identification and characterization of novel nucleoid...

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Doctoral Thesis

Identification and Characterization of Novel Nucleoid Protein

(NNP) in Anaerobic Growth Escherichia coli

Jun Teramoto

Graduate School, Hosei University

Division of Material Chemistry

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CONTENTS

CONTENTS......................................................................................................... I

SUMMARY...................................................................................................... 1

INTRODUCITON................................................................................................ 3

MATERIALS AND METHODS ............................................................................ 8

BACTERIAL STRAINS AND CULTURE CONDITIONS.................................................... 8

PLASMID CONSTRUCTION.................................................................................... 8

PURIFICATION OF NNP PROTEIN.......................................................................... 9

SELEX SEARCH FOR NNP-BINDING SEQUENCES ................................................11

GEL MOBILITY SHIFT ASSAY ............................................................................... 12

DNA PROTECTION ASSAYS AGAINST NUCLEASES................................................. 13

QUANTITATIVE IMMUNOBLOT ANALYSIS ............................................................... 14

INDIRECT IMMUNO-FLUORESCENCE MICROSCOPY ............................................... 15

AFM SAMPLE PREPARATION.............................................................................. 17

AFM IMAGING ................................................................................................. 18

RESULTS......................................................................................................... 19

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ISOLATION OF NNP-BINDING SEQUENCES BY GENOMIC SELEX ........................... 19

TABLES......................................................................................................... 23

IDENTIFICATION OF NNP-BINDING ACTIVITY TO SELEX DNA FRAGMENTS: GEL-SHIFT

ASSAY............................................................................................................. 29

ANALYSIS OF YGIP-BINDING DNA SEQUENCES: DNASE-I FOOT-PRINTING ASSAY ... 32

INFLUENCE OF NNP ON THE NUCLEASE SENSITIVITY OF DNA: DNASE-I................ 37

INFLUENCE OF NNP ON THE NUCLEASE SENSITIVITY OF DNA: EXONUCLEASE III AND

S1 NUCLEASE.................................................................................................. 41

AFM ANALYSIS OF NNP-DNA COMPLEXES ........................................................ 46

INTRACELLULAR LEVEL OF NNP UNDER AEROBIC, HYPOXIC, AND ANAEROBIC

CONDITIONS .................................................................................................... 52

INTRACELLULAR LOCALIZATION OF NNP............................................................. 55

DISCUSSION................................................................................................... 59

ACKNOWLEDGMENTS................................................................................... 67

REFERENCES................................................................................................. 69

PUBLICATION LIST ......................................................................................... 75

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SUMMARY

A systematic search for the regulation targets by YgiP, an

uncharacterized LysR-family transcription factor in E. coli, has been

performed using the genomic SELEX screening. A total of 333 independent

clones have been isolated from 126 different loci in the E. coli genome. No

consensus recognition sequence has been obtained after gel shift and

DNase-I foot-printing assays, implying that YgiP is a DNA-binding protein

with no sequence specificity as the major nucleoid proteins, HU and IHF.

One unique activity of NNP is marked enhancement of the sensitivity of

associated DNA to nucleolytic digestion. The intracellular level of NNP is

very low in E. coli cells grown under aerobic conditions, leaving it hitherto

unidentified as a nucleoid protein, but increased more than 100-fold as

high as those of HU and IHF under anaerobic culture conditions. Taken

together we propose that YgiP is a novel nucleoid protein (hence renamed

to NNP) in anaerobic growth E. coli. When fully induced, NNP is located not

only within the nucleoid but also in the cytoplasm as detected by indirect

immuno-fluorescent microscopy. To get more insight into the mechanism

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of NNP-DNA interaction, we employed atomic force microscopy (AFM) to

directly investigate the structure of NNP-DNA complexes. In the presence

of saturated amounts of NNP, DNA was converted into a compact rod-like

conformation. Thus NNP may play a role of DNA compaction under

anaerobic growth conditions as does Dps in starved cells.

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INTRODUCITON

The complete sequence of Escherichia coli genome has revealed the

presence of 4,452 protein-coding sequences shared between two laboratory

strains, MG1655 and W3110, of K-12 lineage (Riley et al., 2006). The function of

gene product has been experimentally determined or indicated for about half of

the genes, but a large number of genes are still left uncharacterized even though

E. coli has long been used as a model organism in the modern molecular biology.

One major research subject to make a breakthrough is the identification of

functions for all these uncharacterized genes. The difficulty is arisen from the

fact that most of these genes are not expressed under standard laboratory

culture conditions and thus may be needed for E. coli survival under stressful

conditions in nature.

In the E. coli genome, there are approximately 4,500 genes (Riley et al.,

2006), but only a part of the genes are expressed in a given culture condition.

The second subject of post-genome sequence era is to reveal the molecular

basis of regulation underlying expression of the whole set of genes on the E. coli

genome. The genome expression is under the control of a total of about 300

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species of DNA-binding transcription factors (Perez-Rueda and Collado-Vides,

2000; Ishihama, 2000), which altogether determine the distribution of about

2,000 molecules of RNA polymerase within the genome (Ishihama, 1999: 2000).

Of a total of 300 transcriptiton factors, approximately 100 species belong to the

putative regulators that have been predicted, without experimental evidence, on

the basis of presence of DNA-binding and other protein motifs commonly

identified in the known transcription factors. The binding sites of E. coli

transcription factors are generally located within or near the promoters of target

genes or operons. For quick identification of the regulation target genes or

operons by these putative transcription factors, we have established the

genomic SELEX (systematic evolution of ligands by exponential enrichment)

screening system (Shimada et al., 2005) and have been successfully applied for

identification of the whole sets of genes under the control of some known or

unknown factors (Ogasawara et al., 2007a; 2007b; Shimada et al., 2007; Yang

et al., 2007). Once the consensus recognition sequence is predicted after

genomic SELEX screening, one can extend the search for other targets using

the information of whole genome sequence.

One of the putative regulators, YgiP, belongs to the LysR-type

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transcriptional factor family based on its protein sequence. The LysR-type

regulators are the most abundant transcription factor family in E. coli (Ishihama,

2007), and are present in diverse bacterial genera, archaea and algal

chloroplasts and involved in extremely diverse cellular functions including

nitrogen fixation, oxidative stress response and bacterial virulence (Schell, 1993).

LysR family proteins are generally composed of an N-terminal helix-turn-helix

motif, but no consensus sequence has been identified for the DNA-binding

sequences among LysR family members. In this study, a systematic search for

the YgiP-binding sequences has been performed by using the genomic SELEX

system (Shimada et al., 2005). After SELEX screening, we have obtained a

number of different YgiP-binding sequences from a number of loci within the E.

coli genome, but failed to identify the consensus sequence of YgiP binding. Gel

mobility shift and DNase-I foot-printing assays indicated that YgiP is rather

similar to the major nucleoid proteins, HU and IHF, with non-specific DNA

binding activity. However, one significant difference was its marked activity of

enhancing the sensitivity of probe DNA to nucleolytic digestion. The intracellular

level of YgiP is very low in E. coli cells grown under regular aerobic conditions,

leaving YgiP unidentified as a nucleoid protein (Azam and Ishihama, 1999;

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Azam et al., 2000; Ishihama, 2007). Under hypoxic or anaerobic culture

conditions, however, the YgiP level increased more than 100-fold, reaching to

the concentration as high as those of HU and IHF. As in the case of HU and IHF,

YgiP showed a uniform distribution within the entire nucleoid, but under fully

induced state, it also exists in the cytoplasm. Besides, we approached to

elucidate a single-molecule imaging technique (Atomic Force Microscopy: AFM)

(Hansma et al., 1988; Takeyasu et al., 2004; Morikawa et al., 2006) to directly

visualize a single molecule of YgiP-DNA interaction. The result from gel mobility

shift assay and DNase-I foot-printing assays in in vtro, YgiP have indicated

nucleoid protein like diagnostic behavior. Imaging and analyses of DNA-YgiP

complexes at the single molecule level can reveal conformational changes which

are concentration-dependent condensation of DNA by YgiP. The conformational

change has two structural formations, low molecules of YgiP have bond to DNA,

and high molecules of one have formed compaction of DNA. Taken all the

observations together, we will propose that YgiP is a hitherto unidentified

nucleoid protein in E. coli under anaerobic growth conditions, and thus propose

to rename it to NNP (novel nucleoid protein). During the preparation of this report,

Oshima and Biville (2006) reported, based on the location of the ygiP gene, that

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YgiP regulates the expression of neighboring ttdA-ttdB-ygiE operon. The major

nucleoid proteins, HU, IHF, H-NS and Fis, in exponential growth phase E. coli

under aerobic conditions are all known to have functional dichotomy (Ishihama,

2007). Likewise NNP appears to play dual roles, i.e., an architectural role and a

global regulator of transcription.

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MATERIALS AND METHODS

Bacterial strains and culture conditions

The E. coli strains used in this work were: KP7600 (W3110 typeA lacIq

lacZ ΔM15 galK2 galK22) and JD24074, KP7600 nnp, which was constructed by

a transposon insertion method (a gift from T. Miki). Cells were grown at 37℃ in

Luria-Bertani (LB) or M9-0.4% glucose medium. Overnight culture in LB medium

was diluted 1000-fold into fresh LB or M9 medium. For the aerobic culture, the

incubation was carried out at 37OC with a constant sharking to an appropriate

cell density. The cell density was determined by measuring the absorbance at

600 nm. For hypoxic culture, bacteria were suspended in 15 ml centrifuge tubes

with silicone stoppers to make the initial cell density of 0.1 OD600 and anoxia was

achieved to expel any remaining air by chemical duty pump (Millipore) for 60 min.

Cell culture was kept standing at 37OC without shaking.

Plasmid construction

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The promoter assay vector TFP carries two types of fluorescent protein

genes, one for red fluorescent protein (RFP) under the control of reference

promoter lacUV5 and the other for green fluorescent protein (GFP) under the

control of a test promoter (Makinoshima et al., 2002; Shimada et al., 2004). The

SELEX fragment containing ygiP-ttdA sequence upstream from the initiation

codon of ygiP was amplified by PCR using the genomic DNA from E. coli

KP7600 as a template and a pair of primers, H021S (5’-GGCCAGCTATTATGC

ATCGTTAATT-3’) and H021T (5’-GCTTTTTGCAAGATCTCGAACATCG-3’).

These primers contain EcoT22I, BglⅡ sites suitable for cloning into vector pGRP

at the initiation codon of the GFP coding frame. The PCR products were

digested with restriction enzymes and the ligated into the EcoT22I and BglⅡ

sites of TFP. The sequences of the inserted promoter and junction with GFP

coding frame were confirmed by sequencing. The plasmids thus constructed

were named pGRH021. For the construction of plasmid was used AFM analysis.

Purification of NNP protein

For construction of plasmid pNNP for NNP expression, a DNA fragment

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corresponding to the NNP-coding region was amplified by PCR using E. coli

W3110 genome DNA as a template and a pair of primers, which were designed

so as to hybridize upstream or downstream of the NNP-coding sequence. After

digestion with NdeI and NotI, PCP products were cloned into pET21a(+)

(Novagen) between NdeI and NotI sites. The plasmid construct was confirmed

by DNA sequencing. For protein expression, the pNNP plasmid was transformed

into E. coli BL21 (DE3). Transformants were grown in 200 ml of LB medium and

at the cell density of 0.6 at 600 nm, IPTG was added at the final concentration of

1 mM. After 3 hr incubation, cells were harvested by centrifugation, washed with

a lysis buffer (50 mM Tris-HCl, pH 8.0 at 4OC, and 100 mM NaCl), and then

stored at -80 OC until use.

For protein purification, frozen cells were suspended in 3 ml of lysis

buffer containing 100 mM PMSF. Cells were treated with lysozyme and then

subjected to sonication for cell disruption. After centrifugation at 15,000 rpm for

20 min at 4 OC, the resulting supernatant was mixed with 2 ml of 50% Ni-NTA

agarose solution (Qiagen) and loaded onto a column. After washing with 10 ml of

lysis buffer, the column was washed with 10 ml of washing buffer (50 mM

Tris-HCl, pH 8.0 at 4 OC, and 100 mM NaCl), and then 10 ml of washing buffer

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containing 10 mM imidazole. Proteins were then eluted with 2 ml of an elution

buffer (lysis buffer plus 200 mM imidazole), and peak fractions were pooled and

dialyzed against a storage buffer (50 mM Tris-HCl, pH 7.6 at 4 OC, 200 mM KCl,

10 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol), and stored at -80

OC until use. Protein purity was checked on SDS-PAGE.

SELEX search for NNP-binding sequences

The genomic SELEX screening was performed as described previously

(Shimada et al., 2005). In brief, genome DNA of E. coli W3110 was sonicated to

generate fragments of 150 to 300 bp in length. The E. coli DNA library was

constructed after cloning of these DNA fragments into plasmid pBR322 at

EcoRV site. In each experiment, a collection of these DNA fragments was

regenerated by PCR-amplification using the E. coli DNA library plasmids as

templates and a set of primers, EcoRV-F (5’-CTTGGTTATGCCGGTACTGC-3’)

and EcoRV-R (5’-GCGATGCTGTCGGAATGGAC-3’), which hybridize to

pBR322 vector at EcoRV junctions. PCR products thus generated were purified

by 6% polyacrylamide gel electrophoresis (PAGE). For the genomic SELEX

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screening of NNP-binding sequences, 5 pmol of DNA fragments and 10 pmol

His-tagged NNP were mixed in a binding buffer (10 mM Tris-HCl, pH 8.0 at 4OC,

3 mM Mg-acetate, 150 mM NaCl, and 0.1 mM EDTA) and incubated for 30 min

at 37OC. The mixture was applied onto Ni-NTA column and after washing

unbound DNA with the binding buffer containing 10 mM imidazole, DNA-NNP

complexes were eluted with an elution buffer containing 200 mM imidazole. DNA

fragments recovered from the complexes were ligated into pBR322 and

PCR-amplified as above. For sequencing of NNP-bound DNA fragments, PCR

products were cloned into pT7 Blue-T vector (Novagen) and transformed into E.

coli DH5α. Sequencing analysis was carried out using T7-primer

(5’-TAATACGACTCACTATAGGG-3’) with ABI DNA sequencer 3130x.

Gel mobility shift assay

Probes were generated by PCR amplification of NNP-binding

sequences in SELEX using a pair of primers, 5’ fluorescein isothiocyanate

(FITC)–labeled T7-F primer (5’-TAATACGACTACTATAGGG-3’) and T7-R primer

(5’-GGTTTTCCCAGTCACACGACG-3’), the genomic SELEX plasmids

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containing the respective NNP recognition sequences as templates, and Ex Taq

DNA polymerase. PCR products with FITC at their termini were purified by PAGE.

For gel shift assays, 0.5 pmol each of the FITC-labeled probes was incubated at

37OC for 30 min with various amounts of NNP in 15 μl of gel shift buffer

consisting of 10 mM Tris-HCl, pH 7.8 at 4OC, 150 mM NaCl, 3 mM Mg acetate.

After the addition of DNA dye solution, the mixture was directly subjected to 6%

PAGE. Fluorescent-labeled DNA in gels was detected using Pharos FX plus

system (Bio-Rad).

DNA protection assays against nucleases

DNase-I footprinting assay was carried out using FITC-labeled DNA

fragments as described previously (Ogasawara et al., 2007a; 2007b). In brief,

1.0 pmol each of FITC-labeled probes was incubated at 37OC for 30 min with

various amounts of NNP in DNase-I foot-printing buffer consisting of 25 μl of 10

mM Tris-HCl, pH 7.8, 150 mM NaCl, 3 mM magnesium acetate, and 5 mM CaCl2.

After incubation for 30 min, DNA digestion was initiated by the addition of 10 ng

of DNaseⅠ (TaKaRa). After digestion for 30 s at 25OC, the reaction was

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terminated by the addition 25 μl phenol-chloroform. Digested products were

precipitated with ethanol, dissolved in formamide dye, and analyzed by

electrophoresis on a 6% polyacrylamide gel containing 8 M urea. Gel patterns

were recorded using Slab gel DNA sequencer (SHIMADZU).

DNA protection assay against exonuclease III and S1 nuclease was also

carried out using FITC-labeled DNA fragment probes. One pmol each of

FITC-labeled probes was incubated at 37OC for 30 min with various amounts of

NNP. After addition of 1/4 volume of 4x exonuclease III mixture [10x exonuclease III

buffer containing 1 U/μl exonuclease III (TaKaRa)], the digestion reaction was

carried out at 25OC for 1 hr. For DNA protection assay against S1 nuclease, 1/4

volume of 4x S1 nuclease mixture [10x S1 nuclease buffer and 1 U/μl S1

nuclease (TaKaRa)] was added to the NNP-DNA complexes and the S1

digestion was carried out 25OC for 10 min.

Quantitative immunoblot analysis

A quantitative Western blot analysis was carried out by standard method

as described previously (Jishage et al., 1999). In brief, E. coli cells grown in 10

ml of either LB or M9-0.4% glucose medium were harvested by cenrifugation

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and resuspended in 0.3 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl,

5% glycerol, and 1 mM dithiothreitol), and then lysozyme was added to a final

concentration of 20 μg/ml. Total proteins were subjected to 12% SDS-PAGE and

blotted on to PVDF membranes using semi-dry transfer apparatus. Membranes

were first immuno-detected with anti-NNP and then developed with an enhanced

chemiluminescence kit (Amersham-Pharmacia Biotech). The image was

analysed with a LAS-1000 Plus lumino-image analyzer and IMAGE GAUGE

(Fuji Film).

Indirect immuno-fluorescence microscopy

The procedure for indirect immuno-fluorescence microscopy was

essentially the same as previously described (Hiraga et al., 1998; Azam et al.,

2000). In brief, 1 ml of cell culture was added directly into 10 ml of 80% methanol,

mixed very gently by hand (avoiding a vortex), and left for 1 hr at 25 OC. The

fixed cells were collected by centrifugation and resuspended in 0.5-1 ml of 80%

methanol. The methanol-fixed cell suspension (10 μl) was deposited on to a

slide, which was coated with poly L-lysine (1 mg/ml, Mr 150,000-300,000, Wako,

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Japan), and air-dried for 20 min in a clean bench to fix the cells tightly on the

slide. The dried cell area was covered with 50 μl of lysozyme solution (2 mg/ml in

25 mM Tris-HCl, pH 8.0, 50 mM glucose, and 10 mM EDTA) and incubated at

room temperature for 5 min. The slide was covered with 4 ml of 99% methanol

for 1 min, and then with 4 ml of acetone for 1 min until complete dryness. The

dried sample area was surrounded by a paraffin square (23×23 mm), and 50 μl

of PBST (140 mM NaCl, 2 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4 and 0.05%

Tween 20) containing 2% bovine serum albumin (BSA) was placed on the slide

for 15 min for blocking the sample. An aliquot (20-50 μl) of 200~500-fold diluted

anti-NNP antibodies was placed on the sample, and left for 1 hr at room

temperature in a moisture chamber. After washing three times with 5 ml each of

PBST, the sample was treated at room temperature for 1 hr in the moisture

controlled chamber with a 20-50 μl aliquot of 500-fold diluted goat anti-rabbit IgG

antibodies conjugated with a fluorescence compound Cy3. After washing three

times with PBST, the immuno-stained sample was stained with 10 μl of DAPI

(4’,6-diamidino-2-phenylindole dihydrochloride, 10 μg/ml) for 1 min, and covered

with one drop of mountain medium (1 mg/ml p-phenylenediamine, 90% glycerol

in PBS, pH 9.0). The immuno-stained samples were observed with a

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fluorescence and phase-contrast microscope (100×objective lens; Olympus,

Japan) equipped with a chilled, high sensitive digital CCD camera, Regita EXi

(NIPPON ROPER, Japan), connected to a computer. Different filter cassettes

were used corresponding fluorescence compounds. The images were

transferred directly to a Windows XP and processed using Image-Pro Plus

ver.6.0 software. The specificity of the observed immuno-stained fluorescent

signal was verified by the absence of the signal in corresponding

immuno-stained samples prepared using the respective nnp null mutant cells.

AFM sample preparation

Complexes of protein and pGRNNP-10 were formed by incubation of

200 ng of DNA with 10 to 800 ng of NNP, as indicated in the text, in 10 x binding

buffer [50 mM HEPES-KOH (pH 7.6), 1 mM EDTA, 500 mM NaCl, 1 mM DTT ]

for 30 min at 37 OC. This incubation mixture was then fixed 0.1% glutaraldehyde

for 1 hr at 4 OC. After the fixation products were 1/20 dilutions (5 ng/μl of DNA)

[dilution buffer; to final concentration of 5 mM HEPES-KOH (pH 7.6), 2 mM

MgCl2] and pedosited onto a spermidine (1 mM)-treated mica surface. In ten

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minutes, the mica was gently washed with distilled water and dried under

nitrogen gas.

AFM imaging

Imaging was performed in Tapping ModeTM with a MultimodeTM AFM

(Veeco, Santa Barbara, CA) operation with a Nanoscope IIIaTM controller. We

used Olympus silicon cantilevers with spring constants between 36 to 75 N/m.

The scan frequency was typically 1.5 Hz per line and the modulation amplitude

was a few nanometers. Analysis of the DNA-NNP complex was done with the

‘Analyse Section’ function in the Nanoscope software. Two-demensional images

in brightness and contrast were optimized for the purpose of clarity.

Three-dimensional images were created with the Nanoscope software and

exported in TIFF format.

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RESULTS

Isolation of NNP-binding sequences by genomic SELEX

For the identification of DNA sequences that are recognized by E. coli

NNP, we employed the genomic SELEX method (Shimada et al., 2005), which

uses a complete library of E. coli genome DNA fragments, instead of synthetic

oligonucleotides with all possible sequences used in the original SELEX method

(Ellington and Szostak, 1990; Tuerk and Gold, 1990; Singer et al., 1997). First,

we constructed the plasmid library, each carrying a piece of the size-fractionated

DNA fragment (150 to 300 bp in length) from a pool of sonicated E. coli W3110

genome DNA. In each experiment, the DNA fragment mixture was regenerated

after amplification of the inserted DNA fragments by PCR. From a mixture of

these DNA fragments and a fourfold molar excess of the purified His-tagged

NNP protein, the NNP-DNA complexes were affinity purified. In the early stage of

this genomic SELEX cycle, the NNP-bound DNA fragments gave smear bands

on PAGE as did the original genome fragment mixture. After five and six SELEX

cycles, however, the width of the gel band decreased, indicating enrichment of

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specific DNA fragments with NNP-binding activity. The SELEX DNA fragments

were recovered from the gel and cloned into the pT7 Blue plasmid (Novagen) for

sequencing.

A total of 333 independent clones were isolated and classified into two

different groups (Table 1). Group A included 147 independent clones, each

carrying a unique sequence from 31 different spacer regions between two

neighboring genes in the E. coli genome. The most abundant 69 clones were

from nnp-ttdA spacer, followed by 22 clones from ffh-ypjD, 10 clones from

maoC-paaA, 4 clone each from yfaZ-yfaO, ygdH-sdaC, fimB-fimE, 3 clones from

the cadB-cadC, 2 clone each from 7 different loci, and only 1 clone each from

other 17 loci (Table 2). In prokaryotes, the recognition DNA sequences by

transcription regulatory factors are generally located around promoters. If NNP is

such a DNA-binding transcription factor, the regulation targets may be the genes

that are transcribed towards opposite direction from the respective NNP-binding

site (shown in bold in Table 1).

A total of 69 clones from the nnp-ttdA spacer contained two different

segments from a 305 bp-long sequence between nucleotides 3205836 and

3205897 in the revised E. coli genome (Riley et al., 2006). NNP might have a

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strong affinity to this nnp-ttdA spacer region because the number of SELEX

isolates by a test transcription factor correlates with its affinity to the target

sequence (Shimada et al., 2005; Ogasawara et al., 2007a; 2007b). This

sequence with strong affinity to NNP is located upstream of nnp (encoding a

putative transcriptional regulator) [leftward transcription] and upstream of ttdA

(encoding L-tartrate dehydratase subunit) [rightward transcription]. One possible

function of this site is the autogenous control of nnp expression by NNP, but the

expression of the ttdA-ttdB-ygjE operon was shown to be reduced after

disruption of the nnp gene (Oshima and Biville, 2006), indicating that NNP is a

positive regulator of L-tartrate-dependent induction of this operon.

On the other hand, group B included 186 independent clones (Table 1),

which were derived from a total of 96 protein-coding regions, including 19 clones

from nrfE, 15 clones from yhfS, 10 clones from ebgA, 4 clones each from yagM,

mltB, yegB, and yiiG, 3 clones each from kdpB, yegB, and yfcN, and 2 clones

each from 15 different loci (Table 2). By using the genomic SELEX analyses, the

binding DNA sites by transcription factors are often identified within

protein-coding sequences (Shimada et al., 2005; Ogasawara et al., 2007a;

2007b). Possible influence on transcription elongation, termination and/or

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anti-termination remains to be examined.

One unique feature of the genomic SELEX with NNP is the variety of its

recognition sequences along the E. coli genome. A total of 333 independent

clones were isolated from 126 different locations. This may indicate that NNP is

one of the global regulators controlling a number of genes. However, this level

variety of the recognition DNA sequence has never been observed among the

well-characterized global regulators so far examined by the genomic SELEX,

including CRP (Fujita, N., unpubliched data), Cra (Shimada et al., 2005), PdhR

(Ogasawara et al., 2007b), RutR (Shimada et al., 2007) and TyrR (Yang et al.,

2007). This finding supports the concept that NNP plays a role in structuring the

nucleoid under yet uncharacterized environmental conditions.

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TABLES

TABLE 1. NNP-bound DNA fragmentsa

----------------------------------------------------------------------------------------------------------

Group No. of No. of Total no. Group No. of No. of Total no.

clones loci of clones clones loci of clones

----------------------------------------------------------------------------------------------------------

A 69 1 69 B 19 1 19

22 1 22 15 1 15

10 1 10 10 1 10

4 3 12 4 3 12

3 1 3 3 3 9

2 7 14 2 35 70

1 17 17 1 51 51

------------------------------ -------------------------------------------

31 147 95 186

----------------------------------------------------------------------------------------------------------

a Using the genomic SELEX method, a total of 333 DNA fragments have been

isolated as complexes with the purified NNP protein. A total of 147 group A

clones contained the sequences from spacer regions between the neighboring

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genes, while a total of 190 group B clones carried the sequences within the

coding frames. The location of all these SELEX fragments on the genome is

described in Table 2.

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TABLE 2. Location of NNP-binding Sitesa

yjhR → ( 4535728) ( 4535985) ← yjhS1 S

allA → ( 531701 ) ( 531911 ) → allR2 S

yhiM → ( 3635488) ( 3635757) ← yhiN1 SmalS → ( 3738935) ( 3739191) → avtA1 SyicO ← ( 3843357) ( 3843543) → yicP1 SyjbI → ( 4251570) ( 4251774) → ubiC1 S

ompC ← ( 2317295) ( 2317546) → yojN1 SygfJ → ( 3015432) ( 3015694) → ygfK1 SpitB ← ( 3136082) ( 3136260) ← gsp1 SgltD → ( 3360408) ( 3360585) → gltF1 S

ompF ← ( 987209 ) ( 987440 ) ← asnS1 Sdcp ← ( 1629907) ( 1630108) → ydfG1 SfadD ← ( 1892260) ( 1892474) ← yeaY1 SintZ → ( 2558200) ( 2558410) → yffL1 S

proS ← ( 218396 ) ( 218569 ) → yaeB1 SyafT → ( 237914 ) ( 238081 ) ← yafU1 ScyoA ← ( 450685 ) ( 450903 ) ← ampG1 SclpX → ( 457498 ) ( 457687 ) → lon1 S

nanA ← ( 3372949) ( 3373146) ← nanR2 S

ykgC ← ( 318930 ) ( 319113 ) → ykgD2 ScobU ← ( 457498 ) ( 457687 ) → yi522 S

nth → ( 1714950) ( 1715145) → ydgR2 SyejG ← ( 2282437) ( 2282655) ← bcr2 S

ygiP ← ( 3205705) ( 3206011) → ttdA69 Sffh ← ( 2746945) ( 2747146) → ypjD22 S

maoC ← ( 1456223) ( 1456408) → paaA10 SyfaZ ← ( 2369883) ( 2370053) ← yfaO4 SygdH → ( 2927325) ( 2927487) → sdaC4 SfimB → ( 4540962) ( 4541151) → fimE4 ScadB ← ( 4359682) ( 4359901) ← cadC3 SnhaR ← ( 19300 ) ( 19473 ) → insB2 S

No. of clones

Left gene

(Direction) (Direction) Right gene

SELEXfragment

A

yjhR → ( 4535728) ( 4535985) ← yjhS1 S

allA → ( 531701 ) ( 531911 ) → allR2 S

yhiM → ( 3635488) ( 3635757) ← yhiN1 SmalS → ( 3738935) ( 3739191) → avtA1 SyicO ← ( 3843357) ( 3843543) → yicP1 SyjbI → ( 4251570) ( 4251774) → ubiC1 S

ompC ← ( 2317295) ( 2317546) → yojN1 SygfJ → ( 3015432) ( 3015694) → ygfK1 SpitB ← ( 3136082) ( 3136260) ← gsp1 SgltD → ( 3360408) ( 3360585) → gltF1 S

ompF ← ( 987209 ) ( 987440 ) ← asnS1 Sdcp ← ( 1629907) ( 1630108) → ydfG1 SfadD ← ( 1892260) ( 1892474) ← yeaY1 SintZ → ( 2558200) ( 2558410) → yffL1 S

proS ← ( 218396 ) ( 218569 ) → yaeB1 SyafT → ( 237914 ) ( 238081 ) ← yafU1 ScyoA ← ( 450685 ) ( 450903 ) ← ampG1 SclpX → ( 457498 ) ( 457687 ) → lon1 S

nanA ← ( 3372949) ( 3373146) ← nanR2 S

ykgC ← ( 318930 ) ( 319113 ) → ykgD2 ScobU ← ( 457498 ) ( 457687 ) → yi522 S

nth → ( 1714950) ( 1715145) → ydgR2 SyejG ← ( 2282437) ( 2282655) ← bcr2 S

ygiP ← ( 3205705) ( 3206011) → ttdA69 Sffh ← ( 2746945) ( 2747146) → ypjD22 S

maoC ← ( 1456223) ( 1456408) → paaA10 SyfaZ ← ( 2369883) ( 2370053) ← yfaO4 SygdH → ( 2927325) ( 2927487) → sdaC4 SfimB → ( 4540962) ( 4541151) → fimE4 ScadB ← ( 4359682) ( 4359901) ← cadC3 SnhaR ← ( 19300 ) ( 19473 ) → insB2 S

No. of clones

Left gene


SELEXfragment

A

- 26

alsE(yjcU) ← ( 4308156 ) S(alsC ) ( 4308369 ) ← alsA(yjcW)2rpiR ← ( 4312943 ) S(rpiB ) ( 4313143 ) ← phnQ2

ilvE → ( 3954019 ) S(ilvD ) ( 3954145 ) → ilvA2rhaT ← ( 4100341 ) S(sodA )( 4100488 ) → kdgT2iclR ← ( 4225813 ) S(metH )( 4226095 ) → yjbB2

malM → ( 4251570 ) S(yjbI ) ( 4251774 ) → ubiC2

tldD ← ( 3393150 ) S(yhdP )( 3393340 ) ← yhdR2yhdY → ( 3421974 ) S(yhdZ )( 3422181 ) ← rrfF2bcsC ← ( 3689310 ) S(bcsZ )( 3689528 ) ← bcsB2yieE → ( 3894560 ) S(yieF ) ( 3894726 ) ← yieG2

yfcG → ( 2427678 ) S(folX ) ( 2427939 ) → yfcH2evgA → ( 2490983 ) S(evgS )( 2491159 ) ← ｙｆｄE2yfhP ← ( 2662520 ) S(yfhQ )( 2662803 ) → suhB2yghB → ( 3154054 ) S(yqhC )( 3154243 ) → yqhD2

yodC ← ( 2031414 ) S(yedI ) ( 2031645 ) → yedA2sanA → ( 2239167 ) S(yeiT ) ( 2239426 ) → yeiA2ompC ← ( 2319129 ) S(yojN ) ( 2319369 ) → rcsB2yfaQ ← ( 2335728 ) S(yfaS ) ( 2335898 ) ← yfaS2

ompN ← ( 1440538 ) S(ydbK )( 1440791 ) ← hslJ2ydiJ ← ( 1772386 ) S(ydiK ) ( 1772548 ) → ydiL2ynjI ← ( 1848944 ) S(topB ) ( 1849142 ) ← selD2

yecO → ( 1956353 ) S(yecP )( 1956598 ) ← torZ2

aqpZ ← ( 917438 ) S(ybjD ) ( 917699 ) ← ybjX2ymcB ← ( 1048824 ) S(ymcC) ( 1048999 ) ← ymcD2ymfM ← ( 1206523 ) S(ymfN )( 1206714 ) → ymfR2ycjX → ( 1388664 ) S(ycjF ) ( 1388889 ) → tyrR2

secM → ( 109289 ) S(secA )( 109566 ) → mutT2yacG ← ( 111663 ) S(yacF )( 111911 ) ← coaE2panC ← ( 148535 ) S(panB )( 148751 ) ← yadC2mltD ← ( 233850 ) S(gloB ) ( 234048 ) → yafS2tauD → ( 387678 ) S(hemB) ( 387943 ) → yaiT2ybaK ← ( 506372 ) S(ybaP )( 506616 ) → ybaQ2ybaQ → ( 509633 ) S(copA )( 509891 ) → ybaS2ybbS ← ( 531701 ) S(allA ) ( 531911 ) → allR2

kdpC ← ( 725421 ) S(kdpB )( 725635 ) ← kdpA3yegO ← ( 2164860 ) S(yegB )( 2165055 ) → baeS3yfcB ← ( 2455123 ) S(yfcN ) ( 2455386 ) ← yfcO3caiD ← ( 37548 ) S(caiC ) ( 37785 ) ← caiB2

yagL ← ( 293192 ) S(yagM )( 293411 ) ← yagN4ygaD ← ( 2824831 ) S(yegB )( 2825033 ) → srlE4fdhD → ( 4086780 ) S(yiiG ) ( 4086965 ) ← frvR4

ebgR → ( 3224992 ) S(ebgA )( 3224378 ) → ebgC10

nrfD → ( 4294066 ) S(nrfE ) ( 4296753 ) → nrfF19yhfR → ( 3504888 ) S(yhfS ) ( 3505060 ) ← yhfT15

No. of clones

Left gene


SELEXfragment

B

alsE(yjcU) ← ( 4308156 ) S(alsC ) ( 4308369 ) ← alsA(yjcW)2rpiR ← ( 4312943 ) S(rpiB ) ( 4313143 ) ← phnQ2

ilvE → ( 3954019 ) S(ilvD ) ( 3954145 ) → ilvA2rhaT ← ( 4100341 ) S(sodA )( 4100488 ) → kdgT2iclR ← ( 4225813 ) S(metH )( 4226095 ) → yjbB2

malM → ( 4251570 ) S(yjbI ) ( 4251774 ) → ubiC2

tldD ← ( 3393150 ) S(yhdP )( 3393340 ) ← yhdR2yhdY → ( 3421974 ) S(yhdZ )( 3422181 ) ← rrfF2bcsC ← ( 3689310 ) S(bcsZ )( 3689528 ) ← bcsB2yieE → ( 3894560 ) S(yieF ) ( 3894726 ) ← yieG2

yfcG → ( 2427678 ) S(folX ) ( 2427939 ) → yfcH2evgA → ( 2490983 ) S(evgS )( 2491159 ) ← ｙｆｄE2yfhP ← ( 2662520 ) S(yfhQ )( 2662803 ) → suhB2yghB → ( 3154054 ) S(yqhC )( 3154243 ) → yqhD2

yodC ← ( 2031414 ) S(yedI ) ( 2031645 ) → yedA2sanA → ( 2239167 ) S(yeiT ) ( 2239426 ) → yeiA2ompC ← ( 2319129 ) S(yojN ) ( 2319369 ) → rcsB2yfaQ ← ( 2335728 ) S(yfaS ) ( 2335898 ) ← yfaS2

ompN ← ( 1440538 ) S(ydbK )( 1440791 ) ← hslJ2ydiJ ← ( 1772386 ) S(ydiK ) ( 1772548 ) → ydiL2ynjI ← ( 1848944 ) S(topB ) ( 1849142 ) ← selD2

yecO → ( 1956353 ) S(yecP )( 1956598 ) ← torZ2

aqpZ ← ( 917438 ) S(ybjD ) ( 917699 ) ← ybjX2ymcB ← ( 1048824 ) S(ymcC) ( 1048999 ) ← ymcD2ymfM ← ( 1206523 ) S(ymfN )( 1206714 ) → ymfR2ycjX → ( 1388664 ) S(ycjF ) ( 1388889 ) → tyrR2

secM → ( 109289 ) S(secA )( 109566 ) → mutT2yacG ← ( 111663 ) S(yacF )( 111911 ) ← coaE2panC ← ( 148535 ) S(panB )( 148751 ) ← yadC2mltD ← ( 233850 ) S(gloB ) ( 234048 ) → yafS2tauD → ( 387678 ) S(hemB) ( 387943 ) → yaiT2ybaK ← ( 506372 ) S(ybaP )( 506616 ) → ybaQ2ybaQ → ( 509633 ) S(copA )( 509891 ) → ybaS2ybbS ← ( 531701 ) S(allA ) ( 531911 ) → allR2

kdpC ← ( 725421 ) S(kdpB )( 725635 ) ← kdpA3yegO ← ( 2164860 ) S(yegB )( 2165055 ) → baeS3yfcB ← ( 2455123 ) S(yfcN ) ( 2455386 ) ← yfcO3caiD ← ( 37548 ) S(caiC ) ( 37785 ) ← caiB2

yagL ← ( 293192 ) S(yagM )( 293411 ) ← yagN4ygaD ← ( 2824831 ) S(yegB )( 2825033 ) → srlE4fdhD → ( 4086780 ) S(yiiG ) ( 4086965 ) ← frvR4

ebgR → ( 3224992 ) S(ebgA )( 3224378 ) → ebgC10

nrfD → ( 4294066 ) S(nrfE ) ( 4296753 ) → nrfF19yhfR → ( 3504888 ) S(yhfS ) ( 3505060 ) ← yhfT15

No. of clones

Left gene


SELEXfragment

B

- 27

malM → ( 4251570 ) S (yjbI ) ( 4251774 ) → ubiC1yjeB → ( 4406794 ) S (rnr ) ( 4407105 ) → yjfH1yjgR ← ( 4490031 ) S (idnR ) ( 4490283 ) ← idnT1fimH → ( 4549835 ) S (gntP ) ( 4550035 ) → uxuA1yjiJ ← ( 4562158 ) S (yjiK ) ( 4562402 ) ← yjiL1

mcrD ← ( 4577218 ) S (mcrC )( 4577474 ) → mcrB1riml ← ( 4608405 ) S (yjjG ) ( 4608606 ) → prfC1

yicG → ( 3820017 ) S (yicF ) ( 3820274 ) → gmk1wecB ← ( 3971159 ) S (wecC )( 3971344 ) → rffG1mobB ← ( 4040866 ) S (mobA )( 4041046 ) → yihD1glpF ← ( 4117901 ) S (yiiU ) ( 4118115 ) ← menG1

rplN ← ( 3447848 ) S (rpsQ ) ( 3448126 ) ← rpmC1aroB ← ( 3518216 ) S (aroK ) ( 3518461 ) ← hofQ1yhhW ← ( 3579170 ) S (yhhX ) ( 3579419 ) → yhhY1tdh ← ( 3791893 ) S (kbl ) ( 3792174 ) ← htrL1

tdcE ← ( 3262480 ) S (tdcD ) ( 3262763 ) ← tdcC1infB ← ( 3316214 ) S (nusA ) ( 3316430 ) ← yhbC1mdh ← ( 3384291 ) S (argR ) ( 3384490 ) → yhcN1yhdW → ( 3420328 ) S (yhdX ) ( 3420616 ) → yhdY1

ygdR → ( 2971234 ) S (tas) ( 2971469 ) ← ygeD1yghY ← ( 3147858 ) S (yghZ ) ( 3147959 ) ← yahA1ygiC → ( 3181189 ) S (ygiD ) ( 3181476 ) → ygiE1rpsU ← ( 3211261 ) S (dnaG )( 3211424 ) → rpoD1

gyrA ← ( 2345578 ) S (ubiG ) ( 2345831 ) ← yfaL1yfeN → ( 2532516 ) S (yfeR ) ( 2532780 ) → yfeH1yfgC → ( 2617317 ) S (yfgD ) ( 2617460 ) ← yfgE1grpE ← ( 2750781 ) S (ppnK ) ( 2751003 ) → recN1

ydhP ← ( 1741095 ) S (purR ) ( 1741371 ) ← ydhB1yejG ← ( 2283190 ) S (bcr ) ( 2283409 ) ← rsuA1ccmH ← ( 2296502 ) S (dsbE ) ( 2296764 ) ← ccmF1yfaT ← ( 2341705 ) S (yfaA ) ( 2341886 ) ← gyrA1

yciN ← ( 1334499 ) S (topA ) ( 1334720 ) → cysB1yneL ← ( 1594360 ) S (hipA ) ( 1594584 ) ← hipB1speG → ( 1659282 ) S (ynfC ) ( 1659526 ) → ynfD1ydgK → ( 1708310 ) S (rsxA ) ( 1708588 ) → rsxB1

ompX → ( 851728 ) S (ybiP ) ( 851955 ) → mntR1ycaD → ( 947247 ) S (ycaM )( 947555 ) ← ycaN1ymfM → ( 1206523 ) S (ymfN ) ( 1206714 ) → ymfR1hns ← ( 1296454 ) S (tdk) ( 1296693 ) ← ychG1

envY ← ( 586171 ) S (ybcH ) ( 586443 ) ← nfrA1citX ← ( 647089 ) S (citF ) ( 647347 ) ← citE1citF ← ( 649035 ) S (citE ) ( 649263 ) ← citD1ybhH → ( 801991 ) S (ybhI ) ( 802307 ) → ybhJ1

cueO → ( 139751 ) S (gcd ) ( 139930 ) → hpt1lpcA → ( 244095 ) S (yafJ ) ( 244364 ) ← yafK1yagB ← ( 280165 ) S (yagA ) ( 280365 ) → yagE1ybbL → ( 515749 ) S (ybbM )( 516003 ) ← ybbN1

ftsA → ( 105506 ) S (ftsZ ) ( 105778 ) → lpxC1acnB → ( 134201 ) S (yacL ) ( 134432 ) → speD1

yabQ → ( 59764 ) S (rluA ) ( 59966 ) ← hepA1ftsQ → ( 103836 ) S (ftsA ) ( 104038 ) → ftsZ1

malM → ( 4251570 ) S (yjbI ) ( 4251774 ) → ubiC1yjeB → ( 4406794 ) S (rnr ) ( 4407105 ) → yjfH1yjgR ← ( 4490031 ) S (idnR ) ( 4490283 ) ← idnT1fimH → ( 4549835 ) S (gntP ) ( 4550035 ) → uxuA1yjiJ ← ( 4562158 ) S (yjiK ) ( 4562402 ) ← yjiL1

mcrD ← ( 4577218 ) S (mcrC )( 4577474 ) → mcrB1riml ← ( 4608405 ) S (yjjG ) ( 4608606 ) → prfC1

yicG → ( 3820017 ) S (yicF ) ( 3820274 ) → gmk1wecB ← ( 3971159 ) S (wecC )( 3971344 ) → rffG1mobB ← ( 4040866 ) S (mobA )( 4041046 ) → yihD1glpF ← ( 4117901 ) S (yiiU ) ( 4118115 ) ← menG1

rplN ← ( 3447848 ) S (rpsQ ) ( 3448126 ) ← rpmC1aroB ← ( 3518216 ) S (aroK ) ( 3518461 ) ← hofQ1yhhW ← ( 3579170 ) S (yhhX ) ( 3579419 ) → yhhY1tdh ← ( 3791893 ) S (kbl ) ( 3792174 ) ← htrL1

tdcE ← ( 3262480 ) S (tdcD ) ( 3262763 ) ← tdcC1infB ← ( 3316214 ) S (nusA ) ( 3316430 ) ← yhbC1mdh ← ( 3384291 ) S (argR ) ( 3384490 ) → yhcN1yhdW → ( 3420328 ) S (yhdX ) ( 3420616 ) → yhdY1

ygdR → ( 2971234 ) S (tas) ( 2971469 ) ← ygeD1yghY ← ( 3147858 ) S (yghZ ) ( 3147959 ) ← yahA1ygiC → ( 3181189 ) S (ygiD ) ( 3181476 ) → ygiE1rpsU ← ( 3211261 ) S (dnaG )( 3211424 ) → rpoD1

gyrA ← ( 2345578 ) S (ubiG ) ( 2345831 ) ← yfaL1yfeN → ( 2532516 ) S (yfeR ) ( 2532780 ) → yfeH1yfgC → ( 2617317 ) S (yfgD ) ( 2617460 ) ← yfgE1grpE ← ( 2750781 ) S (ppnK ) ( 2751003 ) → recN1

ydhP ← ( 1741095 ) S (purR ) ( 1741371 ) ← ydhB1yejG ← ( 2283190 ) S (bcr ) ( 2283409 ) ← rsuA1ccmH ← ( 2296502 ) S (dsbE ) ( 2296764 ) ← ccmF1yfaT ← ( 2341705 ) S (yfaA ) ( 2341886 ) ← gyrA1

yciN ← ( 1334499 ) S (topA ) ( 1334720 ) → cysB1yneL ← ( 1594360 ) S (hipA ) ( 1594584 ) ← hipB1speG → ( 1659282 ) S (ynfC ) ( 1659526 ) → ynfD1ydgK → ( 1708310 ) S (rsxA ) ( 1708588 ) → rsxB1

ompX → ( 851728 ) S (ybiP ) ( 851955 ) → mntR1ycaD → ( 947247 ) S (ycaM )( 947555 ) ← ycaN1ymfM → ( 1206523 ) S (ymfN ) ( 1206714 ) → ymfR1hns ← ( 1296454 ) S (tdk) ( 1296693 ) ← ychG1

envY ← ( 586171 ) S (ybcH ) ( 586443 ) ← nfrA1citX ← ( 647089 ) S (citF ) ( 647347 ) ← citE1citF ← ( 649035 ) S (citE ) ( 649263 ) ← citD1ybhH → ( 801991 ) S (ybhI ) ( 802307 ) → ybhJ1

cueO → ( 139751 ) S (gcd ) ( 139930 ) → hpt1lpcA → ( 244095 ) S (yafJ ) ( 244364 ) ← yafK1yagB ← ( 280165 ) S (yagA ) ( 280365 ) → yagE1ybbL → ( 515749 ) S (ybbM )( 516003 ) ← ybbN1

ftsA → ( 105506 ) S (ftsZ ) ( 105778 ) → lpxC1acnB → ( 134201 ) S (yacL ) ( 134432 ) → speD1

yabQ → ( 59764 ) S (rluA ) ( 59966 ) ← hepA1ftsQ → ( 103836 ) S (ftsA ) ( 104038 ) → ftsZ1

- 28

a Using the genomic SELEX method, a total of 333 DNA fragments have been

isolated as complexes with the purified NNP protein (see Table 1). For the major

SELEX fragments which were isolated more than 3 times, the location on the E.

coli genome are indicated. Minor clones that ware isolated less than twice are

described in the Supplemental Data. Group A clones contained the sequences

from spacer regions between two neighboring genes as indicated, while group B

clones carried the sequences within the indicated coding frames. The numbers

on both sides of each SELEX (S) fragment indicate the boundaries in the revised

E. coli genome map (Riley et al., 2006). The arrows indicate the direction of

transcription of the neighboring genes.

- 29

Identification of NNP-binding activity to SELEX DNA fragments: Gel-shift

assay

For confirmation of NNP binding to these NNP-binding DNA sequences

identified by the genomic SELEX, we performed the gel mobility shift assay for

six representative DNA sequences, nnp-ttdA, maoC-paaA, yfaZ-yfaO, and

intZ-yffL from group A and ygaD-[mltB]-srlE from group B (see Table 2), and a

reference DNA fragment containing perR-insN spacer sequence, that was not

included in this SELEX clone library.

The NNP binding to the putative NNP box within the nnp-ttdA sequence

may exert either negative control of nnp or positive control of ttdA (see above).

This nnp-ttdA probe, however, formed a ladder of multiple bands on PAGE (Fig.

1A), indicating that multiple molecules of NNP bind to this region in a sequential

manner without significant cooperativity of protein-protein interaction.

Surprisingly similar pattern of NNP binding was observed for all other SELEX

DNA fragments tested, including four group-A probes, maoC-paaA (Fig. 1B),

yfaZ-yfaO (Fig. 1C), intZ-yffL (Fig. 1D) and one group-B probe, ygaD-[mltB]-srlA

(Fig. 1E). The gel shift assay indicates that the sequence selectivity of NNP

- 30

binding is very weak or NNP binds to DNA in non-specific manner. The gel

mobility shift pattern of multi step-complex formation is essentially the same with

those of the major nucleoid proteins, HU and IHF (Azam and Ishihama, 1999),

indicating that the sequence recognition properties of NNP is as broad as that of

these nucleoid structuring proteins. Accordingly one unrelated reference probe,

perR-insN, also formed the ladder of NNP complexes (Fig. 1F), implying that an

increasing number of NNP-binding sequences may be obtained after repeating

the genomic SELEX screening.

- 31

Figure 1. Gel mobility shift assay. Fluorescent-labeled DNA probes, each

containing a SELEX segment from the ygiP-ttdA (A), maoC-paaA (B), yfaZ-yfaO

(C), intZ-yffL (D), and ygaD-[mltB]-srlE (E) regions, or an unrelated reference

fragment from perR-insN (F) were incubated at 37OC for 30 min with the

indicated amounts (lane 1, 0; lane 2, 5 pmol; lane 3, 10 pmol; lane 4, 20 pmol;

and lane 5, 30 pmol) of NNP. The reaction mixtures were directly subjected to

PAGE.

ygiP-ttdA

1 2 3 4 5

A ygiP-ttdA

1 2 3 4 51 2 3 4 5

A maoC-paaA

1 2 3 4 5

B maoC-paaA

1 2 3 4 51 2 3 4 5

B yfaZ-yfaO

1 2 3 4 5

C yfaZ-yfaO

1 2 3 4 51 2 3 4 5

C

intZ-yffL

1 2 3 4 5

D intZ-yffL

1 2 3 4 51 2 3 4 5

D ygaD-[mltB]-srlA

1 2 3 4 5

E ygaD-[mltB]-srlA

1 2 3 4 51 2 3 4 5

E perR-insN

1 2 3 4 5

F perR-insN

1 2 3 4 51 2 3 4 5

F

- 32

Analysis of YgiP-binding DNA sequences: DNase-I foot-printing assay

The gel shift assay indicated the binding of NNP to a number of sites on

each DNA probe. The NNP-binding sites on the nnp-ttdA and maoC-paaA

spacer regions were examined by DNase-I foot-printing assay. After forming

complexes in the presence of a fixed amount of fluorescent-labeled DNA probes

and increasing amounts of NNP, DNase-I was added for a short periodm and the

partially digested DNA products were analyzed by PAGE. On the nnp-ttdA

spacer DNA fragment, clear protection by NNP was found only in the presence

of high concentrations of NNP apparently at two regions (Fig. 2A), a 17-bp

sequence between positions -195 and -211 and a 53-bp sequence between

positions -83 and -135 upstream from the ttdA initiation codon (for sequence see

Fig. 2B). The initiation codon-proximal 53 bp sequence (-83 to -135) overlaps

with a 63-bp segment (-81 and -146 from the ttdA initiation codon) that was

included in all 69 NNP SELEX clones (Table 2), indicating that the genomic

SELEX herewith employed allowed to isolate specific DNA fragments recognized

by NNP. However, there is no homologous sequence between the initiation

codon-proximal and -distal sequences significantly protected by NNP from

- 33

DNase-I digestion.

On the other maoC-paaA spacer DNA fragment, the protected regions

from DNase-I treatment was again observed only in the presence of high

concentrations of NNP at two regions (Fig. 3A), a 21-bp sequence between

positions -12 and -32, and a 25-bp sequence between positions -75 and -99

upstream from the maoC initiation codon (for sequence see Fig. 3B). The

initiation codon-proximal sequence includes the promoter for the maoC operon.

Among all four sequences protected by NNP, we failed to identify the consensus

sequence of putative NNP box.

During this DNase-I protection assay, we realized such an unexpected

strange phenomenon that in the presence of low concentrations of NNP, the

probe DNAs disappeared or were rapidly degraded to short fragments (Fig. 2A

and Fig. 3A). Both the gel mobility shift (Fig. 1) and DNase-I foot-printing assays

(Figs. 2 and 3) were performed at the same input ratio of probe DNA and NNP

protein. When low NNP concentrations (protein/DNA ratio of 6.5:1 and 13:1)

were used in gel mobility shift assay, NNP-complex bands were detected on

PAGE (see Fig. 1). Under the same conditions (NNP/DNA ratio of 6.5:1 and

13:1), probe DNAs were digested upon brief treatment with DNase I (see Figs.

- 34

2A and 3A). In the absence of NNP addition, the ladder of degradation products

by DNase-I was observed (see lane 1 in Figs. 2A and 3A), indicating that the

reaction conditions herein employed are adequate for the DNase-I foot-printing

assay. One possibility is that NNP carries an activity of DNA degradation, but this

is unlikely because by raising the NNP concentration, the regular sequence

ladder reappeared (see lanes 4-6 in Figs. 2A and 3A), allowing the identification

of protected regions from DNase-I digestion. The protection effect of NNP from

DNase-I digestion increased concomitantly with the increase in NNP

concentration. Thus, we interpreted that small amounts of NNP binding

enhances the sensitivity of NNP-associated DNA to DNase-I digestion, but upon

increase of NNP, the DNA surface could be fully covered to interfere with attack

by DNase I. The enhancing effect of DNase-I sensitivity has never been

detected for all the major nucleoid proteins even though the mode of DNA

binding by the major nucleoid proteins, HU and IHF, is similar to that of NNP as

detected by the gel shift assay (Azam and Ishihama, 1999).

Taken these observations together we reached to a prediction that the

binding of small numbers of NNP molecule at low protein concentrations

increases the sensitivity of DNA to digestion by DNase I.

- 35

Figure 2. DNase-I foot-printing of the ygiP-ttdA probe. [A] The

fluorescent-labeled SELEX segment from the ygiP-ttdA region was incubated

with increasing amounts of purified NNP (lane 1, 0 pmol; lane 2, 10 pmol; lane 3,

20 pmol; lane 4, 40 pmol; lane 5, 60 pmol; lane 6, 80 pmol) and subjected to

DNase-I foot-printing assays. Lanes A, T, G, and C represent the respective

sequence ladders. The black bars on right indicate the NNP-binding region. [B]

Two DNase-I foot-print sequences by NNP (NNP-I and NNP-II) are indicated

along the nnp-ttdA spacer region sequence. The initiation codons of the nnp and

ttdA genes are boxed. The numbers indicates the distance (bp) from the ttdA

initiation codon.

A T G CA T G C 1 2 3 4 5 61 2 3 4 5 6

NNPATTCAGCATCGTTAATTATCCGCAGTTGTGATAAGCGCAGTAAGTCGTAGCAATTAATAGGCGACAACACTATTCGCGTC

TCTTTGGCTAAAGGCCATCGATTCAGCATCGTTAATTATCAGAAACCGATTTCCGGTCGATAAGTCGTAGCAATTAATAG

AGCAGTTGTGATAAGCGCAGTGTATTTCGCAAAACATTGCGCGACAACACTATTCGCGTCACATAAAGCGTTTTGTAACG

TACATATTCACGGTGGCAAAAAATATAAAACCACATTTTTATGCATAAGTGCCACCGTTTTTTATATTTTGGTGTAAAAA

AGTGGTAGTTTGTGGCGGTGAATTTTTCCAGACAAATACATCACCATCAAACACCGCCACTTAAAAAGGTCTGTTTATGT

AAAACTGGAGTTGCCATGATGAGCGAAAGTAATAAGCAACTTTTGACCTCAACGGTACTACTCGCTTTCATTATTCGTTG

ttdA

nnp

-195-211 NNP-II

-83

-135

NNP-I

NN

P-I

NN

P-II

- 36

Figure 3. DNase-I foot-printing of the maoC-paaA region. [A] The

fluorescent-labeled ygiP-ttdA region was incubated with increasing amounts of

purified NNP (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 40 pmol;

lane 5, 60 pmol; lane 6, 80 pmol) and subjected to DNaseⅠfoot-printing assays.

Lanes A, T, G, and C represent the sequence ladders. The black bar on the right

indicates the NNP-binding region. [B] Two DNase-I foot-print sequences by NNP

(NNP-I and NNP-II) are indicated along the maoC-paaA spacer region sequence.

Initiation codon of the maoC gene is boxed while the transcription initiation sites

are indicated by dotted arrows. The numbers indicate the distance (bp) from the

maoC initiation codon.

A T G CA T G C 1 2 3 4 5 61 2 3 4 5 6

NNP

TTGTTTTATGAAAGTTACACAATAGTTAAGTAAAATCACAAACAAAATACTTTCAATGTGTTATCAATTCATTTTAGTGT

AATTTTGTATGTTTGAACTGTGACGGATTTCGCACCCTATTTAAAACATACAAACTTGACACTGCCTAAAGCGTGGGATA

TTTTATAAATCATTATTTATCAGTGCATTAATGTTTTCCGAAAATATTTAGTAATAAATAGTCACGTAATTACAAAAGGC

CAGTTGCTATACAGATCGCATAGTTAACATTTCGTTAAAAGTCAACGATATGTCTAGCGTATCAATTGTAAAGCAATTTT

GATCCTTTGCTTTTTATGATTCGCGATTTAACTATTAGCACTAGGAAACGAAAAATACTAAGCGCTAAATTGATAATCGT

ACAGAAATGTGAAACATCTGGAGAGTAGCGATGCAGCAGTTGTGTTTACACTTTGTAGACCTCTCATCGCTACGTCGTCA

maoC

paaA p

maoC p -12

-32

NNP-I

-75-99 NNP-II

NN

P-I

NN

P-II

- 37

Influence of NNP on the nuclease sensitivity of DNA: DNase-I

To confirm the influence of NNP on the sensitivity of DNA against

DNase-I treatment, we next analyzed in details the DNase-I digestion pattern at

various concentrations of NNP and for different time periods. First, we checked

the time course of DNA digestion at fixed concentrations of NNP (10 pmol) and

DNase I (5 mU). In the absence of NNP addition, a low level digestion of DNA

was observed only after 9 min incubation (Fig. 4A, lane 1). In the presence of 10

pmol NNP, the DNA digestion was observed as early as 10 sec (Fig. 4A, lane 2),

indicating about 100-fold increase in the DNase-I sensitivity. Complete digestion

took place within a few minutes (Fig. 4A, lane 6).

Next we performed a systematic analysis of the influence of NNP

concentration on DNA digestion by DNase I. For this purpose, a fixed amount of

DNA and increasing amounts of NNP (up to 80 pmol) were mixed and after

addition of 20 mU DNase I, incubated for 20 sec (Fig. 4B). At NNP

concentrations below 20 pmol (Fig. 4B, lane 7), DNA was almost digested to

smaller fragments, and above 30 pmol (Fig. 4B, lane 8), some protected bands

started to appear. At NNP concentrations above 60 pmol (Fig. 4B, lane 11),

- 38

probe DNA was almost completely protected, indicating that the DNA was fully

covered by NNP, preventing the access of DNase I to the probe DNA.

Using two representative NNP concentrations, i.e., 10 pmol (see Fig. 4B,

lane 6) and 80 pmol (see Fig. 4B, lane 12), the time course of DNA digestion by

5 mU DNase I was repeated. At the low NNP concentrations, the DNA digestion

pattern was essentially the same as that in Fig. 3A. The digestion was observed

as early as at 30 sec (Fig. 4C, lane 2), and long-sized DNA disappeared after

240 (Fig. 4C, lane 5) and 360 sec (Fig. 4C, lane 6) incubation. At the high NNP

concentrations, the sensitivity of probe DNA to DNase I markedly decreased (Fig.

4D). The gel pattern of probe DNA after 10 min incubation (lane 8) was

essentially the same as that in the absence of NNP (lane 2) even though the

intensity of each DNA band was reduced to certain extents.

Taken the time course experiments together we reached to a conclusion

that NNP exerts two different effects on DNA: at low concentrations, DNA

becomes sensitive to DNase-I digestion, while at high concentrations, it

protected DNA from DNase-I digestion.

- 39

Figure 4. DNase-I foot-printing assays of the nnp-ttdA fragment. [A] The

fluorescent-labeled nnp-ttdA spacer region fragment (0.4 μM) was incubated

with 10 pmol NNP for min at 37C and then treated with 5 mU of DNase I for

various times (lanes 1, 9 min; lane 2, 10 sec; lane 3, 20 sec; lane 4, 30 sec; lane

5, 40 sec; lane 6, 5 min; and lane 7, 9 min). The reaction mixture was directly

subjected to PAGE. [B] The fluorescent-labeled nnp-ttdA fragment (0.4 μM) was

incubated for 30 min with the increasing amounts of NNP, and then treated for 2

min (lane 1) or 20 sec (lanes 2 to 11) with 20 mU of DNase I. The amounts of

1 2 3 4 5 6 71 2 3 4 5 6 71 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 111 2 3 4 5 6 7 8 9 10 111 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 81 2 3 4 5 6 7 81 2 3 4 5 6 7 8 1 2 3 4 5 6 7 81 2 3 4 5 6 7 81 2 3 4 5 6 7 8

A B C DTime course(10 pmol NNP)

NNP concentration(120 sec reaction)

Time course(10 pmol NNP)

Time course(80 pmol NNP)

- 40

NNP added were: Lane 1, 0 pmol; lane 2, 0 pmol; lane 3, 1.25 pmol; lane 4, 2.5

pmol; lane 5, 5 pmol; lane 6, 10 pmol; lane 7, 20 pmol; lane 8, 30 pmol; lane 9,

40 pmol; lane 10, 60 pmol and lane 11, 80 pmol. [C] The fluorescent-labeled

nnp-ttdA fragment (0.4 μM) was incubated for 30 min in the absence (lane 1)

or presence of 10 pmol NNP (lanes 2 to 8), and then treated with 5 mU of DNase

I for various times. Lane 1, 10 min; lane 2, 0.5 min; lane 3, 1 min; lane 4, 2 min:

lane 5, 4 min; lane 6, 6 min; lane 7, 8 min; lane 8, 10 min. [D] The

fluorescent-labeled nnp-ttdA fragment (0.4 μM) was incubated for 30 min with 0

pmol (lane 1) or 80 pmol (lanes 2 to 8) NNP, and then treated with 5 mU of

DNase I for various times. Lane 1, 10 min; lane 2, 0.5 min; lane 3, 1 min; lane 4,

2 min: lane 5, 4 min; lane 6, 6 min; lane 7, 8 min; and lane 8, 10 min.

- 41

Influence of NNP on the nuclease sensitivity of DNA: Exonuclease III and

S1 nuclease

The foot-printing assay indicated that NNP has a very distinct feature of

modulating the DNA structure so as to make it sensitive to DNase I, which

degrades both single- and double-strand DNAs to nucleotides in non-specific

manner. To examine whether this effect is specific to DNase I, we next analyzed

possible influence of NNP on the sensitivity of DNA to other nucleases with

different specificity.

Exonuclease III catalyzes the stepwise removal of mononucleotides

from 3’ hydroxyl termini of double-stranded DNA toward 5’ direction. This

enzyme has a strict specificity for double-strand DNA and has been used for

determination of the site of protein-binding site from DNA 3’ terminus. If NNP

binds at various sites in random fashion on the probe DNA, the exonuclease III

treatment should yield various products of different chain length on PAGE. Then

exonuclease III foot-printing was carried out in the presence of various

concentrations of NNP. At low NNP concentrations, the pattern of DNA digestion

by exonucleae III (Fig. 5A and 5B, lanes 3-7) was essentially the same as that in

- 42

its absence (Fig. 5A and 5B, lane 2), implying that NNP is not associated at

specific positions along the DNA. Upon increase of NNP addition, however, the

level of small-sized degradation products decreased (Fig. 5A, lane 8) and

concomitantly, a number of protected DNA bands were detected (Fig. 5B, lane 8),

supporting the prediction that NNP covers the entire surface of probe DNA in a

non-specific manner.

To get insight into the mechanism how NNP induces the conformational

change in DNA, we next analyzed the sensitivity of probe DNA to S1 nuclease.

This enzyme is a single-strand DNA-specific endonuclease releasing

5’-phosphoryl mono- or oligonucleotides, but is also active against

single-stranded regions within double-strand DNA. In the presence of low

concentrations of NNP, the sensitivity of DNA against S1 nuclease increased,

leading to reduction of the intact DNA molecules on the top of gel (Fig. 5C amd

5D, lane 3～5), implying that NNP induced single-stranded regions within the

probe DNA. Since intermediate-sized degradation products were hardly

detected, it appears that the probe DNA is rapidly degraded by S1 nuclease

once DNA opening is induced by NNP. At high NNP concentrations, most of the

DNA probe was again protected from S1 nuclease (Fig. 5C and 5D, lanes 6 and 7).

- 43

Taken all these observations together, we propose that NNP is a novel

hitherto uncharacterized nucleoid protein with the strong activity of inducing the

conformational changes in DNA, presumably inducing the local opening of DNA

duplex.

- 44

Figure 5. DNA protection assay against exonuclease III and S1 nuclease. [A]

The fluorescent-labeled nnp-ttdA fragment (0.4 μM) was incubated for 30 min at

with the increasing amounts of NNP, and then treated with 1 U of exonuclease III

for 60 min. The amounts of NNP added were: Lane 1, 0 pmol (in the absence of

NNP and without exonuclease III treatment); lane 2, 0 pmol (in the absence of

NNP); lane 3, 2.5 pmol; lane 4, 5 pmol; lane 5, 10 pmol; lane 6, 20 pmol; lane 7,

40 pmol; and lane 8, 80 pmol. [B] The fluorescent-labeled nnp-ttdA fragment (0.4

A B C DExonuclease III Exonuclease III S1 nuclease S1 nuclease

1 2 3 4 5 6 7 81 2 3 4 5 6 7 8 1 2 3 4 5 6 7 81 2 3 4 5 6 7 8 1 2 3 4 5 6 7 81 2 3 4 5 6 7 8 1 2 3 4 5 6 7 81 2 3 4 5 6 7 8

- 45

μM) was incubated for 30 min with the increasing amounts of NNP, and then

treated with 1 U of exonuclease III for 60 min. The amounts of NNP added were:

Lane 1, 0 pmol (in the absence of NNP and without exonuclease III treatment);

lane 2, 0 pmol (in the absence of NNP); lane 3, 2.5 pmol; lane 4, 5 pmol; lane 5,

10 pmol; lane 6, 20 pmol; lane 7, 40 pmol; and lane 8, 80 pmol. [C] The

fluorescent-labeled nnp-ttdA fragment (0.4 μM) was incubated for 30 min with

the increasing amounts of NNP, and then treated with 1 U of S1 nuclease for 10

min. The amounts of NNP added were: Lane 1, 0 pmol (in the absence of NNP

and without S1 nuclease treatment); lane 2, 0 pmol (in the absence of NNP);

lane 3, 2.5 pmol; lane 4, 5 pmol; lane 5, 10 pmol; lane 6, 20 pmol; lane 7, 40

pmol; and lane 8, 80 pmol. [D] The fluorescent-labeled nnp-ttdA fragment (0.4

μM) was incubated for 30 min with the increasing amounts of NNP, and then

treated with 1 U of S1 nuclease for 10 min. The amounts of NNP added were:

Lane 1, 0 pmol (in the absence of NNP and without S1 nuclease treatment); lane

2, 0 pmol (in the absence of NNP); lane 3, 2.5 pmol; lane 4, 5 pmol; lane 5, 10

pmol; lane 6, 20 pmol; lane 7, 40 pmol; and lane 8, 80 pmol.

- 46

AFM analysis of NNP-DNA complexes

Atomic force microscopy (AFM) is a powerful tool for imaging DNA and

DNA-proteins complexes (Dame et al., 2000; Ceci et al., 2004; Chan et al.,

2007). AFM analysis provides some distinct advantages over bulk biochemical

assays, particularly when studying proteins that do not interact with specific DNA

sequences. Individual DNA-protein complexes can be observed, leading to

qualitatively categorize the complexes and quantitatively measure the amount of

each complex form. Thus the distribution of complexes with specific features in a

mixture can be determined rather than a bulk average usually determined in

biochemical assays. The mode of NNP binding to DNA was studied using a

circular pTFP plasmid, which contains the ygiP-ttdA spacer region with NNP

binding activity as identified by SELEX (see Table 1). By gel retardation assay,

this sequence formed multiple bands of NNP-DNA complexes at moderate

protein concentrations below the aggregation thresholds (see Fig. 1).

NNP-DNA complexes were observed with AFM in the presence of

various concentrations of NNP (Fig. 6A-6G). At low NNP concentrations below

the NNP/DNA molar ratio of 1/10, NNP bound to various positions along the

- 47

entire plasmid DNA (Fig. 6A-6C). The site of NNP binding is non-specific as

analyzed by DNase-I foot-printing. Upon increase in NNP concentrations above

1/10, the NNP-DNA complexes were converted into rod-like structures (Fig.

6D-6G). The sizes of NNP-DNA complexes were smaller than the plasmid DNA,

indicating that NNP binding induces DNA compaction. The size of rods of

NNP-plasmid complexes, however, appeared to increase concomitantly with

further increase in NNP concentrations. One possibility of the NNP

concentration-dependent increase in the size of NNP-DNA complex rods is the

tail-to-head association of NNP-DNA complexes.

The horizontal and vertical sizes of NNP-DNA complexes were

measured for individual images, and the size distributions are plotted against

NNP concentrations (Fig. 7). The peak of horizontal width as determined for

the different protein concentrations was 23.00~29.50 nm, but the horizontal

width of NNP-DNA rods increased with increase in the NNP/DNA ratio above 1

(Fig. 7A). The peak of vertical length was 0.00~2.00 nm at NNP/DNA ratio of

1/40 and 1/20, but at NNP/DNA ratio between 1/10 and 1/4, the peak of the

height of NNP-DNA complexes shifted to 4.00~6.00 nm with a shoulder of

0.00~2.00 peak (Fig. 7B). Upon increase of NNP above 1/2, only a single peak

- 48

of rod height was observed at the same level of 4.00~6.00 nm, which may

indicate the size of plasmid DNA fully covered with NNP.

The peak of DNA length was 2.2~3.3 μm at low NNP/DNA ratio (1/40

and 1/20), but multiple peaks with longer length appeared upon increase of NNP

addition (Fig. 7C). The NNP concentration-coupled change in the contour map of

horizontal width (Fig. 7A), vertical height (Fig. 7B) and length of NNP-DNA

complexes (Fig. 7C) indicates the structural transitions of NNP-DNA complexes

concomitantly with increase in the level of associated NNP.

- 49

A

B

C

D

NNP/DNA= 1/40

NNP/DNA= 1/20

NNP/DNA= 1/10

NNP/DNA= 1/4

E

F

G

NNP/DNA= 1/0.25

NNP/DNA= 1/1

NNP/DNA= 1/2

- 50

Figure 6. AFM images of NNP–DNA complexes at different NNP/DNA ratios. The

ratio of NNP/DNA is ng distinction. (A)~(G) of NNP-DNA complexes is the ratio of

NNP/DNA= 1/40, 1/20, 1/10, 1/4, 1/2, 1/1, and 1/0.25, respectively. All close-up

images of condensed molecules show a 500 x 500 nm surface area. (A)~(F) of

the color scale range from 0.0 to 5.0 nm, and (G) range from 0.0 to 25.0 nm

(from dark to bright).

- 51

Figure 7. Distribution curve of measurements performed on NNP-DNA

complexes. (A) Distribution of the horizontal length of NNP/DNA complexes. (B)

Distribution of the vertical length of NNP/DNA complexes. (C) DNA contour

length distributions of NNP/DNA complexes length.

0.00～

2.00

2.00～

4.00

4.00～

6.00

6.00～

8.00

8.00～

10.0

10.0～

12.0

12.0～

14.0

14.0～

16.0

16.0～

18.0

18.0～

20.0

20.0～

22.0

22.0～

24.0

24.0～

26.0

26.0～

28.0

28.0～

30.0

30.0～

32.0

32.0～

34.0

34.0～

36.0

36.0～

38.0

38.0～

40.0

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Rel

ativ

e Fr

eque

ncy

(nm)

NNP/DNA = 1/40 (n=668)NNP/DNA = 1/20 (n=442)NNP/DNA = 1/10 (n=188)NNP/DNA = 1/4 (n=425)NNP/DNA = 1/2 (n=565)NNP/DNA = 1/1 (n=842)NNP/DNA = 1/0.25 (n=258)

0.00～

2.00

2.00～

4.00

4.00～

6.00

6.00～

8.00

8.00～

10.0

10.0～

12.0

12.0～

14.0

14.0～

16.0

16.0～

18.0

18.0～

20.0

20.0～

22.0

22.0～

24.0

24.0～

26.0

26.0～

28.0

28.0～

30.0

30.0～

32.0

32.0～

34.0

34.0～

36.0

36.0～

38.0

38.0～

40.0

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Rel

ativ

e Fr

eque

ncy

0.00～

2.00

2.00～

4.00

4.00～

6.00

6.00～

8.00

8.00～

10.0

10.0～

12.0

12.0～

14.0

14.0～

16.0

16.0～

18.0

18.0～

20.0

20.0～

22.0

22.0～

24.0

24.0～

26.0

26.0～

28.0

28.0～

30.0

30.0～

32.0

32.0～

34.0

34.0～

36.0

36.0～

38.0

38.0～

40.0

0.00～

2.00

2.00～

4.00

4.00～

6.00

6.00～

8.00

8.00～

10.0

10.0～

12.0

12.0～

14.0

14.0～

16.0

16.0～

18.0

18.0～

20.0

20.0～

22.0

22.0～

24.0

24.0～

26.0

26.0～

28.0

28.0～

30.0

30.0～

32.0

32.0～

34.0

34.0～

36.0

36.0～

38.0

38.0～

40.0

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Rel

ativ

e Fr

eque

ncy

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Rel

ativ

e Fr

eque

ncy

(nm)


NNP/DNA = 1/40 (n=668)NNP/DNA = 1/40 (n=668)NNP/DNA = 1/20 (n=442)NNP/DNA = 1/20 (n=442)NNP/DNA = 1/10 (n=188)NNP/DNA = 1/10 (n=188)NNP/DNA = 1/4 (n=425)NNP/DNA = 1/4 (n=425)NNP/DNA = 1/2 (n=565)NNP/DNA = 1/2 (n=565)NNP/DNA = 1/1 (n=842)NNP/DNA = 1/1 (n=842)NNP/DNA = 1/0.25 (n=258)NNP/DNA = 1/0.25 (n=258)

0.00～

550550

～1100

1100～

16501650

～2200

2200～

27502750

～3300

3300～

38503850

～4400

4400～

49504950

～5500

5500～

60506050

～6600

6600～

71507150

～7700

7700～

82508250

～8800

8800～

93509350

～9900

9900～

1045010450

～11000

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

(nm)


0.00～

550550

～1100

1100～

16501650

～2200

2200～

27502750

～3300

3300～

38503850

～4400

4400～

49504950

～5500

5500～

60506050

～6600

6600～

71507150

～7700

7700～

82508250

～8800

8800～

93509350

～9900

9900～

1045010450

～11000

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

0.00～

550550

～1100

1100～

16501650

～2200

2200～

27502750

～3300

3300～

38503850

～4400

4400～

49504950

～5500

5500～

60506050

～6600

6600～

71507150

～7700

7700～

82508250

～8800

8800～

93509350

～9900

9900～

1045010450

～11000

0.00～

550550

～1100

1100～

16501650

～2200

2200～

27502750

～3300

3300～

38503850

～4400

4400～

49504950

～5500

5500～

60506050

～6600

6600～

71507150

～7700

7700～

82508250

～8800

8800～

93509350

～9900

9900～

1045010450

～11000

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

(nm)



A B

C

10.00～

16.50

16.50～

23.00

23.00～

29.50

29.50～

36.00

36.00～

42.50

42.50～

49.00

49.00～

55.50

55.50～

62.00

62.00～

68.50

68.50～

75.00

75.00～

81.50

81.50～

88.00

88.00～

94.50

94.50～

101.0101.0

～107.5

107.5～

114.0

114.0～

120.5

120.5～

127.0

127.0～

133.5

133.5～

140.0

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

(nm)


10.00～

16.50

16.50～

23.00

23.00～

29.50

29.50～

36.00

36.00～

42.50

42.50～

49.00

49.00～

55.50

55.50～

62.00

62.00～

68.50

68.50～

75.00

75.00～

81.50

81.50～

88.00

88.00～

94.50

94.50～

101.0101.0

～107.5

107.5～

114.0

114.0～

120.5

120.5～

127.0

127.0～

133.5

133.5～

140.0

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

10.00～

16.50

16.50～

23.00

23.00～

29.50

29.50～

36.00

36.00～

42.50

42.50～

49.00

49.00～

55.50

55.50～

62.00

62.00～

68.50

68.50～

75.00

75.00～

81.50

81.50～

88.00

88.00～

94.50

94.50～

101.0101.0

～107.5

107.5～

114.0

114.0～

120.5

120.5～

127.0

127.0～

133.5

133.5～

140.0

10.00～

16.50

16.50～

23.00

23.00～

29.50

29.50～

36.00

36.00～

42.50

42.50～

49.00

49.00～

55.50

55.50～

62.00

62.00～

68.50

68.50～

75.00

75.00～

81.50

81.50～

88.00

88.00～

94.50

94.50～

101.0101.0

～107.5

107.5～

114.0

114.0～

120.5

120.5～

127.0

127.0～

133.5

133.5～

140.0

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Rel

ativ

e Fr

eque

ncy

(nm)



- 52

Intracellular level of NNP under aerobic, hypoxic, and anaerobic conditions

The protein composition of nucleoids has been studied in details for E.

coli grown under laboratory culture conditions (Azam et al., 1999; Azam and

Ishihama, 1999), but NNP has not been identified. We then measured the

intracellular concentration of NNP by using the quantitative Western blot analysis.

When E. coli cells were grown under aerobic conditions in either rich (LB) or

poor (M9 containing 0.4% glucose) medium, the level of NNP was less than 10%

the level of RNA polymerase α subunit (Fig. 8A). Under the same culture

conditions, the level of RpoA is approximately 5,000 molecules per genome

equivalent of DNA (Ishihama, 1990), while the major nucleoid proteins (Fis, HU,

IHF and H-NS) in exponentially growing E. coli cells range from 10,000 to

100,000 molecules per genome (Azam et al., 2000). Thus, a relatively small

amount of NNP (less than 1% of the major nucleoid proteins) must have been

left unidentified.

Next we measured the NNP level in E. coli cells grown under a hypoxias

condition. Surprisingly, the level of NNP was 1.5- to 2.0-fold higher than the

RpoA level in both rich and poor media and from exponential growth to stationary

- 53

phase (Fig. 8B). On the basis of RpoA level (approximately 5,000 molecules per

genome equivalent DNA) (Ishihama, 1999: 2000), the level of NNP was

estimated to range 7,000-9,000 molecules per genome. In late stationary phase,

the NNP level was significantly higher in the poor medium than in the rich

medium (Fig. 8B, 96 to 144 hr culture). This finding suggests that NNP plays a

role in either maintenance of the nucleoid architecture or expression of the

nucleoid function under the anaerobic conditions.

NNP

RpoA

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

NNP

RpoA

A1 B1 1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 12

LB mediumM9 medium LB mediumM9 medium

Culture time (hr)2 2.5 4 24 48 722 2.5 4 24 48 72

0

0.5

1.0

1.5

2.0

2.5

NN

P/R

poA

2 2.5 4 24 48 722 2.5 4 24 48 722 2.5 4 24 48 722 2.5 4 24 48 720

0.5

1.0

1.5

2.0

2.5

NN

P/R

poA

0

0.5

1.0

1.5

2.0

2.5

NN

P/R

poA

Culture time (hr)

24 48 72 96 120 14424 48 72 96 120 14424 48 72 96 120 14424 48 72 96 120 1440

0.5

1.0

1.5

2.0

2.5

NN

P/R

poA

0

0.5

1.0

1.5

2.0

2.5

NN

P/R

poA

LB mediumM9 medium LB mediumM9 medium

A2 B2

- 54

Figure 8. Western blot analysis of whole-cell lysates with anti-NNP serum. [A1]

Wild-type E. coli KP7600 and its nnp disruptant JD24074 were grown in LB and

M9-0.4% glucose media at 37OC for various times under aerobic conditions.

Cell lysates were subjected to Western blots analysis againt anti-NNP and

anti-RpoA antibodies. Lanes 1 and 7, OD600=0.3; lanes 2 and 8, OD600=0.6;

lanes 3 and 9, OD600=1.0; lanes 4 and 10, 24 hr; lane 5 and 11, 48 hr; lanes 6

and 12, 72 hr. [A2] The intensity of immuno-blot bands was measured with. [B1]

Wild-type E. coli KP7600 and its nnp disruptant JD24074 were grown in LB and

M9-0.4% glucose media at 37OC for various times under hypoxic culture

conditions. Cell lysates were subjected to Western blots analysis against

anti-NNP and anti-RpoA antibodies. Lanes 1 and 7, OD600=0.3; lanes 2 and 8,

OD600=0.6; lanes 3 and 9, OD600=1.0; lanes 4 and 10, 24 hr; lane 5 and 11, 48 hr;

lanes 6 and 12, 72 hr. [B2] The intensity of immuno-blot bands was measured

with a LAS-1000 Plus lumino-image analyzer and IMAGE GAUGE (Fuji Film).

- 55

Intracellular localization of NNP

Previously we analyzed the intracellular localization of nucleoid proteins

by indirect immno-fluorescent labeling, and classified them into two groups: one

group proteins including HU, IHF, H-NS and Dps are uniformly distributed within

the nucleoid; and another group proteins including Fis are located at specific loci

within the nucleoid (Azam et al., 2000). Consequently, we observed the

intracellular distribution of NNP in E. coli grown under aerobic and hypoxic

conditions. The intracellular level of NNP within the aerobically grown wild-type

KP7600 cells is very low (see Fig. 8), showing a faint staining with anti-NNP

antibodies (Fig. 9A, W1-W4). This faint staining was not detected for the mutant

JD24074 lacking the nnp gene (Fig. 9A, M1-M4).

Under the hypoxic conditions, NNP was markedly induced, and formed

highly stained dots (Fig. 9B, W1-W4; Fig. 9D, W), indicating that NNP belongs to

the group-II nucleoid proteins, including Fis, Rob (CbpA), CbpB and IciA, which

show irregular distribution within the nucleoid (Azam et al., 2000). The

distribution pattern is most similar to that of Fis, a bifuctional nucleoid protein

with the regulatory activity of a set of growth-related genes. Under the highly

- 56

induced wild-type E. coli cells under the hypoxic conditions, NNP is enriched

near the interface between the nucleoid and cytoplasm (or the surface of

nucleoid), but it is also present in the cytoplasm, implying that some of the

over-produced NNP stays in the cytoplasm. In the nnp mutant, no

immuno-stained NNP dots were not detected (Fig. 9B, M1-M4; Fig. 9D, M),

indicating that NNP could be detected by the immuno-staining method and with

use of the anti-NNP antibody.

Taken all these results we conclude that NNP is a nucleoid protein

specific in anaerobically grown E. coli cells. As in the case of other major

nucleoid proteins (Ishihama, 2007), NNP may have functional dichotomy,

controlling both the structure and function of the nucleoid. In fact, NNP was

proposed to regulate the neighboring ttdA-ttdB-ygiE operon (Oshima and Biville,

2006).

- 57

A

B

C

D

W1

W1

W

W

M1

M1

W2

W2

W

W

M2

M2

W3

W3

M

M

M3

M3

W4

W4

M

M

M4

M4

- 58

Figure 9. Intracellular localization of the NNP protein. Wild-type E. coli KP7600

and its nnp mutant JD24074 were grown in M9-0.4% glucose media for 24 hr at

37OC under hypoxic conditions, and subjected to the indirect

immuno-fluorescent microscopy according to the standard procedure described

in Experimental procedures. [A] Wild–type strain KP7600 (W1-W4) and the nnp

mutant JD24074 (M1-M4) were grown under aerobic [A] or hypoxic [B]

conditions. W1 and M1 represent; W2 and M2, Indirect immuno-immunoblot

against anti-NNP; W3 and M3, DAPI-staining; W4 and M4, merged images of

DAPI and immuno-stained patterns. [C] and [D], the area shown by dotted

square are expanded. Anti-NNP antibody was raised in rabbits using the purified

NNP as an antigen. Cys3-labeled anti-rabbit IgG antibody was used as the

secondary antibody. White bars indicate 5 μm.

- 59

DISCUSSION

DNA binding specificity of NNP: The aim of this research was conducted

for the identification of target genes or promotes by an uncharacterized putative

transcription factor NNP (renamed from YgiP) of LysR family. A collection of DNA

fragments isolated after the genomic SELEX screening included a number of

different sequences, indicating that NNP is a non-specific DNA binding protein.

The non-sequence specific nature of DNA recognition is similar to those of the

major nucleoid proteins, HU and IHF. The lack of co-operatively in DNA binding

is also similar to these nucleoid proteins (Azam et al., 1999). Accordingly, NNP

gave a concentration-dependent multiple ladders in gel shift assay (see Fig. 2).

This mode of DNA binding is similar to those of the major nucleoid proteins, HU

and IHF, of growing E. coli cells.

Enhancement of DNase-I sensitivity by NNP: One unique feature of NNP is

the enhancement of DNase-I sensitivity of associated DNA. In the presence of

low concentrations of NNP, DNase I completely digested the probe DNA at

concentrations as low as that did not degrade the DNA in the absence of NNP

(see Figs. 2-4). The influence of NNP on the sensitivity of exonuclese III was,

- 60

however, not so striking (see Fig. 5), indicating that the influence of NNP on the

DNA conformation is local within a neighboring narrow region. As predicted from

this hypothesis, the sensitivity to S1 nuclease is enhanced (see Fig. 5), implying

that NNP induces a local opening of DNA. Once the probe DNA was fully

saturated, however, the probe DNA became resistant to DNase I and S1

nuclease digestion. In DNase I foot-printing assays, some apparently protected

regions were detected at high NNP concentrations. However, the possibility

cannot be excluded that the apparently protected regions represent the

sequences, which are converted to highly sensitive to DNase I due to NNP

binding surrounding these regions.

Two forms of NNP-DNA complexes: In agreement with the biochemical

analyses of NNP-DNA interaction, the AFM images indicated NNP

concentration-dependent conformational transition of NNP-DNA complexes

(see Fig. 8). At low NNP concentrations below 1/10 NNP/DNA ratio, NNP is

associated at various sites on the substrate pTFP plasmid, supporting the

non-specific mode of NNP binding. At NNP-associated sites, clamp structures

were observed, that is not inconsistent with the local melting model of

double-strand DNA, generating high sensitivity to DNase I and S1 nuclease. At

- 61

high NNP concentrations, rod-like structures were formed (see Fig. 8), which

were elongated upon further addition of NNP supposedly by forming tail-to-head

linkages between a unit rod of NNP-saturated plasmid DNA. The NNP mediate

conformational transition agrees with the formation of totally retarded aggregates

in the GMSA (see Fig. 1 and Fig. 6).

After statistical analysis of AFM images formed with increasing

concentrations of NNP, we propose two forms of NNP-DNA complexes (Fig. 10).

At low NNP concentrations, NNP binds to various positions on plasmid DNA in

non-specific manner (Form 1) and upon increase in the NNP concentration,

more NNP molecules binds through protein-protein interactions, ultimately

forming the NNP-saturated complexes (Form 2).

Conformation of NNP-saturated DNA: The contour distribution of vertical

length of NNP-DNA complexes is rather constant independent of the protein

concentrations, but that of horizontal length increased with the increase in

protein concentration (see Fig. 7). This extension in the contour length of horizon

suggests the hierarchy of condensed architectures of DNA by NNP, probably by

forming tail-to-head joining of the unit rod-like structure of NNP-plasmid DNA

complex (see Fig. 10 for the model). After measurement of the vertical and

- 62

horizontal lengths and the total length for a number of Form-2 NNP-DNA

complexes, we reached to figure its conformation of native rod in solution as

follows: The width and the height are 26±3 nm and 6±1 nm, respectively, and

the length of unit rod is about 2 μm. The conformation of NNP-DNA rods is

similar to that of TMV capsid with the diameter of 18 nm. The section dimension

of NNP-DNA rod is about 150 nm2 while that of TMV capsid is about 250 nm2.

The length of NNP-DNA is smaller than that of pTFP plasmid, supporting our

proposal that NNP plays a role in DNA compaction.

Direct observation of nucleoid protein-DNA complexes with AFM has

been successfully employed for analysis of protein-DNA complexes with E. coli

H-NS (Dame et al., 2000), Dps (Ceci et al., 2004) and Fis (Maure et al., 2006) as

well as HU-like protein HCc3 of dianoflagellate (Chan et al., 2007), The pattern

of DNA compaction by NNP is rather similar to that of HCc3 from dianoflagellate,

which lacks histones and retains permanently condensed chromosome (Chan et

al., 2007). Both NNP and HCc3 proteins form bundle architectures for DNA

compaction in protein concentration-dependent manner.

Induction of NNP under anaerobic conditions: NNP is apparently similar to

the major nucleoid proteins, but it has never been identified in the isolated

- 63

nucleoids from E. coli cells grown under laboratory culture conditions (Azam and

Ishihama, 2000; Ishihama, 2007). After testing various culture conditions, we

reached to the finding that NNP is highly expressed in cells grown under hypoxic

or anaerobic conditions. This finding indicates that NNP plays a role in

structuring the nucleoid in cells under anaerobic conditions as in the cases of Fis

in rapidly growing cells and Dps in stationary-phase cells. Fis is synthesized

preferentially in growing cells (Ball et al., 1992; Azam and Ishihama, 2000) and

plays an essential role for transcription of the growth-related genes (Ishihama,

2007). Fis is one of the key global regulators of a number of genes for adaptation

to external conditions such as the availability of oxygen and nutrients (Nilsson et

al., 1990; Ross et al., 1990; Ishihama, 2007). On the other hand, Dps

(DNA-binding protein form starved cells) is the major nucleoid protein only in

starved stationary-phase cells (Almiron et al., 992; Azam et al., 1999) and plays

roles in protecting of resting bacterial cells from environmental stresses such as

a high level of toxic iron. Transformation of the nucleoids from exponential

growth-specific fibrous structures to stationary phase-specific rod forms (Kim et

al., 2004) depends on the association of Dps to the genome DNA. This group of

nucleoid proteins may be stored on the nucleoid surface, forming dots as

- 64

detected by indirect immuno-staining (Azam et al., 2000; also see Fig. 9).

Since NNP is highly expressed under anaerobic and starved conditions, it may

play a role in protection of the genome DNA from oxidization stress and nutrient

starvation. NNP contains two sets of Cys-Cys pair, one consisting of Cys170 and

Cys193, and another consisting of Cys288 and Cys296. Since the Cys-Cys pair is

involved in redox sensing (Zheng et al., 1998), NNP may also play a role in

redox sensing. This motif is also involved in metal chelating, implying a role in

resistance of metals as did Dps against iron. A group of Gram-positive bacteria

such as Bacillus, Clostridium, Sporosarcina, and Anphibacillus form spores for

survival under harmful stresses, but Gram-negative bacteria such as E. coli must

harbour another system for survival under stressful conditions. NNP and Dps

play essential roles for protection of the genome during dormant stage.

Regulatory role of NNP: In prokaryotes, the genes encoding transcription

factors are generally located on the genome adjoining to the target genes for

regulation. Relying on this rule, Oshima and Biville (2006) identified that YgiP,

here renamed to NNP, is involved in regulation of the adjacent ttdA-ttdB-ygiE

operon. Interestingly, tartrate dehydratase encoded by ttdA-ttdB for tartrate

uptake is required for anaerobic growth on glycerol as carbon source in the

- 65

presence of tartrate. Our findings extend this concept and further indicate that

NNP is a bifunctional nucleoid protein under anaerobic growth, playing dual roles

in both structuring the nucleoid and regulation of a set of genes needed for

growth under anaerobiosis. Along this line, the genes under the control of NNP

must be present besides the ttdA-ttdB-ygiE operon, which altogether contribute

to the E. coli surviral under the anaerobiotic conditions such as in animals.

- 66

Figure 10. A diagram showing the structural change of NNP-induced DNA

condensation. NNP-DNA complexes formed in the presence of increasing

concentrations of NNP were observed with AFM. Typical figures are shown in

panel A-D. Based on the AFM pattern analysis, we propose two distinct forms of

NNP-DNA complex (see text). The diagrams shown below the AFM figures

represent the NNP concentration-dependent transition model between two forms.

DNA strand is shown by a black line while NNP is shown by orange colour.

NNP/DNA =1/4NNP/DNA =1/4 NNP/DNA =1/1NNP/DNA =1/1

NNP/DNA =1/0.25NNP/DNA =1/0.25

NNP/DNA =1/0.25NNP/DNA =1/0.25

NNP/DNA =1/2NNP/DNA =1/2 NNP/DNA =1/1NNP/DNA =1/1NNP/DNA =1/2NNP/DNA =1/2 NNP/DNA =1/1NNP/DNA =1/1

DNA

DNA DNA

DNA

DNA

DNA

A B

C D

NNP/DNA =1/10NNP/DNA =1/40NNP/DNA =1/10NNP/DNA =1/40

- 67

ACKNOWLEDGMENTS

I wish to express my sincerest gratitude to Professor Akira Ishihama,

Graduate School of Division of Material Chemistry, Hosei University, for kind

guidance, valuable suggestion and discussions, and continuous encouragement

throughout the course of this work and critical reading of the manuscript.

I am grateful to Assistant Professor Shigehiro Yoshimura and Professer

Kunio Takeyasu, School of Biosciences, University of Kyoto, for valuable

suggestions and discussions throughout the word of AFM analysis. I would also

thank to the members of Professor Takeyasu’s laboratory for kindness help.

I am also deeply indebted to Dr. Kaneyoshi Yamamoto, Hosei University

for valuable suggestions and discussions throughout the work.

I would also like to thank to Dr. Takenori Miki for providing the E. coli

mutant lacking the nnp gene.

Many thanks are due to Miss Kiyo Hirao, Miss Ayako Kori, Mr. Yoshito

Ogawa, Miss Kayoko Yamada, Dr. Hiroshi Ogasawara, Mr. Tomohiro Shimada,

Miss. Minami Naruse, Mr. Naoki Kobayashi and other members of Division of

Material Chemistry, Hosei University.

- 68

Finally, very special thanks are forwarded to my parents for their continuous

support and cooperation, without which this work was never possible.

- 69

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Publication list

Acceptance Treatise

1) Negative Regulation of DNA Repair Gene (ung) Expression by the

CpxR/CpxA Two-Component System in Escherichia coli K-12 and Induction

of Mutations by Increased Expression of CpxR

Ogasawara. H., Teramoto. J., Hirao. K., Yamamoto. K., Ishihama. A.,

Utsumi. R. 2004. 186(24). Journal of Bacteriology.

2) Negative regulation of DNA repair gene (uvrA) expression by ArcA/ArcB

two-component system in Escherichia coli

Ogasawara. H., Teramoto. J., Yamamoto. S., Hirao. K., Yamamoto. K.,

Ishihama. A., Utsumi. R. 2005. 251. FEMS Microbiology Letters.

3) Transcription Factor-Promoter Interaction Networks

A. Ishihama., T. Shimada., H. Ogasawara., J. Teramoto., K. Yamamoto. 2005.

Micro- and Nano-Mechatronics and Human Science.

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J. Bacteriol., submmitted for preparation.

1) Novel Nucleoid Protein (NNP) with Enhancing Activity of DNA Sensitivity to

Nucleases in Anaerobic Growth E. coli.

Teramoto. J and A. Ishihama.

In preparation

1) Novel Nucleoid Protein (NNP) from Escherichia coli: Mode of NNP-DNA

interaction.

Teramoto, J., S. H. Yoshimura. and A. Ishihama.

Verbal & Poster Presentation

1) 日本ゲノム微生物学会第 1 回：かずさアーク

口頭演題「大腸菌単一細胞のプロモーター強度の測定」

寺本潤、長谷川明子、曲山幸生、Elshimy Haitham、

市川明彦、新井史人、福田敏男、石浜明

- 77

2) 18th IEEE International Symposium on Micro-NanoMechatronics and Human

Science

Multi-Scale Genetics of Transcription Network: Understanding the Regulator

All 300 Transcription Factors from a Single Organism Escherichia Coli.

A. Ishihama, H. Ogasawara, T. Shimada, J. Teramoto, A. Hasegawa, Y.

Umezawa, K. Yabuki, Y. Ishida, T. Inaba, A. Kori, K. Yamada, Y. Kitai, N.

Kobayashi, D. Kato and K. Yamamoto.

3) 特定領域女性研究者第一回ワークショップ

大腸菌単一細胞観測系の構築とプロモーター強度測定

長谷川明子、寺本潤、曲山幸生、新井史人、福田敏男、石浜明

4) 第 30 回日本分子生物学会・第 80 回日本生化学会

「大腸菌新規核様体タンパク質の同定」

寺本潤、吉村成弘、郡彩子、竹安邦夫、石浜明


「大腸菌単一細胞観測系の構築とプロモーター強度測定」

- 78

長谷川明子、寺本潤、郡彩子、曲山幸生、El-Shimy H. Magdy、中島正博、林

育菁、新井史人、福田敏男、石浜明


「大腸菌転写因子 TorR の新規標的遺伝子群の探索と制御」

稲葉達也、寺本潤、三木健良、石浜明

identification and characterization of novel nucleoid...

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