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VirB1 Orthologs from Brucella suis and pKM101 Complement Defects of the Lytic Transglycosylase Required for Efficient Type IV Secretion from Agrobacterium tumefaciens. Christoph Höppner, 2004.Type IV secretion systems mediate conjugative plasmid transferas well as the translocation of virulence factors from various gram-negative pathogens to eukaryotic host cells . The translocation apparatus consists of 9 to 12 components, and the componentsfrom different organisms are believed to have similar functions.However, orthologs to proteins of the prototypical type IV system,VirB of Agrobacterium tumefaciens, typically share only 15 to30% identical amino acids, and functional complementation between components of different type IV secretion systems has not been achieved . We here report a heterologous complementation in thecase of A . tumefaciens virB1 defects with its orthologs from Brucella suis [VirB1s] and the IncN plasmid pKM101 [TraL] . In contrast, expression of the genes encoding the VirB1 orthologsfrom the IncF plasmid [open reading frame 169] and from the Helicobacter pylori cag pathogenicity island [HP0523] did not complement VirB1 functions . The complementation of VirB1 activitywas assessed by T-pilus formation, by tumor formation on woundedplants, by IncQ plasmid transfer, and by IncQ plasmid recipientassay . Replacement of the key active-site Glu residue by Alaabolished the complementation by VirB1 from B . suis and by TraL, demonstrating that heterologous complementation requires anintact lytic transglycosylase active site . In contrast, theVirB1 active-site mutant from A . tumefaciens retained considerable residual activity in various activity assays, implying thatthis protein exerts additional effects during the type IV secretion process. Effects of an Antibiotic Cycling Program on Antibiotic Prescribing Practices in an Intensive Care Unit. Liana R. Merz, 2004.Various interventions have been proposed to combat the increase of antibiotic resistance and influence antibiotic prescribing practices . A prospective cohort study in a medical intensive care unit was conducted to determine the effect of an antibiotic cycling program on patterns of antibiotic use and to determine patient factors associated with cycling adherence . Four major classes of antibiotics for empirical therapy of suspected gram-negative bacterial infections were rotated at 3- and 4-month intervals . During the study, 1,003 patients received antibiotic therapy with at least one of the study drugs; of the 792 receiving cycle antibiotics during the cycling period, 598 (75.5%) received an on-cycle drug . Compared to the baseline, cycling recommendations increased the use of the target cycle agent: the use of cephalosporins increased during cycle 1 (56 to 64% of total antibiotic days, P < 0.001), fluoroquinolone use increased in cycle 2 (24 to 55%, P < 0.001), carbapenem use increased during cycle 3 (14 to 38%, P < 0.001), and use of extended-spectrum penicillins increased in cycle 4 (5 to 36%, P < 0.001) . Overall, 48% of total cycle antibiotic days were compliant with the cycling protocol . On average, 8.8 days per patient were spent receiving on-cycle drugs (range, 1 to 109) . Cycle periods that specified carbapenem and fluoroquinolone use had the highest number of off-cycle days (62 and 64%) . Predictors of on-cycle antibiotic use were increased severity of illness, as measured by an acute physiology and chronic health evaluation II score, and greater length of intensive care unit stay . In conclusion, the successful implementation of this cycling protocol increased antibiotic heterogeneity over time in the study unit . Noninvasive Pigment Identification in Single Cells from Living Phototrophic Biofilms by Confocal Imaging Spectrofluorometry. M. Roldán, 2004.A new imaging technique for the analysis of fluorescent pigments from a single cell is reported . It is based on confocal scanning laser microscopy coupled with spectrofluorometric methods . The setup allows simultaneous establishment of the relationships among pigment analysis in vivo, morphology, and three-dimensional localization inside thick intact microbial assemblages . Cinnamate:Coenzyme A Ligase from the Filamentous Bacterium Streptomyces coelicolor A3(2). Masafumi Kaneko, 2003.4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins . Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2) . The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity . The optimum pH and temperature of the reaction were pH 8.0 and 30°C, respectively . The Km value for 4-coumarate and kcat were determined as 131 ± 4 µM and 0.202 ± 0.007 s-1, respectively . The Km value was comparable to those of plant 4CLs . The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs . The enzyme efficiently converted cinnamate (Km, 190 ± 2 µM; kcat, 0.475 ± 0.012 s-1), which is a very poor substrate for plant 4CLs . Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs . The enzyme was therefore named ScCCL for S . coelicolor A3(2) cinnamate CoA ligase . To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated . Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala294 and Ala318 are involved in substrate recognition . Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme . Biodiversity, Community Structural Shifts, and Biogeography of Prokaryotes within Antarctic Continental Shelf Sediment. John P. Bowman, 2003.16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region . Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions) . The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs) . At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes . At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria . Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core . Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment .
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