1 Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela. Caracas, Venezuela
2 Department of Medical Microbiology and Immunology, 1-63 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
Correspondence
Diane E. Taylor
diane.taylor{at}ualberta.ca
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ABSTRACT |
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Tables listing oligonucleotide primers (Table S1) and showing SD values for the effect of temperature and H-NS protein on the expression of tra promoters (Table S2) are available as supplementary data with the online version of this paper.
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INTRODUCTION |
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Conjugative plasmids belonging to the incompatibility group HI1 (IncHI1) encode multiple antibiotic resistances; they have been implicated as a significant factor in the persistence and re-emergence of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and therefore contribute to the persistence of typhoid fever worldwide (Kariuki et al., 2004; Wain & Kidgell, 2004
). The continued prevalence of IncHI1 plasmids can be partially attributed to the conjugative transfer functions of these plasmids, since horizontal gene transfer maintains a plasmid within a bacterial population. Therefore, the conjugative system is a promising drug target to inhibit the dissemination of plasmid-encoded antibiotic resistance.
IncHI1 plasmids are low-copy-number, self-transmissible plasmids that have an unusual thermosensitive process of conjugation. Transfer occurs optimally between 22 °C and 30 °C, but is negligible at 37 °C (Rodriguez-Lemoine et al., 1975; Taylor & Levine, 1980
). Despite the fact that this phenomenon was known three decades ago, the molecular basis for this temperature dependence has not been widely studied.
Plasmid transfer requires the expression of plasmid-encoded transfer genes (tra genes). R27, the prototypical IncHI1 plasmid, has been sequenced (Sherburne et al., 2000), and the transfer genes were found to be clustered in two separate regions on the plasmid, Tra1 and Tra2, which are separated by 64 kb. The Tra1 region contains the origin of transfer and 14 genes, nine of which are essential for transfer and encode Mpf components and the coupling and relaxosome proteins (Lawley et al., 2002
) (see Fig. 1a
). The Tra2 region contains 24 genes, including 11 which are essential for transfer and encode Mpf proteins (Lawley et al., 2003a
; Rooker et al., 1999
) (see Fig. 2a
). Although tra genes have been identified by sequencing and functional and mutational analyses, transcriptional regulation has not been examined and the promoter regions have not yet been determined.
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Studies on other temperature-dependent expression systems, mainly virulence genes, have shown that bacterial genes usually become derepressed on a shift from an ambient low temperature to body temperature upon entering a human host (Madrid et al., 2002; Prosseda et al., 2002
; Umanski et al., 2002
). In contrast, the effect of temperature on transfer of IncHI plasmids is the reverse, i.e. conjugation is repressed at 37 °C.
The goal of the present study was to analyse transcription of R27 transfer genes at 30 °C and 37 °C and to identify the operon organization and transcriptional start points. In addition, the promoters and regulatory regions were cloned and tested for expression. Our results suggest that R27 tra genes are controlled at the transcriptional level. This work provides new insights into the genetic structure, transcriptional organization and regulation of transfer genes on the R27 plasmid.
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METHODS |
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DNA manipulations.
R27 plasmid DNA was isolated using the Qiagen Plasmid Purification Kit. Standard recombinant DNA methods were performed as described by Sambrook & Russell (2001). Restriction endonucleases and T4 DNA ligase were used according to the manufacturers' instructions (Invitrogen, Roche Diagnostics) and digested DNA was analysed by agarose gel electrophoresis. DNA fragments were isolated from agarose gels using the QIAquick Gel Extraction Kit (Qiagen).
RNA extraction.
E. coli cultures harbouring R27 were grown at either 30 °C or 37 °C. Cells were inoculated from overnight cultures grown at the same temperature. Cells were harvested when the culture reached 0·3 OD600 units. Total bacterial RNA was purified using the RNeasy Midi Kit (Qiagen), in accordance with the manufacturer's directions. To ensure that the RNA was devoid of contaminating DNA, the preparation was treated with Turbo DNase (Ambion). The RNA was precipitated with ethanol, dissolved in an appropriate volume of diethyl-pyrocarbonate-treated water, and quantified using an Ultraspec 4000 spectrophotometer (Pharmacia).
RT-PCR.
For RT-PCR amplification of all transcripts, 2 µg total RNA was used as a template. RNA was retrotranscribed into cDNA utilizing the SuperScriptII RT with the random hexamers included in the kit (Invitrogen), generally according to the manufacturer's instructions. One-tenth of each product was subjected to PCR. Primer pairs specific for each two adjacent genes (see Table S1, provided as supplementary data with the online version of this paper) were designed using the R27 annotated sequence (GenBank accession no. AF250878). Typically, the annealing temperature was 5 degrees below the Tm of the primers. The PCR programme used was as follows: pre-incubation at 94 °C for 2 min, denaturing at 94 °C for 30 s, annealing for 30 s and extension at 72 °C for 1 min for every 1 kb of target sequence for 30 cycles, followed by 10 min further extension at 72 °C. Negative controls without template and negative control with RNA that had not been subjected to retrotranscription were included in each assay. A positive control using R27 DNA as the template was prepared with each pair of primers, generating products of the predicted sizes. The E. coli pfkA housekeeping gene, which encodes phosphofructokinase, was included as a positive control for the bacterial transcripts. Amplification products were resolved on an agarose gel and stained with ethidium bromide. RT-PCR amplifications were performed at least twice with total RNA preparations obtained from a minimum of three independent extractions and similar results were obtained in all experiments.
Rapid amplification of cDNA ends (RACE).
To determine the transcription initiation sites, 5'RACE experiments were performed using the protocol provided by the manufacturer (Invitrogen). DNase-treated total RNA (4 µg) was reverse transcribed by using SuperscriptII RT with specific primers for each operon (see supplementary Table S1). cDNAs were tailed by adding cytosine residues (or adenosine when necessary) at the 3' end by using Terminal Transferase and amplified by PCR with a second operon-specific primer. The PCR conditions used were 30 cycles consisting of 1 min denaturation at 94 °C, 1 min annealing at the appropriate temperature, and 1 min extension at 72 °C. The primary PCR products were amplified using a nested gene-specific primer. To precisely determine the 5' ends of the transcripts, DNA sequencing reactions were done in the DNA Services Laboratory, Department of Biological Sciences, University of Alberta. At least 20 different final nested-PCR products for each one of the six promoters were sequenced.
Computer analysis.
Prediction of intrinsic curvature and DNA sequence analysis was performed using the LASERGENE software package (DNASTAR, Inc.). Prediction of bacterial protein subcellular localization was performed using the PSORT program (Gardy et al., 2003). Prediction of DNA-binding domains was done using DBS-PRED (Ahmad et al., 2004
). The BLAST program (Altschul et al., 1990
) was used for homology searches of amino acid sequences within the SWISS-PROT, GenBank/EMBL and PIR databases.
Construction of CAT-transcriptional fusions.
Fragments containing each promoter were amplified by PCR (30 cycles) using R27 DNA as the template. The primer sequences used for cloning, as well as the sizes of the cloned regions, are listed in supplementary Table S1. The PCR products, which included terminal BamHIHindIII sites, were double digested and cloned in the low-copy-number vector pKK232-8 (Pharmacia LKB Biotechnology), which contains a promoterless chloramphenicol acetyltransferase (CAT) gene, digested with the same enzymes. Transcriptional fusion constructs were transformed into E. coli cells, after which ampicillin-resistant colonies were selected. The resulting plasmids (Table 1) were sequenced to confirm the fidelity of the PCR amplification.
Two serial deletion constructions of the R operon upstream region were created by PCR (Table 1, Fig. 4
). The PCR products were digested with BamHI/HindIII and the resulting fragments were cloned in the pKK232-8 vector, leading to the construction of plasmids pR300 and pR200. These clones included the ATG initiation codon. The resulting plasmids were sequenced and transformed into E. coli cells and also into E. coli cells harbouring R27. CAT expression of the resulting transformants was determined.
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Conjugation assay.
Conjugal transfer of derepressed R27 (drR27) was performed as previously described (Taylor & Levine, 1980). A derepressed donor strain (Whelan et al., 1994
) was used to achieve a satisfactory degree of reproducibility, since consistently reproducible numbers of transconjugants were not obtained when a wild-type repressed donor was used. As transfer of R27 is dependent on the temperature at which the donor cells have been grown prior to mating, both donor and recipient cultures were pre-grown overnight at the appropriate temperature. Overnight cultures of E. coli donor and recipient strains were diluted 20-fold and incubated for 3 h with aeration at 30 °C or 37 °C. A mating mixture consisting of 0·1 ml of the donor, 0·4 ml of the recipient culture and 0·5 ml culture medium was incubated overnight at the tested temperature. Transconjugants were diluted and plated in duplicate on LB medium supplemented with the appropriate antibiotics. Each set of matings was repeated three times. Transfer frequencies were expressed as transconjugants per donor cell.
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RESULTS |
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Within the Tra1 region, the following three operons were found: (1) The F operon, containing trhF, trhH and trhG genes, (2) the R operon, which contains trhR and trhY genes, and (3) the H operon, which comprises traH, orf121, traI, traG, orf118, traJ, orf116 and orf115 (Fig. 1a). The F operon and the R operon are transcribed in the same direction and away from oriT. The H operon is transcribed in the opposite direction and contains genes that encode relaxosome proteins, the coupling protein and other proteins not essential for transfer. These results were in agreement with the deduced operon organization assigned to this region by sequence analysis (Lawley et al., 2002
). Nevertheless, there was a discrepancy with the trhX gene, whose transcript could not be detected under the conditions used in the present work. Primer sets 1-3, 1-4 and 1-5 failed to amplify a product in the RT-PCR reaction (Fig. 1
), supporting the assignment of the above three operons.
In the Tra2 region, the following three operons were found: (1) the AC operon, containing trhA, trhL, trhE, trhK, orf30, trhB, orf28, orf27, trhV and trhC genes, (2) the Z operon, which contains trhO, orf16, orf17 and trhZ genes, and (3) the AN operon, which contains htdA, htdF, htdK, orf9, trhP, trhW, trhU and trhN genes (Fig. 2a). The AC and AN operons encode proteins responsible for mating pair formation and pilus biosynthesis and are transcribed in the same direction. The Z operon, encoding entry exclusion proteins (J. Gunton & D. E. Taylor, unpublished), is transcribed in the opposite direction.
Transcriptional expression
Identification of tra operons enabled us to perform transcriptional studies for all the transfer genes. To determine if transcriptional control is responsible for temperature-sensitive R27 transfer, RT-PCR was carried out using RNA isolated from bacteria harbouring R27 grown at both 30 °C and 37 °C, using the same primer sets as above.
Results obtained from the F and R operons in the Tra1 region (Fig. 3a) and the AC operon in the Tra2 region (Fig. 3b
) revealed that tra transcripts were consistently present at higher levels at 30 °C than 37 °C. The constitutively expressed E. coli pfkA gene, encoding phosphofructokinase, was used as a control. Transcripts of tra genes from cells growing at 37 °C were lower when compared to the levels of RNA isolated from cells grown at 30 °C. Although the control, pfkA (Fig. 3b
, lane A), had significantly less of the total RT-PCR product loaded, this did not affect the result that the tra operons are transcriptionally downregulated at 37 °C.
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Transcriptional start sites
To characterize each of the six tra operon promoters and putative regulatory regions, we verified the transcriptional start site by the 5'RACE procedure. The 5' ends of these six transcripts, corresponding to the respective transcription initiation sites, were identified (Fig. 4). A single G residue was detected as the start site for the AC and AN operons, whereas the other four operons had single A residues as the start site. The 5' untranslated regions (UTR) are 1434 bp long, except those of the R and AN operons, which are 136 bp and 137 bp, respectively (Fig. 4b, f
). Analysis of the upstream regions of the R, H and AN operons revealed the presence of putative 35 and 10 boxes, with three to five of the six positions matching the E. coli consensus sequence. The positions of the transcriptional start sites are consistent with the locations of the deduced promoter sequence elements. The 35 and 10 boxes are separated by 17 or fewer base pairs, except those of the H and AN operons, which have longer spacer regions, 21 and 20 bp respectively (Fig. 4c, f
). For the F, AC and Z operons, the promoter boxes could not be definitely assigned. The absence of consensus sequences and/or canonical spacing between the 10 and 35 boxes could have implications for the regulation of operon expression. The F and AC operons contain genes that encode major structural proteins; therefore these genes must be tightly regulated without canonical promoter regions. Also, the presence of several upstream AT tracts was found in all the sequences analysed.
Interestingly, the origin of transfer is contained within the 5' UTR mRNA of trhRY (Fig. 4b). The TrhR and TrhY proteins are required for conjugation (Lawley et al., 2002
). These proteins have homologous counterparts in the IncHI2 R478 plasmid (GenBank accession no. BX664015), but show no homology to any component of other known conjugative systems. They may represent T4SS components that are unique to IncHI plasmids. Since genes that regulate conjugative transfer are generally present around oriT and are the least well-conserved components of transfer systems, trhR and trhY may play a role in regulating conjugative transfer. Consistent with this idea, overexpression of the trhR and trhY genes increased the transfer frequency of R27 (Lawley et al., 2002
). With this in mind, we searched for domains in TrhR and TrhY proteins. trhR encodes a protein, predicted to be an inner-membrane protein, with 50 % identity to the Actinobacillus pleuropneumoniae ApfC protein (GenBank accession no. AA064353), which is essential for type IV pilus production. In Legionella pneumophila, the homologous protein PilC has increased expression at 30 °C as opposed to 37 °C (Liles et al., 1998
). These proteins with functional similarity failed to complement mutants in related organisms (Johnston et al., 1998
), suggesting that they could be system-specific. trhY encodes a protein predicted to be a cytoplasmic protein with 85·5 % probability to bind DNA. TrhY shows 50 % identity with the transcriptional repressor of the xylose operon from Bacillus halodurans (GenBank accession no. F83994).
The origin of transfer is surrounded by the divergent promoters of the R and H operons, with A/T-rich regions, positioned as tracts. The presence of A tracts has been proposed to bend DNA in a way that is sensitive to temperature. The bend is stable below 30 °C, whereas the stability is reduced above 33 °C (Chan et al., 1993; Falconi et al., 1998
; Rohde et al., 1999
). Analysis of upstream promoter sequences revealed potential H-NS and FIS binding sites in the region between the divergent promoters of the R and H operons. In silico analysis of the remaining upstream tra promoter regions demonstrated the presence of DNA curve regions in the three Tra1 operons and in the AC and Z operons (Fig. 4
). The upstream region of the AN operon appears to be deficient in regions containing bends. Binding of H-NS to the oriT region has been recently reported (Forns et al., 2005
).
Functional analysis of the promoter regions
Identification of the transcriptional start sites of the tra operons enabled cloning of the promoters and potential regulatory regions. To address the question of how promoters are regulated, we used a CAT reporter gene fused to each tra promoter, including upstream regions (as described in Methods, Fig. 4). These clones were transformed into E. coli cells and E. coli cells harbouring the R27 plasmid grown at 30 °C or 37 °C, and CAT expression was determined. The expression values at 30 °C were normalized to 1 for comparative purposes (Table 2
). At 37 °C, the constructs exhibited the same (AC and AN) or lower (R, F and Z) expression level, except for the H operon (relaxosome operon), which showed higher expression, compared to that seen at 30 °C.
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Characterization of the upstream promoter regions containing intrinsic DNA curvature suggests that nucleoid-associated proteins can bind at this position. To test the influence of these proteins, transcriptional fusions were introduced into an isogenic E. coli hns mutant strain and also into the hns mutant carrying the R27 plasmid, and the transcriptional activities were measured (Table 2). Except for the Z promoter, in the presence of pR27, little difference was observed in transcription levels between 30 °C and 37 °C in the H-NS-deficient E. coli strain, compared with that obtained in wild-type background. These results indicated that the H-NS protein is involved in negative regulation of tra genes transcription, in agreement with recently published data (Forns et al., 2005
). The effect of the hns mutation is more obvious in the presence of the R27 plasmid than in its absence, except for the Z operon, whose transcription seemed to be H-NS independent. These results confirm that the H-NS protein is required for the thermosensitive repression activity of R27.
In the presence of R27, both AN and H operons exhibited higher expression at 37 °C than that observed at 30 °C in the wild-type host background. These results are contradictory to those obtained using RT-PCR. However, analysis of transcription directed by the cloned region excluded the possibility of detection of global regulation, or post-transcriptional processing or degradation. The increased expression of both operons at the temperature that is non-permissive for mating suggests that some factors, encoded by any one of the tested operons, are required to inhibit conjugation in a temperature-dependent fashion. The AN operon contains the htdA gene, which has been suggested to be a negative regulator (Whelan et al., 1994). Nonetheless, the negative effect of pAN expression is influenced by the presence of H-NS protein, the temperature shift and the presence of R27, as well as perhaps some cis or trans element.
To determine if curved DNA is implicated in transcriptional regulation, we generated deletions that would eliminate the potential bend in the DNA. Two different deletions in the upstream region of the R operon were analysed (Fig. 4). Deletions of the curved regions caused derepression of transcription at the non-permissive temperature (Table 2
), indicating that temperature sensitivity was lost. This occurred even in the presence of the parental plasmid, suggesting the presence of negative regulatory sites between bases 167 and +1. To investigate if H-NS might be involved in the loss of temperature-sensitive regulation due to deletions of the R operon upstream region, the constructs were also introduced into hns-deficient cells (Table 2
). The results showed that H-NS repression is lost.
Some promoters that bind nucleoid-associated proteins are extremely sensitive to variations in the superhelical density of the DNA. To determine if DNA supercoiling is related to H-NS and curved DNA regulation, transcriptional analysis was performed using cells grown in media with subinhibitory concentrations of DNA gyrase inhibitor novobiocin. The tra promoters were inhibited to some extent at the same level at both temperatures (data not shown), but still responded to temperature regulation, indicating that DNA supercoiling does not modulate temperature-dependent regulation.
Mating
To confirm the in vitro results, we determined the frequency of transfer using different conditions known to play a role in the regulation of other bacterial genes and evaluated if these conditions altered the transfer frequency at both temperatures. The results showed that acidic pH, phase of growth, anaerobiosis, high osmolarity, as well as inhibition of DNA gyrase and presence of quorum-sensing molecules (homoserine lactone), had no significant effect on transfer frequency at either temperature (Table 3). When the hns or fis mutant harbouring R27 was used as the donor in mating assays, the transfer frequency was slightly reduced.
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DISCUSSION |
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Like those of the plasmid RK2 (incompatibility group P) and the Ti plasmids (Farrand et al., 1996; Zatyka et al., 1994
) the transfer genes of R27 are clustered into two regions, and they also share the same organization of two divergently expressed operons flanking the oriT region. One of these operons encodes TrhR and TrhY proteins, which might be a part of the regulatory system, involved in the control of transcription of the tra genes. Further studies are required to confirm or refute this idea.
The data presented here suggest that regulation of gene expression by nucleoid-structuring proteins may be more widespread in bacteria than previously documented. H-NS directly or indirectly affects the expression of several genes that are regulated in response to temperature or other environmental conditions (reviewed by Dorman, 2004). A recent publication has implicated Hha and H-NS proteins, both plasmid and chromosomal, in temperature-sensitive conjugative transfer of R27 (Forns et al., 2005
). Virulence and flagellar genes in Yersinia enterocolitica (Pepe et al., 1994
; Rohde et al., 1994
), virulence genes in Shigella (Falconi et al., 1998
; Prosseda et al., 2004
, 2002
) and in enteropathogenic E. coli (Umanski et al., 2002
), lipid A myristoyl transferase and haemolysin in E. coli (Madrid et al., 2002
; Yoon et al., 2004
) and the urease gene cluster in Proteus mirabilis (Poore & Mobley, 2003
) are also regulated by H-NS. Usually thermosensitive proteins that modulate expression bind DNA at low temperatures and permit expression at higher temperatures; however, the opposite pattern of thermosensitivity was observed in R27.
DNA superhelicity has been proposed to serve as a global regulator of gene expression (Hatfield & Benham, 2002). The absence of H-NS in the cell results in an increased degree of negative supercoiling in both plasmid and chromosomal DNA. An association between changes in DNA topology regulated by H-NS protein, regulation of gene expression and environmental conditions has been reported (Asakura et al., 2004
; Gallant et al., 2004
; Krishnan et al., 2004
). In other systems, DNA supercoiling is promoted by high temperature, high osmolarity and anaerobic conditions, and the addition of novobiocin and homoserine lactone. The data presented here show that these conditions do not significantly affect the transfer frequency of R27 plasmid at 37 °C.
The assembly of the transfer apparatus and coordinated transfer of plasmid DNA represent a complex biological process that is undoubtedly energetically demanding. It is therefore not surprising that conjugation is a tightly regulated process, which responds to environmental changes. Our data suggest that the tra promoters are strongly regulated, and their expression was repressed in the presence of plasmid R27. Literature reports suggest that the nucleoid-associated proteins do not usually exert regulatory functions alone. Each has at least one additional regulator. This is in agreement with our findings, which show that a putative R27-encoded repressor protein could be involved in the inhibition of the promoters at 37 °C. In plasmid RK2, it has been reported that relaxosome proteins repress oriT (Zatyka et al., 1994). One of these proteins, TraK, binds to the oriT region, increasing the bending of this stretch of DNA. In plasmid R1162, promoters of the mob genes, required for conjugative mobilization, are negatively regulated at oriT by the Mob proteins.
Steck et al. (1993) showed that the length of the spacer region between the 35 and 10 elements is related to the optimal expression for promoters. Those promoters which have a maximum expression at high levels of supercoiling commonly have spacers that are larger than 17 bp. The H and AN promoters have the larger spacing region between the 35 and 10 elements identified (Fig. 4
) and also exhibit higher expression at 37 °C (Table 2
). While further investigation is required, it is tempting to suggest that any of the proteins encoded by these operons can repress the expression of other proteins essential for R27 transfer.
Bacteria harbouring H plasmids have been found in clinical environments as well as in soil, sewage and fresh water. These plasmids were shown to be promiscuous and to transfer at environmental temperatures (Maher & Taylor, 1993; Smith et al., 1978
). In addition to antibiotic resistance, other temperature-sensitive conjugative H plasmids encode determinants for resistance to a range of antimicrobial agents, including heavy metals, tellurium and channel-forming colicins (Alonso et al., 2002
; Rodriguez-Lemoine, 1992
). This confers considerable selective advantages upon host cells, allowing for survival in hostile and rapidly changing environmental conditions and enhancing the dissemination of resistance among pathogenic and indigenous bacterial species in water and soil environments (Maher & Taylor, 1993
). Plasmid- and host-encoded proteins, such as H-NS, seem be part of the regulatory mechanism of IncH plasmid conjugation in response to temperature changes. This has implications for successful IncHI1 plasmid transfer and maintenance in the population. Knowledge of the regulation mechanism of plasmid transfer could be used to help reduce the transfer of conjugal plasmids in their natural niche.
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ACKNOWLEDGEMENTS |
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Received 10 June 2005;
revised 17 August 2005;
accepted 13 September 2005.
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