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Dps Protects Cells against Multiple Stresses during Stationary Phase. Sudha Nair, 2004.Dps, the nonspecific DNA-binding protein from starved cells, is the most abundant protein in stationary-phase Escherichia coli . Dps homologs are found throughout the bacteria and in at least one archaeal species . Dps has been shown to protect cells from oxidative stress during exponential-phase growth . During stationary phase, Dps organizes the chromosome into a highly ordered, stable nucleoprotein complex called the biocrystal . We show here that Dps is required for long-term stationary-phase viability under competitive conditions and that dps mutants have altered lag phases compared to wild-type cells . We also show that during stationary phase Dps protects the cell not only from oxidative stress but also from UV and gamma irradiation, iron and copper toxicity, thermal stress, and acid and base shock . The protective roles of Dps are most likely achieved through a combination of functions associated with the protein-DNA binding and chromosome compaction, metal chelation, ferroxidase activity, and regulation of gene expression . Mechanism of Resistance to Several Antimicrobial Agents in Salmonella Clinical Isolates Causing Traveler's Diarrhea. Roberto Cabrera, 2004.The evolution of antimicrobial resistance in Salmonella isolates causing traveler's diarrhea (TD) and their mechanisms of resistance to several antimicrobial agents were analyzed . From 1995 to 2002, a total of 62 Salmonella strains were isolated from stools of patients with TD . The antimicrobial susceptibility to 12 antibiotics was determined, and the molecular mechanisms of resistance to several of them were detected as well . The highest levels of resistance were found against tetracycline and ampicillin (21 and 19%, respectively), followed by resistance to nalidixic acid (16%), which was mainly detected from 2000 onward . Molecular mechanisms of resistance were analyzed in 16 isolates . In these isolates, which were resistant to ampicillin, two genes encoding ß-lactamases were detected: oxa-1 (one isolate) and tem-like (seven isolates [in one strain concomitantly with a carb-2]) . Resistance to tetracycline was mainly related to tetA (five cases) and to tetB and tetG (one case each) . Resistance to chloramphenicol was related to the presence of the floR and cmlA genes and to chloramphenicol acetyltransferase activity in one case each . Different genes encoding dihydrofolate-reductases (dfrA1, dfrA12, dfrA14, and dfrA17) were detected in trimethoprim-resistant isolates . Resistance to nalidixic acid was related to the presence of mutations in the amino acid codons 83 or 87 of the gyrA gene . Further surveillance of the Salmonella spp . causing TD is needed to detect trends in their resistance to antimicrobial agents, as we have shown in our study with nalidixic acid . Moreover, such studies will lead to better treatment and strategies to prevent and limit their spread . Double-Blind, Randomized Study of the Efficacy and Safety of Oral Pharmacokinetically Enhanced Amoxicillin-Clavulanate (2,000/125 Milligrams) versus Those of Amoxicillin-Clavulanate (875/125 Milligrams), Both Given Twice Daily for 7 Days, in Treatment of Bacterial Community-Acquired Pneumonia in Adults. T. M. File Jr., 2004.This randomized, double-blind, noninferiority trial was designed to demonstrate that pharmacokinetically enhanced amoxicillin-clavulanate (2,000/125 mg) was at least as effective clinically as amoxicillin-clavulanate 875/125 mg, both given twice daily for 7 days, in the treatment of community-acquired pneumonia in adults . In total, 633 clinically and radiologically confirmed community-acquired pneumonia patients (intent-to-treat population) were randomized to receive either oral amoxicillin-clavulanate 2,000/125 mg (n = 322) or oral amoxicillin-clavulanate 875/125 mg (n = 311) . At screening, 160 of 633 (25.3%) patients had at least one typical pathogen isolated from expectorated or invasive sputum samples or blood culture (bacteriology intent-to-treat population) . Streptococcus pneumoniae (58 of 160, 36.3%), methicillin-susceptible Staphylococcus aureus (34 of 160, 21.3%), and Haemophilus influenzae (33 of 160, 20.6%) were the most common typical causative pathogens isolated in both groups in the bacteriology intent-to-treat population . Clinical success in the clinical per protocol population at test of cure (days 16 to 37), the primary efficacy endpoint, was 90.3% (223 of 247) for amoxicillin-clavulanate 2,000/125 mg and 87.6% (198 of 226) for amoxicillin-clavulanate 875/125 mg (treatment difference, 2.7; 95% confidence interval, –3.0, 8.3) . Bacteriological success at test of cure in the bacteriology per protocol population was 86.6% (58 of 67) for amoxicillin-clavulanate 2,000/125 mg and 78.4% (40 of 51) for amoxicillin-clavulanate 875/125 mg (treatment difference, 8.1%; 95% confidence interval, –5.8, 22.1) . Both therapies were well tolerated . Amoxicillin-clavulanate 2,000/125 mg twice daily was shown to be as clinically effective as amoxicillin-clavulanate 875/125 mg twice daily for 7 days in the treatment of adult patients with community-acquired pneumonia, without a noted increase in the reported rate of adverse events . Enzymatic Synthesis of High-Molecular-Mass Poly-  -Glutamate and Regulation of Its Stereochemistry.Makoto Ashiuchi, 2004.For the first time, we succeeded in synthesizing in vitro poly- -glutamate (PGA) with high molecular masses (>1,000 kDa) by the use of enzyme-associated cell membranes from Bacillus subtilis subsp . chungkookjang . The activity for PGA synthesis, however, was readily lost in the presence of critical concentrations of detergents tested in micelles . The optimum pH for the reaction was found to be
 7.0 . We examined the effects of some divalent cations on PGA synthesis and found that Mg2+ was essential in catalysis and that Zn2+ additionally boosted the activity . In contrast, Fe2+ and Ca2+ acted as inhibitors . Mn2+ did not apparently influence the in vitro formation of PGA . DL-Glutamate (D isomer content, 60 to 80%) apparently served as the best substrate; D-Glutamate was preferable to the L isomer as a substrate . When D- and L-glutamate were used for the reaction, the elongated chains of PGAs were composed of the D- and L-isomers, respectively . Our results suggest that the stereochemical properties of enzymatically synthesized PGAs substantially depend on the stereochemistry (DL ratio) of glutamate as the substrate . Furthermore, genetic analysis indicated that all the pgsB, -C, and -A gene products, which are responsible for PGA production by B . subtilis cells, were also indispensable for enzymatic PGA synthesis .
Inactivation of Cytochrome o Ubiquinol Oxidase Relieves Catabolic Repression of the Pseudomonas putida GPo1 Alkane Degradation Pathway. M. Alejandro Dinamarca, 2002.Expression of the alkane degradation pathway encoded by the OCT plasmid of Pseudomonas putida GPo1 is regulated by two control systems . One relies on the transcriptional regulator AlkS, which activates expression of the pathway in the presence of alkanes . The other, which is a dominant global regulation control, represses the expression of the pathway genes when a preferred carbon source is present in the growth medium in addition to alkanes . This catabolite repression control occurs through a poorly characterized mechanism that ultimately regulates transcription from the two AlkS-activated promoters of the pathway . To identify the factors involved, a screening method was developed to isolate mutants without this control . Several isolates were obtained, all of which contained mutations that mapped to genes encoding cytochrome o ubiquinol oxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions . Elimination of this terminal oxidase led to a decrease in the catabolic repression observed both in rich Luria-Bertani medium and in a defined medium containing lactate or succinate as the carbon source . This suggests that catabolic repression could monitor the physiological or metabolic status by using information from the electron transport chain or from the redox state of the cell . Since inactivation of the crc gene also reduces catabolic repression in rich medium (although not that observed in a defined medium), a strain was generated lacking both the Crc function and the cytochrome o terminal oxidase . The two mutations had an additive effect in relieving catabolic repression in rich medium . This suggests that crc and cyo belong to different regulation pathways, both contributing to catabolic repression . Transposition of DEH, a Broad-Host-Range Transposon Flanked by ISPpu12, in Pseudomonas putida Is Associated with Genomic Rearrangements and Dehalogenase Gene Silencing. Andrew J. Weightman, 2002.Pseudomonas putida strain PP3 produces two hydrolytic dehalogenases encoded by dehI and dehII, which are members of different deh gene families . The 9.74-kb DEH transposon containing dehI and its cognate regulatory gene, dehRI, was isolated from strain PP3 by using the TOL plasmid pWW0 . DEH was fully sequenced and shown to have a composite transposon structure, within which dehI and dehRI were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats . The flanking repeat unit, designated ISPpu12, had the structure of an insertion sequence in that it was bordered by 24-bp near-perfect inverted repeats and contained four open reading frames (ORFs), one of which was identified as tnpA, putatively encoding an ISL3 family transposase . A putative lipoprotein signal peptidase was encoded by an adjacent ORF, lspA, and the others, ISPpu12 orf1 and orf2, were tentatively identified as a truncated cation efflux transporter gene and a PbrR family regulator gene, respectively . The orf1-orf2 intergenic region contained an exact match with a previously described active, outward-orientated promoter, Pout . Transposition of DEH-ISPpu12 was investigated by cloning the whole transposon into a suicide plasmid donor, pAWT34, and transferring the construct to various recipients . In this way DEH-ISPpu12 was shown to transpose in a broad range of Proteobacteria . Transposition of ISPpu12 independently from DEH, and inverse transposition, whereby the vector DNA and ISPpu12 inserted into the target genome without the deh genes, were also observed to occur at high frequencies in P . putida PaW340 . Transposition of a second DEH-ISPpu12 derivative introduced exogenously into P . putida PP3 via the suicide donor pAWT50 resulted in silencing of resident dehI and dehII genes in about 10% of transposition transconjugants and provided a genetic link between transposition of ISPpu12 and dehalogenase gene silencing . Database searches identified ISPpu12-related sequences in several bacterial species, predominantly associated with plasmids and xenobiotic degradative genes . The potential role of ISPpu12 in gene silencing and activation, as well as the adaptation of bacteria to degrade xenobiotic compounds, is discussed . Antimicrobial-Resistant Campylobacter Species from Retail Raw Meats. Beilei Ge, 2003.The antimicrobial susceptibilities of 378 Campylobacter isolates were determined . Resistance to tetracycline was the most common (82%), followed by resistance to doxycycline (77%), erythromycin (54%), nalidixic acid (41%), and ciprofloxacin (35%) . Campylobacter coli displayed significantly higher rates of resistance to ciprofloxacin and erythromycin than Campylobacter jejuni, and Campylobacter isolates from turkey meat showed a greater resistance than those from chicken meat .
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