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PubMed2006

Bacteriocin production, plasmid content and plasmid location of enterocin P structural gene in enterococci isolated from food sources.

AIMS: To characterize bacteriocin production, antimicrobial spectrum and plasmid content in bacteriocinogenic enterococci from foods. METHODS AND RESULTS: Bacteriocinogenic Enterococcus faecium (14 isolates) and Enterococcus faecalis (three isolates) showed two different patterns of bacteriocin production in liquid broth: exponential-phase and stationary-phase production. Bacteriocin concentrates from all enterococci were inactivated by trypsin, but seldom by heat (100-117 degrees C), extremes of pH (2.0 to 9.0) or reducing agents (such as dithiothreitol). All bacteriocin concentrates were active against Listeria innocua and Listeria monocytogenes, and most were also active against many Ent. faecalis and Ent. faecium isolates. Enterococci clustered in three main groups according to their plasmid content (which included plasmids from 2.0 to 53 kb). Several isolates from different foods showed almost identical plasmid profiles. The enterocin P structural gene (entP) was detected by hybridization on plasmids of c. 19, 26 and/or 35-38 kb. CONCLUSIONS: Enterococci from food show different patterns of bacteriocin production and different plasmid content in spite of carrying similar bacteriocin-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the diversity of bacteriocinogenic enterococci from food sources carrying apparently similar enterocin genes.

Bacteriocins↗

PubMed1988

RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase.

The nucleotide sequence of the type II sulfonamide resistance dihydropteroate synthase (sulII) gene was determined. The molecular weight determined by maxicells was 30,000, and the predicted molecular weight for the polypeptide was 28,469. Comparison with the sulI gene encoded by Tn21 showed 57% DNA similarity. The sulII-encoded polypeptide has 138 of 271 amino acids in common with the polypeptide encoded by sulI. The sulII gene is located on various IncQ (broad-host-range) plasmids and other small nonconjugative resistance plasmids. Detailed restriction maps were constructed to compare the different plasmids in which sulII is found. The large conjugative plasmid pGS05 and the IncQ plasmid RSF1010 contained identical nucleotide sequences for the sulII gene. This type of sulfonamide resistance is very frequently found among gram-negative bacteria because of its efficient spread to various plasmids.

Amino Acid Sequence↗

PubMed1996

Integration of pT181-like tetracycline resistance plasmids into large staphylococcal plasmids involves IS257.

Four large staphylococcal plasmids ranging in size from 31 to 82 kbp have been shown to mediate tetracycline resistance via an integrated copy of the tet(K)-encoding plasmid pT181 which was flanked by copies of the insertion element IS257. In two cases, IS257 elements interrupted the repC reading frame of pT181 and an 8-bp sequence from within the repC gene was duplicated at the interrupted site. In the third plasmid, the IS257 elements interrupted the pT181 DNA immediately upstream of the repC coding sequence with an 8-bp duplication. In the fourth case, the IS257 elements flanked a pT181-like plasmid with one IS257 in the repC coding sequence and the other within the recombinase (pre) coding sequence, so that a section of the pT181 sequence was deleted. All four integration sites detected in this study differ from those previously described for the IS257-mediated integration of pT181-like plasmids into large plasmids or into the chromosomal DNA.

Blotting, Southern↗

PubMed1997

High-level plasmid-mediated gentamicin resistance and pheromone response of plasmids present in clinical isolates of Enterococcus faecalis.

Eleven pheromone-responding plasmids encoding erythromycin or gentamicin resistance were isolated from multiresistant clinical Enterococcus faecalis isolates. The plasmids were classified into six types with respect to their pheromone responses. The three erythromycin resistance plasmids responded to different pheromones. Of the eight gentamicin resistance plasmids, four plasmids responded to same pheromone. Southern hybridization studies showed that the genes involved in regulation of the pheromone response were conserved in the drug resistance plasmids.

Anti-Bacterial Agents↗

PubMed2004

Characterization of plasmid pOR1 from Ornithobacterium rhinotracheale and construction of a shuttle plasmid.

The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.

Animals↗

PubMed1980

Transfer of a CFA/I-ST plasmid promoted by a conjugative plasmid in a strain of Escherichia coli of serotype O128ac:H12.

Escherichia coli strains belonging to serotype O128ac:H12 and producing heat-stable enterotoxin (ST) and colonization factor CFA/I were found in Sao Paulo in children with diarrhea, but not in normal children. Segregants occurred in such strains with a frequency of about 10%, which have lost the ability to produce ST and CFA/I at the same time. From one strain, both properties were transformed jointly in matings to an E. coli K-12 strain. All such ST+ CFA/I+ progeny had received two plasmids of length 97 and 64 kilobases in the matings. Insertion of a transposon, Tn5, carrying a gene for kanamycin resistance, into the two plasmids enabled us to select for kanamycin-resistant progeny in further matings. Analysis of such progeny strains in terms of plasmid content and production of ST and CFA/I revealed that the larger plasmid carries the genes for St and CFA/I and is not self-transmissible, whereas the smaller plasmid does not carry any recognizable phenotypic traits, but is conjugative and promotes cotransfer of the larger plasmid with a frequency of about 30%.

Colony-Forming Units Assay↗

PubMed1981

Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis.

Yersinia enterocolitica isolates harboring a particular species of plasmid deoxyribonucleic acid showed a high degree of lethality for gerbils and caused the detachment of HEp-2 tissue cell monolayers. Strains cured of their plasmid deoxyribonucleic acid showed loss of these properties. However, invasiveness of HEp-2 cells was shown not to be a plasmid-mediated property. The expression of plasmid-associated properties, including at least three major outer membrane polypeptides, occurred during growth at 37 but not at 25 degrees C and was related to the concentration of calcium in the growth medium. The plasmid species associated with these properties ranged in molecular mass from 40 x 10(6) to 48 x 10(6) daltons and comprised a family of related plasmids.

Animals↗

PubMed1984

Serum resistance encoded by colicin V plasmids in Escherichia coli and its relationship to the plasmid transfer system.

Eight colicin V plasmids were conjugated into a plasmidless Escherichia coli (K-12) strain that was susceptible to the bactericidal effects of normal rabbit serum. The resulting colicin V-positive strains were examined for their capacity to resist the lethal effects of serum. Serum resistance was assessed as growth of the bacterial strain in medium containing 5% normal rabbit serum inoculated from a culture in the early exponential phase. Only three of the eight colicin V plasmids were found to confer the serum resistance phenotype on the host strain. Derepression of the transfer system was associated with serum resistance in two of the plasmids. Two other derepressed plasmids did not confer serum resistance on the host bacterium. Therefore, such derepression alone was insufficient to produce serum resistance. The factor(s) encoded by colicin V plasmids and responsible for the serum resistance of the bacterial strains bearing the plasmids was shown to be a property associated with the cell and not an extracellular factor excreted into the growth medium.

Animals↗

PubMed1978

Evolution of Pseudomonas R-plasmids: consequences of Tn1 insertion and resultant partial diploidy to chromosome and Tra- R-plasmid mobilization.

Tn1 transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tn1 by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition of Tn1 results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid. These aggregates have lost at least part of Tn1 from their parent FP2:Tn1 component, but now they mobilize the tra- R-plasmid from a recombination-deficient (Rec-) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.

Chromosomes, Bacterial↗

PubMed1978

Replication of colicin E1 plasmid DNA in vivo requires no plasmid-encoded proteins.

A derivative of bacteriophage lambda containing a colicin E1 plasmid replicon was constructed by recombinant DNA techniques. This phage, lambdacol100, has two functional modes of DNA replication; it can replicate via either plasmid or phage replication systems. lambdacol100 has been used to introduce the colicin E1 plasmid replicon into Escherichia coli previously treated with chloramphenicol to block protein synthesis. Under these conditions, lambdacol100 DNA is replicated normally as a colicin E1 plasmid. This suggests that colicin E1 plasmid replication in vivo does not require any plasmid-encoded proteins.

Bacterial Proteins↗

PubMed1979

Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.

A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.

Anti-Bacterial Agents↗

PubMed1981

Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids.

Ten plasmids were isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant thermophilic bacteria. Of the 10 plasmids tested, 2 could transform Bacillus subtilis, yielding resistance to specific antibiotics. Plasmid pTB20 (2.8 X 10(6) daltons, approximately 24 copies per chromosome) specifies resistance to tetracycline (Tcr), whereas pTB19 (17.2 X 10(6) daltons, approximately 1 copy per chromosome) renders the host resistant to both kanamycin and tetracycline (KMrTcr). Three plasmids were not self-transmissible. The restriction endonuclease cleavage maps of the two plasmids, pTB19 and pTB20, were constructed. pTB19 and pTB20, both of which were originally isolated from thermophilic bacilli, were tested for stability in B. subtilis. Digestion of pTB19 followed by ligation yielded deletion plasmids pTB512 (Kmr), pTB52 (Tcr), and pTB53 (KmrTcr). Determinants of Kmr, Tcr, and DNA replication were associated with EcoRI fragments R1b (4.2 X 10(6) daltons), R3 (2.8 X 10(6) daltons), and R1a (4.2 X 10(6) daltons), respectively. Restriction endonuclease cleavage maps of pTB51, pTB52, and pTB53 were constructed. Tetracycline resistance of pTB20 was confirmed to be in the EcoRI fragment (1.85 X 10(6) daltons).

Bacillus subtilis↗

PubMed1982

Relationship between Nif plasmids of fast-growing Rhizobium species and Ti plasmids of Agrobacterium tumefaciens.

By use of the Southern blot hybridization technique the extent of DNA homology was determined between the Nif plasmid of a number of fast-growing Rhizobium species and Ti plasmids of the octopine (pTiAch5) and nopaline (pTiC58) type. DNA sequences common to these plasmids were located on functional maps of the Ti plasmids. No homology between Nif plasmids and the T region of Ti plasmids was detected.

Arginine↗

PubMed1982

Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis.

A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.

Bacillus subtilis↗

PubMed1986

Conjugative transfer of the naturally occurring plasmids of Acetobacter xylinum by IncP-plasmid-mediated mobilization.

Broad-host-range plasmids and cloning vectors were conjugatively transferred to Acetobacter xylinum. One of the plasmids, RP4::Mu cts61, was used for the insertion of Tn1 into the 16-, 44-, and 64-kilobase-pair plasmids of A. xylinum. The Tn1-labeled plasmids could be mobilized by a helper plasmid. Many of the Tn1 insertions affected the copy number of the plasmids.

Cellulose↗

PubMed1987

Replication properties of pIM13, a naturally occurring plasmid found in Bacillus subtilis, and of its close relative pE5, a plasmid native to Staphylococcus aureus.

A naturally occurring plasmid from Bacillus subtilis, pIM13, codes for constitutively expressed macrolide-lincosamide-streptogramin B (MLS) resistance, is stably maintained at a high copy number, and exists as a series of covalent multimers. The complete sequence of pIM13 has been reported (M. Monod, C. Denoya, and D. Dubnau, J. Bacteriol. 167:138-147, 1986) and two long open reading frames have been identified, one of which (ermC') is greater than 90% homologous to the ermC MLS resistance determinant of the Staphylococcus aureus plasmid pE194. The second reading frame (repL) shares homology with the only long open reading frame of the cryptic S. aureus plasmid pSN2 and is probably involved in plasmid replication. The map of pIM13 is almost a precise match with that of pE5, a naturally occurring, stable, low-copy-number, inducible MLS resistance plasmid found in S. aureus. pIM13 is unstable in S. aureus but still multimerizes in that host, while pE5 is unstable in B. subtilis and does not form multimers in either host. The complete sequence of pE5 is presented, and comparison between pIM13 and pE5 revealed two stretches of sequence present in pE5 that were missing from pIM13. It is likely that a 107-base-pair segment in the ermC' leader region missing from pIM13 accounts for the constitutive nature of the pIM13 MLS resistance and that the lack of an additional 120-base-pair segment in pIM13 that is present on pE5 gives rise to the high copy number, stability, and multimerization in B. subtilis. The missing 120 base pairs occur at the carboxyl-terminal end of the putative replication protein coding sequence and results in truncation of that protein. It is suggested either that the missing segment contains a site involved in resolution of multimers into monomers or that the smaller replication protein causes defective termination of replication. It is concluded that pIM13 and pE5 are coancestral plasmids and it is probable that pIM13 arose from pE5.

Amino Acid Sequence↗

PubMed1990

General organization of the conjugal transfer genes of the IncW plasmid R388 and interactions between R388 and IncN and IncP plasmids.

The complete conjugal transfer gene region of the IncW plasmid R388 has been cloned in multicopy vector plasmids and mapped to a contiguous 14.9-kilobase segment by insertion mutagenesis. The fertility of the cloned region could still be inhibited by a coresident IncP plasmid. The transfer region has been dissected into two regions, one involved in pilus synthesis and assembly (PILW), and the other involved in conjugal DNA metabolism (MOBW). They have been separately cloned. PILW also contains the genes involved in entry exclusion. MOBW contains oriT and the gene products required for efficient mobilization by PILW. MOBW plasmids could also be mobilized efficiently by PILN, the specific pilus of the IncN plasmid pCU1, but not by PILP, the specific pilus of the IncP plasmid RP1.

Cloning, Molecular↗

PubMed1990

Mobilization of closely related plasmids pUB110 and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame beta.

Genetic analysis of the closely related nonconjugative plasmids pUB110 and pBC16 has demonstrated that the open reading frame beta (ORF-beta) region in pUB110 and the corresponding homologous region in pBC16 are essential for mobilization of these plasmids by pLS20 or its derivatives. Deletions in this region or insertions that interrupted ORF-beta severely impaired or eliminated the mobilization of pUB110::pUC18 and pBC16::pUC18 hybrids. In contrast, a hybrid in which pUC18 was inserted into pBC16 at a point outside ORF-beta transferred at a frequency comparable to that of intact pUB110 or pBC16 (10(-4) transcipients per donor cell). The defect of most transfer-deficient (Mob-) hybrid plasmids could be complemented by an intact sister plasmid (i.e., pBC16 for pUB110::pUC18 Mob- hybrids). The inability to complement certain constructs suggested that the origin of transfer might be located in an area 5' to ORF-beta. Furthermore, cloning the region 5' to ORF-beta onto a nonmobilizable pC194::pUC18 construct resulted in a hybrid plasmid, pUCCoriTBC16, that could be mobilized with complementation. These results indicate that mobilization of pUB110 and pBC16 by conjugative helper plasmids requires ORF-beta in trans and at least one other region, including the RSA sequence, which presumably functions as an origin of transfer, in cis.

Bacillus↗