BACKGROUND
attenuated salmonella are being developed as vaccines to protect against typhoid fever  <cit> .
there are also endeavors employing salmonella as delivery vectors for therapeutic molecules.
one strategy utilizes attenuated salmonella, which expresses a gene or gene fragment encoding a protective antigen as vaccine against bacterial pathogens  <cit> .
the heterologous genes can be expressed from the salmonella chromosome, or, more often, from a multi-copy plasmid  <cit> .
another strategy exploits salmonella as a delivery vector of dna vaccine against viral pathogens  <cit> .
the later strategy is also used to deliver dna encoding tumor antigen or cytokine for therapeutic applications in oncology  <cit> .
in addition, salmonella is used to deliver small interfering rnas   <cit> , ribozymes  <cit>  and large dna molecules encoding a viral genome  <cit> .
for instance, in vivo delivery of an artificial bacterial chromosome  carrying the viral genome of the murine cytomegalovrirus  by salmonella typhimurium led to a productive virus infection in mice and resulted in elevated titers of specific antibodies against lethal mcmv challenge  <cit> .
most vaccine designs utilize salmonella delivery vectors carrying a single plasmid for expression of a single antigen or of a fusion protein carrying epitopes from more than one antigen  <cit> .
to induce broader immunity against a particular pathogen or various pathogens, one might need to express multiple antigens from a single plasmid carrying different antigen cassettes or from multiple plasmids in a single cell, each expressing one or more relevant antigens.
co-delivery of plasmids encoding tumor antigens and cytokines by salmonella has been successfully demonstrated to improve protective immunity against cancer  <cit> .
in the case where multiple plasmids are carried in the same salmonella vector strain, there are most likely regions of homology between the plasmids, since the widely used puc- and pbr-based plasmids have origins of replication that are nearly identical and both share regions of homology with the p15a ori.
additionally, commonly used promoter sequences, transcriptonal terminators and other expression plasmid components may also be present on plasmids coexisting in the same bacterial cell.
the presence of these similar or identical dna sequences would serve to facilitate undesirable interplasmid recombination.
in some cases the bacterial vector may intentionally harbor multiple copies of the same dna sequence, which may lead to plasmid instability.
recently, we encountered such a situation during the development of a bacterial based influenza vaccine.
we constructed a single plasmid carrying eight head-to-tail connected influenza cdna cassettes  <cit> .
the plasmid was intended for delivery into host cells by an attenuated salmonella strain.
the multiple repetitive sequences residing in the plasmid make its stability within the attenuated salmonella an important concern because any intraplasmid recombination event results in deletion of one or more influenza gene cassettes.
recent work in our laboratory has focused on developing new strategies for attenuated salmonella vaccine strains, with features including regulated delayed in vivo attenuation  <cit> , regulated delayed in vivo antigen synthesis  <cit> , and programmed delayed in vivo cell lysis  <cit> .
for all of these systems, one or more chromosomal and/or plasmid genes are placed under the control of the arac pbad promoter.
eventually, our goal is to combine all of these features into a single salmonella vaccine vector strain.
such a strain will therefore carry multiple chromosomal and plasmid copies of arac pbad, providing sites for potential recombination, which could lead to unwanted chromosomal or plasmid rearrangements.
however, to our knowledge, there have been no published studies specifically designed to evaluate plasmid recombination in salmonella enterica.
deletions of several escherichia coli genes are known to reduce the frequency of plasmid recombination, including the reca, rece, recf and recj genes  <cit> .
the reca gene encodes the general recombinase reca, involved in nearly all forms of recombination in the cell  <cit> .
the rece, recf and recj proteins play a role in plasmid recombination and recombination repair  <cit> .
the reca, recf and recj proteins are highly homologous between e. coli and s. enterica, therefore they may play similar roles in dna recombination.
despite these possible similarities, the recombination systems in the two organisms differ somewhat, as s. enterica does not encode rece  <cit> .
based on these concerns, we decided to determine the effect of rec gene deletions on intraplasmid recombination, interplasmid recombination, intrachromosomal recombination and plasmid integration in s. enterica.
in this work, we examine the effect of Δreca, Δrecf and Δrecj mutations on dna recombination frequencies in three serovars of salmonella enterica currently relevant to vaccine development.
our results show that the effect of these mutations on recombination can vary among salmonella serovars and with previously published results in e. coli.
RESULTS
plasmid construction
we constructed a series of plasmids  encoding various truncated teta genes to assay plasmid recombination frequencies using the strategies similar to those described previously  <cit> .
restoration of a functional teta gene via intra- or intermolecular recombination resulted in a change of the bacterial phenotype from tetracycline sensitive to tetracycline resistant, and served as a marker allowing us to measure the frequency of recombination events .
* cat: chloramphenicol resistance gene; teta: tetracycline resistance gene; amp: ampicillin resistance gene; kan: kanamycin resistance gene; 3'tet: 3' portion of the teta gene; 5'tet: 5' portion of the teta gene together with its promoter; aacc1: 3-n-aminoglycoside acetyltransferase.
plasmids pya <dig> and pya <dig> were constructed to test intraplasmid recombination .
plasmid pya <dig> carries two truncated teta genes , which have 466-bp of tandemly repeated sequence.
an intramolecular recombination event can delete one of the repeats resulting in an intact teta gene, thereby recreating the structure of plasmid pacyc <dig> .
theoretically, intermolecular recombination may occur between two pya <dig> molecules to form a plasmid dimer with a functional teta gene .
plasmid pya <dig> contains a 602-bp teta sequence duplication separated by a 1041-bp kan cassette.
the intramolecular recombination product is equivalent to pacyc <dig>  the intermolecular recombination product is a dimer plasmid containing an intact teta gene .
plasmids pya <dig> and pya <dig> carry the 3'tet gene and 5'tet gene, respectively .
the rec+ salmonella strain χ <dig> carrying either plasmid individually was sensitive to tetracycline.
there is 751-bp of teta dna in common between the two truncated teta genes.
recombination between the two plasmids creates a hybrid plasmid containing an intact teta gene .
intraplasmid recombination products
to verify the recombination products, plasmid dna was prepared from tetracycline resistant  single colonies derived from χ <dig>  χ <dig> and χ <dig>  plasmids extracted from tcr clones of χ <dig> were digested with xbai and sali.
theoretically, xbai/sali digestion of pya <dig> will yield two fragments , pacyc <dig> will yield two fragments  and pya <dig> dimer will yield four fragments .
the results  showed that digestion of all  <dig> tcr clones yielded a 721-bp band, indicating either a pya <dig> dimer or a plasmid equivalent to pacyc <dig>  three clones  yielded the pya <dig> dimer-specific 1653-bp band.
therefore, we conclude that the other  <dig> clones recombined to form the pacyc184-like structure.
of note, several clones  also yielded the 1187-bp pya4463-specific band, suggesting that the original plasmid  and its recombination product  could coexist in the same bacterial cell.
plasmids extracted from tcr clones of χ <dig> were digested with kpni and ecori.
theoretically, plasmid pya <dig> will be digested into two fragments , plasmid pacyc <dig> will be linearized  and the pya <dig> plasmid dimer will be digested into four fragments .
examination of the restricted dna  showed that only one clone  had the pya <dig> dimer-specific 1643-bp band.
the most prominent band in the other lanes was a 4245-bp band expected for pacyc184-like recombination products.
nine clones contained a mixture of pacyc <dig> and pya <dig> .
interplasmid recombination products
plasmids extracted from tcr clones of χ <dig> were digested with ncoi and bglii.
both pya <dig> and pya <dig> are linearized into a dna fragment about  <dig> kb.
therefore, in cells containing each or both monomeric plasmids, the digested product will be a single band.
the pya4464-pya <dig> hybrid will be cut into two fragments .
all four of the tcr clones we isolated and examined showed recombination product specific bands and the 4-kb band expected when each plasmid exists separately in the cell.
four tetracycline sensitive  isolates were examined and only a single band was observed, as expected .
these results suggest that interplasmid recombination occurred in the tcr cells and that both dimer and individual monomers corresponding to at least one of the two starting plasmids can coexist in the same bacterial cell.
we performed a similar experiment in s. typhi strain ty <dig> and obtained identical results .
construction of rec deletion strains
we constructed a series of strains for these studies carrying deletions in either reca, recf or recj in s. typhimurium uk- <dig>  s. typhi ty <dig> and s. paratyphi a .
we also constructed ΔrecaΔ recf and Δrecj Δ recf double mutants in s. typhimurium.
deletion of reca, recf and recj results in an increase in sensitivity to uv irradiation  <cit> .
to verify the presence of these deletions phenotypically in our strains, the uv sensitivity of the s. typhimurium mutant strains was measured.
the Δrecf and Δrecj mutants showed significantly lower surviving fractions than the wild type strain after the same exposure dose .
by contrast, after five seconds of uv exposure  to  <dig>  ×  <dig> cfu of the Δreca <dig> mutant , we were unable to recover any surviving cells .
uv resistance similar to the wild-type strain χ <dig> was restored to s. typhimurium Δreca and Δrecf mutants strains after introduction of reca plasmid  or either recf plasmid , respectively.
transformation of either mutant strain with vector plasmid pya <dig> did not restore uv resistance .
* kan: kanamycin resistance gene; 5'tet: 5' portion of the teta gene together with its promoter; 3'tet: 3' portion of the teta gene.
effect of rec deletions on intraplasmid recombination
to examine the influence of Δreca, Δrecf and Δrecj mutations on intraplasmid recombination frequencies, plasmid pya <dig>  or pya <dig>  were introduced into salmonella rec mutants and their parental strains and analyzed as described in the methods section.
the recombination frequency of plasmid pya <dig> was approximately  <dig> - <dig>  × 10- <dig> in rec+ typhimurium, typhi and paratyphi a .
in s. typhimurium and paratyphi a, most of the rec deletions had no effect on the intraplasmid recombination frequency of plasmid pya <dig> except that a small, but statistically significant decrease in recombination was observed in the Δreca mutant of paratyphi a. however, in both s. typhi strains, both Δrecf mutations resulted in approximately 10-fold decrease in recombination frequency , while the Δreca and Δrecj mutations resulted in a 2-3-fold reduction .
in the complementation test, the recombination frequency of plasmid pya <dig> in s. typhi χ <dig> was restored to  <dig>  ±  <dig>  × 10- <dig> and  <dig>  ±  <dig>  × 10- <dig> by introduction of plasmid pya <dig> encoding s. typhimurium recf gene and pya <dig> encoding the s. typhi recf gene, respectively .
aintraplasmid recombination without intervening sequence .
b intraplasmid recombination with a 1041-bp intervening sequence .
c interplasmid recombination.
d not assayed.
** p <  <dig> , relative to the parental rec+ strain.
the results with plasmid pya <dig> were also variable among strains.
the recombination frequency in rec+ s. typhimurium and s. paratyphi a strains was approximately 2- <dig> × 10- <dig> and in both s. typhi strains, the frequency was 3-fold higher, at  <dig>  × 10- <dig>  and  <dig>  × 10- <dig> .
in s. typhimurium and s. typhi ty <dig>  the Δ reca and Δrecf mutations reduced the recombination frequency of plasmid pya <dig> by 5-20-fold .
the results were similar for s. paratyphi a, though the Δrecf mutation only led to 3-fold lower plasmid pya <dig> recombination .
the Δrecj mutation had no effect in s. typhimurium and resulted in a 2-3-fold decrease in recombination in both s. typhi ty <dig> and s. paratyphi a. combining the Δreca Δrecf mutations in s. typhimurium led to a recombination frequency similar to the frequencies observed for both mutations individually, indicating no additive effect.
in the complementation test, plasmid pya <dig>  which encodes s. typhimurium reca, was transformed into s. typhimurium Δreca mutant χ <dig> and s. typhi Δreca mutant χ <dig>  their respective recombination frequencies were  <dig>  ±  <dig>  × 10- <dig> and  <dig>  ±  <dig>  × 10- <dig>  which were comparable to the corresponding wild type strains  .
the recf-encoding plasmids pya <dig> and pya <dig> were transformed into recf mutant strains χ <dig> and χ <dig>  respectively.
the respective recombination frequencies were increased to  <dig>  ±  <dig>  × 10- <dig> and  <dig>  ±  <dig>  × 10- <dig>
effect of rec deletions on interplasmid recombination
to evaluate interplasmid recombination, plasmids pya <dig> and pya <dig> were co-electroporated into the wild-type and rec deletion strains.
electroporants from each test strain were grown in lb broth containing both ampicillin and chloramphenicol to maintain selection for both plasmids.
the frequency of recombination was determined as described in the methods section.
the interplasmid recombination frequency was 1- <dig> × 10- <dig> for rec+ s. typhimurium, s. typhi and s. paratyphi a strains .
for typhimurium and paratyphi a, the Δreca and each Δrecf mutation reduced the interplasmid recombination frequency by about 3-10-fold .
in contrast, the Δreca mutation had no effect on interplasmid recombination in s. typhi ty <dig>  the Δrecf mutations did not reduce interplasmid recombination in either of the typhi strains.
surprisingly, introduction of the Δrecf <dig> mutation into s. typhi ty <dig> resulted in significantly higher interplasmid recombination .
note that we performed this analysis in eight independent experiments and observed a higher recombination frequency of interplasmid recombination each time.
the Δrecj mutation had no significant effect in s. typhi, and a small  but significant effect in s. typhimurium and s. paratyphi a. the recombination frequencies were also determined in s. typhimurium strains Δreca Δrecf and Δrecf Δrecj double deletions.
no additive effect between the two mutations was observed with respect to each single mutation.
effect of rec deletions on chromosome related recombination
to measure intrachomosomal recombination frequencies, we introduced the pya4590-derived dna sequence containing two truncated teta genes  into the s. typhimurium chromosome at cysg.
the two truncated teta genes had  <dig> bp of overlapping sequence.
intrachromosomal recombination deletes the kanamycin resistance cassette and restores one intact copy of the teta gene .
deletion of reca resulted in a 5-fold reduced recombination frequency compared to the rec+ strain χ <dig> , while the recf or recj deletions had no effect, indicating that recf and recj are not involved in this process .
a mean ± std from 3- <dig> assays were shown in the table.
b upon introduction of pkd <dig> , the frequency was  <dig>  ±  <dig>  × 10- <dig> .
** p <  <dig> , relative to the parental rec+ strain.
to examine plasmid integration, the 5'tet gene was introduced into the s. typhimurium chromosome at cysg.
the resulting strains were transformed with plasmid pya <dig>  .
the  <dig> bp of overlapping sequence between 5'tet on the chromosome and the 3'tet on the plasmid could result in plasmid integration into the chromosome, generating an intact teta gene .
deletion of reca had a profound effect, reducing the integration frequency to less than  <dig> × 10- <dig>  which was below the limits of detection in this assay , indicating a strict requirement for reca in this process.
introduction of plasmid pkd <dig>  which encodes the λ red recombinase, into χ <dig>  carrying pya <dig> restored the integration frequency to the level of the rec+ strain χ <dig>  deletion of recf reduced the frequency of integration less than 3-fold  and the Δrecj deletion had no effect.
effect of rec deletions on the virulence of s. typhimurium
balb/c mice were orally inoculated with the highly virulent s. typhimurium strain χ <dig> and its rec mutant derivatives.
the ld50s of χ <dig>  χ <dig>  and χ <dig>  were similar,  <dig>  ×  <dig>   <dig>  ×  <dig> and  <dig>  ×  <dig> cfu, respectively .
the ld <dig> of the Δrecf Δrecj double mutant was approximately 100-fold higher than χ <dig>  at  <dig>  ×  <dig> cfu.
all mice inoculated with  <dig>  ×  <dig> cfu of the Δreca mutant survived, indicating that the ld <dig> was >  <dig>  ×  <dig> cfu.
two months following the initial inoculation with the Δ reca mutant strain, surviving mice were challenged with either  <dig>  ×  <dig> or  <dig>  ×  <dig> cfu of wild-type strain χ <dig>  all mice survived the challenge, indicating that Δ reca mutant strain χ <dig> was both attenuated and immunogenic.
discussion
we began our studies using information gathered in e. coli as a reference point.
in e. coli, reca-dependent homologous recombination relies on the recbcd pathway, the recfor pathway  and the rece pathway  <cit> .
the recbcd pathway is important in conjugational and transductional recombination  <cit> , and may also be involved in the recombination of plasmids containing one or more chi sites  <cit> .
recombination in small plasmids lacking a chi sequence is primarily catalyzed by the recfor pathway  <cit> .
recf, reco, and recr bind to gaps of ssdna and displace the single-strand dna binding proteins to allow reca to bind  <cit> .
the recj ssdna exonuclease acts in concert with recfor to enlarge the ssdna region when needed.
strand exchange is then catalyzed by reca  <cit> .
because of their prominent role in plasmid recombination in e. coli, we analyzed the effect of mutations in recf, recj and reca on plasmid recombination in salmonella.
attenuated s. typhi strains have been developed as antigen delivery vectors for human vaccine use.
due to the host restriction phenotype of s. typhi, preliminary work is typically done in s. typhimurium using mice as the model system to work out attenuation and antigen expression strategies.
recently, we have also been investigating attenuated derivatives of the host-restricted strain s. paratyphi a as a human vaccine vector.
therefore, it was of interest to evaluate and compare the effects of rec mutations in these three salmonella serovars.
we selected s. typhi strain ty <dig> as exemplary of this serovar because most of the vaccines tested in clinical trials to date have been derived from this strain  <cit> .
s. typhi strain isp <dig> has also been evaluated in clinical trials  <cit>  and we therefore included it in some of our analyses.
we found that, for some dna substrates, the effects of Δreca and Δrecf deletion mutations differed among salmonella enterica serotypes.
in particular, we found that deleting reca, recf or recj in s. typhi ty <dig> and deleting recf in strain isp <dig> had significant effects  on the recombination frequency of our direct repeat substrate, pya <dig> .
no or very limited effect  was observed for our s. typhimurium and s. paratyphi a strains, consistent with results reported for e. coli indicating that recombination of this type of substrate is reca-independent  <cit> .
in contrast, the Δreca and Δrecf mutations resulted in lower interplasmid recombination in typhimurium and paratyphi a but not in typhi strains.
deletion of recj led to a reduction in intraplasmid recombination frequencies in s. typhi, while no effect was seen in s. typhimurium.
the Δrecj mutation also affected plasmid recombination frequencies for two of the three substrates tested in s. paratyphi a. taken together, these results suggest that the recombination system in s. typhi, or at least in strains ty <dig> and isp <dig>  is not identical to the recombination system in s. typhimurium and s. paratyphi a.
to investigate the mechanism responsible for the observed differences, we analyzed the genome sequences of s. typhimurium uk- <dig> , s. paratyphi a   <cit>  and s. typhi ty <dig>   <cit> .
no paralogs of the reca, recf and recj genes were found in the three strains.
the s. typhimurium uk- <dig> has reca, reco and recr protein sequences identical to typhi ty <dig>  and recf and recj protein sequences with over 99% identity.
plasmids expressing typhimurium recf or typhi recf complemented the Δrecf <dig> mutation in typhi, as evidenced by the uv sensitivity profile  and intraplasmid recombination of pya <dig> .
therefore, the basis for these differences are not clear and indicates that there may be other genes or gene products involved.
a more detailed analysis of this phenomenon is under investigation.
plasmid recombination frequencies were higher in our salmonella strains than those reported in e. coli.
we observed intra- and interplasmid recombination frequencies on the order of  <dig> × 10- <dig> in rec+ salmonella, whereas measurements made in e. coli strain ab <dig> using a similar plasmid system  revealed a basal frequency around 10-fold lower, approximately  <dig> × 10- <dig> for both types of substrates  <cit> .
interestingly, the effect of a recf mutation in e. coli was to reduce the recombination frequency of intra- and interplasmid recombination approximately 30-fold, to roughly the same frequencies we observed for s. typhimurium .
however, consistent with the results in e. coli, the effects of reca, recf, and reca recf mutations were similar, indicating that the mutations are epistatic.
recf has been shown previously to play a role in recombinational repair of chromosomal dna in response to dna damaging agents  <cit> , including a major role in homologous recombination between direct repeats in the chromosome of s. typhimurium.
in our study, we did not observe any effect of recf on intrachromosomal recombination, although it did have an effect on the frequency of plasmid integration .
this discrepancy can be explained by the fact that we did not use dna damaging agents in our study.
these agents lead to single stranded stretches of dna that represent substrates for recf .
our observation that recf did affect plasmid integration may reflect the presence of stretches of ssdna in the plasmid, presumably due to supercoiling effects.
to induce strong primary and memory immune responses, salmonella delivery vectors should be sufficiently invasive and persistent to allow antigen expression in targeting organs, while maintaining a high degree of safety.
this requires the use of mutations that attenuate the salmonella vector without impairing its antigen delivery ability.
many attenuating mutations impair invasion and colonization ability.
in our study, we confirmed a previous report that recf is not required for s. typhimurium virulence in mice  <cit> , indicating that the recf mutant remains invasive and replicates well in colonized organs.
therefore, including a Δrecf mutation in a salmonella vaccine strain is unlikely to affect its immunogenicity.
our results with the s. typhimurium Δreca strain are consistent with two previous, independent studies showing that reca mutations reduce salmonella virulence  <cit> .
to evaluate the potential effect of Δreca mutation on immunogenicity, mice inoculated with the reca mutant were challenged with a lethal dose of virulent wild-type s. typhimurium.
all the challenged mice survived, indicating that a Δreca mutant retains immunogenicity and therefore may be suitable for use in a vaccine.
however, since it does not affect virulence, inclusion of a Δrecf mutation into a salmonella vector that has been attenuated by other means to reduce the frequency of intra- and interplasmid recombination, may be more desirable than a Δreca mutation.
studies are currently underway to investigate these possibilities.
our data show that Δreca and Δrecf mutations resulted in reduced frequencies of intraplasmid recombination in all salmonella strains tested, which included three serovars, when there was an intervening sequence between the direct duplications .
our results also show that it is likely that deletions in reca, recf or recj will not be useful for reducing interplasmid recombination in s. typhi vaccine strains, since we did not observe any reduction in interplasmid recombination frequency.
this result was disappointing, since the majority of human trials with live salmonella vaccines have focused on s. typhi.
in the case of s. typhi, it appears that the best approach to preventing interplasmid recombination will be in the careful design of each plasmid, avoiding any stretches of homology.
however, for vaccines based on s. typhimurium or s. paratyphi a, introduction of a Δrecf mutation into attenuated salmonella vaccine strains carrying multiple plasmids is a useful approach to reduce unwanted plasmid/plasmid or plasmid/chromosome recombination without further attenuating the strain or negatively influencing its immunogenicity.
the Δreca mutation had a similar or more pronounced effect on reducing various classes of recombination and it clearly had an effect on virulence.
we did not examine the effect of a Δreca mutation on the immunogenicity of a vectored antigen.
based on its effect on virulence, it may affect the immunogenicity of the vectored antigen in some attenuation backgrounds and therefore may not be applicable for all attenuation strategies.
CONCLUSIONS
in this study we showed that Δreca and Δrecf mutations reduce intraplasmid recombination in s. typhimurium, s. typhi and s. paratyphi while there is an intervening sequence between the duplicated sequences.
the Δreca and Δrecf mutations reduce interplasmid recombination in s. typhimurium and s. paratyphi but not in s. typhi.
the Δrecf mutations also sharply reduce intraplasmid recombination between direct duplications in s. typhi.
since Δreca mutation results in an avirulent salmonella strain, the Δrecf mutation is ideal for reducing plasmid recombination in salmonella delivery vectors without impairing the virulence.
the intrachromosomal recombination and plasmid integration are 2- <dig> orders lower than plasmid recombination, therefore are less concerned.
these information help develop salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy.

 attenuated salmonella are being developed as vaccines to protect against typhoid fever. there are also endeavors employing salmonella as delivery vectors for therapeutic molecules. one strategy utilizes attenuated salmonella, which expresses a gene or gene fragment encoding a protective antigen as vaccine against bacterial pathogens. the heterologous genes can be expressed from the salmonella chromosome, or, more often, from a multi-copy plasmid. another strategy exploits salmonella as a delivery vector of dna vaccine against viral pathogens. the later strategy is also used to deliver dna encoding tumor antigen or cytokine for therapeutic applications in oncology. in addition, salmonella is used to deliver small interfering rnas, ribozymes and large dna molecules encoding a viral genome. for instance, in vivo delivery of an artificial bacterial chromosome carrying the viral genome of the murine cytomegalovrirus by salmonella typhimurium led to a productive virus infection in mice and resulted in elevated titers of specific antibodies against lethal mcmv challenge. most vaccine designs utilize salmonella delivery vectors carrying a single plasmid for expression of a single antigen or of a fusion protein carrying epitopes from more than one antigen. to induce broader immunity against a particular pathogen or various pathogens, one might need to express multiple antigens from a single plasmid carrying different antigen cassettes or from multiple plasmids in a single cell, each expressing one or more relevant antigens. co-delivery of plasmids encoding tumor antigens and cytokines by salmonella has been successfully demonstrated to improve protective immunity against cancer. in the case where multiple plasmids are carried in the same salmonella vector strain, there are most likely regions of homology between the plasmids, since the widely used puc- and pbr-based plasmids have origins of replication that are nearly identical and both share regions of homology with the p15a ori. additionally, commonly used promoter sequences, transcriptonal terminators and other expression plasmid components may also be present on plasmids coexisting in the same bacterial cell. the presence of these similar or identical dna sequences would serve to facilitate undesirable interplasmid recombination. in some cases the bacterial vector may intentionally harbor multiple copies of the same dna sequence, which may lead to plasmid instability. recently, we encountered such a situation during the development of a bacterial based influenza vaccine. we constructed a single plasmid carrying eight head-to-tail connected influenza cdna cassettes. the plasmid was intended for delivery into host cells by an attenuated salmonella strain. the multiple repetitive sequences residing in the plasmid make its stability within the attenuated salmonella an important concern because any intraplasmid recombination event results in deletion of one or more influenza gene cassettes. recent work in our laboratory has focused on developing new strategies for attenuated salmonella vaccine strains, with features including regulated delayed in vivo attenuation, regulated delayed in vivo antigen synthesis, and programmed delayed in vivo cell lysis. for all of these systems, one or more chromosomal and/or plasmid genes are placed under the control of the arac pbad promoter. eventually, our goal is to combine all of these features into a single salmonella vaccine vector strain. such a strain will therefore carry multiple chromosomal and plasmid copies of arac pbad, providing sites for potential recombination, which could lead to unwanted chromosomal or plasmid rearrangements. however, to our knowledge, there have been no published studies specifically designed to evaluate plasmid recombination in salmonella enterica. deletions of several escherichia coli genes are known to reduce the frequency of plasmid recombination, including the reca, rece, recf and recj genes. the reca gene encodes the general recombinase reca, involved in nearly all forms of recombination in the cell. the rece, recf and recj proteins play a role in plasmid recombination and recombination repair. the reca, recf and recj proteins are highly homologous between e. coli and s. enterica, therefore they may play similar roles in dna recombination. despite these possible similarities, the recombination systems in the two organisms differ somewhat, as s. enterica does not encode rece. based on these concerns, we decided to determine the effect of rec gene deletions on intraplasmid recombination, interplasmid recombination, intrachromosomal recombination and plasmid integration in s. enterica. in this work, we examine the effect of Δreca, Δrecf and Δrecj mutations on dna recombination frequencies in three serovars of salmonella enterica currently relevant to vaccine development. our results show that the effect of these mutations on recombination can vary among salmonella serovars and with previously published results in e. coli.  plasmid construction we constructed a series of plasmids encoding various truncated teta genes to assay plasmid recombination frequencies using the strategies similar to those described previously. restoration of a functional teta gene via intra- or intermolecular recombination resulted in a change of the bacterial phenotype from tetracycline sensitive to tetracycline resistant, and served as a marker allowing us to measure the frequency of recombination events. * cat: chloramphenicol resistance gene; teta: tetracycline resistance gene; amp: ampicillin resistance gene; kan: kanamycin resistance gene; 3'tet: 3' portion of the teta gene; 5'tet: 5' portion of the teta gene together with its promoter; aacc1: 3-n-aminoglycoside acetyltransferase. plasmids pya and pya were constructed to test intraplasmid recombination. plasmid pya carries two truncated teta genes, which have 466-bp of tandemly repeated sequence. an intramolecular recombination event can delete one of the repeats resulting in an intact teta gene, thereby recreating the structure of plasmid pacyc. theoretically, intermolecular recombination may occur between two pya molecules to form a plasmid dimer with a functional teta gene. plasmid pya contains a 602-bp teta sequence duplication separated by a 1041-bp kan cassette. the intramolecular recombination product is equivalent to pacyc the intermolecular recombination product is a dimer plasmid containing an intact teta gene. plasmids pya and pya carry the 3'tet gene and 5'tet gene, respectively. the rec+ salmonella strain χ carrying either plasmid individually was sensitive to tetracycline. there is 751-bp of teta dna in common between the two truncated teta genes. recombination between the two plasmids creates a hybrid plasmid containing an intact teta gene. intraplasmid recombination products to verify the recombination products, plasmid dna was prepared from tetracycline resistant single colonies derived from χ χ and χ plasmids extracted from tcr clones of χ were digested with xbai and sali. theoretically, xbai/sali digestion of pya will yield two fragments, pacyc will yield two fragments and pya dimer will yield four fragments. the results showed that digestion of all tcr clones yielded a 721-bp band, indicating either a pya dimer or a plasmid equivalent to pacyc three clones yielded the pya dimer-specific 1653-bp band. therefore, we conclude that the other clones recombined to form the pacyc184-like structure. of note, several clones also yielded the 1187-bp pya4463-specific band, suggesting that the original plasmid and its recombination product could coexist in the same bacterial cell. plasmids extracted from tcr clones of χ were digested with kpni and ecori. theoretically, plasmid pya will be digested into two fragments, plasmid pacyc will be linearized and the pya plasmid dimer will be digested into four fragments. examination of the restricted dna showed that only one clone had the pya dimer-specific 1643-bp band. the most prominent band in the other lanes was a 4245-bp band expected for pacyc184-like recombination products. nine clones contained a mixture of pacyc and pya. interplasmid recombination products plasmids extracted from tcr clones of χ were digested with ncoi and bglii. both pya and pya are linearized into a dna fragment about kb. therefore, in cells containing each or both monomeric plasmids, the digested product will be a single band. the pya4464-pya hybrid will be cut into two fragments. all four of the tcr clones we isolated and examined showed recombination product specific bands and the 4-kb band expected when each plasmid exists separately in the cell. four tetracycline sensitive isolates were examined and only a single band was observed, as expected. these results suggest that interplasmid recombination occurred in the tcr cells and that both dimer and individual monomers corresponding to at least one of the two starting plasmids can coexist in the same bacterial cell. we performed a similar experiment in s. typhi strain ty and obtained identical results. construction of rec deletion strains we constructed a series of strains for these studies carrying deletions in either reca, recf or recj in s. typhimurium uk- s. typhi ty and s. paratyphi a. we also constructed ΔrecaΔ recf and Δrecj Δ recf double mutants in s. typhimurium. deletion of reca, recf and recj results in an increase in sensitivity to uv irradiation. to verify the presence of these deletions phenotypically in our strains, the uv sensitivity of the s. typhimurium mutant strains was measured. the Δrecf and Δrecj mutants showed significantly lower surviving fractions than the wild type strain after the same exposure dose. by contrast, after five seconds of uv exposure to × cfu of the Δreca mutant, we were unable to recover any surviving cells. uv resistance similar to the wild-type strain χ was restored to s. typhimurium Δreca and Δrecf mutants strains after introduction of reca plasmid or either recf plasmid, respectively. transformation of either mutant strain with vector plasmid pya did not restore uv resistance. * kan: kanamycin resistance gene; 5'tet: 5' portion of the teta gene together with its promoter; 3'tet: 3' portion of the teta gene. effect of rec deletions on intraplasmid recombination to examine the influence of Δreca, Δrecf and Δrecj mutations on intraplasmid recombination frequencies, plasmid pya or pya were introduced into salmonella rec mutants and their parental strains and analyzed as described in the methods section. the recombination frequency of plasmid pya was approximately - × 10- in rec+ typhimurium, typhi and paratyphi a. in s. typhimurium and paratyphi a, most of the rec deletions had no effect on the intraplasmid recombination frequency of plasmid pya except that a small, but statistically significant decrease in recombination was observed in the Δreca mutant of paratyphi a. however, in both s. typhi strains, both Δrecf mutations resulted in approximately 10-fold decrease in recombination frequency, while the Δreca and Δrecj mutations resulted in a 2-3-fold reduction. in the complementation test, the recombination frequency of plasmid pya in s. typhi χ was restored to ± × 10- and ± × 10- by introduction of plasmid pya encoding s. typhimurium recf gene and pya encoding the s. typhi recf gene, respectively. aintraplasmid recombination without intervening sequence. b intraplasmid recombination with a 1041-bp intervening sequence. c interplasmid recombination. d not assayed. ** p <, relative to the parental rec+ strain. the results with plasmid pya were also variable among strains. the recombination frequency in rec+ s. typhimurium and s. paratyphi a strains was approximately 2- × 10- and in both s. typhi strains, the frequency was 3-fold higher, at × 10- and × 10-. in s. typhimurium and s. typhi ty the Δ reca and Δrecf mutations reduced the recombination frequency of plasmid pya by 5-20-fold. the results were similar for s. paratyphi a, though the Δrecf mutation only led to 3-fold lower plasmid pya recombination. the Δrecj mutation had no effect in s. typhimurium and resulted in a 2-3-fold decrease in recombination in both s. typhi ty and s. paratyphi a. combining the Δreca Δrecf mutations in s. typhimurium led to a recombination frequency similar to the frequencies observed for both mutations individually, indicating no additive effect. in the complementation test, plasmid pya which encodes s. typhimurium reca, was transformed into s. typhimurium Δreca mutant χ and s. typhi Δreca mutant χ their respective recombination frequencies were ± × 10- and ± × 10- which were comparable to the corresponding wild type strains. the recf-encoding plasmids pya and pya were transformed into recf mutant strains χ and χ respectively. the respective recombination frequencies were increased to ± × 10- and ± × 10- effect of rec deletions on interplasmid recombination to evaluate interplasmid recombination, plasmids pya and pya were co-electroporated into the wild-type and rec deletion strains. electroporants from each test strain were grown in lb broth containing both ampicillin and chloramphenicol to maintain selection for both plasmids. the frequency of recombination was determined as described in the methods section. the interplasmid recombination frequency was 1- × 10- for rec+ s. typhimurium, s. typhi and s. paratyphi a strains. for typhimurium and paratyphi a, the Δreca and each Δrecf mutation reduced the interplasmid recombination frequency by about 3-10-fold. in contrast, the Δreca mutation had no effect on interplasmid recombination in s. typhi ty the Δrecf mutations did not reduce interplasmid recombination in either of the typhi strains. surprisingly, introduction of the Δrecf mutation into s. typhi ty resulted in significantly higher interplasmid recombination. note that we performed this analysis in eight independent experiments and observed a higher recombination frequency of interplasmid recombination each time. the Δrecj mutation had no significant effect in s. typhi, and a small but significant effect in s. typhimurium and s. paratyphi a. the recombination frequencies were also determined in s. typhimurium strains Δreca Δrecf and Δrecf Δrecj double deletions. no additive effect between the two mutations was observed with respect to each single mutation. effect of rec deletions on chromosome related recombination to measure intrachomosomal recombination frequencies, we introduced the pya4590-derived dna sequence containing two truncated teta genes into the s. typhimurium chromosome at cysg. the two truncated teta genes had bp of overlapping sequence. intrachromosomal recombination deletes the kanamycin resistance cassette and restores one intact copy of the teta gene. deletion of reca resulted in a 5-fold reduced recombination frequency compared to the rec+ strain χ, while the recf or recj deletions had no effect, indicating that recf and recj are not involved in this process. a mean ± std from 3- assays were shown in the table. b upon introduction of pkd, the frequency was ± × 10-. ** p <, relative to the parental rec+ strain. to examine plasmid integration, the 5'tet gene was introduced into the s. typhimurium chromosome at cysg. the resulting strains were transformed with plasmid pya. the bp of overlapping sequence between 5'tet on the chromosome and the 3'tet on the plasmid could result in plasmid integration into the chromosome, generating an intact teta gene. deletion of reca had a profound effect, reducing the integration frequency to less than × 10- which was below the limits of detection in this assay, indicating a strict requirement for reca in this process. introduction of plasmid pkd which encodes the λ red recombinase, into χ carrying pya restored the integration frequency to the level of the rec+ strain χ deletion of recf reduced the frequency of integration less than 3-fold and the Δrecj deletion had no effect. effect of rec deletions on the virulence of s. typhimurium balb/c mice were orally inoculated with the highly virulent s. typhimurium strain χ and its rec mutant derivatives. the ld50s of χ χ and χ were similar, × × and × cfu, respectively. the ld of the Δrecf Δrecj double mutant was approximately 100-fold higher than χ at × cfu. all mice inoculated with × cfu of the Δreca mutant survived, indicating that the ld was > × cfu. two months following the initial inoculation with the Δ reca mutant strain, surviving mice were challenged with either × or × cfu of wild-type strain χ all mice survived the challenge, indicating that Δ reca mutant strain χ was both attenuated and immunogenic. discussion we began our studies using information gathered in e. coli as a reference point. in e. coli, reca-dependent homologous recombination relies on the recbcd pathway, the recfor pathway and the rece pathway. the recbcd pathway is important in conjugational and transductional recombination, and may also be involved in the recombination of plasmids containing one or more chi sites. recombination in small plasmids lacking a chi sequence is primarily catalyzed by the recfor pathway. recf, reco, and recr bind to gaps of ssdna and displace the single-strand dna binding proteins to allow reca to bind. the recj ssdna exonuclease acts in concert with recfor to enlarge the ssdna region when needed. strand exchange is then catalyzed by reca. because of their prominent role in plasmid recombination in e. coli, we analyzed the effect of mutations in recf, recj and reca on plasmid recombination in salmonella. attenuated s. typhi strains have been developed as antigen delivery vectors for human vaccine use. due to the host restriction phenotype of s. typhi, preliminary work is typically done in s. typhimurium using mice as the model system to work out attenuation and antigen expression strategies. recently, we have also been investigating attenuated derivatives of the host-restricted strain s. paratyphi a as a human vaccine vector. therefore, it was of interest to evaluate and compare the effects of rec mutations in these three salmonella serovars. we selected s. typhi strain ty as exemplary of this serovar because most of the vaccines tested in clinical trials to date have been derived from this strain. s. typhi strain isp has also been evaluated in clinical trials and we therefore included it in some of our analyses. we found that, for some dna substrates, the effects of Δreca and Δrecf deletion mutations differed among salmonella enterica serotypes. in particular, we found that deleting reca, recf or recj in s. typhi ty and deleting recf in strain isp had significant effects on the recombination frequency of our direct repeat substrate, pya. no or very limited effect was observed for our s. typhimurium and s. paratyphi a strains, consistent with results reported for e. coli indicating that recombination of this type of substrate is reca-independent. in contrast, the Δreca and Δrecf mutations resulted in lower interplasmid recombination in typhimurium and paratyphi a but not in typhi strains. deletion of recj led to a reduction in intraplasmid recombination frequencies in s. typhi, while no effect was seen in s. typhimurium. the Δrecj mutation also affected plasmid recombination frequencies for two of the three substrates tested in s. paratyphi a. taken together, these results suggest that the recombination system in s. typhi, or at least in strains ty and isp is not identical to the recombination system in s. typhimurium and s. paratyphi a. to investigate the mechanism responsible for the observed differences, we analyzed the genome sequences of s. typhimurium uk-, s. paratyphi a and s. typhi ty. no paralogs of the reca, recf and recj genes were found in the three strains. the s. typhimurium uk- has reca, reco and recr protein sequences identical to typhi ty and recf and recj protein sequences with over 99% identity. plasmids expressing typhimurium recf or typhi recf complemented the Δrecf mutation in typhi, as evidenced by the uv sensitivity profile and intraplasmid recombination of pya. therefore, the basis for these differences are not clear and indicates that there may be other genes or gene products involved. a more detailed analysis of this phenomenon is under investigation. plasmid recombination frequencies were higher in our salmonella strains than those reported in e. coli. we observed intra- and interplasmid recombination frequencies on the order of × 10- in rec+ salmonella, whereas measurements made in e. coli strain ab using a similar plasmid system revealed a basal frequency around 10-fold lower, approximately × 10- for both types of substrates. interestingly, the effect of a recf mutation in e. coli was to reduce the recombination frequency of intra- and interplasmid recombination approximately 30-fold, to roughly the same frequencies we observed for s. typhimurium. however, consistent with the results in e. coli, the effects of reca, recf, and reca recf mutations were similar, indicating that the mutations are epistatic. recf has been shown previously to play a role in recombinational repair of chromosomal dna in response to dna damaging agents, including a major role in homologous recombination between direct repeats in the chromosome of s. typhimurium. in our study, we did not observe any effect of recf on intrachromosomal recombination, although it did have an effect on the frequency of plasmid integration. this discrepancy can be explained by the fact that we did not use dna damaging agents in our study. these agents lead to single stranded stretches of dna that represent substrates for recf. our observation that recf did affect plasmid integration may reflect the presence of stretches of ssdna in the plasmid, presumably due to supercoiling effects. to induce strong primary and memory immune responses, salmonella delivery vectors should be sufficiently invasive and persistent to allow antigen expression in targeting organs, while maintaining a high degree of safety. this requires the use of mutations that attenuate the salmonella vector without impairing its antigen delivery ability. many attenuating mutations impair invasion and colonization ability. in our study, we confirmed a previous report that recf is not required for s. typhimurium virulence in mice, indicating that the recf mutant remains invasive and replicates well in colonized organs. therefore, including a Δrecf mutation in a salmonella vaccine strain is unlikely to affect its immunogenicity. our results with the s. typhimurium Δreca strain are consistent with two previous, independent studies showing that reca mutations reduce salmonella virulence. to evaluate the potential effect of Δreca mutation on immunogenicity, mice inoculated with the reca mutant were challenged with a lethal dose of virulent wild-type s. typhimurium. all the challenged mice survived, indicating that a Δreca mutant retains immunogenicity and therefore may be suitable for use in a vaccine. however, since it does not affect virulence, inclusion of a Δrecf mutation into a salmonella vector that has been attenuated by other means to reduce the frequency of intra- and interplasmid recombination, may be more desirable than a Δreca mutation. studies are currently underway to investigate these possibilities. our data show that Δreca and Δrecf mutations resulted in reduced frequencies of intraplasmid recombination in all salmonella strains tested, which included three serovars, when there was an intervening sequence between the direct duplications. our results also show that it is likely that deletions in reca, recf or recj will not be useful for reducing interplasmid recombination in s. typhi vaccine strains, since we did not observe any reduction in interplasmid recombination frequency. this result was disappointing, since the majority of human trials with live salmonella vaccines have focused on s. typhi. in the case of s. typhi, it appears that the best approach to preventing interplasmid recombination will be in the careful design of each plasmid, avoiding any stretches of homology. however, for vaccines based on s. typhimurium or s. paratyphi a, introduction of a Δrecf mutation into attenuated salmonella vaccine strains carrying multiple plasmids is a useful approach to reduce unwanted plasmid/plasmid or plasmid/chromosome recombination without further attenuating the strain or negatively influencing its immunogenicity. the Δreca mutation had a similar or more pronounced effect on reducing various classes of recombination and it clearly had an effect on virulence. we did not examine the effect of a Δreca mutation on the immunogenicity of a vectored antigen. based on its effect on virulence, it may affect the immunogenicity of the vectored antigen in some attenuation backgrounds and therefore may not be applicable for all attenuation strategies.  in this study we showed that Δreca and Δrecf mutations reduce intraplasmid recombination in s. typhimurium, s. typhi and s. paratyphi while there is an intervening sequence between the duplicated sequences. the Δreca and Δrecf mutations reduce interplasmid recombination in s. typhimurium and s. paratyphi but not in s. typhi. the Δrecf mutations also sharply reduce intraplasmid recombination between direct duplications in s. typhi. since Δreca mutation results in an avirulent salmonella strain, the Δrecf mutation is ideal for reducing plasmid recombination in salmonella delivery vectors without impairing the virulence. the intrachromosomal recombination and plasmid integration are 2- orders lower than plasmid recombination, therefore are less concerned. these information help develop salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy. 
as the carrier for target gene, the cargo plasmid should be stable in the bacterial vector. this information will be useful for developing salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy. salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. the frequencies of intrachromosomal recombination and plasmid integration into the chromosome were and logs lower than plasmid recombination frequencies in rec+ strains. a Δreca mutation reduced both intrachromosomal recombination and plasmid integration frequencies. plasmid recombination has been reduced in e. coli by mutating several genes including the reca, rece, recf and recj. however, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in salmonella enterica. the effect of reca, recf and recj deletions on dna recombination was examined in three serotypes of salmonella enterica. the Δreca and Δrecf mutations can reduce plasmid recombination frequencies in salmonella enterica, but the effect can vary between serovars. we found that intraplasmid recombination between direct duplications was recf-independent in typhimurium and paratyphi a, but could be significantly reduced in typhi by a Δreca or Δrecf mutation; in all three salmonella serotypes, both Δreca and Δrecf mutations reduced intraplasmid recombination when a bp intervening sequence was present between the duplications; Δreca and Δrecf mutations resulted in lower frequencies of interplasmid recombination in typhimurium and paratyphi a, but not in typhi; in some cases, a Δrecj mutation could reduce plasmid recombination but was less effective than Δreca and Δrecf mutations.
 salmonella has been employed to deliver therapeutic molecules against cancer and infectious diseases. as the carrier for target gene, the cargo plasmid should be stable in the bacterial vector. plasmid recombination has been reduced in e. coli by mutating several genes including the reca, rece, recf and recj. however, to our knowledge, there have been no published studies of the effect of these or any other genes that play a role in plasmid recombination in salmonella enterica.  the effect of reca, recf and recj deletions on dna recombination was examined in three serotypes of salmonella enterica. we found that intraplasmid recombination between direct duplications was recf-independent in typhimurium and paratyphi a, but could be significantly reduced in typhi by a Δreca or Δrecf mutation; in all three salmonella serotypes, both Δreca and Δrecf mutations reduced intraplasmid recombination when a bp intervening sequence was present between the duplications; Δreca and Δrecf mutations resulted in lower frequencies of interplasmid recombination in typhimurium and paratyphi a, but not in typhi; in some cases, a Δrecj mutation could reduce plasmid recombination but was less effective than Δreca and Δrecf mutations. we also examined chromosome-related recombination. the frequencies of intrachromosomal recombination and plasmid integration into the chromosome were and logs lower than plasmid recombination frequencies in rec+ strains. a Δreca mutation reduced both intrachromosomal recombination and plasmid integration frequencies.  the Δreca and Δrecf mutations can reduce plasmid recombination frequencies in salmonella enterica, but the effect can vary between serovars. this information will be useful for developing salmonella delivery vectors able to stably maintain plasmid cargoes for vaccine development and gene therapy. 