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.     

Identification of new sex pheromone plasmids in Enterococcus faecalis.

Summary We describe the identification of the following new sex pheromone plasmids inEnterococcus faecalis: a haemolysin-bacteriocin plasmid, pIP964; three R plasmids, pIP1017, pIP1438 and pIP1440; and two cryptic conjugative plasmids, pIP1141 and pMV120. The identification was based on the formation of cell aggregates on filter membranes during conjugation, on efficient transfer in broth matings, and on a positive clumping reaction of cells carrying these plasmids. In addition these plasmids hybridized with DNA probes specific for sex pheromone-induced structural genes encoding surface proteins required for conjugative transfer of the plasmids.     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

Molecular analysis of a composite chromosomal conjugative element (Tn3701) of Streptococcus pyogenes.

The plasmid-free Streptococcus pyogenes A454 contains a conjugative element, Tn3701, encoding resistance to erythromycin (Emr), tetracycline (Tcr), and minocycline (Mnr). We have mapped a 50-kilobase (kb) chromosomal region of A454 corresponding to the internal part of Tn3701. Tn3701 includes a 19.7-kb structure, designated Tn3703, on which the Emr Tcr Mnr determinants were localized. Tn3703 was very similar in structure to Tn916. Translocation of the Emr Tcr Mnr markers from A454 onto pIP964, an Enterococcus faecalis hemolysin plasmid, yielded different pIP964 derivatives. When the inserts of four of these derivatives were aligned with the 50-kb region of Tn3701, three of them were found to result from the transposition of Tn3703 and one resulted from the insertion of a 44.0-kb portion of Tn3701, including Tn3703. Tn3701 inserted, apparently without changing its structure, in the chromosomes of various streptococcal transconjugants, as well as in one of the 12 E. faecalis transconjugants studied. Tn3703 inserted at different chromosomal sites in four E. faecalis transconjugants, and one copy of Tn3701 plus an additional copy of Tn3703 were detected in the chromosomes of seven transconjugants.     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group.

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Heterogeneric conjugal transfer of the pheromone-responsive plasmid pIP964 (IncHlyI) of Enterococcus faecalis in the apparent absence of pheromone induction

Abstract Erythromycin-resistant derivatives of the pheromone-responsive plasmid pIP964 from Enterococcus faecalis were constructed to study its host range. This was done by inserting the integrative vector pAT112 and the related replicon pTCR1 harboring oriR of the broad host range plasmid pAMβ1 into the hemolysin-bacteriocin operon of pIP964, to give pTCR2 and pTCR3, respectively. Plasmid pTCR2 was transferred by filter matings from E. faecalis to Enterococcus faecium and Listeria monocytogenes at frequencies of 2×10⁻⁷ and 5×10⁻⁷ per donor, respectively, in the apparent absence of pheromone induction and cellular aggregation. In these hosts, pTCR2 remained intact as a self-replicating element and maintained its transfer capabilities. Plasmid pTCR3, but not pTCR2, was transferred at similar frequencies from E. faecalis to Lactococcus lactis and Streptococcus agalactiae . Thus, the transfer system of pIP964 possesses a broader host-range than its replication system.     

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.     

An alternative approach for evaluating the phenotypic virulence factors of pathogenic Escherichia coli

Escherichia coli is a recognized zoonotic food-borne pathogen; however, the use of polymerase chain reaction (PCR) in the underdeveloped countries to differentiate pathogenic from non-pathogenic E. coli is a problematic issue. Our grail was to assess the phenotypic virulence markers motility, hemolysin, congo red agar, embryo lethality assay and serum resistance for pathogenic E. coli (PEC) correlated to PCR tests which is currently used world-wide to evaluate the PEC. The 448 strains of Escherichia coli that were isolated from different sources, were characterized for phenotypic virulence factors such as motility, hemolysin, Congo red binding, Embryo Lethality assay (ELA) and serum resistance, as well as antibiotic susceptibility using disc diffusion method to 23 antibiotics. Results exhibited 100% motility and Congo red binding, 97.1% for hemolysin production and 90.2% in the ELA. As a result, we were able to hypothetically conclude that the aforementioned virulence markers are plain, straightforward, economical, rapid, more dynamic, uncomplicated methodology, duplicatable and cost next to nothing when compared to the molecular PCR. Their implementation in a diagnostic microbiology laboratory for vetting is a rewarding task in the underdeveloped countries. It augments endeavors to minimize the use of PCR in our investigations especially during epidemiological and outbreak investigations of PEC.     

Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens

The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined. The plasmid genome comprises 10,207 bp and has a dA + dT content of 75%. Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified. The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content. Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.     

Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035.

The large conjugative plasmid pIP501 was originally isolated from Streptococcus agalactiae. To study the molecular basis of pIP501 replication we determined the nucleotide sequence of a 2.2 kb DNA segment which is essential and sufficient for autonomous replication of pIP501 derived plasmids, in Bacillus subtilis cells. This region can be divided into two functionally discrete segments: a 496 bp region (oriR) that acts as an origin of replication, and a 1488 bp segment coding for an essential replication protein (RepR). The RepR protein, which has a molecular mass of 57.4 kDa, could complement in trans a thermosensitive replicon bearing the pIP501 origin. Chimeric Rep proteins and replicons were obtained by domain swapping between rep genes of closely related streptococcal plasmids belonging to the inc18 group (pIP501, pAM beta 1 and pSM19035). The chimeras were functional in B. subtilis.     

Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.     

Detection of brain-specific gene expression in brain cells in primary culture: a novel promoter assay based on the use of a retrovirus vector.

Promoter activities of the brain-specific genes for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were investigated in brain cells in primary culture with the use of a novel retrovirus vector, pIP200. With this vector, promoter activity can be expressed in terms of beta-galactosidase activity. Differentiation of the primary brain cells to mature glial cells was not affected by treatment with the pIP200 virus vector. The 256-bp 5'-flanking region of the GFAP gene directed astrocyte-specific expression of lacZ. It was silent in fibroblasts, even in multiple copies. The 1.3-kb 5'-flanking region of the MBP gene exhibited strict tissue (oligodendrocyte) specificity under the present assay method but showed some leakiness when integrated into the chromosome in multiple copies. Promoter regions conferring cell type specificity in brain were effectively identified by the present method.     

Silkworm Apolipophorin Protein Inhibits Hemolysin Gene Expression of Staphylococcus aureus via Binding to Cell Surface Lipoteichoic Acids

We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.     

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.     

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.     

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.     

Nucleotide sequence of the chloramphenicol resistance determinant of the streptococcal plasmid pIP501

We have sequenced the chloramphenicol resistance determinant (cat) of plasmid pIP501 from Streptococcus agalactiae to investigate its relationship with other cognate cat determinants. Sequence analysis revealed that it exhibits a high degree of similarity with the cat genes of plasmids pC221 and pUB112 from Staphylococcus aureus and pSCS1 from Staphylococcus intermedius. These genes, however, display several differences in their regulatory and coding regions. These results demonstrate that the cat determinant of plasmid pIP501 belongs to the pC221 subgroup of CAT variants.