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We previously reported that a region of the Escherichia coli chromosome at 18 min increased E E activity when cloned in multicopy (J. Mecsas, P. E. Rouviere, ...
JOURNAL OF BACTERIOLOGY, Feb. 1995, p. 799–804 0021-9193/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 177, No. 3

Identification and Characterization of an Outer Membrane Protein, OmpX, in Escherichia coli That Is Homologous to a Family of Outer Membrane Proteins Including Ail of Yersinia enterocolitica JOAN MECSAS,1† RODNEY WELCH,2 JIM W. ERICKSON,1‡ 1

AND

CAROL A. GROSS1*

2

Department of Bacteriology and Department of Medical Microbiology, University of Wisconsin— Madison, Madison, Wisconsin 53706 Received 20 April 1994/Accepted 15 November 1994

We previously reported that a region of the Escherichia coli chromosome at 18 min increased EsE activity when cloned in multicopy (J. Mecsas, P. E. Rouviere, J. W. Erickson, T. J. Donohue, and C. A. Gross, Genes Dev. 7:2618–2628, 1993). In the present report, we identify and characterize the gene responsible for the increase in EsE activity. This gene is in a monocistronic operon with two promoters and a rho-independent terminator. Sequence analysis of this gene indicated that it encodes an outer membrane protein which is 83% identical to OmpX in Enterobacter cloacae, leading us to name this gene ompX. There are four other proteins that are homologous to OmpX. Several of these proteins, Ail of Yersinia enterocolitica and Rck and PagC of Salmonella typhimurium, have properties that allow bacteria to adhere to mammalian cells, survive exposure to human serum, and/or survive within macrophages. We therefore characterized strains deleted for ompX for their growth phenotypes, EsE activity, serum resistance, and adherence to mammalian cells. No differences in growth rates, serum resistance, or adherence to mammalian cells were observed; however, EsE activity was dependent on expression of OmpX in certain strain backgrounds. The outer membrane (OM) of gram-negative bacteria contains a number of proteins. The functions of these OM proteins (OMPs) include forming channels allowing passive diffusion of water-soluble moieties (23), carrying out enzymatic functions such as by acting as proteases (8), and serving as protein ushers involved in assembly of complex structures protruding from the OM such as pili (13). Specific OMPs are often highly regulated in response to growth, nutrient, and environmental conditions (22). sE is one of two alternative sigma factors in Escherichia coli whose activity is induced by heat and ethanol (6, 33). Thus far, RNA polymerase containing sE (EsE) is known to recognize two promoters: the promoter of the degP (htrA) gene, which encodes a periplasmic protease; and the P3 promoter of rpoH, the gene encoding s32, the other heat- and ethanol-inducible sigma factor (6). Recently, OMP production was shown to affect the activity of EsE (19). When OmpC, OmpF, and OmpT, as well as a previously unidentified OMP, encoded by a gene located at 18 min on the E. coli chromosome were overproduced, EsE activity increased (19). In this report, we characterize the gene encoding this OMP. We find that this OMP belongs to a family of recently discovered OMPs encoded by bacteria and phage.

This screen identified genes that, when present on a multicopy plasmid, increased expression of EsE activity at the rpoHP3 and degP promoters. A plasmid library of E. coli DNA partially digested with Sau3A was cloned into the unique BamHI site of pBR322 (gift of P. Kung and E. A. Craig). pJE100 contained a 4-kb insert which has subsequently been shown to contain the dps gene (1) in addition to ompX. Subcloning this region into plasmid pBR322 (New England Biolabs, Woburn, Mass.), pGEM-7Zf (Promega, Madison, Wis.), or M13mp18 and M13mp19 (New England Biolabs) was done by standard methods (17). For physiology experiments, pJE100 and JE102 were used because cells with these plasmids were healthier than cells with the smaller plasmid pJE105. We deleted the ompX coding sequences and replaced them with an insertion carrying a gene conferring resistance to chloramphenicol, using a three-step cloning strategy. First, the 0.5-kb PstI-SphI fragment from pJM13 was cloned into pUC19, resulting in pJM16. Next, a 0.5-kb DNA fragment containing sequences immediately upstream of ompX and encoding the first 14 amino acids of the signal peptide was amplified from pJM12, using the universal M13 primer and OmpX377-356/367A. This primer has a 1-bp mismatch within ompX gene sequences which creates a new PstI site. The amplified DNA fragment was cleaved with PstI and XbaI and cloned into pJM16. The resulting plasmid, pJM18, had a 370-bp deletion within ompX with a unique PstI site in the ompX sequences and 400 bases of E. coli DNA flanking DompX. A gene conferring chloramphenicol resistance (Camr) with transcriptional and translational stop sequences at each end from pHP45V-Cm (7, 26) was cloned into the unique PstI site, generating pJM19. The synthetic oligodeoxynucleotides made for this study (by Genosys, Woodlands, Tex.) were OmpX201-217, OmpX373-389, OmpX547-563, OmpX224-207, OmpX377-356/367A, OmpX533-516, and OmpX997-983. They are numbered according to the DNA sequence in Fig. 1. Oligonucleotides numbered from increasing to decreasing order are homologous to the noncoding strand. DNA sequencing. Single-stranded DNA templates were used for sequencing with a Sequenase kit (U.S. Biochemical Corp., Cleveland, Ohio) as instructed by the manufacturer, using the dideoxynucleotide chain termination method. Ambiguous regions were sequenced by using Sequitherm (Epicentre Technologies, Madison, Wis.) and pJM12 or pJM13 as a template as instructed by the manufacturer. Both strands were sequenced, and the data were analyzed by using Genetics Computer Group programs (5). Both GenBank and EMBL databases were searched for homologous amino acid sequences by computer, using the program TFASTA. RNA synthesis, purification, and mapping. In vivo RNA isolation, purification, and S1 mapping were performed as described previously (19). The probes used for determining the operon structure of ompX were generated by cleavage of pJE100 with PstI and labeled at either the 59 or 39 end with T4 polynucleotide kinase or Klenow fragment (New England Biolabs), respectively. For mapping the start site of transcription of ompX, DNA probes were generated by 59 end labeling OmpX377-356/367A and OmpX533-516 and then using them in PCRs with M13 universal primer, Tfl (Epicentre Technologies) (a thermostable DNA

MATERIALS AND METHODS Media, strains, and plasmids. All media were prepared as described by Miller (19a). Strains and plasmids are listed in Table 1. Plasmid pJE100, encoding OmpX, was initially isolated in a screen for positive regulators of EsE activity (5a, 19).

* Corresponding author. Present address: Department of Stomatology, University of California, San Francisco, San Francisco, CA 94143. Phone: (415) 476-1493. Fax: (415) 476-4204. † Present address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5402. ‡ Present address: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720. 799

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TABLE 1. Strains and plasmids used Strain or plasmid

Phenotype

E. coli strains MC1061a araD139 D(ara, leu)7697 DlacX74 galU galK hsr hsm strA MG1655 Prototrophic E. coli K-12 PSM2 K-12, thi metC glnPo J98 JC7623 recB21 recC22 sbcB15 arg ara his leu pro thr Plasmids pJE100 4-kb insert of partial E. coli Sau3A digest in BamHI site in pBR322 pJE101 1.0-kb DEcoRV-EcoRV of pJE100 pJE102 1.3-kb DNcoI-NcoI of pJE100 pJE103 2.5-kb DEcoRV-NcoI of pJE100 pJE104 1.8-kb DEcoRV-NsiI of pJE100 pJE105 2.7-kb DEcoRV-XbaI of pJE100 pJE106 2.5-kb DNsiI-SalI of pJE100 pJE107 1.5-kb DXbaI-SalI of pJE100 pJM12 0.8-kb XbaI-PstI fragment of pJE105 in pGEM-7Zf pJM13 0.5-kb PstI-SphI fragment of pJE105 in pGEM-7Zf pJM16 0.5-kb PstI-SphI fragment of pJE105 in pUC19 pJM18 0.5-kb XbaI-PstI PCR amplified with M13 universal primer and OmpX377-356/ 367A of pJM12 in pJM16 pJM19 Camr gene of pHP45VCM in PstI site of pJM18 pHP45 Camr gene surrounded by transcriptionVCM translation terminators pVM102 Encodes Ail a b

Reference or source

4 CGSGb 18 12 34 P. Kung This This This This This This This This

study study study study study study study study

This study This study This study This study 7 3

E. coli K-12 except for the ara regions. CGSC, E. coli Genetic Stock Center, Yale University.

polymerase), and pJM12. The sizes of the S1 mapped products were compared against a sequencing ladder run in parallel lanes generated by cycle sequencing with Sequitherm, the OmpX377-356/367A or OmpX533-516 primer, and a pJM12 DNA template. pHtrA (14) was used as a source of probe for determining the effect of OmpX expression on degP transcription. In vitro transcription reactions were carried out on linear DNA templates prepared by using PCR with Tfl polymerase, pJM12 as a template, M13 universal primer, and OmpX533-516. PCR products were gel isolated and purified by using Gene Clean. Transcription reactions were carried out as previously described (6) in a variety of salt concentrations ranging from 20 to 100 mM NaCl or 30 mM KCl. Construction of an DompX strain. The strategy used to generate a chromosomal deletion of ompX involved homologous recombination of a linearized DNA fragment from pJM19 (containing DompX::Camr) electroporated into JC7623 (recBC sbcB), a strain which cannot rapidly degrade linear DNA. Camr colonies were selected and then screened for loss of the ompX gene sequences by assaying the sizes of fragments amplified from chromosomal DNA by using PCR. To determine if any major chromosomal rearrangements had occurred after introduction of the Camr marker, the linkage between the Camr marker and two Tn10 insertions located on either side of ompX was tested by P1 mapping (29). Briefly, P1 phage were grown on strain CAG18493 and CAG12034, which contain Tn10 insertions and either zbh-29 at 17.75 min (CAG18493) or zbi-3058 at 18.75 min (CAG12034). The resulting P1 lysates were then used to infect the JC7623 Camr strain. Tetracycline-resistant (Tetr) colonies were selected and then screened for Camr. In both cases, the expected linkage was obtained, which indicated that no major chromosomal arrangements had occurred. To confirm that ompX was not an essential gene, P1 phage was grown on a JC7623 Camr strain containing the Tn10::zhb-29 insertion to test whether the deletion of ompX was easily transferable to other strains, including MC1061 (4), MG1655 (E. coli Genetic Stock Center, Yale University), PSM2 (4), J96 (12), and J198 (12). Strains infected with this lysate were first selected for Tetr and then screened for Camr. In all cases but one, 40 to 60% of the Tetr cells were also converted to Camr, indicating that ompX is not essential. In J198, a wild-type isolate, the two resistance genes were not cotransducible. However, Camr colonies were obtained when selected for directly. PCR analysis indicated that ompX coding sequences were missing. Thus, these loci are no longer linked in J198.

Complement resistance and cell adherence assays. Bacterial resistance of complement killing was determined essentially by the method of Bliska and Falkow (3). Three-milliliter overnight cultures in LB were harvested by centrifugation, washed twice in saline with 5 mM MgCl2 (SM), and resuspended in 1 ml of SM. Aliquots of the bacterial suspension were mixed with equal volumes of normal human serum (NHS) or 20% dilutions of NHS. The cell-serum mixtures were incubated for 60 min at 378C. E. coli HB101 harboring plasmid pVM102 (3), the kind gift of Virginia Miller, was used as a positive control for Ailmediated complement resistance. Heat-inactivated NHS (568C, 30 min) was used as an addition control. The CFU present in the different mixtures were determined by making serial dilutions and plating them onto LB agar plates. The ability of E. coli to adhere to epithelial cells in the absence or overproduction of OmpX was determined. Isogenic strains of J96 or MC1061 that were either overexpressing or deleted for ompX were grown overnight in LB; 106 bacteria were spun onto HEp-2 cells that had been seeded into 24-well plates at a density of 105 cells per well. The mixture was incubated at 378C for 2 h, washed five times with phosphate-buffered saline, and then lysed with 1% Triton. To determine the number of bacteria that remained tightly associated with the HEp-2 cells, the CFU of bacteria in the wells were determined. Nucleotide sequence accession number. The ompX sequence has been assigned GenBank accession number L37088.

RESULTS Sequence of gene that induces EsE activity. We recently showed that multicopy plasmids containing DNA from the 18-min region of the E. coli chromosome increase EsE activity five- to eightfold (19). To characterize the gene causing this phenotype, the DNA was subcloned on the basis of its ability to increase EsE activity (Fig. 1). The gene was localized to a 1.3-kb XbaI-to-Sau3A fragment (pJE105). Further subclones of pJE105 containing either a 0.8-kb XbaI-PstI fragment (pJM12) or 0.5-kb PstI-SalI fragment (pJM13) failed to stimulate EsE activity. The sequence of the gene (Fig. 2) and the predicted protein sequence both showed strong similarity to a family of OMPs that have recently been identified in gramnegative bacteria (Table 2). In fact, this gene was 83% identical at the protein level to the Enterobactor cloacae ompX member of this family (30). Hence, we have called the E. coli gene ompX. The predicted amino acid sequence of the protein indicates that it possesses a signal sequence at its N terminus and that the molecular masses of the unprocessed and processed proteins are 18.6 and 16.3 kDa, respectively. In other work (19), we have shown that ompX is localized to the OM. Operon structure. S1 mapping analysis of in vivo-synthesized RNA indicated two transcription start sites located approximately 220 (P1) and 50 (P2) bases upstream of the start codon at position 336 (Fig. 3A). The protein sequence indicated that there are three overlapping open reading frames of 15 to 22 amino acids starting with AUG in the region between the promoters and the start of ompX; however, they have poor Shine-Dalgarno sequences and are unlikely to be translated. One transcription stop site (T1) was located 30 bases downstream of the stop codon immediately following an inverted repeat (Fig. 3B). The level of ompX mRNA remained constant at 30 and 428C. Thus, ompX is probably in a monocistronic operon with two promoters and a rho-independent terminator. To further define the promoters, S1 mapping was repeated with shorter probes and run in parallel with sequencing reactions (Fig. 4). Transcription initiates from promoter P1 at nucleotides 95 to 96, located 240 nucleotides upstream of the translation start. Transcription from P2 initiates at nucleotides 303 to 304, located 32 nucleotides upstream of the translation start site (Fig. 2). To determine if Es70 recognizes these promoters, in vitro transcription assays were carried out with Es70 on linear DNA templates containing the promoter region of ompX. The transcripts were then mapped with S1 nuclease to see if their sizes correlated with those of transcripts detected in vivo (Fig. 4). We detected a transcript initiated by Es70 at P2 under conditions of low salt (either 20 mM NaCl or 30 mM

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FIG. 1. Physical map of the E. coli chromosome at 18 min and the regions of E. coli DNA that increase EsE activity when cloned in multicopy. Abbreviations for restriction enzymes: P2, PvuII; RV, EcoRV; Nc, NciI; X, XbaI; Ps, PstI.

KCl) but not high salt (data not shown). This result indicates that P2 is a bona fide Es70 promoter in vivo and is not a degradation product of P1. No transcription at P1 with Es70 was detected under any conditions tested. Deletion phenotype. A search of the literature indicated that a 20-kb region encompassing glnP to moeBA had been deleted in several derivatives of the E. coli K-12 strain PSM2 (18, 24). The location of these markers made it likely that the deletion included ompX (Fig. 1). Using a variety of primers to the ompX gene in PCRs, we demonstrated that the ompX sequences were missing in these deletion strains but were present in the parental PSM2 strain (data not shown). This finding indicated that a DompX strain was probably viable; however, to study additional phenotypes of OmpX, we made a specific insertion/ deletion in ompX (see Materials and Methods). Since OmpX is a monocistronic operon, any phenotypes of the DompX strain are due to the lack of OmpX. Growth of the DompX strain was indistinguishable from that of the parental strain at either 30 or 428C, both in defined media and in LB medium. Since ompX was initially identified because overproducing OmpX protein increased EsE activity, we tested whether deletion of ompX reduced EsE activity. Surprisingly, we found that the effect of the ompX deletion was strain dependent. In PSM2, a deletion of ompX caused about a fourfold reduction in EsE activity at the degP promoter, while no difference was observed in MC1061 at either the degP promoter (Fig. 5) or rpoHP3 (data not shown). Transcription from an rpoHP3-galK (6) reporter plasmid in another K-12 strain, C600, was also unaffected by DompX (data not shown), suggesting that the phenotype in the PSM2 strain is most likely due to an addi-

tional alteration in that strain background. As predicted, overproduction of OmpX from a pBR322 plasmid increased EsE activity in both strain backgrounds (Fig. 5). Several of the proteins homologous to OmpX appear to have roles in virulence. Specifically, Ail of Yersinia enterocolitica and Rck of Salmonella typhimurium protect cells from exposure to serum (3, 9, 10), and Ail allows bacteria to adhere to epithelial cells (21). We tested whether expression of OmpX changes the ability of E. coli to survive exposure to serum in a virulence strain, J96, and in MG1655. Isogenic strains containing a multicopy plasmid encoding OmpX (pJE102) did not have increased resistance to either 10 or 50% NHS compared with the parental strains. In these same experiments, HB101 cells with a multicopy plasmid encoding Ail had a 104- to 106-fold increased survival in serum. Likewise, introduction of the DompX allele into these strains did not increase its sensitivity to exposure to either 10 or 50% NHS. The ability of OmpX to promote adherence to epithelial cells was also tested. Isogenic strains of J96 or MC1061 either overexpressing or deleted for ompX were incubated with HEp-2 cells. No differences were observed between the wildtype and DompX strains in the ability to adhere to HEp-2 cells, indicating that OmpX is not involved in adherence. The J96 strain overproducing OmpX had a slightly lower ability to adhere to or survive in epithelial cells than wild-type strain J96. This could be due to destabilization of the OM caused by overproduction of OmpX. Thus, we are unable to demonstrate any role for E. coli OmpX in serum survival or adherence to mammalian cells.

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FIG. 3. S1 mapping of in vivo-synthesized transcripts containing ompX. RNA was isolated from PSM2 at 308C and 10 min after a shift to 428C. A constant amount of RNA was used in each reaction and hybridized to a 59-end-labeled ompX probe (A) or 39-end-labeled ompX probe (B). The size of the protected DNA probe was compared against molecular weight markers from pBR322 cleaved with HpaII. Sizes are indicated in nucleotides.

FIG. 2. Nucleotide sequence of the ompX operon. Putative 235 and 210 regions of P2 are underlined twice. Transcription start sites are in boldface. The Shine-Dalgarno sequence is underlined and indicated by S.D. The DNA sequence of the open reading frame is divided into codons, with the corresponding amino acid indicated beneath each codon. The putative signal sequence is underlined with dashes, and site of cleavage is indicated by the /. The inverted repeat is underlined. Numbering on the left refers to nucleotide sequence; numbering on the right refers to amino acid sequence, with 11 as the first amino acid of the mature peptide as predicted from reference 30. The signal sequence starts at 223 (M) and ends at 21 (A). Bars above the sequence indicate positions of the oligonucleotides used in constructing the knockout (OmpX377-356/367A) and the runoff templates for transcription assays (OmpX356-377/367A and OmpX516-533). The T at position 367 represents a mismatch between the oligonucleotide and the wild-type sequence at this position. Dashed underlines indicate the native and engineered PstI sites at nucleotides 740 and 366, respectively.

DISCUSSION On the basis of the ability to induce EsE activity, we have identified an OMP, OmpX, that is highly homologous to a family of proteins recently identified in gram-negative bacteria and their associated phages (Table 2). OmpX is 25 to 83% identical with the five other members of this family. Stoorvogel et al. (30) have proposed a model for the topology of OmpX in

E. cloacae on the basis of the predictions from the KyteDoolittle hydropathy plot. This structure has eight membranespanning regions, four surface-exposed loops with 8 to 21 amino acids, and three periplasmic loops with five or fewer amino acids. Comparisons of the sequences between OmpX of E. coli and E. cloacae show that all of the changes between the two occur in regions predicted to be exposed to the cell surface or periplasm, with the exception of one conservative change of methionine to isoleucine at position 113 of the mature E. coli OmpX protein. The largest area of divergence occurs at the third cell surface-exposed loop from amino acids 95 to 102 of the mature peptide. In this region there are no identical residues between the two OmpXs. In addition, E. coli OmpX has two fewer amino acids in this loop than E. cloacae. A thorough sequence alignment of the first five members (ail, pagC, rck, lom, and ompX of E. cloacae) of this family has been carried out (10). A signature sequence of this family of proteins, identified as G[X]N[X]KYRYE (10, 28), is present in OmpX at amino acids 23 to 31. In addition, we noticed that with the exception of lom (the l-encoded member of this family), these proteins have in common the YAQS amino acids at positions 9 to 12. Expression of ail and rck has been shown to increase the ability of bacteria to survive exposure to serum (3, 9, 10, 25). Ail also allows bacteria to adhere to epithelial cells (21). pagC mutants are less able to survive within cultured macrophages and are 1,000-fold less virulent in mice (20). Our experiments indicate that the expression of OmpX does not protect E. coli from exposure to serum or enhance its ability to associate with epithelial cells. The largest areas of divergence between the OmpX proteins and the proteins with functions in virulence, Ail, Rck, and PagC, occur in the predicted surfaceexposed residues. In particular, the two OmpXs have gaps in the surface-exposed residues in regions 2 and 3. Possibly regions 2 and 3 are involved in repelling the action of complement and/or adhering to epithelial cells. Further experiments involving mutational analysis, as well as epitope swapping be-

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TABLE 2. Functions and percentages of identity of outer membrane proteins homologous to OmpX of E. coli Bacterium

E. coli E. cloacae Y. enterocolitica S. typhimurium S. typhimurium E. coli l phage a b

Gene

% Identitya

Function(s)

ompX ompX ail rckb pagC lom

83 40 37 32 24

Unknown Adhesion to mammalian cells, serum resistance Serum resistance Virulence survival in macrophages Expressed during lysogenic infection

Reference(s)

30, 31 3, 21, 25 8, 9 28 2

Protein identity. Plasmid encoded.

tween E. coli OmpX and these proteins, would test this hypothesis. Since PagC of S. typhimurium is only 35% identical to OmpX of E. coli, whereas many homologs between these two bacteria are 70 to 95% identical (15, 27), we speculate that PagC and OmpX have different functions. We observed that the ompX deletion affected EsE activity in one strain but not in others. In a previous report, we showed that EsE activity is modulated by varying levels of OMP produced (19). When OMPs were overproduced, EsE activity increased, whereas when OMPs were underproduced, EsE activity decreased. Furthermore, EsE activity was induced in a DdsbA strain (which leads to misfolded proteins in the periplasm and OM) and by expression of OmpCtd, an OmpC

FIG. 4. S1 mapping of in vivo and in vitro transcripts to determine the start site of promoters. In vivo or in vitro-synthesized Es70-containing RNA was hybridized to 59-end-labeled probes as indicated and then run in parallel with DNA sequencing reactions to determine the start site of transcription.

mutant that is secreted but poorly inserted in the OM. We proposed that EsE activity is modulated either by the amount of OMPs in the OM, by the amount of OMPs passing through the secretion pathway to the OM, or by misfolded or mislocalized OMPs. Given this context, there are many possible explanations for the different effects of the DompX mutation on EsE activity in different strain backgrounds. For example, OmpX could be a major OMP in PSM2 but not MC1061. Although we have not directly examined the proteins in the OM of PSM2, transcriptional analysis indicates that this is unlikely because the levels of ompX transcription in MC1061 were higher than in PSM2 (data not shown). A second explanation is that either an intragenic or extragenic mutation in PSM2 causes OmpX to be misfolded or poorly inserted in the OM. Thus, its expression could induce EsE activity, and a deletion of the gene could reduce EsE activity in PSM2. Finally, there may be a protein with a redundant or overlapping function with OmpX. A strain lacking both OmpX and this other protein may have lower EsE activity. ompX is probably in a monocistronic operon and is transcribed in exponentially growing cells at 30 and 428C from two promoters, P1 and P2. Since the level of transcription did not change after a shift from 30 to 428C, OmpX is probably not involved in a temperature-regulated function. P2 is transcribed by Es70; however, P1 was not transcribed by Es70 in vitro

FIG. 5. S1 mapping of in vivo-synthesized degP transcripts in two strains. Constant amounts of RNA from wild-type (WT) MC1061 (lane 1) and PSM2 (lane 4) and otherwise isogenic strains containing either pJE100 (lanes 2 and 5) or DompX (lanes 4 and 6) were analyzed. A constant amount of RNA from MC1061 grown at 308C was hybridized to a 59-end-labeled probe for degP and digested with S1 nuclease.

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although sequences around the start site of transcription show some match to those of Es70 promoters. Possibly transcription from P1 requires a supercoiled template. Alternatively, either another form of RNA polymerase may recognize P1 or a transcriptional activator may be needed for P1 expression, although sequence scanning did not provide evidence for either possibility. We did not find consensus binding sequences for other forms of RNA polymerase (except Es38, whose promoter recognition sequences are similar to those of Es70 [32]), nor did we detect consensus binding sequences for OmpR, a transcriptional activator that functions at promoters for other OMPs (16), or either direct or inverted repeats which are often recognized by transcriptional activators. phmB encodes a pHinducible, secreted protein and maps to 18 min of the E. coli chromosome (11). If ompX and phmB are identical, P1 may be induced at high pH. The expression of two members of this family of proteins is regulated at the transcriptional level. ail mRNA is regulated by both temperature and growth conditions. In exponentially growing cells, ail RNA levels are higher at 30 than 378C, while in stationary-phase cells, ail RNA levels are higher at 37 than 308C (25). pagC of S. typhimurium is under positive control of the transcriptional activator phoP (20). Understanding the regulation of the P1 promoter may aid in elucidating the function of OmpX. ACKNOWLEDGMENTS We thank Willy Walter for help in preparing the figures. This work was supported by Public Health Service grant GM36278 from the National Institute of General Medical Sciences. REFERENCES 1. Almiron, M., A. J. Link, D. Furlong, and R. Kolter. 1992. A novel DNAbinding protein with regulatory and protective roles in starved Escherichia coli. Genes Dev. 6:2646–2654. 2. Barondess, J. J., and J. Beckwith. 1990. A bacterial virulence determinant encoded by lysogenic coliphage l. Nature (London) 346:871–874. 3. Bliska, J. B., and S. Falkow. 1992. Bacterial resistance to complement killing mediated by the Ail protein of Yersinia enterocolitica. Proc. Natl. Acad. Sci. USA 89:3561–3565. 4. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179–207. 5. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12:387–395. 5a.Erickson, J. Unpublished data. 6. Erickson, J. W., and C. A. Gross. 1989. Identification of the sigma E subunit of Escherichia coli RNA polymerase: a second alternate sigma factor involved in high-temperature gene expression. Genes Dev. 3:1462–1471. 7. Fellay, R., J. Frey, and H. Krisch. 1987. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene 52:147–154. 8. Grodberg, J., M. D. Lundrigan, D. L. Toledo, W. F. Mangel, and J. J. Dunn. 1988. Complete nucleotide sequence and deduced amino acid sequence of the ompT gene of Escherichia coli K12. Nucleic Acids Res. 16:1209. 9. Hackett, J., P. Wyk, P. Hasegawa, and V. Mathan. 1987. Mediation of serum resistance in Salmonella typhimurium by an 11 kilodalton polypeptide encoded by the cryptic plasmid. J. Infect. Dis. 155:540–549. 10. Heffernan, E. J., J. Harwood, J. Fierer, and D. Guiney. 1992. The Salmonella typhimurium virulence plasmid complement gene rck is homologous to a family of virulence-related outer membrane protein genes, including pagC and ail. J. Bacteriol. 174:84–91. 11. Heyde, M., J.-L. Coll, and R. Portalier. 1991. Identification of Escherichia coli genes whose expression increases as a function of external pH. Mol. Gen. Genet. 229:197–205.

12. Hull, R. A., R. Gill, P. Hsu, B. Minshew, and S. Falkow. 1981. Construction and expression of recombinant plasmids encoding type I or D-mannoseresistant pili from a urinary tract infection Escherichia coli isolate. Infect. Immun. 33:933–938. 13. Jones, C. H., F. Jacob-Dubuisson, K. Dodson, M. Kuehn, L. Slonim, R. Striker, and S. J. Hultgren. 1992. Adhesion presentation in bacteria requires molecular chaperones and ushers. Infect. Immun. 60:4445–4451. 14. Lipinska, B., S. Sharma, and C. Georgopoulos. 1988. Sequence analysis and regulation of the htrA gene of Escherichia coli: a s32-independent mechanism of heat-inducible transcription. Nucleic Acids Res. 16:10053–10067. 15. Lonetto, M., M. Gribskov, and C. A. Gross. 1992. The s70 family: sequence conservation and evolutionary relationships. J. Bacteriol. 174:3843–3849. 16. Maeda, S., Y. Ozawa, T. Mizuno, and S. Mizushima. 1988. Sterospecific positioning of the cis-acting sequence with respect to the canonical promoter is required for activation of the ompC gene by positive regulator, OmpR, in Escherichia coli. J. Mol. Biol. 202:433–441. 17. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 18. Masters, P. S., and J.-S. Hong. 1981. Genetics of the glutamine transport system on Escherichia coli. J. Bacteriol. 147:805–819. 19. Mecsas, J., P. E. Rouviere, J. W. Erickson, T. J. Donohue, and C. A. Gross. 1993. The activity of sE, an Escherichia coli heat-inducible s-factor, is modulated by expression of outer membrane proteins. Genes Dev. 7:2618–2628. 19a.Miller, J. H. 1973. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 20. Miller, S. I., A. M. Kukral, and J. J. Mekalanos. 1989. A two component regulatory system (phoP and phoQ) controls Salmonella typhimurium virulence. Proc. Natl. Acad. Sci. USA 86:5048–5058. 21. Miller, V. L., and S. Falkow. 1988. Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect. Immun. 56:1242–1248. 22. Mizuno, T., and S. Mizushima. 1990. Signal transduction and gene regulation through the phosphorylation of two regulatory components: the molecular basis for the osmotic regulation of the porin genes. Mol. Microbiol. 4:1077–1082. 23. Nikaido, H. 1992. Porins and specific channels of bacterial outer membranes. Mol. Microbiol. 6:435–442. 24. Nohno, T., T. Saito, and J.-S. Hong. 1986. Cloning and complete nucleotide sequence of the Escherichia coli glutamine permease operon (glnHPQ). Mol. Gen. Genet 205:260–269. 25. Pierson, D. E., and S. Falkow. 1993. The ail gene of Yersinia enterocolitica has a role in the ability of the organism to survive serum killing. Infect. Immun. 61:1846–1852. 26. Prenti, P., and H. M. Krisch. 1984. In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313. 27. Puente, J. L., V. Alvarez-Scherer, G. Gosset, and E. Calva. 1989. Comparative analysis of Salmonella typhi and Escherichia coli ompC gene. Gene 83:197–206. 28. Pulkkinen, W. S., and S. I. Miller. 1991. A Salmonella typhimurium virulence protein is similar to a Yersinia enterocolitica invasion protein and a bacteriophage lambda outer membrane protein. J. Bacteriol. 173:86–93. 29. Singer, M., T. A. Baker, G. Schnitzler, S. M. Deischel, M. Goel, W. Dove, K. J. Jaacks, A. D. Grossman, J. W. Erickson, and C. A. Gross. 1989. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol. Rev. 53:1–24. 30. Stoorvogel, J., M. J. A. W. M. van Bussel, J. Tommassen, and J. A. M. van de Klundert. 1991. Molecular characterization of an Enterobacter cloacae outer membrane protein (OmpX). J. Bacteriol. 173:156–160. 31. Stoorvogel, J., M. J. A. W. M. van Bussel, and J. A. M. van de Klundert. 1991. Biological characterization of an Enterobacter cloacae outer membrane protein (OmpX). J. Bacteriol. 173:161–167. 32. Tanaka, K., Y. Takayanagi, N. Fujita, A. Ishihama, and H. Takahashi. 1993. Heterogeneity of the principle s factor in Escherichia coli: the rpoS gene product, s38, is a second principle s factor of RNA polymerase in stationaryphase Escherichia coli. Proc. Natl. Acad. Sci. USA 90:3511–3515. 33. Wang, Q. P., and J. M. Kaguni. 1989. A novel sigma factor is involved in expression of the rpoH gene of Escherichia coli. J. Bacteriol. 171:4248–53. 34. Zhou, Y.-N., N. Kusukawa, J. W. Erickson, C. A. Gross, and T. Yura. 1988. Isolation and characterization of the Escherichia coli mutants that lack the heat shock sigma factor s32. J. Bacteriol. 170:3640–3649.