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The RecA Protein of Helicobacter pylori Requires a Posttranslational Modification for Full Activity. Wolfgang Fischer, 2004.The RecA protein is a central component of the homologous recombination machinery and of the SOS system in most bacteria . In performing these functions, it is involved in DNA repair processes and plays an important role in natural transformation competence . This may be especially important in Helicobacter pylori, where an unusually high degree of microdiversity among strains is generated by homologous recombination . We have suggested previously that the H . pylori RecA protein is subject to posttranslational modifications that result in a slight shift in its electrophoretic mobility . Here we show that at least two genes downstream of recA are involved in this modification and that this process is dependent on genes involved in glycosylation and lipopolysaccharide biosynthesis . Site-directed mutagenesis of a putative glycosylation site results in production of an unmodified RecA protein . This posttranslational modification is not involved in membrane targeting or cell division functions but is necessary for the full function of RecA in DNA repair . Thus, it might be an adaptation to the specific requirements of H . pylori in its natural environment . Characterization of an rRNA Operon (rrnB) of Mycobacterium fortuitum and Other Mycobacterial Species: Implications for the Classification of Mycobacteria. M. C. Menendez, 2002.Mycobacteria are thought to have either one or two rRNA operons per genome . All mycobacteria investigated to date have an operon, designated rrnA, located downstream from the murA gene . We report that Mycobacteriun fortuitum has a second rrn operon, designated rrnB, which is located downstream from the tyrS gene; tyrS is very close to the 3" end of a gene (3-mag) coding for 3-methylpurine-DNA-glycosylase . The second rrn operon of Mycobacterium smegmatis was shown to have a similar organization, namely, 5" 3-mag-tyrS-rrnB 3" . The rrnB operon of M . fortuitum was found to have a single dedicated promoter . During exponential growth in a rich medium, the rrnB and rrnA operons were the major and minor contributors, respectively, to pre-rRNA synthesis . Genomic DNA was isolated from eight other fast-growing mycobacterial species . Samples were investigated by Southern blot analysis using probes for murA, tyrS, and 16S rRNA sequences . The results revealed that both rrnA and rrnB operons were present in each species . The results form the basis for a proposed new scheme for the classification of mycobacteria . The approach, which is phylogenetic in concept, is based on particular properties of the rrn operons of a cell, namely, the number per genome and a feature of 16S rRNA gene sequences . A Novel IS Element, IS621, of the IS110/IS492 Family Transposes to a Specific Site in Repetitive Extragenic Palindromic Sequences in Escherichia coli. Sunju Choi, 2003.An Escherichia coli strain, ECOR28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome . This insertion sequence (named IS621) has one large open reading frame encoding a putative protein that is 326 amino acids in length . A computer-aided homology search using the DNA sequence as the query revealed that IS621 was homologous to the piv genes, encoding pilin gene invertase (PIV) . A homology search using the amino acid sequence of the putative protein encoded by IS621 as the query revealed that the protein also has partial homology to transposases encoded by the IS110/IS492 family elements, which were known to have partial homology to PIV . This indicates that IS621 belongs to the IS110/IS492 family but is most closely related to the piv genes . In fact, a phylogenetic tree constructed on the basis of amino acid sequences of PIV proteins and transposases revealed that IS621 belongs to the piv gene group, which is distinct from the IS110/IS492 family elements, which form several groups . PIV proteins and transposases encoded by the IS110/IS492 family elements, including IS621, have four acidic amino acid residues, which are conserved at positions in their N-terminal regions . These residues may constitute a tetrad D-E(or D)-D-D motif as the catalytic center . Interestingly, IS621 was inserted at specific sites within repetitive extragenic palindromic (REP) sequences at 10 loci in the ECOR28 genome . IS621 may not recognize the entire REP sequence in transposition, but it recognizes a 15-bp sequence conserved in the REP sequences around the target site . There are several elements belonging to the IS110/IS492 family that also transpose to specific sites in the repeated sequences, as does IS621 . IS621 does not have terminal inverted repeats like most of the IS110/IS492 family elements . The terminal sequences of IS621 have homology with the 26-bp inverted repeat sequences of pilin gene inversion sites that are recognized and used for inversion of pilin genes by PIV . This suggests that IS621 initiates transposition through recognition of their terminal regions and cleavage at the ends by a mechanism similar to that used for PIV to promote inversion at the pilin gene inversion sites . Effect of Plasterboard Composition on Stachybotrys chartarum Growth and Biological Activity of Spores. Timo Murtoniemi, 2003.The effects of plasterboard composition on the growth and sporulation of Stachybotrys chartarum as well as on the inflammatory potential of the spores were studied . S . chartarum was grown on 13 modified plasterboards under saturated humidity conditions . The biomass was estimated by measuring the ergosterol content of the S . chartarum culture while the spore-induced cytotoxicity and production of nitric oxide (NO), tumor necrosis factor alpha (TNF-  ), and interleukin-6 in mouse macrophages was used to illustrate the bioactivity of spores . The ergosterol content of S . chartarum correlated with the number of spores collected from plasterboards . The growth and sporulation decreased compared to that of the reference board in those cases where (i) the liner was treated with biocide, (ii) starch was removed from the plasterboard, or (iii) desulfurization gypsum was used in the core . Spores collected from all the plasterboards were toxic to the macrophages . The biocide added to the core did not reduce the growth; in fact, the spores collected from that board evoked the highest cytotoxicity . The conventional additives used in the core had inhibitory effects on growth . Recycled plasterboards used in the core and the board lacking the starch triggered spore-induced TNF- production in macrophages . In summary, this study shows that the growth of a strain of S . chartarum on plasterboard and the subsequent bioactivity of spores were affected by minor changes to the composition of the core or liners, but it could not be totally prevented without resorting to the use of biocides . However, incomplete prevention of microbial growth by biocides even increased the cytotoxic potential of the spores .
Microaerophilic Cooperation of Reductive and Oxidative Pathways Allows Maximal Photosynthetic Membrane Biosynthesis in Rhodospirillum rubrum. Hartmut Grammel, 2003.The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions) . R . rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose . Employing a unique partial O2 pressure (pO2) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect . A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions . Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose . Fructose was utilized via the Embden-Meyerhof-Parnas pathway . High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures . No glucose-6-phosphate dehydrogenase activity was detected under any conditions . We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions . The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions . Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions . We propose a model by which the primary "switching" of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R . rubrum .
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