Journal of Bacteriology, November 2002, p . 5926-5934, Vol . 184, No . 21

DNA Inversion on Conjugative Plasmid pVT745

Jinbiao Chen,¹ Donald J . Leblanc,^2, and Dominique M . Galli^1*

School of Dentistry, Indiana University, Indianapolis, Indiana 46202,¹ Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285²

Received 6 March 2002/ Accepted 31 July 2002

Resolvases and invertases have been identified on a variety of bacterial plasmids of gram-positive and gram-negative origins . They have been shown to play a role in plasmid partition (reviewed in reference 17) and plasmid replication (3, 7) . In addition, phase variation in prokaryotes has been associated with DNA inversions catalyzed by site-specific recombinases encoded on phage, chromosomal, or plasmid DNA (for reviews see references 14 and 19) . Complex DNA rearrangements in which multiple DNA segments can invert independently or in groups have been described for p15B (16), as well as for IncI1 and IncI2 plasmids (for review see reference 19) . One of the best-characterized multiple-DNA inversion systems is the shufflon of conjugative plasmid R64, which controls mating frequencies of the plasmid to different recipients by changing a component of the pilus (19) .

Plasmid pVT745 is one of few plasmids isolated from the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans (20) . DNA nucleotide base sequence analysis revealed functions related to conjugation, replication, and replicon stability on the 25-kb size plasmid (12) . Conjugative transfer of pVT745 from its host, VT745, to other A . actinomycetemcomitans strains or Escherichia coli was shown at a low frequency of 10^-5 to 10^-6 (12) . Despite its conjugative properties, the plasmid has not been found in any clinical isolates of A . actinomycetemcomitans other than VT745 . However, remnants of pVT745 DNA were detected on the chromosomes of various A . actinomycetemcomitans strains (22) . Analysis of the DNA sequence of pVT745 identified the presence of a gene, AA01, which showed a high degree of similarity to the Din family of site-specific recombinases (12) . The objective of this study was to determine the functional and biological role of the pVT745-specific recombinase .

 
DNA preparations and recombinant DNA techniques. Plasmid DNA was isolated from A . actinomycetemcomitans and E . coli as described previously (11) . Restriction endonucleases, Klenow polymerase I, and T4 DNA ligase were used in accordance with the manufacturer's instructions (Gibco-BRL) . Standard recombinant DNA techniques were performed as described by Sambrook et al . (23) . DNA-DNA hybridization conditions and transformation by electroporation have been described previously (11) . Standard three-step PCR experiments were performed with Taq polymerase from Promega (Madison, Wis.) . All PCR primers used are listed in Table 2 . PCR amplification products were cloned into pGEM-T Easy (Promega) according to the manufacturer's instructions .

 
RNA isolation and RT-PCR. Total RNA was isolated from A . actinomycetemcomitans by use of the RNaqueous kit (Ambion) according to the manufacturer's instructions . Residual DNA was removed by treatment with RNase-free DNase (Gibco-BRL) for 30 min at 37°C . Reverse transcriptase PCR (RT-PCR) was performed with the Qiagen OneStep RT-PCR kit (Qiagen) as described by the manufacturer . Reverse transcription was performed at 55°C with primers P11 and P12 (invertase gene) and at 65°C with primers P13 and P14 (leukotoxin gene) (Table 2) . Amplification products were visualized after electrophoresis in 0.8% agarose gels .

DNA sequencing. Nucleotide sequencing was performed with custom-made primers (P1, P2, P3, and P4) (Table 2) flanking the junctions of the invertible element on pDMG21B by use of the femtomole DNA Cycle sequencing system (Promega) .

Construction of a recA-deficient ATCC 29522 strain. The nucleotide sequence of the recA gene was retrieved from the genome of ATCC 700685 (http://www.genome.ou.edu/act.html) and was used to design two oligonucleotides flanking the gene on the 5' (P5) and 3' (P6) ends (Table 2) . These primers were then used in a PCR experiment to amplify the corresponding gene in ATCC 29522 . The amplification product of 4.2 kb was ligated to vector pGEM-T Easy (Promega) and was introduced into E . coli JM109 . However, the intact gene recombined into the E . coli genome . Therefore, a 1.8-kb internal XhoII fragment of recA was removed from the 4.2-kb PCR product and was cloned into pUC19 linearized with BamHI . The new construct was purified from E . coli JM109 and was digested with MluI . The latter restriction site was unique and was located in the center of the 1.8-kb PCR insert . The linearized vector was then blunt ended with the large fragment of Klenow polymerase I (Gibco-BRL) and was ligated to a ca . 2-kb fragment excised from pMK3, which carried the gene for STR resistance . The latter, a ClaI fragment, was blunt ended as well . The resulting construct of 6.5 kb was purified from E . coli and was used to transform strain ATCC 29522Rif by electroporation . Since pUC19 does not replicate in A . actinomycetemcomitans, transformants resistant to RIF and STR indicated that all or part of the recA construct had inserted into the A . actinomycetemcomitans chromosome via homologous recombination .

Construction of pVT745 derivatives: pDMG27, pDMG28, pDMG30, and pDMG32. All recombinant constructs used for allelic replacement were obtained in E . coli strain JM109 . The following cloning strategy was used to construct pDMG27 in which the gene for the site-specific recombinase (inv) on pDMG21A is replaced with an STR resistance cassette via allelic exchange by homologous recombination (pDMG27) . First, vector pUC19 was modified by eliminating its unique EcoRI and AvaI sites located in the multiple-cloning site . This was accomplished by digesting pUC19 with the corresponding restriction enzymes, treatment of the restriction sites with the large fragment of Klenow polymerase I (Gibco-BRL), and religation of the free ends of the vector . Subsequently the 2.9-kb BamHI-HincII fragment of pVT745, which carried the gene for the site-specific recombinase (12), was cloned into the modified pUC19 vector digested with the same restriction enzymes . The resulting construct was double digested with EcoRI-AvaI to remove an internal 400-bp fragment from the target gene . The linearized construct was then blunt ended with Klenow polymerase I and was ligated with a 2-kb blunt-ended fragment carrying the STR resistance cassette from pMK5 . This final pUC19 derivative was purified from E . coli JM109 and was transferred into VT745:pDMG21A via electroporation . Since pUC19 does not replicate in this host, only transformants that had the stm gene integrated into the resident plasmid, pDMG21A, by homologous recombination were able to grow in the presence of SPT . The pUC19 construct allowed for a single- or a double-crossover event to occur . Plasmid DNA isolated from the transformants was analyzed by restriction enzyme digestion and Southern blot hybridizations using pUC19 or the STR resistance gene as probes . Results from these experiments confirmed that both, a single-crossover event and a double-crossover event, had occurred . One of the pDMG21A derivatives obtained by double crossover, pDMG27, was subsequently used in conjugation assays .

To construct pDMG28 and pDMG30, an additional selective marker, SPT (spc), was inserted into pDMG21A and pDMG27 at an XbaI site, which is located outside the invertible segment (Fig . 1A) . This was accomplished as follows: primers were designed (P7 and P8 [Table 2]) and were used to amplify an 870-bp fragment of pVT745 by PCR . The target sequence corresponded to a region upstream of the putative oriV and contained the XbaI site into which spc was to be inserted (12) . To one of the primers the restriction site for EcoRI and to the other primer the restriction site for HindIII were added to allow for cloning of the PCR amplification product into pUC19 digested with both of these enzymes . The resulting pUC19 construct was digested with XbaI; the free ends were treated with the large fragment of Klenow polymerase I (Gibco-BRL) and were ligated with a 1.1-kb spc cassette . The latter had been retrieved from pGEM7Zf(-)/spc (8) as a 1.1-kb XbaI-BamHI fragment and had been blunt ended as well . The final construct was purified from E . coli strain JM109 and used to transform strains VT745:pDMG21A and VT745:pDMG27 by electroporation . Plasmid DNA was isolated from SPT-resistant transformants and analyzed by restriction enzyme digest and Southern blot hybridization . Recombinant plasmids into which the spc gene had inserted into the target XbaI site via single crossover were labeled pDMG28 (for the pDMG21A construct) and pDMG29 (for the pDMG27 construct) .

 
Construct pDMG32 was obtained similar to pDMG28 with the spc gene inserted into an EcoRI located 3' of oriV (Fig . 1A) . Again, the regions adjacent to the target EcoRI site were amplified by PCR (primers P9 and P10 [Table 2]) and the 2-kb amplification product was cloned into a pUC19 derivative from which the EcoRI site had been removed by treatment with the large fragment of Klenow polymerase I . The new construct was then digested with EcoRI to cleave this unique site located in the center of the PCR product, blunt ended, and ligated with the spc cassette . Again, the final product was purified from JM109 and introduced into VT745 via electroporation . Recombinant SPT-resistant transformants were obtained in which the spc gene had inserted into the target EcoRI site of pDMG21A via single and double crossover . One of the pDMG21A derivatives obtained by double crossover, pDMG32, was used subsequently in mating assays .

Construction of invertase expression vector pJC20. The invertase gene was amplified from pVT745 by PCR with custom-made primers P11 and P12 (Table 2) . The latter primer included a HindIII site . The amplification product of 815 bp was then cloned into pGEM-T Easy (Promega) and was subsequently retrieved as a SalI-HindIII fragment . The latter fragment was cloned into expression vector pNP3 (P . Permpanich, personal communication) cut with the same enzymes . The final construct, pJC20, was purified from E . coli JM109 and used to transform various strains of A . actinomycetemcomitans via electroporation .

Analysis of segregational stability of plasmids. Plasmid stability was assessed as described by Galli et al . (10) . In short, strains were grown in antibiotic-free TSBYE broth for 7 days (equivalent to 90 to 100 cell doublings) while being diluted 1:100 in fresh medium once a day to maintain the cultures in exponential growth phase . Thereafter, serial dilutions of each culture were spread on antibiotic-free TSBYE plates . Percentages of plasmid-containing cells were obtained by transfer of 100 colonies from these plates to selective and nonselective plates with sterile toothpicks . Experiments were performed at least twice .

Mating experiments. Conjugative surface matings were performed between A . actinomycetemcomitans strains as described previously for 6 h unless noted otherwise (12) . Transfer frequencies were expressed as the number of transconjugants per donor cell . At least 10 selected transconjugants were examined for the presence of plasmid DNA after each mating experiment . Spontaneous RIF mutations occurred at a frequency of less than 10^-9 and did not interfere with low-frequency matings .

Characterization of RecA^- transformants. Recombination-deficient mutants (RecA^- cells) were identified based on their sensitivity to UV light and methyl methanesulfonate (MMS) . Parental ATCC 29522 cells and potential RecA^- transformants were grown in broth to late exponential phase and were then streaked in parallel onto TSBYE agar plates . Plates were irradiated with UV light with increasing doses (500, 1,000, 2,000 and 4,000 µJ) (UV Stratalinker 1800; Stratagene, La Jolla, Calif.), and their contents were then incubated for 2 days .

The sensitivity of RecA^- cells to the chemical MMS was tested by adding MMS to TSBYE agar plates in various concentrations, and a comparison of cell growth on these plates to that of parental strain ATCC 29522 grown under the same conditions was made .

 
The DNA inversion is recA independent. To determine if recA was involved in the structural rearrangement of pDMG21A, a recombination-deficient mutant of strain ATCC 29522Rif was constructed . Allelic replacement of recA via a double-crossover event with a mutated copy carrying an STR resistance cassette was confirmed by restriction enzyme analysis and Southern blot hybridization (not shown) . Sensitivity to 0.001% MMS and UV light (2,000 µJ) confirmed the recA-deficient phenotype (not shown) .

Strain ATCC 29522RifrecA was then used as a recipient in the conjugative transfer of pDMG20A and pDMG21A from VT745 . Again, all transconjugants tested harbored the rearranged version of the transferred plasmids, indicating that the DNA inversion event was independent of the recA status of the recipient (Table 3) .

 
The DNA inversion is caused by the pVT745-encoded site-specific recombinase. Nucleotide sequence analysis of pVT745 revealed that the plasmid carried a gene, AA01, which showed strong homology to site-specific DNA recombinases (12) . Indeed, the AA01 gene product was 45% identical to Min, a DNA inversion enzyme of plasmid p15B, which is a member of the Din family of site-specific recombinases (Fig . 3A) (16) . Conserved regions included the N-terminal end with the active serine residue required for the formation of covalent intermediates with DNA at the crossover sites (Fig . 3A) (14) . To elucidate the role of the pVT745-specific recombinase in the DNA inversion event, the gene was replaced with an STR resistance cassette as described in Materials and Methods . The resulting construct, a derivative of pDMG21A, was designated pDMG27 . When pDMG27 was transferred from VT745 to ATCC 29522Rif via conjugation, rearrangements on the plasmid were no longer observed (Table 3) . This was true for the recA⁺ and recA mutant recipient strain . This result clearly demonstrated that the site-specific recombinase was involved in the DNA inversion event . Therefore, it was decided to rename gene AA01 encoding the site-specific recombinase inv for DNA invertase .

 
Comparison of restriction enzyme profiles of pDMG21A and pDMG21B located the junctions of the invertible segment to a region just upstream of the site-specific recombinase and between AA10 and AA11 (Fig . 1A) (12) . Custom-made oligonucleotide primers were used to sequence the junction fragments . Analysis of the resulting DNA nucleotide sequence revealed the presence of a 22-bp repeat (IR22) with dyad symmetry in opposite orientation at the junctions (Fig . 1A and 3B) . These two recombination sites (DNA crossover sequences) were similar but not identical to the conserved crossover sites of the Min and Din systems (Fig . 3B) (14, 24) . The main difference was in the region containing the central asymmetric dinucleotide, where Min introduces a 2-bp staggered cut . The central nonsymmetric A-A dinucleotide is conserved in the family of Din invertases (14) . Its absence in the Inv-specific crossover sites could indicate that, despite the high degree of homology, there is a difference in target specificity for Inv and Min . The inversion of the 9-kb segment did not result in the creation of new open reading frames or the connection or disconnection of parts of open reading frames (Fig . 1B) as described for other systems .

The DNA inversion is host dependent. Since no rearrangement had ever been observed on pVT745 or one of its derivatives in the original host, VT745, it was possible that the inversion was triggered by the mating event . Therefore, pDMG20A was introduced into ATCC 29522Rif by electroporation . Again, DNA inversion took place . The transformants screened all harbored pDMG20B . This raised the possibility that a host-specific factor was involved in the inversion event . The following possibilities were postulated: (i) an accessory factor is required for inversion in addition to the site-specific recombinase, and such a factor is present in all A . actinomycetemcomitans strains but VT745 . (ii) An accessory factor is required for inversion in addition to the site-specific recombinase, and such a factor is present in all A . actinomycetemcomitans strains but JP2, of which VT745 is a member (15) . (iii) VT745 or any JP2 strain harbors a factor that is absent in all other A . actinomycetemcomitans strains and that maintains the plasmid in the preferred arrangement "A." The second assumption could be eliminated when conjugative transfer of pDMG20A and pDMG21A to ATCC 700685Rif, a JP2-like strain, resulted in the inversion of the 9-kb segment (Table 3) . To test the third assumption strain, ATCC 29522Rif was used as a donor to transfer pDMG21B to VT745 (Table 3) . The latter harbored pDMG4 (10), an SPT-resistant replicon that is neither conjugative nor mobilizable, to allow for selection of transconjugants . No reversion of pDMG21B to pDMG21A was observed in VT745:pDMG4 . Also, transformation of VT745 by electroporation with pDMG20B did not result in the reversion of the plasmid to pDMG20A . Therefore, it was concluded that a specific host factor other than RecA is necessary in addition to the site-specific recombinase to catalyze the inversion . Such a factor is either missing or inactive in VT745 . However, the absence of this specific host factor is not a distinct feature of JP2-like strains to which VT745 belongs, since DNA inversion was observed in ATCC 700685Rif, another JP2-like strain .

The invertase does not contribute to the segregational stability of pVT745. Strain VT745 carrying the various pVT745 derivatives was grown in the absence of selective pressure to determine if inversion of the 9-kb segment on pDMG21B or the loss of invertase activity on pDMG27 affected segregational stability of the plasmids . The percentage of cells within a given population that still harbored the plasmids after 100 and 200 generations, respectively, is shown in Table 4 . Since pDMG27 was as stable as pDMG21A, it is unlikely that the site-specific recombinase is required for efficient partitioning of pVT745 . However, the rearrangement of the invertible region seems to affect the segregational stability of the plasmid as demonstrated by the rapid loss of pDMG21B in the absence of selective pressure (Table 4) . This was true for pDMG21B in VT745 as well as in ATCC 29522Rif .

 
The invertase affects pVT745 conjugation frequencies. While the various mating experiments described above were being conducted, it was observed that significantly higher frequencies of transfer were obtained for pDMG27 than for pDMG21A . Therefore, actual conjugation frequencies per donor cell were determined using VT745 as a donor strain and the ATCC 29522Rif recA⁺ and recA mutant recipient strains (Table 5) . Surprisingly, pDMG27 appeared to transfer at a frequency several orders of magnitude higher than did pDMG21A . However, it was unlikely that the plasmids were transferred at different rates, since they differed only in their invertase activity and since electroporation experiments had indicated that Inv was neither active in the host VT745 nor activated by conjugation alone . Therefore, the difference in number of selectable transconjugants had to be associated with an event that took place in the recipient cells after completion of the actual plasmid transfer . A possible explanation for the low number of detectable pDMG21 transconjugants was that the invertible segment and its associated KAN-resistant phenotype had been excised from the plasmid in recipient cells after the mating event . The resulting truncated pVT745 derivative would then only be detectable if a second marker located outside the invertible region was present . Therefore pDMG28 and pDMG32 were constructed, both derivatives of pDMG21A with an additional spc cassette inserted 5' or 3' to oriV (Fig . 1A) to allow for replication and selection of any potential deletion derivative . Insertion of spc was in a noncoding region . An spc marker was also inserted into pDMG27 to confirm that the addition of a new cassette and the target location for this marker did not affect the mating frequencies of the inv mutant derivative . This last construct was designated pDMG30 .

 
Mating experiments were then carried out with all new constructs . Selection for transconjugants was on either KAN or SPT . Again, mating frequencies for both pDMG21A derivatives, pDMG28 and pDMG32, were several orders of magnitude lower than for pDMG30, which carried a mutated inv gene in addition to the spc cassette (Table 5) . However, whereas the numbers of transconjugants obtained for pDMG28 were similar to the ones obtained for pDMG21A, mating frequencies were increased for pDMG32 (Table 5) . Doubling times for A . actinomycetemcomitans range between 1.5 and 2 h . Therefore, mating experiments were performed for 6 h as described previously (13) and for 0.5 h to confirm that the decrease in number of transconjugants was not the result of segregational instability of the plasmid . The significant decrease in actual mating times had no effect on the outcome of the experiment . Also, all KAN-resistant transconjugants were resistant to SPT and vice versa, indicating that neither marker had been lost as was to be expected with a deletion derivative . Results of restriction enzyme analysis with plasmid DNA isolated from selected transconjugants confirmed the absence of any truncated version of either pDMG28 or pDMG32 (not shown) . Surprisingly, up to 30% of the pDMG32 transconjugants screened carried the invertible 9-kb region in orientation A (not shown) .

Expression of the invertase gene alone does not trigger the inversion event. The fact that the invertible segment appeared to remain stable in orientation A or B once the pVT745 derivatives were established in the recipient cells suggested that the invertase gene was no longer expressed after the inversion event . Also, lack of inversion in VT745 could be explained by the absence of inv expression . To test these assumptions, the presence of inv-specific mRNA was determined in strains VT745:pDMG21A and -B, ATCC 29522Rif:pDMG21B, and ATCC 29522Rif:pDMG32A . Results of RT-PCR experiments confirmed the absence of an inv-specific transcript in all strains tested . In contrast, an RT-PCR product was detected in strain ATCC 29522Rif:pJC20, which expressed the invertase gene under the control of the spc promoter (not shown) . Integrity of the RNA preparations was confirmed by the detection of the A . actinomycetemcomitans-specific leukotoxin transcript in VT745:pDMG21A and -B by RT-PCR (data not shown) .

If indeed inversion was regulated by inv expression, the presence of a constitutively expressed invertase would result in the rearrangement of pVT745 derivatives during normal cell growth . Such an event could then be detected by the presence of pVT745 derivatives in the A and B orientation in the same plasmid preparation . To test this assumption, pJC20, a pMMB67 derivative that expresses the invertase, was transferred into VT745:pDMG21A and -B, ATCC 29522Rif:pDMG21B, and ATCC 29522Rif:pDMG32A by electroporation . Plasmid DNA isolated from selected transformants was then screened for the orientation of the invertible segment . In all cases, DNA inversion was not observed for any of the pVT745 derivatives, although expression of inv from pJC20 was confirmed by RT-PCR .

Once a pVT745 derivative was established within the recipient cell, subsequent DNA inversion was never observed . The possibility that inversion occurred in response to the mating event itself was ruled out since the transfer of a pVT745 derivative into ATCC 29522Rif via electroporation also resulted in rearrangement of the invertible segment . However, it is conceivable that DNA entry into the cell, regardless of entry mechanism, triggers the inversion . Regulation of invertase expression alone did not appear to be sufficient to turn the inversion event on or off . Constitutive expression of inv in trans during normal cell growth did not result in the inversion of the 9-kb segment regardless of its orientation on the various pVT745 derivatives used . Therefore, one or more additional factors that are either plasmid or host encoded are required to activate the inversion event . It is likely that such a factor(s) is expressed transiently when new DNA enters the cell .

Also, inversion of the 9-kb fragment on pVT745 was never observed in VT745 regardless of the transfer mechanism used . Again, the likeliest explanation is that one or more specific host factor(s) is required to activate the inversion event and that such a factor(s) is absent in the native strain . The Din family of recombinases requires Fis (factor for inversion stimulation), which binds to specific cis-acting enhancer sequences for efficient inversion (18) . These conserved Fis-binding sites are located in the 5' end of the recombinase genes within the first 90 nucleotides of the coding frame . Consensus sequences for Fis host factor binding sites are present in the 5' end of the pVT745-specific inv gene (J . Chen and D . M . Galli, unpublished data) . Future work will include elucidation of the role of Fis or other host factors in the promotion of inversion on pVT745 .

It is not yet known if Inv of pVT745 has a resolvase activity in addition to its function as an invertase . Such activity would play a role in plasmid partitioning by catalyzing the conversion of dimers and higher oligomers into monomers . It was shown in the present study that pDMG27, the pVT745 derivative with an inactivated inv, was still segregationally stable in the original host strain after 200 generations . This result is in line with the notion that recombination between two repeats in direct orientation, which would cause the deletion of the intervening DNA segment, is inefficient in the Din family of inversion systems (24) .

In contrast to most other systems, at this point no biological function can be linked to the inversion event . Neither are new open reading frames created nor are existing open reading frames connected or disconnected . In future studies we will attempt to show if the transcriptional expression of the open reading frames flanking the two IR22 sites has been altered by the inversion of the 9-kb fragment . The only obvious consequence of invertase activity was a significant decrease in the number of selectable transconjugants upon transfer of the pVT745 derivatives . The reason for the loss of transconjugants in the presence of a functional invertase is not clear at this point . In two plasmid systems of Clostridium perfringens, the activity of a site-specific recombinase has been associated with the excision of a conjugative transposon, Tn4451, and associated resistance cassettes (1, 5, 6) . The transposon was consequently lost in C . perfringens transconjugants or integrated into the chromosome of E . coli recipient cells at a low frequency (2) . However, it is unlikely that a transposon or some other putative element present on pVT745 was excising after conjugative transfer . The insertion of a spc marker upstream (pDMG28) or downstream of oriV (pDMG32) would have permitted selection for deletion derivatives of either construct that might have lost the KAN cassette . However, the number of retrievable transconjugants did not increase when selection was for SPT . Also, the few SPT-resistant transconjugants obtained still carried the KAN resistance marker, suggesting that excision of only a defined fragment was not occurring . The loss of all resistance markers at a high frequency regardless of their location on the plasmids was an indication of the complete loss of the plasmid in recipient cells . Surprisingly, the transfer of pDMG32 increased numbers of transconjugants obtained by 1 order of magnitude when compared to that of pDMG21A and pDMG28, although all three constructs harbor an intact inv gene . Furthermore, during screening of selected transconjugants for plasmid content, pDMG32 was isolated with the invertible element in orientations A and B . Therefore, it appears that an additional plasmid-encoded element that acts either in cis or in trans affects inversion of the 9-kb segment . Such an element has to be located in the vicinity of the EcoRI site into which spc was inserted in the pDMG32 construct . Its interaction with the invertase gene or gene product appears to be associated with the plasmid loss observed in transconjugants . Likewise, this factor could be involved in regulating the inversion event when pVT745 DNA enters the cell as discussed above .

At this point the presence of the inv gene on pVT745 would appear to be a disadvantage for the effective dissemination of the plasmid via conjugation . In addition, the rearranged version is segregationally unstable, as shown with pDMG21B . It remains to be seen if a true biological role can be associated with inv or if the gene is the remnant of a functional element, which once was part of pVT745 but subsequently lost .

This study was supported by NIH grant R01 DE12107 to D.J.L . and D.M.G .

Present address: Pharmacia Corporation, Kalamazoo, MI 49001 .

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What Is Yeast?, What Is Pcr?, What Is Molecular Biology?, What Is MIC?, What Is Genetics?, e, Bacteria, e, Microbiology, s, Microorganism, e, Microorganisms, i, Microbes, e, Phage, c, Enterobacteriacea, i, Lactobacillus, c, Bacteriophages, c, Escherichia coli, i, S. cerevisiae, a, Gram positive, c, Clostridia, n, Bacillus anthracis, s, Microorganism, n, Antibiotics, a, Staphylococcus aureus, c, Clostridia, e, Amikacin, e, Antibiotics, i, Yeasts, a, Antimicrobial, i, Schizosaccharomyces, e, Escherichia coli, c, Lactobacillus, i, Biodegradation




 

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