714 MOLECULAR GENETICS 180]

This saves time and decreases the risk of degradation by nucleases. The DNA can then be isolated on a CsCI gradient or deproteinized and puri- fied according to classic procedures.

Yields of 1 mg of DNA can be expected from 2 liters of culture having approximately 10 s cells/ml.

[80] M u t a g e n e s i s o f C y a n o b a c t e r i a b y Class ica l a n d

G e n e - T r a n s f e r - B a s e d M e t h o d s

By SUSAN S. G O L D E N

Introduction

One of the most powerful approaches to the study of cellular phenom- ena is the analysis of mutants which are altered in particular functions. The utility of cyanobacterial mutants is enhanced by the recent advances in genetic transfer techniques for many species, 1-3 which afford the op- portunity of identifying genes by complementation. This chapter outlines methods which have been used successfully to obtain mutants in a variety of cyanobacteria, with a description of the system in which each method was developed. In many cases the protocol can be modified to apply to other cyanobacterial species. Classical methods are described, including use of chemical mutagens and UV irradiation, which are applicable to both unicellular and filamentous species. Transposon mutagenesis has been demonstrated only in a transformable strain, but it should also be possible in filamentous strains which can receive plasmids by conjugation from Escherichia coli. Techniques for both site-directed and random mu- tagenesis are also described which, at this time, have been shown to be useful only in transformable unicellular strains. These methods are based on recombination between the chromosome and added DNA which pos- sesses sequence similarity to the chromosomal DNA. This chapter also discusses some procedures that are necessary when working with fila- mentous species to separate mutagenized from wild-type cells on the same filament. The following protocols for mutagenesis and mutant en- richment should provide a starting point for developing the appropriate selection and screening methods to obtain a desired cyanobacterial mutant.

R. D. Porter, this volume [78]. 2 F. Joset, this volume [79]. 3 j . Elhai and C. P. Wolk, this volume [83].

Copyright © 1988 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 167 All rights of reproduction in any form reserved.

[80] MUTAGENESIS OF CYANOBACTERIA 715

Basic Reagents, Supplies, and Techniques for Mutagenesis of Cyanobacteria

Liquid and Solid Media

Basic media and culture conditions for diverse cyanobacterial species have been established and are described elsewhere in this volume. 4 Sev- eral methods for preparing agar plates to optimize cyanobacterial plating efficiencies have been described. These include the following: (1) prepar- ing twice-concentrated solutions of medium and agar (in water) which are autoclaved separately and mixed when cooled to pouring temperature, 5 (2) adding sterile sodium thiosulfate to the mixture to a final concentration of 1 mM before pouring, 6 (3) using agar that has been purified by the method of Braun and Wood, 7 and (4) preparing plates with only 1% agar. 8 All of these precautions are probably of some benefit, and some can be combined. Separate sterilization of agar and mineral salts is strongly rec- ommended.

At least some cyanobacterial strains do not survive quantitatively when plated directly on medium containing a selective agent to which the cells should be rcsistant. 6 The following technique for underlaying the selective agent improves plating efficiency.

Procedure

1. Spread 100- to 150-/.d aliquots of each culture sample on 100-mm plates containing 40 ml of basal medium solidified with 1.5% agar.

2. Incubate the plates under standard illuminated growth conditions for 4-6 hr prior to the addition of the required selective agent.

3. Add the appropriate selective agent by lifting the agar slab with an ethanol-flamed spatula and dispensing 400/~l of a 100× concen- trated stock underneath. Distribute the solution by rotating the spatula under the agar and removing it gently to reseat the agar slab.

4. Continue incubation of the plates until colonies form (4-7 days).

Another method of plating cells and adding selective agents is to use soft agar overlays. 5 Inocula can be added to 2 ml portions of 0.7-1.0% agar melted and cooled to 48 °, mixed, and poured onto the surface of the

4 R. Castenholz, this volume [3]. 5 M. M. Allen, J. Phycol. 4, 1 (1968).

S. S. Golden and L. A. Sherman, J. Bacteriol. 158, 36 (1984). 7 A. C. Braun and H. N. Wood, Proc. Natl. Acad. Sci. U.S.A. 48, 1776 (1962). 8 T. C. Currier, J. F. Haury, and C. P. Wolk, J. Bacteriol. 129, 1556 (1977).

716 MOLECULAR GENETICS [80]

agar. A second layer containing a selective agent can be poured over the first at a later time.

Heterocystous strains which are otherwise able to grow in the absence of combined nitrogen may plate poorly on nitrogen-free media after soni- cation 9 (see Special Considerations for Mutagenesis of Filamentous Cyanobacteria). This may be because cells deplete their supplies of stored nitrogen before filaments have reached a critical length necessary for heterocyst differentiation.l° Therefore the following protocols should in- clude a combined nitrogen source in the basal medium to avoid starvation for nitrogen.

Centrifugation to Harvest Cells

Conditions for harvesting and washing cyanobacterial cultures vary somewhat with different strains. Cells can usually be pelleted by centrifu- gation at 1000-4000 g for 5-10 min at 10-20 °. Some strains are cold sensitive and should not be subjected to refrigeration during the harvest- ing. n Filamentous strains may not form a tight pellet at these or higher centrifugal forces. Use of conical tubes and a swinging-bucket rotor im- proves the pellet compaction.

Classical M e t h o d s o f M u t a g e n e s i s

Chemical Mutagenesis

Classical methods of mutagenesis, such as treatment with chemical mutagens, have been used successfully for both unicellular ~2 and filamen- tous 8 cyanobacterial species. In both types of strains, chemical mutagens provide a means of producing a variety of lesions at random loci. A recent report by Chapman and Meeks 9 describes a careful study of parameters that affect mutagenesis frequency induced by N-methyl-N'-nitro-Nonitro- soguanidine (MNNG) in Anabaena ATCC 29413. Their measure of muta- genesis was the frequency of obtaining colonies that are resistant to the analog 5'-fluorocytosine, for which there is a positive selection. The cen- tral findings of this study are that concentration and exposure time are not important variables in themselves, but that a combination of the two

9 j. S. Chapman and J. C. Meeks, J. Gen. Microbiol. 133, 111 (1987). 10 M. Wilcox, G. J. Mitchison, and R. J. Smith, in "Microbiology--1975" (D. Schlessinger,

ed.), p. 453. Am. Soc. Microbiol., Washington, D.C., 1975. n R. Rippka, J. Deruelles, J. B. Waterbury, M. Herdman, and R. Y. Stanier, J. Gen.

Microbiol. U l , 1 (1979). 12 L. A. Sherman and J. Cunningham, Plant Sci. Lett. 8, 319 (1977).

[80] MUTAGENESIS OF CYANOBACTERIA 717

which achieves 99% lethality produces the highest frequency of mutants. The pH of the incubation was found to strongly influence mutation fre- quency, with an optimum at pH 6.0. Another important factor was the duration of the expression period after mutagenesis to allow segregation of mutant and wild-type chromosomes. The following procedure is adapted from the findings of Chapman and Meeks, and is similar to the procedure used by others. 9,12 Mutants should be obtained at a frequency of 1-4 x 10 -4 in Anabaena ATCC 29413,9 as compared to a spontaneous rate of 1-2 × 10 -7.

Reagents for MNNG Mutagenesis

MNNG stock solution: 10 mg/ml in glass-distilled water. Filter-steril- ize and freeze 2.0-ml aliquots. Do not refreeze. Note that MNNG is a hazardous compound; follow the supplier's handling instruc- tions carefully.

Sterile I0 mM citrate buffer, pH 6.0

Procedure

1. Grow 50 ml of cyanobacterial culture to 106-107 cells/ml in liquid basal medium under standard conditions.

2. If working with a filamentous strain, fragment filaments to an average length of 2 cells (see Special Considerations for Muta- genesis of Filamentous Strains).

3. Wash the fragmented culture by centrifugation twice with basal medium and resuspend at 1 x 106-2 × 10 7 cells/ml in 20 ml of 10 mM citrate buffer, pH 6.0.

4. Treat with MNNG at 1.0 mg/ml for 15 min (or at a lower concen- tration for a period of time which results in 99% killing) in the light at room temperature.

5. Remove the mutagen by three centrifugal washes and resuspend cells in 50 ml basal medium.

6. Incubate under standard growth conditions for 6 days. Note that some mutants grow better at room temperature than at 30 ° (J. C. Meeks, personal communication).

7. Repeat fragmentation of filaments (if appropriate). 8. If the desired mutant is expected to have impaired growth, include

steps for mutant enrichment (see below). 9. Plate survivors on solid medium containing appropriate nutritional

supplements or selective agents to screen or select for desired mutant phenotypes. Concentrate cells to approximately 1 × 108 cells/ml and plate 200/zl/plate on 4-10 plates prior to adding a selective agent. To screen for a nonselectable phenotype, plate

718 MOLECULAR GENETICS [80]

dilutions of mutagenized cells on a permissive master plate. This may contain nutritional supplements for auxotrophs or may be incubated at a permissive temperature for conditional mutants.

10. Incubate plates under standard growth conditions until colonies form. Replica plate if necessary for mutant screening.

Similar frequencies of mutation can be achieved by treating 20 ml of cell suspension with 5-20/zl of diethyl sulfate (DES) for 30 rain. 9 A major drawback of both MNNG and DES is the tendency of both compounds to produce multiple clustered lesions. 13 Nitrous acid is more likely to pro- duce single site mutations, but this agent induces mutations at a much lower frequency than the other compounds. A 5-min treatment of cells with 50 mM nitrous acid resulted in a mutation frequency of 1.5 × 10-5; increasing the exposure time reduced viability without increasing the mu- tation frequency. 9

Mutagenesis by Irradiation with Ultraviolet Light

Ultraviolet (UV) radiation has been used successfully to increase the incidence of mutation 25- to 1000-fold in the unicellular species Synecho- cystis PCC 671414 (Aphanocapsa 6714) and Synechocystis PCC 680315 (Aphanocapsa 6803), as measured by the frequency of colonies resistant to p-fluorophenylalanine or fructose, respectively. Thiel and Leone ~6 ob- tained mutants of the filamentous species Anabaena variabilis ATCC 29413 following exposure to germicidal UV light which caused greater than 99% killing. However, Thiel and E. Levine report that this strain, as well as the other filamentous strains Anabaena PCC 7118, Anabaena PCC 7120, and A nabaena sp. M-13 (University of Tokyo) are all quite resistant to UV (T. Thiel, personal communication). Wolk and colleagues 17 used UV irradiation to obtain mutants of Anabaena PCC 7120 which are phe- notypically nitrogen-fixation defective. All of these researchers report that the cyanobacteria that have been tested have active photoreactiva- tion systems. For this reason an important requirement for obtaining mutants is a period of growth under nonphotoreactivating conditions. In strains which can not grow heterotrophically in the dark, this condition can be met by growth in yellow light. 17 The following procedure was

13 N. Guerola, J. L. Ingraham, and E. Cerda-Olmedo, Nature (London), New Biol. 230, 122 (1971).

14 C. Astier, F. Joset-Espardellier, and I. Meyer, Arch. Microbiol. 120, 93 (1979). 15 E. Flores and G. Schmetterer, J. Bacteriol. 166, 693 (1986). ~6 T. Thiei and M. Leone, J. Bacteriol. 168, 769 (1986). 17 C. P. Wolk, E. Flores, G. Schmetterer, A. Herrero, and J. Elhai, Proc. Int. Syrup.

Nitrogen Fixation, 6th, p. 491 (1985).

[80] MUTAGENESIS OF CYANOBACTERIA 719

compiled from the conditions used in the reports summarized above and from additional suggestions of some of these researchers.

Supplies for UV Mutagenesis

30-W germicidal UV light source, prewarmed for 30 min Yellow lights for cyanobacterial growth (e.g., General Electric Bug-

lites)

Procedure

I. If working with a filamentous strain, fragment the filaments to 1-3 cell lengths (see Special Considerations for Mutagenesis of Fila- mentous Cyanobacteria).

2. Suspend cyanobacterial cells in basal medium at 1-2 × 107 cells/ ml. Remove 1 ml as a control sample for steps 4-5.

3. Irradiate 5 ml of cells in a 100-mm petri dish at a distance of 45 cm from a 30-W germicidal UV light. Remove 1-ml portions at 2-min intervals.

4. Incubate aliquots for at least 36 hr under yellow lights at the same intensity used for growth in white light. Facultative chemohetero- trophs can be incubated in the dark with an appropriate carbon source.

5. Plate serial dilutions of mutagenized cells on appropriate basal or nutrient-enriched solid medium to assess survival.

6. Plate the remainder of each aliquot by spreading or by incorporat- ing in a soft agar overlay.

7. If a selective agent is needed, incubate plates under white light for at least 24 hr before adding the agent, either in a soft agar overlay or underneath the agar.

8. Continue incubating under normal growth conditions until colonies form.

G e n e Inac t iva t ion by Inser t ion of Se lec tab le He te ro logous Sequences

Mutagenesis by Transposition of Tn901

Cyanobacterial transposons have not yet been described, but a cyano- bacterial gene can be tagged and cloned by transposition of an E. coli element. Tandeau de Marsac et al. 18 identified a gene involved in methionine biosynthesis by transposition of transposon Tn90I (encoding

~s N. Tandeau de Marsac, W. E. Borrias, C. J. Kuhlemeier, A. M. Castets, G. A. van Arkel, and C. A. M. J. J. van den Hondel, Gene 20, 111 (1982).

720 MOLECULAR GENETICS [80]

ampicillin resistance) into the chromosome of Synechococcus PCC 7942 (Anacystis nidulans R2). They introduced the transposon into the cyano- bacterium by transformation with the plasmid pCH 1,~9 which replicates in this organism and carries Tn901. All of the transformed cells were resis- tant to the antibiotic ampicillin, encoded by the transposon. The mutant was detected by screening the total population of ampicillin-resistant transformants for methionine auxotrophs which were induced by inser- tion of the transposon into a structural gene. The inactivated methionine gene was isolated by cloning DNA from the mutant in E. coli and selecting ampicillin resistance encoded by the transposon. In turn the wild-type gene was identified from a library of Synechococcus PCC 7942 DNA by sequence similarity to the segment flanking the Tn901 element.

Although TngO1 transposition occurs at a low frequency, this tech- nique may be applicable in filamentous strains that can receive conjugat- able shuttle vectors from E. coli but that have not yet been shown to recombine foreign DNA into the chromosome. Discovery of conditions that induce transposition at a higher frequency or identification of ele- ments tha~t readily transpose in cyanobacterial strains would enhance the usefulness of this procedure. Dr. W. E. Borrias reports that similar exper- iments using Tn5 (kanamycin resistance) showed instability of the inser- tion sequences at the ends of the element during replication in the cyano- bacterium and resulted in an immobile kanamycin-resistance trait. Mutagenesis by transposition of elements from nonreplicating "suicide" vectors to the chromosome has not been reported for any of the conjugat- able or transformable strains. However, transposition from a nonreplicat- ing E. coli plasmid to an endogenous plasmid in Synechococcus PCC 7942 has been reported for two different transposons.~9,2°

Procedure. Plasmid pCH1 is an autonomously replicating plasmid for Synechococcus PCC 7942 which carries Tn901 and confers resistance to 1 /zg/ml ampicillin to the cyanobacterium. 19 Other shuttle vectors for trans- formation or conjugation between E. coli and a cyanobacterium should be suitable for carrying the transposon into the cell.

1. Introduce the transposon-containing plasmid into the cell by trans- formation, selecting for the transposon antibiotic-resistance marker and/or markers on the replicating vector.

2. Grow transformants to 108 cells/ml and culture for at least 10 gener- ations in liquid medium in the presence of 1 /xg/ml ampicillin (for

19 C. A. M. J. J. van den Hondel, S. Verbeek, A. van der Ende, P. J. Weisbeek, W. E. Borrias, and G. A. van Arkel, Proc. Natl. Acad. Sci. U.S.A. 77, 1570 (1980).

2o L. A. Sherman and P. van de Putte, J. Bacteriol. 150, 410 (1982).

[80] MUTAGENES1S OF CYANOBACTERIA 721

Tn901) and any nutritional supplement required by the expected mutant.

3. Wash cells by two sequential centrifugation steps, resuspending in growth medium at each step. Resuspend the final cell pellet at 5 × 106 cells/ml in growth medium and continue with a 50-ml sample.

4. If working with a filamentous strain, disrupt filaments as described below (section on Special Considerations for Mutagenesis of Fila- mentous Strains).

5. If the desired mutant should have impaired growth under normal, unsupplemented conditions, include steps for mutant enrichment (see below).

6. Harvest the cells and resuspend in 5 ml medium. 7. Plate cells onto solid medium with appropriate nutritional supple-

ments (for an expected auxotroph). Plate 100/.d of undiluted cells, as well as 10 -~, 10 -2, and 10 -3 dilutions to ensure obtaining a master plate with many well-separated colonies.

8. Incubate plates approximately 1 week, until colonies are visible. 9. Replica plate onto solid media with or without nutritional supple-

ments, or otherwise under conditions that allow detection of mu- tant colonies.

Recombinational Methods of Mutagenesis

Some methods of mutagenesis are uniquely applicable to the trans- formable unicellular cyanobacterial strains, 2 typified by Synechococcus PCC 7942 (Anacystis nidulans R2), Synechococcus PCC 7002 (Agmenel- lure quadruplicatum PR-6), and Synechocystis PCC 6803 (Aphanacapsa). Successful mutagenesis of each of these strains by integration of foreign DNA into the chromosome has been reported in the literature. 2~-23 It is likely that these methods will be more widely applicable as protocols for genetic transfer in other strains are developed or improved. The following paragraphs provide strategies rather than protocols for recombinational mutagenesis. The requirements for use of these procedures are (1) a trans- formable host which can incorporate nonreplicating DNA, (2) purified DNA from the host, and (3) an antibiotic-resistance gene "cassette" which is appropriate for selection in the host. Transformation protocols vary from strain to strain and are discussed elsewhere in this volume. 1,2 A

21 S. S. Golden, J. Brusslan, and R. Haselkorn, EMBO J. 5, 2789 (1986). 22 j. S. Buzby, R. D. Porter, and S. E. Stevens, Science 230, 805 (1985). 23 j . G. K. Williams, this volume [85].

722 MOLECULAR GENETICS [80]

recent chapter in this series deals specifically with these methods and describes techniques for the analysis of nucleic acids from the resulting mutant transformants. 24

Ectopic Mutagenesis

Ectopic mutagenesis was used by Buzby et al. z2 to produce mutations at random sites in the chromosome of Synechococcus PCC 7002. The basis of this techni~lue is the ligation of random fragments of chromo- somal DNA from the host to a DNA fragment bearing an antibiotic- resistance gene. The ligation is performed in such a manner that the ligated molecules remain linear and the DNA lacks sequences for replica- tion in the cyanobacterial host. The entire ligation mix is used to trans- form the host to antibiotic resistance. Transformation occurs by an un- known mechanism which results in the integration of the marker into the chromosome, presumably at the locus of the linked host DNA. These authors used random fragments of Synechococcus PCC 7002 DNA, par- tially digested with Sau3A, ligated to a BamHI/PvulI fragment carrying an ampicillin-resistance gene. The compatability of Sau3A and BamHI sticky ends allowed ligation to form linear ligated molecules, which could not circularize by the remaining Sau3A and PvulI ends. (Circular mole- cules bearing a single region of sequence similarity to the chromosome may integrate by an apparent single crossover which results in duplication of the chromosomal lOCUS. 24) The ectopic mutagenesis method is useful when a particular phenotype is desired which requires mutation at an unknown locus. The resulting antibiotic-resistant transformants can be screened for that phenotype, and the mutated locus is marked by the presence of heterologous DNA. The interrupted gene, and the corre- sponding wild-type gene, can then be cloned by the same strategy used to isolate a met gene by Tn901 insertion ~8 (see Mutagenesis by Transposition of Tn901).

Inactivation o f Specific Genes

Another insertional mutagenesis method which is applicable in the transformable strains is the inactivation of a known gene in the chromo- some by replacement with an interrupted allele. This procedure differs from that of ectopic mutagenesis in that the former method uses random site insertion to obtain a desired phenotype whereas the following method targets a particular locus to assess the phenotype which results from gene

24 S. S. Golden, J. Brusslan, and R. Haselkorn, this series, Vol. 153, p. 215.

[80] MUTAGENESIS OF CYANOBACTERIA 723

inactivation. This method was used recently to show that all three of the psbA genes in Synechococcus PCC 7942 are functional. 2~

A cyanobacterial gene cloned in an E. coli plasmid is digested with a restriction enzyme that cuts within the open reading frame of the gene to be inactivated and does not otherwise cut the plasmid. An antibiotic- resistance gene, hereafter referred to as the inactivation cassette, is li- gated to the ends to recircularize the plasmid. The desired plasmid is selected in E. coli by the antibiotic-resistance markers on the vector and from the inactivation cassette. Alternatively, the cyanobacterial gene can be interrupted by transposon insertion while it is maintained on an E. coli plasmid. 25 A preparation of the recombinant plasmid is then used to trans- form the cyanobacterial host to antibiotic resistance encoded by the inac- tivation cassette. The resulting transformants will have replaced the wild- type gene in the chromosome with the inactivated allele, and the (nonreplicating) plasmid vector sequences will be lost from the cell. Southern analysis is necessary to determine the new restriction pattern at that locus and thereby confirm inactivation of the gene. If a marker on the plasmid vector is selected, the transformants will be the result of a single crossover event which integrates the plasmid and causes a duplication at the site of chromosomal insertion. In Synechococcus PCC 7942, the dou- ble-crossover or gene conversion event which replaces the chromosomal gene occurs at a sufficiently higher frequency than the single-crossover event that plasmid integration is not observed unless specifically selected. Inactivation of presumed essential genes has been shown to result in the apparent selection of cells carrying a mixed population of chromosomes, so that the selectable marker and uninterrupted gene are both present. 2~

Site-Directed Mutagenesis

Another method made possible by recombination of cloned genes with the chromosome is site-directed mutagenesis, causing a single nucleotide mutation in a gene of interest. Site-directed mutagenesis of genes cloned in E. coli, by repair of a single strand hybridized to a mismatched oligo- mer, has been described. 26 When this method is used to alter a cloned cyanobacterial gene, the mutant gene can be returned to the cyanobacte- rial host by DNA-mediated transformation. Strategies for detecting inte- gration of the mutated DNA into the chromosome at its native locus are (1) direct selection for an expected phenotype, 27 (2) restoration of some

25 G. B. Ruvkun and F. M. Ausubel, Nature (London) 289, 85 (1981). 26 T. A. Kunkel, Proc. Natl. Acad. Sci. U.S.A. 82, 488 (1985). 27 S. S. Golden and R. Haselkorn, Science 229, 1104 (1985).

724 MOLECULAR GENETICS 180]

level of function (pseudorevertants) in a host which has had that gene previously inactivated, 23 (3) screening for loss of an antibiotic-resistance marker in a gene-inactivated strain, 23 and (4) selection for a linked marker. In the last case, an antibiotic-resistance gene is inserted within a cloned (cyanobacterial DNA) fragment as in the gene inactivation scheme. In this case the marker insertion is made at a restriction site outside of the coding region of the gene. Cyanobacterial DNA flanking the heterologous marker again directs it to the appropriate locus in the chromosome. Evidence that this strategy is feasible is provided by the insertion of Tn5 downstream of a herbicide-resistance allele of the Synechococcus PCC 7942 psbAl gene. 21 Ninety-six percent of the kanamycin-resistant transformants carried the linked, single nucleotide mutation conferring herbicide resistance. As is the case in the gene inacti- vation procedure, heterologous DNA outside of the borders of the cloned cyanobacterial DNA is lost during the recombination event unless the vector is specifically selected.

Special Considerations for Mutagenesis of Filamentous Cyanobacteria

Mutagenesis of filamentous strains poses a special problem in that the colony forming unit is a filament, whereas the target of mutagenesis is a chromosome in a single cell. This means that the resulting colony follow- ing mutagenesis can be a mixture of mutant and wild-type cells. If undis- rupted filaments are used for mutagenesis and plating, the phenotype of a single mutant cell in a filament would be lost at the level of screening colonies. Physical disruption of filaments to I-3 cell lengths prior to muta- genesis, and again after mutagenized cells have been allowed to recover and divide, minimizes the incidence of mixed colonies, a,28

Cavitation in a sonic cleaning bath, or by a sonic cell disruptor equipped with a microprobe tip, is useful for breaking filaments. Because there is considerable variability between individual sonicators, and be- cause tube dimensions and volume will affect cavitation, it is not practical to provide a specific protocol here. Treatment times in the literature vary from less than 15 sec to at least 15 rain. With either type of apparatus there are two parameters, filament length and cell survival, which should be assessed during treatment to establish a protocol. Microscopic exami- nation of aliquots is necessary to determine the number of cells per fila- ment and whether the cell suspension is being disrupted uniformly. It should be possible to achieve an average length of 1-3 cells and still maintain close to 100% plating efficiency. 9,28

28 C. P. Wolk and E. Wojciuch, Arch. Mikrobiol. 91, 91 (1973).

[80] MUTAGENESIS OF CYANOBACTERIA 725

Nonuniformity of filament length is a potential problem when using a sonicator microprobe tip which is placed into the cell suspension, from which most of the energy is directed downward such that filaments at the meniscus receive less exposure. To use this type of apparatus, the probe can be sterilized by immersing in 70% ethanol and rinsing in sterile dis- tilled water. To maximize exposure, the depth of the probe in the cell suspension should be the minimum penetration which does not cause splattering. Care should be taken to avoid heating during the sonication. Delivering the energy in measured pulses will help to control and repro- duce the exposure. Gentle mixing at intervals during treatment will im- prove uniformity. Using a sonic cleaning bath requires fewer precautions. The sample, in a sterile Erlenmeyer flask, can be placed directly in the bath. Intermittent swirling of the culture will improve uniform disruption.

The following simple procedure for estimating cell survival was devel- oped by Dr. D. Parker. Because it does not depend on counting colony forming units, survival can be determined independent of filament length. The assay is also appropriate for estimating survival from treatments other than sonication.

Estimated Survival Curve for Sonicated Samples

1. Make serial 2-fold dilutions of the unsonicated culture in basal medium, up to a 128-fold dilution. Spot 10/xl of each dilution and of the undiluted culture onto a basal medium-l% agar plate.

2. Remove two 10-~1 samples from the culture after different times of sonication. Spot one 10-/.d sample of each time point and a 4-fold dilution of the other onto the agar plate.

3. Incubate under normal growth conditions for 2.5-3 days until growth is evident.

4. Score plates as early as possible. Compare the appearance of each spot of sonicated cells with the dilutions of unsonicated cells. A sonicated spot that is equivalent to the 2-fold dilution of unsoni- cated cells represents approximately 50% survival, one equivalent to the 4-fold dilution represents 25% survival, etc.

Mutant Enrichment Methods

Antibiotic Selection of Growth-Impaired Mutants

The antibiotics penicillin, ampicillin, and cycloserine can be used to kill wild-type cells selectively in a mutagenized cyanobacterial culture as with chemoheterotrophic bacteria. It is necessary to carry out this enrich-

726 MOLECULAR GENETICS [80]

merit procedure under conditions in which the desired mutants have im- paired growth and are less susceptible to the drug. For auxotrophs or nitrogen fixation-deficient mutants, this is achieved by a starvation period in which the needed nutrient is deleted from the medium for a number of generations. Temperature-sensitive mutants can be treated following an incubation at the restrictive temperature to halt cell division. The follow- ing procedure is recommended as an addition to the classical and transpo- son-mediated mutagenesis methods,

Procedure

1. Mutagenize cells and continue with the chosen protocol through a postmutagenesis growth period to allow segregation of mutant and wild-type chromosomes. Fragment filaments if working with a fila- mentous strain (see Special Considerations for Mutagenesis of Fila- mentous Cyanobacteria).

2. Transfer the culture to restrictive conditions for 24 hr as a pre- enrichment step. For auxotrophs, wash cells twice with basal me- dium to remove nutritional supplements and carry out this incu- bation under normal growth conditions in basal medium. For temperature-sensitive mutants, incubate at the restrictive tempera- ture under standard illumination.

3. Add 150/xg/ml ampicillin or cycloserine ~s or 200 U/ml penicillin G 8 and incubate under illuminated, restrictive conditions for 16-24 hr or at least two generations.

4. Remove the antibiotic by two centrifugal washes. Resuspend cells for plating; see mutagenesis protocol for recommended plating den- sities.

Metronidazole Enrichment o f Photosynthetically Impaired Mutants

Guikema and Sherman 29 have developed a protocol for the isolation of temperature-sensitive photosynthesis mutants of Synechococcus ce- drorum (UTEX 1191, IU 1191) using the redox-active drug metronidazole (2-methyl-5-nitroimidazole-l-ethanol). Approximately one-half of the temperature-sensitive mutants (30 ° permissive/40 ° restrictive) examined were impaired in photosynthetic function, with abnormalities throughout the photosynthetic electron transport chain. Metronidazole is an effective electron acceptor of photosystem I and is specifically toxic to photosyn- thesis-proficient cells. Incubation of S. cedrorum cells in the presence of the drug caused an 80% reduction of viable cells in the dark, and killing was enhanced five orders of magnitude by illumination. Inhibitors of elec-

29 j . A. Guikema and L. A. Sherman, J. Bioenerg. Biomembr. 12, 277 (1980).

[80] MUTAGENESIS OF CYANOBACTERIA 727

tron t ransport such as DCMU block this enhancement of toxicity. Be- cause maximum toxicity is electron transport dependent , metronidazole enrichment is primarily useful for conditional photosynthesis mutants in facultative photohetero t rophs and chemoheterotrophs as well as obligate photoautotrophs.

Reagents for Metronidazole Enrichment

Metronidazole (Sigma) stock solution: prepare a fresh 50 m M solu- tion in glass-distilled water and filter sterilize before use

Procedure

1. Mutagenize cells as previously described and incubate under per- missive conditions to allow segregation of mutant chromosomes.

2. Transfer cells to restrictive conditions for one doubling to allow cessation of electron transport in photosynthetically impaired mu- tants.

3. Add metronidazole to 1 mM and incubate 6 hr under illuminated restrictive conditions.

4. Remove the drug by two centrifugal washes and resuspend in basal medium at a concentrat ion which, assuming 10 -5 survival, will yield 1-2 x 103 cells/ml.

5. Plate 100-/zl aliquots and incubate in permissive illuminated condi- tions for approximately 1 week, until colonies form. Replica plate to screen for a photosynthesis-impaired phenotype.

Acknowledgments

This chapter is a synthesis of methods which were worked out in the laboratories of the researchers who are referenced here. I gratefully acknowledge their contributions to this chapter. I especially thank Drs. Gerard van Arkel, Mies Borrias, Jack Meeks, Dorothy Parker, Terry Thiel, and Peter Wolk, who provided me with protocols and advice.