Molecular Characterization of a 21.4 Kilobase Antibiotic Resistance Plasmid from an α-Hemolytic Escherichia coli O108:H- Human Clinical Isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes

DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA

DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight.</jats:p

Molecular construction and characterization of nif mutants of the obligate methanotroph Methylosinus sp. strain 6

We describe here a method for constructing mutants in bacteria that are not amenable to mutant isolation by conventional means. A one-step marker exchange procedure was used to construct nitrogen fixation (nif) mutants of the obligate methane-utilizing bacterium Methylosinus sp. strain 6, using transposon 5 (Tn5)-containing nif genes cloned into pBR325. The resultant mutants appeared to contain defects in nif structural genes, and DNA hybridization analysis showed that although one out of five had apparently been produced as a result of double-crossover homologous recombination, a variety of molecular events had led to the production of the other four mutants.</jats:p