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In Vivo Phosphorylation of Partner Switching Regulators Correlates with Stress Transmission in the Environmental Signaling Pathway of Bacillus subtilis. Tae-Jong Kim, 2004.Exposure of bacteria to diverse growth-limiting stresses inducesthe synthesis of a common set of proteins which provide broadprotection against future, potentially lethal stresses . AmongBacillus subtilis and its relatives, this general stress responseis controlled by the  B
transcription factor . Signals of environmentaland energy stress
activate
B
through a multicomponent networkthat functions via a partner
switching mechanism, in which protein-proteininteractions are
governed by serine and threonine phosphorylation.Here, we tested a
central prediction of the current model forthe environmental
signaling branch of this network . We usedisoelectric focusing and
immunoblotting experiments to determinethe in vivo phosphorylation
states of the RsbRA and RsbS regulators,which act in concert to
negatively control the RsbU environmentalsignaling phosphatase . As
predicted by the model, the ratioof the phosphorylated to
unphosphorylated forms of both RsbRAand RsbS increased in response
to salt or ethanol stress . However,these two regulators differed
substantially with regard to theextent of their phosphorylation
under both steady-state andstress conditions, with RsbRA always the
more highly modified.Mutant analysis showed that the RsbT kinase,
which is requiredfor environmental signaling, was also required for
the in vivophosphorylation of RsbRA and RsbS . Moreover, the T171A
alterationof RsbRA, which blocks environmental signaling, also
blockedin vivo phosphorylation of RsbRA and impeded phosphorylation
of RsbS . These in vivo results corroborate previous genetic
analyses and link the phosphorylated forms of RsbRA and RsbSto the
active transmission of environmental stress signals.
CO2- and Anaerobiosis-Induced Changes in Physiology and Gene Expression of Different Listeria monocytogenes Strains. Anne-Marie Jydegaard-Axelsen, 2004.Although carbon dioxide (CO2) is known to inhibit growth of most bacteria, very little is known about the cellular response . The food-borne pathogen Listeria monocytogenes is characterized by its ability to grow in high CO2 concentrations at refrigeration temperatures . We examined the listerial responses of different strains to growth in air, 100% N2, and 100% CO2 . The CO2-induced changes in membrane lipid fatty acid composition and expression of selected genes were strain dependent . The acid-tolerant L . monocytogenes LO28 responded in the same manner to CO2 as to other anaerobic, slightly acidic environments (100% N2, pH 5.7) . An increase in the expression of the genes encoding glutamate decarboxylase (essential for survival in strong acid) as well as an increased amount of branched-chain fatty acids in the membrane was observed in both atmospheres . In contrast, the acid-sensitive L . monocytogenes strain EGD responded differently to CO2 and N2 at the same pH . In a separate experiment with L . monocytogenes 412, an increased isocitrate dehydrogenase activity level was observed for cells grown in CO2-containing atmospheres . Together, our findings demonstrate that the CO2-response is a partly strain-dependent complex mechanism . The possible links between the CO2-dependent changes in isocitrate dehydrogenase activity, glutamate metabolism and branched fatty acid biosynthesis are discussed . Genome-Wide Dynamic Transcriptional Profiling of the Light-to-Dark Transition in Synechocystis sp . Strain PCC 6803. Ryan T. Gill, 2002.We report the results of whole-genome transcriptional profiling of the light-to-dark transition with the model photosynthetic prokaryote Synechocystis sp . strain PCC 6803 (Synechocystis) . Experiments were conducted by growing Synechocystis cultures to mid-exponential phase and then exposing them to two cycles of light/dark conditions, during which RNA samples were obtained . These samples were probed with a full-genome DNA microarray (3,169 genes, 20 samples) as well as a partial-genome microarray (88 genes, 29 samples) . We concluded that (i) 30-min sampling intervals accurately captured transcriptional dynamics throughout the light/dark transition, (ii) 25% of the Synechocystis genes (783 genes) responded positively to the presence of light, and (iii) the response dynamics varied greatly for individual genes, with a delay of up to 120 to 150 min for some genes . Four classes of genes were identified on the basis of their dynamic gene expression profiles: class I (108 genes, 30-min response time), class II (279 genes, 60 to 90 min), class III (258 genes, 120 to 150 min), and class IV (138 genes, 180 min) . The dynamics of several transcripts from genes involved in photosynthesis and primary energy generation are discussed . Finally, we applied Fisher discriminant analysis to better visualize the progression of the overall transcriptional program throughout the light/dark transition and to determine those genes most indicative of the lighting conditions during growth . Whole-Genome DNA Microarray Analysis of a Hyperthermophile and an Archaeon: Pyrococcus furiosus Grown on Carbohydrates or Peptides. Gerrit J. Schut, 2003.The first complete-genome DNA microarray was constructed for a hyperthermophile or a nonhalophilic archaeon by using the 2,065 open reading frames (ORFs) that have been annotated in the genome of Pyrococcus furiosus (optimal growth temperature, 100°C) . This was used to determine relative transcript levels in cells grown at 95°C with either peptides or a carbohydrate (maltose) used as the primary carbon source . Approximately 20% (398 of 2065) of the ORFs did not appear to be significantly expressed under either growth condition . Of the remaining 1,667 ORFs, the expression of 125 of them (8%) differed by more than fivefold between the two cultures, and 82 of the 125 (65%) appear to be part of operons, indicating extensive coordinate regulation . Of the 27 operons that are regulated, 5 of them encode (conserved) hypothetical proteins . A total of 18 operons are up-regulated (greater than fivefold) in maltose-grown cells, including those responsible for maltose transport and for the biosynthesis of 12 amino acids, of ornithine, and of citric acid cycle intermediate products . A total of nine operons are up-regulated (greater than fivefold) in peptide-grown cells, including those encoding enzymes involved in the production of acyl and aryl acids and 2-ketoacids, which are used for energy conservation . Analyses of the spent growth media confirmed the production of branched-chain and aromatic acids during growth on peptides . In addition, six nonlinked enzymes in the pathways of sugar metabolism were regulated more than fivefold—three in maltose-grown cells that are unique to the unusual glycolytic pathway and three in peptide-grown cells that are unique to gluconeogenesis . The catalytic activities of 16 metabolic enzymes whose expression appeared to be highly regulated in the two cell types correlated very well with the microarray data . The degree of coordinate regulation revealed by the microarray data was unanticipated and shows that P . furiosus can readily adapt to a change in its primary carbon source . Oxygen Consumption by Anaerobic Saccharomyces cerevisiae under Enological Conditions: Effect on Fermentation Kinetics. Eric Rosenfeld, 2003.The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs) . A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated . We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth . However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period . Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects . As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively) . Both mutants exhibited normal anaerobic fermentation kinetics . However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate . In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions . Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis . These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics .
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