|
|
|
|
|
|
Single-Nucleotide Polymorphism Mutation Spectra and Resistance to Quinolones in Salmonella enterica Serovar Enteritidis with a Mutator Phenotype. Dan D. Levy, 2004.Resistance to quinolone antibiotics has been associated with single-nucleotide polymorphisms (SNPs) in the quinolone resistance-determining region (QRDR) of gyrA . Mutations in the gyrA gene were compared by using mutant populations derived from wild-type Salmonella enterica serovar Enteritidis and its isogenic mutS::Tn10 mutator counterpart . Spontaneous mutants arising during nonselective growth were isolated by selection with either nalidixic acid, enrofloxacin, or ciprofloxacin . QRDR SNPs were identified in approximately 70% (512 of 695) of the isolates via colony hybridization with radiolabeled oligonucleotide probes . Notably, transition base substitution SNPs in the QRDR were dramatically increased in mutants derived from the mutS strain . Some, but not all, antibiotic-resistant mutants lacking QRDR SNPs were resistant to tetracycline and chloramphenicol, consistent with alterations in nonspecific efflux pumps or other membrane transport mechanisms . Changing the selection conditions shifted the mutation spectrum . Selection with ciprofloxacin was least likely to yield a mutant harboring either a QRDR SNP or chloramphenicol resistance . Selection with enrofloxacin was more likely to yield mutants containing Ser83  Phe mutations, whereas selection with ciprofloxacin or nalidixic acid favored recovery of Asp87Gly mutants . Fluoroquinolone-resistant Salmonella strains isolated from veterinary or clinical settings frequently display a mutational spectrum with a preponderance of transition SNPs in the QRDR, the pattern found in vitro among mutS mutator mutants reported here . Both the preponderance of transition mutations and the varied mutation spectra reported for veterinary and clinical isolates suggest that bacterial mutators defective in methyl-directed mismatch repair may play a role in the emergence of quinolone and fluoroquinolone resistance in feral settings .
Heterologous Production of Clostridium cellulovorans engB, Using Protease-Deficient Bacillus subtilis, and Preparation of Active Recombinant Cellulosomes. Koichiro Murashima, 2002.In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex . Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a "designer" cellulosome, or a recombinant cellulosome with a specific function . We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units . The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli . The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis . Depletion of Free 30S Ribosomal Subunits in Escherichia coli by Expression of RNA Containing Shine-Dalgarno-Like Sequences. Mary V. Mawn, 2002.We have constructed synthetic coding sequences for the expression of poly(  ,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli . These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs . An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA . Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs . Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient . Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin . The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA . We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
