The rpoH gene encoding σ32 homolog of Vibrio cholerae

Gene 189 (1997) 203–207

The rpoH gene encoding s32 homolog of Vibrio cholerae Gautam Kumar Sahu, Rukhsana Chowdhury, Jyotirmoy Das * Biophysics Division, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India Received 7 August 1996; received in revised form 4 November 1996; accepted 4 November 1996; Received by J. Wild

Abstract The Vibrio cholerae rpoH gene coding for the heat-shock sigma factor, s32, has been cloned and shown to functionally complement Escherichia coli rpoH mutants. The nt sequence of the gene has been determined and the deduced aa sequence is more than 80% homologous to the E. coli rpoH gene product. Downstream of the V. cholerae rpoH gene, an unidentified dehydrogenase gene (udhA) is present on the opposite strand facing rpoH. The predicted secondary structure of the 5∞-proximal region of V. cholerae rpoH mRNA is apparently different from the conserved secondary structures of the rpoH mRNA reported for several bacterial species. The ‘RpoH box’, a stretch of 9 aa (QRKLFFNLR) unique to s32 factors, and the ‘downstream box’ sequence complementary to a part of the 16S rRNA, have been detected. © 1997 Elsevier Science B.V. Keywords: Heat shock response; Sigma factor; RpoH box; mRNA secondary structure; Bidirectional transcription terminator; udhA gene

1. Introduction In Vibrio cholerae, a Gram− non-invasive enteric bacterium and the causative agent of the diarrheal disease cholera, the expression of several virulence genes including the cholera toxin gene (ctx) can be modulated by growth temperature and other environmental factors (Miller and Mekalanos, 1988; Parsot and Mekalanos, 1991). The toxR gene of V. cholerae, encoding the central transcriptional regulator of a number of virulence genes is expressed at significantly reduced levels at elevated temperatures (Parsot and Mekalanos, 1991). It has been suggested that the induction of the heat shock (HS) response and consequent increase in the divergent transcription of a htpG-like HS gene located immediately upstream of the toxR, leads to a proportionate decrease * Corresponding author. Tel. +91 33 4730350; Fax +91 33 4730350/4730284; e-mail: iicb%sirnetc@sirnetd ernet.in Abbreviations: aa, amino acid(s); Ap, ampicillin; bp, base pair(s); CT, cholera toxin; ctx, gene encoding CT; HS, heat shock; HSP, HS protein; kb, kilobase(s) or 1000 bp; Km, kanamycin; LB agar, Luria Bertani medium containing 1.5% agar; livJ, gene for leucine, isoleucine, valine binding protein; MCS, multiple cloning site(s); nt, nucleotide(s); ORF, open reading frame; re, recombinant; rRNA, ribosomal RNA; rpoH, gene encoding s32; SD, Shine Dalgarno (sequence); ts, temperature sensitive; udhA, gene coding for an unidentified dehydrogenase; V, Vibrio. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 49 - 9

in the expression of toxR (Parsot and Mekalanos, 1991). The consensus sequence for E. coli HS promoters recognised by RNA polymerase containing s32, the positive regulator of HS, was detected upstream of the V. cholerae htpG-like gene. Expression of this gene is induced in E. coli by elevation of growth temperature and also by increase in the level of the E. coli rpoH gene product, s32, at a given temperature (Parsot and Mekalanos, 1991). Moreover, E. coli s32 protein can induce synthesis of V. cholerae HS proteins (HSPs) following introduction of the E. coli rpoH gene, in a multicopy plasmid into V. cholerae. These observations suggests that a s32-like factor may be responsible for the induction of HSPs in V. cholerae (Sahu et al., 1994). The HS response has been characterised in this organism and 16 HSPs which are induced in response to small and large elevations of temperature have been identified in the hypertoxinogenic strain 569B (Sahu et al., 1994). The maximum induction of HSPs occurs at about 20 min after temperature-upshift in V. cholerae, in contrast to only 5 min in E. coli and some other organisms (Bukau, 1993; Yura et al., 1993; Sahu et al., 1994). To investigate the regulation of HS response as well as to examine the role of s32 in virulence of V. cholerae, the cloning of rpoH gene was necessary. The present report suggests that the predicted mRNA secondary structure of the rpoH gene of V. cholerae has some difference

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from that reported for E. coli and several other bacterial species.

2. Experimental 2.1. Cloning of the rpoH gene Chromosomal DNA of the hypertoxinogenic strain 569B was digested with the enzymes EcoRI or HindIII, Southern blotted and hybridized with labeled 0.9-kb HindIII-PvuII fragment of plasmid pKV7 carrying the E. coli rpoH gene ( Zhou et al., 1988) at 60°C for 16 h. The E. coli rpoH gene hybridized with EcoRI and HindIII-digested V. cholerae DNA in the 5.5- and 3.5-kb regions, respectively. The DNA fragments in the 5.5-kb region of EcoRI digested V. cholerae DNA were eluted from the gel, further digested with HindIII and hybridized with the same probe as above. The probe hybridized with HindIII digested 5.5-kb EcoRI DNA fragments in the 3.5-kb region. Using DNA fragments of this region, a mini bank was constructed in plasmid pACYC177, and the library was maintained in the E. coli strain XL1-Blue. KmSApR colonies were selected as clones harboring the recombinant (re) plasmids. This procedure allowed an enrichment of the rpoH gene-containing fragments in the library. The re plasmids were screened by Southern blot hybridization using E. coli rpoH gene as probe. Out of only 50 re plasmids screened, two hybridized with the probe. Both plasmids carried a 3.5-kb HindIII fragment. The plasmids were individually transferred to the E. coli rpoH mutant strain, K165 [rpoH(am) trp(am) lac(am) mal(am) pho(am) supC(ts) strC ] (Cooper and Ruettinger, 1975). Both re plasmids could suppress ts phenotype of the E. coli rpoH mutant. One of them was selected for further study and was designated pGS350 (Fig. 1). The 3.5-kb insert from plasmid pGS350 was digested with HincII producing 1.5- and 2-kb fragments. These two fragments were separately ligated to HindIII+PvuII digested pBR322 and transformed into E. coli strain K165. The plasmid carrying the 1.5-kb DNA fragment alone could suppress the ts phenotype of the E. coli rpoH mutant strain, K165. The re plasmid carrying the V. cholerae 1.5-kb DNA fragment will be referred to as pGS150 ( Fig. 1). A restriction map of the 1.5-kb DNA fragment was constructed using several restriction enzymes (Fig. 1). 2.2. The nt sequence of the rpoH gene The complete nt sequence of the 1.2-kb HindIII-XmnI fragment ( Fig. 1) was determined (Fig. 2). The sequence contained an ORF of 858 nt coding for a protein of 286 aa located within the 0.9-kb HindIII-PstI fragment

Fig. 1. Construction of re plasmids carrying the rpoH gene of V. cholerae. The thin lines represent vector DNA and thick lines, chromosomal inserts. The solid bar represents the 1.5-kb fragment carrying rpoH of which the 0.9-kb HindIII-PstI fragment containing coding region was cloned into pUC19 to give the plasmid pGS90. The plasmid pGS90 was used to construct a library of ordered deletion clones using the Nested deletion kit (Pharmacia Biotech, UK ). The MCS in the re plasmid pGS90 is indicated. A, AccI; B, BamHI; C, ClaI; E, EcoRI; Ev, EcoRV; H, HindIII; Hn, HincII; P, PstI; Pv, PvuII; S, SacI; Sm, SmaI; X, XmnI;

( Fig. 1). This ORF has more than 80% similarity at the aa level with E. coli rpoH gene product ( Yura et al., 1984). The 0.9-kb HindIII-PstI fragment was cloned into plasmid pUC19 and the re plasmid designated pGS90 ( Fig. 1) could complement the ts phenotype of the E. coli rpoH mutant. A consensus SD sequence, GAGGA is present 5 bp upstream of the initiation codon. The aa sequence deduced from the nt sequence showed a stretch of 9 aa ‘QRKLFFNLR’ (residues 132–140, Fig. 2) identical to the ‘RpoH box’ reported for s32 homologs of other bacterial species (Nakahigashi et al., 1995). This box is highly conserved in all s32

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Fig. 2. Nucleotide and deduced aa sequences of the rpoH gene of V. cholerae (GenBank accession No. U44432). A putative SD sequence and downstream box (DS-Box) are indicated. The translation stop codons are underlined with asterisks under the sequence. DNA binding regions 2.4 (aa 109–131) and 4.2 (aa 251–280) are underlined. The ‘RpoH box’ and a putative bidirectional transcriptional terminator are represented by shaded and open boxes, respectively. The arrow indicates the direction of transcription of the udhA gene. HindIII and PstI sites spanning the ORF are shown. Dideoxy sequencing was performed on a set of ordered deletion clones using Sequenase version 2.0 ( US Biochemical, Cleveland, OH, USA)

homologs but is not present in other s factors. It has been postulated that this region may be involved in the DnaK-DnaJ mediated translational control and in imparting instability to s32 (Nagai et al., 1994; Nakahigashi et al., 1995). The DNA-binding domains in the regions 2.4 and 4.2 were detected (Fig. 2) which are thought to be involved in the binding of RNA polymerase holoenzyme ( Es32) to −10 and −35 regions of HS promoters (Lonetto et al., 1992). Downstream of the termination codon, an inverted repeat sequence starting with AAAs and ending with TTTs which has been found to be present in putative bidirectional transcription terminators (Postle and Good, 1985), was detected. The presence of such a putative bidirectional terminator downstream of the rpoH coding sequence ( Fig. 2) suggests the possibility of the presence of an ORF in the opposite strand facing the rpoH gene. Consistent with this idea, computer analysis of the nt sequence revealed a region on the opposite strand coding for 46 aa which has more than 90% similarity to the C-terminus of the E.coli udhA gene product (Blattner et al., 1993). Thus, downstream of the V. cholerae rpoH gene, the udhA gene is present on

the opposite strand facing towards rpoH (Fig. 2). It may be noted that the rpoH gene is followed by the livJ gene on the same strand both in E. coli and Citrobacter freundii (Landick et al., 1984; Garvin and Hardies, 1989). 2.3. Analysis of rpoH mRNA secondary structure The secondary structure of the 5∞-proximal region of the rpoH mRNA has been predicted for several Gram− organisms and shown to exhibit some apparently conserved features (Nakahigashi et al., 1995). The primary feature of the rpoH mRNA secondary structure at low temperatures is the sequestering of the initiation codon and downstream box by base pairing which may restrict ribosome entry leading to the repression of translation ( Yuzawa et al., 1993). Using the computer program used to analyze the mRNA secondary structures for other organisms, the mRNA secondary structure of the 5∞-proximal portion (nt −20 to +214) of the V. cholerae rpoH coding sequence has been predicted ( Fig. 3). The structure is apparently different from that reported for other organisms. In E. coli, there is various

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Fig. 3. Predicted secondary structure of the 5∞-region of rpoH mRNA. The 5∞-region of rpoH mRNA (nt −20 to +214) was analysed by MFOLD (Jaeger et al., 1990) and the resulting secondary structure with minimum free energy was visualised by LOOPVIEWER (Gilbert, 1990). The start codon (ATG) and the SD sequence are boxed. The insert shows a putative ‘downstream box’ (DS-Box) in the rpoH sequence (capital letters) complementary to a part of the 16S rRNA (small letters) of E. coli and V. cholerae. The position of the DS-Box in the rpoH mRNA secondary structure is indicated. $, G-T pairs.

evidence to suggest that the rpoH mRNA secondary structure may play a role in the thermoregulation of its translation ( Yuzawa et al., 1993; Nagai et al., 1994). In the predicted secondary structure of V. cholerae rpoH mRNA, the downstream box (nt 6–20) complementary to a part of the 16S rRNA (Fig. 3, Kita-Tsukamoto et al., 1993) forms basepairing and probably is not available for interaction with ribosomes. Unlike in rpoH mRNA structures of several other bacteria reported, the ATG initiation codon is free in the secondary structure of V. cholerae rpoH mRNA (Fig. 3). However, the upstream region including the SD sequence forms a

stem. These features of V. cholerae rpoH mRNA secondary structure suggest that, although the predicted structure is different from that of other bacterial species, the model proposed for mRNA secondary structure-based translational repression in other organisms may still be valid in V. cholerae. However, the possibility of an alternative mechanism for the induction of HS response in V. cholerae can not be ruled out in view of the fact that the maximum induction of HSPs occurs 20 min after temperature up-shift as opposed to 5 min for other organisms (Bukau, 1993; Yura et al., 1993; Sahu et al., 1994).

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3. Discussion The present report describes the cloning, sequencing and analysis of the predicted mRNA secondary structure of the rpoH gene of V. cholerae. The deduced aa sequence showed more than 80% similarity with the E. coli rpoH gene product. While in E. coli the rpoH gene is followed by the livJ gene on the same strand, the V. cholerae gene is followed by the udhA gene on the opposite strand. In contrast to 5 min in E. coli, the maximum induction of HSPs occurs about 20 min after temperature-upshift in V. cholerae (Bukau, 1993; Yura et al., 1993; Sahu et al., 1994). The rapid induction of HS response in E. coli is primarily due to an increase in the rate of translation of the rpoH mRNA as well as increased stability of the s32 at elevated temperatures ( Yura et al., 1993). It has been postulated that the secondary structure of rpoH mRNA is involved in the translational repression during steady-state growth and disruption of the structure upon HS leads to an increase in rpoH mRNA translation ( Yuzawa et al., 1993). The rpoH genes from several bacterial species have been cloned and the predicted mRNA secondary structures have been shown to be significantly similar, indicating that the regulatory mechanism of the HS response may be conserved (Nakahigashi et al., 1995). Although the significance of the observed difference in the secondary structure of rpoH mRNA of V. cholerae remains to be elucidated, it might reflect an alternative mechanism for regulation of HS response in V. cholerae, especially in view of the fact that the maximum induction of HSPs occurs 20 min after temperature up-shift as opposed to 5 min for other organisms (Sahu et al., 1994).

Acknowledgement We are grateful to Prof. Takashi Yura, HSP Research Institute, Kyoto, Japan, for analyzing the V. cholerae rpoH mRNA secondary structure and for the gift of the plasmid pKV7. We also thank Prof. Frederick C. Neidhardt, Department of Microbiology and Immunology, University of Michigan, USA for E. coli K165 strain. This work was supported by the Department of Biotechnology (Grant No. BT/TF/15/03/91), Government of India, to J.D. G.K.S. is grateful to Council of Scientific and Industrial Research for a predoctoral fellowship.

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