ORF3 of pFNL10 confers plasmid stability in the absence of selection
Our approach to E. coli-Francisella
shuttle vector construction was to build vectors from the “bottom up”, starting with the minimal requirement for a Francisella
replicon taken from the cryptic plasmid pFNL10. Our first generation were not stable in the absence of antibiotic selection, but introduction of the ORF4–ORF5 region from pFNL10 resulted in stable second generation plasmids. A puzzling observation was that one widely used shuttle vector, pFNLTP1, lacks an intact ORF4–ORF5 region but is still stable in the absence of selection (Maier, et al., 2004
). A major difference between pFNLTP1 and our vectors is that pFNLTP1 has an intact copy of the ORF3 gene of pFNL10. This gene encodes a hypothetical protein with homology to putative integrases/resolvases found in other plasmids but its function in plasmid biology is unknown (Pomerantsev, et al., 2001a
, Pomerantsev, et al., 2001b
We tested the role of ORF3 in plasmid maintenance by cloning a PCR product bearing ORF3 from pFNL10 into the unstable plasmid pMP527. The construction was designed to recapitulate the ORF3 location in pFNL10 within the context of the pFNL10 components of pMP527 (). We found that the new plasmid, pMP716, was capable of efficient transformation of F. tularensis strains LVS and Schu similar to that of pMP527, and was stable in the absence of antibiotic selection in both strains.
Shuttle vector copy number
The function of the ORF3 product is unknown, but it may play a role in the separation of concatamers as suggested by its homology to putative plasmid integrases/resolvases. However, this function was hypothesized to be the role of the ORF2 gene product, a putative helicase, since plasmids lacking ORF2 form higher-order concatamers (Pomerantsev, et al., 2001a
, Pomerantsev, et al., 2001b
). None of our plasmids that lack ORF3 form multimers (data not shown).
Alternatively, ORF3 might affect plasmid copy number, which could increase the chance of proper segregation of plasmids into daughter cells. To explore this, we determined the average copy number of these plasmids in LVS (). We found that the unstable plasmid pMP527 has an average copy number of 12 copies/genome, while that of pMP716, the stable derivative of pMP527 bearing ORF3 is 26/genome. We also determined that pFNLTP1 has an average copy number of 171 copies/genome (). The instability of pMP527 is puzzling, as one would not expect that a plasmid with a copy number of ~12 to be easily lost. Thus, there may be additional factors that contribute to plasmid segregation in this organism. However, the presence of ORF3 doubled the copy number of pMP716 compared to its parent pMP527, which could explain why the former plasmid is stable in the absence of selection.
Plasmid copy number determinations
To test this idea, we mutagenized ORF3 in pFNLTP1 by taking advantage of a unique ClaI restriction endonuclease site situated near the middle of the gene. The plasmid was digested with ClaI, blunted and self ligated to generate pMP773. This modification generated a frameshift mutation resulting in termination in the middle of the ORF3 coding sequence. As shown in , the mutation decreased the copy number of pMP773 to 60 copies/genome. This is still higher than the ORF3 bearing plasmid pMP716 and, consistent with the view that higher copy number plasmids are difficult to lose, we found that pMP773 is stable in the absence of antibiotic selection.
The addition of ORF3 to the unstable plasmid pMP527 doubled the copy number, while mutation of ORF3 in the high copy number plasmid pFNLTP1 decreased the copy number by almost three-fold but still within the range that ensures stability in the absence of selection. Variations in DNA structure, supercoiling, and subsequent replication dynamics may explain why pFNLTP1 and pMP773 have higher copy numbers compared to pMP527 and pMP716.
We previously reported that pFNLTP1 transformants of F. tularensis
Schu grow more slowly on plates than transformants bearing pMP527 derivatives, and we hypothesized that this might be due to presence of ORF3 in pFNLTP1 (LoVullo, et al., 2006
). However, we have found that Schu transformants bearing pMP716 (pMP527 with ORF3) grow as well as clones bearing pMP527, but that transformants bearing pMP773 (pFNLTP1 with a mutated ORF3) still have a growth defect (data not shown). Perhaps the relatively higher copy number of pFNLTP1 and pMP773 contributes to the growth phenotype of Schu clones transformed with these plasmids.
Shuttle vector improvements
In this work, we describe new, third generation shuttle vectors (shown in ), derived from the HygR
plasmids pMP529 (unstable), pMP633 (stable) or the KmR
plasmid pMP607 (stable) (LoVullo, et al., 2006
). In the case of the stable plasmids, all are second-generation vectors bearing the ORF4–ORF5 toxin-antitoxin genes. We chose to use these stable plasmids instead of the stable ORF3-bearing plasmids because of concern for the higher copy number of the ORF3 plasmids. We thought it best to refrain from using the ORF3-bearing vector as the primary platform for new vectors as its higher copy number might be detrimental in the cloning of genes with potentially toxic products. The copy number of the ORF4–ORF5 plasmid could not be formally measured because the amount of plasmid DNA in the nucleic acid preparations was too low to yield the necessary data needed to calculate copy number. We noted that DNA preparations from cells bearing the ORF4–ORF5 plasmid had less DNA than samples prepared from cells containing the unstable, low copy number vector pMP527 (data not shown). We think that the lower yield is due to a decrease in copy number brought about by the presence of the hyg
gene, which has a higher G+C content than that found in the genome of Francisella
and could possibly affect plasmid replication dynamics. Thus, we chose the ORF4–ORF5 HygR
plasmid as the backbone upon which new vectors would be built.
The first modification is the inclusion of the promoter region of the F. tularensis blaB
gene. We chose this particular promoter because we have found that it is not recognized in E. coli
(data not shown) and would therefore be beneficial for the cloning of F. tularensis
genes that are toxic to E. coli
. This promoter region was modified by the addition of the multiple cloning site (mcs) taken from our earlier sacB
vector pMP590 (LoVullo, et al., 2006
). A cassette consisting of PblaB
-mcs was introduced into a set of three intermediate plasmids (pMP656, pMP658, and pMP622, see ) that were used for subsequent modifications.
The multiple cloning site of the PblaB-mcs cassette was altered to include the recognition site for the endonuclease RsrII. This is an unusual enzyme that recognizes the sequence CGGWCCG, where W is either a T or A. Because of this particular specificity, it is possible to use this single site for directional cloning of PCR fragments (). We took advantage of this since there is only one RsrII site in the genomes of F. novicida Utah112, LVS, and F. tularensis Schu S4 located in the gene FTT_1110 (or relevant homologs). Thus, the novel nature of this enzyme allows one to use it for the directional cloning of almost all Francisella genes. To incorporate this recognition site into the multiple cloning site of our HygR plasmids, we had to remove three RsrII sites present within the hyg gene by site-directed mutagenesis, as described in the methods section. The RsrII site was engineered into the mcs of the PblaB-mcs plasmid set to generate pMP822 (stable, HygR), pMP823 (unstable, HygR), and pMP814 (stable, KmR). Maps of these plasmids are shown in .
Additional plasmids were constructed, each of which contained the RsrII-mcs but lacked the PblaB promoter. These were built for use in cases where the PblaB promoter would not be desirable, for example, in situations where screening for gene expression in E. coli is required, or when the native promoter is more appropriate for the particular gene in question. These plasmids, pMP831 (stable, HygR), pMP829 (unstable, HygR), and pMP828 (stable, KmR) are shown in . The modifications to these plasmids do not affect their ability to transform E. coli or Francisella bacteria (data not shown).
We have shown here that the biology of Francisella plasmids is complex. There are at least two ways to stabilize shuttle plasmids in this organism: one can use the toxin-antitoxin genes to maintain the plasmid in the population, or one can increase the copy number of the plasmid by adding the ORF3 gene and presumably improve segregation into daughter cells. In addition, our results support the idea that high-copy number plasmids attenuate the growth of F. tularensis subsp. tularensis. We envision that the improved shuttle vectors described in this manuscript will be helpful to others working on the genetics of Francisella bacteria.