Excision dynamics of Vibrio pathogenicity island-2 from Vibrio cholerae: role of a recombination directionality factor VefA
© Almagro-Moreno et al; licensee BioMed Central Ltd. 2010
Received: 19 August 2010
Accepted: 30 November 2010
Published: 30 November 2010
Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758) that belongs to the tyrosine recombinase family. IntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate.
We determined the conditions and the factors that affect excision of VPI-2 in V. cholerae N16961. We demonstrate that excision from chromosome 1 is induced at low temperature and after sublethal UV-light irradiation treatment. In addition, after UV-light irradiation compared to untreated cells, cells showed increased expression of three genes, intV2 (VC1758) , and two putative recombination directionality factors (RDFs), vefA (VC1785) and vefB (VC1809) encoded within VPI-2. We demonstrate that along with IntV2, the RDF VefA is essential for excision. We constructed a knockout mutant of vefA in V. cholerae N16961, and found that no excision of VPI-2 occurred, indicating that a functional vefA gene is required for excision. Deletion of the second RDF encoded by vefB did not result in a loss of excision. Among Vibrio species in the genome database, we identified 27 putative RDFs within regions that also encoded IntV2 homologues. Within each species the RDFs and their cognate IntV2 proteins were associated with different island regions suggesting that this pairing is widespread.
We demonstrate that excision of VPI-2 is induced under some environmental stress conditions and we show for the first time that an RDF encoded within a pathogenicity island in V. cholerae is required for excision of the region.
VPI-1 and VPI-2 do not share any genes in common but do share some functional characteristics such as the ability to integrate into the chromosome, specifically at a tRNA site using an integrase belonging to the tyrosine recombinase family [16, 18, 23, 26, 28]. VPI-2 integrates into chromosome 1 at a tRNA-serine locus, whereas VPI-1 is located at the tmRNA locus. Both regions are flanked by direct repeats (DRs) named attL and attR[16, 18, 23, 26, 28]. These integrases, IntV1 (VC0847) and IntV2 (VC1758), are believed to mediate insertion into the host chromosome through site specific recombination between an attachment site attP, present in the pathogenicity island, and attB, present in the bacterial chromosome.
Pathogenicity islands have been shown to excise from their host genome in pathogenic Escherichia coli and Yersinia species [29–36]. In E. coli strain 536, a uropathogenic isolate, Hacker and colleagues have identified six PAIs, all of which encode a tyrosine recombinase integrase and are flanked by DRs [31, 33, 36–39]. They demonstrated that PAI-I, II, III and V can excise from the chromosome by site-specific recombination involving their respective DRs (attL and attR) [31, 33]. The PAIs were shown to excise at different frequencies depending on the growth conditions [31, 33]. Likewise, both VPI-1 and VPI-2 have been shown to excise from their host chromosome [23, 28]. Rajanna and colleagues demonstrated that VPI-1 can excise from V. cholerae N16961 at very low rates . They determined that the integrase IntV1 (VC0847) was not essential for excision since a transposase within the region appeared to compensate for an IntV1 knockout . Recently, Murphy and Boyd demonstrated that VPI-2 from V. cholerae N16961 can excise from chromosome 1, which also occurred at very low frequency under optimal growth conditions . Their study showed that IntV2 (VC1758) was essential for excision and the formation of a circular intermediate (CI) . Pathogenicity islands from both E. coli and V. cholerae are non-self mobilizable, they do not encode any proteins such as those for phage structural proteins or conjugation systems needed for cell to cell mobility [23, 28, 31, 33, 36–39]. The mechanism of transfer for most pathogenicity islands remains to be elucidated but likely involves hitchhiking with plasmids, conjugative transposons, Integrative and Conjugative Elements (ICEs), or generalized transducing phages or uptake by transformation.
It is known that for some mobile and integrative genetic elements (MIGEs) the presence of a recombination directionality factor (RDF)/excisionase is required for excision [40, 41]. For instance, Xis is required for the excision of the ICE SXT from V. cholerae, Hef from the High Pathogenicity Island of Yersinia pestis, and Rox from the Shigella Resistance Locus (SRL) of Shigella flexneri. RDFs are small basic proteins that bind and bend DNA on the recombination sites attL and attR triggering excision by coordinating the assembly of the excisive intasome [43–45]. In addition, some RDFs have been found to inhibit reintegration of the CI by converting attP into a catalytically inactive structure and are thought to stabilize the appropriate positioning of the integrase within the excisive intasome [46–48]. To date, no RDFs have been identified in E. coli or V. cholerae pathogenicity islands.
Here, we report the environmental conditions that induce excision of VPI-2. We examined the VPI-2-encoded factors that are required for VPI-2 excision, determining that V. cholerae cells subjected to stress conditions showed an increase in the excision levels of VPI-2 compared to cell grown at optimal conditions. Bioinformatic analysis of the VPI-2 region identified two open reading frames (ORFs) VC1785 and VC1809 that show homology to previously described RDFs, which we named VefA and VefB. We examined the role of these genes in VPI-2 excision.
Bacterial strains and growth conditions
Bacterial strains and plasmids used in this study.
Genotype and/or phenotype
O1 El Tor, VPI-2 +, SmR
N16961, ΔVC1758, SmR
RAM-1, pIntV2, SmR CmR
N16961, ΔVC1785, SmR
N16961, ΔVC1809, SmR
SAM3, pVefA, SmR CmR
N16961, pBAD33, SmR CmR
RAM-1, pBAD33, SmR CmR
SAM-3, pBAD33, SmR CmR
Suicide plasmid, CmR, SacB
Expression plasmid, Ara, CmR
vc1758 cloned into pBAD33
ΔVC1785 cloned into pDS132
ΔVC1809 cloned into pDS132
vc1785 cloned into pBAD33
Determination of VPI-2 excision rate
Oligonucleotide primers used in this study.
Sequence (5'-3'). Restriction site underscored
attB (VPI-2) (F-R)
BLAST search was performed using Xis (ABA87014), an RDF from V.cholerae SXT ICE element required for excision, AlpA, a well known RDF from E. coli (AAA18418) and the Hef protein (NP_405464) from Y. pestis pathogenicity island, as seeds on the genome sequence of V. cholerae N16961 . DNA sequences from putative RDFs were downloaded from GenBank and the sequences were aligned using ClustalW . Next, the protein sequences of characterized RDFs were used as seeds to perform BLASTN and BLASTP searches against Vibrio genomes sequences in the database . The retrieved sequence must give an e-value below 10-3, relative to known RDFs.
RNA extraction and Real-Time quantitative PCR (QPCR)
Total RNA from V. cholerae N16961 was extracted 12 hours post-inoculation in LB broth from one group treated with sub-lethal UV-irradiation and one group untreated as follows. The cells from 5 mLs 11 hours growth cultures were pelleted and resuspended in 5 mLs of PBS. A 100 uL aliquot was taken from each sample prior to treatment to calculate colony forming units (CFUs). Each 5 mL sample was placed in a plastic Petri dishes without a cover and one set of samples was irradiated with a sublethal dose of 25 J/m2 of UV irradiation in a Fisher Scientific UV cross linker (FB-UVXL-1000) and the other 5 mL set of samples was left untreated as previously described by others . The cells from both UV treated and untreated samples were recovered, pelleted, resuspended in 5 mLs of LB broth and grown for 1 hour at 37°C. A 100 uL aliquot was taken from each sample to calculate CFUs post treatment from both sets of samples. The CFU counts pre and post treatment were identical at ~9.75 × 109/ml as expected. Every experiment was performed in triplicate. Total RNA was extracted from each culture using RNAprotect Bacteria reagent (Qiagen, Valencia, CA) and an RNeasy mini kit (Qiagen) according to the manufacturer's protocols. RNA purity and the presence of genomic DNA were assessed using an ND-1000 NanoDrop UV-Vis spectrophotometer (NanoDrop Technologies) giving values of A260/A280 > 2.0 and A260/A230 > 2.0 indicating of no protein and solvent contamination, respectively. In addition, 1 μg of each sample of RNA was run on a 1% agarose gel in 1× TBE buffer to examine quality of the samples. RNA was measured to calculate the volume of sample to be added to perform a reverse transcriptase (RT) reaction using SuperScript II Reverse Transcriptase and random hexamers following manufacturer's instructions (Invitrogen). The purity and quantity of cDNA was examined using an ND-1000 NanoDrop UV-Vis spectrophotometer as above. QPCR was performed using standard protocol using primer pairs for vc1758, vc1785, vc1809 and vc0432 (intV2, vefA, vefB and mdh, respectively) listed in Table 2 using SYBR green PCR Master Mix (Invitrogen) on an Applied Biosystems 7000 Real Time PCR System (Foster City, CA). To confirm that primer pairs only amplified target genes to assure accurate quantification of the results, non-template controls were included in each replicate. The intV2, vefA, vefB and mdh PCR products were visually checked on agarose gels. The melting curves of PCR products were used to ensure the absence of primer dimers, contamination with genomic DNA and non-specific homologous sequences. The data was analyzed using ABI PRISM 7000 SDS software (Applied Biosystems). Differences in the gene ratios were extrapolated using the delta-delta Ct method . Every sample was assayed in triplicate and each experiment was performed using a minimum of three different samples.
Construction of mutant strains
To construct the mutant strains, primers were designed to conduct Splice Overlap Extension (SOE) PCR followed by allelic exchange . SOE PCR primers were designed to produce non-functioning constructs of the 204-bp vefA and the 228-bp vefB genes. The size of the regions removed from vefA and vefB is 169-bp and 191-bp, respectively and were constructed in V. cholerae strain N16961 to create mutant strains V. cholerae SAM-3 and SAM-4, respectively (Table 1). Primer pairs SOEVC1785A/SOEVC1785B and SOEVC1785C/SOEVC1785 D were used to amplify PCR products from VC1785 from V. cholerae strain N16961 (Table 2). The ligated product was amplified with primer pair SOEVC1785A and SOEVC1785 D, which was restricted with enzymes, XbaI and SacI and ligated with pDS132 (New England Biolabs) resulting in pΔ1785. pΔ1785 was transformed into E. coli strain DH5αλpir, plasmid purified and then transformed into E. coli β2155 cells. E. coli β2155 transformants were conjugated with N16961. V. cholerae cells were passaged in LB-suc to cure them of the integrated pΔ1785. PCR was used to screen for V. cholerae strains in which the wild type gene was replaced by the mutant gene, which was confirmed by sequencing. The Δ1785 strain was designated V. cholerae strain SAM-3. A knockout mutant of VC1809 was constructed in N16961 as described above using primer pairs listed in Table 2. Complementation of RAM-1 and SAM-3 mutant strains were generated by creating pIntV2 and pVefA, by cloning intV2 (VC1758) and vefA (VC1785), respectively into the SacI/XbaI sites of the expression plasmid pBAD33 (New England Biolabs) using standard cloning protocol (Table 1 and 2).
Results and Discussion
VPI-2 excision rates under different growth conditions
It was previously shown that the four pathogenicity islands identified in V. cholerae N16961 can excise from chromosome 1 and form circular intermediates (CI) [23, 28]. The excision of VPI-1 and VPI-2 occurs at very low levels suggesting that excision is tightly controlled, although it may also suggest that the excision event is inefficient, possibly due to poor expression of the regulatory genes, an altered regulatory circuit, or mutations that might occur in these sequences as the region become evolutionarily integrated into the host chromosome [23, 28]. First, we quantified the excision levels of VPI-2 in cultures of V. cholerae N16961 grown for 12 hours in LB at 37°C (standard conditions) by measuring the presence of attB, the locus present on the chromosome after VPI-2 excises (Figure 1), and comparing it with the housekeeping gene mdh using QPCR. We used attB as a surrogate for VPI-2 excision measurements since the copy number of attP in the CI is minuscule compared to attB, which replicates along with the rest of the chromosome unlike excised VPI-2. We compared the presence of attB with mdh since all cells encode one functional copy of the latter. PCR products of attB and mdh were visually checked on an agarose gel and their melting temperature analyzed to ensure we had the correct PCR products. The reference gene was assayed both separately and in the same reaction. Both primer pairs used were tested by comparing the results obtained using previously quantified cloned copies of mdh and attB and gave comparable results. We found that attB was present in 1 in every 1.6 (±0.2) × 106V. cholerae cells under optimal growth conditions.
VPI-2 encodes two novel recombination directionality factors
IntV2 and VefA are essential for the excision of VPI-2
Next, we determined whether one, both, or neither of the putative RDFs uncovered by our bioinformatic analysis are required for VPI-2 excision. To do this, we constructed in-frame deletion mutations in each gene to create mutant strain SAM-3 (ΔvefA) and SAM-4 (ΔvefB). The two mutant strains and the wild-type N16961 were each inoculated into LB and all three strains grew similarly indicating that the mutant constructs did not have any general growth defect (data not shown). We determined the attB levels using QPCR in strain SAM-3 compared to the wild-type strain grown under the same conditions. We found that no VPI-2 excision occurs in SAM-3 cells when compared with the wild type, indicating that a functional copy of vefA is essential for efficient excision of VPI-2 (Figure 5). We complemented SAM-3 with a functional copy of vefA (SAM-5) and measured attB levels in these cells with the wild type levels both under standard conditions, to find that some excision occurred, but it was less than in wild-type cells (Figure 5). In our vefB mutant strain (SAM-4), we found no difference in VPI-2 excision levels compared to wild-type grown under the same conditions, which demonstrates that vefB is not essential for excision (Figure 5). From these data it appears that vefA is the cognate RDF for VPI-2 excision. In our control experiments, transformation of SAM-3 with pBAD33 alone (resulting in strain SAM-13) did not affect attB levels (data not shown).
Vibrio species island-encoded integrases with corresponding RDFs
Locus tags for integrases and corresponding RDFs identified in this study.
Vibrio cholerae N16961*
Vibrio cholerae TM 11079-80
Vibrio cholerae TMA21
Vibrio cholerae 12129(1)
Vibrio cholerae V51
Vibrio cholerae 1587
Vibrio cholerae CT 5369-93
Vibrio cholerae RC385
Vibrio cholerae TMA 21
Vibrio cholerae MZO-3
Vibrio cholerae 12129(1)
Vibrio cholerae N16961*
Vibrio cholerae MZO-3
Vibrio vulnificus YJO16*
Vibrio vulnificus YJ016*
Vibrio vulnificus YJO16*
Vibrio furnissii CIP 102972
Vibrio furnissii CIP 102972
Vibrio coralliilyticus ATCC BAA-450
Vibrio sp. Ex25
Vibrio sp. RC341
Vibrio sp. MED222
Vibrio splendidus 12B01
Vibrio parahaemolyticus AQ3810
Vibrio parahaemolyticus K5030
Vibrio parahaemolyticus AQ3810
Vibrio harveyi HY01
From our analysis, no RDF was identified within the VPI-1 or the VSP-I regions in N16961 or within homologous regions in the other 27 sequenced strains of V. cholerae in the database. Both the VPI-1 and VSP-I regions have been shown to excise from their chromosome location, and VPI-1 encodes a tyrosine recombinase with homology to IntV2, thus they may therefore use an alternative mechanism of excision or perhaps co-opt an RDF from another region on the genome. Overall our data indicates that the presence of both an integrase and a cognate RDF pairing is a relatively conserved feature but not an essential one.
In this study, we analyzed the excision dynamics of VPI-2 encoded within V. cholerae N16961. Our results indicate that excision is controlled by at least two conserved factors within the island, an integrase encoded by intV2 and an RDF encoded by vefA, whose expression is induced by environmental stimuli similar to other MIGEs such as prophages, ICEs and integrons. We identified two putative RDFs and found that of the two we identified, only one VefA is essential for the efficient excision of VPI-2. We determined the occurrence of RDFs among the genomes of sequenced Vibrio species and found 27 putative RDFs that also had a homologue of IntV2 associated with it, which suggests that requirement for both an RDF and a corresponding integrase is a relatively common feature.
This research was supported by National Science Foundation CAREER award DEB-0844409 to E.F.B.
The authors declare no conflicts of interest.
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