SummaryThe intracellular metabolism of Listeria monocytogenes was studied by 13 C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-
Listeria monocytogenes is able to efficiently utilize glycerol as a carbon source. In a defined minimal medium, the growth rate (during balanced growth) in the presence of glycerol is similar to that in the presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed high-level transcriptional upregulation of the genes known to be involved in glycerol uptake and metabolism (glpFK and glpD) in the presence of glycerol (compared to that in the presence of glucose and/or cellobiose). Levels of expression of the genes encoding a second putative glycerol uptake facilitator (GlpF 2 ) and a second putative glycerol kinase (GlpK 2 ) were less enhanced under these conditions. GlpK 1 but not GlpK 2 was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while the loss of GlpK 1 affected replication in Caco-2 cells less than did the loss of GlpK 2 and GlpD. Additional genes whose transcription levels were higher in the presence of glycerol than in the presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression control. Transcriptional downregulation in the presence of glycerol (compared to those in the presence glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N metabolism, and the biosynthesis of branched-chain amino acids. The highest level of transcriptional upregulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions, a low level of HPr-Ser-P and a high level of HPr-His-P were present in the cells, suggesting that all enzyme IIA (EIIA) (or EIIB) components of the phosphotransferase system (PTS) permeases expressed will be phosphorylated. These and other data suggest that the phosphorylation state of PTS permeases correlates with PrfA activity.Listeria monocytogenes is known as a facultative intracellular pathogen that can cause severe systemic infections in humans (for recent reviews, see references 15 and 47). This bacterial pathogen has therefore been extensively studied in the last decades preferentially with respect to its virulence genes and the encoded virulence factors. The virulence factors identified were shown to be involved mainly in the intracellular (cytosolic) growth cycle, and their genes were highly expressed under intracellular growth conditions (26). Most of the virulence genes are under the control of the transcription activator PrfA, whose expression is regulated at the transcriptional and the posttranscriptional levels (for recent reviews, see references 21 and 27). In addition, the activity of the PrfA protein is modulated by an as-yet-unknown factor(s) whose production appears to be linked to the metabolism of L. monocytogenes.
The genome of the gastric pathogen Helicobacter pylori harbors a remarkably low number of regulatory genes, including three and five open reading frames encoding two-component histidine kinases and response regulators, respectively, which are putatively involved in transcriptional regulation. Two of the response regulator genes, hp1043 and hp166, proved to be essential for cell growth, and inactivation of the response regulator gene hp1021 resulted in a severe growth defect, as indicated by a small-colony phenotype. The sequences of the receiver domains of response regulators HP1043 and HP1021 differ from the consensus sequence of the acidic pocket of the receiver domain which is involved in the phosphotransfer reaction from the histidine kinase to the response regulator. Using a genetic complementation system, we demonstrated that the function of response regulator HP166, which is essential for cell growth, can be provided by a mutated derivative carrying a D52N substitution at the site of phosphorylation. We found that the atypical receiver sequences of HP1043 and HP1021 are not crucial for the function of these response regulators. Phosphorylation of the receiver domains of HP1043 and HP1021 is not needed for response regulator function and may not occur at all. Thus, the phosphorylation-independent action of these regulators differs from the well-established two-component paradigm.
Helicobacter pylori is a human gastric pathogen which is extremely well adapted to its unique habitat. Crucial for the survival under the acidic conditions prevailing in the stomach is the enzyme urease. Transcriptome analysis has shown that transcription of a large number of genes responds to the exposure of H. pylori to acid including the genes encoding the urease subunits UreA and UreB as well as several genes which have been previously identified as target genes of the two-component system HP166-HP165. Here, we provide genetic evidence that a stimulus perceived by the histidine kinase HP165 is pH since increased transcription at acidic pH from the promoters controlling the expression of the ORFs hp119 and hp1432 which belong to the HP166-HP165 regulon is strictly dependent on the presence of histidine kinase HP165. Furthermore, we show that the basal transcription from the promoter of the ureA gene is modulated by the HP166-HP165 two-component system in response to acidic pH. On the other hand, the acid-induced increase in transcription of the promoter directing the expression of the orphan response regulator HP1021 is not controlled by the HP166-HP165 two-component system, nor is it mediated by HP1021 itself.
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