Glycerol Metabolism and PrfA Activity inListeria monocytogenes

Abstract

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.

Modulation of PrfA activity in Listeria monocytogenes upon growth in different culture media

“…PrfA activity is much higher in the presence of non-PTS carbon substrates such as glycerol (Joseph et al, 2008;Mertins et al, 2007). High PrfA activity is also observed in L. monocytogenes replicating inside mammalian host cells (Renzoni et al, 1999), where non-PTS carbon sources are used (Eylert et al, 2008;R.…”

“…Our recent data (Joseph et al, 2008;Mertins et al, 2007) rather suggest that the phosphorylation state of the PTS permeases required for the uptake of the above-mentioned carbohydrates seems to correlate with the PrfA activity. Based on these data we proposed a model (Joseph et al, 2008) which assumes that the unphosphorylated PTS permease (which is the principal state during active sugar transport, as the phosphate group is transferred to the sugar) may inhibit PrfA activity, possibly by binding directly to PrfA.…”

“…In the presence of high PrfA*, the expression of these genes was generally even slightly lower. With glycerol as carbon source, more up-and downregulation of genes for several PTSs was observed, which may reflect the fact that these PTS genes are under CCR control, and CCR is relieved to a large extent in L. monocytogenes grown in the presence of glycerol (Joseph et al, 2008).…”

“…Based on these data we proposed a model (Joseph et al, 2008) which assumes that the unphosphorylated PTS permease (which is the principal state during active sugar transport, as the phosphate group is transferred to the sugar) may inhibit PrfA activity, possibly by binding directly to PrfA. The phosphorylated PTS permease (the principal state in the absence of sugar transport) may either activate PrfA or release this transcription factor from the inactive PTS/PrfA complex, and the released PrfA becomes transcriptionally active.…”

Refinement of the Listeria monocytogenes σB regulon through quantitative proteomic analysis

“…lmo0398 and lmo0399 were previously proposed to play a role in s B -dependent regulation of carbohydrate metabolism during low nutrient energy stress conditions encountered during stationary phase (Chaturongakul et al, 2008). Interestingly, the gene encoding Lmo1539, a Group D protein involved in the uptake of glycerol, which L. monocytogenes can utilize as a carbon source for growth (Joseph et al, 2008;Premaratne et al, 1991), was found to be negatively regulated by s B in the 10403S microarray analyses conducted by Raengpradub et al (2008) and Oliver et al (2010) (FC50.2 and 0.5, respectively; P,0.01 in both studies), but was not found to be significantly differentially expressed in the microarray comparison of a prfA* and prfA*DsigB strain (Ollinger et al, 2009). However, lmo1539 was found to be upregulated during intracellular replication (Chatterjee et al, 2006) and in a s B -dependent manner in the intestine (Toledo-Arana et al, 2009) using L. monocytogenes strain EGD-e, during stationary phase stress using L. monocytogenes strains FSL J1-194, FSL J2-071 and FSL J1-208 (Oliver et al, 2010), and showed evidence for positive regulation by s B using L. monocytogenes strain 10403S when FPSS (fluorophenylstyrene sulfonamide) was used to inhibit s B activation (Palmer et al, 2011).…”