Cloning, analysis and expression of an rpoS homologue gene from Pseudomonas aeruginosa PAO1

Gene, 150 (1994) 81-85 0 1994 Elsevier Science B.V. All rights reserved.

0378-l 119/94/$07.00

81

GENE 08318

Cloning, analysis and expression of an rpoS homologue gene from Pseudomonas aeruginosa PA0 1 (RNA polymerase; sigma factor; rpoD Homologue gene; stationary phase; pcm; Escherichia coli)

Kan Tanaka and Hideo Takahashi Molecular Genetics and Breeding, Institute ofMolecular and Cellular Biosciences, The University of Tokyo, Tokyo 113, Japan Received by A. Nakazawa:

3 May 1994; Revised/Accepted:

30 June/l

July 1994; Received at publishers:

4 August

1994

SUMMARY

A homologue of the rpoS gene of Escherichia coli was cloned from Pseudomonas aeruginosa PA01 by hybridization with an oligodeoxyribonucleotide probe designed from an amino-acid stretch conserved among the principal o factors of eubacteria. Two open reading frames, the pcm gene and the orf-297 of unknown function, were found in the upstream region of rpoS, and in the same order as in E. coli. The rpoS gene of P. aeruginosa was expressed in E. coli and complemented the catalase deficiency of the rpoS mutant of E. coli. The RpoS protein of P. aeruginosa was identified by Western blot analysis in both P. aeruginosa (Pa) and the transformed E. coli. Levels of RpoS of Pa increased drastically at the onset of the stationary growth phase.

INTRODUCTION

The rpoS gene product of E. coli was shown to be an RNA polymerase sigma factor, 03* (or IS’) (Tanaka et al., 1993). It is required for the expression of many stationary-phase-specific genes, such as the katE encoding HP11 catalase and the xthA encoding exonuclease III (Lange and Hengge-Aronis, 1991; Hengge-Aronis, 1993). Moreover, rpoS defective mutants survive rather poorly under starvation conditions (Siegele and Kolter, 1992; Correspondence to: Dr. H. Takahashi, Institute

of Molecular

and

Cellular

Molecular

Genetics

Biosciences,

The

and Breeding, University

Hengge-Aronis, 1993). Therefore, the rpoS gene product regulates the gene expression at the stationary phase through the modulation of the promoter recognition specificity of RNA polymerase. Because 038 has a promoter-recognition specificity similar to that of the major sigma factor, o”, it has been suggested that 038 is a second principal sigma factor at the stationary growth phase (Tanaka et al., 1993).

EXPERIMENTAL

AND DISCUSSION

of

Abbreviations: A, absorbance (1 cm); aa, amino acid(s); bp, base pair(s); E., Escherichia; kb, kilobase or 1000 bp; MCS, multiple cloning site(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF (orf), open read-

(a) Cloning of the second rpoD homologue gene of Pa Using the oligo rpoD probe (Tanaka et al., 1988), we identified two rpoD homologue genes in the genome of Pa (Tanaka and Takahashi, 1991). Analyses of the first rpoD homologue gene, corresponding to the 2.3-kb PstI

ing frame; P., Pseudomonas; Pa, P. aeruginosa; pcm, gene encoding protein carboxyl methyltransferase in E. cob; PAGE, polyacrylamide-gel electrophoresis; rpol), gene encoding principal cr factor in E. coli; rpoS, gene encoding stationary phase-specific o factor (era* or crs) in E. co/i; SDS, sodium dodecyl sulfate; [I, denotes plasmid-carrier state.

fragment, was described previously (Tanaka and Takahashi, 1991; Fujita et al., 1992), and proved to encode the principal sigma factor. We first called this gene rpoDA (Tanaka and Takahashi, 1991; Fujita et al., 1992),

Tokyo, Bunkyo-ku,

Tokyo

113, Japan.

Tel. (81-3) 3812-2111,

ext. 7825;

Fax (81-3) 3813-0539.

SSDI 0378-l

119(94)00529-X

82 the region downstream from rpoS, two typical Rhoindependent transcriptional terminators were found (tl and t2 in Figs. 1 and 2). In the spacer regions between pcm and orf-297, and between orf-297 and rpoS, no terminator-like sequence was found. Although there is an inverted repeat structure in the downstream from pcm, we would not predict a termination site because of the absence of the T-stretch sequence. This sequence analysis suggests that the three ORFs may comprise an operon.

but renamed it rpoD because of the functional identity with the rpoD gene of E. cob. The further hybridization analyses localized the second rpoD homologue on a 1.8-kb KpnI-Hind111 fragment (data not shown). Therefore, we cloned this fragment into the MCS of pTZ18R and pTZ19R (Pharmacia, Uppsala, Sweden) plasmid vectors as described (Tanaka et al., 1988), and named the resulting plasmids pDB18R and pDB19R, respectively. We also cloned 4.5 and 1.9-kb BamHI fragments overlapping with the 1.8-kb KpnI-Hind111 fragment onto pTZ18R for the gene walking purpose, and named them pASAl and pASA3, respectively. The physical map of this region is shown in Fig. 1. The DNA region corresponding to the rpoD probe coincided with the BumHI site, which indicated the presence of the rpoD box sequence (Tanaka et al., 1988).

(c) Expression of the Pa rpoS gene in E. coli To test whether the putative rpoS gene of Pa had RpoS activity in E. coli, we performed a complementation analysis using the rpoS defective strain of E. coli K-12. As a host strain we used JM103F. This strain was constructed by the Pl transduction (Miller, 1992) of the rpoS13::Tn10 allele of UM122 strain (Loewenet al., 1985) to JM103 (Yanisch-Perron et al., 1985). JM103F showed very weak catalase activity in the semi-quantitative assay using H,O, on LB agar plates (Zambrano et al., 1993). When JM103F strain was transformed with pDB18R or pDB19R, strong catalase activities were observed. Therefore, we concluded that the rpoS gene of Pa substituted for rpoS of E. coli, and that it is a functional homologue of rpoS of E. coli. The expression of rpoS of Pa in E. coli was further confirmed by Western blot analysis (Fig. 4) using anti-RpoS (E. coli) antiserum (Tanaka et al., 1993). When the rpoS13::TnIO allele was transferred to JM103, the RpoS signal vanished completely (Fig. 4, lane 3). Introduction of a plasmid containing the rpoS gene of E. coli led to the re-appearance of the signal at the same position (Fig. 4, lane 5). Discrete signals at the slightly larger molecular weight positions were found when pDB18R or pDB19R plasmids were introduced into JM103F (Fig. 4, lanes 6 and 7). These positions were precisely identical with the RpoS signal obtained from the lysate of Pa (Fig. 4, lane 8). This clearly indicated

(b) Structure of the rpoS gene of Pa

The nt sequence of 2910-bp BumHI-EcoRI fragment (Fig. 1) was determined and analyzed (Fig. 2). As expected, an ORF of 334 aa highly homologous to bacterial sigma factors (Helmann and Chamberlin, 1988; Lonetto et al., 1992) was found. The aa sequence comparison revealed high similarity to the rpoS gene of E. coIi (Fig. 3C) (Tanaka et al., 1993; Mulvey and Loewen, 1989; Takayanagi et al., 1994). We propose this ORF to be the rpoS gene of Pa, and named as such. In the upstream region of rpoS, we could find two additional ORFs in the same direction as rpoS (Figs. 1 and 2). These ORFs were also found in the upstream region of rpoS of E. coli (Takayanagi et al., 1994). Thus, the organization of these genes appeared to be conserved between E. coli and Pa. We named these ORFs pcm and orf-297, according to the corresponding ORFs of E. coli (Fu et al., 1991; Takayanagi et al., 1994). Thus far, we do not have information on the function of the orf-297. Sequence alignments of the respective ORFs were shown in Fig. 3. In

pcm BamHI

orf-297 KpnI

200 bp

rpoS ClaI SmaI

Hind111 EC&I

HinclI BamHI

! t

t t1

rpoD box

k

L I1.9-kb BarnHI fmgment cloned in pASA3

II/I

BamHI

i

j,T I, ,,

4

45kb BanHI fragment cloned in pASAl >

* 1.8-kb KpnI-HiidIII fragment cloned in pDB18R and pDB19R

Fig. 1. Physical map and schematic representation of the Pa rpoS region. A restriction enzyme map around the rpoS gene of Pa is shown. The sequenced 2910-bp BarnHI-EcoRI region is indicated by a bold line. The positions of the rpoD box and the two Rho-independent transcriptional terminators (tl and t2) are shown by arrows. The directions and the sizes of the predicted ORFs are also indicated. DNA regions cloned in plasmids are indicated below the restriction map.

x3 . . . . Bm?HI * ~pcm . . . 1 ~AGGCGTTGCAGGACAAGGCCAAGGAGCGTCTTGC~A~T~CCTGCGTMTGTTGTCTTTCGTTGG~CGATGGCT~GAGGGTTGGTCGGCGTTG~TCCCTAC~T~~ 1 IQALQDKAKERLAELNLRNVVFRWGDGWEGWSALAPYNGI

.

l

.

121 TCATCGTCACCGCGGCGGCCACGGAAGTCCC~AGTCGTTGCT~ACCAGTTG~GCCTG~GGGCGCCTGGTGATCCCGGTC~TG~GGCGA~TCCAGC~CTGATGCTGATCGTCC 41 IVTAAATEVPQSLLDQLAP GGRLVIPVGGGEVQQLMLIVR 241 GCACCGAGGACGGGTTCAGCCGCCAGGTACTCGACTCGACTCGGTACGTTTCGTCCC~T~TCMCGGCCCGATC~CTGAGCCGGC ~T$GSZU.CcGATTG~cGAAAGcG~G~ 81 TEDGFSRQVLDSVRFVPLLNGP I A ter + Lxf-297 361 AGATGGATAAGGGGGAAGGATTGAGGCTAGCAGCGACCCTGCG~~T~ACTCG~TCTACGGTGGCTGCCACCTGTTGCTCG~~CGTCGTCTGTTCCCTT~T~GC~~~TGTT~GT 1 MDKGEGLRLAATLRQWT RLYGGCHLLLGAVVCSLLAACSS 481 ~GT~GC~TCCCGGCGGGGTCAAGGTCGTCGATCGCAACGGTTCCGCGCCTGCCGCTGCGCGGCGTACGCCGGTCACCA~GGGCAGTACATCGTGCGCCGCGGCGACACC~TGTATT~~A 41 SPPGGVKVVDRNGSAPAAARRTPVTSGQYIVR RGDTLYSI 601 TTGCCTTCCGCTTCGGCTGGGACTGGAAAGCCCT~CCGCGCGC~TGGCATC~~CGCCCTATACCATCCAGGTC~TC~GCTATCC~TTTGGT~GCGGGCATCGACGC~~~GT 81 AFRFGWDWKALAARNGIAPPYT IQVGQAI QFGGRASTQPS K&mI 721 CCGTCGCGAAAARCACGCCGGTCGTCGCTGCGCCCGTG~GACC~GCCTACCCCGGTTCCACCCGCAGTTAGTACGTC~T~C~GCCCGCACCGGCT~CGGC~TCGA~~A~GA 121 VAKNTPVVAAPVATKPTPVP PAVSTSVPAKPAPAPASTTT 841 C~~CACC~AG~AG~GG~GCGA~TC~~GT~GT~G~GG~~T~~T~G~~~GGTG~ATGG~~TG~GAG~GGTA~T~TGAT~G~~GTTTTGCCT~~~GG~GTTTG~T~GGGA 161 P P s SGATPVVAGPAVGGWAWPASGTLIGRFASNGSLNKGI 961 TTGATATAGCCGGTCAATTGGGCCAGCCTGTCCTG~TGCGTCTGGTGGGACCGTTGTATACGCC~TAGTGGTTTGCGG~CTAC~CGAGTTGGTCATCATC~CAC~CGAGACCT 201 DIAGQLGQPVLAASGGTVVYAGSGLRGYGELVI IKHNETY CldI 1081 ACGTGAGTGCCTACGGTCACAACCGCAGGCTGCTGGTGCGGGC ~TG~CGAGATGGG~T~~A~AGGGT~TGCA~TT~G VSAYGHNRRLLVREGQQVKVGQSIAEMGSTGTDRVKLHFE 241 1201 AGATTCGCCGCCAGGGTACTGTCGATCCACTGC~TATTT~CACGTCGCTGACCG~AGTTCGCCCGCCCACATCATGTA~TGA~GGGTCCGGGCGTGTCCAGCGGG~GG~ I RR Q G K P VD P L Q Y L P R Rter 281 SmaI -_)rpos 1321 TCGCCCGGGCTTGAGTCGAACTCATGCA.&~ATAACGACATGGCACTC AAAAAAGAAGGGCCGGAGTTTGACCACGATGATGAAGTGCTCCTCCTGGAGCCCGGCATCATGCTGGACGA MALKKEGPEFDHDDEVLLLEPGIMLDE 1441 GTCGTCTGCCGACGAGCAGCCTTCTCCCCGGGCRACTCCACGCAGCTGTATCT 28 S SAD E Q P S P RA T P KA T T S F S S K Q H K H ID Y T R A L D A HincII 1561 CAACGRAATCGGTTTCTCGCCCCT~~CCG~GAGG~GTCCACTTCGCTCGTCTGGCGCAG~GGGCGATCCCGCTGGTC~~GCGGATGATCGAGAGC~CCTGC~TTGGT 68 NE I G F L P L L T P E E E V H F A R L A Q K G D P A G R K R M I E S

T

Q

L

Y

L

N

L

R

L

V

R

G

F

R

F

2041 AGA~GTCT~TCTTGGTCCGGACTCGGACAAGACCCCTGCTGGATACGCTCACCGACGATCGCCCCACCGATCCGTGCGAGCTGCTGCAGGATGACGATCTCAGCG~GCATCGACCAGTG I D Q 228D V S L G P D S D K T L L D T LTDDRPTDPCELLQDDDLSES

W

1681 GGTGAAGATCGCCCGGCGCTATGTCAATCGCGGACTGTCCCT~TCGACCTGATCGAGG~G~~CCTAGGCCTGATCC~~CGTGGAG~GTTCGATCC~AGC~~ATTCC~TT 108VK I AR R YVNRG L S L L D L I E E G N L G I. I RAVE K F D P E BdmHI 1801 CTCGACCTACGCCACCTGGT~~CAGACCATCGA~GG~CATCATG~CCAGACCCGGACCATTC~TTGCCGATCCATGTGGTC~GGAGCTC~CGTCTACCT~GT~~C 148 S T YA T WW I RQ T I E RA I MNQ T R T I R L P IHVVKELNVYLRAA

1921 GCGGGAACTGACCCACAAGCTCGACCACGAACCTTCACCTTCACCCG~G~TCGCC~CCTGCTGGAG~GCCGGTCGCCGAGGTC~GCGCATGCTCGGCCTG~CG~CGGGTGACTTCGGT 188 R E L T H K L D HE P S P E E IANLLEKPVAEVKRMLGLNERVTSV

2161 GcTGAcGG~cTcAccGACAGCAGCGTGAGGTGAGGTGGTGATTC~C~TTCGGCTTGCGCGGTCACG~GCAGCACGCTGG~GAGGTCGGCCAGG~TCGGCCTGAcCCGcGAGcGGGT IGLTRERV VVIRRFGLRGHESSTLEEVGQE 268 L T E L T D K Q RE 2281 TCGTCAGATCCAGGTCGAGGCGCTGAAGCGCGCCTGC~GAGATTCTGGAG~G~TGGCCTGTCGAGTGACGCGCTGTTCCAGTGACGG~CCTTAGA~CCACT~T~ G L S S DAL F Q ter 308 R Q I Q V E A L K R L R E I L E KN 2401 GCCGGGTTTTTTGTGTCTG~T~TTGT~GCATT~CTTACACG~GTGTGAGCCT~GTAGAT~TGCCC~GAGGTTTGCC~CGCTCGGTG~TTT~GT~CTTATTG~TT Hind111 TTTTTGCCTGCCGTTTTACTCGTCGCCAAAGAAAAG

2641 GATCGGGAAGGGACGTCGCCAG~A~CGATTCATCAGGATGATGACGA~ACTG~GAGT~

2761 GCCcGCTGATTGCGCGGTTGcGTATCAGCGCTCCAGATGCTGGA~TTGCCGGCACGCCGTCCCATTCCTCGGcGTcGGGCAGGGcATcCTTCTTCTcGGTGATGTTCGGcCAGACTTc EcoRI 2881 CGCCAGCTCGCTGTTCAGCTCGATW 2911

Fig. 2. The nt sequence Nested deletion clones

of the Pa rpoS region. The sequence of 2910 bp corresponding to the BarnHI-EcoRI fragment of the rpoS region is presented. for sequencing were constructed from plasmids pDBlSR, pASAl and pASA3 using the exonuclease III directed methods

(Yanisch-Perron et al., 1985). Sequencing reactions were performed with Sequenase TMVer. 2 (US Biochemical, Cleveland, OH, USA) and [WEEP] dATP (Amersham), or Model 373A-18 DNA sequencer (Applied Biosystems Japan, Tokyo, Japan) as indicated by the suppliers. Inverted repeat structures are indicated by arrows. Putative ribosome-binding sites of orf--297 and the rpoS gene are shown by dotted underlines. A nt sequence portion complementary

to the rpoD probe

(rpoD box, Tanaka

et al., 1988) is indicated

by a double

underline.

The GenBank

accession

No. for this sequence

is D26134.

that the rpoS gene of Pa could direct the synthesis of a protein in E. coli that was similar to that seen in Pa. (d) Synthesis of the RpoS protein in Pa Synthesis of the RpoS protein in Pa was monitored by Western blot analysis. Pa PA01 was grown in LB medium at 37”C, and sampled periodically from the mid-

logarithmic phase to the stationary phase. The cells were lysed in SDS-sample buffer, and used for the analysis as in Fig. 4. The RpoS protein increased drastically at the onset of the stationary growth phase (Fig. 5). RpoS levels in E. coli become elevated in stationary phase cells (Tanaka et al., 1993). Therefore, the rpoS genes of E. coli and Pa appear to be similarly regulated, and may play

84 A

pcm

[49.0%/104 aal . . . . . . . . . . Pa IQALQD~ERLAELNLRR~DG~GWSALAPYNGIfVTAAATEVPQSLLDQLAPGGRLVIPVGGGEVQQLMLIVRTEDGFSRQVLDS~~LLNGPIA104 l * *t,** * * * l . * * . .. l *****. * .* l .*t tm l * .*.*.** * ****.*t * l *...*t*t** l .* l . l , EC IKGLQWQARRRLI(NLDLHNST~GDGWQGWPARAPFDAIIVTMPPEIPTALMTQLDEGGILVLPV-GEEHQYLKRVRRRGGEFIIDTVEAVRFVPLVKGELA 210 B

orf-297/orf-281

[41.9%/265 aa . . . . . . . . . . . Pa MDKGEGLRLAATLRQWTRLYGGCHLLLGAWCSLLRACSSSPPGGVKVVDRNGSAPAAARRTPVTSGQYIVRRGDTLYSIAFRFGWDWKALMRNGIAPPYTIQV~AIQ---------F . *.. ** l * * l * ..*t* .* * l ,.****. l *. . . ~NGRIVYNRQYGNIPKGSYSGSTYTVKKGDTLFYIAWITANDFRDLAQRNNIQAPYALNVMTLQVGNASGTPIT EC Pa GGRASTQPSVAKNTPW------AAPVATKP------TPVPPAVSTSVP-AKPAPAP--ASTTTPPSSGATPW----AGPAVGGWAWPASGTLIGRFASNGSLNKGIDIAMLGQPVLA l ** t .* *. l * t t* t**t**** t*. * .* *t l ** * l * * *. EC GGNAITQADAAE~WIKPAQNSTVAVASQPTITYS~~SG~QS~~LP~KPTATT~~VT~TASTTEPTVSSTSTSTP~ST~~TEGKVIETFGASE~NKGIDIAGSK~AIIA2S1 Pa ASGGTVVYAGSGLRGYGELVIIKHNETWSAYGHNRRLLVR297 .***t *.** ** ** **et** * ***t* .t****.***t**t. l .****t* .*****t tt *,** EC TADGRWYAGNALRGYGNLIIIKHNDDYLSAYAHNDTMLVQQE~~KIATMGSTGTSSTRLHFEIRYKGKSVNPLRYLPQR c

l

**

. 111 76 297

** ..*

l

281

rpos

[76.2%/277 aal .I____-___._ 1.2 ___._________.] . . . . . . [- 2.1.--l Pa MALKKEGPEFDHDDEVLLLEPGI~ESSADEQPSPRATPKATTSFSSKQHKHIDYTRALDATQLYLNEIGFSPLLTPEEEVHFARLAQKGDPAGRKRMlESNLRLVVKIARRYRGLS t ***t**** ***.***** l *** *** * .** * l ~**t****t**t****t* MSQNTLKVHDLNEDAEFDENOVEVFDEKALVELEPSDNDLLA115 EC

. 120 t***

[____________________ 3 _____________________] [____ 2.2 ____][______ 2.3 _____I [_____2,4 ________] Pa LLDLIEEGNLGLIRAVEKFDPERGFRFSTYATWWIRQTIERAIMNQTRTIRLPIHVVKELNWLRAARELTHKLDHEPSPEEIANLLEKPVAEVKRMLGLNERVTSVDVSLGPDSDKTLL 240 **t******t***********t******tt**t***********"**************~********* t***.******** ****. t.*** .* *** ****.t**+ ** **.* *t EC LLDLIEEGNLGLIRliVEKFPERGFRPSTYAT~IRQTIERAIPINQTRTIRLPIHIVKELNVYLRTARELSHKLDHEPSAEEIAEQLDKPVDDVSRMLRLNERITSMTPW;GDSEKALL 235 [_____ 4.1 _____] [__________ 4.2 ___________] Pa DTLTDDRPTDPCELLQDDDLSESIDQWLTELTDKQREWIRRFGLRGHESSTLEEVGQEIGLTRER~QIQVEALKRLRILEKNGLSSDALFQ * * *.. t _ t**+. .** ** ** **t**. ****t * * ***.*t *********t*t***.*.******. .t* EC DItAoE~NGPEDTTQDDD~QSIVKWLFELNAI(PREVLGLLGYEAATLEDVGREIGLTRERVRQIQVEGLRRLRIITPGLNIEALFRE

Fig. 3. The aa sequence using a computer

comparisons

program

of the predicted

(SDC-GENETYX,

gene products

Software

of Z? aeruginosa

Development,

Tokyo,

Japan).

334 .*** 330

(Pa) and E. cob (EC). The deduced Identical

and conserved

aa residues

aa sequences are indicated

were aligned by asterisks

and dots, respectively. Conserved aa changes are defined as any within the following groups: (I, L, M, V), (H, K, R), (D, E, N, Q), (A, G), (F, Y, W), (S, T), (P) and (C). The lengths of the homologous regions and the percentage of the identical residues in that portions are shown in brackets. (A) pcm The predicted

104-aa C-terminal

sequence

of the Pa encoded

by

pcmwas compared

with the corresponding

sequence

of E. coli. (Fu et al., 1991)

(B) orf-297/o+281: The aa sequences of the orf-297 of Pa and the orf-281 were aligned. The M, values of these ORFs were calculated and 29971, respectively. (C) rpoS: The aa sequences of RpoS of Pu (334 aa) and E. coli (330 aa) (Tanaka et al., 1993) were compared The proposed

conserved

regions

are shown

above

the sequences.

The M, values

of these gene products

were calculated

to be 30 835 and aligned.

to be 38 235 and 37 956.

respectively.

important roles in stationary phase gene expression in both organisms (Tanaka et al., 1993; Takayanagi et al., 1994). (e) Conclusions (I ) The rpoS gene of Pa PA01 encoding a 38-kDa protein has been cloned and sequenced. Two open reading frames, the pcm gene and the orf-297 of unknown function, were found in the upstream region of rpoS, and in the same order as in E. coli. (2) The rpoS gene of Pa was able to complement catalase deficiency of an E. coli rpoS mutant. (3) The gene product was identified in Pa, and shown to increase at the stationary-growth phase, indicating the similar function as in E. coli.

ACKNOWLEDGEMENTS

The authors thank Akiko Nishimura (Genetic Stocks Research Center, National Institute of Genetics,

Mishima, Japan) for providing a strain. We also thank Yukio Ohashi for technical assistance. This work was supported in part by grants for scientific research from the Ministry of Education, Science, and Culture of Japan and from the ‘Biodesign Research Program’ of RIKEN.

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Lanes: 1, MC4100

endA

supE sbcBC thi-1 rpsL A(&pro;

JM103F

(like

JM103

JM103F[pKTFl]; PA01

rpoS13::TnlO);

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(Pa). Methods: Bacterial

were sampled gation.

but

Yanisch-Perron

grown

phase,

Cells were lysed in SDS-PAGE corresponding

5,

in LB medium

and collected

8, Pa at 37°C

by centrifu-

sample buffer, and heated for 2

min at 95°C. Cell mass was estimated samples

4, JM103F[pTZ18R]; 7, JM103F[pDB19R];

cultures

at the early stationary

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from A,,,

of the culture,

to the same cell mass (equivalent

and

to the mass

in 10 ul of culture of AG6,,= 1.0) were analysed in 0.1% SDS-12% PAGE and used for the immunoblot analysis with the anti-RpoS (E. coli-Ec) antiserum

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Nucleotide protein

sequence

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is a novel cr transcription

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Y., Tanaka,

upstream

P.C.: KatF

K. and

Takahashi,

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Fig. 5. Growth-phase-dependent synthesis of the RpoS protein in Pa. Pa cells were grown in LB medium and sampled periodically at every 30 min from the mid-exponential constant

the anti-RpoS bance

phase

to the stationary

mass of the cells were used for the immunoblot (E. coli) antiserum

(AscO) of the culture

phase. analysis

The with

(see the legend to Fig. 4). The absor-

used in experiments

for each lane was as

follows: lane 1, 0.24; lane 2, 0.45; lane 3, 1.0; lane 4, 1.5; lane 5, 1.8; lane 6, 1.95; lane 7, 2.3; lane 8, 3.7 (overnight

culture).

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