Conjugation is necessary for a bacterial plasmid to survive under protozoan predation

Horizontal gene transfer by conjugative plasmids plays a critical role in the evolution of antibiotic resistance. Interactions between bacteria and other organisms can affect the persistence and spread of conjugative plasmids. Here we show that protozoan predation increased the persistence and spread of the antibiotic resistance plasmid RP4 in populations of the opportunist bacterial pathogen Serratia marcescens . A conjugation-defective mutant plasmid was unable to survive under predation, suggesting that conjugative transfer is required for plasmid persistence under the realistic condition of predation. These results indicate that multi-trophic interactions can affect the maintenance of conjugative plasmids with implications for bacterial evolution and the spread of antibiotic resistance genes.

Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

ABSTRACTThe plasmid pGKE75-catA138T, which comprises pUC18 and thecatA138Tgene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as anEscherichia coli-Geobacillus kaustophilusshuttle plasmid that confers moderate chloramphenicol resistance onG. kaustophilusHTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138Tin an error-prone thermophile, generating the mutant plasmid pGKE75αβ-catA138Tresponsible for substantial chloramphenicol resistance at 65°C. pGKE75αβ-catA138Tcontained no mutation in thecatA138Tgene but had two mutations in the pUC replicon, even though the replicon has no apparent role inG. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75αβ-catA138Tis attributable to increases in intracellular CATA138Tand acetyl-coenzyme A following a decrease in incomplete forms of pGKE75αβ-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced inG. kaustophilus. It is noteworthy thatG. kaustophiluswas transformed with pGKE75αβ-catA138Tusing chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated inE. coliat a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes forG. kaustophilusandE. coli.

Synergistically Active and Safe Fixed Dose Combination of Meropenem and Sulbactam

Meropenem is a third generation broad spectrum antibiotic. Emergence of meropenem resistance has been reported due to development of mutant plasmid mediated metallo-β-lactamases (IMP-6) and AmpC β–lactamases. Sulbactam, a stable β-lactamase inhibitor, increase antimicrobial activity of meropenem by inhibiting the enzyme β-lactamase. Fixed dose combination of Meropenem-sulbactam in the proportion of 1:1, 1:2, 1:3, 2:1 and 3:1 were evaluated for the antimicrobial activity. Combination of meropenem and sulbactam in the ratio of 2:1 exhibited the synergistic activity. This combination was checked for the subchronic toxicity on wistar rats and no change in biochemical and physiological parameters was observed.

Structural Maintenance of Chromosomes Protein of Bacillussubtilis Affects Supercoiling In Vivo

ABSTRACT Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.

Fine Structure Recombinational Analysis of Cloned Genes Using Yeast Transformation

ABSTRACT We describe a general method for analyzing the genetic fine structure of plasmid-borne genes in yeast. Previously we had reported that a linearized plasmid is efficiently rescued by recombination with a homologous restriction fragment when these are co-introduced by DNA-mediated transformation of yeast. Here, we show that a mutation can be localized to a small DNA interval when members of a deletion series of wild-type restriction fragments are used in the rescue of a linearized mutant plasmid. The resolution of this method is to at least 30 base pairs and is limited by the loss of a wild-type marker with proximity to a free DNA end. As a means for establishing the nonidentity of two mutations, we determined the resolution of two-point crosses with a mutant linearized plasmid and a mutant homologous restriction fragment. Recombination between mutations separated by as little as 100 base pairs was detected. Moreover, the results indicate that exchange within a marked interval results primarily from one of two single crossovers that repair the linearized plasmid. These approaches to mapping the genetic fine structure of plasmids should join existing methods in a robust approach to the mutational analysis of gene structure in yeast.