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Genomic Structure of the Salmonella enterica Serovar Typhimurium DT 64 Bacteriophage ST64T: Evidence for Modular Genetic Architecture. Princess T. Mmolawa, 2003.The complete sequence of the double-stranded DNA genome of a serotype-converting temperate bacteriophage, ST64T, was determined . The 40,679-bp genomic sequence of ST64T has an overall GC content of 47.5% and was reminiscent of a number of lambdoid phages, in particular, P22 . Inferred proteins of ST64T which exhibited a high degree of sequence similarity to P22 proteins (>90%) included the functional serotype conversion cassette, integrase, excisionase, Abc1, Abc2, early antitermination (gp24), NinD, NinH, NinZ, packaging (gp3 and gp2), head (with the exception of gp26, gp7, gp20, and gp16), and tail proteins . The putative immunity genes were highly related to those of Salmonella enterica serotype Typhimurium phage L, whereas the lysis genes were almost identical to those of S . enterica serovar Typhimurium PS3 . Levofloxacin-Resistant Invasive Streptococcus pneumoniae in the United States: Evidence for Clonal Spread and the Impact of Conjugate Pneumococcal Vaccine. Mathias W. R. Pletz, 2004.The emergence of fluoroquinolone resistance in sterile-site isolates of Streptococcus pneumoniae is documented in this study characterizing all invasive levofloxacin-resistant (MIC,  8 mg/liter) S . pneumoniae isolates (n = 50) obtained from the Centers for Disease Control and Prevention Active Bacterial Core Surveillance from 1998 to 2002 . Resistance among all isolates increased from 0.1% in 1998 to 0.6% in 2001 (P = 0.008) but decreased to 0.4% in 2002, while resistance among vaccine serotypes continued to increase from 0.3% in 1998 to 1.0% in 2002, suggesting that fluoroquinolones continue to exert selective pressure on these vaccine serotypes . Only 22% of resistant isolates were not covered by the conjugate vaccine serogroups . Multilocus sequence typing revealed that 58% of resistant strains were related to five international clones identified by the Pneumococcal Molecular Epidemiology Network, with the Spain23F-1 clone being most frequent (16% of all isolates) . Thirty-six percent of the isolates were coresistant to penicillin, 44% were coresistant to macrolides, and 28% were multiresistant to penicillin, macrolides, and fluoroquinolones . Fifty percent of the isolates were resistant to any three drug classes . Ninety-four percent of the isolates had multiple mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes . In 16% of the isolates, there was evidence of an active efflux mechanism . An unusual isolate was found that showed only a single parE mutation and for which the ciprofloxacin MIC was lower (2 mg/liter) than that of levofloxacin (8 mg/liter) . Our results suggest that invasive pneumococcal isolates resistant to levofloxacin in the United States show considerable evidence of multiple resistance and of clonal spread .
Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp.. Hiroko Tokunaga, 2004.It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained . We have addressed this question by identifying the gene responsible for multidrug resistance . A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp . strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane . The mutant produced an elevated level of the 58-kDa outer membrane protein . This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did . An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts . The hrdC gene encoding the 58-kDa outer membrane protein has been cloned . Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr . The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC . Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells . These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium . Properties of 2-Oxoglutarate:Ferredoxin Oxidoreductase from Thauera aromatica and Its Role in Enzymatic Reduction of the Aromatic Ring. Edith Dörner, 2002.Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA . The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor . In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme . KGOR activity was increased 10- to 50-fold in T . aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates . The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 µmol min-1 mg-1 . The native enzyme had a molecular mass of 200 ± 20 kDa (mean ± standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (  ß)2 composition . The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 ± 0.5 mol of Fe, 7.2 ± 0.5 mol of acid-labile sulfur, and 1.6 ± 0.2 mol of thiamine diphosphate (TPP) per mol of protein . The high specificity for 2-oxoglutarate and the low Km for ferredoxin ( 10 µM) indicated that both are the in vivo substrates of the enzyme . KGOR catalyzed the isotope exchange between 14CO2 and C1 of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases . The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway . Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T . aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S]1+/2+ clusters/monomer . Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro . This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring .
Elimination of Channel-Forming Activity by Insertional Inactivation of the p13 Gene in Borrelia burgdorferi. Yngve Östberg, 2002.P13 is a chromosomally encoded 13-kDa integral outer membrane protein of the Lyme disease agent, Borrelia burgdorferi . The aim of this study was to investigate the function of the P13 protein . Here, we inactivated the p13 gene by targeted mutagenesis and investigated the porin activities of outer membrane proteins by using lipid bilayer experiments . Channel-forming activity was lost in the p13 mutant compared to wild-type B . burgdorferi, indicating that P13 may function as a porin . We purified native P13 to homogeneity by fast performance liquid chromatography and demonstrated that pure P13 has channel-forming activity with a single-channel conductance in 1 M KCl of 3.5 nS, the same as the porin activity that was lost in the p13 mutant . Further characterization of the channel formed by P13 suggested that it is cation selective and voltage independent . In addition, no major physiological effects of the inactivated p13 gene could be detected under normal growth conditions . The inactivation of p13 is the first reported inactivation of a gene encoding an integral outer membrane protein in B . burgdorferi . Here, we describe both genetic and biophysical experiments indicating that P13 in B . burgdorferi is an outer membrane protein with porin activity . ISLpl1 Is a Functional IS30-Related Insertion Element in Lactobacillus plantarum That Is Also Found in Other Lactic Acid Bacteria. Hervé Nicoloff, 2003.We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum . ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335 . By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome . The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1 . When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L . plantarum strains as well as in Pediococcus strains . The ISLpl1 pattern changed over many generations of the strain L . plantarum NCIMB 1406 . This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L . plantarum . Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains . Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes . In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance . ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment .
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