Promoter Region of the Escherichia coli O7-Specific Lipopolysaccharide Gene Cluster: Structural and Functional Characterization of an Upstream Untranslated mRNA Sequence

Cristina L Marolda; Miguel A Valvano

doi:10.1128/jb.180.12.3070-3079.1998

. 1998 Jun;180(12):3070–3079. doi: 10.1128/jb.180.12.3070-3079.1998

Promoter Region of the Escherichia coli O7-Specific Lipopolysaccharide Gene Cluster: Structural and Functional Characterization of an Upstream Untranslated mRNA Sequence

Cristina L Marolda ¹, Miguel A Valvano ^1,^*

PMCID: PMC107806 PMID: 9620955

Abstract

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wb_EcO7). Typical −10 and −35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wb_EcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wb_EcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wb_EcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg²⁺ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wb_EcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Lipopolysaccharide (LPS) is a complex cell surface glycolipid that consists of three regions: lipid A, core oligosaccharide, and the O-specific polysaccharide chain or O antigen (50). LPS is the most abundant lipid in the outer leaflet of the outer membrane in the majority of gram-negative bacteria. While the lipid A is embedded in the membrane, the O antigen extends towards the surface and, in pathogenic organisms, contributes to the evasion of the lytic action of serum complement (20). The biosynthesis of LPS takes place in two separate pathways, one resulting in the formation of the lipid A core and another one resulting in the formation of the O antigen (49, 50). The O antigen intermediates are synthesized attached to undecaprenyl phosphate, and the complete O subunit is translocated across the inner membrane, polymerized, and transferred onto the lipid A core to complete the LPS molecule (49).

The synthesis of LPS involves a large number of genes, many of which are parts of various clusters located on different regions of the bacterial chromosome and, in some organisms, also in plasmids (40). Therefore, it is conceivable that LPS gene expression has to be coordinated to ensure that all necessary components are available at any given time. However, the regulation of LPS synthesis is not well understood. It has been shown recently that certain chemical modifications of lipid A are regulated by the Mg²⁺ concentration through the phoP-phoQ regulon (16). Regulation of LPS biosynthesis by amino acid starvation has also been reported (18), but there is no information on how this regulation is accomplished.

Gene expression of the core biosynthetic gene cluster is regulated by the RfaH protein (11, 34) and also by the heat shock response (21). RfaH is a homolog of the NusG factor that regulates gene expression of the hemolysin operon (6, 23, 24, 31), polysaccharide capsule genes (43), and genes for the transfer of the F plasmid (8). RfaH regulation is exerted at the level of elongation of mRNA (5) and depends on cis-acting sequences known as ops (for operon polarity suppressor) located upstream of the coding regions of the RfaH-regulated operons (4, 23, 31). One such operon is waaQGPSBIJYZK, which includes 10 genes of the core LPS gene cluster (10, 34). Similar, presumably untranslated, leader mRNA segments containing ops exist in the O polysaccharide gene clusters of Escherichia coli, Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica (17), but no direct evidence exists on their role in regulating gene expression.

Regulation of the O-specific polysaccharide gene expression has not been systematically investigated despite the availability of completely sequenced O polysaccharide gene clusters. In at least one case, O polysaccharide gene expression is regulated by temperature via changes in DNA supercoiling (39), and in another case it is regulated by osmolarity (1). Posttranscriptional regulation occurs via the cld (rol) determinant, whose product controls the length distribution of O polysaccharide chains by a mechanism that is not completely understood (7, 12, 47).

We are using the E. coli O7 polysaccharide as a model system to understand the synthesis, assembly, and regulation of O polysaccharide gene expression (2, 27, 28, 30, 45). We have previously reported the DNA sequence and gene organization of the upstream portion of the E. coli O7 antigen biosynthesis gene cluster, wb_EcO7 (previously called rfb; see reference 37 for a description of the new nomenclature adopted for bacterial polysaccharide genes), containing a promoter region and four biosynthetic genes, rlmBADC, which are involved in the biosynthesis of the nucleotide sugar precursor dTDP-rhamnose (27). In this work we report the characterization of this promoter and also of an upstream leader segment with several predicted stem-loop structures. These regions appear to be important as a site for the regulation of the elongation of the wb_EcO7 mRNA transcript in a RfaH-dependent manner. We also show that the wb_EcO7 promoter activity is expressed constitutively.

MATERIALS AND METHODS

Bacterial strains, plasmids, and general methods.

The E. coli strains and plasmids used in this study are described in Table 1. CLM13 and CLM12 are rfaH⁺ and rfaH-deficient derivatives constructed by P1 transduction of strain ED3869 by using a lysate prepared from strain PN130 (fadA::Tn10). The presence of the rfaH mutation was confirmed by examining the lipid A core banding pattern (38) as well as by sensitivity to bacteriophage C21 and concomitant resistance to bacteriophage U3. Bacteria were cultured in Luria broth (LB) supplemented with ampicillin (100 μg/ml), kanamycin (50 μg/ml), and tetracycline (20 μg/ml) as appropriate. For some experiments bacteria were cultured on MacConkey agar plates. LPS was extracted as previously described (30) and analyzed by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (9). Tricine SDS-PAGE gels (16%) were purchased from Novex, San Diego, Calif. DNA sequencing of plasmid constructs was carried out with an automated sequencer at MOBIX, McMaster’s University, Hamilton, Ontario.

TABLE 1.

E. coli strains and plasmids used in this study

Strain or plasmid	Relevant properties^a	Source or reference
Strains
DH5α	F⁻ φ80lacZΔM15 endA recA hsdR(r_K⁻m_K⁻) supE thi gyrA relA? Δ(lacZYA-argF)U169	Laboratory stocks
CLM4	lacZ trp Δ(sbcB-rfb) upp rel rpsL recA	28
CLM12	ED3869 rfaH11 fadA::Tn10	This work
CLM13	ED3869 fadA::Tn10	This work
ED3869	Δ(codB-lac)3 tsx-80 galK λ^d^e^f trp-52 lys-30 rpsL160(Str^r) rfaH11 malA30	8
JT4000	SG20250 Δlon-510	41
MV103	VW187 Δlac	46
PN130	LE392 fadA30 (fadA::Tn10)	P. Black
SG20250	Δlac rpsL relA1 araD139 flbB lon⁺	41
Plasmids
pEX1	Expression vector; Ap^r	33
pFZY1	Promoterless cloning vector; Ap^roriF	22
pGEM3	Cloning and sequencing vector; Ap^r	Promega
pKZ17	6.8-kb BamHI fragment containing rfaH gene cloned into pBR322; Ap^r	38
pTL61T	Promoterless cloning vector; Ap^r	25
pCM10	8.1-kb HindIII fragment containing wb_EcO7 promoter region cloned into pACYC184; Cm^r	26
pCM111	5.6-kb EcoRI fragment containing wb_EcO7 promoter region cloned into pGEM3; Ap^r	This work
pCM117	1.2-kb EcoRI-HindIII fragment from pCM10 cloned into pTL61T; Ap^r Lac⁺	27
pCM131	1.2-kb EcoRI-HindIII fragment from pCM117 cloned into pFZY1; Ap^r Lac⁺	This work
pCM141	1.2-kb EcoRI-HindIII fragment containing wb_EcO7 promoter region with a 71-bp internal deletion cloned into pTL61T; Ap^r Lac⁺	This work
pCM143	412-bp amplicon (primers 25-26) from pCM111 cloned into pGEM3; Ap^r	This work
pCM144	1.2-kb EcoRI-HindIII fragment from pCM141 cloned into pFZY1; Ap^r Lac⁺	This work
pCM145	1.2-kb EcoRI-HindIII fragment containing wb_EcO7 promoter region with an 8-bp internal deletion cloned into pTL61T; Ap^r Lac⁺	This work
pCM147	1.2-kb EcoRI-HindIII fragment containing wb_EcO7 promoter region with a 26-bp internal deletion cloned into pTL61T; Ap^r Lac⁺	This work
pCM148	1.2-kb EcoRI-HindIII fragment from pCM145 cloned into pFZY1; Ap^r Lac⁺	This work
pCM152	1.2-kb EcoRI-HindIII fragment from pCM147 cloned into pFZY1; Ap^r Lac⁺	This work
pCM153	1.2-kb EcoRI-HindIII fragment containing wb_EcO7 promoter region with a 5-bp internal deletion cloned into pTL61T; Ap^r Lac⁺	This work
pCM154	1.2-kb EcoRI-HindIII fragment from pCM153 cloned into pFZY1; Ap^r Lac⁺	This work
pCM160	490-bp amplicon (primers 50-51) from pKZ17 containing rfaH gene cloned into pEX1; Ap^r	This work
pCM162	Km^r cassette inserted into Ap^r gene of pCM160; Km^r Ap^s	This work
pCM163	606-bp amplicon (primers 28-42) from pCM10 cloned into SmaI site from pTL61T; Ap^r Lac⁺	This work
pCM164	588-bp amplicon (primers 28-44) from pCM10 cloned into SmaI site from pTL61T; Ap^r Lac⁺	This work
pCM166	572-bp amplicon (primers 28-29) from pCM10 cloned into SmaI site from pTL61T; Ap^r Lac⁺	This work
pCM169	653-bp amplicon (primers 28-60) from pCM10 cloned into SmaI site from pTL61T; Ap^r Lac⁺	This work
pCM173	632-bp EcoRI-HindIII fragment from pCM166 cloned into pFZY1; Ap^r Lac⁺	This work
pCM174	648-bp EcoRI-HindIII fragment from pCM164 cloned into pFZY1; Ap^r Lac⁺	This work
pCM175	666-bp EcoRI-HindIII fragment from pCM163 cloned into pFZY1; Ap^r Lac⁺	This work
pCM176	713-bp EcoRI-HindIII fragment from pCM169 cloned into pFZY1; Ap^r Lac⁺	This work
pCM180	737-bp EcoRI-HindIII fragment from pCM177 cloned into pFZY1; Ap^r Lac⁺	This work
pCM182	410-bp amplicon (primers 74-76) from strain CLM4 containing waaQ promoter region cloned into pTL61T; Ap^r Lac⁺	This work
pCM183	310-bp amplicon (primers 74-75) from strain CLM4 containing waaQ promoter region cloned into pTL61T; Ap^r Lac⁺	This work
pCM186	423-bp EcoRI-HindIII fragment from pCM182 cloned into pFZY1; Ap^r Lac⁺	This work
pCM187	323-bp EcoRI-HindIII fragment from pCM183 cloned into pFZY1; Ap^r Lac⁺	This work
pCM188	561-bp amplicon (primers 28-89) from pCM10 cloned into SmaI site from pTL61T; Ap^r Lac⁺	This work
pCM190	574-bp EcoRI-HindIII fragment from pCM188 cloned into pFZY1; Ap^r Lac⁺	This work
pCM192	1.2-kb EcoRI-HindIII fragment containing wb_EcO7 promoter region with a 24-bp internal deletion cloned into pTL61T; Ap^r Lac⁺	This work
pCM193	1.2-kb EcoRI-HindIII fragment containing wb_EcO7 promoter region with a 99-bp internal deletion cloned into pTL61T; Ap^r Lac⁺	This work
pCM194	1.2-kb EcoRI-HindIII fragment from pCM192 cloned into pFZY1; Ap^r Lac⁺	This work
pCM195	1.2-kb EcoRI-HindIII fragment from pCM193 cloned into pFZY1; Ap^r Lac⁺	This work
pCM196	1.2-kb EcoRI-HindIII fragment containing wb_EcO7 promoter region with a 130-bp internal deletion cloned into pFZY1; Ap^r Lac⁺	This work
pCM197	0.8-kb fragment containing sequences downstream from −35 region of wb_EcO7 promoter to HindIII site on rlmB cloned into pTL61T	This work

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Str, streptomycin; Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin. The locations and sequences of the primers are indicated in Fig. 2 and Table 2, respectively.

Internal and nested deletions of the wb_EcO7 and waaQ untranslated leader sequences.

Internal deletions of the wb_EcO7 leader sequence were constructed by using a PCR strategy described elsewhere (3). Briefly, two independent PCRs were conducted with primers flanking the deletion endpoints. After phosphorylation with T4 polynucleotide kinase, both amplicons were ligated and the ligation mix was further amplified with the primers annealing to the distal ends of the deletion junction. The product of the second amplification was cleaved with EcoRI and HindIII, purified from a 0.7% agarose gel with the QIAquick gel extraction kit (Qiagen, Chatsworth, Calif.), and ligated to the same sites in pTL61T, followed by transformation into E. coli DH5α. Recombinant plasmids were characterized by restriction endonuclease analysis and PCR amplification, and the appropriate constructs were verified by DNA sequencing. The primers used are shown in Table 2. pCM10 and pCM111 (Table 1) were used as DNA templates for these experiments. To maintain the same levels of expression as found in the chromosome, each construct was cleaved with EcoRI and HindIII, and the fragments containing the various deletions were cloned into the single-copy-number vector pFZY1. Constructs in pFZY1 were verified by PCR with internal primers and a primer corresponding to the upstream sequence of the lacZ gene.

TABLE 2.

Primers used in construction of plasmids

Primer	Sequence	Source
25	5′-CTGGAAGTGTCGGTCAGTA	wb_EcO7
26	5′-TGACGAACAACAGCAGAACC	wb_EcO7
28	5′-AGTCAGCCGCATTGTTG	wb_EcO7
29	5′-TTCCACGATAGTGACGAG	wb_EcO7
30	5′-CTGGAGCAGTCTATTTCAC	wb_EcO7
31	5′-TTCCATGCGCGAACCAA	wb_EcO7
41	5′-CGTGCATTAATGCATC	wb_EcO7
42	5′-CCTGGCTTAACAG	wb_EcO7
43	5′-CTGTTAAGCCAGG	wb_EcO7
44	5′-AGAGCACTGCGTACAT	wb_EcO7
50	5′-ATGCAATCCTGGTAT	rfaH
51	5′-GTAGAGTTTGCGGAA	rfaH
52	5′-GGTAAGACAATTAGCGT	wb_EcO7
57	5′-TGGCAAACAGAGATTGTG	galK
58	5′-TCATGGTCATAGCTGTT	lacZ
60	5′-GACTGCTCCAGATTTGAT	wb_EcO7
74	5′-GCTGACTTATGG	waaQ
75	5′-ACGCACTTACTTTGACG	waaQ
76	5′-TTCCACTAGCGACTCTT	waaQ
89	5′-CGTGCCAAATCCGAAA	wb_EcO7
90	5′-AATTCAAACGCTAATTGTC	wb_EcO7
95	5′-GGAAATGTACGCAGTGCT	wb_EcO7
110	5′-GGCTATATGGAATAAAAAAGTGAAGATAC	wb_EcO7
117	5′-CTACAGCTGTTTGGTAAG	wb_EcO7

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Nested deletions of the untranslated leader sequences in the wb_EcO7 and waaQ promoter regions were also constructed by a PCR strategy. For deletions along the wb_EcO7 promoter region, PCR amplifications were carried out with primer sets 28-29, 28-44, 28-42, and 28-60 (Table 2). The products were phosphorylated and cleaved with EcoRI followed by ligation in pTL61T digested with EcoRI and SmaI. For deletions of the waaQ leader sequence, PCR amplifications were carried out with primer sets 74-75 and 74-76 (Table 2). After phosphorylation the products were cloned into pTL61T digested with SmaI. As in the construction of the internal deletions, all the fragments containing the nested deletions were verified by DNA sequencing and cloned in pFZY1. All PCRs were carried out with a Hybaid Omnigene temperature cycler (Interscience, Markham, Ontario, Canada) with PwoI DNA polymerase (Boehringer Mannheim, Dorval, Quebec, Canada), since this enzyme does not have a terminal transferase activity and possesses a high polymerization fidelity.

β-Galactosidase assays.

The units of β-galactosidase were determined as described by Putnam and Koch (36). Each sample was analyzed in triplicate during at least three independent experiments. To compare results from different experiments, the units of activity were corrected for the background level determined in cells containing the negative control pFZY1, which were included in every experiment. The values were expressed as a percentage of the activity of the construct containing the wild-type promoter region (pCM131).

RNase protection assay.

The RNA probe was prepared in vitro with plasmid pCM143 linearized with HindIII as a template. The in vitro RNA synthesis reaction mixture consisted of 0.7 μg of DNA template, 10 mM dithiothreitol, 800 μM ribonucleoside 5′-triphosphate, 40 U of RNasin (Promega), 100 μg of bovine serum albumin, 20 μCi of [α³²P]CTP, and 40 U of T7 RNA polymerase. The mixture was incubated for 30 min at 37°C followed by the addition of 2 U of RNase-free DNase. After incubation for another 30 min, the probe was purified with a Bio-Spin chromatography column (Bio-Rad, Mississauga, Ontario, Canada). The radiolabeled RNA probe was used for an RNase protection assay performed with the ribonuclease protection kit (United States Biochemical Corp., Cleveland, Ohio). Lysates containing the target RNA were prepared from exponential cultures of strain CLM4(pCM117) and also from strain CLM4(pGEM3) as a negative control. Probe (10⁶ cpm) was added to 45 μl of cell lysate, and the hybridization was carried out overnight at 37°C. Samples were processed and analyzed on a sequencing gel as indicated by the supplier.

Analysis of mRNA secondary structure.

The secondary structures of the leader untranslated mRNA sequences were predicted with the program Mfold version 2.3 (19) and the on-line Mfold server at http://www.ibc.wustl.edu/~zuker/rna/form1.cgi. The default parameters (37°C, 1 M NaCl, no divalent ions, and 5% suboptimality) and the energy parameters given by Walter et al. (48) were used for the predictions.

Transcriptional regulation of the wb_EcO7 promoter.

In all of the following experiments, aliquots were taken from cultures at various times and examined for β-galactosidase activity. The following conditions were investigated.

(i) Temperature.

Overnight cultures of DH5α cells carrying either pCM131 or pFZY1 were inoculated into fresh LB medium and incubated at 22, 37, or 42°C until the cells reached the stationary phase. In some experiments, cells were first incubated at 37°C for 2 h, at which time the cultures were rapidly shifted to either 22 or 42°C.

(ii) Osmolarity.

Osmolarity experiments were carried out with MV103(pCM131) and MV103(pFZY1) cells grown in M9 medium (35). An overnight culture was diluted into fresh medium containing 50, 100, 200, 400, or 800 mM NaCl and incubated for 2 h, at which time the levels of β-galactosidase activity were measured.

(iii) Regulation by Mg²⁺ concentration.

MV103(pCM131) and MV103(pFZY1) bacterial cells grown in N medium (42) with 40 μM MgCl₂ were washed three times with N salts, inoculated into fresh N medium containing no magnesium or 0.05, 0.25, 1.25, or 6.25 mM MgCl₂, and incubated for 2 h.

(iv) DNA supercoiling.

SG20250(pCM131) and SG20250(pFZY1) cells were used for DNA supercoiling experiments. The cells were inoculated into LB with 0, 12.5, 25, 50, 75, and 100 μg of novobiocin/ml (13) and incubated for 2 h at 37°C before their β-galactosidase levels were determined.

(v) Effect of the lon mutation.

In the absence of the Lon protease, there is a higher availability of a positive regulator, RcsA, which increases the production of colanic acid capsule (15). To test whether the wb_EcO7 promoter is regulated by this mechanism, we examined β-galactosidase production over the growth curve in SG20250(pCM131), SG20250(pFZY1), JT4000(pCM131), and JT4000 (pFZY1) cells.

RESULTS

Characterization of the wb_EcO7 promoter region.

The intergenic region between galF and the first gene of the O7-specific LPS gene cluster, rlmB, in E. coli O7:K1 consists of a 367-bp segment (Fig. 1) and contains a promoter (27). To localize the promoter element precisely, we determined the site of transcription initiation by an RNase protection assay. This involved the in vitro synthesis of a radiolabeled antisense RNA probe that was derived from pCM143 (Fig. 2). The probe was used for hybridization with total RNA isolated from strains CLM4(pCM117) and CLM4(pGEM3) as a negative control. Strain CLM4, containing a deletion of the E. coli K-12 wb cluster, was utilized to rule out detection of protected RNA fragments from transcripts initiated at the chromosomal wb_EcK12 promoter region, which is almost identical to that of wb_EcO7 (51). Three protected fragments of 225, 220, and 210 bp were identified (Fig. 3), which corresponded to transcripts initiated at two G residues (positions +1 and +5) and an A residue (position +16), respectively (Fig. 1 and 3). A fourth, 180-bp fragment was also observed (Fig. 3), corresponding to a transcript initiated at an A residue at position +45. This pattern of RNase protection was reproducible, since the same four protected fragments were detected in two independent experiments.

FIG. 2 — Internal and nested deletions of the wb_EcO7 promoter region. A partial restriction map of the 1.2-kb *Eco*RI-*Hin*dIII fragment containing part of *galF* and part of *rlmB* is shown. The arrows indicate the locations and directions (upper arrows, sense; lower arrows, antisense) of the primers used for construction of the deletions. The primers are numbered, and their DNA sequences are indicated in Table 2. “PA probe” indicates the boundaries of the insert in pCM143 that was used for the RNase protection experiments. The −35 and −10 regions of P1, the initiation of transcription (shaded arrow), and the locations of the *ops1* and *ops2* sequences are indicated. E, *Eco*RI; Ps, *Pst*I; Pv, *Pvu*II; Sp, *Sph*I; H, *Hin*dIII. The region indicated with roman numerals corresponds to the predicted stem-loop structures I, II, III, and IV (Fig. 5) in the transcribed but untranslated leader sequence, and the numbers in parentheses indicate the boundaries of each structure, starting with 1 to indicate the first transcribed nucleotide. The boundaries of the deletions in the various constructs are also indicated. The β-galactosidase activities mediated by each deletion construct were determined in *rfaH*⁺ and *rfaH* backgrounds and are expressed as percentages of the values obtained for the wild-type construct pCM131 in the *rfaH*⁺ strain. The means and standard deviations were calculated based on data from four determinations.

FIG. 3 — RNase protection assay. The left side of the figure shows an autoradiograph of a 4% polyacrylamide DNA-sequencing gel. Lane PA contains the RNase-protected fragments. The bases corresponding to each protected fragment are indicated with asterisks.

Upstream of the G at the +1 position, −10 and −35 regions, also predicted by the computer algorithm of O’Neill (32), were found, suggesting that transcription is initiated at this nucleotide. A putative second promoter was identified upstream of the A at +16 (Fig. 1), which could explain initiation of transcription at this site. The putative −35 sequence of the second promoter was located just upstream of the −10 sequence shown in Fig. 1. The existence of this second promoter was investigated by the construction of plasmid pCM197 (Table 1), which harbors a fusion to a promoterless lac operon and lacks the −35 sequence indicated in Fig. 1 but contains the intact putative second promoter region. The level of β-galactosidase activity mediated by this plasmid was identical to that of the vector control (data not shown), ruling out the existence of a second promoter overlapping the one indicated in Fig. 1. Therefore, we conclude that the three protected fragments of 220, 210, and 180 bp shown in Fig. 3 may have been created by enzymatic processing near the 5′ end of the mRNA. This interpretation is plausible, considering that the 5′ segment of this mRNA remains untranslated and has a good probability of folding into several stem-loops (see below). The sizes of these three smaller protected fragments were consistent with those expected for possible endonucleolytic cleavage sites in AT-rich regions between predicted stem-loops.

To investigate the level of expression of the promoter in its normal gene dosage, the 1.2-kb EcoRI-HindIII fragment from pCM117 was subcloned into the single-copy-number vector pFZY1, resulting in pCM131 (Table 1 and Fig. 2). DH5α cells containing this plasmid expressed around 250 U of β-galactosidase, indicating that the wb_EcO7 promoter has a relatively low activity in vivo at its normal dosage.

The wb_EcO7 promoter is regulated by the product of the rfaH gene.

The localization of the transcription initiation sites in the wb_EcO7 promoter also served to confirm that there is an untranslated mRNA segment of 173 bp (Fig. 1). The untranslated segment includes a 39-bp sequence that has been found to be conserved in many other polysaccharide clusters and has been designated JUMPStart (17). Recently, an 8-bp subsequence within JUMPStart has been shown to be involved in increasing the transcription of distal genes of the E. coli hemolysin gene cluster (4, 31), and it has been designated ops. This sequence is present at one end of the JUMPStart sequence in wb_EcO7 (ops1 [Fig. 1]), whereas a partial ops containing only 5 bp is found near the other end (ops2 [Fig. 1]). ops sequences are widely recognized in the untranslated regions of RfaH-regulated gene clusters (4, 23, 43). Work by several laboratories suggested that RfaH is involved in controlling the elongation of bacterial transcripts, probably acting as a transcriptional antiterminator (4, 5, 11, 34). Also, it has been shown that the RfaH-mediated effect requires an intact ops (4, 5, 23). To investigate whether rfaH plays any role in the regulation of the wb_EcO7 promoter, we constructed the isogenic strains CLM12 and CLM13, the former containing a mutated rfaH gene (rfaH11 allele). We used these strains to compare the levels of expression of β-galactosidase directed by plasmid pCM131. Figure 4 shows that in the presence of the rfaH mutation the expression of the wb_EcO7 promoter (contained in pCM131) is reduced about sevenfold with respect to wild-type levels, reaching values similar to those found in strain CLM12(pFZY1). The expression of β-galactosidase was restored in cells containing pCM162, which carries a functional E. coli K-12 rfaH gene cloned in the vector pEX1. In this case, the expression was higher than that mediated by pCM131 alone, probably due to the higher gene dosage of RfaH, as has been shown previously with the hemolysin system (4). From these experiments we concluded that the expression of wb_EcO7 is regulated by the RfaH protein.

FIG. 4 — Regulation of the wb_EcO7 and *waaQ* promoters by RfaH. β-Galactosidase assays were conducted on the indicated *E. coli* strains carrying various plasmids. The results are expressed as percentages of the activity of the wild-type promoter fusion in CLM13(pCM131) and represent the means of three determinations.

Interestingly, a deletion that eliminated the JUMPStart region, including both ops sequences (pCM173 [Fig. 2]) displayed a low level of transcriptional activity irrespective of the presence of RfaH (Fig. 4). This result was unexpected, since the wb_EcO7 promoter region as well as the site of transcription initiation remained intact in this plasmid. As a control, we constructed analogous transcriptional fusions with the waaQ promoter region of the lipopolysaccharide core gene cluster (10), which is also regulated by RfaH (34) and contains a JUMPStart sequence with ops elements (17). The data in Fig. 4 show that a deletion of most of the waaQ untranslated region, leaving only the first 15 bp (pCM187, Δ+15 [Fig. 5]), resulted in a level of β-galactosidase expression higher than that determined by pCM186 (Δ+110), containing an intact JUMPStart (Fig. 5). The deletion endpoint in pCM187 is roughly similar to that of pCM173 in the wb_EcO7 promoter region (Fig. 2 and 5). Also, β-galactosidase expression by the fusion containing the entire waaQ promoter region and JUMPStart sequences (pCM186) was regulated in a RfaH-dependent fashion (Fig. 4), although the expression dropped only 1.5-fold in the rfaH mutant strain. In contrast, the expression of β-galactosidase by pCM187 was RfaH independent (Fig. 4). These results suggest that the JUMPStart sequence is important for the RfaH-mediated effect. Also, since both the wb_EcO7 and waaQ promoters are cloned in the same cloning vector and determine similar levels of β-galactosidase activity, these results cannot be explained by differences in promoter strength. Therefore, other differences between these two apparently similar RfaH-regulated promoter regions may exist.

FIG. 5 — Predicted RNA secondary structure of the untranslated leader sequences of the wb_EcO7 and *waaQ* promoter regions calculated with the program Mfold. The asterisk in each structure indicates the first transcribed nucleotide. Double lines above the UGGA sequence in wb_EcO7 indicate a probable Shine-Dalgarno sequence. The predicted stem-loops are indicated with roman numerals. The endpoints of nested deletions as well as the number of nucleotides remaining after each deletion are also indicated. The plasmids containing the deletions are indicated in parentheses and are shown in Fig. 2. The boxed sequences in both regions denote the JUMPStart sequences. The individually boxed nucleotides indicate the *ops1* and *ops2* subsequences.

Prediction of the secondary structure of the wb_EcO7 untranslated leader mRNA.

Although a relationship has been established between the JUMPStart sequence and regulation of transcription by RfaH (4, 23), the untranslated mRNA regions in RfaH-regulated genes usually contain other sequences in addition to JUMPStart. These sequences could potentially involve other cis-regulatory elements specific for each system that could explain the differences we observed between the expression of wb_EcO7 and waaQ transcriptional fusions. We examined the structural properties of the wb_EcO7 173-bp upstream leader sequence with the program Mfold in an attempt to develop a model for the analysis of this sequence. Mfold predicted four stem-loop structures, with an overall minimal free energy of −46.6 ± 2.3 kcal (Fig. 5). Of these, stem-loop I is probably the least stable structure, since it contains only one G-C pair. In contrast, structures II, III, and IV are likely to be more stable and probably occur in vivo, since they contain many paired G-C bases. These structures consistently appeared under different conditions predicted to be suboptimal. The JUMPStart sequence spanned both the stem and the loops of the predicted structure III (Fig. 5). The ops1 subsequence was located partly in the stem and contained three paired G-C bases, while the five bases of ops2 formed part of the predicted loop (Fig. 5).

For comparison we also modelled the secondary structure of the untranslated mRNA of the waaQ promoter region. In this case, the JUMPStart sequence begins at position +18 (Fig. 5) instead of position +61, as in the case of the wb_EcO7 promoter region (Fig. 1 and 5). The secondary structure analysis of the untranslated mRNA of waaQ predicts a structure similar to stem-loop III of the wb_EcO7 promoter (Fig. 5, stem-loop I), while there is no structure similar to stem-loop II.

Deletion analysis of the upstream untranslated leader sequences in wb_EcO7 and waaQ.

To investigate the possible role of the predicted stem-loops in transcription by the wb_EcO7 promoter, we constructed a series of nested deletion derivatives spanning the region from bases +25 to +134 that were transcriptionally fused to a promoterless lac operon (Fig. 2 and 5). The deletions were constructed as described in Materials and Methods and were subcloned into the single-copy-number vector pFZY1. The levels of expression of β-galactosidase mediated by the resulting recombinant plasmids in rfaH and rfaH⁺ backgrounds, as well as the effects of the deletions on the predicted stem-loops shown in Fig. 5, are indicated in Fig. 2. Only pCM176 expressed a β-galactosidase level comparable with that of the wild-type promoter in pCM131, and it was the only nested deletion whose expression was regulated by RfaH (Fig. 2). This deletion contained 134 of the 173 bases of the untranslated segment and completely spanned the predicted stem-loops I, II, and III (Fig. 2 and 5). Therefore, we concluded that stem-loop IV does not play a significant role in transcriptional regulation of the expression of the wb_EcO7 promoter under these experimental conditions. However, stem-loop IV could be important in modulating the translation of rmlB, since the Shine-Dalgarno sequence is folded within the stem (Fig. 5).

The other deletions, resulting in the elimination of ops1 (Δ+87; pCM175), ops2 (Δ+69; pCM174), and both ops1 and ops2 as well as the complete stem-loop III (Δ+53; pCM173) (Fig. 2 and 5), caused dramatic reductions in the expression of β-galactosidase as well as loss of RfaH-mediated regulation (Fig. 2). These observations could be explained by an effect of the JUMPStart element on transcription initiation or by premature transcription termination at predicted stem-loops I and II (Fig. 5). pCM190 (Fig. 2), containing a deletion-eliminating structure II (Δ+25 [Fig. 5]) determined a level of β-galactosidase expression twofold higher than the expression mediated by the wild-type promoter regions in pCM131 and deletion plasmid pCM176. Since pCM190 contains the complete predicted stem-loop I, we concluded that this structure does not play a role in transcription termination and, since it has only one G-C pair, may not even exist in vivo. In contrast, the increased level of β-galactosidase expression when predicted stem-loop II is disrupted suggests this region alone is important for determining a reduction in β-galactosidase expression, which is consistent with premature termination of the initiated transcript. In conclusion, the experiments with the nested deletions show that predicted stem-loop II may serve as a site for premature termination of transcription and that a complete stem-loop III, including the complete JUMPStart sequence, is required for wild-type levels of transcriptional activity. This interpretation is also consistent with the fact that a stem-loop II-like structure is absent in the waaQ leader sequence (Fig. 5) and may explain the higher expression of β-galactosidase detected with pCM186 and pCM187 even in the absence of RfaH (Fig. 4).

Relative contributions of stem-loop structures and ops sequences to wb_EcO7 promoter expression.

Although the nested deletion experiments permitted us to detect a functional role for the predicted stem-loop II in the wb_EcO7 promoter, they did not address the relative contribution of each predicted stem-loop to the promoter activity. For this purpose, we constructed a series of internal deletions (Fig. 2). The cloning strategy was similar to that described above except that the deletions contained the remainder of the untranslated leader sequence and 512 bp of the coding region of the rlmB gene. In this manner, the results could be better compared with those for the plasmid pCM131. The predicted stem-loop structures affected by each deletion are also indicated in Fig. 2. The internal deletion of the predicted stem-loop II (pCM194) resulted in an expression level 30% higher than that mediated by pCM131 containing the wild-type wb_EcO7 promoter. In contrast, the internal deletion of stem-loop III (pCM144) caused a 65% reduction in β-galactosidase activity (Fig. 2).

Other internal deletions eliminating specific sequences within the predicted stem-loop III were constructed. Figure 2 shows that deletion of ops1 alone (pCM148) caused a 70% reduction in the expression of β-galactosidase with respect to wild-type levels while the deletion of ops2 (pCM154) caused a 40% reduction. As expected, deletion of both ops1 and ops2 and the intervening sequences (pCM152) also resulted in a marked decrease in β-galactosidase production, and they all lost RfaH-mediated regulation (Fig. 2). From these experiments, we concluded that the ops1 subsequence, and to a lesser degree ops2, is a critical cis-regulatory element involved in the transcriptional activity of the wb_EcO7 promoter region. Furthermore, the effects of these deletions on the expression of β-galactosidase were similar to the effect of the rfaH mutation on the transcriptional activity of the wild-type wb_EcO7 promoter (Fig. 4), suggesting that at least an intact ops1 subsequence is required for the RfaH-mediated function.

Interestingly, a deletion removing both predicted structures II and III (pCM195) gave a level of transcriptional activity higher than that of pCM144, again suggesting that structure II may play a role in premature termination. Also, sequences downstream from structure III may be important for the stability of the message, since removing the majority of the untranslated segment (pCM196) resulted in a level of expression similar to that of the wild-type promoter region in pCM131, albeit the expression was not regulated by RfaH (Fig. 2).

Expression of the wb_EcO7 promoter region under different conditions.

The cloning and characterization of the promoter region as a transcriptional fusion in a single-copy-number vector allowed us to examine the possible roles of different conditions of growth in the transcriptional activity of this promoter. We tested the effects of growth temperature, Mg²⁺ concentration, and osmolarity because these conditions have been shown to be important for regulation of LPS in other systems (1, 16, 39). We also investigated whether the wb_EcO7 promoter expression changes under conditions affecting the DNA topology, especially DNA supercoiling (39), as well as in the presence of the lon mutation that is known to increase the expression of colanic acid capsule (15). No differences were observed in the expression of β-galactosidase under any of the conditions tested (data not shown). Therefore, we conclude that wb_EcO7 promoter activity is expressed constitutively.

DISCUSSION

In the present work we have characterized the promoter region of the wb_EcO7 gene cluster and confirmed the existence of a relatively large untranslated leader sequence. Secondary structure prediction analysis combined with transcriptional fusions served to define several regions, possibly folding into complex stem-loop structures, some of which may be functional in vivo. The leader sequence contains a predicted stem-loop that spans the JUMPStart sequence and carries two ops elements. We demonstrated that the ops elements, especially ops1, are sufficient for an RfaH-mediated regulation of wb_EcO7 promoter activity. This is in agreement with previous studies by other laboratories of other RfaH-regulated gene clusters (6, 23, 24, 31, 43). However, unlike previous results of others (6, 31), we demonstrate that the partial ops sequence (ops2) also plays a role in the RfaH-mediated function. Although it is clear that RfaH and ops sequences cooperate to control elongation of the initiated transcript, the actual mechanism of this interaction remains to be elucidated. Recently, Bailey et al. (5) discussed several models to explain how RfaH and the ops element interact. These authors suggest that the ops element may recruit RfaH and possibly other factors to the transcription complex, resulting in a modification of the RNA polymerase into a termination-resistant form. It has been shown by others (11) that rfaH mutants can be suppressed by a mutation in the Rho termination factor. Therefore, it is possible that in addition to RfaH, Rho and another factor(s) may be required for the continuation of the elongation of the transcript. It is not clear from the published literature whether RfaH interacts directly with DNA. We were unable to determine direct binding of purified RfaH in vitro to a synthetic RNA oligonucleotide containing the JUMPStart segment (29), suggesting that either more factors are needed or the binding takes place in the context of the entire transcription complex and perhaps involves single-stranded DNA.

We also showed in this work that RfaH acts on the leader segment of the waaQ operon of the core oligosaccharide synthesis cluster. More importantly, deletion of this region is associated with an expression of promoter activity higher than wild-type levels, as determined by production of β-galactosidase. This is in agreement with recent observations by Leeds and Welch (23), suggesting that the JUMPStart region is a site for the RNA polymerase to pause. In contrast to the observations with the waaQ leader segment, deletions of JUMPStart and ops1 in the corresponding leader sequence of wb_EcO7 were associated with dramatic decreases in transcriptional activity. These findings could not be explained by differences in the strengths of the two promoters. Experiments using nested deletion-fusion suggested the existence in the wb_EcO7 leader region of a short segment with a predicted stem-loop structure located upstream of the JUMPStart sequence (predicted stem-loop II [Fig. 5]). There was no similar predicted stem-loop in the corresponding region in waaQ. Deletion of this segment in the wb_EcO7 leader sequence resulted in an increased level of transcription. Internal deletion-fusion experiments revealed that untranslated sequences downstream from the JUMPStart region (corresponding to predicted stem-loop IV [Fig. 5]) are also important to maintain a level of transcriptional activity similar to that of the wild-type wb_EcO7 leader. Interestingly, the effect of the regions flanking the ops sequences on the expression of the transcriptional fusions is apparent only when the ops sequences are deleted or in the rfaH mutant.

We propose a working model (Fig. 6) where the predicted stem-loop II and IV regions may serve as sites for transcription termination, but only in the absence of RfaH or in the presence of a mutation(s) compromising the ops sequences contained in stem-loop III. In the presence of RfaH, the ops sequences presumably would serve to bring RfaH itself or another transcription factor(s) together with the RNA polymerase elongation complex, and the binding of all these factors may prevent the formation of stable stem-loops II and IV. Since the putative stem-loop IV includes the predicted Shine-Dalgarno sequence of rlmB, it is possible that in the presence of the elongation complex this sequence is exposed to the ribosome and the stem-loop does not form. Further studies using site-directed mutagenesis are required to confirm the existence of the proposed structures and their roles as sites for premature transcription termination.

In support of our hypothesis, structures similar to stem-loop II, also located upstream of the JUMPStart sequence and ops elements, can be modelled in the case of the Y. enterocolitica wb gene cluster (29). To our knowledge, this is the only other O polysaccharide gene cluster in which the boundaries of the leader sequence have been determined experimentally (52). Also, the structures are present in the untranslated regions of O polysaccharide gene clusters of S. flexneri, E. coli K-12, and S. enterica, which are very similar to the wb_EcO7 untranslated region (29). We propose that these structures play a general role in maintaining a tight regulation of the transcription of O polysaccharide genes in the absence of RfaH. The lack of equivalent structures not only in the E. coli K-12 waaQ but also in all the other E. coli core types and the core region of S. enterica (28) would explain why the expression of core lipid A is not completely blocked in rfaH mutants. Our hypothesis is consistent with the general view that core lipid A is more important for the bacterial cell than the O-specific polysaccharide chain. In fact, expression of O polysaccharide in the absence of core would be energetically unfavorable and also may lead to a rapid consumption of the bactoprenyl phosphate that is essential for the synthesis of peptidoglycan (14).

Interestingly, the leader sequences in capsule polysaccharide clusters, such as colanic acid capsule and region II of K5 capsular polysaccharide genes (43, 44), are also longer and more complex than the waaQ leader region. This may be related to the fact that these genes may be subjected to additional regulatory controls, such as RcsA and RcsB in the case of the colanic acid capsule and other yet-undiscovered factors in the case of the K5 capsule.

Attempts to identify regulatory factors other than RfaH acting on the wb_EcO7 promoter were unsuccessful. The wb_EcO7 promoter was not upregulated in a lon mutant, suggesting that it is not under the control of the RcsA and RcsB regulators. Likewise, the wb_EcO7 promoter was not regulated by DNA supercoiling, temperature shifts, osmolarity, and the Mg²⁺ concentration, suggesting that this promoter is constitutive in terms of transcription initiation and is regulated only at the level of mRNA elongation by RfaH, at least under our experimental conditions. These findings do not preclude the existence of regulation at the level of translation of gene products or enzyme feedback interactions. Further experiments are required to examine these possibilities as well as other regions within the wb_EcO7 cluster that may play a role in the regulation of gene expression.

ACKNOWLEDGMENTS

We are grateful to the colleagues mentioned in the text or referenced in Table 1 for kindly supplying strains and plasmids. Special thanks are due to the members of the lab for useful discussions and to C. Whitfield and D. E. Heinrichs for supplying us with the sequences of the E. coli core clusters for core chemotypes 1, 2, 3, and 4 prior to publication.

This investigation was supported by grant MT10206 from the Medical Research Council of Canada.

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PERMALINK

Promoter Region of the Escherichia coli O7-Specific Lipopolysaccharide Gene Cluster: Structural and Functional Characterization of an Upstream Untranslated mRNA Sequence

Cristina L Marolda

Miguel A Valvano

Abstract