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Phosphorylation of the Lipid A Region of Meningococcal Lipopolysaccharide: Identification of a Family of Transferases That Add Phosphoethanolamine to Lipopolysaccharide. Andrew D. Cox, 2003.A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined . Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria . When grown and isolated under appropriate conditions, N . meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues . In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue . Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A . Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species . We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A." RelA Protein Is Involved in Induction of Genetic Competence in Certain Bacillus subtilis Strains by Moderating the Level of Intracellular GTP. Takashi Inaoka, 2002.We found that the ability to develop genetic competence of a certain relaxed (relA) aspartate-auxotrophic strain of Bacillus subtilis is significantly lower than that of the isogenic stringent (relA+) strain . Transcriptional fusion analysis utilizing a lacZ reporter gene indicated that the amount of the ComK protein, known as the key protein for competence development, is greatly reduced in the relaxed strain than in the stringent strain . We also found that the addition of decoyinine, a GMP synthetase inhibitor, induces expression of a competence gene (comG) in the relaxed strain, accompanied by a pronounced decrease in the level of intracellular GTP as measured by high-performance liquid chromatography . The transformation efficiency of the relaxed strain increased 100-fold when decoyinine was added at t0 (the transition point between exponential to stationary growth phase) . Conversely, supplementation of guanosine together with decoyinine completely abolished the observed effect of adding decoyinine on competence development . Furthermore, the impaired ability of the relaxed strain for competence development was completely restored by disrupting the codY gene, which is known to negatively control comK expression . Our results indicate that the RelA protein plays an essential role in the induction of competence development at least under certain physiological conditions by reducing the level of intracellular GTP and overcoming CodY-mediated regulation . Transcription of Quorum-Sensing System Genes in Clinical and Environmental Isolates of Pseudomonas aeruginosa. Ségolène Cabrol, 2003.Quorum sensing (QS)-based transcriptional responses in Pseudomonas aeruginosa have been defined on the basis of increases in transcript levels of QS-controlled genes such as lasB and aprA following the hierarchical transcriptional increases of central controllers such as the lasR gene . These increases occur at high bacterial concentrations such as early-stationary-phase growth in vitro . However, the extent to which the increases occur in a variety of clinical and environmental isolates has not been determined nor is there extensive information on allelic variation in lasR genes . An analysis of the sequences of the lasR gene among 66 clinical and environmental isolates showed that 81% have a sequence either identical to that of strain PAO1 or with a silent mutation, 15% have nucleotide changes resulting in amino acid changes, and 5% have an insertion sequence in the lasR gene . Using real-time PCR to quantify transcript levels of lasR, lasB, and aprA in the early log and early stationary phases among 35 isolates from bacteremia and pneumonia cases and the environment, we found most (33 of 35) strains had increases in lasR transcripts in early stationary phase but with a very wide range of final transcript levels per cell . There was a strong correlation (r2 = 0.84) between early-log- and early-stationary-phase transcript levels in all strains, but this finding remained true only for the 50% of strains above the median level of lasR found in early log phase . There were significant (P < 0.05) but weak-to-modest correlations of lasR transcript levels with aprA (r2 = 0.2) and lasB (r2 = 0.5) transcript levels, but again this correlation occurred only in the 50% of P . aeruginosa strains with the highest levels of lasR transcripts in early stationary phase . There were no differences in distribution of lasR alleles among the bacteremia, pneumonia, or environmental isolates . Overall, only about 50% of P . aeruginosa strains from clinical and environmental sources show a lasR-dependent increase in the transcription of aprA and lasB genes, indicating that for about 50% of clinical isolates this regulatory system may not play a significant role in pathogenesis .
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