Stefanie Müller-Altrock

Glycerol Metabolism and PrfA Activity in Listeria monocytogenes

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.

Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes

Analysis of Listeria monocytogenes ptsH, hprK, and ccpA mutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. The hprK mutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing medium whereas expression of these genes was even slightly down-regulated in the ccpA mutant compared to the wild-type strain. The ptsH mutant could only grow in a rich culture medium, and here the PrfA-dependent genes were up-regulated as in the hprK mutant. As expected, HPr-Ser-P was not produced in the hprK and ptsH mutants and synthesized at a similar level in the ccpA mutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity when L. monocytogenes was grown in minimal medium with different phosphotransferase system (PTS) carbohydrates. Comparison of the transcript profiles of the hprK and ccpA mutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in the hprK mutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that it is not the component(s) of the CCR or the common PTS pathway but, rather, the component(s) of subsequent steps that seem to be involved in the modulation of PrfA activity.

SigB-Dependent In Vitro Transcription of prfA and Some Newly Identified Genes of Listeria monocytogenes Whose Expression Is Affected by PrfA In Vivo

Recent studies have identified several new genes in Listeria monocytogenes which are positively or negatively affected by PrfA and grouped into three classes (E. Milohanic et al., Mol. Microbiol. 47:1613-1625, 2003). In vitro transcription performed with promoters of some class III genes showed strict SigB-dependent but PrfA-independent transcription initiation. Transcription starting at the prfA promoter PprfA2 was also optimal with SigB-loaded RNA polymerase, suggesting a direct link between SigB- and PrfA-dependent gene expression.

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

PrfA is the major transcriptional activator of most virulence genes of Listeria monocytogenes. Its activity is modulated by a variety of culture conditions. Here, we studied the PrfA activity in the L. monocytogenes wild-type strain EGD and an isogenic prfA deletion mutant (EGDDeltaprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDDeltaprfApPrfA and EGDDeltaprfApPrfA*) in response to growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented with one of the three phosphotransferase system (PTS) carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type strain in BHI and LB with all of these carbon sources, while PrfA activity was high in minimal medium in the presence of glycerol. EGDDeltaprfApPrfA*, expressing a large amount of PrfA* protein, showed high PrfA activity under all growth conditions. In contrast, strain EGDDeltaprfApPrfA, expressing an equally high amount of PrfA protein, showed high PrfA activity only when cultured in BHI, and not in LB or MM (in the presence of any of the carbon sources). A ptsH mutant (lacking a functional HPr) was able to grow in BHI but not in LB or MM, regardless of which of the four carbon sources was added, suggesting that in LB and MM the uptake of the used PTS carbohydrates and the catabolism of glycerol are fully dependent on the functional common PTS pathway. The BHI culture medium, in contrast, apparently contains carbon sources (supporting listerial growth) which are taken up and metabolized by L. monocytogenes independently of the common PTS pathway. The growth rates of L. monocytogenes were strongly reduced in the presence of large amounts of PrfA (or PrfA*) protein when growing in MM, but were less reduced in LB and only slightly reduced in BHI. The expression of the genes encoding the PTS permeases of L. monocytogenes was determined in the listerial strains under the applied growth conditions. The data obtained further support the hypothesis that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases.

Overexpression of PrfA Leads to Growth Inhibition of Listeria monocytogenes in Glucose-Containing Culture Media by Interfering with Glucose Uptake

Listeria monocytogenes strains expressing high levels of the virulence regulator PrfA (mutant PrfA* or wild-type PrfA) show strong growth inhibition in minimal media when they are supplemented with glucose but not when they are supplemented with glucose-6-phosphate compared to the growth of isogenic strains expressing low levels of PrfA. A significantly reduced rate of glucose uptake was observed in a PrfA*-overexpressing strain growing in LB supplemented with glucose. Comparative transcriptome analyses were performed with RNA isolated from a prfA mutant and an isogenic strain carrying multiple copies of prfA or prfA* on a plasmid. These analyses revealed that in addition to high transcriptional up-regulation of the known PrfA-regulated virulence genes (group I), there was less pronounced up-regulation of the expression of several phage and metabolic genes (group II) and there was strong down-regulation of several genes involved mainly in carbon and nitrogen metabolism in the PrfA*-overexpressing strain (group III). Among the latter genes are the nrgAB, gltAB, and glnRA operons (involved in nitrogen metabolism), the ilvB operon (involved in biosynthesis of the branched-chain amino acids), and genes for some ABC transporters. Most of the down-regulated genes have been shown previously to belong to a class of genes in Bacillus subtilis whose expression is negatively affected by impaired glucose uptake. Our results lead to the conclusion that excess PrfA (or PrfA*) interferes with a component(s) essential for phosphotransferase system-mediated glucose transport.

A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins.

Listeria ivanovii differs from the human pathogen Listeria monocytogenes in that it specifically affects ruminants, causing septicaemia and abortion but not meningo‐encephalitis. The genetic characterization of spontaneous L. ivanovii mutants lacking the virulence factor SmcL (sphingomyelinase) led us to identify LIPI‐2, the first species‐specific pathogenicity island from Listeria. Besides SmcL, this 22 kb chromosomal locus encodes 10 internalin (Inl) proteins: i‐InlB1 and ‐B2 are large/surface‐associated Inls similar to L. monocytogenes InlB; i‐InlE to –L are small/excreted (SE)‐Inls, i‐InlG being a tandem fusion of two SE‐Inls. Except i‐inlB1, all LIPI‐2 inl genes are controlled by the virulence regulator, PrfA. LIPI‐2 is inserted into a tRNA locus and is unstable – half of it deleting at ≈10−4 frequency with a portion of contiguous DNA. The spontaneous mutants were attenuated in vivo in mice and lambs and showed impaired intracellular growth and apoptosis induction in bovine MDBK cells. Targeted knock‐out mutations associated the virulence defect with LIPI‐2 genes. The region between the core genome loci ysnB‐tRNAarg and ydeI flanking LIPI‐2 contained different gene complements in the different Listeria spp. and even serovars of L. monocytogenes, including remnants of the PSA bacteriophage int gene in serovar 4b, indicating it is a hot spot for horizontal genome diversification. LIPI‐2 is conserved in L. ivanovii ssp. ivanovii and londoniensis, suggesting an early acquisition during the species’ evolution. LIPI‐2 is likely to play an important role in the pathogenic and host tropism of L. ivanovii.

In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA‐dependent and ‐independent promoters that are differentially activated by GTP

Most known virulence genes of Listeria monocytogenes are regulated by the transcriptional factor PrfA. Using our recently established in vitro transcription system, we have studied the PrfA‐dependent promoter (PinlC) regulating the expression of the small, secreted internalin C. PrfA‐dependent and PrfA‐independent transcription is observed starting from PinlC in vitro and in vivo, suggesting the presence of two apparently overlapping promoters both of which use the same −10 box. Although the PrfA‐dependent transcription requires, as expected, the PrfA‐box, PrfA‐independent transcription depends on a −35 box located directly downstream of the PrfA‐box. PrfA‐independent transcription starts at A, 7 bp downstream of the common −10 box (A7), and is strongly inhibited by PrfA because of the close proximity of the PrfA binding site to the −35 box. PrfA‐dependent transcription starts preferentially at G5 but, in the absence of this start nucleotide, alternative start sites at A positions 7 or 8 bp downstream of the −10 box can also be used. The −35 box of the PrfA‐independent promoter can be functionally inactivated without affecting PrfA‐dependent transcription as long as the distance between the PrfA‐box and the −10 box remains fixed to 22 (or 23) bp. Vice versa, the PrfA‐box can be deleted without affecting PrfA‐independent transcription from PinlC, which is no longer inhibited by PrfA. The PrfA‐dependent transcription initiation needs, in contrast to the PrfA‐independent one, the presence of a high concentration of GTP (and ATP) but not of CTP and UTP. Overlapping PrfA‐dependent and PrfA‐independent promoter activity was also demonstrated for the mpl promoter (Pmpl). Again, PrfA‐dependent transcription starting at Pmpl is dominant at high GTP concentration and PrfA‐independent transcription at low GTP. Here too, the PrfA‐dependent and the PrfA‐independent promoters share the same −10 box characteristic of SigA‐loaded RNA polymerase. High GTP concentration also appears to be necessary for transcription initiation at other PrfA‐dependent promoters (Phly, PactA) but not at the PrfA‐independent promoter PinlC‐m8.

Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes

PrfA, the master regulator of LIPI-1, is indispensable for the pathogenesis of the human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. PrfA is also present in the apathogenic species Listeria seeligeri, and in this study, we elucidate the differences between PrfA proteins from the pathogenic and apathogenic species of the genus Listeria. PrfA proteins of L. monocytogenes (PrfA(Lm) and PrfA*(Lm)), L. ivanovii (PrfA(Li)), and L. seeligeri (PrfA(Ls)) were purified, and their equilibrium constants for binding to the PrfA box of the hly promoter (Phly(Lm)) were determined by surface plasmon resonance. In addition, the capacities of these PrfA proteins to bind to the PrfA-dependent promoters Phly and PactA and to form ternary complexes together with RNA polymerase were analyzed in electrophoretic mobility shift assays, and their abilities to initiate transcription in vitro starting at these promoters were compared. The results show that PrfA(Li) resembled the constitutively active mutant PrfA*(Lm) more than the wild-type PrfA(Lm), whereas PrfA(Ls) showed a drastically reduced capacity to bind to the PrfA-dependent promoters Phly and PactA. In contrast, the efficiencies of PrfA(Lm), PrfA*(Lm), and PrfA(Li) forming ternary complexes and initiating transcription at Phly and PactA were rather similar, while those of PrfA(Ls) were also much lower. The low binding and transcriptional activation capacities of PrfA(Ls) seem to be in part due to amino acid exchanges in its C-terminal domain (compared to PrfA(Lm) and PrfA(Li)). In contrast to the significant differences in the biochemical properties of PrfA(Lm), PrfA(Li), and PrfA(Ls), the PrfA-dependent promoters of hly (Phly(Lm), Phly(L)(i), and Phly(L)(s)) and actA (PactA(Lm), PactA(L)(i), and PactA(L)(s)) of the three Listeria species did not significantly differ in their binding affinities to the various PrfA proteins and in their strengths to promote transcription in vitro. The allelic replacement of prfA(Lm) with prfA(Ls) in L. monocytogenes leads to low expression of PrfA-dependent genes and to reduced in vivo virulence of L. monocytogenes, suggesting that the altered properties of PrfA(Ls) protein are a major cause for the low virulence of L. seeligeri.

Supportive and inhibitory elements of a putative PrfA-dependent promoter in Listeria monocytogenes.

Elements essential for PrfA‐dependent transcription were analysed on two promoters of Listeria monocytogenes, the PrfA‐dependent promoter of the phospholipase gene plcA (PplcA) and a putative promoter of the aroA gene (ParoA2) which contains a similar PrfA‐binding site and a similar −10 box as PplcA but does not function as PrfA‐dependent promoter. We constructed a series of hybrid plcA‐aroA promoters by exchanging corresponding sequence elements of these two ‘promoters’. The results showed that the two critical elements of PrfA‐dependent promoters, the PrfA‐box and the −10 box, can be functionally exchanged as long as the distance in between is maintained to 22 or 23 bp. However, the interspace sequence and the sequence downstream of the −10 box of ParoA2 were strongly inhibitory for PrfA‐dependent transcription. A detailed analysis of these two sequences revealed that the RNA polymerase binding site being part of the actual in vivo and in vitro used aroA promoter (ParoA1) and a sequence immediately downstream of the putative −10 site, possibly blocking the formation of the open complex, were responsible for the inhibition of PrfA‐dependent transcription from ParoA2. Taking into consideration the lessons learned from this study we were able to construct a functional PrfA‐dependent aroA promoter.