Regulatory links between carbon and nitrogen metabolism.

The metabolism of carbon- and nitrogen-containing compounds is fundamental to all forms of life. To cope with changing environmental conditions, bacteria have to sense the nutrient supply and adapt their metabolism accordingly. In addition to nutrient- and pathway-specific responses, they integrate information from the different branches of metabolism to coordinate the control of the expression of many metabolic genes. Two major players interconnecting carbon and nitrogen regulation are the PII proteins and the phosphotransferase system. Moreover, several DNA-binding transcription regulators sense signals are derived from both carbon and nitrogen metabolism. The regulatory networks enable the bacteria to make the appropriate metabolic responses to changing nutrient availabilities in the environment.

How seryl-phosphorylated HPr inhibits PrfA, a transcription activator of Listeria monocytogenes virulence genes.

Listeria monocytogenes PrfA, a transcription activator for several virulence genes, including the hemolysin-encoding hly, is inhibited by rapidly metabolizable carbon sources (glucose, fructose, etc.). This inhibition is not mediated via the major carbon catabolite repression mechanism of gram-positive bacteria, since inactivation of the catabolite control protein A (CcpA) did not prevent the repression of virulence genes by the above sugars. In order to test whether the catabolite co-repressor P-Ser-HPr might be involved in PrfA regulation, we used a Bacillus subtilis strain (BUG1199) containing L. monocytogenes prfA under control of pspac and the lacZ reporter gene fused to the PrfA-activated hly promoter. Formation of P-Ser-HPr requires the bifunctional HPr kinase/phosphorylase (HprK/P), which, depending on the concentration of certain metabolites, either phosphorylates HPr at Ser-46 or dephosphorylates P-Ser-HPr. The hprKV267F allele codes for an HprK/P leading to the accumulation of P-Ser-HPr, since it has normal kinase, but almost no phosphorylase activity. Interestingly, introducing hprKV267F into BUG1199 strongly inhibited transcription activation by PrfA. Preventing the accumulation of P-Ser-HPr in the hprKV267F mutant by replacing Ser-46 in HPr with an alanine restored PrfA activity, while ccpA inactivation had no effect. Interestingly, disruption of ccpA in the hprK wild-type strain BUG1199 also led to inhibition of PrfA. The lowered lacZ expression in the ccpA strain is probably also due to elevated amounts of P-Ser-HPr, since it disappeared when Ser-46 in HPr was replaced with an alanine. To carry out its catalytic function in sugar transport, HPr of the phosphotransferase system (PTS) is also phosphorylated by phosphoenolpyruvate and enzyme I at His-15. However, P-Ser-HPr is only very slowly phosphorylated by enzyme I, which probably accounts for PrfA inhibition. In agreement with this concept, disruption of the enzyme I- or HPr-encoding genes also strongly inhibited PrfA activity. PrfA activity therefore seems to depend on a fully functional PTS phosphorylation cascade.

Isolation of lacZ fusions to Proteus mirabilis genes regulated by intercellular signaling: potential role for the sugar phosphotransferase (Pts) system in regulation.

Using a mini-Tn5lacZ1 reporter transposon, lacZ fusions have been identified in Proteus mirabilis that are activated by the accumulation of self-produced extracellular signals. Genes identified by this approach include putative homologs of pgm, nlpA and two genes of unknown function. The extracellular signal(s) involved in activation were resistant to the effects of acid and alkali. The signal required for activation of (nlpA) cma482::lacZ was sensitive to protease, suggesting the signal is a peptide or small protein. The signals behaved as polar molecules and were not extractable with ethyl acetate. A mini-Tn5Cm insertion was identified in a probable ptsI homolog that blocked activation of the cma134::lacZ fusion by an extracellular signal. The ptsI mutation did not alter extracellular signal production and may have a role in signal response.

Bacillus subtilis mutant LicT antiterminators exhibiting enzyme I- and HPr-independent antitermination affect catabolite repression of the bglPH operon.

The Bacillus subtilis antiterminator LicT regulates the expression of bglPH and bglS, which encode the enzymes for the metabolism of aryl-beta-glucosides and the beta-glucanase BglS. The N-terminal domain of LicT (first 55 amino acids) prevents the formation of rho-independent terminators on the respective transcripts by binding to target sites overlapping these terminators. Proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulate the antitermination activity of LicT by phosphorylating histidines in its two PTS regulation domains (PRDs). Phosphorylation at His-100 in PRD-1 requires the PTS proteins enzyme I and HPr and the phosphorylated permease BglP and inactivates LicT. During transport and phosphorylation of aryl-beta-glucosides, BglP is dephosphorylated, which renders LicT active and thus leads to bglPH and bglS induction. In contrast, phosphorylation at His-207 and/or His-269 in PRD-2, which requires only enzyme I and HPr, is absolutely necessary for LicT activity and bglPH and bglS expression. We isolated spontaneous licT mutants expressing bglPH even when enzyme I and HPr were absent (as indicated by the designation "Pia" [PTS-independent antitermination]). Introduced in a ptsHI(+) strain, two classes of licT(Pia) mutations could be distinguished. Mutants synthesizing LicT(Pia) antiterminators altered in PRD-2 still required induction by aryl-beta-glucosides, whereas mutations affecting PRD-1 caused constitutive bglPH expression. One of the two carbon catabolite repression (CCR) mechanisms operative for bglPH requires the rho-independent terminator and is probably prevented when LicT is activated by P approximately His-HPr-dependent phosphorylation in PRD-2 (where the prefix "P approximately " stands for "phospho"). During CCR, the small amount of P approximately His-HPr present in cells growing on repressing PTS sugars probably leads to insufficient phosphorylation at PRD-2 of LicT and therefore to reduced bglPH expression. In agreement with this concept, mutants synthesizing a P approximately His-HPr-independent LicT(Pia) had lost LicT-modulated CCR.

Potential virulence role of the Legionella pneumophila ptsP ortholog.

We previously identified the Legionella pneumophila ptsP (phosphoenolpyruvate phosphotransferase) ortholog gene as a putative virulence factor in a study of signature-tagged mutagenesis using a guinea pig pneumonia model. In this study, we further defined the phenotypic properties of L. pneumophila ptsP and its complete sequence. The L. pneumophila ptsP was 2,295 bases in length. Its deduced amino acid sequence had high similarity with ptsP orthologs of Pseudomonas aeruginosa, Azotobacter vinelandii, and Escherichia coli, with nearly identical lengths. Here we show that while the mutant grew well in laboratory media, it was defective in both lung and spleen multiplication in guinea pigs. It grew slowly in guinea pig alveolar macrophages despite good uptake into the cells. Furthermore, there was minimal growth in a human alveolar epithelial cell line (A549). Transcomplementation of the L. pneumophila ptsP mutant almost completely rescued its growth in alveolar macrophages, in A549 cells, and in guinea pig lung and spleen. The L. pneumophila ptsP mutant was capable of evasion of phagosome-lysosome fusion and resided in ribosome-studded phagosomes. Pore formation activity of the mutant was normal. The L. pneumophila ptsP mutant expressed DotA and IcmX in apparently normal amounts, suggesting that the ptsP mutation did not affect dotA and icmX regulation. In addition, the mutant was resistant to serum and neutrophil killing. Taken together, these findings show that L. pneumophila ptsP is required for full in vivo virulence of L. pneumophila, most probably by affecting intracellular growth.

Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation.

The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon. LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine-containing protein (HPr). We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr. The phosphoryl group transfer between HPr and LicT is reversible. Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide-urea gels. These bands probably correspond to one-, two- and threefold phosphorylated LicT. After phosphorylation of LicT with [32P]-PEP, enzyme I and HPr, proteolytic digestion of [32P]-P-LicT, separation of the peptides by reverse-phase chromatography, mass spectrometry and N-terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT. These three histidyl residues (His-159, His-207 and His-269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators. Phosphorylation of LicT in the presence of serylphosphorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylation in the presence of HPr. The slower phosphorylation in the presence of P-Ser-HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon.

Control of glucose metabolism by the enzymes of the glucose phosphotransferase system in Salmonella typhimurium.

The quantitative role of the phosphoenolpyruvate:glucose phosphotransferase system (glucose phosphotransferase system) in glucose uptake and metabolism, and phosphotransferase-system-mediated regulation of glycerol uptake, was studied in vivo in Salmonella typhimurium. Expression plasmids were constructed which contained the genes encoding enzyme I (ptsI), HP (ptsH), IIAGlc (crr), and IICBGlc (ptsG) of the glucose phosphotransferase system behind inducible promoters. These plasmids allowed the controlled expression of each of the glucose phosphotransferase system proteins from about 30% to about 300% of its wild-type level. When enzyme I, HPr or IIAGlc were modulated between 30% and 300% of their wild-type value, hardly any effects on the growth rate on glucose, the glucose oxidation rate, the rate of methyl alpha-D-glucopyranoside (a glucose analog) uptake or the phosphotransferase-system-mediated inhibition of glycerol uptake by methyl alpha-D-glucopyranoside were observed. Employing the method of metabolic control analysis, it was shown that the enzyme flux control coefficients of these phosphotransferase system components on the different measured processes were close to zero. The enzyme flux control coefficient of IICBGlc on growth on glucose or glucose oxidation was also close to zero. In contrast, the enzyme flux control coefficient of IICBGlc on the flux through the glucose phosphotransferase system (transport and phosphorylation) was 0.72. The experimentally determined enzyme flux control coefficients allowed us to calculate the flux control coefficients of the phosphoenolpyruvate/pyruvate and methyl alpha-D-glucopyranoside/methyl alpha-D-glucopyranoside 6-phosphate couples and the process control coefficients of the phosphotransfer reactions of the glucose phosphotransferase system. We discuss the implications of these values and the possible control points in the glucose phosphotransferase system.

Mechanism of catabolite repression of tryptophanase synthesis in Escherichia coli.

Repression of tryptophanase (tryptophan indole-lyase) by glucose and its non-metabolizable analogue methyl alpha-glucoside has been studied employing a series of isogenic strains of Escherichia coli lacking cyclic AMP phosphodiesterase and altered for two of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), Enzyme I and Enzyme IIAGlc. Basal activity of tryptophanase was depressed mildly by inclusion of glucose in the growth medium, but inducible tryptophanase synthesis was subject to strong glucose repression in the parental strain, which exhibited normal PTS enzyme activities. Methyl alpha-glucoside was without effect in this strain. Loss of Enzyme I decreased sensitivity to repression by glucose but enhanced sensitivity to repression by methyl alpha-glucoside. Loss of Enzyme IIAGlc activity largely abolished repression by methyl alpha-glucoside but had a less severe effect on glucose repression. The repressive effects of both sugars were fully reversed by inclusion of cyclic AMP in the growth medium. Tryptophan uptake under the same conditions was inhibited weakly by glucose and more strongly by methyl alpha-glucoside in the parental strain. Inhibition by both sugars was alleviated by partial loss of Enzyme I. Inhibition by methyl alpha-glucoside appeared to be largely due to energy competition and was not responsible for repression of tryptophanase synthesis. Measurement of net production of cyclic AMP as well as intracellular concentrations of cyclic AMP revealed a good correlation with intensity of repression. The results suggest that while basal tryptophanase synthesis is relatively insensitive to catabolite repression, inducible synthesis is subject to strong repression by two distinct mechanisms, one dependent on enzyme IIAGlc of the PTS and the other independent of this protein. Both mechanisms are attributable to depressed rates of cyclic AMP synthesis. No evidence for a cyclic-AMP-independent mechanism of catabolite repression was obtained.