Cambridge University Department of Pathology, Tennis Court Road, Cambridge CB2 1QP, UK1
Author for correspondence: Colin Hughes. Tel: +44 1223 333732. Fax: +44 1223 333327. e-mail: ch{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Keywords: flagella, LRP, toxin, virulence gene expression
a The GenBank accession number for the sequence determined in this work is AJ250100.
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INTRODUCTION |
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Several transcriptional regulators of swarming differentiation have been identified, including the FlhD2C2 activator complex encoded by the flagellar hierarchy master operon flhDC (Furness et al., 1997 ; Claret & Hughes, 2000a
, b
), the leucine-responsive regulatory protein (Lrp) (Hay et al., 1997
), and the cell envelope Umo proteins, especially UmoB (Dufour et al., 1998
). It seems that these proteins might act as part of a regulatory network that integrates a number of stimuli and co-regulates virulence and flagellar gene expression (Fraser & Hughes, 1999
). While signals that govern expression of the flagellar gene hierarchy are assumed to funnel through flhDC, this need not be the case for the co-regulated virulence genes. In particular, Lrp appears to regulate both flhDC and hpmBA, as a non-swarming lrp::Tn5 mutant is non-haemolytic even when its hyper-flagellation and swarming is recovered by overexpression of flhDC (Hay et al., 1997
). In this report, we characterize hpmBA transcription, and investigate the influence of the swarm regulators Lrp, FlhD2C2 and UmoB upon expression of hpmBA and also zapA.
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METHODS |
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Promoter mapping by primer extension.
Oligonucleotides A1 (5'-CGCCCGGAGGGTGAAAGTTTAAG-3', complementary to nucleotides 15 to 38 of hpmA), B1 (5'-CAGCTTAATAGTGTTAATAAAAC-3', complementary to nucleotides 16 to 38 of hpmB), and B2 (5'-CCAGTTACTCTTTAATTG-3', complementary to nucleotides -304 to -321 5' of hpmB) were 5' end-labelled with [-32P]ATP (Amersham) by T4 polynucleotide kinase. Primer extension reactions were performed as described by Dufour et al. (1998
).
Luciferase reporter assays.
Transcriptional fusion assays were performed as previously described (Gygi et al., 1995 ). P. mirabilis wild-type and the flhDC
strains carrying derivatives of the reporter plasmid pQF120 (Ronald et al., 1990
) containing transcriptional fusions of putative hpmBA promoter regions to the luxAB genes of Vibrio fischeri were monitored in a Bio-Orbit 1253 single-tube luminometer in the presence of 0·02% (v/v) dodecanal (Aldrich). The baseline luciferase activities of wild-type cells carrying the pQF120 parental vector were subtracted from the activities produced by cells containing hpmBAluxAB fusions. Each data collection point was replicated five times and the mean and standard error were calculated.
Purification of N-terminally His-tagged Lrp protein.
The lrp gene was PCR-amplified with Pfu Polymerase (Promega) from P. mirabilis U6450 chromosomal DNA using the mutagenizing oligonucleotide primers Lrp1 (5'-GAAAGAGAGTATACATATGATTGATAAT-3'; NdeI site) and Lrp2 (5'-GCACTAACCTACCAGCAGGCTTAGCGTG-3'; BamHI site). The PCR fragment was inserted into pET15b (Novagen). N-His-Lrp was produced in E. coli BL21(DE3) (Studier & Moffart, 1986 ), by induction at mid-exponential phase with 1 mM IPTG for 3 h. Protein purification was carried out under non-denaturing conditions using His-Bind Quick 900 columns (Novagen) as described by the manufacturer.
Gel retardation assays.
DNA fragments were PCR-amplified with Pfu Polymerase (Promega) using pGF74 as a template. The downstream oligonucleotide primer B1 (5'-CAGCTTTAATAGTGTTAATAAAA-3') is internal to the hpmB gene. The upstream oligonucleotide primers hpm AccI (5'-GATTTTATTTGTCGACAACGCTTATTCTC-3') and hpm ClaI (5'-CGTTTATTGATCGATAATACTAAAAAAAG-3') were used to amplify the 239 bp fragment spanning nucleotides 473 to 711 and the 183 bp fragment from nucleotide 529 to 711 (see Fig. 4), respectively. PCR products were digested with XhoI, dephosphorylated with calf intestinal phosphatase and labelled using T4 polynucleotide kinase and [-32P]dATP. The control 157 bp labelled DNA fragment was obtained as described by Claret & Hughes (2000b
). The 183 bp, 239 bp and 157 bp fragments were purified on a polyacrylamide gel as described by Sambrook et al. (1989)
. Labelled fragments (0·02 pmol) in 10 µl buffer A (20 mM Tris/HCl pH 7·4, 50 mM NaCl, 0·1 mM EDTA, 0·1 mM DTT, 20%, v/v, glycerol, 100 µg BSA ml-1) were incubated with 030 ng purified N-His-Lrp. After 20 min incubation at room temperature, samples were run on a 6% polyacrylamide gel (2 h, 130 V, room temperature) containing 0·5x TBE; the gel was dried and autoradiographed.
Generation of the P. mirabilis flhDC null mutant.
A 250 bp fragment between nucleotides 62 and 312 (HinP1I and HincII sites, respectively) of the 351 bp P. mirabilis flhD gene was removed and replaced by the 2 kbp spectinomycin resistance omega () cassette (Fellay et al., 1987
), creating the plasmid pRF10
. The approximately 4·6 kbp flhDC
locus of pRF10
was inserted into the suicide vector pGP704 (Miller & Mekalanos, 1988
). Conjugal transfer of the suicide plasmid from E. coli SM10::
pir to P. mirabilis U6450, and isolation of flhDC
mutants, were carried out as described by Swihart & Welch (1990)
. Colony blots were probed with an approximately 1·4 kbp BamHI fragment of pGP704 and an approximately 2·0 kbp HindIII fragment of the
cassette.
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RESULTS |
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A single extension product terminating 70 bp 5' of hpmB and 8 bp 3' of the -10 hexamer of the putative hpmB 70 promoter was detected in the swarm-cell RNA but not in that from the vegetative cells (Fig. 1a
). No extension product was evident immediately 3' of the putative
28 promoter, and this was verified by carrying out primer extension with an oligonucleotide primer complementary to sequence 5' of the
70 consensus (not shown). Several swarm-cell-specific primer extension products terminated in the region 5' of hpmA (Fig. 1b
), but none was compatible with transcription initiation from the putative hpmA
70 promoter. These data indicate that the
70 promoter 5' of the hpmB gene is the only promoter active and upregulated during swarming. They suggest that the hpmA mRNA might arise from processing of a bicistronic hpmBA transcript.
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Three derivative reporter fusions, pLUX415, pLUX227 and pLUX183, contained truncations of the 712 bp hpmB upstream region (Fig. 5a). The largest, pLUX415, contained a 5' truncation to 415 bp, retaining the putative
28 consensus sequence, the four putative-Lrp-binding sites and the hpmB
70 promoter. In wild-type cells it produced a peak luciferase activity comparable to pLUX712 (Fig. 5a
), suggesting that the sequences required for full transcriptional upregulation during swarming are within this 415 bp region. Further 5' truncation to 227 bp removed the potential
28 -35 consensus and the putative Lrp1 site to generate pLUX227, which in the wild-type generated a peak luciferase activity about 50% of that produced by pLUX425 and pLUX712 (Fig. 5a
).
Nevertheless, pLUX227 peak activity in wild-type was about sixfold higher than that in the flhDC mutant, indicating that the hpmB promoter on pLUX227 was still substantially upregulated in response to differentiation. Further truncation of the hpmB promoter region to 183 bp in pLUX183 removed the putative Lrp1 and Lrp2 sites and caused severe reduction in the promoters activity, such that wild-type and flhDC cells carrying pLUX183 produced comparably low peak luciferase activities (Fig. 5a). In addition to these hpmB
70 promoter fusions, a luxAB transcriptional fusion was constructed containing a 413 bp region encompassing the hpmB
28 consensus but not the
70 promoter (pLUXD413). Peak luciferase activities in the wild-type and the flhDC mutant carrying pLUXD413 were comparably low (Fig. 5a
), in agreement with the primer extension data (Fig. 1a
, b
), which indicated that the putative
28 consensus is not an active promoter. A second transcriptional reporter plasmid containing the 230 bp region immediately 5' of hpmA (pLUXA230, not shown) was also constructed; it generated very low activities in wild-type and flhDC mutant cells, again indicating that there is probably not an independently regulated promoter immediately 5' of hpmA
Multiple Lrp binding to the hpmB promoter region
The fusion analysis suggested that sequences 5' of the hpmB 70 promoter are needed for the transcriptional upregulation of the hpmBA operon during swarming. In particular, a region spanning two putative Lrp-binding sites (Lrp1 and Lrp2, Fig. 4
) appears to be essential. To establish if Lrp does indeed bind to this hpmB promoter region, interaction was assessed in band-shift experiments. Soluble N-terminally His-tagged Lrp protein was purified under non-denaturing conditions on a nickel affinity cartridge. It was incubated with a 183 bp DNA region spanning nucleotides 529 to 711 (Fig. 4
) encompassing the putative Lrp sites 3 and 4, and with a 239 bp DNA fragment containing nucleotides 473 to 711 (Fig. 4
) encompassing the putative Lrp sites 2, 3 and 4. Incubation was carried out in the presence of a 1000-fold excess of poly(dI-dC) non-specific competitor DNA. We estimated the concentration of His-Lrp needed for half saturation of the labelled DNA in repeated band-shift assays. For the smaller fragment (-123 to +60) this was 17 nM, for the larger fragment (-179 to +60) 5 nM. Fig. 6
shows that migration of the shorter fragment was retarded by increasing amounts of His-Lrp, indicating the formation of a nucleoprotein complex, while the larger fragment generated two retarded bands, indicating formation of two different complexes. Interactions of His-Lrp with a still larger fragment, the 416 bp region spanning nucleotides 297 to 711 (containing all four putative Lrp sites shown in Fig. 4
), indicated the formation of a complex that, due to its large size, remained in the wells of the polyacrylamide gel after electrophoresis (data not shown). No binding was seen when a control shift assay was performed with a 157 bp DNA fragment carrying the upstream promoter region of the P. mirabilis flhB gene (Claret & Hughes, 2000b
). These data indicate that His-Lrp binds specifically to both the 183 bp and 239 bp fragments, but there may be multiple interactions in each case; the data show that the 239 bp fragment contains at least one more site than the 183 bp fragment.
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DISCUSSION |
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During differentiation, the steady-state levels of hpmA mRNA were considerably higher than the levels of hpmBA mRNA. This was somewhat unexpected, as the primer extension data suggest that the hpmB transcript is abundant. However, abundance of the hpmB cDNA extension product does not necessarily reflect the stability of the hpmBA messenger, as a successful primer extension would only require hpmB mRNA fragments containing the first 200 bases. When considered together, the primer extension and Northern blot data indicate that an unstable full-length hpmBA transcript is processed to produce a stable hpmA mRNA and an unstable hpmB mRNA. Similar processing of polycistronic transcripts has been widely observed: e.g. the E. coli ftsZA and malEFG transcripts, the Salmonella histidine transport operon, and the extensively studied Rhodobacter capsulatus puf operon (Cam et al., 1996 ; Newbury et al., 1987
; Stern et al., 1988
; Klug, 1993
). In the case of the R. capsulatus pufQBALMX transcript, which encodes proteins involved in photosynthesis, processing of the mRNA produces transcript segments with different stabilities and this subsequently influences translation and the stoichiometry of the proteins in the photosynthetic complexes (Klug, 1993
). Similarly, processing of the P. mirabilis hpmBA transcript might be one of the mechanisms that regulates the ratio of the HpmA haemolysin and its cognate exporter/activator protein, HpmB. These proteins are highly similar to ShlA and ShlB of Serratia marcescens. ShlB has been shown to form pores in artificial lipid bilayer membranes, and it is thought to form a channel in the bacterial outer membrane through which the ShlA haemolysin is exported (Konninger et al., 1999
). It seems unlikely that the HpmB (ShlB) exporter and HpmA (ShlA) haemolysin would be required in equimolar amounts, and limiting the production of the pore-forming outer-membrane exporter while producing large amounts of exported haemolysin might bring maximal benefits to the bacterial cell.
As hpmBA is most likely transcribed as an operon, we investigated the role of sequences upstream of hpmB in the modulation of its transcription during the swarm cycle. Analysis of luxAB transcriptional fusions to the non-coding region upstream of hpmB showed that this 697 bp region can be truncated to 415 bp without affecting maximal promoter activity in swarm cells. This 415 bp region encompasses four putative consensus sequences for Lrp (Fig. 4, Lrp1Lrp4). Further truncation of the hpmB upstream region, removing the Lrp1 site, reduced maximal promoter activity to 50%, and additional removal of the Lrp2 site abolished upregulation of promoter activity during swarming. This suggests that while the putative Lrp1 site enhances, but is not essential for, upregulation of the hpmB
70 promoter, the region spanning the Lrp2 site is absolutely required. Binding of Lrp to the hpmB upstream region was confirmed by in vitro band-shift experiments, and the data indicate that there are at least two Lrp-binding sites in the 239 bp region encompassing the putative Lrp2, Lrp3 and Lrp4 consensus sequences, and at least one binding site in the 183 bp region that includes the Lrp3 and Lrp4 sequences. There may be further Lrp-binding sites in the larger 415 bp region which carries all four of the putative Lrp consensus sequences, as this fragment formed a very large nucleoprotein complex that did not migrate into the native polyacrylamide gel. Previously characterized examples of Lrp binding to promoter regions (Gazeau et al., 1994
; Cui et al., 1995
; Rhee et al., 1996
) show that Lrp often binds at multiple sites, and that binding sometimes shows a degree of cooperativity. Therefore, we cannot exclude the possibility that the 183 bp hpmB upstream fragment contains more than one Lrp-binding site, and the 239 bp region contains more than two sites. Our results suggest that Lrp acts directly to activate the transcription of this operon. As the Lrp-binding sites 2, 3 and 4 upstream of hpmB are within 30 bp of each other, it seems possible that they bind Lrp cooperatively.
Lrp is only one component of a regulatory network that also includes the FlhD2C2 and UmoB proteins, both of which have key roles in the control of flagellar biogenesis during swarming (Furness et al., 1997 ; Dufour et al., 1998
). Less is known about their involvement in regulating the production of virulence factors such as the HpmA haemolysin. We investigated the influence of Lrp, FlhD2C2 and UmoB on the transcription of hpmBA during the swarm cycle and found that they were all essential, as the hpmA transcript was not detected in any of the regulator mutants. In wild-type P. mirabilis, the peak level of hpmA transcript could be moderately increased by overexpression of lrp, flhDC and umoB, and while hpmA expression could be recovered in the regulator mutants by direct complementation (not shown), it was not restored by cross-complementation by the other regulators, e.g. by overexpression of flhDC in the lrp null mutant, which overcomes the cell elongation and swarming defects of this mutant but does not restore haemolytic activity (Hay et al., 1997
). This suggests that while Lrp, FlhD2C2 and UmoB can positively influence transcription of hpmBA, there is not a single pathway that controls haemolysin expression during swarming. Rather, regulation of hpmBA appears to be complex and multifactorial. The present study and previously published data suggest that Lrp directly regulates transcription of hpmBA while FlhD2C2 and UmoB could be acting indirectly. In parallel with our studies of hpmBA expression, we investigated briefly the effects of Lrp, FlhD2C2 and UmoB on transcription of the zapA protease gene. Unlike the HpmA haemolysin, which is maximally produced in actively swarming cells, peak production of the Zap protease occurs slightly later in the swarm cycle, in differentiated cells that are about to enter the consolidation phase (Walker et al., 1999
). We found that zapA regulation is markedly different to that of hpmBA, as Lrp is not required for zapA upregulation during swarming; instead, zapA appears to be primarily regulated by UmoB.
The data presented outline several components of hpmBA regulation, including differential mRNA stability, and indicate direct and indirect roles for several proteins in upregulating the single 70 operon promoter during swarming differentiation. Taken with the initial characterization of zapA expression, the data strengthen a view of swarming as a major physiological shift, involving a complex network of regulation.
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Received 13 February 2002;
revised 26 March 2002;
accepted 27 March 2002.