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Cysteinyl-tRNACys Formation in Methanocaldococcus jannaschii: the Mechanism Is Still Unknown. Benfang Ruan, 2004.Most organisms form Cys-tRNACys, an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS) . However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS . It was reported that M . jannaschii prolyl-tRNA synthetase (C . Stathopoulos, T . Li, R . Longman, U . C . Vothknecht, H . D . Becker, M . Ibba, and D . Söll, Science 287:479-482, 2000; R . S . Lipman, K . R . Sowers, and Y . M . Hou, Biochemistry 39:7792-7798, 2000) or the M . jannaschii MJ1477 protein (C . Fabrega, M . A . Farrow, B . Mukhopadhyay, V . de Crécy-Lagard, A . R . Ortiz, and P . Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNACys formation . These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene . Here we show that E . coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the Km for cysteine and a ninefold reduction in the kcat for ATP . While transformants of E . coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E . coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium . An E . coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D . radiodurans DR0705 gene . Construction of a D . radiodurans DR0705 deletion strain showed this gene to be dispensable . However, attempts to delete D . radiodurans cysS failed, suggesting that this is an essential Deinococcus gene . These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNACys synthesis in M . jannaschii . Thus, the mechanism of Cys-tRNACys formation in M . jannaschii still remains to be discovered . Oxygen Limitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosa in Biofilms. Giorgia Borriello, 2004.The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro . Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h . It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions . Oxygen penetrated about 50 µm into the biofilms, which averaged 210 µm thick . The region of active protein synthesis was visualized by using an inducible green fluorescent protein . This zone was also limited to a narrow band , approximately 30 µm wide, adjacent to the air interface of the biofilm . The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h–1 . These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state . In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air . When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically . Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested . Nitrate amendment stimulated the growth of untreated control P . aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics . Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P . aeruginosa biofilms causing infections in vivo . Adaptation of Escherichia coli O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid. Hyun-Gyun Yuk, 2004.The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E . coli (NPEC, ATCC 25922) . Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min . Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1  7c) in the membrane lipids of all strains . The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity . HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 108 CFU/ml . In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity . These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion .
The Streptomyces Genome Contains Multiple Pseudo-attB Sites for the  C31-Encoded Site-Specific Recombination System.Patricia Combes, 2002.The integrase from the Streptomyces phage C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of
 or its relatives . Moreover,
C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species .
C31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome . The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions . In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB . Strains of Streptomyces coelicolor and S . lividans were constructed with a deletion of the attB site ( attB), and pSET152 was introduced into these strains by conjugation . Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome . The sequences of the integration sites had similarity to those of attB . Analysis of the insertions of pSET152 into both attB+ and
attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant .
Molecular Characterization of Brucella abortus Chromosome II Recombination. Georgios Tsoktouridis, 2003.Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution . The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp . Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B . melitensis 16M and Brucella abortus 2308 . Analysis of 45 SSH clones revealed several deletions on chromosomes of B . abortus and B . melitensis that encoded proteins of various metabolic pathways . A 640-kb inversion on chromosome II of B . abortus has been reported previously (S . Michaux Charachon, G . Bourg, E . Jumas Bilak, P . Guigue Talet, A . Allardet Servent, D . O'Callaghan, and M . Ramuz, J . Bacteriol . 179:3244-3249, 1997) and is further described in this study . One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293 . The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp . As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region . The splitting of the PDEA gene most likely inactivated the function of this enzyme . A recombination mechanism responsible for this inversion is proposed .
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