Incompatibility group Y member relationships: pIP231 and plasmid prophages P1 and P7

The plasmid pIP231 exhibits stronger incompatibility with both plasmid prophages P1 and P7 than P1 and P7 exhibit with each other. DNA-DNA hybridization experiments showed that pIP231 is strongly homologous with both P1 and P7 in the central region of the prophage genomes which contains genes determining replication, incompatibility, and maintenance functions. In addition, a region on the left of P7 which contains Tn902 showed some homology with pIP231, as did the region of P1 carrying IS1.     

Tn1525, a kanamycin R determinant flanked by two direct copies of IS15

We have isolated plasmid pIP112 (IncI1) from Salmonella panama and characterized by restriction endonucleases analysis and by recombinant DNA techniques a transposable element designated Tn1525. This 4.44 kilobase (kb) transposon confers resistance to kanamycin by synthesis of an aminoglycoside phosphotransferase (3') (5") type I and contains two copies of IS15 (1.5 kb) in direct orientation. The modular organisation of Tn1525 offers the possibility for intramolecular homologous recombination between the two terminal direct repeats and thus accounts for the in vivo structural lability of plasmid pIP112: instability of kanamycin resistance and tandem amplification of the kanamycin determinant. Other transposons mediating resistance to kanamycin by the same enzymatic mechanism were analysed by agarose and polyacrylamide gel electrophoresis, following digestion with restriction endonucleases, and by Southern hybridizations. These comparisons indicate that, although the structural genes for the phosphotransferases are homologous, Tn1525 differs from Tn903 and Tn2350 and is closely related but distinct from Tn6. Using the same techniques Tn1525 was detected on plasmids belonging to different incompatibility groups and originating from various species of Gram-negative clinical isolates. These results indicate that Tn1525 is representative of a new family of class I composite transposons already spread in diverse pathogenic bacterial genera.     

Stimulation of IS1 excision by bacteriophage P1 ref function.

Lysogenization by a c1ts variant of coliphage P1, P1c1.100, markedly increased the frequency of reversion of a galT::IS1 mutation. The formation of Gal+ colonies presumably occurs by microhomologous recombination between the 9-base-pair repeats in galT (CGCCGCTAC) generated by the transposition of IS1. The responsible P1 gene, ref, has been cloned and sequenced. ref encodes a 22.8-kilodalton protein and is located near the P1 site-specific recombination function, cre. Expression of ref was repressed by P1 c+. The absence of a distinctive ribosome-binding site is consistent with a poor translation of ref from an expression vector in vivo. Placement of a ribosome-binding site before ref resulted in the extensive synthesis of the Ref protein. Ref stimulated precise excision in recB or himA cells, but not in recA mutants. Ref was active in lexA3 mutants, suggesting that the recombination activity of RecA was directly involved in the reaction. We have constructed a P1c1.100 ref::Tn10 mutant. The absence of Ref did not appear to restrict dramatically the ability of P1 to grow lytically or to form lysogens. Thus, the role of ref in the physiology of P1 remains to be determined.     

Translocation of sequences encoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez

Summary Salmonella ordonez strain BM2000 carries kanamycin (Km), ampicillin (Ap), spectinomycin (Sp), chloramphenicol (Cm), tetracyline (Tc), and sulfonamide (Su) resistance and production of colicin Ib (Cib). The Km and Cib characters were carried by a 97kb IncI1 plasmid (pIP565). In addition to the Km and Cib traits, all or part of the other antibiotic resistance (R) determinants could be transferred by conjugation from S. ordonez to Escherichia coli where all the acquired characters are borne by an IncI1 plasmid, designated complete or partial composite plasmid respectively. DNA from pIP565 and composite plasmids and total DNA from strain BM2000 were studied by agarose and polyacrylamide gel electrophoresis following digestion with restriction endonucleases, and by Southern hybridization. These comparative analyses enabled us a) to show that acquisition by pIP565 of resistance to all or some of the antibiotics was due to the insertion of a single DNA fragment into the receptor plasmid; b) to detect two types of composite plasmids with regard to the specificity of insertion into pIP565 and the mapping of the inserts; c) to demonstrate that the ApCmSpSuTc resistance determinants were integrated into S. ordonez BM2000 chromosomal DNA; d) to map the restriction fragments of the translocatable sequence integrated into strain BM2000 chromosome or into pIP565.The results obtained suggest that two distinct mechanisms for the translocation of the R determinants coexist in S. ordonez BM2000. Recombination between two of the four directly repeated copies of the IS-like sequence (IS1522) present in S. ordonez chromosome leads to the circularisation of all or part of the AmCmSpSuTc R determinants and is followed by either 1) a second recombination with the copy of IS1522 in pIP565 (Type I composite plasmids), or 2) transposition of precise groups of characters in various sites of pIP565 (Type II composite plasmids).     

Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant.

A recombinant plasmid carrying the recA gene of Aeromonas caviae was isolated from an A. caviae genomic library by complementation of an Escherichia coli recA mutant. The plasmid restored resistance to both UV irradiation and to the DNA-damaging agent methyl methanesulfonate in the E. coli recA mutant strain. The cloned gene also restored recombination proficiency as measured by the formation of lac+ recombinants from duplicated mutant lacZ genes and by the ability to propagate a strain of phage lambda (red gam) that requires host recombination functions for growth. The approximate location of the recA gene on the cloned DNA fragment was determined by constructing deletions and by the insertion of Tn5, both of which abolished the ability of the recombinant plasmid to complement the E. coli recA strains. A. caviae recA::Tn5 was introduced into A. caviae by P1 transduction. The resulting A. caviae recA mutant strain was considerably more sensitive to UV light than was its parent. Southern hybridization analysis indicated that the A. caviae recA gene has diverged from the recA genes from a variety of gram-negative bacteria, including A. hydrophila and A. sobria. Maxicell labeling experiments revealed that the RecA protein of A. caviae had an Mr of about 39,400.     

The CAM-OCT plasmid enhances UV responses of Pseudomonas aeruginosa recA mutants.

The effect of the CAM-OCT plasmid on responses to UV irradiation of Pseudomonas aeruginosa recA mutants was characterized. Mutant alleles examined included rec-1, rec-2, and recA7::Tn501. The plasmid substantially enhanced both survival and mutagenesis of RecA- cells after treatment with UV light. Survival of the RecA-(CAM-OCT) cells after UV irradiation was intermediate between that seen in the wild-type P. aeruginosa PAO1 and the increased survival seen in PAO1(CAM-OCT) cells. Mutability was quantitated by the reversion to carbenicillin resistance of strains carrying a bla(Am) mutation on a derivative of plasmid RP1. UV-induced mutagenesis of CAM-OCT carrying recA mutants occurred at levels comparable to that seen in PAO1(CAM-OCT). The ability of CAM-OCT plasmid to suppress the recombination deficiency in recA mutants was tested by assaying for bacteriophage F116L-generalized transduction of a Tn7 insertion in the alkane utilization genes of CAM-OCT. Transduction of the Tn7 insertion was not detected in RecA-(CAM-OCT) strains but was easily seen in PAO1(CAM-OCT), indicating that the plasmid does not encode a recA analog. The results indicate that the CAM-OCT UV response genes are expressed in RecA- cells, which differs from results seen with other UV response-enhancing plasmids. The results suggest that CAM-OCT either encodes several UV responses genes itself or induces chromosomal UV response genes by an alternate mechanism.     

Nucleotide sequence of the kanamycin resistance determinant of the pneumococcal transposon Tn1545: evolutionary relationships and transcriptional analysis of aphA-3 genes

The nucleotide sequence of the kanamycin resistance determinant aphA-3 encoded by transposon Tn1545 from Streptococcus pneumoniae was determined and compared to those of plasmids pJH1 and pIP1433 from Streptococcus faecalis and Campylobacter coli, respectively. The three sequences were found to be identical and differed by two substitutions and the deletion of a codon from that of plasmid pSH2 from Staphylococcus aureus. Comparison of the 5' noncoding sequences indicated that the regions containing the aphA-3 gene in pJH1 and in Tn1545 evolved independently by deletion from a sequence similar to that found in pIP1433. In the latter plasmid, aphA-3 is transcribed from a promoter, P1, which is flanked by two 12-base pair direct repeats. The rearrangement observed in pJH1 removed one of these recombinogenic sites and altered the -10 and 3' flanking sequences of P1. The promoter thus generated. P1', allows expression of similar level of kanamycin resistance as P1. However, fusion experiments carried out with a promotorless chloramphenicol acetyltransferase gene indicated that the canonical promoter P1 is significantly less efficient than P1'. From analysis of the thermodynamic properties of these promoters, we conclude that this difference in strength reflects the melting properties of the -10 sequences. The transition from pIP1433 to pJH1 may correspond to the progression of a molecule structurally unstable to a more stable one combined with the need to maintain an efficient promoter upstream of the aphA-3 gene. The deletion event in Tn1545, which occurred between the two 12-base pair directly repeated sequences, removed P1 in its entirety.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficient plasmid mobilization by pIP501 in Lactococcus lactis subsp. lactis.

pIP501 is a streptococcal conjugative plasmid which can be transmitted among numerous gram-positive strains. To identify a minimal mobilization (mob) locus of pIP501, DNA fragments of pIP501 were cloned into nonconjugative target plasmids and tested for mobilization by pIP501. We show that nonmobilizable plasmids containing a specific fragment of pIP501 are transmitted at high frequencies between Lactococcus lactis subsp. lactis strains if transfer (tra) functions are provided in trans by a pIP501 derivative. Independent transfer of the mobilized plasmid was observed in up to 44% of transconjugants. A 2.2-kb segment containing mob was sequenced. This DNA segment is characterized by three palindromes (palI, palII, and palIII) and a 202-amino-acid open reading frame (ORFX) of unknown function. The smallest DNA fragment conferring high frequency mobilization was localized to a 1.0-kb region (extending from pIP501 coordinates 3.60 to 4.60 on the 30.2-kb map) which contains palI (delta G = -27 kcal/mol [ca. -110,000 J/mol]). A 26-bp sequence identical to palI is present on pIP501, upstream of the plasmid copy control region. Further homologies with the palI sequence are also found with the related Enterococcus faecalis conjugative plasmid pAM beta 1. The region containing mob maps outside the previously described segment mediating pIP501 conjugation. Our results with recA strains indicate that the mob site is a hot spot for cointegrate formation.     

Studies of a plasmid coding for tetracycline resistance and hydrogen sulfide production incompatible with the prophage P1

Summary The plasmid pIP231, determining tetracycline resistance and hydrogen sulfide production is shown to belong to incompatibility group Y and to code for a restriction and modification system. Unlike the IneY plasmids, P7 and P15B, plasmid pIP231 shows only little genetic and physical homology with P1 prophage.     

Cloning and expression of plasmid DNA sequences involved in Salmonella serotype Typhimurium virulence

A 22kb region of the 90kb virulence-associated plasmid, pIP1350, of Typhimurium strain C52 was cloned into the mobilizable vector pSUP202, yielding plasmid pIP1352. This recombinant plasmid restored full virulence to plasmidless strain C53 in a mouse model. Transposon Tn5 insertion mutagenesis demonstrated the existence of two DNA sequences in pIP1352 designated VirA and VirB, both of which are essential for the expression of virulence. A recombinant plasmid containing only the VirA and VirB regions markedly increased the virulence of the plasmidless strain C53, but did not confer full virulence. These results suggested that a third virulence-associated region might be present on pIP1352. Eleven proteins encoded by the 22kb insert sequence of pIP1352 were identified in Escherichia coli SE5000 maxicells. The VirA region encoded at least two proteins with apparent molecular weights of 71,000 and 28,000 and the VirB region encoded two proteins of 43,000 and 38,000.     

Recombination in recA cells between direct repeats of insertion element IS1.

The IS1 sequences that flank the Tn9 chloramphenicol acetyltransferase gene as direct repeats recombine after transformation into an Escherichia coli recA strain. The recombination requires the lambda pL promoter on the plasmid. A plasmid that contains mutant IS1 elements does not recombine. These results indicate that this recombination requires an IS1-specific gene product. The recombinational activity of IS1 may resolve transient cointegrates formed during the transposition of IS1. I discuss a possible role for the lambda pL promoter.     

Convenient construction of strains useful for transducing recA mutations with bacteriophage P1

Abstract The generalized transducing phage P1 grew well on heterozygous Escherichia coli carrying recA srlC 300::Tn 10 on the chromosome and recA ⁺ on a pBR322-derived plasmid. Because of the close linkage of Tn 10 to recA mutations, including recA 1, recA 13, recA 56, recA deletion and recA allele of E. coli BNG30, the latter can be moved to other strains in transductional crosses for selective resistance to tetracycline.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

Reversible translocation of antibiotic resistance determinants in Salmonella ordonez.

Summary Salmonella ordonez (BM 2000) codes for kanamycin (Km, aphA), ampicillin (Ap), streptomycin (SmSp: aadA and Sm: aphC), chloramphenicol (Cm), tetracycline (Tc) and sulfornamide (Su) resistances and for production of colicin Ib (Cib). Genetical analysis by incompatibility testing, conjugation, transformation and physical studies using electron microscopy, agarose gel electrophoresis, led us to associate the Km and Cib characters to a 98.7 kilobase (kb) IncII plasmid (pIP565), and the Sm (aphC) and Su determinants to a 8.3 kb plasmid (pIP605). The ApCmSmSp(aadA)SuTc determinants were not associated in BM2000 S. ordonez with a plasmid structure. Following conjugation of S. ordonez to E. coli, the ApCmSmSpSuTc determinants were found stably associated with a single plasmid structure (pIP173, 127.5 kb) belonging to IncII group. Agarose gel electrophoresis of plasmid DNA restriction endonuclease digests and electron microscopy heteroduplex analysis showed that the acquisition of the ApCmSmSpSuTc determinants resulted from the insertion into pIP565 of a 28.8 kb DNA sequence. This sequence coding for ApCmSmSpSuTc resistances in S. ordonez could be translocated either to pIP565 plasmid or to several IncII plasmids but never to plasmids belonging to IncW, IncP or IncFII, suggesting the existence of specific sequences on the IncII receptor plasmids. Mereover, R-determinants were translocated back en bloc from pIP173 to the chromosome of a susceptible S. ordonez. The results were consistent with the presence in BM2000 S. ordonez chromosomal DNA of an integrated translocatable sequence encoding ApCmSmSpSuTc resistances. Such a structural association could account for the stability of these resistances in the Salmonella ordonez serotype.     

Transposon-facilitated recombination in Vibrio cholerae

Summary Improved Vibrio cholerae donors were constructed by introducing the ampicillin transposon, Tn1, into both the conjugative plasmid, P, and the bacterial chromosome to provide portable regions of homology. The resulting Tfr (Transposon-facilitated recombination) donors transferred genes at high frequency from origins specified by the chromosomally inserted Tn1 copies. Tn1 was transposed into the chromosome from a deleted P::Tn1 vector, which was eliminated from the cells by superinfection with a thermosensitive P::Tn9 (chloramphenicol) mutant plasmid. After eliminating the thermosensitive plasmid, the chromosomally resistant isolates were converted into donors with a P::Tn1 conjugative plasmid. Tfr donors were also obtained by isolating Tn1 insertion mutations in a gene for thymine biosynthesis. Chromosomal sites of Tn1 relative to bacterial genes were determined by measuring gene transfer frequencies and genetic linkage. In one case, linkage of the amp gene to the chromosomal genes that defined its location was demonstrated. Chromosomal transfer by Tfr donors was reversed by isolating P:Tn1 plasmids that contained Tn1 inserted in the opposite orientation.     

Translocation of ampicillin transposon Tn1 in Escherichia coli cells during sexual reproduction

The efficiency of Tn1 transposition has been shown to increase considerably in course of bacterial conjugation. Usually, the frequency of Tn1 transposition from plasmid pSA2001, a derivative of RP4, into the chromosome never exceeded 0.1% per cell. Percentage of His+ transconjugants, marked by transposon Tn1 during conjugation between Hfr donor, carrying plasmid pSA2001, and auxotrophic recipient, was about 30%. Transposon Tn1 transfer into the recipient cells does not depend on the recA+ gene function in donor cells or on conjugative transfer of plasmid pSA2001. The transfer requires the recA+ gene function in recipients as well as the Hfr function in donor cells. Southern's blot-hybridization revealed the insertion of transposon Tn1 into the different sites of the chromosome of His+ transconjugants. The transposon inserted during conjugation retains the ability to potential further translocation into new sites on the chromosomal DNA.     

Genetic and physical studies of recombinant plasmids formed between an R plasmid of compatibility group FI and sex factor F of HfrH.

Recombinant plasmids between an R plasmid of the FI group (R162/3) and the sex factor F or HfrH were produced after the conjugal transfer of this R plasmid into HfrH. Three types of recombinant plasmids were identified after the mating of HfrH (R162/3) with recA and rec+ recipients. One specimen of each type (pIP218, pIP222, pIP226) was studied in this report. All three recombinant plasmids carry the same genetic information for resistance to antibiotics (CSSuT) retained from R162/3. pIP218 retained all the other properties from F of HfrH: derepression for pilus synthesis, mobilization of the chromosome for the proximally transferred HfrH genes (thr, leu, proA), interference with T7 propagation, and ability to be cured by acridine orange. pIP222 retained from F of HfrH the derepression for pilus synthesis and the same polarity of chromosome transfer (thr, leu, proA), while pIP226 retained the interference with T7 propagation and acridine orange curing. Physical studies revealed that replication control and/or recovery of F and pIP218 as covalent circles of deoxyribonucleic acid are similar, and are different from R162/3. The new plasmids are more likely the result of a substitutive recombination event than a fusion. We propose genetic maps of these recombinant plasmids, showing the unequal participation of the parental plasmids in their formation.     

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.     

A site-specific recombination function in Staphylococcus aureus plasmids.

All known small staphylococcal plasmids possess one or two recombination sites at which site-specific cointegrate formation occurs. One of these sites, RSA, is present on two small multicopy plasmids, pT181 and pE194; it consists of 24 base pairs of identity in the two plasmids, the "core," flanked by some 50 base pairs of decreasing homology. Here we show that recombination at RSA is recA independent and is mediated by a plasmid-encoded, trans-acting protein, Pre (plasmid recombination). Pre-mediated recombination is site specific in that it occurs within the core sequence of RSA in a recA1 host. Recombination also occurs between two intramolecular RSA sites. Unlike site-specific recombination systems encoded by other plasmids, Pre-RSA is not involved in plasmid maintenance.