Newly identified genes involved in the signal transduction of Escherichia coli K-12

Gene, 140 (1994) 73377 0 1994 Elsevier Science B.V. All rights reserved.

73

0378-l 119/94/$07.00

GENE 07677

Newly identified genes involved in the signal transduction Escherichia coli K- 12 (Two-component

system; bagA; bugs; eugA; eugS; environmental

R. Utsumi, S. Katayama, H. Tanabe and M. Noda

M. Taniguchi,

Department

Kinki

of Agricultural

Received by A. Nakazawa:

Chrmistry

University,

of

adaptation)

T. Horie, M. Ikeda, S. Igaki, H. Nakagawa,

A. Miwa,

Naru 631. Japan

12 July 1993; Revised/Accepted:

20 August/l3

September

1993; Received at publishers:

28 October

1993

SUMMARY

We cloned and sequenced two Escherichia coli genes which are members of a family of an environmentally responsive two-component system. The nucleotide (nt) and deduced amino-acid sequences of these two genes were found to be homologous to those of the Bordetella pertussis bugA and hugs genes. They were mapped at 51 min (clones 6B9 to 7G9 of the Kohara miniset library of the E. coli chromosome). Both proteins, deduced from their nt sequences, were identified in the coupled in vitro transcription-translation system; their molecular masses were consistent with BvgA and BvgS (23 and 135 kDa, respectively). Furthermore, when these genes were expressed on a multicopy plasmid in an enuZ deletion strain, ompC expression was induced. This expression was found to be regulated by low temperature, MgSO, and nicotinic acid, factors known to control the virulence of B. pertussis via BvgA and BvgS. These results indicate that the newly cloned genes were structually and functionally similar to hugA and bugS, and we designated these genes evgA and eugS.

INTRODUCTION

To respond to wide varieties of environmental stimuli, cells use signal-transduction mechanisms which involve pairs of proteins that communicate with each other by protein phosphorylation (two-component system)

Correspondence to: Dr. R. Utsumi, Department Chemistry, Kinki University, 33277204 Nakamachi, Tel. (81-742) 43-1511; Fax (81-742) 43-2970.

of Agricultural Nara 631, Japan.

Abbreviations: aa, amino acid(s); Ap, ampicillin; PGal, b-galactosidase; B., Bordrtella; bp, base pair(s); BvgA, protein which is phosphorylated by BvgS and binds to specific DNA sequences to regulate transcription; BvgS, sensor protein modulated by environmental signals: hvgA and hugs; B. pertussis virulence genes encoding BvgA and BvgS, respectively; A, deletion; enuZ, gene encoding inner membrane protein EnvZ; kb. kilobase or 1000 bp; nt, nucleotide(s); ompC, gene encoding outer membrane protein OmpC; ompR, gene encoding DNA-binding protein OmpR which regulates transcription of ompC gene; ORF, open reading frame; [I, denotes plasmid-carrier state. SSDI

0378-1119(93)E0673-2

(Albright et al., 1989; Stock et al., 1989). Generally, a two-component regulatory system consists of a membrane receptor and a cytoplasmic DNA-binding protein. Environmental information gathered by the receptor is passed to the DNA-binding protein (regulatory protein) via phosphorylation, which changes the properties of the regulatory protein, resulting in an alteration of gene expression (Igo et al., 1990). For example, the expression of omp regulon of Escherichia coli, which includes the genes for the major outer membrane porin proteins OmpF and OmpC, is controlled by changes in osmolarity. This regulation is governed by the inner membrane protein EnvZ (a receptor kinase) and the DNA-binding protein OmpR, a transcriptional regulator (Forst et al., 1988; Norioka et al., 1986). Recently, a fully functional chimeric transducer was constructed by recombining the sensor domain of the Tar chemoreceptor and the kinase domain of EnvZ (Utsumi et al., 1989). This chimeric receptor can activate

74 OmpR

to transcribe

the ompC gene in response

ligand ‘aspartate’ and is highly similar to intact exhibiting the activities of both self-phosphorylation phosphate transfer to OmpR (Rampersaud Utsumi et al., 1991); that provides direct evidence

that EnvZ

acts as a sensor and modulator. that expression

production

proteins

regulatory

a Gene Mapping

EnvZ and

Japan, was used. The nylon membrane was subjected to hybridization with the probe DNA, and the hybridized probe was detected by the enzyme-linked immunoassay

et al., 1991; experimental

the other hand, it is well known of the

to the

NtrC,

On

or overOmpR,

DctD, CheY, UhpA, PgtA, FixJ and PhoR in the absence of their sensor partners, elicits a target response that, in most cases, is constitutive observations

might

(Albright

be explained

et al., 1989). These

by low-level

cross-talk

and a subsequent

Membrane

supplied

enzyme-catalyzed

a non-radioactive DNA labeling (Boehringer-Mannheim, Mannheim, probe DNAs were labeled ration

of digoxygenin-labeled

by Takara

Syuzo,

color reaction

using

and detection Germany).

by randomly

primed

kit The

incorpo-

deoxyuridine-triphosphate.

The 2.1-kb HindIII-AvaI fragment of pSKOO1 was labeled with digoxigenin. It was hybridizing to the 1 miniset phages

6B9 (Noda

et al., 1991) (#412 serial number

of

from the sensors of the other system. Alternatively, the regulatory protein may have some inherent activity in

the miniset) from the Kohara library (Kohara et al., 1987). On the other hand, the phage 7G9 as well as 6B9

the absence

were hybridizing with the 2.0-kb MluI fragment of pSK001. These results indicated that the newly cloned

of modification/phosphorylation.

The aim of

the present study was to isolate a new two-component system by using such an in vivo cross-talk. In this report, we describe a new set of E. coli genes that can activate the ompC transcription when present in multi-copy state.

region was mapped at 51 min and spans the clones 6B9 to 7G9 on Kohara’s physical map of E. coli K-12. (b) Sequence analysis The sequences of 5083 bp on both strands

EXPERIMENTAL

AND DISCUSSION

(a) Cloning of new genes as multicopy suppressors of enuZ mutation Genomic DNA was prepared from E. co/i AT141 (AompR and AenvZ strain of E. coli MC4100; Mizuno et al., 1987) as previously reported (Kawamukai et al., 1991). It was partially digested with HindIII, ligated to HindIII-digested pUC19, and the ligation mixture was used to transform E. coli RU1012 (AenvZ::KmRof E. coli MH225) (Utsumi et al., 1989). Transformants were selected on the MacConkey plates containing lactose and Ap. One clearly red colony was selected, and its plasmid was named pSK001. Similar strategy had previously been used to clone the chimeric receptor-encoding sequence taz (Utsumi et al., 1989) and the barA gene which encodes a novel sensor-regulator protein belonging to a homologous family of signal-transduction proteins involved in adaptive responses in E. coli (Nagasawa et al., 1992). The BGal activity of RU1012[pSKOOl] and RU1012[pUC19] was 340 and 40 units, respectively. After digestion of pSKOO1 DNA with HindIII, a 5.6-kb Hind111 fragment derived from E. coli AT141 was detected by agarose gel electrophoresis. In order to localize the cloned genes on the E. coli chromosome (Nishimura et al., 1992; Noda et al., 1991)

were deter-

mined by an autosequencer (Fig. 1). Analyses using MacMolly DNA analysis computer software revealed two ORFs. The homologus sequences were searched for and aligned against the entries in the SwissProt, NBRFPDB, GenBank, DDBJ and EMBL-GDB databases (supplied by SDC Software, Japan). Two ORFs, ORFl of 204 aa and 0RF2 of 1197 aa (Fig. 1) were found to be homologous to BvgA and BvgS of Bordetella pertussis (Arico et al., 1991; Stibitz et al., 1991) respectively. The optimized score with BvgA and BvgS was 518, while the scores were about 200 for other two-component systems. The alignment between ORFl and BvgA indicated that ORFl has highly conserved aa in regulatory components of the two component systems (see single asterisk in Fig. 1). A helix-turn-helix motif previously reported (Brennan et al., 1989, Arico et al., 1991) was found at aa 161 to 180 of ORFl (underlined in Fig. 1). The alignment between ORF2 and BvgS showed that 0RF2 belongs to the family of sensor components. EvgS contains hydrophobic regions at aa 3 to 19 (FLPYIFLLCCGLWSTIS), aa 379 to 394 (VLFAEAFITTPYVFVM) and aa 536 to 552 (FYIVTTLSVLLVGSSLL). These results suggest that ORFl and 0RF2 constitute a two-component system. The ORFl and 0RF2 were named EvgA and EvgS, respectively. To identify the protein products of EvgA and EvgS,

Fig. 1. The nt sequence and the predicted aa sequence of prg:A and ergS. The asterisks indicate the conserved aa residues (*) of the two component system (Stock et al., 1989) and the stop codons (***). The helix-turn-helix motif (Arico et al.. 1991; David et al., 1988; Freidrich et al., 1987; Brennan et al., 1989) is underlined. The nt sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nt sequence data bases under the accession Nos. D11142 and D14008.

75 1 TGmrrTAmGAATTAA;CTGrCCAC;ATTATCTCT;ATTTCTCAT~GATGAAA~~ATGAATTGA~TGAAAAGAG~A~CA~C~CATATCAAA~ACAGTGCAT~TGTTGCACA~ TATCCCAGGAACGTTGGAGGCCTAATTTAAATGAGGTGCAAAAAAAACAACAGAGGACTAAACCGTGGC~TGCAATACAA~C~ACGCCTGTAGGA~AGTAAGAAGAC~ATAGTG CCAACTTGAAACTATAAATCATCGGTACAATCCCTGA~TA~G~GACATTTCA~ATGCCGACTA~ATATGGTATAC~GTCGAA~ATC~AAAGGAAG~CAGAT~TC~A ~ATTGAGAAAATGAGATGACGCC~ATGTCTGTATTACTACAGGGAGAAGGGAGATGCTTCATTGCAAAGGGAATAATCTATGAACGCAATAATTATTGATGACCATCCTCTTGCT EvgAMNAIIIDD*HPLA ATCGCAGCAATTCGTAATTTATTGATCAAAAACGATATTGCAGCGGGTGGAAACACTTAAGCCTGATATCGTCATCATTGAT IAAIRNLLIKNDIEILAELTEGGSAVQRVETLKPDIVIID* GTCGATATCCCCGGAGrrAACGGTATCCAGGTGTTAGAAACGCTGAGGAAGCGCCAATATAGCGGAA~A~A~ATCGTCTCCGCTAAAAATGACCA~ACGGGAAACA~GTGCT VDIPGVNGIQVLETLRKRQYSGIIIIVSAKNDHFYGKHCA GATGCTGGCGCTAATGGTrrCGTGAGTAAAAAAGAAGGCATGAACAATATCA~GCGGCTA~GAAGCTGCAAAAAATGGCTACTGCTA~CCCC~CTCTCTCAACCGG~TGTTGGA DAGANGFVSK*KEGMNNIIAAIEAAKNGYCYFPFSLNRFVG AGTTTAACGTCCGACCAGCAAAAACTCGACTCCTTATCGAAACAAGAAATTAGTGTCATGCGGTATATTCTTGATGGCAAGGATAATAATGACATTCCTGAAAAAATGTTCATCAGCAAC SLTSDQQKLDSLSKQEISVMRYILDGKDNNDIAEKMFISN AAAACTGTCAGCACTTATAAAAGTCGCCTGATGGAAAAATTAGAATGTAAATCACTGATGGATC~ACACA~CGCACAACGTAACAAAATCGGCTAACCACATGAAG~ACCCTAlO80 M K F L P KTV 5 T Y K 5 R L M E K L E C K S L M D L YT F A QR N K I G *** TA~~CTTCTCTGTTGTGGTCTTTGGTCGACCATAAG~CGCAGACGAAGATTACATCGAATATCGTGGCATCAGTAGTAACAACCGTGTCACACTTGATCCACTACGTCTGAGCAA1200 I F L L C C G L W 5 T IS F A D E D Y I E Y R G IS S N N R V T L D P L R L S CAAGGAATTACGrrGGTTAGCGAGCAAAAAAAATCTTGTGA~GCAGTACATAAGTCCCAAACGGCTACGTTGTTGCATACCGATTCGCAGCAACGGGTTCGTGGTATTA1320 KELRWLASKKNLVIAVHKSQTATLLHTDSQQRVRGINADY mAAATCTmAAAAAGAGCGTTAAATATCAAATTAACACTCCGGGAATACGCAGATCATCAAAAAGCAATGGACGCGC~GCAGAAGGTGAAGTCGATATAGTGTTATCACA~TAGT1440 L N L L K R A L N I K L T L R E Y A D H Q K A M D A LA E G E V D I V L S H L TACTTCGCCGCCTCTTAATAATGACATTGCTGCTGCAACCAAACCA~GATAATTACC~CCGGCGCTGGTAACCACC~CACGACTCAATGCGACCGCTTACCTCACCAAAACCAGTAAA1560 T 5 P P L N N D I A AT K P L I IT F PA L VT T F H D S M R P L T S P K P V TATTGCTCGGGTAGCAAATTACCCCCCAGACCTAATTACAAA~ATATCAGGCATTAGCATCCGTCTCAGCTGGGCACAAl680 I AR V A NY P P D E V I H Q 5 F P KAT I I S F T N L Y QA LA S V S A G H TGATTAC~TATTGGTAGTAACATCATTACCAGCAGTATGAmCCCCCGCTATTTCACTCACTCCTTAAATGTAGTGAAATA~ATAACTCGCCGCGTCAATATAATCCTCTCTTGACCAG1800 D Y F I G S N I IT S S M IS RY F T H S L N V V KYY N S P RQY N P L L T AAAAGAATCTGTCATTCTTAATGAAGTACTCAATACAmGTCGCTATGAAGTATCACAAAATCGGCTTGATACAGGAAACCTGGCCmCTGAACAACAA1920 K E 5 VI L NE V L N RF V DA L T N E V R Y E V S QN R L DT G N LA F L N ACCATTAGAACTCACTGAACATGAAAAACAGTGGATTAAGCAGCATCCCAA~AAAGGTGCTGGAAAATCC~ACTCGCCCCCCTA~CTATGACGGATGAAAATGGCTCGG~CGGGG2~0 P L E L T E H E KQW I KQH P N L KV L E N PY S P PY 5 MT D E N G S V R CGTTATGGGGGACATTCTTAATA~ATTACCTTGCAAACAGG~AAA~CTCCGATCACCG~CACACAATATCCATGCTGGAACACAGCTTAGCCCCGGAGGATGGGATATAAT2160 VM G D I L N I IT L QT G L N F 5 PIT V 5 H N I HAG T Q L 5 P G G WD I ACCTGGCGCTATTTATAGTGAAGATCGAGAAAATAATGT~A~GCTGAAGCCTTCATAACAACGCCTTACG~GTCATGCAAAAAGCGCCTGACAGTGAACAAACA~AAAAAA2280 P GA I Y S E D R E N N V L F A E A F I TT P Y V F V MQKA P D S E QT L K AGGAATGAAAGTTGCCATTCCATATTACTATGAGCTGCAT~CGAA~AAAAGAGATGTATCCGGAGGTTGAATGGATACAGGTCGATAATGCCAGCGCTGCA~CACAAGGTTAAGGA2400 GM KV A I PYY Y E L H F E L K E MY P E V E W I QV D N A SAA F H KV K AGGTGAAC~GATGCTCTGGTCGCGACACAGCTAAATTCGCGTTACATGATCGATCATTACTATCCTAATGAACTTTATCAT~C~A~CCCGGCG~CCGAATGCATCACTTTCG~2520 G E L DA L V A T Q L N 5 R Y M I D H YY P N E L Y H F L I P G V P N A S L S CGCTTTTCCTCGCGGAGAACCGGAACTTAAGGATATTATTAATAAAGCACTGAATGCAATTCCCCCAAGCGAAGTTCTGCGCCTGACGGAAAAATGGATTAAAATGCCCAATGTGACCAT2640 A F P R G E P E L K D I I N K A L N A I P P S E V L R L T E KW I KM P N V T

120 240 360 480 12 600 52 720 92 840 132 960 172 Y EvgS N

46 86

V126 N166 N

206

R246 K286 G

326

1366 K406 E

446

F

486

1526

TGACACATGGGACCTATATAGCGAGCAATTTTATATTGTTACGACATTATCCGTTAGTTGGCAGTAGCCTTTTATGGGGATTCTACCTGTTACGCTCAGTTCGTCGTCGTAAAGT2760 DT W D L Y S E Q F Y I V TT L 5 V L L V G S S L L W G F Y L L R S V R RR K V 566 CATTCAGGGTGArrTAGAAAACCAAATATCATTCCGAAAAGCACTCTCGGATTCCTGA2880 I Q G D L E N Q IS F R K A L 5 D S L P N P T Y V V N WQ G NV I S H N S A F E 606 ACATTATTTCACTGCGGATTACTACAAAAATGCAATGrrAAGAAAACAGTGACTCACCCTTTAAAGATGTTTmCTAATGCGCATGAAGTCACAGCAGAAACGAAAGAAAATCG3000 H Y F T A D Y Y K N A M L P L E N S D S P F K D V F 5 N A H E V T A E T K E N R 646 AACAATATACACACAGGTATTGAAArrGATAATGGCATCGAGAAAAGATGCATTAATCACTGGCATACA~ATGCAATCTTCCTGCAAGTGACAATGCAGTATATATTTGTGG~GGCA3120 T I Y T QV F E I D N G I E K R C I N H W H T L C N L PA S D NAVY I C G W Q686 AGATATTACTGAAACGCGTGATCTAATTAATGCACTCGAGGTAGAAAAAAATAAAGCGATAAAGGCTACCCTCTGGCAACGATGAGTCACGAAATAAGAACACC3240 DITETRDLINALEVEKNKAIKATVAKSQFLATMSH*EIRTP726 AATAAGCTCTATTATGGGCTCCTGGAACrrCTGTCGGA~CTGGTCTTAGCAAGGAGCAACGGGTGGAGGCGAT~CACTTGCCTACGCCACCAAACAATCAGTCCTCGGC~AATTGG3360 I S S I M G F L E L L S D S G L S K E Q R V E A I S LAY AT K Q S V L G L I G 766 TGAAATCCTTGATGTCGACAAAATTGAATCGGGTAACTATCAACTTCAACCACAATGGGTCGATATCCCTAC~AGTCCAGAACAC~GTCACTC~CGGTGCGA~GCTGCAAGCAA3480 E I L DV D K I E S G N YQ L QP QWV D I PT L V QNT C H S F GA I AA S K806 ATCGATCGCATTAAGTTGCAGCAGTACGmCCTGAACA~ACCTGGTTAAGATCGACCCTCAGGCG~AAGCAGGTC~ATCAAA~GCTGAGTAATGCTCTCAAA~TACCACCGA3600 SIALSCSSTFPEHYLVKIDPQAFKQVLSNLLSN*ALKFTTE846 GGGGGCAGTAAAAATTACGACCTCCCTGGGTCACATTGATGACAACCACGCTGTTATCAAAATGACGATTATGGA~CTGGAAGTGGA~A~GCAGGAAGAACAACAACAACTGTTTAA3720 GAVKITTSLGHIDDNHAVIKMTIMD*SG*SG*LLQEEQQQLFK886 ACGCTACAGCCAAACAAGTGCAGGTCGTCAGCAAACAGGTTGTCATTAGAAAGTCATCCAGGCAT3840 RYSQTSAGRQQTGSG*LG*LMICKELIKNMQGDLSLESHPGI926 AGGAACAACAT~ACGATCACAATCCCGGTAGAAATTAGCCAGCAAGTGGCGACTGTCGAGGCAAAAGCAGAACAACCCATCACACTACCTGAAAAGTTGAGCATA~AATCGCGGA3960 G T T F T I T I P V E I S QQV A T V E A K A E Q P IT L P E K L S I L I ADD 966 TCATCCGACCAACAGGCTATACTCAAACGCCAGCTAAATCTA~AGGATATGATGTTGATGAAGCCACTGATGGTGTGCAAGCGCTACACAAAGTCAGTATGCAACATTACGATCTGCT4080 HPTNRLLLKRQLNLLGYDVDEATDGVQALHKVSMQHYDLL1006 TATTACTGACGTTAATATGCCGAATATGGATGGTmGAGrrACCCATCTGGGGGCTTACAGCCAACGCACAGGCTAACGAACATGA4200 ITDVNMPNMDGFELTRKLREQNSSLPIWGLTANAQANEHElO46 AAAAGGGTTAAGTTGTGGCATGAACTTATGTTTGTTCAAACCG~GACCCTGGATGTACTGAAAACACAT~AAGTCAG~GTACCAAG~GCGCATATTGCACCTCAGTATCGCCACCT4320 KGLSCGMNLCLFKPLTLDVLKTHLSQLYQVAHIAPQYRHL1086 TGATATCGAAGCCCTGAAAAATAATACGGCGAACGATCTACAACTGATGCAGGAGA~CTCATGAC~TCCAGCATGAAACGCATAAAGATCTACCCGCTGCG~CAAGCACTAGAAGC4~0 DIEALKNNTANDLQLMQEILMTFQHETHKDLPAAFQALEA1126 TGGCGATAACAGAACTTTCCATCAGTGTATTCATCGCATCCACGGTGCGGCTAACATCCTGAAmGCAAAAGTTGA~AATATTAGCCATCAGTTAGAAATAACACCTGTTTCAGATGA4~0 GDNRTFHQCIHRIHGAANILNLQKLINISHQLEITPVSDD1166 CAGTAAGCCTGAAATTCTTCAGTTGCTGAACTCTGTAAAAGAGCACATTGCAGAGCTGGACCAGGAGA~GCTG~~CTGTCAGAAAAATGACTAAATAGCGGCTCCCACAATGmAA4680 SKPEILQLLNSVKEHIAELD Q E I A V F C Q K N D ***1197 ATGTGGGAGCTATTTACCAGCACATCTTACATTTATGATGAGAACTCCTGGCGAA~G~CCCCATGCTGGTCTGATGTTATGCTCCTGGCATAACATTGGGTCCGTGCAGCCGCTGAT4800 mTATCGGATTACCCGGCATCATGATTCCCCCGGC~CGATCAACATATTTCTCGCTTCAGmGTCCCAATTTAATGGCAA~TAATGGCCTCAGCCACACTTAATAACGGCGCGACGGGTACACCAAC4920 TTCATGTATTCTGGCTAACCAAACTTCAGCTGCCTCCCTTGTACGCGTAAGATATTGCTGCTAAATCGGGGATCATT5040 AACCAGTTCCGTAAGCTCCAGTGCCTGGCATAACGCAGAAAAA

76 plasmid

pSKOOl-encoded

proteins

were

labelled

with

[35S]methionine, using in vitro-coupled translation system supplied by Promega

transcription (Madison, WI,

USA) and analyzed

polyacrylamide

gradient

using a SDS- lo-20%

gel (Daiichikagaku,

Japan).

As deduced

from

evgA and evgS nt sequences, 23-kDa and 135-kDa protein bands were visualized by autoradiography with Fuji RX

ompC expression environmental RU1012

be controlled independent

et al., 1991).

pertussis

It is well known is repressed

that the virulence

by nicotinic

acid, MgSO,,

gene of B. pevtussis or low temperature

(Miller et al., 1989). The effects of such environmental factors on the ompC expression mediated through the EvgA and EvgS system were investigated (Fig. 2). When the proteins were expressed from a multicopy plasmid, pSK001, in an envZ deletion strain, RU1012, in which lacZ+ was placesd under the ompC promoter, ompC expression was found to be regulated by nicotinic acid, MgSO,, or low temperature. In K1108 (an ompR mutant of RU1012) (Utsumi et al., 1989) containing pSK001, 500

and haemolysin,

of the Bordetella jexneri,

400

two-component

the

of epithelial suggest

regulatory which

are

cells (Bernardini

that EvgA and EvgS system similar

to the

system.

(d) Conclusions ( 1) We have cloned and sequenced two E. coli K-12 genes involved in signal transduction. The 5083-nt sequences encoding ORFs of 204 aa and 1197 aa were determined. They were mapped at 51 min on the E. coli search revealed that the two ORFs to hvgA and bugs, respectively. The predicted from their nt sequences were an in vitro transcription-translation

system. (3) When these two ORFs were expressed from a multicopy plasmid in an envZ deletion strain, ompC ex-

300

F I

pression was regulated by nicotinic acid, MgSO, or low temperature, the factors that control the virulence of B.

200

B 5 f a Q

the vir genes,

can work as a signal-transduction BvgA-BvgS

along

species (Gross et al., 1989). two-component

controls

for invasion

protein products detected, using

5

3 0

the

are scattered

proteins, adenylate

chromosome.

=

r" +

which

et al., 1990). These results

BvgA and BvgS trans-

haemagglutinin,

In

responsible

In fact, such a cross-

outer membrane

filamentous

OmpR-EnvZ

via the Evg

with EvgA and EvgS,

and EnvZ.

chromosome system

in

could

with Bvg system and E. coli OmpR

many genes encoding toxin,

such

that

the ompC expression

and a by-pass

of OmpR

Shigella

without

suggest

in two ways: by a cross-talk

(2) A computer were homologous 3

data

(Gross et al., 1993). Furthermore,

cyclase ompC expression via EvgA and EvgS

repressed

These

pSK001,

talk can be observed activates

factors on regulation of the

containing

system and OmpR

film. These molecular masses of EvgA and EvgS were identical with BvgA and BvgS (Arico et al., 1991; Stibitz

(c) Effect of environmental

was significantly signals.

pertussis via BvgA and BvgS. 100

C

ACKNOWLEDGEMENTS Fig. 2. Effect of nicotinic

acid, MgSO,

and temperature

on ompC ex-

pression. After RU1012[pSK001] (A), K1108[pSKOOl] (B), RU1012 [pAT428] (C), and RU1012[pUC19] (D) were cultured exponentially in a modified Davis minimal medium at 37-C (filled bars), 28 C (hatched bars), and in the presence of 10 mM MgSO, (stippled bars) or 10 mM nicotinic acid (open bars), flGa1 assays were performed (units according to Miller, 1972). Modified Davis minimal medium contains: 7 g K,HP0,/2 g KH,PO,/I g (NH,),HPO,/O.l g MgSOJO.5 g Na,.citrate/4 g glucose per liter. plasmid pAT428 (Mizuno et al., 1982) is a derivative of pBR322 containing ornpR+ and ent’Z+. Plasmid was constructed in this study. pSKOO1 containing e~gA+ and rqS+ RU1012 (Utsumi et al., 1989) is the AerzoZ::KmR derivative of MH225, which in turn is a derivative of MC4100 containing (ompClacZt)10~25[pl(209)] (Hall et al.. 1979). K1108 (Utsumi et al.. 1989) is an ompR mutant of RU1012.

We thank Dr. Y. Kohara for providing the ordered gene bank of E. coli and for his critical reading of the manuscript as well as Dr. A. Ishihama and Dr. A. Noda for providing the gene mapping membrane and Dr. T. Mizuno for his helpful discussion and providing us with E. coli AT141. This work was supported by the Naito Foundation, the Kato Memorial Bioscience Foundation, and a Grant-in-Aid for Scientific Research from the Environmental Science Research Institute, Kinki University and the Ministry of Education, Science and Culture, Japan.

77 Mizuno, T., Wurtzel, E.T. and Inouye, M.: Cloning of the regulatory genes (ompR and enuZ) for the matrix proteins of Escherirhia co/i

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