTATGTACACCACTCGTCGCTATGCTGCTTGCATCCGTGACCTyrSerAspIleThrArgProGlyGlyAsnMetTyrThrThrThrArgArgTyrAlaAlaCysIleArgAspL
1191 TGGACTACTACCTCCGCTATGCTACCTATGCTATGCTGGCTGGCGATCCTTCCATCCTGGATGAGCGTGTeuAspTyrTyrLeuArgTyrA1aThrTyrAlaMetLeuAlaGlyAspProSerIleLeuAspGluArgVa
1261 ACTCAATGGCCTGAAAGAAACTTATAACTCTCTTTGGGTGTACCCATTGGCGCTACCGTTC~GCTATCC~lLeuAsnGlyLeuLysGluThrTyrAsnSerLeuGlyValProIleGlyAlaThrValGl~laIleGln
1331 GCTATGAAAGAAGTTACTGCTGGCTTAGTTGGTGCTGATGCTGGT~GG~TGGGCATTTACTTTGATTAlaMetLysGluValThrAlaGlyLeuValGlyAlaAspAetGlyIleTyrPheAspT
1401 ACATCTGCTCTGGCTTGAGCTAAGACTGCTCACTGCTCACAGAGG~GCTAG~TGTAGTCATCCCCTTTGG~~yrIleCysSerGlyLeuSer***
1471 CCTACAGTCTTGGTTCTTCATTCCTATAAACTTAGGGCCGTGAGTGCTAGACCGC1541 CAAAGGCTTGTCTGTATCATTGATAAGTTTTTAGCGAGCTAGTATTGGCTTATGACTCCCGGCCTTTAGTC1611 ATTTGATAAATATTACTGTCAAATACTGTCAAAATTGCTGTC~TTGCTGACTT~CTCAGGAGC~GAT~TCATGAGA
MetArg1681 GTTTTCAAAGTAACAGCTTGCGTTCCCAGCCAAACACGGATACCT
ValPheLysValThrAlaCysValProSerGlnThrArgIleArgThrGl~rgGluLeuGl~snThrT1751 ATTTCACTAAGCTGGTTCCCTATGACAACTGGTTCAGAGACAGCGG
yrPheThrLysLeuValProTyrAspAsnTrpPheArgGluGlnGl~rg
7 0
140210280350420
490 t
560
630
700
770
840
910
980
1050
1120
1190
1260
1330
1400
1470
154016101680
1750
Figure 4.8 The nucleotide and deduced amino acid, sequences of the
complete apcAB and partial apcC gene of 5’. platensis Cl.
The underlining sequences indicate the putative palindromic
motif of terminator. This sequence data has been submitted
to the EMBL/GenBank nucleotide sequence database, and
the accession number is X95898. (+l indicates the putative
of transcription start site);I’
9 1
AP a subunits
Syn elon MSWTKSIVNADAEARYLSPGELDRIKNFVSTGERRLRIAQTLTENRERIVKQAGDQLFQ 60Cyan0 MSIVTKSIVNADAEARYLSPGELDRIKSFAASGERRLRIAEAGQQLFQ6714 _~~IVTKSI~~AEARYLSPGELD~I~F~GG~RLRIAETLTGSRETI~QAGDRLFQ.- = ~sp Cl MSIVTKSIVNADAEARYLSPGELDRIKSFVTSGERRVRIATGARERIIKEAGNQLFQ
** ************************ * * * *** * ** * ** ***
Syn elon KRPDWSPGGNAYGEEMTATCLRDLDrYLRLVTYGIVAGDVPIEEIGLVGVREMYNSLG 120Cyan0 KRPDIVSPGGNAYGEEMTATCLRDLDrYLRLVTYGWAGDASLG6714 KRPDIVSPGGNAYGEEMTATCLRDMDYYLRLVTYGWSGDVTPIEEIGLVGVREMYRSLGsp Cl KRPDWSPGGNAYGEEMTATCLRDLDYYLRLITYGIVAGDVKSLG
**** ****************If** ****** *** * ** ******* *** *** ***
Syn elon TPIPAVAEGIRAMKNVACSLLSAEDAAEAGSYFDFVIGAMQ 161Cyan0 TPVAAVAEGVRSAKSVATGLLSGDDAAEAGSYFDWIAALQ6714 TPIEAVAQSVREMKEVASGLMSSDDAAEASAYFDFVIGAMSsp Cl TPIEAVAEGVRAMKSVATSLLSGEDAAEAGAYFDYLIGAMS
** *** * * ** * * ***** *** * *
AP f3 subunits
Syn elonCyan06714sp Cl
Syn elonCyan06714sp Cl
Syn elonCyan06714sp Cl
MQDAITAVINASDVQGKYLDTAAMEKLKAYFATGELRVRASVISANAANIVKEAVAKSLM Q D P I T A V I N A A D V Q G K Y L D T A S V E K L K S Y F Q T G E L R V R A S LMQDAITAVINSADVQGKYLDGAAMDKLKNYFASGELRVRAASVISANAATIVKEAVAKSLMQDAITSVINSSDVQGKYLDRSAIQKLKAYFATGELRVRASL*** ** *** ******** *** ** ******** * ** * ********
LYSDITRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDPSILDERVLNGLKETYNSLGLYSDITRPGGNMYTTRRYAACIRDLDrrVRYATYAMLAGDTSILDERVLNGLKETYNSLGLYSDVTRPGGNMYTTRRYAACIRDLDWLRYATYAMLAGDASILDERVLNGLKETYNSLGLYSDITRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDPSILDERVLNGLKETYNSLG**** *********************** *********** *******************
VPIAATVQAIQAMKEVTASLVGADAGKEMGIYFDYICSGLS 161VPVGATIQAIQAAKEVTAGLVGPDAGREMGIYYDYISSGLGVPISSTVQAIQAIKEVTASLVGADAGKEMGVYLDYICSGLSVPIGATVQAIQAMKEVTAGLVGADAGKEMGIYFDYICSGLS** * ***** ***** *** *** *** * *** ***
60
120
Figure 4.9 The alignment of the amino acid sequence from apcAB gene
product of 5’. platensis Cl (sp. Cl) and apcAB gene products
of Synechococcus elongatus (Syn elon), Cyanophora
paradoxa (Cyano) and Synechocystis PCC6714 (6714). The
identical amino acids are indicated by asterisks. :1
92
Lc subunits
Syn elon MRMFKITACVPSQTRIRTQRELQNTYFTKLVPYENWFREQQ 41
6714 MRMFRITACVPSQTRIRTQRELQNTYFTKLVPYDNWFREQQ-- - -- : ~
Fre dip RLFKVTACVPSQTRfRTQRELQNTYpTItLVPFENWFREQQ
sp Cl MRVFKVTACVPSQTRIRTQRELQNTYFTKLVPYDNWFREQQ
* * ************************** *******
Figure 4.10 The alignment of the partial amino acid sequence from
apcC gene product of S. platensis Cl (sp. Cl) and apcC
gene products of Synechococcus elongatus (Syn elon),
Synechocystis PCC6714 and Fremyella diplosiphon (Fre
dip) The identical amino acids are indicated by asterisks.
93
The AP a coding sequence extends from nucleotide 368 to
nucleotide 853 while the AP /3 coding sequence extends from nucleotide
937 to nucleotide 1,423. The predicted masses using the program
GENETY-X-MAC -XI+ the APC-m subunit apoprotein are -17,39 1, and
17,414, daltons for APCa and APCP, respectively.The AP a- and AP p-
subunit genes are in the same orientation with APCa located upstream
from the APCp gene. This organization is opposite to that found for the
phycocyanin (PC) genes of Synechococcus PCC7002, in which PCP is
upstream from PCs [32].
Data from genomic Southern hybridization analysis with 1 .l
kbp EcoRI-EcoRV homologous probe of S. platensis Cr (Fig.4.11) and
1.8 kbp EcoRI fragment homologous probe (Fig.4.12), and mapping
studies (Fig.4.13) support the idea that the apcAB genes of S. platensis
C1 are present only a single copy in the genome. Single copy of
phycobiliprotein genes has been found in most in cyanobacteria and
eukaryotic algae. A notable exception is F. diplosiphon of which these
are three sets of cpc genes [32]. Synechococcus sp. strain PCC6301 also
has two sets of cpc genes [61].
A comparison of the promoter sequences found upstream of the
transcription start sites of apcABC from Synechocystis PCC6714,
Calothrix PCC760 1, Synechococcus PCC630 1, Synechococcus
PCC7002 and a putative promoter of S. platensis Cl is shown in
Fig.4.14. The result showed that the untranslated region of S. platensis
Cl apcABC operon extends 271 bp upstream of the apcA start codon.
This region is considerably larger than those found in four organisms
mentioned above and does not appear to be any similarity to the E.coZi’
94
consensus sequences. In the -10 region (with respect to transcription
start site (+l), see Fig.4.14) of the cyanobacteria there are two types of
conserved sequences. The first sequence is C(T/G)GAGAAT(A/G)
found in-Synechom =PCC6714 and Caldrix PCC7601, while the
second one is C(T/G)CAAAAT(G/A) found in Synechococcus
PCC6301, Synechococcus PCC7002 and S. platensis Cr. There are no
sequences conserved in the -35 region. In addition, the consensus
sequence TTA(A/C)AAA(C/A)T(G/A)TTA (in the -50 region) is
conserved among all these five organisms. Although there are no Ecoli
consensus, it does appear that the apcABC genes may be under the same
transcriptional control in all five organisms.
Hypothetical palindromic sequences, possibly the terminator
sequence, could be saught within the DNA sequences of apcAB
presented in Fig.4.8, using the program GENETYX-MAC. It was
apparent that the stem and loop structure could be drawn from
nucleotide 1,446 to 1,478 (AATGTAGTCATCCCCTTTGGAAATACC
TACAGT) with the percentage of matching was 72.72% (as shown in
Fig.4.15). This structure is found within the intergenic region between
apcB and apcC and is not followed by a stretch of T’s characteristic of a
rho-independent terminator. This finding is in accordance to that found
in Synechocystis PCC6714 [7]. DiMagno and Haselkorn (1993) [7] have
reported that the obtained operon apcABC of Synechocystis PCC67 14
has two possible termination sites, one following apcB and the other
following apcC. As such, it seems likely that there is another possible
terminator 5’ to the apcC of S. platensis Cl.
95
A B C D E F G H
-_ -
21226 bp
5 148,4973
4268
3530
2027
Figure 4.11 Hybridization of the 1.1 kb EcoRI-EcoRV fragment of pMG
004 carrying part of the apcAB gene to total genomic S.
platensis Ci DNA. Restriction endonuclease digests of S.
platensis Ci total DNA were electrophoresed, transferred to
nitrocellulose membrane. Lanes: A, h marker digested with
HindIII-EcoRI; B, HincII; C, EcoRI; D, EcoRV; E, EcoRI-
EcoRV; F, EcoRI-HincII; G, EcoRV-HincII; H, uncut DNA.
I
96
A B C
21226
- - -
5 148,4973
4268
3530
1904,2027
1584, 1375
947,83 1
bp
Figure 4.12 Hybridization of the 1.8 kb EcoRI fragment of pMG 004
carrying apcABC gene to total genomic 5’. platensis Cl
DNA. Restriction endonuclease digests of 5’. platensis C,
total D N A w e r e electrophoresed, transferred to
nitrocellulose membrane. Lanes: A, h marker digested with
Hi&III-EcoRI; B, C, total DNA of 5’. platensis Cl digested
with EcoRV, EcoRV-HincII, respectively.
97
Figure 4.13 Restriction map of a 2 1 kbp EcoRI fragment of S. platensis
Ci composed of the apt gene region. Complete open
reading frames of apcA, apcB are shown as solid boxes,
whereas the partial open reading frame of apcC is shown as
an open box. Abbreviation: R, EcoRI; Hc, Hⅈ Rv,
EcoRV; C, CZaI. (- single lines indicates the size of
flanking area of apt genes with known distance, ---- broken
lines indicate the ones with unknown distance). The size of
the genes are shown by the bar denoted 200 bp.
98
SP.CI
Syn 6114
Cal 7601
Syn 6301
Syn 7002
SP.CI
Syn 6714
Cal 7601
Syn 6301
Syn 7002
-60 -40 -20
+1
CGA--GGTAGTCATG
-GGCAGGTTGCT---
CGGAAGGTAACT-GA
CGAAAGGTAATTCG-
CGGATGGCTGTAGGA** **
-2
CATTT
GTTTG
TTTTG
-ATTC
-TTTG** 3
Figure 4.14 Alignment of the putative of transcription start sites (+l)
and promoters (-10, -50 regions in boxes) of apcABC gene
of 5’. platensis Cl to those from Synechocystis PCC6714
(Syn 6714), Calothrix PCC7601 (Cal 7601), Synechococcus
PCC6301 (Syn 6301) and Synechococcus PCC7002 (Syn
7002) [7] Identical nucleotides are indicated by asterisks.
99
[GENE&X-MAC: Hairpin Loop and Stem Parts]1996.05.02Filename : UntitledSequence Size : 1799Sequence Position: 1 - 1799Start Position : 1446StemPafts Size 7 K-
- ~. .-Loop Parts Size : 11Matting Percent : 72.727 %
T T T
C G
C G
C A
C A- A
AT-c :
L :A --TT- A
CT-T - :
GGAAGCTAGk- FCTTGGTTCTTCAATTCCTA
1446 1478
Figure 4.15 The putative hairpin loop and stem part at the 3’end of the p
subunit of apt of 5’. platensis Cl using GENETYX-MAC
program.
Chapter 5
Conclusion and Suggestion
5.1 Conclusion
The complete apcAB and partial apcC genes from 5’. platensis
Cl were cloned by PCR and sequenced. The deduced amino acid
sequences of apcA, apcB and apcC genes of S. platensis Cl were
compared with those of the corresponding known phycobilisome
components. Sequence identities within each class of allophycocyanin
(AP) subunits were very high (80-85% for the a subunits, 84-93% for p
subunits and 92% for Lc polypeptide).
The nucleotide sequences of a 1.8 kb PCR fragment revealed
the coding sequences of genes for the a-, p- subunits and partial small
core linker protein of S. platensis Cr. Two complete open reading
frames of apcA and apcB (16 1 amino acids for each gene), and a partial
one of apcC (36 amino acids) were found. The organization of genes is
in an order as followed: apcA, apcB and apcC. The predicted masses of
the apt subunit apoprotein are 17,391 and 17,414 daltons for apt a and
p, respectively. These genes form an operon, apcABC, with a single
putative transcription start site and one putative termination site,
downstream of apcAB. The apt genes appear to be present in only one
copy per genome. The promoter region, like those of the apcABC
operons of other cyanobacteria, does not resemble the consensus;
promoter sequences of E. coli.
101
5.2 Suggestion
1. To confirm whether the obtained gene is the apt gene of S.
platensis Cl, an--immunological method, -utilizing antibodies to-
allophycocyanin, can be used to screen E.coZi colonies containing
fragment of apt gene, as in the case of phycocyanin. In addition, it can
be simply done by the transformation of the gene into transformable
cyanobacterium, Synechococcus sp., which it has not expression of
allophycocyanin. The occurrence of the allophycocyanin will be
indicator to prove that the obtained genes is the allophycocyanin gene of
S. platensis Cl.
2. Sl nuclease mapping and northern analysis should be
performed to analyse the apcABC operon whether it is composed of one
promoter and two terminators, of which one following apcB another
following apcC.
3. From the knowledge of gene organization of the known
allophycocyanin gene clusters, ie. apcEABC, it is possible to clone apcE
from 5’. platensis Ci by subcloning a DNA fragment upstream of apcA
in the 9 kb EcoRI fragment obtained from the h genomic library.
Moreover, the complete apcC can also be obtained by screening or
chromosome walking the genomic library using the partial apcC as a
probe.
References
1.
2.
3.
4.
5 .
6.
7.
8 .
Vonshak, A., 1990, “Recent Advances in Microalgal
Biotechnology,? Biotechnological Advances, Vol. 8, pp. 709-727.-
Cohen, Z., Vonshak, A. and Richmond, A., 1987, “Fatty Acid
Composition of Spirulina Strains Under Various Environmental
Conditions,” Phytochemistry, Vol. 26, pp. 2255-2258.
Santillan, C., 1982, “Mass production of Spirulina,” Experientia,
Vol. 38, pp. 40-43.
Jassby, A., 1988, Spirulina : a model for microalgae as human
food, New York, Cambridge University, pp. 149-179.
Grossman, A.R., Schaefer, M.R., Chiang, G.G. and Collier, J.L.,
1993, “The Phycobilisome, a Light-Harvesting Complex
Responsive to Environmental Conditions,” Microbiological
Reviews, Vol. 57, No. 3, pp. 725-749.
Olaizola, M. and Duerr, E.O., 1990, “Effects of light intensity and
quality on the growth rate and photosynthetic pigment content of
Spirulina platensis,” Journal of Applied Phycology, Vol. 2, pp.
97-104.
DiMagno, L . and Hase lkorn , R . , 1993, “ Iso la t ion and
characterization of the genes encoding allophycocyanin subunits
and two linker proteins from Synechocystis 6714,” Plant
Molecular Biology, Vol. 21, pp. 835-845.
Lundell, D.J., Yamanaka, G. and Glazer, A.N., 1981, “A terminal
energy acceptor of the phycobilisome: the 75,000 Dalton
polypeptide of Synechococcus 630 1 phycobilisome-A ne\li,,
biliprotein,” Journal of Cell Biology, Vol. 91, pp. 3 15-3 19.
103
9 .
1 0 .
1 1 .
1 2 .
13.
1 4 .
15.
1 6 .
Brejc, K., Ficner, R., Huber, R. and Steinbacher, S., 1995,
“Isolation, Crystallization, Crystal Structure Analysis and
Refinement of Allophycocyanin from the Cyanobacterium
S’ndina plcrterzs& -at 23 A- Resolution,‘? Journal of Molecular.-
Biology, Vol. 249, pp. 424-440.
Jung, T.M. and Dailey, M.O., 1989, “A novel and inexpensive
source of allophycocyanin for multicolor flow cytometry,”
Journal of Immunological Methods, Vol. 121, pp. 9-l 8.
Garnier, F. Dubacq, J.P. and Thomas, J.C., 1994, “Evidence for a
Transient Association of New Proteins with the Spirulina maxima
Phycobilisome in Relation to Light Intensity,” Plant Physiology,
Vol. 106, pp. 747-754.
Johnson, T.R., Haynes II, J.I., Wealand, J.L., Yarbrough, L.R. and
Hirchberg, R., 1988, “Structure and Regulation of Genes
Encoding Phycocyanin and Allophycocyanin from Anabaena
variabilis ATCC 29413,” Journal of Bacteriology, Vol. 170, No.
4, pp. 1858-1865.
S&ram, B.L . and Kroes , H .H. , 1971 , “S t ruc tu re o f
Phycocyanobilin,” Europeon Journal of Biochemistry, Vol. 19,
pp. 581-594.
Richmond, A., 1986, CRC Handbook of Microalgal Mass
Culture, Boca Raton, CRC Press Inc., pp. 56-66.
Gantt, E., 1994, Supramolecular Membrane Organization,
Dordrecht, Kluwer Academic, pp. 119- 13 8.
Trinor, F.R., 1978, Introductory Phycology, New York, John
Wiley & Sons, pp. 9-15.:
I
104
17.
1 8 .
19.
20.
21.
22.
23.
24.
25.
Nierzwicki-Bauer, S.A., Balkwill, D.L. and Stevens, S.E.Jr.,
1983, “Three dimensional ultrastructure of a unicellular
cyanobacterium,” Journal of Cell Biology, Vol. 97, pp. 713-720.
Houmard, W _Tandeau- de Marsac;, N.,- 1988, “Cyanobacterial-
Genitic Tools: Current Status,” Method in Enzymology, Vol. 167,
No. 2, pp. 808-847.
Ciferri, 0. and Tiboni, O., 1985, “The biochemistry and industrial
potential of Spirulina,” Annual Reviews Microbiology, Vol. 39,
No. 3, pp. 503-526.
Ciferri, O., 1983, “Spirulina : The Edible Microorganism,”
Microbiological Review, Vol. 47, pp. 55 l-578.
Richmond, A., 1986, CRC Handbook of Microalgal Mass
Culture, Boca Raton, CRC Press Inc., pp. 212-230.
Chiu, R.J., Liu, H.-I. and Soong, P., 1978, “Mass production and
development of the blue-green alga, Spirulina,” Cooperative
Science Program Seminar on Cultivation and Utilization of
Economic Algae, Taiwan, pp. 87-94.
Vonshak, A. and Richmond, A., 1988, “Mass Production of the
Blue-green Alga Spirulina : An Overview,” Biomass, Vol. 15,
No. 4, pp. 233-247.
Richmond, A., 1988, Spirulina In Borowitzka, A. and
Borowitzka, L., Microalgal Biotechnology, New York,
Cambridge University Press, pp. 83- 12 1.
Tandeau de Marsac, N., Bazire, G.C., 1977, “Molecular
composition of cyanobacterial phycobilisomes,” Proceeding of
The National Academy of Science of The United States ofi,
America, Vol. 74, No. 4, pp. 1635-1639.
105
26. Bryant, D.A., 1991, “Cyanobacterial Phycobilisomes: Progress
toward Complete Structural and Functional Analysis via
Molecular Genetics,” Genetic of Plants, Vol. 78, pp. 257-299.
27. Bhalerao, R.P., =1993, A mutational- analysis of structure,-
assembly and energy transfer, University of Umea, Sweden, pp.
4-79.
28. Giddings, T.H., Wasman, C. and Staehelin, L.A., 1983, “Structure
of the thylakoids and envelope membranes of the cyanelles of
Cyanophora paradoxa,” Plant Physiology, Vol. 71, pp. 409-419.
29. Glazer, A.N., 1984, “Phycobilisome: A micromolecular Complex
Optimized for light transfer,” Biochimica et Biophyseca Acta,
Vol. 768, pp. 29-51.
30. Glazer, A.N., Lundell, D.J., Yamanaka, G. and William, R.C.,
1983, “The structure of a ‘simple’ phycobilisome,” Annual
Microbiology (Inst Pasteur), Vol. 134B, pp. 159-l 80.
31. Houmard, J., Capuano, V., Coursin, T. and Tandeau de Marsac,
N., 1988, “Gene Encoding Core Components of the
Phycobilisome in the Cyanobacterium Calothrix sp. Strain PCC
7601: Occurrence of a Multigene Family,” Journal of
Bacteriology, Vol. 170, No. 12, pp. 55 12-5521.
32. Conley, P.B., Lemaux, P.G. and Grossman, A., 1988, “Molecular
Characterization and Evolution of Sequences Encoding Light-
harvesting Components in the Chromatically Adapting Cyano-
bacter ium Frenzyella diplosiphon,” Journal of Molecular
Biology, Vol. 199, pp. 447-465.
106
33.
34.
35.
36.
37.
38.
39.
40.
Grossman, A.R., 1990, “Chromatic adaptation and the events
involved in phycobilisome biosynthesis,” Plant Cell
Environment, Vol. 13, pp. 65 l-666.
Tandeau de-Ma.rsac, N., 1977, “l)c~currence and nature of.-
chromatic adaptation in cyanobacteria,” Journal of Bacteriology,
Vol. 130, pp. 82-91.
Tandeau de Marsac, N., Mazel, D., Damerval, T., Guglielmi, G.,
Capuano, V. and Houmard, J., 1988, “Photoregulation of gene
expression in the filamentous cyanobacterium Calothrix sp. PCC
760 1: light-harvesting complexes and cell differentiation,”
Photosynthesis research, Vol. 18, pp. 99-l 32.
Sidler, W.A., 1994, Phycobilisome and Phycobiliprotein
Structure, Dordrecht, Kluwer Academic, pp. 139-2 16.
Bryant, D.A., Guglielmi, G., Tandeau de Marsac, N., Castet, A.M.
and Cohen-Bazire, G., 1979, “The structure of cyanobacterial
phycobilisomes: A model,” Archives of Microbiology, Vol. 123,
pp. 113-127.
Zuber, H., 1987, The structure of light-harvesting pigment-
protein complexes, Amsterdam, Elsevier Biomedical,
pp. 157-259.
Grossman, A.R., Schaefer, M.R., Chiang, G.G. and Collier, J.L.,
1994, The Responses of Cyanobacteria to Environmental
Conditions: Light and Nutrients, Dordrecht, Kluwer
Academic, pp. 64 l-675.
Glazer, A.N., 1985, “Light harvesting by phycobilisomes,”
Annual Review Biophysical Chemistry, Vol. 14, pp. 47-77.:i’
107
41. Gantt, E., Lipschultz, CA. and Zilinskas, B.A., 1976, “Further
evidence for a phycobilisome model from selective dissociation
fluorescence emission immunoprecipitation and electron
mi-croscopy+B-iochimica et Biophysics Acta, Vol. 430, pp. 375 =
388.
42. Fuglistaller P. Widmer, H., Sidler, W., Frank, G. and Zuber, H.,
198 1, “Isolation and characterization of phycoerythrocyanin and
chromatic adaptation of the thermophilic cyanobacterium
Mastigocladus laminosus,” Archives of Microbiology, Vol. 129,
pp. 268-274.
43. Babu, T.S., Kumar, A. and Varma, A.K., 1991, “Effect of Light
Quality on Phycobilisome Components of the Cyanobacterium
Spirulina platensis,” Plant Physiology, Vol. 95, pp. 492-497.
44. Houmard, J., Mazel, D., Moguet, C., Bryant, D.A. and Tandeau
de Marsac, N, 1986, “Organization and nucleotide sequence of
genes encoding core components of the phycobilisomes from
Synechococcus 6301,” Molecular General Genetic, Vol. 205, pp.
404-410.
45. Glazer, A.N., 1984, “Phycobilisomes: A Macromolecular
complex optimized for light energy transfer,” Biochimica et
Biophysics Acta, Vol. 768, pp. 29-5 1.
46. Liu, B.and Troxler, R.F., 1993, “A Cyanidium caldarium
Allophycocyanin p Subunit Gene,” Plant Physiology, Vol. 103,
pp. 293-294.
108
47. Bryant, D.A., 1988, Genetic analysis of phycobilisome
biosynthesis, assembly, structure, and function in the
cyanobacterium Synechococcus sp PCC 7002, Rockville,
American Society of Plant~Physiologists, pp. 62-90. .-
48. Houmard, J., Capuano, V., Colombano M.V., Coursin, T. and
Tandeau de Marsac, N., 1990, “Molecular characterization of the
terminal energy acceptor of cyanobacterial phycobilisome,”
Proceeding of The National Academy of Science of The United
States of America, Vol. 87, pp. 2152-2156.
49. Capuano, V., Braux, A., Tandeau de Marsac, N. and Houmard, J.,
1991, “The ‘anchor polypept ide’ of cyanobacterial
phycobilisomes. Molecular characterization of the Synechococcus
sp. PCC 6301 apcE gene,” The Journal of Biological Chemistry,
Vol. 266, pp. 7239-7247.
50. Offner, G.D. and Troxler, R.F., 1983, “Primary Structure of
Allophycocyanin from the Unicellular Rhodophyte, Cyanidium
Caldarium,” The Journal of Biological Chemistry, Vol. 258, No.
16, pp. 993 l-9940.
51. Glazer, A.N. and Bryant, D.A., 1975, Allophycocyanin B (h,,
671, 618 nm). A new cyanobacterial phycobiliprotein,” Archives
of Microbiology, Vol. 104, pp. 15-22.
52. Bhalerao, R.P., Gillbro, T. and Gustafsson, P., 1991, “Structure
and energy transfer of the phycobilisome in a linker protein
replacement mutant of cyanobacterium Synechococcus 7942,”
Biochimica et Biophysics Acta, Vol. 1060, pp. 59-66.
109
53. Anderson, L.K. and Grossman, A.R., 1990, “Genes for
Phycocyanin Subunits in Synechocystis sp. Strain PCC 6701 and
Assembly Mutant UV16,” Journal of Bacteriology, Vol. 172, No.
3,pp. 1289=U%,~ _ - ~ _ z-
54. Lagarias, J.C., Klotz, A.V., Dallas, J.L., Glazer, A.N., Bishop,
J.E. and O’Connell, J.F., 1988, “Exclusive A-ring linkage for
singly attached phycocyanobilins and phycoerythrobilins in
phycobiliproteins,” The Journal of Biological Chemistry, Vol.
263, pp. 12977-12985.
55. Williams, V.P. and Glazer, A.N., 1978, “Structural studies on
phycobiliproteins. I. Bilin-containing peptides o f
C-phycocyanin,” The Journal of Biological Chemistry, Vol. 253,
pp. 202-211.
56. Glazer, A.N. and Hixson, C.S., 1975, “Characterization of R-
Phycocyanin. Chromophore content of R-phycocyanin and C-
phycoerythrin,” The Journal of Biological Chemistry, Vol. 25, pp.
5487-5495.
57. Ong, L.J. and Glazer, A.N., 1987, “R-phycocyanin II, a new
phycocyanin occurring in marine Synechococcus species.
Identification of the terminal energy acceptor bilin in
phycocyanins,” The Journal of Biological Chemistry, Vol. 262,
pp. 6323-6327.
58. Ong, L.J., Glazer, A.N. and Waterbury, J.B., 1984, “An unusual
phycoerythrin from a marine cyanobacterium,” Science, Vol. 224,
pp. 80-83.
110
59. Ong, L.J. and Glazer, A.N., 1988, Structural studies of
phycobiliproteins in unicellular marine cyanobacteria, Rockville,
American Society of Plant Physiologists, pp. 102- 12 1.
60. Conley, P.B.,-Lemaux, P.-G.,- Lomax, T-L and Grossman, A.R., ~-
1986, “Genes encoding major light-harvesting polypeptides are
clusters on the genome of the cyanobacterium Fremyella
diplosiphon,” Proceeding of The National Academy of Science of
The United States of America, Vol. 83, pp. 3924-3928.
61. Grossman, A.R., Lemaux, P.G. and Conley, P.B., 1986,
“Regulated Synthesis of Phycobilisome Components,”
Photochemistry and Photobiology, Vol. 44, No. 6, pp. 827-837.
62. Bryant, D.A., Glazer, A.N. and Eiserling, F.A., 1976,
“Characterization and structural properties of the major
biliproteins of Anabaena sp.,” Archives of Microbiology, Vol.
110, pp. 61-75.
63. Bryant, D.A., 1982, “Phycoerythrocyanin and phycoerythrin:
Properties and occurrence in cyanobacteria,” Journal of General
Microbiology, Vol. 128, pp. 835-844.
64. Lundell, D.J., Williams, R.C. and Glazer, A.N., 1981, “Molecular
architecture of a light-harvesting antenna. In vitro assembly of the
rod substructures of Synechococcus 630 1 phycobilisomes,” The
Journal of Biological Chemistry, Vol. 256, pp. 3580-3592.
65. Redlinger, T. and Gantt, E., 1982, “A M, 95,000 polypeptide in
Porphyridium cruentum phycobilisomes and thylakoids: Possible
function in linkage of phycobilisomes to thylakoids and in energy
transfer,” Proceeding of The National Academy of Science of The;,
United States of America, Vol. 79, pp. 5542-5546.
111
66. Ducret, A., Sidler, W., Frank, G. and Zuber, H., 1994, “The
complete amino acid sequence of R-phycocyanin-I a and p
subunits from the red alga Porphyridium cruentum,”
Europeon Journalof Biochemistry, VoL221, pp. 563-580. .T
67. Pilot, T.J. and Fox, J.L., 1984, “Cloning and sequencing of the
genes encoding the a and p subunits of C-phycocyanin from the
cyanobacterium Agmenellum quadruplicatum,” Proceeding of
The National Academy of Science of The United States of
America, Vol. 81, pp. 6983-6987.
68. Mazel, D., Guglielmi, G., Houmard, J., Sidler, W., Bryant,
D.A. and Tandeau de Marsac, N., 1986, “Green light induces
transcription of the phycoerythrin operon in the
cyanobacterium Calothrix 760 1,” Nucleic Acids Research, Vol.
14, pp. 8279-8290.
69. Anderson, L.K. and Grossman, A.R., 1990b, “Structure and
light-regulated expression of phycoerythrin genes in wild-type
and phycobilisome assembly mutants of Synechocystis sp.
strain PCC7601,” Journal of Bacteriology, Vol. 172, pp. 1297-
1305.
70. Herdman, M., Janvier, M., Rippka, R. and Stanier, R.Y., 1979,
“Genome Size of Cyanobacteria,” Journal of General
Microbiology, Vol. 111, pp. 73-85.
71. Lemaux, P.G. and Grossman, A.R., 1984, “Isolation and
characterization of a gene for a major light-harvesting
polypeptide from Cyanophora paradoxa,” Proceeding of The
National Academy of Science of The United States of America,’
Vol. 81, pp. 4100-4104.
112
72. Buttarelli, F.R., Calogero, R.A., Tiboni, O., Gualerzi, C.O. and
Port, C.L., 1989, “Characterization of the str operon genes from
Spirulina platensis and their evolutionary relationship to those of
other proka+&+‘l Molecular General Genetic, Vol,217, pp. 97- s
104.
73. Nelissen, B., Wilmotte, A., Necfs, J.M. and De Wachter, R.,
1994, “Phylogenetic relationships among filamentous helical
cyanobacteria investigated on the basis of 16s ribosomal RNA
gene sequence analysis,” Systemic Applied Microbiology, Vol.
17, pp. 206-210.
74. Salvi, S., Trinei, M., Lanfaloni, L. and Pon, C.L., 1994, “Cloning
and characterization of the gene encoding an esterase from
Spirulina platensis,” Molecular General Genetic, Vol. 243, pp.
124-126.
75. Steinemann, D. and Lill, H., 1995, “Sequence of the gamma-
subunit of Spirulina platensis: a new principle of thiol
modulation of FOFl ATP synthase?,” Biochimica et Biophysics
Acta., Vol. 1230, pp. 86-90.
76. Sanangelantoni, A.M. and Tiboni, O., 1993, “The chromosomal
location of genes for elongation factor Tu and ribosomal protein
S 10 in the cyanobacterium Spirulina platensis provides clues to
the ancestral organization of the str and S 10 operons in
prokaryotes,” Journal of General Microbiology, Vol. 139, pp.
2579-2584.
77. Sanangelantoni, A.M., Calogero, R.C., ‘Buttarelli, F.R.,Gualerzi,
C.O. and Tiboni, O., 1990, “Organization and nucleotidej
sequence of the genes for ribosomal protein S2 and elongation
113
factor Ts in Spirulina platensis,” FEMS Microbiology Letters,
Vol. 66, pp. 141-146.
78. Milano, A., Riccardi, G., De Rossi, E., Barbierato, L. and Ceferri,
0,. 1991, ‘%&olecular characterization. of the genes encoding ~-
aceto-hydroxy acid synthase in the cyanobacterium Spirulina
platensis,” Journal of General Microbiology, Vol. 138, pp. 1399-
1408.
79. Bini, F., De Rossi, E., Barbierato, L. and Riccardi, G., 1992,
Molecular cloning and sequencing of the beta-isopropylmalate
dehydrogenase gene from the cyanobacterium Spirulina
platensis,” Journal of General Microbiology, Vol. 138, pp. 493-
498.
80. Riccardi, G., De Rossi, E., Valle, G.D. and Ciferri, O., 1985,
“Cloning of the glutamine synthetase gene from Spirulina
platensis,” Plant Molecular Biology, Vol. 4, pp. 133- 136.
81. Deshnium, P., 1992, Molecular cloning of the gene for Al2
desaturase from Spirulina platensis Italy strain, Thesis, Master
of Science, Biotechnology Programe, King Mongkut’s Institute of
Technology Thonburi, 80 p.
82. Elhai, J. and Wolk, C.P., 1988, “A versatile class of positive-
selection vectors based on the nonviability of palindrome-
containing plasmids that allows cloning into long polylinkers,”
Gene, Vol. 68, pp. 119-138.
83. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular
Cloning. A Laboratoty Manual. 2nd ed., New York, Cold Spring:
Harbor Laboratory Press, pp. 1.25-l .30. 1
114
84.
85.
86.
87.
88.
89.
90.
91.
Zarrouk, C., 1966, Construction a 1’ Etude d’ une Cyanophycae
Influence de Divers Facteurs Physiques et Chimi ques sur las
Croissance et la Photosysthese de Spirulina maxima, Thesis,
University of-Paris84 p. - ~ _ -
Davis, L.G., Dibner, M.D. and Battey, J.F. 1986, Basic methods
in Molecular Biology, New York, Elsevier, 388 p.
Sanger, F., Nicklen, S. and Coulson, A.R., 1977, “DNA
sequencing with chain-terminating inhibitors,” Proceeding of The
National Academy of Science of The United States of America,
Vol. 74, No. 5463-5467.
Zhou, M.Y., Xue, D., Gomez-Sanchez, E.P. and Gomez-Sanchez,
C.E., 1994, “Improved Downward Capillary Transfer for Blotting
of DNA and RNA,” Biotechniques, Vol. 16, No. 1, pp. 58-59.
Glatron, M.F. and Rapoport, G., 1972, “Biosynthesis of the
parasporal inclusion of Bacillus thuringiensis : half-life of its
corresponding messenger RNA,” Biochimie, Vol. 54, pp. 1291-
1301.
Bryant, D.A., de Lorimier, R., Lambert, D.H., Dubbs, J.M.,
Stirewalt, V.L., Stevens, S.E., Porter, R.D., Tam, J. and Jay, E.,
1985, “Molecular cloning and nucleotide sequence of the a and p
subunits of allophycocyanin from the cyanelle genome of
Cyanophora paradoxa,” Proceeding of The National Academy of
Science of The United States of America, Vol. 82, pp. 3242-3246.
Old, R.W. and Primrose, S.B., 1989, Analysing DNA
Sequences, 4th ed., Oxford, Blackwell Scientific, pp. 99-107.
Fuglistaller, P., Rumbelli, R., Suter, F. and Zuber, H., 1984,:
“Minor polypeptides from the phycobi l i some of the
115
cyanobacterium Mastigocladus laminosus. Isolation,
characterization and amino-acid sequences of a colourless 8.9-
kDa polypeptide and of a 16.2~kDa phycobiliprotein,” Hoppe-
Seyler’s Z Physiological Chemistry, Yol, 365, pp. 1085-1096.-
Appendix
Restriction enzyme of allophycocyanin gene in S. platensis Cl
1 0 2 0 3 0 4 0 5 0 6 05' CC'k!dTCCCTti~T-ficTTTTT?CC-CATGAAA%TT AAAAACTATTACAAATATACT
h
Fin17 0 8 0 9 0 100 110 120
AATAATGTGAACATAATGCCTCAAAATACATTTCGAGGTACATTTCGAGGTAGTCATGTCATGAGGTTTCAT
130 140 150 160 170 180TTGGGGGACCAAATAGGGACACCCGAAACTCGTGGCGTGGCGGCGTAT~TC~TACGCCCGAT
h h h
Fin1 Fin1AflIAsuIAvaIICfrl31Eco471NspIVSau961Sin1190 200 210 220 230 240
CGCGATCGATAAATGACTCGGC~TCTTGGT~TAGCC~GTTGCCTGCTCAGGAG~AA h A
XorII XorIIPVUI PVUI
NruIClaIBan111
PleI250 260 270 280 290 300
GTTGCCTGCTAACCGCCACCTGTGGCAGGTTAAATGGTACGCCA,.. h
BspMI EcoNI310 320 330 340 350 360
CGACACCGGGCTGACCGAAAGTCGTAGGCTTCATCATGAACCCTGTGA
TaqII370 380 390 400 410 420
GTAGAAAATGAGTATCGTTACC~TCCATCGTCAATGGCGCGTTATCTh
AlwNI430 440 450 460 470 480
GAGCCCTGGTGAATTAGATCGGATCAAATCCTTTGTTACCTTTGTTACCTCTGGCG~CGCCGGGTTCGA A
AocIIBan11Bsp12861EcoT381NspIISduI
SecI
117
490 500 510 520 530 540GATTGCTGAAACCATGACAGGTGCTCGTGAGCGCATCATC~GG~GCAGG~CC~CT
HgiEII AocII SfaNIBsp12861HgiAINspII
-. - - _~_ SduI - .~
550 560 570 580 590 600TTTCCAAAAACGTCCTGATGTAGTCTCTCCCGGTGGAAATTGAC
Ah A
SstIII BsmAIMae11
610 620 630 640 650 660TGCCACCTGCCTGCGGGATCTAGACTACTACCTGCGTCTGATCACCTACGG~TTGTTGC
AA A A h A
BspMIXbaIHgaI HphI BspMIBstYI BclIMflIXhoII
670 680 690 700 710 720TGGCGATGTTACCCCCATTGAAGAAATCGGGGGGTTGTAGGTGTTCGCG~TGTAC~TC
A
NruI730 740 750 760 770 780
TCTTGGTACTCCCATCGAAGCAGTAGCTGAAGGTGTACGGTGTACGGGCTATG~GAGTGTAGCCACA A A
AlwNI Ksp6321Ear1
790 800 810 820 830 840TTCCCTGCTGTCTGGAGAAGACGCAGCCGAAGCAGGTGCAGGTGCTTACTTCGACTACCT~TTGG
A A A
GsuI BbvIIBspMI
850 860 870 880 890 900TGCAATGTCATAAGCACTGGCGATTATCTCTTATTAATCG
A
VspIAseI
910 920 930 940 950 960TTCCTAGATCAAGCGACCATTAGCAAACGAAACCATCATGTCACTTCCGT
970 980 990 1000 1010 1020AATCAACTCCTCTGACGTTCAAGGTAAATACCTGGATCGTAGCGCTATCCACTGAA
A h A
SstIII Eco47111Mae11
1030 1040 1050 1060 1070 1080AGCCTATTTCGCTACTGGTGAACTGCGCGTTCGTGCGCATGC
h
Eco47111 :
118
1090 1100 1110 1120 1130 1140AGCTAACATCGTTAAGGAAGCAGTTGCTAAGTCTCTCTGCTGTACTCCGATATCACCCGTCC
h A AA A
BsmAI HphI ECORV Fin1HgiEII
1150 1160 1170 1180 1190 1200CGGTGGTAATATGTACACCACTCGTCGCTATGCTGCTTGCATCCGTGACCTGGACTACTA
-_ - - --A A.~ --~ +. A
BbvI TthlllII SfaNITsp451
1210 1220 1230 1240 1250 1260CCTCCGCTATGCTACCTATGCTATGCTGGCTGGCGATCCTTCCATCCTGGATGAGCGTGT
A
AlwIBin1
1270 1280 1290 1300 1310 1320ACTCAATGGCCTGAAAGAAACTTATAACTCTCTTTGGGTGTACCCATTGGCGCTACCGTTCA
h ,.. A
FokI HaeI TaqII1330 1340 1350 1360 1370 1380
AGCTATCCAAGCTATGAAAGAAGTTACTGCTGGCTTACTGCTGGCTTAGTTGGTGCTGATGCTGGT~GGAA
SfaNI1390 1400 1410 1420 1430 1440
AATGGGCATTTACTTTGATTACATCTGCTCTGGCTTGAGCT~GACTGCTCACAGAGG~
1450 1460 1470 1480 1490 1500GCTAGAATGTAGTCATCCCCTTTGG~TACCTACAGTCTTGGTTCTTC~TTCCTAT~
A
Mb0111510 1520 1530 1540 1550 1560
ACTTAGGGCCAGGGAAGGTCTGAAGTGAGTGAGTGCTAGACCGCC~GGCTTGTCTGTATCATA *
AsuICfrl31NspIVSau961
SecI1570 1580 1590 1600 1610 1620
TGATAAGTTTTAGCGAGCTAGTATTGGCTTATGACTCCCGGCCTTTAGTCATTTGAT~A
PleI1630 1640 1650 1660 1670 1680
TATTACTGTCAAATACTGTCAAZlATTGCTGACTTAACTCTCATGAGAh
SspI1690 1700 1710 1720 1730 1740
GTTTTCAAAGTILACAGCTTGCGTTCCCAGCCAAACACGGGTTAA
TthlllII1750 1760 1770 1780 1790 1800
CAAAATACCTATTTCACTAAGCTGGTTCCCTATGACAACTCAGCGGh
NspBII