Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters.

ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor. We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors. Furthermore, transcripts from three tandem promoters in ompC were identified in vitro. We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene. The levels of beta-galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters. The function of OmpR appears to be the enhancement of a basal level of ompC expression. From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC.

* This work was supported by United States Public Health Service Grant GM19043 from the National Institute of General Medical Sciences and by Grant NB3871 from the American Cancer Society (to M. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Recipient of a Fogarty International Fellowship from the National Institutes of Health during the course of this work. Present address: Inst. for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan.
As a direct test of this hypothesis, we have undertaken in vitro transcriptional studies and found that RNA polymerase can indeed transcribe ompC without the aid of positive factors. Additionally, we found transcription to occur from multiple tandem promoters, which proved to be consistent with the sequence of the region. We have used site-directed mutagenesis to separate these promoters and have tested them individually for transcriptional activity in vitro and in vivo. Our results show that ompC is expressed at a low constitutive basal level that is subject to activation by OmpR, and the site of this interaction appears to be the upstream tandem promoters.

EXPERIMENTAL PROCEDURES
Materials-Restriction endonucleases were obtained from Bethesda Research Laboratories or from International Biotechnologies, Inc., New Haven, CT. Hind11 10-mer and XbaI 8-mer linkers were from New England Biolabs, Beverly, MA. Ba131, T4 polynucleotide kinase, T4 DNA ligase, and Klenow enzyme were from Bethesda Research Laboratories. E. coli (14) was used as vector for oligonucleotide-directed sitespecific mutagenesis. pYM140 is a plasmid carrying the lpp gene under control of two promoters and was used to generate control transcripts in in vitro transcription reactions. Media-Cells were grown and maintained in Luria broth. Nutrient broth was supplemented with 20% sucrose and used as high osmolarity medium for assay of ,%galactosidase activity. Ampicillin was added at 50 pg/ml. Construction of Plasmids-As shown in Fig. L4, pMY 150, a plasmid clone of ompC, was cut at the unique BgZII site and digested with Bak31. Subsequent ligation with a HindIII linker generated plasmid pBH110. An XbaI linker was ligated at this HindIII site to convert it into an XbaI site, and the 440-bpz XbaI fragment carrying the ompC promoter along with the micF gene was inserted into plasmid pKM005. This generated plasmid pGR111, which now carried a ompC-&Z gene fusion. The MspI site in the ompCpromoter fragment was further replaced with an XbuI site, and the 350-bp XbaI fragment thus obtained carrying the ompC promoter and part of the micF gene was inserted into pKM005 to obtain a second ompC-&Z fusion plasmid, pKL007. pGRll1 and pKL007 have varying lengths of DNA present upstream of the ompC promoter (see Fig. 2) with the micF gene functional only in the pGRl11 plasmid. Because of the manipulations involved, pKL007 also has 4 additional base pairs at the ompC-lac2 fusion site.
The 350-bp XbaI fragment carried in pKL007 was also cloned into vector pJDC406 to generate plasmid pKI0641 which was subsequently employed in site-directed mutagenesis. . Synthetic oligonucleotides were employed to generate the mutant pKI0643, in which a C residue at -94 from the start of transcription (see Fig. 4) was replaced by an A residue, thus generating a restriction site for the enzyme AhaIII. pKI0643 served as parent plasmid for the generation of two additional mutants, pKI0644 in which a C at -131 is substituted with a G to generate a NruI site and pKI0645 in which a C at -63 replaced with a G generates a BglII site (see Fig. 4).
In order to test in vivo activities of the dissected ompC promoter, each of the mutant plasmids pKI0643, pKI0644, and pKI0645 was digested with XbaI and the particular enzyme whose site was introduced by mutagenesis, and the fragments carrying the start of transcriptional of the ompC gene were cloned into pKM007 to produce plasmids pKI0743, pKI0744, and pKI0745, respectively (Fig. 1C). These plasmids are ompC-lacZ fusions essentially identical to pKL007 at the site of fusion but carrying varying lengths of the 5' region of the ompC promoter (see Fig. 2).
Oligonucleotide-directed Site-specific Mutagenesis-Oligodeoxynucleotides for creating the mutations were synthesized by automated phosphoramidate chemistry (15) in a Systec Microsyn 1450 DNA synthesizer. The protocol for introducing mutations at specific sites was essentially as described (16). Heteroduplexes were made by heat denaturation and annealing between plasmids pJDC406 cut at the XbaI site and pKI0641 cut at PstI. The oligomer Ei'GCATTTAAATTTTGA3' was used to introduce the Aha111 site (TTTAAA) and pKI0643 thus constructed. Plasmid pKI0643 was used with pJDC406 to generate, in a similar manner, plasmids pKI0644 and pKI0645. pKI0644 carries a NruI site (TCGCGA) introduced by the oligomer 5'AT"CGCGATTCCGC3' in addition to AhaIII, and the oligomer VTTAAGATCTTTCATTB' was used to introduce the BglII site (AGATCT) of plasmid pKI0645. The positions of the mutations in the ompC promoter and the resulting plasmids are indicated in Fig. 4.
In Vitro Transcription-DNA fragments were used as template in in vitro transcription reactions essentially as described (17) and analyzed on 8% polyacrylamide-urea gels.
8-Galactosidase Assays-Cells carrying various plasmids were grown overnight in Luria broth and transferred at a 1:lOO dilution into nutrient broth containing 20% sucrose. 8-Galactosidase activity was assayed as described (18) after 2-3 h of growth with shaking at 37 "C.

RESULTS
I n Vitro Transcriptwnal Analysis-Plasmid pompCPA5 is an ompC-lac2 gene fusion plasmid and represented in Fig. 2. The HpaI site upstream of ompC is changed to XbaI in the plasmids pompCPA5 and pGR111. In order to examine the transcriptional capacity of the ompC promoter, defined in vitro transcription reactions were carried out using the XbaI-BamHI fragment of pompCPA5 as template. A 390-bp HinfIdigested pYM140 (a lpp clone) fragment was used as control to generate RNA markers of 160 and 245 bases (a and b, respectively, in Fig. 3). Analysis of transcripts on a denaturing gel indicated three major transcripts (labeled I, 2, and 3 in Fig. 3A, lane I). When the shorter XbaI-Hind111 fragment of pompCPA5 was used as template, all three major bands now migrated faster (Fig. 3A, lane 2) identifying them as run-off transcripts in the ompC direction. Consistent with this, a MspI-HindIII fragment (template cut at the 5' end) produced an identical set of three transcripts as XbaI-Hind111 (data not shown). The three bands, 1, 2, and 3, were therefore clearly transcribed in the direction of ompC. Further shortening of the, 3' end of the template by using the XbaI fragment of  pGRll1 resulted in predictably faster migrating bands corresponding to transcripts 1, 2, and 3 (Fig. 3A, lane 3). The shortest transcript 1 is estimated to be only about 30 bases in length and therefore the bands appear rather faint. A 4-base addition at the 3' end of the above template was effected when the XbaI fragment of plasmid pKL007 was used as template; the predicted resultant increase in the sizes of the three transcripts was observed (Fig. 3A, lane 4).
The transcript 1 corresponds to the ompC transcript identified by S1 mapping of in uivo synthesized RNA (12) and is believed to be directed by the P1 promoter ((12) see Fig. 4).
This particular band is more intense when a corresponding fragment carrying an up-promoter mutation of ompC is used as template; this mutation allows a high ompC expression independent of mutations in ompR? The two additional transcripts, 2 and 3, suggest the presence of additional promoters in the system.

Identification of Three Tandem
Promoters-The sequence of the ompC promoter region was scrutinized in order to identify any possible upstream promoter sequences that could initiate transcription and generate transcripts 2 and 3 described above. Two overlapping putative promoters (P2 and P3, Fig. 4) were identified with possible -35 and -10 regions in good agreement with the consensus sequences of TTGACA and TATAAT, respectively (19). The -10 region of P1 (TAT-CAT) is separated from the -35 region (ATGAAA) by 17 bases, and the P3 -10 region (AAACAT) is separated by 18 bases from the -35 region (TTGAAA) (Fig. 4).
In order to enable dissection of these promoters, the XbaI fragment of pKL007 was subcloned into pJDC406 (plasmid pKI0641) and used as the target for site-directed mutagenesis. In this manner, an Aha111 site was introduced immediately upstream of the -35 region of promoter P3 (plasmid pKI0643). The XbaI fragments of pKI0641 and pKI0643 were used as templates for in vitro transcription and showed an essentially identical transcription pattern (Fig. 3A, lanes 4  and 5). The introduction of the mutation, therefore, did not significantly alter the generated transcript pattern; an enhancement of band 2 was observed, but the reason for this is not clear.
In order to perform a finer dissection of the promoter region, additional restriction sites were created in pKI0643, as shown in Fig. lB, and Fig. 4, generating pKI0644 that has a NruI site and pKI0645 that has a BglI site. When the 3' NruI-XbaI fragment from pKI0644 was used as a template in transcription, the three transcripts, 1,2, and 3, were observed K. Ikenaka and G . Ramakrishnan  and 4 indicate bands that are full length template DNA fragment labeled by addition of ribonucleotides a t their 3"OH ends. (Fig. 3B, lane 2), identical to the pKI0643 fragment (Fig. 3B,  lane 1); however, the bands are relatively weaker, probably because the shorter DNA fragment is a poor template for transcription.
When the 3' AhuIII-XbaI ompC fragment of pKI0643 was used at the template, only two identifiable transcripts, 1 and 2, were generated. Production of both transcripts, however, t t

P2(-35)1 P3(-35) P3(-10)
-51  was very poor. This was expected as the Aha111 site is too close to the -35 region of promoter P3 to allow efficient transcription. The BglII-XbaI fragment pKI0645 does not carry either of the promoters P2 and P3; and as expected, only transcript 1 was observed when the fragment was used in an in vitro transcription assay (Fig. 3B, lane 4). These results clearly demonstrate that there are three functional promoters for ompC transcription in uitro.

A T A A A G C C A T A T A A C A~G T T A A T A A C A T G A A
I n Vivo Analysis of Tandem Promoters-In order to test if the P2 and P3 promoters, in addition to P1, are functional in uiuo, the fragments used for in uitro transcription in Fig. 3B were inserted into pKM007, a promoter-proving vector, as shown in Fig. 1C. The resulting plasmids pKI0743,0744, and 0745 carry ompC-lac2 fusions of the fragments derived from pKI0643,0644, and 0645, respectively. MC4100 (ompB+) and MH1160 (ompRI) cells were transformed with these plasmids, and the @-galactosidase produced by the fusion was then assayed after growth in nutrient broth supplemented with 20% sucrose; ompC is normally expressed at a high level in this medium. Cells were transformed with vector pKM005 as control for background production of @-galactosidase. Table I lists the @-galactosidase activities of MC4100 cells transformed with various plasmids. Comparison of the activities of pGR111 and pKL007 shows a 66% reduction in activity following shortening of the promoter fragment at the 5' end. pKI0744 and pKI0743 have essentially the same activity, similar to pKL007, although they have different 5' ends to the ompC promoter. pKI0745 which lacks promoters P2 and P3 shows another 80% reduction in activity.
MH1160 is an ompR mutant cell strain that is phenotypically OmpF-OmpC- (6). We measured the @-galactosidase activities of the various ompC-lac2 fusion plasmids in this strain in order to determine if any of the fusions still retained a response to OmpR. Table 1 shows that pGR111, pKI0743, and pKI0745 in MH1160 cells have essentially the same low @-galactosidase activity as pKI0745 in MC4100, the ompR' strain. pKI0743 has all three promoters, P1, P2, and P3, while pKI0745 has only P1. It is important to note that pKI0743 is capable of activation by OmpR whereas pKI0745 is not. This result indicates that the region of the P2 and P3 promoters is required for OmpR function.

DISCUSSION
In this paper, we have presented results showing that, in a defined transcription system, three distinct transcripts are produced from the DNA fragment carrying the ompC promoter. Since the lengths of these transcripts were equally shortened by shortening the DNA fragment at the 3' end ( Fig. 3A), we concluded that these transcripts were produced from three tandem promoters, P1, P2, and P3 (Fig. 4). The P1 promoter has been previously identified by S1 mapping of the ompC mRNA produced in vivo (12). Promoters P2 and P3 have been identified upstream of PI. The -35 and -10 regions of these promoters are separated by 17 bp in the case of P2 and 18 bp in the case of P3, which is consistent with the 17 f 1-bp spacing for the E. coli promoter (19). There is an overlap of the -10 region of P3 and the -35 region of P2. The promoters P2 and P3 lie 36 and 56 bp, respectively, upstream of the promoter P1, and the sizes of the transcripts generated in uitro are consistent with this spacing.
DNase I protection studies of the ompC promoter fragments with RNA polymerase show this entire stretch of three tandem promoter sequences to be protected in the absence of any external factors.' These results suggest that the ompC gene can be transcribed at a basal level without requirement for OmpR. Similar attempts at in uitro transcription with the ompF promoter failed to yield the predicted mapped tran-s~r i p t .~ This indicates that a positive factor is absolutely required for activation of ompF transcription, whereas ompC gene transcription occurs, although at a low level, in the absence of factors besides RNA polymerase. The role played by the ompB products must therefore be different in regulation of the two porin genes. These results are consistent with the observation that ompC, when cloned into multicopy plasmids, is expressed in ompR mutants of OmpC-phenotype, whereas OmpF is not similarily produced in ompR mutants of OmpF-phenotype (20).' There was a stepwise decrease in ompC expression in uiuo, as measured by @-galactosidase activity, when the length of the region upstream of the ompC promoter was shortened. pKL007 had 33% of the activity of pGR111, which is probably due to the absence of a functional micF gene in pKL007. In the cells harboring pGR111, in which micF is active, OmpF synthesis should decrease (12), which then may make conditions favorable for ompC expression. The plasmids pKI0744 and pKI0743 showed essentially the same activity as pKL007 (Table I). However, pKI0745 showed a further 80% reduction in @-galactosidase activity, indicating that the promoters P2 S. Norioka, G. Ramakrishnan Multiple promoters are commonly found to be present preceding genes and operons in E. coli that are subject to regulation, such as gal (21), uurB (22), rrnE (23), rpsA (23), carAB (24,25), lcrc (26), and g l d (27). It has been proposed that the activities of the different promoters are sensitive to different signals and thus coordinate the control of gene expression in response to multiple factors (22). It is possible that in a similar manner the different promoters of the ompC gene render it responsive to multiple factors. The region upstream of the transcriptional start is also the probable site for interaction with the regulatory factor OmpR as has been suggested previously for ompF (28). /3-Galactosidase activities in ompR' or ompR1 cells harboring an ompC-lucZ fusion plasmid with only the P1 promoter were similar to each other. However, when o m p C -h Z fusion plasmids with all three promoters (pKI0743 or the plasmids with longer fragments in Fig. 2) were used, the activities were much higher in ompR+ cells than in ompR1 cells (Table I). These results are consistent with the notion that OmpR interacts with the P3 and/or P 2 region. We have purified the OmpR protein and found it to specifically bind at the P3 region and to specifically inhibit the production of the P3 transcript i n vitro.' Interaction of OmpR with P3 in vivo might result in the activation of transcription from the downstream promoter(s). This would explain the finding that the major ompC transcript in uiuo was found by S1 mapping to be directed by the P1 promoter (12). In the absence of OmpR, all three promoters may be active in transcription i n uiuo.
There are other possibilities for OmpR function. OmpR may be involved in removal of some inhibitor of transcription from either of the promoters. Re-examination of the DNA sequence between -50 and -80 (Fig. 4) reveals several interesting aspects of this region. First, sequences "TAGCGA" (-78 to -73) and "AAAGTT" (-56 to -51) are quite similar to "TAGCGA and "AAAGAT," which are the -35 and -10 sequences of ompF (28), respectively. Second, a sequence "TTTTGAAC" overlapping the -35 region of P3 is also present at about 70 bp upstream of the ompF transcription initiation site. This common sequence may be important for recognition by OmpR. We are currently investigating the importance of these sequences to OmpR binding and action.