Chromosomally Located fosA7 in Salmonella Isolates From China

This study aimed to investigate the prevalence of fosfomycin fosA7 in Salmonella enterica isolates from food animals and retail meat products in China and the impact of fosA7 on bacterial fitness. A total of 360 Salmonella isolates collected from 11 provinces and cities in China were detected for fosA7. All fosA7-positive Salmonella isolates were determined minimum inhibitory concentrations (MICs) and sequenced by Illumina Hiseq. The fosA7 gene of S. Derby isolate HA2-WA5 was knocked out. The full length of fosA7 was cloned into vector pBR322 and then transformed into various hosts. MICs of fosfomycin, growth curves, stability, and fitness of fosA7 were evaluated. The fosA7 gene was identified in S. Derby (ST40, n = 30) and S. Reading (ST1628, n = 5). MICs to fosfomycin of 35 fosA7-positive isolates were 1 to 32 mg/L. All fosA7 were located on chromosomes of Salmonella. The deletion of fosA7 in HA2-WA5 decreased fosfomycin MIC by 16-fold and slightly affected its fitness. The acquisition of plasmid-borne fosA7 enhanced MICs of fosfomycin in Salmonella (1,024-fold) and Escherichia coli (16-fold). The recombinant plasmid pBR322-fosA7 was stable in Salmonella Typhimurium, S. Pullorum, S. Derby, and E. coli, except for Salmonella Enteritidis, and barely affected on the growth of them but significantly increased biological fitness in Salmonella. The spread of specific Salmonella serovars such as S. Derby ST40 will facilitate the dissemination of fosA7. fosA7 can confer high-level fosfomycin resistance and enhance bacterial fitness in Salmonella if transferred on plasmids; thus, it has the potential to be a reservoir of the mobilized fosfomycin resistance gene.

qnrD, a Novel Gene Conferring Transferable Quinolone Resistance in Salmonella enterica Serovar Kentucky and Bovismorbificans Strains of Human Origin

ABSTRACT In a previous study, four Salmonella isolates from humans in the Henan province of China showed reduced susceptibility to ciprofloxacin (MIC, 0.125 to 0.25 μg/ml) but were susceptible to nalidixic acid (MIC, 4 to 8 μg/ml). All isolates were negative for known qnr genes (A, B, and S), aac(6′)Ib-cr, and mutations in gyrA and parC. Plasmid DNA was extracted from all four isolates and transformed into Escherichia coli TG1 and DH10B cells by electroporation, and transformants were selected on 0.06 μg/ml ciprofloxacin containing brain heart infusion agar plates. Resistance to ciprofloxacin could be transferred by electroporation, and a similar 4,270-bp plasmid was found in all transformants. By sequence analysis, the plasmid was found to carry an open reading frame that had similarities to other qnr genes and that encoded a 214-amino-acid pentapeptide repeat protein. This gene, designated qnrD, showed 48% similarity to qnrA1, 61% similarity to qnrB1, and 41% similarity to qnrS1. Further subcloning of the qnrD coding region into the constitutively expressed tetA gene of vector pBR322 showed that the gene conferred an increase in the MIC of ciprofloxacin by a factor of 32 (from an MIC of 0.002 to an MIC of 0.06 μg/ml). For comparison, qnrA1 and qnrS1 were also subcloned into pBR322 and transformed into DH10B cells, conferring MICs of 0.125 and 0.5 μg/ml, respectively. A phylogenetic analysis of all known qnr sequences was performed and showed that qnrD was more closely related to the qnrB variants but formed an independent cluster. To our knowledge, this is the first description of this qnrD gene.

Dependence on pH of substrate binding to a mutant lactose carrier, lacYun, in Escherichia coli. A model for H+/lactose symport

The lacYun gene, which encodes a lactose carrier showing the uncoupled phenotype of substrate transport in Escherichia coli [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414], was cloned on a plasmid vector, pBR322. The binding of a substrate, p-nitrophenyl alpha-galactoside, to the lacYun carrier in membranes from the strain harbouring the lacYun clone showed a pH-dependence different from its binding to the wild-type lactose carrier. This finding indicated that the lacYun mutation confers higher affinity for H+ on the carrier, exerting its effect on the less efficient dissociation of substrate inside cells. The result coincides with the proposal [Yamato & Rosenbusch (1983) FEBS Lett. 151, 102-104] that the proton affecting the substrate binding is the coupling proton of the proton/lactose symport reaction, which allows only the ordered mechanism of binding of substrate to an H+-carrier binary complex. From the simplest model of the symport reaction, constructed on the basis of these results, the coupling site of energy in the carrier cycle of the transport reaction can be identified at the substrate-dissociation step inside cells.

Amplified RNase H Activity in Escherichia coli B/r Increases Sensitivity to Ultraviolet Radiation

ABSTRACT Strains of E. coli B/r transformed with the plasmid pSK760 were found to be sensitized to inactivation by ultraviolet radiation (UV) and to have elevated levels of RNase H activity. Strains transformed with the carrier vector pBR322 or the plasmid pSK762C derived from pSK760 but with an inactivated rnh gene were not sensitized. UV-inactivation data for strains having known defects in DNA repair and transformed with pSK760 suggested an interference by RNase H of postreplication repair: uvrA cells were strongly sensitized, wild-type and uvrA recF cells were moderately sensitized and recA cells were not sensitized; and minimal medium recovery was no longer apparent in sensitized uvrA cells. Biochemical studies showed that post-UV DNA synthesis was sensitized and that the smaller amounts of DNA synthesized after irradiation, while of normal reduced size as indicated by sedimentation position in alkaline sucrose gradients, did not shift to a larger size (more rapidly sedimenting) upon additional incubation. We suggest an excess level of RNase H interferes with reinitiation of DNA synthesis on damaged templates to disturb the normal pattern of daughter strand gaps and thereby to inhibit postreplication repair.

Stable maintenance in chemostat-grown Escherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene*

Plasmid stability was studied in antibiotic-free chemo-stat cultures. Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoRl/EcoRV region of the cloning vector pBR322 or in the HindIII]BamHl region of pACYCI84 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.

Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae.

A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating a alpha cells and asporogenous alpha alpha cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the a alpha sporulation probe relative to the alpha alpha control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, a alpha, and alpha alpha cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified. Transcripts complementary to these genes are present only in a alpha cells after transfer to sporulation medium. Three of these clones contain two sporulation-specific genes. Three genes have been identified that are expressed in all cell types during vegetative growth and only in a alpha cells in sporulation medium.

Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae

A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating a alpha cells and asporogenous alpha alpha cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the a alpha sporulation probe relative to the alpha alpha control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, a alpha, and alpha alpha cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified. Transcripts complementary to these genes are present only in a alpha cells after transfer to sporulation medium. Three of these clones contain two sporulation-specific genes. Three genes have been identified that are expressed in all cell types during vegetative growth and only in a alpha cells in sporulation medium.