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Transcription Regulation of ezrA and Its Effect on Cell Division of Bacillus subtilis. Kuei-Min Chung, 2004.The EzrA protein of Bacillus subtilis is a negative regulator for FtsZ (Z)-ring formation . It is able to modulate the frequency and position of Z-ring formation during cell division . The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P . A . Levin, I . G . Kurster, and A . D . Grossman, Proc . Natl . Acad . Sci . USA 96:9642-9647, 1999) . We have studied the regulation of ezrA expression during the growth of B . subtilis and its effects on the intracellular level of EzrA as well as the cell length of B . subtilis . With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping  A-type
promoters, P1 and P2, located about 100 bp upstream of the initiation
codon of ezrA, have been identified . P1, supposed to be an
extended –10 promoter, was responsible for most of the ezrA
expression during the growth of B . subtilis . Disruption of
this promoter reduced the intracellular level of EzrA very
significantly compared with disruption of P2 . Moreover, deletion of
both promoters completely abolished EzrA in B . subtilis . More
importantly, the cell length and percentage of filamentous cells of
B . subtilis were significantly increased by disruption of the
promoter(s) . Thus, EzrA is required for efficient cell division
during the growth of B . subtilis, despite serving as a
negative regulator for Z-ring formation .
Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa Isolated from a Lung of a Patient with Cystic Fibrosis. Franz von Götz, 2004.The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms . In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P . aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population . Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins . This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type . The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS . Thus, the prevailing assumption that P . aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants . Mechanisms of Bactericidal Action of Cinnamaldehyde against Listeria monocytogenes and of Eugenol against L . monocytogenes and Lactobacillus sakei. Alexander O. Gill, 2004.The spice oil components eugenol and cinnamaldehyde possess activity against both gram-positive and gram-negative bacteria, but the mechanisms of action remain obscure . In broth media at 20°C, 5 mM eugenol or 30 mM cinnamaldehyde was bactericidal (>1-log reduction in the number of CFU per milliliter in 1 h) to Listeria monocytogenes . At a concentration of 6 mM eugenol was bactericidal to Lactobacillus sakei, but treatment with 0.5 M cinnamaldehyde had no significant effect . To investigate the role of interference with energy generation in the mechanism of action, the cellular and extracellular ATP levels of cells in HEPES buffer at 20°C were measured . Treatment of nonenergized L . monocytogenes with 5 mM eugenol, 40 mM cinnamaldehyde, or 10 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 5 min prevented an increase in the cellular ATP concentration upon addition of glucose . Treatment of energized L . monocytogenes with 40 mM cinnamaldehyde or 10 µM CCCP caused a rapid decline in cellular ATP levels, but 5 mM eugenol had no effect on cellular ATP . Treatment of L . sakei with 10 mM eugenol prevented ATP generation by nonenergized cells and had no effect on the cellular ATP of energized cells . CCCP at a concentration of 100 µM had no significant effect on the cellular ATP of L . sakei . No significant changes in extracellular ATP were observed . Due to their rapidity, effects on energy generation clearly play a major role in the activity of eugenol and cinnamaldehyde at bactericidal concentrations . The possible mechanisms of inhibition of energy generation are inhibition of glucose uptake or utilization of glucose and effects on membrane permeability . Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut. Satoko Noda, 2003.Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists . Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters . In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N . koshunensis . The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population . The absence of cluster II spirochetes in N . koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I . Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp . in N . koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes . The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes . The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment . Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species . The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists . Nitric Oxide Reductase (norB) Genes from Pure Cultures and Environmental Samples. Gesche Braker, 2003.A PCR-based approach was developed to recover nitric oxide (NO) reductase (norB) genes as a functional marker gene for denitrifying bacteria . norB database sequences grouped in two very distinct branches . One encodes the quinol-oxidizing single-subunit class (qNorB), while the other class is a cytochrome bc-type complex (cNorB) . The latter oxidizes cytochrome c, and the gene is localized adjacent to norC . While both norB types occur in denitrifying strains, the qnorB type was also found in a variety of nondenitrifying strains, suggesting a function in detoxifying NO . Branch-specific degenerate primer sets detected the two norB types in our denitrifier cultures . Specificity was confirmed by sequence analysis of the norB amplicons and failure to amplify norB from nondenitrifying strains . These primer sets also specifically amplified norB from freshwater and marine sediments . Pairwise comparison of amplified norB sequences indicated minimum levels of amino acid identity of 43.9% for qnorB and 38% for cnorB . Phylogenetic analysis confirmed the existence of two classes of norB genes, which clustered according to the respective primer set . Within the qnorB cluster, the majority of genes from isolates and a few environmental clones formed a separate subcluster . Most environmental qnorB clones originating from both habitats clustered into two distinct subclusters of novel sequences from presumably as yet uncultivated organisms . cnorB clones were located on separate branches within subclusters of genes from known organisms, suggesting an origin from similar organisms .
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