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PubMed1993

Biochemical, immunological and ultrastructural characterization of aggregation substances encoded by Enterococcus faecalis sex-pheromone plasmids.

The sex-pheromone system of Enterococcus faecalis can be viewed as a unique and highly efficient plasmid-collection mechanism. The contact needed for transfer of the conjugative sex-pheromone plasmids is mediated by an adhesin, called aggregation substance, which is encoded by these plasmids. We show here that for 17 of the 18 sex-pheromone plasmids (pAM373 being the exception) described to date, their adhesins are immunologically related to each other. In each case, we observed the presence of an N-terminal fragment of about 78 kDa in addition to the 137-kDa form of mature aggregation substance. The cross-reactions were different for the various plasmids. In the case of pPD1 the 78-kDa fragment reacted only weakly. The aggregation substance encoded by sex-pheromone plasmid pAD1 (Asa1) was characterized in detail. The conditions used for SDS/PAGE had a drastic influence on the migration behavior of mature aggregation substance and differently migrating, interconvertible forms were identified. Preliminary data indicate that Asa1 might be a glycoprotein. Antibodies were isolated which are directed against the N- and C-terminal parts of aggregation substance. They showed about the same reactivity on Western blots; however, only antibodies directed against the N-terminal part of the aggregation substance could inhibit the bacterial cell/cell contact. The reactions of the two antibody preparations with induced cells of E. faecalis was analyzed by transmission electron microscopy. The results indicated that especially the N-terminal part of aggregation substance is exposed on the cell surface of E. faecalis; the C-terminal part seems to be much less exposed.

Antibodies↗

PubMed2005

Drug resistance of Enterococcus faecium clinical isolates and the conjugative transfer of gentamicin and erythromycin resistance traits.

Drug resistance and the transferability of resistance were examined in 218 Enterococcus faecium clinical isolates obtained from in-patients of a Japanese university hospital between 1990 and 1999. One hundred and sixty one isolates (73.9%) were drug-resistant and 127 (58.2%) isolates were resistant to two or more drugs. Vancomycin resistant E. faecium (VRE) was not isolated. The transferability of drug-resistance to an E. faecium strain was examined by broth or filter mating. Six (12.5%) of the 48 gentamicin resistance traits, and fifty (50%) of the 101 erythromycin resistance traits were transferred by filter mating. The gentamicin resistance traits of five isolates and the erythromycin resistance traits of four isolates were transferred to the recipient strains by both broth mating and filter mating at a frequency of about 10(-6) and 10(-5) per donor cell, respectively. The five gentamicin resistant strains were shown to harbor pMG1-like plasmids on the basis of their Southern hybridization with pMG1 (65.1 kbp, Gm(r)), which transfers efficiently between enterococci by broth mating. Each of the four erythromycin resistant transconjugants obtained by broth mating harbored a large conjugative plasmid (more than 100 kbp). The plasmids showed no homology with well-characterized enterococcal conjugative plasmids such as pAD1, pPD1, pAM(beta)1, pIP501 and pMG1 by Southern hybridization. Of the erythromycin resistance traits that transferred only by filter mating, it was found that the erythromycin resistance trait was conferred by a 47-kbp transposable element that transferred from the chromosome of the donor strain to different sites within the pheromone responsive plasmid pAD1 (60 kbp) of the recipient strain, suggesting that the erythromycin resistance trait was encoded on a conjugative transposon, which was named Tn950.

Anti-Bacterial Agents↗

PubMed1986

Gentamicin-resistance plasmids in an intensive care unit.

To better understand the role of plasmids and their importance in the endemic antibiotic resistance of Enterobacteriaceae, we began a prospective study of our combined medical intensive care/coronary care unit. An initial culture survey of the patients, ward staff, and environment was followed by a prospective sampling of 139 consecutive new admissions at the time of admission to the unit, and at regular intervals thereafter for the remainder of their stay. All cultures were planted on agar-containing gentamicin. Of the 147 patients studied, 12 (8.2%) were colonized with 20 strains of gentamicin-resistant gram-negative bacilli (GRGNB) at 29 sites. An additional four GRGNB were isolated from the environment. Of the 24 GRGNB strains, 7 (29%) Enterobacteriaceae carried plasmids shown to carry the gentamicin-resistance determinant. Plasmids were further characterized by restriction endonuclease digestion profiles of plasmid DNA purified from E. coli C600 transconjugants or transformants. A 93 kb plasmid introduced to the unit by a Serratia liquefaciens colonizing a patient transferred from another area in the hospital was identical to 93 kb plasmids carried by a C. amalonaticus and an E. aerogenes subsequently colonizing another patient on the unit. A 60 kb plasmid, first isolated from a S. marcescens colonizing a sink drain was later isolated from an E. cloacae colonizing a patient. Our results indicate that spread of specific R-plasmids may be one mechanism for dissemination of antibiotic resistance on our MICU/CCU.

Drug Resistance, Microbial↗

PubMed1985

Overproduction of staphylokinase in Escherichia coli and its characterization.

A recombinant plasmid which directs the overproduction in Escherichia coli of staphylokinase from Staphylococcus aureus has been constructed by placing the staphylokinase gene, sak, under the control of bacteriophage lambda PR promoter in the plasmid. When an E. coli strain having the plasmid was induced, the staphylokinase activity in the periplasmic fraction increased about 60-fold and the 15.5-kDa protein corresponding to the mature form reached about 25% of the periplasmic proteins. At the same time the 18.5-kDa protein corresponding to the precursor form was accumulated in the membrane fraction, showing that the processing and translocation of the sak gene product were restricted during high level of its synthesis. By using this strain, the mature staphylokinase has been easily purified to near homogeneity. The purification steps consisted of extraction of the periplasmic proteins by osmotic shock and CM-cellulose column chromatography. Two species of staphylokinase were identified after CM-cellulose column chromatography. Although their isoelectric points and NH2-terminal amino acid sequences were different, their specific activities were almost equal. These results strongly suggest that the NH2-terminal portion of staphylokinase is not important for its activity.

Chromatography, Ion Exchange↗

PubMed1981

Molecular nature of a plasmid specifying beta-lactamase production in Haemophilus ducreyi.

We characterized pJB1, the plasmid previously reported to mediate beta-lactamase production in Haemophilus ducreyi. We studied its relationship to pMR0360 and RSF0885, the plasmids responsible for beta-lactamase production in Neisseria gonorrhoeae and Haemophilus parainfluenzae, respectively. Although pJB1 was maintained as a multicopy pool in Escherichia coli, it was not stably maintained in the absence of antibiotic selection. Electron microscope heteroduplex studies showed that it carried 100% of the transposable ampicillin resistance sequence TnA. This sequence was transposed to plasmid pUB307 at a low rate. Heteroduplexes between pMR0360 and pJB1 showed that they contained 3.3 megadaltons of homologous sequences. Two sets of nonhomologous sequences, one a TnA sequence and the other a non-TnA sequence, took the form of insertion loops. For plasmids pMR0360 and RSF0885, previously shown to be highly related, the nonhomologous sequences took the form of a substitution loop. We concluded that all three plasmids shared major portions of their sequences but differed in discrete segments. pJB1 was the first such plasmid to have a physically and functionally intact TnA sequence.

Ampicillin↗

PubMed2006

Essential elements of a licensed, mammalian plasmid origin of DNA synthesis.

We developed a mammalian plasmid replicon with a formerly uncharacterized origin of DNA synthesis, 8xRep*. 8xRep* functions efficiently to support once-per-cell-cycle synthesis of plasmid DNA which initiates within Rep*. By characterizing Rep*'s requirements for acting as an origin, we have uncovered several striking properties it shares with DS, the only other well-characterized, licensed, mammalian plasmid origin of DNA synthesis. Rep* contains a pair of previously unrecognized Epstein-Barr nuclear antigen 1 (EBNA1)-binding sites that are both necessary and sufficient in cis for its origin activity. These sites have an essential 21-bp center-to-center spacing, are bent by EBNA1, and recruit the origin recognition complex. The properties shared between DS and Rep* define cis and trans characteristics of a mammalian, extrachromosomal replicon. The role of EBNA1 likely reflects its evolution from cellular factors involved in the assembly of the initiation machinery.

Base Sequence↗

PubMed1986

A cryptic plasmid from Nocardia orientalis NRRL 2452, a vancomycin producer.

A plasmid was found in Nocardia orientalis (formerly Streptomyces orientalis). Physical characterization of the plasmid DNA indicates a size of 33.5 kb and a single cleavage site for EcoR I. The presence of plasmid, and variation in its copy member, did not directly affect vancomycin resistance or production levels. The plasmid represents the first to be isolated and characterized from a glycopeptide-producing nocardia.

Chromosome Mapping↗

PubMed1998

Characterization of the earliest known Staphylococcus aureus plasmid encoding a multidrug efflux system.

The staphylococcal qacB-encoding multidrug resistance plasmid pSK156, isolated from a clinical strain dating from 1951, was characterized. Comparison of the regions flanking qacB with other qacA- and qacB-encoding plasmids provided insights into the evolution and dissemination of these multidrug efflux genes and led to the detection of the earliest known copy of the insertion sequence IS257.

Bacterial Proteins↗

PubMed1994

Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358.

The narrow spectrum mercury-resistant (mer) operons of transposons Tn21 and Tn501 are inducible by inorganic mercury salts. The major regulatory gene merR is transcribed divergently from the other mer genes, which are cotranscribed. The MerR protein represses its own expression, as well as the expression of the other mer genes in the absence of the inducers. The synthesis of the polycistronic mer message is stimulated by MerR in the presence of the inducers. The MerRBS protein encoded by the broad spectrum mer operon of plasmid pDU1358 was characterized as a novel organomercurial receptor, distinguishing it from the narrow spectrum MerRNS proteins, described above. Several organomercurial compounds directly effected cellular activation of the mer operon transcription via the receptor protein MerRBS, but not by MerRNS. The merR gene from pDU1358 was cloned under the tac promoter, and the overexpressed MerRBS protein was soluble in buffer solutions containing 0.5 M NaCl at pH 7.5, but precipitated when NaCl concentration was reduced to 0.1 M (MerRBS concentrations at or above 0.1 mg/ml). MerRBS was purified to near homogeneity by selective precipitation and solubilization by varying the salt concentration in buffer solutions, followed by Sephadex G-75 column chromatography. Both MerRBS and Tn21-encoded MerRNS bound with DNA fragments containing the pDU1358 mer operator sequence with comparable affinities. In vitro run-off transcription studies revealed that MerRBS activated mer operon expression in the presence of Hg2+ or phenylmercuric acetate. Phenylmercuric acetate did not induce mer operon expression when the MerRNS was used in the assay.

Bacterial Proteins↗

PubMed2005

Generation and molecular characterization of new temperature-sensitive plasmids intended for genetic engineering of Pasteurellaceae.

Temperature-sensitive (TS) plasmids were generated through chemical mutagenesis of a derivative of the streptomycin resistance parent plasmid pD70, isolated from Mannheimia hemolytica serotype 1. Three TS plasmids which failed to replicate at or above 42 degrees C in M. hemolytica but which were fully functional below 31 degrees C were selected for further analysis. Two of the TS plasmids were shown by sequencing to possess unique single-base-pair mutations. The third TS plasmid contained a unique base pair substitution and a second mutation that had been previously identified. These mutations were clustered within a 200-bp region of the presumed plasmid origin of replication. Site-directed single-nucleotide substitutions were introduced into the wild-type pD70 origin of replication to confirm that mutations identified by sequencing had conferred thermoregulated replication. Deletion analysis on the wild-type pD70 plasmid replicon revealed that approximately 720 bp are necessary for plasmid maintenance. Replication of the TS plasmids was thermoregulated in Pasteurella multocida and Haemophilus somnus as well. To consistently transform H. somnus with TS plasmid, in vitro DNA methylation with commercially available HhaI methyltransferase was necessary to protect against the organism's restriction enzyme HsoI (recognition sequence 5'-GCGC-3') characterized herein.

Animals↗

PubMed1998

Cloning and molecular characterization of a multicopy, linear plasmid-carried, repeat motif-containing gene from Borrelia turicatae, a causative agent of relapsing fever.

Borrelia turicatae is one of several spirochete species that can cause relapsing fever. Here, we describe the identification and characterization of a gene from B. turicatae and other relapsing-fever spirochetes that exhibits homology with the rep+ and ORF-E gene families of the Lyme disease spirochetes. This gene, which we have designated repA, encodes a putative protein of 30.2 kDa with an isoelectric point of 4.69. The central region of RepA harbors a series of amino acid repeat motifs which exhibit homology with casein kinase 2 phosphorylation sites. Through Southern hybridization analyses, we demonstrate that repA (or a closely related sequence) is multicopy in the relapsing-fever spirochetes and is carried on variably sized linear plasmids in both Borrelia parkeri and B. turicatae. Transcriptional analyses demonstrate that repA is expressed, albeit at low levels, during in vitro cultivation of B. turicatae. Transcriptional start site analysis revealed that repA is preceded by a consensus ribosomal binding site and an appropriately spaced promoter element. The sequence conservation, unique features, and multicopy status of repA and its homologs suggest that RepA may play an important genus-wide role in the biology of the Borrelia.

Amino Acid Sequence↗

PubMed1992

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.

Amino Acid Sequence↗

PubMed2004

Evolution of multi-resistance plasmids in Australian clinical isolates of Escherichia coli.

Plasmids allow the movement of genetic material, including antimicrobial resistance genes, between bacterial species and genera. They frequently mediate resistance to multiple antimicrobials and can result in the acquisition by a pathogen of resistance to all or most clinically relevant antimicrobials. Unfortunately, there are still large gaps in our understanding of how new multi-resistance plasmids evolve. Five Australian clinical institutions collaborated in this study of multi-resistance plasmids in clinical isolates of Escherichia coli. We characterized 72 resistance plasmids in terms of the antimicrobial resistance profile they conferred, their size and their incompatibility group. Restriction fragment length polymorphisms were used to determine the genetic relationships between the plasmids. Relationships between the host cells were determined using multi-locus enzyme electrophoresis. A lack of correlation between the evolutionary history of the host cells and their plasmids suggests that the horizontal transfer of resistance plasmids between strains of E. coli is common. The resistance plasmids were very diverse, with a wide range of resistance profiles and a lack of discrete evolutionary lineages. Multi-resistance plasmids did not evolve via the co-integrative capture of smaller resistance plasmids; rather, the roles of recombination and the horizontal movement of mobile genetic elements appeared to be most important.

Anti-Bacterial Agents↗

PubMed1977

Plasmid-mediated resistance to gentamicin in Staphylococcus aureus.

Two strains isolated from a recent outbreak of infections by gentamicin-resistant Staphylococcus aureus were examined to determine whether genetic control of this resistance is plasmid or chromosomally mediated. Curing techniques indicated a plasmid location in both strains. Physical isolation and characterization of the plasmid deoxyribonucleic acid from one strain revealed that the determinant for gentamicin resistance resides on a 50S plasmid.

DNA, Bacterial↗

PubMed1982

Mutants of plasmid prophage P1 and elevated copy number: isolation and characterization.

A series of mutations of the P1 plasmid prophage that lead to increased copy number was isolated and analyzed. The copy number of the mutants was elevated at least five- to eightfold relative to wild-type P1, as determined by single-cell resistance to antibiotics, activity of enzymes, content of superhelical DNA, and reassociation kinetics. The copy number of two of the mutants was temperature dependent. Based on dominance tests, the mutants fell into two classes, cis specific and recessive. The latter class included a temperature-sensitive copy mutant. The existence of a class of recessive mutants suggests that the replication of the P1 plasmid is negatively regulated.

Ampicillin↗

PubMed1990

Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria.

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu) strains of the pathogen were isolated. The Cu genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu genes on pXV10A. A probe containing subcloned Cu genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.

Journal Article↗

PubMed2000

Construction of a promoter-rescue plasmid for Butyrivibrio fibrisolvens and its use in characterization of a flagellin promoter.

The Butyrivibrio fibrisolvens/Escherichia coli shuttle vector pBHerm has been modified to produce a plasmid (pBHE) that can be used for the identification and characterization of promoters in B. fibrisolvens. pBHE allows the insertion of a test promoter immediately upstream of a promoterless erythromycin resistance gene (ermAM). The efficacy of the pBHE plasmid in isolating and characterizing promoters was tested by inserting the flagellin gene (flaA) promoter from B. fibrisolvens OR77. Transcription of the ermAM gene from the flaA promoter was significantly higher than that observed when the ermAM gene was under the control of its own promoter. The flagelling gene of OR77 appears to be transcribed from two different promoters that produce transcripts initiating approximately 130 bp apart. Two mutant flaA promoter constructs, containing mutations in the -10 and -35 regions of either of the two putative promoter regions, showed drastic alterations in both the origin and amounts of the two transcripts produced. Mutations in either promoter affected transcription from both promoters, indicating that both regions contribute to gene expression.

DNA, Bacterial↗

PubMed1992

Enterobacterial tetracycline resistance in relation to plasmid incompatibility.

Sixty-eight well-characterized antibiotic resistance (R) plasmids belonging to 19 Incompatibility groups were screened with probes representing heterologous classes (A-E) of the enterobacterial tetracycline resistance (TcR) determinant. The Class B determinant was shown to be predominant in the IncF and IncH complexes and the IncC group. The Class A determinant was shown to be predominant in the IncP and IncM groups. There was no correlation between distribution of the class of TcR gene and the genus or the species of the host bacterial strain. The plasmids R714a (IncFI) and pHH1465 (IncC) contained TcR determinants which had no homology with any of these five probes.

Enterobacteriaceae↗