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Growth Phase-Dependent Regulation and Stringent Control of fis Are Conserved Processes in Enteric Bacteria and Involve a Single Promoter (fis P) in Escherichia coli. Prabhat Mallik, 2004.The intracellular concentration of the Escherichia coli factor for inversion stimulation (Fis), a global regulator of transcription and a facilitator of certain site-specific DNA recombination events, varies substantially in response to changes in the nutritional environment and growth phase . Under conditions of nutritional upshift, fis is transiently expressed at very high levels, whereas under induced starvation conditions, fis is repressed by stringent control . We show that both of these regulatory processes operate on the chromosomal fis genes of the enterobacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris, strongly suggesting that the physiological role of Fis is closely tied to its transcriptional regulation in response to the nutritional environment . These transcriptional regulatory processes were previously shown to involve a single promoter (fis P) preceding the fis operon in E . coli . Recent work challenged this notion by presenting evidence from primer extension assays which appeared to indicate that there are multiple promoters upstream of fis P that contribute significantly to the expression and regulation of fis in E . coli . Thus, a rigorous analysis of the fis promoter region was conducted to assess the contribution of such additional promoters . However, our data from primer extension analysis, S1 nuclease mapping, ß-galactosidase assays, and in vitro transcription analysis all indicate that fis P is the sole E . coli fis promoter in vivo and in vitro . We further show how certain conditions used in the primer extension reactions can generate artifacts resulting from secondary annealing events that are the likely source of incorrect assignment of additional fis promoters . Rgg Coordinates Virulence Factor Synthesis and Metabolism in Streptococcus pyogenes. Michael S. Chaussee, 2003.Streptococcus pyogenes is a human-specific pathogen that relies on its host for metabolic substrates . Rgg-like proteins constitute a family of transcriptional regulators present in several gram-positive bacteria . In S . pyogenes, Rgg influences the expression of several virulence-associated proteins localized to the cell wall and extracellular environment . Secreted enzymes may degrade host macromolecules, thereby liberating metabolic substrates . To determine if Rgg regulation of exoprotein expression is associated with altered metabolism, the catabolic activities of S . pyogenes strain NZ131 (serotype M49) and an isogenic rgg mutant strain were analyzed during growth with complex and defined media . As expected, the wild-type strain preferentially used glucose and produced lactic acid during the exponential phase of growth . In contrast, the rgg mutant fermented arginine in the exponential phase of growth, even in the presence of glucose . Arginine degradation was associated with a neutral culture pH and excretion of NH3 and ornithine . Arginine, serine, and asparagine were depleted from mutant cultures during growth . The addition of arginine and serine to culture media increased the growth yield and NH3 production of mutant but not wild-type cultures . Addition of asparagine had no effect on the growth yield of either strain . Altered metabolism of arginine and serine in the mutant was associated with increased transcript levels of genes encoding arginine deiminase and a putative serine dehydratase . Thus, Rgg coordinates virulence factor synthesis and catabolic activity and may be important in the pathogen's adaptation to changes in the availability of metabolic substrates . Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae. Michael B. Cooley, 2003.Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce . Under appropriate conditions, these bacteria will grow on and invade the plant tissue . We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens . Under sterile conditions and at 100% humidity, S . enterica serovar Newport and E . coli O157:H7 grew to 109 CFU g-1 on A . thaliana roots and to 2 x 107 CFU g-1 on shoots . Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition . Inoculation with green fluorescent protein-labeled S . enterica and E . coli O157:H7 showed invasion of the roots at lateral root junctions . Movement was eliminated and invasion decreased when nonmotile mutants of S . enterica were used . Survival of S . enterica serovar Newport and E . coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days . Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil . Enterobacter asburiae was isolated from soil-grown A . thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions . Seed and chaff harvested from contaminated plants were occasionally contaminated . The rate of recovery of S . enterica and E . coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation . Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025) . This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique . However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat .
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