Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation. (1/44)

The filamentous soil bacterium Streptomyces coelicolor undergoes a complex cycle of morphological differentiation involving the formation of an aerial mycelium and the production of pigmented antibiotics. We have developed a procedure for generating insertional mutants of S. coelicolor based on in vitro transposition of a plasmid library of cloned S. coelicolor DNAs. The insertionally mutated library was introduced into S. coelicolor, and transposon insertions were recovered at widely scattered locations around the chromosome. Many of the insertions revealed previously uncharacterized genes, and several caused novel mutant phenotypes, such as altered pigment production, enhanced antibiotic sensitivity, delayed or impaired formation of aerial hyphae, and a block in spore formation. The sporulation mutant harbored an insertion in one of three adjacent genes that are apparently unique to Streptomyces but are each represented by at least 20 paralogs at dispersed locations in the chromosome. Individual members of the three families often are found grouped together in a characteristic arrangement, suggesting that they have a common function.  (+info)

Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype typhimurium phage type DT104. (2/44)

PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 422 veterinary isolates of Salmonella enterica serotype Typhimurium. The identities of extra-integron genes encoding resistance to streptomycin, gentamicin, kanamycin, and apramycin were evaluated. Gentamicin resistance was conferred by the aadB gene. Kanamycin resistance was encoded by either the aphA1-Iab gene or the Kn gene. Apramycin resistance was determined by the aacC4 gene. Analysis of gene distribution did not reveal significant differences with regard to phage type, host species, or region except for the Kn gene, which was found mostly in nonclinical isolates. The data from this study indicate that pentaresistant DT104 does not acquire extra-integron genes in species- or geography-related foci, which supports the hypothesis that clonal expansion is the method of spread of this organism.  (+info)

Effects of apramycin, a novel aminoglycoside antibiotic on bacterial protein synthesis. (3/44)

1. The novel aminoglycoside antibiotic apramycin is shown to be a potent inhibitor of protein synthesis in bacteria both in vivo and in vitro. 2. In cell-free systems from Escherichia coli programmed with poly(U), apramycin induces translation errors, as assayed by incorporation of leucine, isoleucine and serine, although this effect occurs only to a limited extent. 3. Apramycin inhibits the translocation step of protein synthesis both in vivo, in protoplasts of Bacillus megaterium, and in vitro, in cell-free systems from E. coli. It is proposed that this is the primary inhibitory effect of the drug.  (+info)

Acquisition and epidemiology of antibiotic-resistant Escherichia coli in a cohort of newborn calves. (4/44)

OBJECTIVES: The acquisition of antibiotic-resistant commensal Escherichia coli was examined in a cohort of newborn calves. METHODS: Faecal samples were collected weekly from calves over a 4 month period and screened for E. coli resistant to ampicillin, apramycin and nalidixic acid at concentrations of 16, 8 and 8 mg/L, respectively. E. coli viable counts were performed on samples from a subset of calves. RESULTS: All calves acquired ampicillin- and nalidixic acid-resistant E. coli, while only 67% acquired apramycin-resistant E. coli during the study. Sixty-seven per cent of samples were resistant to at least one of the three antibiotics. Prevalence of ampicillin and nalidixic acid resistance was high initially and declined significantly with age (P < 0.001). No temporal or age-related pattern was observed in the prevalence of apramycin resistance. Housing the cohort had a significant effect on the prevalence of nalidixic acid resistance (P < 0.001). Total and ampicillin- and nalidixic acid-resistant E. coli counts declined with calf age (P < 0.001), with the rate of decline in ampicillin-resistant counts being greater than that for total counts (P < 0.001). The proportion of total E. coli counts that were resistant to ampicillin or nalidixic acid also declined with age (P < 0.001). CONCLUSIONS: Cohort calves rapidly acquired antibiotic-resistant bacteria within days of birth. Carriage of resistant bacteria was associated with both age and housing status of the cohort.  (+info)

High frequency transfer and horizontal spread of apramycin resistance in calf faecal Escherichia coli. (5/44)

OBJECTIVES: The aminoglycoside apramycin has been used extensively in animal husbandry in the UK since 1978. This study aimed to determine both whether calves that had never been treated with aminoglycoside antibiotics harboured apramycin-resistant (apr(R)) commensal Escherichia coli, and the mode of spread of the resistance gene. METHODS: Apr(R) E. coli from weekly calf faecal samples were typed by pulsed-field gel electrophoresis, antibiotic resistance phenotype, plasmid restriction profiles and plasmid transfer frequencies. RESULTS: During 4 months of weekly sampling, six of 11 calves were found to harbour apr(R) E. coli. All apr(R) E. coli (45) were cross-resistant to gentamicin and tobramycin, which are both used in human medicine. Resistance was conferred by the aac(3)IV gene, present on three different conjugative plasmids. Two of these plasmids also mediated tetracycline and streptomycin resistance. One plasmid demonstrated very high transfer frequencies and was found in three different genotypes. CONCLUSIONS: We report the presence of apr(R) commensal E. coli in cattle that have never been treated with aminoglycosides. The presence of one conjugative plasmid in three different genotypes is evidence of horizontal spread of this plasmid. This is the first report of a very high transfer frequency of apr(R) plasmid, demonstrating horizontal spread in the commensal flora of food animals.  (+info)

Apramycin resistance plasmids in Escherichia coli: possible transfer to Salmonella typhimurium in calves. (6/44)

An outbreak of salmonellosis in calves was monitored for persistence of Salmonella typhimurium excretion in faeces and the effect of treatment with apramycin. Prior to treatment apramycin-resistant Escherichia coli were present but all S. typhimurium isolates were sensitive. Following the treatment of six calves with apramycin, apramycin-resistant S. typhimurium were isolated from two treated calves and one untreated calf. Plasmid profiles of E. coli and S. typhimurium were compared and plasmids conferring resistance to apramycin and several other antibiotics were transferred by conjugation in vitro from calf E. coli and S. typhimurium isolates to E. coli K-12 and from E. coli to S. typhimurium. The plasmids conjugated with high frequency in vitro from E. coli to S. typhimurium, and hybridized to a DNA probe specific for the gene encoding aminoglycoside acetyltransferase 3-IV (AAC(3)-IV) which confers resistance to apramycin, gentamicin, netilmicin and tobramycin.  (+info)

The kinetic mechanism of AAC3-IV aminoglycoside acetyltransferase from Escherichia coli. (7/44)

The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.  (+info)

Two conformational states in the crystal structure of the Homo sapiens cytoplasmic ribosomal decoding A site. (8/44)

The decoding A site of the small ribosomal subunit is an RNA molecular switch, which monitors codon-anticodon interactions to guarantee translation fidelity. We have solved the crystal structure of an RNA fragment containing two Homo sapiens cytoplasmic A sites. Each of the two A sites presents a different conformational state. In one state, adenines A1492 and A1493 are fully bulged-out with C1409 forming a wobble-like pair to A1491. In the second state, adenines A1492 and A1493 form non-Watson-Crick pairs with C1409 and G1408, respectively while A1491 bulges out. The first state of the eukaryotic A site is, thus, basically the same as in the bacterial A site with bulging A1492 and A1493. It is the state used for recognition of the codon/anticodon complex. On the contrary, the second state of the H.sapiens cytoplasmic A site is drastically different from any of those observed for the bacterial A site without bulging A1492 and A1493.  (+info)