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Cell Density-Dependent Gene Contributes to Efficient Seed Colonization by Pseudomonas putida KT2440. Manuel Espinosa-Urgel, 2004.We have characterized the expression pattern of a gene, ddcA, involved in initial colonization of corn seeds by Pseudomonas putida KT2440 . The ddcA gene codes for a putative membrane polypeptide belonging to a family of conserved proteins of unknown function . Members of this family are widespread among prokaryotes and include the products of a Salmonella enterica serovar Typhimurium gene expressed during invasion of macrophages and psiE, an Escherichia coli phosphate starvation-inducible gene . Although its specific role is undetermined, the presence of ddcA in multicopy restored the seed adhesion capacity of a KT2440 ddcA mutant . Expression of ddcA is growth phase regulated, being maximal at the beginning of stationary phase . It is independent of RpoS, nutrient depletion, or phosphate starvation, and it is not the result of changes in the medium pH during growth . Expression of ddcA is directly dependent on cell density, being also stimulated by the addition of conditioned medium and of seed exudates . This is the first evidence suggesting the existence of a quorum-sensing system in P . putida KT2440 . The potential implication of such a signaling process in seed adhesion and colonization by the bacterium is discussed . RtsA and RtsB Coordinately Regulate Expression of the Invasion and Flagellar Genes in Salmonella enterica Serovar Typhimurium. Craig D. Ellermeier, 2003.Salmonella enterica serovar Typhimurium encounters numerous host environments and defense mechanisms during the infection process . The bacterium responds by tightly regulating the expression of virulence genes . We identified two regulatory proteins, termed RtsA and RtsB, which are encoded in an operon located on an island integrated at tRNAPheU in S . enterica serovar Typhimurium . RtsA belongs to the AraC/XylS family of regulators, and RtsB is a helix-turn-helix DNA binding protein . In a random screen, we identified five RtsA-regulated fusions, all belonging to the Salmonella pathogenicity island 1 (SPI1) regulon, which encodes a type III secretion system (TTSS) required for invasion of epithelial cells . We show that RtsA increases expression of the invasion genes by inducing hilA expression . RtsA also induces expression of hilD, hilC, and the invF operon . However, induction of hilA is independent of HilC and HilD and is mediated by direct binding of RtsA to the hilA promoter . The phenotype of an rtsA null mutation is similar to the phenotype of a hilC mutation, both of which decrease expression of SPI1 genes approximately twofold . We also show that RtsA can induce expression of a SPI1 TTSS effector, slrP, independent of any SPI1 regulatory protein . RtsB represses expression of the flagellar genes by binding to the flhDC promoter region . Repression of the positive activators flhDC decreases expression of the entire flagellar regulon . We propose that RtsA and RtsB coordinate induction of invasion and repression of motility in the small intestine . Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism. Christine M. Beatty, 2003.The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation . Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2 . In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by "focusing" RNA polymerase to acsP2 . We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism . Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II . Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II . The locations of these DNA sites for CRP (centered at positions -69.5 and -122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions . In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the  subunit of RNA polymerase holoenzyme (-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the
-CTD . Other surface-exposed residues in the
-CTD contributed to acs transcription, suggesting that the
-CTD may interact with at least one protein other than CRP .
Formation of 4-Hydroxy-2,5-Dimethyl-3[2H]-Furanone by Zygosaccharomyces rouxii: Identification of an Intermediate. Tobias Hauck, 2003.The formation of the important flavor compound 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDMF; Furaneol) from D-fructose-1,6-bisphosphate by the yeast Zygosaccharomyces rouxii was studied with regard to the identification of intermediates present in the culture medium . Addition of o-phenylenediamine, a trapping reagent for  -dicarbonyls, to the culture medium and subsequent analysis by high-pressure liquid chromatography with diode array detection revealed the formation of three quinoxaline derivatives derived from D-fructose-1,6-bisphosphate under the applied growth conditions (30°C; pH 4 to 5) . Isolation and characterization of these compounds by tandem mass spectrometry and nuclear magnetic resonance spectroscopy led to the identification of phosphoric acid mono-(2,3,4-trihydroxy-4-quinoxaline-2-yl-butyl) ester (Q1), phosphoric acid mono-[2,3-dihydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q2), and phosphoric acid mono-[2-hydroxy-3-(3-methyl-quinoxaline-2-yl)-propyl] ester (Q3) . Q1 and Q2 were formed independently of Z . rouxii cells, whereas Q3 was detected only in incubation systems containing the yeast . Identification of Q2 demonstrated for the first time the chemical formation of 1-deoxy-2,3-hexodiulose-6-phosphate in the culture medium, a generally expected but never identified intermediate in the formation pathway of HDMF . Since HDMF was detected only in the presence of Z . rouxii cells, additional enzymatic steps were presumed . Incubation of periplasmic and cytosolic protein extracts obtained from yeast cells with D-fructose-1,6-bisphosphate led to the formation of HDMF, implying the presence of the required enzymes in both extracts .
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