A Conserved Helicase Processivity Factor Is Needed for Conjugation and Replication of an Integrative and Conjugative Element

Integrative and conjugative elements (ICEs) are agents of horizontal gene transfer and have major roles in evolution and acquisition of new traits, including antibiotic resistances. ICEs are found integrated in a host chromosome and can excise and transfer to recipient bacteria via conjugation. Conjugation involves nicking of the ICE origin of transfer (oriT) by the ICE–encoded relaxase and transfer of the nicked single strand of ICE DNA. For ICEBs1 of Bacillus subtilis, nicking of oriT by the ICEBs1 relaxase NicK also initiates rolling circle replication. This autonomous replication of ICEBs1 is critical for stability of the excised element in growing cells. We found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication of ICEBs1. Our results indicate that this gene, helP (formerly ydcP), encodes a helicase processivity factor that enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA. HelP was required for both conjugation and replication of ICEBs1, and HelP and NicK were the only ICEBs1 proteins needed for replication from ICEBs1 oriT. Using chromatin immunoprecipitation, we measured association of HelP, NicK, PcrA, and the host-encoded single-strand DNA binding protein Ssb with ICEBs1. We found that NicK was required for association of HelP and PcrA with ICEBs1 DNA. HelP was required for association of PcrA and Ssb with ICEBs1 regions distal, but not proximal, to oriT, indicating that PcrA needs HelP to progress beyond nicked oriT and unwind ICEBs1. In vitro, HelP directly stimulated the helicase activity of the PcrA homologue UvrD. Our findings demonstrate that HelP is a helicase processivity factor needed for efficient unwinding of ICEBs1 for conjugation and replication. Homologues of HelP and PcrA-type helicases are encoded on many known and putative ICEs. We propose that these factors are essential for ICE conjugation, replication, and genetic stability.


Introduction
Integrative and conjugative elements (ICEs), also known as conjugative transposons, are mobile genetic elements that play a significant role in bacterial evolution and the acquisition of new traits [1]. They contribute significantly to the spread of antibiotic resistances in pathogenic bacteria. ICEs or putative ICEs are found in all major bacterial clades [2]. They reside integrated in a host genome and are propagated along with the host chromosome. Under certain conditions, an ICE can excise from the chromosome, form a double-stranded DNA (dsDNA) circle, and transfer to a recipient. Like conjugative plasmids, ICEs encode a multicomponent mating pore complex that mediates their transfer from donors to recipients. Most ICEs are thought to transfer linear ssDNA. Transfer is through a type IV secretion system in Gram negative bacteria [3,4], or its counterpart in Gram positive bacteria [5]. ICEs that transfer ssDNA were generally thought to lack the ability to undergo autonomous replication. However, recent work [6][7][8] and findings presented here indicate that autonomous replication is a property of many ICEs and that the mechanisms are conserved.
ICEBs1 is approximately 20 kb and normally found integrated in the tRNA gene trnS-leu2 of Bacillus subtilis [9,10]. ICEBs1 gene expression and excision can be induced in .90% of cells in a population by overproduction of the activator and cell signaling regulator RapI [9]. Following induction, ICEBs1 undergoes autonomous plasmid-like rolling circle replication [6]. Replication of ICEBs1 is needed for stability of the element after excision [6].
ICEBs1 replication and conjugation both begin with nicking of the ICEBs1 origin of transfer, oriT, by the ICEBs1-encoded relaxase NicK. The nicked DNA is then unwound by the hostencoded helicase PcrA, rather than the replicative helicase (B. subtilis DnaC) [6]. During rolling-circle plasmid replication, the free 39-OH of the nicked strand acts as a primer for replication by the host DNA polymerase, followed by recircularization of and complementary strand synthesis from the unwound single strand. By analogy to other conjugative systems, the single strand of ICEBs1 DNA covalently attached to the relaxase can also be targeted to the mating machinery by the putative coupling protein ConQ and transferred into recipient cells. Although unwinding of ICEBs1 DNA by the PcrA helicase is essential for both replication and conjugation of ICEBs1, replication of the element in donor cells is not required for its transfer to recipients [6].
PcrA-type helicases (PcrA from Gram positive bacteria and UvrD and Rep from E. coli) are required for rolling circle replication of many different plasmids and phages. The PcrA-type proteins are efficient and processive DNA translocases, but typically have poor helicase activity. For many of the characterized phages and plasmids, the element-encoded relaxase that is needed for initiation of replication interacts with the PcrA-type helicase to stimulate DNA unwinding {reviewed in [11,12,13]}.
Unlike these other relaxases, we found that the ICEBs1 relaxase NicK was not sufficient for ICEBs1 replication. In addition to nicK, a second ICEBs1 gene (helP, previously ydcP) was necessary for replication from ICEBs1 oriT. Expression of both nicK and helP in B. subtilis was sufficient to support replication from oriT. helP encodes a protein of previously unknown function and is conserved in many ICEs. We found that HelP is required for both mating and replication of ICEBs1, and that it stimulates the function of the helicase PcrA. We also found that the E. coli helicase UvrD (a homologue of PcrA) can substitute for PcrA in B. subtilis, to support both cell viability and ICEBs1 conjugation and replication. Based on in vivo and in vitro analyses, HelP is a helicase processivity factor that is needed for efficient unwinding of ICEBs1.
helP homologues are found in many ICEs, often in a module with genes encoding the relaxase and the putative coupling protein, indicating that these ICEs may also be capable of autonomous replication. PcrA homologues are also found on many extrachromosomal elements, either separately or as a helicase domain attached to the relaxase, indicating that these elements all share a need for DNA unwinding that is met in different ways.

Results
Two ICEBs1 genes, nicK and helP, are sufficient for autonomous replication from the ICEBs1 oriT in B. subtilis Studies of several rolling-circle plasmids have shown that only one plasmid gene, encoding the plasmid relaxase, is required for replication from the cognate origin of replication {reviewed in [12]}. In contrast, we found that the ICEBs1 relaxase NicK was not sufficient for autonomous replication from the ICEBs1 origin of replication oriT. Instead, a second ICEBs1 gene, helP, was also needed.
To determine which ICEBs1 genes are needed for replication from oriT, we constructed a series of plasmids that carry the ICEBs1 oriT along with various candidate ICEBs1 genes. We then tested each plasmid for its ability to replicate in B. subtilis. The parent plasmid, pUS19 [14], carries a pUC-derived origin of replication that is not functional in B. subtilis, but is functional in E. coli, allowing purification of each test plasmid from E. coli. pUS19 also carries spcE, which allowed us to transform each plasmid into B. subtilis and select for spectinomycin-resistant transformants that stably acquired the plasmid. Transcription of the ICEBs1 genes was driven from the ICEBs1 promoter Pxis that was cloned onto the plasmid. Pxis is derepressed in cells lacking ICEBs1.
After analyzing various plasmids containing different combinations of candidate ICEBs1 genes (data not shown), we found that helP and nicK were sufficient to support replication from ICEBs1 oriT (Table 1). A plasmid containing oriT, nicK, and helP (pCAL1255) was capable of transforming a B. subtilis strain lacking ICEBs1 (Table 1, pCAL1255). The plasmid copy number was between 25-30 (Table 1) as indicated by the amount of spcE DNA (plasmid) relative to ydbT, a chromosomal gene adjacent to the ICEBs1 attachment site attB.
Replication of pCAL1255 (oriT, Pxis-helP-nicK) in B. subtilis was dependent on expression of helP and nicK from Pxis. We repressed expression from Pxis by transforming pCAL1255 into B. subtilis carrying an intact integrated ICEBs1, which expresses the ICEBs1 repressor ImmR. We were still able to obtain spectinomycinresistant transformants of pCAL1255 in the ICEBs1-containing cells. However, in these transformants, the plasmid copy number was one, indicating that the plasmid was likely integrated into the chromosomal ICEBs1 by homologous recombination.
We found that autonomous plasmid replication from oriT was dependent on both helP and nicK. We did not obtain any It has recently been recognized that some, and perhaps many, ICEs undergo autonomous replication after excision from the host chromosome and that replication is important for stability and propagation of these ICEs in growing cells. Using ICEBs1, an ICE from Bacillus subtilis, we found a conserved and previously uncharacterized ICE gene that is required for conjugation and replication. We found that this gene, helP, encodes a helicase processivity factor that associates with ICEBs1 DNA and enables the host-encoded helicase PcrA to unwind the double-stranded ICEBs1 DNA, making a template for both conjugation and DNA replication. Homologues of helP are found in many ICEs, indicating that this mechanism of unwinding is likely conserved among these elements.
spectinomycin-resistant transformants in B. subtilis cells lacking ICEBs1 from plasmids containing nicK without helP (Table 1, pJT151) or helP without nicK (Table 1, pCAL1260). However, we were able to transform these plasmids into strains that expressed the missing gene from a chromosomal locus, showing that the replication defects could be complemented. pJT151 (oriT, Pxis-nicK) was able to replicate and had a copy number of 70-80 in a strain expressing helP from the chromosome. pCAL1260 (oriT, Pxis-helP) was able to replicate and had a copy number of approximately 4 in a strain expressing nicK from the chromosome ( Table 1). The different copy numbers of pCAL1255 (oriT, Pxis-helP-nicK) and the plasmids missing helP or nicK but complemented from a chromosomal copy of the gene may be due to different expression levels of HelP and NicK from the plasmid versus the chromosome, or the effects of the different plasmid sequences on replication efficiency. The low copy number when nicK was expressed from the chromosome is also consistent with the notion that the relaxase functions preferentially in cis [15]. Based on these results, we conclude that, unlike many rolling circle plasmid replicons, the ICEBs1 relaxase NicK is not the only element-encoded protein needed for autonomous replication. ICEBs1-encoded NicK and HelP are both needed to support replication from oriT in the absence of other ICEBs1 products.
helP is required for autonomous replication of ICEBs1 We found that helP is required for replication of ICEBs1. We constructed an in-frame markerless deletion of helP (DhelP) that removed its entire coding sequence from ICEBs1 ( Figure 1B). After inducing ICEBs1 gene expression, we measured ICEBs1 copy number by quantitative real time PCR (qPCR). The copy number of ICEBs1 relative to the chromosome is expressed as the relative amount of DNA from ICEBs1 oriT compared to ydbT, as described previously [6]. Consistent with previous findings, the relative copy number of ICEBs1 oriT was 3-4 per cell one hour after induction of ICEBs1 gene expression ( Table 2, line 1). Under similar conditions, the copy number of the ICEBs1 DhelP mutant was approximately 0.5 ( Table 2, line 2). A copy number less than one is consistent with previous findings that replicationdefective ICEBs1 is progressively lost from a population of dividing cells [6].
helP is required for ICEBs1 conjugation and functions primarily in donor cells In order to determine the stage at which HelP functions in ICEBs1 replication, we tested whether helP is required for transfer of ICEBs1 into a recipient. Although ICEBs1 replication is not required for mating, the initial steps of nicking and unwinding of ICEBs1 DNA are common to both mating and replication [6,15]. Wild type ICEBs1 had a mating efficiency of 3.7% (3.7 transconjugants per 100 donors) ( Table 2, line 1), similar to previous results [9,[16][17][18]. In contrast, transfer of the ICEBs1 DhelP mutant was undetectable (Table 2, line 2). Expression of helP from a truncated ICEBs1 integrated into the chromosome at thrC (strain JT335; Figure 1) largely restored conjugation, indicating that the primary mating defect was due to loss of helP and not due to polarity on downstream genes ( Table 2 line 3).
Although HelP primarily functions in the donor, it also appears to play a role in the recipient. We found that expression of helP in the recipient from the IPTG-inducible promoter Pspank(hy) increased the mating efficiency to 1.4%, from the 0.11% efficiency when helP was provided only in the donor. There was no detectable transfer of the ICEBs1 DhelP mutant unless helP was also expressed in the donor, and no increase in mating efficiency of wild type ICEBs1 when helP was also expressed in the recipient. We suspect that HelP is required for autonomous replication and increases the stability of ICEBs1 in recipient cells prior to its integration into the recipient chromosome.
Since HelP was required for both conjugation and replication of ICEBs1, we expected it to be involved in nicking or unwinding of ICEBs1 DNA. There was still nicking of the proper site in oriT in the absence of helP (data not shown), consistent with previous findings that NicK is the only ICEBs1-encoded protein needed for nicking of oriT [15]. Therefore we suspected that HelP was involved in the unwinding of ICEBs1 DNA by the host-encoded helicase PcrA, a DNA translocase that has very limited processivity as a helicase [19]. The solution structure of a HelP homologue, SAG0934 from Streptococcus agalactiae [20], which is identical to Orf22 of Tn916 from Enterococcus faecalis, indicates that HelP contains an oligonucleotide/oligosaccharide binding fold (OBfold) that is present in many ssDNA binding proteins [21], consistent with a possible role in binding ICEBs1 ssDNA. Although HelP homologues and some Ssb proteins share the The antibiotic resistance marker (kan) that is inserted in rapI-phrI is not shown. A. Genetic map of ICEBs1. The linear integrated form of ICEBs1 is shown.
Open arrows indicate open reading frames and the direction of transcription. The black rectangles at the ends of ICEBs1 represent the 60 bp direct repeats that contain the site-specific recombination sites in the left and right attachment sites, attL and attR. Some gene names are indicated. The genes encoding the ICEBs1 repressor immR and anti-repressor immA are indicated by R and A, respectively. The origin of transfer (oriT) is indicated by a black line in the 59 end of nicK. oriT is needed for ICEBs1 transfer [15] and replication [6]. Primers used in ChIP-PCR experiments hybridize to nicK (oriT), and conE. B. Schematic of the DhelP155 mutation in ICEBs1. Thin horizontal lines represent the regions of ICEBs1 that are present in the mutant. The gap in the line represents the in-frame markerless deletion of helP. C-D. Diagram of the truncated ICEBs1 derivatives that were used to test complementation of DhelP donors. Both constructs are integrated at thrC and contain ICEBs1 genes represented by the horizontal lines, from attL to ydcO (C) or helP (D). Both derivatives contain cat (chloramphenicol resistance) in place of the part of ICEBs1 that is deleted, and neither can excise from the chromosome due to the absence of attR. doi:10.1371/journal.pgen.1003198.g001 OB-fold, there appear to be no other significant sequence similarities between these proteins.

HelP associates with ICEBs1 oriT
Using crosslinking and immunoprecipitation (ChIP), we found that HelP was associated with ICEBs1 oriT in vivo ( Figure 2). Following induction of ICEBs1 gene expression, protein and DNA were crosslinked with formaldehyde and HelP was immunoprecipitated with polyclonal anti-HelP antibodies. Preliminary experiments with DNA microarrays (ChIP-chip) indicated that HelP was strongly associated with the excised and replicating ICEBs1 DNA and that there was little or no specific association with the chromosome (data not shown). We then measured association of HelP with ICEBs1 DNA following ChIP using quantitative real time PCR (ChIP-PCR) with primers specific to ICEBs1 oriT and normalized to the amount of DNA from a chromosomal region (Materials and Methods). oriT DNA was enriched .500-fold in the anti-HelP immunoprecipitates ( Figure 2A) indicating association of HelP with ICEBs1 oriT in vivo. The immunoprecipitation was specific to HelP as ICEBs1 oriT was not significantly enriched in a strain deleted for helP (2.4-fold relative association in DhelP mutant compared to .500-fold in wild type).

Association of HelP with ICEBs1 oriT depends on the ICEBs1 relaxase NicK
We found that in vivo, association of HelP with ICEBs1 oriT required the activity of the relaxase NicK. In a strain containing a nicK deletion, and a functional oriT [15], association of HelP with ICEBs1 was abolished ( Figure 2A). To test if NicK nicking activity was required for association of HelP at oriT, we made a mutation in the catalytic site of NicK that changes the conserved tyrosine at position 195 to phenylalanine (nicKY195F). We also incorporated a C-terminal 36 c-Myc tag onto the wild type and Y195F mutant NicK proteins to use in ChIP experiments with monoclonal antibodies to c-Myc. Both NicKY195F-Myc NicK-Myc were associated with ICEBs1 oriT as determined by ChIP-PCR experiments (4062-fold enrichment and 4464-fold enrichment, respectively). As expected, there was no detectable nicking of oriT by NicKY195F-Myc (,0.05%60.03% nicked oriT) whereas there was normal nicking of oriT by NicK-Myc (32%63.7% nicked oriT), as determined by primer extension assays [15]. The NicK-Myc was also functional in conjugation (Materials and Methods).
We measured the association of HelP with ICEBs1 oriT in vivo in strains expressing either NicK-Myc or NicKY195F-Myc. As expected, HelP was associated with ICEBs1 oriT in the strain expressing functional NicK-Myc (data not shown). In contrast, HelP was not detectably associated with ICEBs1 oriT in the strain expressing NicKY195F-Myc ( Figure 2A). These results indicate that the presence of NicK at oriT is not sufficient and that nicking of oriT is required for recruitment of HelP to oriT. We also found that HelP was associated with the rolling circle replicating plasmid pBS42 (data not shown), consistent with the notion that HelP does not require specific interaction with NicK.
HelP is not required for recruitment of the helicase PcrA to ICEBs1 oriT in vivo The host-encoded helicase PcrA is required for both conjugation and replication of ICEBs1 [6]. Since HelP is also required for conjugation and replication of ICEBs1, we tested for effects of HelP on PcrA. We measured association of PcrA with ICEBs1 oriT by ChIP-PCR using polyclonal anti-PcrA antibodies and primers specific to oriT as described above. PcrA association with oriT was not significantly affected in a DhelP mutant, compared to wild type ( Figure 2B). We conclude that association of PcrA with ICEBs1 oriT is not dependent on HelP.
In contrast, we found that association of PcrA with ICEBs1 oriT appeared to be largely, but not entirely, dependent on nicking of oriT by NicK. Enrichment of oriT in the PcrA immunoprecipitates was reduced but not abolished in the nicKY195F-myc mutant (approximately 6-fold enrichment in nicKY195F-myc vs 65-70-fold enrichment with nicK-myc). Association of PcrA with ICEBs1 DNA in the nicK catalytic site mutant indicates that there might be interactions between ICEBs1 NicK and the host PcrA, as there are with other replicative rolling circle relaxases and PcrA [22,23]. The immunoprecipitation was specific for PcrA; in a pcrA recF double mutant [6], ICEBs1 sequences were not significantly enriched (approximately 1.1-fold relative enrichment of oriT compared to 12.5-fold enrichment in the recF parent). (Loss of recF suppresses the lethality caused by loss of pcrA [24,25]).

HelP is required for progression of the helicase PcrA through ICEBs1 DNA in vivo
Unwinding and replication of ICEBs1 DNA by PcrA proceeds unidirectionally from the nicked oriT [6]. Replication of ICEBs1 is also accompanied by association of the host-encoded single stranded DNA binding protein (Ssb) to the ICEBs1 DNA [6]. To examine the location and role of HelP during unwinding and replication of ICEBs1 DNA from the nicked oriT, we compared association of HelP, PcrA and Ssb with an oriT-proximal (Figure 2A-2C) to an oriT-distal region ( Figure 2D-2E).
We found that although HelP was not needed for the initial recruitment of PcrA to oriT ( Figure 2B), it was required for the association of PcrA with oriT-distal regions ( Figure 2E). Association of HelP and PcrA with the oriT-distal (conE) region of ICEBs1 DNA was readily detectable in wild type (helP + ) cells ( Figure 2D, 2E). In contrast, association of PcrA with ICEBs1 DNA in the Mating efficiencies and copy numbers (averages from at least 3 independent experiments 6 standard deviation) were measured one hour after induction of ICEBs1 gene expression by overproduction of RapI from amyE::{Pspank(hy)-rapI spc}. Copy number of oriT is expressed relative to a control region ydbT adjacent to ICEBs1. Wild type and all derivates of ICEBs1 contained D(rapI-phrI)::kan. The ICEBs1 constructs at thrC contained the left end of ICEBs1 and extend to immediately before helP (thrC::helpD; Figure 1C) or to the end of helP (thrC::helP + ; Figure 1D). doi:10.1371/journal.pgen.1003198.t002 DhelP mutant was greatly reduced in this region ( Figure 2E). This reduction was due to loss of helP and not an unexpected secondary effect due to polarity or alterations in the DNA because association was restored when helP was expressed in trans from an ectopic locus ( Figure 2E). Together, our results indicate that HelP is not needed for the initial association of PcrA with ICEBs1 oriT, but that HelP is needed for PcrA to become associated with distal regions, perhaps by affecting helicase processivity. We monitored association of Ssb with ICEBs1 DNA (indicative of unwound ssDNA) using an Ssb-GFP fusion and ChIP-PCR with anti-GFP antibodies, essentially as described previously [6]. We found that Ssb-GFP was associated with both the oriT-proximal and oriT-distal (conE) region of ICEBs1 ( Figure 2C, 2E). In contrast, there was little or no association of Ssb-GFP with the oriT-distal region in the helP mutant ( Figure 2E), and association was reduced, but still appreciable, in the oriT region ( Figure 2C). Association of Ssb with ICEBs1 DNA was restored by expression of helP from an ectopic locus ( Figure 2C, 2E), indicating that the defect in the helP mutant was due to loss of helP and not some unexpected effect. Based on these results, we conclude that HelP is required for both PcrA and Ssb to associate with oriT-distal sequences in ICEBs1, and that HelP is likely needed for the processive unwinding of ICEBs1 DNA. This function would be sufficient to explain the requirement for helP in both conjugation and replication of ICEBs1.

HelP stimulates helicase activity of the PcrA homologue UvrD in vitro
We wished to test directly the ability of HelP to facilitate unwinding of duplex DNA by PcrA. Our many different preparations of B. subtilis PcrA were of low concentration and rapidly lost helicase activity. Since most structural and biochemical analyses of PcrA have been done with a homologue from another organism {reviewed in [26]}, we decided to test for effects of HelP on UvrD from E. coli. UvrD is a well-studied homologue of PcrA and has 41% identity with B. subtilis PcrA. Like PcrA, UvrD is required for replication of several rolling circle replicating plasmids [27]. We found that expression of E. coli uvrD from the IPTGinducible promoter Pspank (Pspank-uvrD) in B. subtilis suppressed the lethality caused by loss of pcrA. This suppression occurred in both the absence and the presence of IPTG, indicating that, in the absence of induction, expression from Pspank was leaky and sufficient levels of UvrD were produced. In addition, UvrD was able to support replication and conjugation of ICEBs1 nearly as well as PcrA (Table 3). In cells missing pcrA but expressing uvrD, the mating efficiency was approximately 1% (transconjugants per donor) and ICEBs1 was capable of replication and had a copy number of 2-3 (Table 3 line 3). Based on these results, we conclude that E. coli uvrD can replace pcrA in B. subtilis and provides the functions of PcrA needed for cell viability and those needed for ICEBs1 conjugation and replication.
We found that HelP stimulated the ability of UvrD to unwind a partial duplex DNA substrate in vitro. We purified hexahistidinetagged UvrD (his-UvrD) and HelP (his-HelP) and used two different partial duplex DNA substrates to monitor unwinding (Materials and Methods). The substrates had either a small (22 nucleotides) or large (81 nucleotides) fluorophore-labeled oligonucleotide hybridized to single-stranded M13mp8 DNA. Helicase activity was measured by release of the labeled oligonucleotide, detected by gel electrophoresis and fluorometry. HelP alone had no effect on the partial duplex substrate. In both cases, ,0.5% of the substrate duplex was unwound after 45 minutes. UvrD alone was able to unwind the 22 bp duplex: approximately 20% of the substrate was unwound after 45 minutes ( Figure 3A). The addition of HelP increased the amount of substrate that was unwound to approximately 40% ( Figure 3A). In contrast, there was little or no release of the large (81-mer) oligonucleotide from the partial duplex by UvrD alone (,2% unwound after 45 minutes). In the presence of HelP and UvrD, .25% of the 81 bp duplex substrate was unwound by 45 minutes (Figure 3B). Based on the increase in unwinding and the difference in stimulation between the 22 bp and 81 bp duplex substrates, we conclude that HelP stimulates the ability of UvrD, and likely PcrA, to processively unwind duplex DNA. In addition, since there is no relaxase in this assay, relaxase, and specifically NicK, is not required, at least in vitro, for the function of HelP.

Conservation of helP in many ICEs
The well characterized ICE Tn916 encodes two HelP homologues, Orf22 and Orf23. Comparison of HelP to each of these indicates that HelP is more similar to each protein than they are to each other ( Figure 4). In addition, Orf23 is about 20 amino acids shorter at the C-terminus than Orf22 and HelP.
The gene organization in ICEBs1 near helP and in Tn916 near orf22 and orf23 is similar ( Figure 5). helP and its homologues are usually grouped with two other genes: 1) a relaxase gene, nicK in ICEBs1 and orf20 in Tn916, and 2) a gene encoding the predicted coupling protein, conQ in ICEBs1 [18] and orf21 in Tn916 ( Figure 5). The coupling protein targets the relaxosome complex that is linked to ssDNA to the mating pore [28]. This genetic arrangement reflects a functional relationship as the relaxase and likely HelP are part of the relaxosome that interacts with the cognate coupling protein. When present, the majority of helP homologues are found in pairs, although ICEBs1 has only one helP.
helP homologues are found in at least 72 ICEs or putative ICEs, primarily in firmicutes [29]. Phylogenic analysis revealed that these HelP homologues fall into seven clades ( Figure 6). When an ICE encodes two HelP homologues, each one is in a separate clade: one clade contains the longer HelP homologue and other clade contains the shorter HelP homologue. For example the clade labeled ''Tn916 Orf22'' contains most of the longer HelP homologues and the clade labeled ''Tn916 Orf23'' contains most of the shorter HelP homologues. ICEBs1 HelP is similar in size to the longer Orf22, but appears to be almost equidistant from the Orf22 and Orf23 clades ( Figure 6). Together, our results indicate that HelP proteins are encoded by many different ICEs from Firmicutes. If the function of these homologues is conserved, then HelP proteins act as helicase processivity factors for many ICEs and these ICEs likely undergo autonomous rolling circle replication.

Discussion
We found that the ICEBs1 helP gene product stimulates unwinding of ICEBs1 DNA by the helicase PcrA in vivo. In vitro, HelP stimulated processivity of the PcrA-like helicase UvrD from E. coli. Our findings indicate that HelP is a helicase processivity factor that is required for conjugation and replication of ICEBs1. HelP may represent a new family of helicase processivity factors as helP homologues are found in many other known and putative ICEs. Together, our results indicate that the order of events leading to ICEBs1 replication and conjugation include: 1) nicking Induction of ICEBs1 gene expression was carried out by overproduction of RapI from amyE::{Pxyl-rapI spc} for two hours followed by mating as described in Materials and Methods. Data presented are averages from two experiments 6 standard deviation. Wild type and all derivates of ICEBs1 contained D(rapI-phrI)::kan. All strains were also thrC mutants, with either thrC::cat or E. coli uvrD cloned and integrated into thrC (thrC::Pspank-uvrD mls). doi:10.1371/journal.pgen.1003198.t003

DNA translocase and helicase activities of PcrA-family proteins
PcrA and its well-characterized homologues in E. coli, UvrD and Rep, are members of superfamily 1 (SF1) of non-hexameric helicases {reviewed in [13,26]}. These proteins are highly processive 39-59 ssDNA translocases that bind to and move along strands of ssDNA. They are also weak 39-59 helicases that bind and destabilize dsDNA. They are involved in multiple cellular processes, including several types of DNA repair, replication restart, and clearing recombination proteins from DNA {reviewed in [26,30]}. Strains lacking SF1 helicases are usually nonviable [24,31], presumably due to loss of the DNA translocase activity of these proteins [31][32][33].
Although PcrA, Rep and UvrD are efficient and processive DNA translocases, they have very poor helicase activity on their (bottom sequence) were aligned using Clustal X. Markings above and below the HelP sequence indicate which amino acids are identical (asterisk), highly similar (colon) or weakly similar (period) to Tn916 Orf22 (marks above) and Tn916 Orf23 (marks below). Markings for the pairwise comparison of Orf22 to Orf23 are shown below the Tn916 Orf23 sequence. Needleman-Wunsch alignment scores indicate that HelP is more similar to Orf22 and Orf23 (N-W scores = 142 and 104, respectively) than Orf22 is to Orf23 (N-W score = 31). ICEBs1 HelP is 126 amino acids, Tn916 Orf22 is 128 amino acids, and Tn916 Orf23 is 104 amino acids. doi:10.1371/journal.pgen.1003198.g004  The homologues were analyzed using CLUSTAL X and grouped into clades if they were within a distance of ,0.22. The diagram is of an unrooted tree with the number of homologues in each clade shown in parentheses. Branch lengths indicate relative phylogenic distances. The width and length of branch tips indicate the number of homologues and the relative phylogenic distance between homologues in the clade, respectively. Of the seven clades, six are named for a representative ICE and its HelP homologue and one is named ''Orf23-like'' to reflect its close relationship to the Tn916 Orf23 clade. Twelve homologues in the ICE6013 Orf14 clade have the identical 106 amino acid sequence. The two homologues in the ICEBs1 HelP clade only differ by 5 of 126 amino acids. The two homologues in the Orf23-like clade only differ by 7 of 108 amino acids. Sixteen ICEs encode a single homologue of HelP. These sixteen homologues are from three clades -ICEBs1 HelP (2), ICE6013 Orf14 (12)  own [19,34,35]. Helicase activity requires oligomerization [36][37][38], and these helicases interact with accessory factors that stimulate activity. For example, the DNA mismatch repair protein MutL facilitates loading and processivity of UvrD in E. coli [39] and the double-strand break repair protein Ku interacts with and stimulates processivity of UvrD1 in Mycobacterium smegmatis [40]. PcrA processivity is stimulated in vitro by YxaL from B. subtilis [41], although the role of YxaL in vivo is not known.

Plasmid-and phage-encoded helicase processivity factors
Although they are poorly processive, PcrA, UvrD, and Rep are used by many plasmids and phages that undergo rolling-circle replication. In E. coli, UvrD is needed for rolling circle replication of some plasmids [27,42] and Rep is used for replication of several ssDNA phages {reviewed in [11]}. In some Gram positive bacteria, PcrA is used for replication of many different rolling circle plasmids [12,31,32]. In addition, PcrA is required for unwinding of ICEBs1 DNA for both conjugation and replication [6].
For rolling circle replicating elements that use a host-encoded SF1-type helicase, efficient unwinding of duplex DNA is often stimulated by interaction with the element-encoded replication relaxase. Relaxases introduce a nick into dsDNA and mark the site for recruitment of the helicase for unwinding and replication of the plasmid or phage DNA. Replicative relaxases of ssDNA phages from E. coli, including gpA (cisA) from øX174 and the product of gene II of the f1 family of phages, increase Rep helicase processivity {reviewed in [11]}. The replicative relaxases of rolling circle replicating plasmids pT181 and the related pC221, RepC and RepD, respectively, appear to interact with and stimulate PcrA [23,[43][44][45][46][47]. In the case of RepD, this stimulation is thought to occur by increasing the affinity of PcrA for its DNA substrate and potentially by decreasing its rate of dissociation, rather than by altering its kinetic properties [46,48].
HelP, a conserved helicase processivity factor As far as we are aware, all known examples of plasmid or phageencoded helicase-stimulating proteins are relaxases. By analogy to the related plasmid relaxases, we expected that the only ICEBs1 product needed for ICEBs1 rolling circle replication would be its relaxase NicK. However, we found that the ICEBs1 gene product HelP is a helicase processivity factor that is required, in addition to NicK, for replication and conjugation of ICEBs1. We do not yet know how HelP stimulates helicase activity, but it appears to act at a step after association of PcrA with ICEBs1 oriT DNA. HelP could stimulate dimerization of PcrA since it is the dimer and not the monomer that has helicase activity [19]. HelP could also decrease the rate of dissociation of PcrA from DNA during unwinding, analogous to the activity demonstrated for RepD [46,48]. HelP could accomplish one or a combination of these stimulatory functions by direct protein-protein contact with PcrA. Alternatively, HelP could remodel the DNA substrate during unwinding, promoting helicase activity.
helP homologues are found primarily in firmicutes. There appear to be .300 homologues in the non-redundant protein database, and at least 128 of these are found in known or putative ICEs that are included in the ICEberg database [29], and we suspect that most of the others are in unrecognized ICEs. Although ICEBs1 has only one helP, most, but not all, helP homologues are found in pairs, with one member of the pair approximately 20 amino acids longer than the other. It is possible that each member of a HelP pair stimulates processivity of a dissimilar range of helicases, thereby broadening the host range of the mobile element. If the function of HelP homologues is conserved, then the ICEs with helP homologues likely undergo autonomous replication and that HelP proteins are important for stability and transfer of many ICEs.

Other strategies for DNA unwinding of extrachromosomal elements
In contrast to the many ICEs and plasmids that utilize a hostencoded SF1-type helicase for replication and transfer and rely on an accessory factor to facilitate unwinding, some ICEs and plasmids encode their own SF1-type helicase. PcrA-like helicase domains are found attached to some plasmid relaxases. For example, TraI of the E. coli F plasmid and TrwC of R388, have a C-terminal PcrA-like helicase domain that is required for conjugation [49][50][51]. The helicase activity is highly processive, potentially because it is tethered to the relaxosome {reviewed in [52]}. In addition, many known and putative ICEs encode discrete PcrA-like helicases [29]. It is not known if they are processive or require accessory proteins, although some of those ICEs also contain a helP homologue. An element that encodes its own helicase might have a broader host range by avoiding reliance on a host-encoded helicase. That HelP homologues, relaxases that also function as processivity factors, and PcrA homologues are encoded on conjugative elements indicates that the need for DNA unwinding can be met in different ways.

Strains and alleles
All B. subtilis strains ( Table 4) are derivatives of JH642 (trpC2 pheA1) and were constructed by natural transformation. Strains are either cured of ICEBs1 (designated ICEBs1 0 ) or contain ICEBs1 Figure 7. Model for association of the relaxase, helicase, HelP, and Ssb with ICEBs1 DNA. Following excision from the chromosome (not shown), the double-stranded circular ICEBs1 DNA is nicked at oriT by the relaxase NicK (filled circle). By analogy to related relaxases, NicK likely becomes covalently attached to the 59 end of the nicked DNA on the strand that is transferred during conjugation. HelP (open circles) and the hostencoded helicase PcrA (gray packman) associate with the nicked ICEBs1 at oriT. HelP facilitates processive unwinding of ICEBs1 by PcrA and subsequent association of the host-encoded Ssb (open triangles). Unwinding of ICEBs1 by PcrA and HelP is required for replication and conjugation of ICEBs1. HelP is depicted as binding to both single strands of ICEBs1 DNA adjacent to PcrA, although it is possible that HelP associates only with one of the two single strands. doi:10.1371/journal.pgen.1003198.g007 marked with the D(rapI-phrI)342::kan allele to monitor conjugative transfer [9]. ICEBs1 gene expression was induced by overproduction of rapI from amyE::{(Pspank(hy)-rapI) spc}, amyE::{(Pxyl-rapI) spc}, or lacA::{(Pxyl-rapI) tet}. Strains used as recipients in mating experiments contained the streptomycin resistance allele (str-84) [9]. Other mutations that were previously described include: the deletioninsertion DpcrA1021::cat [6]; thrC1165::cat [6], used as a control for threonine auxotrophy and chloramphenicol resistance; and thrC329::{(Pxis-nicK) mls} [15], used for ectopic expression of nicK.
helP. DhelP155 is an unmarked 413-bp deletion that removes the entire coding sequence and the 35 bp helP-ydcQ intergenic region (Figure 1), constructed using the same method as for the DnicK306 allele [15]. Complementation of DhelP155 was tested using a truncated ICEBs1 derivative integrated at thrC, thrC229::{(ICEBs1-1637 DconQ-attR::cat) mls} that is missing all of the ICEBs1 genes downstream from helP ( Figure 1D), and contains helP and all the upstream ICEBs1 genes [18]. The control that does not complement DhelP was essentially the same ICEBs1 insertion at thrC, but with a deletion that removes helP ( Figure 1C), thrC229::{(ICEBs1-1635 DhelP-attR::cat) mls}.
Expression of helP from LacI-repressible-IPTG-inducible promoter Pspank(hy) [53] in B. subtilis was from a fusion at thrC, thrC148::{(Pspank(hy)-helP) mls}. This was made by amplifying helP by PCR from B. subtilis genomic DNA, digesting with Nhe1 and ligating into Nhe1-cut pCAL215 [16] to give pCAL890. The Pspank promoter of pCAL890 was altered to Pspank(hy) by Quikchange mutagenesis to give the plasmid pJT146 which was then used to integrate Pspank(hy)-helP mls into the thrC locus by double-crossover recombination.
pcrA is essential in B. subtilis [24,31]. The presence of thrC1642::{(Pspank-uvrD) mls} suppressed the lethality caused by loss of pcrA. That is, DpcrA::cat could be transformed into competent cells containing the Pspank-uvrD, but could not be transformed into cells without uvrD. Growth of the Pspank-uvrD DpcrA mutant was independent of IPTG, indicating that the leaky (uninduced) expression from Pspank-uvrD was sufficient to compensate for the absence of pcrA.
nicK. The nicK-myc allele was constructed by allelic replacement using essentially the same method as for the DhelP155 allele.  An approximately 2.1 kb DNA insert containing nicK, a Cterminal 36 c-myc tag and 897 bp of ICEBs1 sequence downstream of nicK were cloned into the plasmid pCAL1422 by isothermal assembly [54] to give the plasmid pJT296. pCAL1422 contains cat (chloramphenicol-resistance in B. subtilis) and E. coli lacZ under control of the constitutive promoter Ppen [55]. The tyrosine codon at position 195 of nicK-myc in pJT296 was changed to a phenylalanine codon, nicKY195F-myc, by quick-change mutagenesis (Stratagene), giving plasmid pJT318. pJT296 and pJT318 were used to replace nicK on ICEBs1 with nicK-myc and nicKY195F-myc, respectively, by first integrating by single crossover and then screening for loss of the plasmid by virtue of loss of lacZ, and then testing by PCR for introduction of the indicated allele, essentially as described [15]. Wild type NicK fused to the Myc-tag (NicK-Myc) was active as judged by normal conjugation efficiencies (approximately 1.2% transconjugants per donor). In contrast, the nicKY195F-myc mutant was defective in conjugation (,0.00008% transconjugants per donor).
pCAL1255 (oriT, Pxis-helP-nicK) contains the xis promoter (Pxis) driving transcription of helP and nicK. The Pxis-helP-nicK insertion is comprised of three non-contiguous segments of ICEBs1: 1) a 527 bp segment containing 254 bp of the 59-end of immR and the entire 273 bp intergenic region between immR and xis; 2) a 419 bp segment that contains helP and extends 35 bp downstream of the helP stop codon; and 3) a 1076 bp segment containing nicK (and oriT) and extending 17 bp downstream of the nicK stop codon. The junctions between the DNA segments were designed to allow translation initiation of helP from the xis ribosome binding site and translation initiation of nicK from the conQ ribosome binding site, as the start codons of helP and of nicK were placed the same distance downstream of the Pxis and the helP-conQ intergenic regions as the native start codons of xis and conQ, respectively. Transcription of the Pxis-helP-nicK insertion is in the same orientation as the spectinomycin-and ampicillin-resistance genes on the vector backbone.
pCAL1260 (oriT, Pxis-helP) contains the same Pxis-helP-nicK insertion present in pCAL1255, but with an additional T inserted between the 5 th and 6 th codon of nicK. The single base insertion in pCAL1260 leads to premature termination after the 10 th codon of the nicK ORF. pJT151 is essentially pCAL1255 (oriT, Pxis-nicK) but with the entire helP coding sequence deleted.
N-terminal hexahistidine tagged HelP (his-HelP) and UvrD (his-UvrD) were overproduced in and purified from E. coli. helP was amplified by PCR from B. subtilis (strain JH642) chromosomal DNA, digested with BamHI and HindIII and ligated into the same sites of the T7-expression vector pET28b (Novagen) to give plasmid pCAL1297. uvrD was amplified from E. coli strain MC1061and cloned, by isothermal assembly, into the XbaI and BamHI sites of pET28b to give plasmid pJT370.

Conjugation experiments
For conjugation experiments, all donor strains contained ICEBs1 with the D(rapI-phrI)342::kan allele and ICEBs1 gene expression was induced by overproduction of the regulatory protein RapI from an ectopic locus [9]. When donors carried the Pspank(hy)-rapI allele, all strains were grown in rich medium and induction was for one hour with 1 mM IPTG. When the Pxyl-rapI was used, strains were grown in minimal S750 medium containing 1% arabinose and induction was for 2 hours after the addition of 1% xylose [56]. Recipients were streptomycin resistant (str-84). Donors and recipients were mixed in a 1:1 ratio, filtered onto nitrocellulose membranes and incubated on agar containing minimal salts for 3 hours, essentially as described [9]. The mating efficiency was determined as the number of colony forming units (CFU) of streptomycin-and kanamycin-resistant transconjugants per CFU of the donor.

Chromatin immunoprecipitation, copy number, and qPCR
Association of HelP, PcrA, NicK-Myc and Ssb-GFP with ICEBs1 DNA was measured by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) essentially as described [6,57]. Polyclonal antibodies from rabbits (Covance) were used to precipitate HelP, PcrA and Ssb-GFP and monoclonal antibodies to c-Myc (Invitrogen) were used to precipitate NicK-Myc.
Plasmid copy number was determined using primers oJT168, 59-GTGGAATCAT CCTCCCAAAC-39, and oJT169, 59-AAT GGCTCTT CTCACATCAG-39, that are specific to spcE found on the plasmids and not in the chromosome.

Protein expression and purification
his-HelP and his-UvrD were purified using similar conditions. Expression strains were grown in 2 liters of 26YT medium supplemented with 0.4% glycerol, 20 mM sodium phosphate buffer pH 7.0, and 10 mM MgSO 4 at 30uC until they reached an OD600 of 0.8. Protein expression was induced by the addition of 0.2% arabinose and 1 mM IPTG followed by incubation for 3 hours. Cells were harvested by centrifugation, washed with 100 ml of phosphate-buffered saline, resuspended in 25 ml lysis buffer {50 mM Tris-HCl ph 8.0, 0.3 M NaCl, 10 mM imidazole, 1 mg/ml lysozyme, 5 mg/ml DNase I and 16 CellLytic Express (Sigma-Aldrich)} and lysed by incubation at room temperature with gentle inversions. The lysate was cleared by centrifugation at 15,0006 g and the cleared lysate was applied to 1 ml Ni-NTA agarose resin, washed with Tris-NaCl (50 mM Tris-HCl pH 8.0, 0.3 M NaCl) containing 10 and 20 mM imidazole and eluted with the same buffer containing 250 mM imidazole. The eluted protein was dialyzed against 10 mM MOPS, pH 7.5, 200 mM NaCl and 1 mM tris(2carboxyethyl)phosphine. his-UvrD was approximately 93% pure and his-HelP was approximately 97% pure.

Identification and analysis of HelP homologues
We identified 128 homologues of ICEBs1 helP (including helP) in 72 bacterial ICEs using the HMMER3 and WU-BLAST2 search tools in ICEberg (http://db-mml.sjtu.edu.cn/ICEberg/), the webbased resource for bacterial ICEs . The ICEberg database (last updated on August 14, 2011) contains sequence information for 431 known and putative ICEs. All 130 homologues were aligned using ClustalX 2.1 [58] using multiple-alignment mode with default parameters. The neighbor-joining method was used to generate the phylogenic tree from the ClustalX PHYLIP output file at the Interactive Tree of Life (http://itol.embl.de/index. shtml) [59]. Seven clades were defined by grouping together homologues that had an average distance of ,0.22.
An additional HMMER search [60] with default search settings (hmmer.janelia.org) identified .300 HelP homologues in the nonredundant protein database. All but five of these were in Firmicutes. The exceptions were one homologue in a Mycoplasma, Ureaplasma urealyticum, two in an uncultured bacterium MID12, and two homologues in Klebsiella pneumoniae which carries Tn916 (usually associated with Firmicutes) on the composite ICE Tn6009.
Global sequence similarities between HelP and the two HelP homologues from Tn916 were analyzed by the Needleman-Wunsch alignment method [61].