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1.
The gene cluster adjacent to the sequence of rpoN (encoding sigma factor sigma54) of Pseudomonas putida has been studied with respect to the C source regulation of the Pu promoter of the upper TOL (toluene catabolism) operon. The region includes four open reading frames (ORFs), two of which (named ptsN and ptsO genes) encode proteins similar to components of the phosphoenolpyruvate:sugar phosphotransferase system. Each of the four genes was disrupted with a nonpolar insertion, and the effects in the inhibition caused by glucose on Pu activity were inspected with a lacZ reporter system. Although cells lacking ORF102, ORF284, and ptsO did not display any evident phenotype under the conditions tested, the loss of ptsN, which encodes the IIANtr protein, made Pu unresponsive to repression by glucose. The ptsN mutant had rates of glucose/gluconate consumption identical to those of the wild type, thus ruling out indirect effects mediated by the transport of the carbohydrate. A site-directed ptsN mutant in which the conserved phospho-acceptor site His68 of IIANtr was replaced by an aspartic acid residue made Pu blind to the presence or absence of glucose, thus supporting the notion that phosphorylation of IIANtr mediates the C source inhibition of the promoter. These data substantiate the existence of a molecular pathway for co-regulation of some sigma54 promoters in which IIANtr is a key protein intermediate.  相似文献   

2.
The genome of Pseudomonas putida KT2440 encodes only five recognizable proteins belonging to the phosphoenolpyruvate (PEP)-carbohydrate phosphotransferase system (PTS). Two of these PTS constituents (FruA and FruB) form a complete system for fructose intake. The other three products, encoded by ptsP (EI(Ntr)), ptsO (NPr), and ptsN (EIIA(Ntr)), comprise a branch of the system unrelated to sugar traffic but thought to have an influence on coordination of N and C metabolism. We used a genetic approach to clarify the course of high-energy phosphate through this reduced set of PTS proteins. To this end, we monitored the phosphorylation state in vivo of the EIIA(Ntr) enzyme in various genetic backgrounds and growth conditions. Our results show that the source of phosphate available to the system is PEP and that the primary flow of phosphate through the N/C-sensing PTS proceeds from PEP to EI(Ntr) to NPr to EIIA(Ntr). We also found that in the presence of fructose, unlike in the presence of succinate, EIIA(Ntr) can be phosphorylated in a ptsP strain but not in a ptsP fruB double mutant. This result revealed that the fructose transport system has the ability to cross talk in vivo with the N-related PTS branch. The data reported here thus document an unexpected connection in vivo between the sugar-dependent and sugar-independent PTSs.  相似文献   

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While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen-metabolic phosphotransferase system (nitrogen-metabolic PTS). To elucidate the physiological role of the nitrogen-metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K-12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen-metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIA(Ntr)) mutant became extremely sensitive to leucine-containing peptides (LCPs), while both ptsP (encoding enzyme I(Ntr)) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho-form of enzyme IIA(Ntr) was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho-form of enzyme IIA(Ntr) is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched-chain amino acids.  相似文献   

5.
The genome of Pseudomonas putida KT2440 encodes five proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system. Two of these (FruA and FruB) form a dedicated system for fructose intake, while enzyme I(Ntr) (EI(Ntr); encoded by ptsP), NPr (ptsO), and EII(Ntr) (ptsN) act in concert to control the intracellular accumulation of polyhydroxyalkanoates, a typical product of carbon overflow.  相似文献   

6.
The ptsN gene of Pseudomonas putida encodes IIA(Ntr), a protein of the phosphoenol pyruvate:sugar phosphotransferase (PTS) system which is required for the C source inhibition of the sigma(54)-dependent promoter Pu of the TOL (toluate degradation) plasmid pWW0. Using two-dimensional gel electrophoresis, we have examined the effect of ptsN disruption on the general expression pattern of P. putida. To this end, cells were grown in the presence or absence of glucose, and a 1,117-spot subset of the P. putida proteome was used as a reference for comparisons. Among all gene products whose expression was lowered by this carbon source (247 spots [about 22%]), only 6 behaved as Pu (i.e., were depressed in the ptsN background). This evidenced only a minor role for IIA(Ntr) in the extensive inhibition of gene expression in P. putida caused by glucose. However, the same experiments revealed a large incidence of glucose-independent effects brought about by the ptsN mutation. As many as 108 spots (ca. 9% of the cell products analyzed) were influenced, positively or negatively, by the loss of IIA(Ntr). By matching this pattern with that of an rpoN::OmegaKm strain of P. putida, which lacks the sigma(54) protein, we judge that most proteins whose expression was affected by ptsN were unrelated to the alternative sigma factor. These data suggest a role of IIA(Ntr) as a general regulator, independent of the presence of repressive carbon sources and not limited to sigma(54)-dependent genes.  相似文献   

7.
The cellular levels of the alternative sigma factor sigma(54) of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of sigma(54) were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the sigma(54)-dependent Pu promoter of the TOL plasmid. Our results show that approximately 80 +/- 26 molecules of sigma(54) exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of sigma(54) determine adaptation and differentiation when cells face starvation, sigma(54) in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of sigma(54) molecules per cell in P. putida is barely above the predicted number of sigma(54)-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other sigma(54)-dependent systems) may become amenable to physiological control.  相似文献   

8.
9.
The activity of the toluene-responsive sigma(54) Pu promoter of the pWW0 TOL plasmid of Pseudomonas putida is down-regulated in vivo during exponential growth in rich medium and also by the presence of glucose in the culture. Although the Pu promoter already performs poorly during log growth in minimal medium when amended with casamino acids, the addition of glucose further decreased by two- to threefold the accumulation of beta-galactosidase in a Pu-lacZ reporter P. putida strain. Since Pu was still down-regulated during exponential growth regardless of glucose addition, it appeared that the carbohydrate separately influenced promoter activity. This notion was supported by the growth-dependent induction pattern of Pu in a ptsN mutant of P. putida, the loss of which makes Pu no longer responsive to repression by glucose. On the other hand, overexpression of the sigma factor sigma(54), known to partially alleviate the exponential silencing of the promoter, did not affect glucose inhibition of Pu. These data indicated that exponential silencing and carbon source-dependent repression are two overlapping but genetically distinguishable mechanisms that adapt Pu to the physiological status of the cells and nutrient availability.  相似文献   

10.
The nitrogen-related branch of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) of Pseudomonas putida includes the ptsN gene encoding the EIINtr (PtsN) enzyme. Although the implication of this protein in a variety of cellular functions has been observed in diverse bacteria, the physiological signals that bring about phosphorylation/dephosphorylation of the PtsN protein are not understood. This work documents the phosphorylation status of the EIINtr enzyme of P. putida at various growth stages in distinct media. Culture conditions were chosen to include fructose (the uptake of which is controlled by the PTS) or glucose (a non-PTS sugar in P. putida) in minimal medium with casamino acids, ammonia, or nitrate as alternative nitrogen sources. To quantify the relative ratio of PtsN/PtsN approximately P in live cells, we resorted to the in situ electrophoresis of whole bacteria expressing an E-epitope-tagged EIINtr followed by the fractionation of the thereby released native proteome in a non-denaturing gel. Although the PtsN species phosphorylated in amino acid His68 was detected under virtually all growth scenarios, the relative levels of the non-phosphorylated form varied dramatically depending on the growth phase and the nutrients available in the medium. The share of phosphorylated PtsN increased along growth in a fashion apparently independent of any trafficking of sugars. The large variations of non-phosphorylated PtsN in different growth conditions, in contrast to the systematic excess of the phosphorylated PtsN form, suggested that the P-free PtsN is the predominant signaling species of the protein.  相似文献   

11.
Bradyrhizobium japonicum transports oligopeptides and the heme precursor delta-aminolevulinic acid (ALA) by a common mechanism. Two Tn5-induced mutants disrupted in the lysC and ptsP genes were identified based on the inability to use prolyl-glycyl-glycine as a proline source and were defective in [(14)C]ALA uptake activity. lysC and ptsP were shown to be proximal genes in the B. japonicum genome. However, RNase protection and in trans complementation analysis showed that lysC and ptsP are transcribed separately, and that both genes are involved in oligopeptide transport. Aspartokinase, encoded by lysC, catalyzes the phosphorylation of aspartate for synthesis of three amino acids, but the lysC strain is not an amino acid auxotroph. The ptsP gene encodes Enzyme I(Ntr) (EI(Ntr)), a paralogue of Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. In vitro pull-down experiments indicated that purified recombinant aspartokinase and EI(Ntr) interact directly with each other. Expression of ptsP in trans from a multicopy plasmid complemented the lysC mutant, suggesting that aspartokinase normally affects Enzyme I(Ntr) in a manner that can be compensated for by increasing the copy number of the ptsP gene. ATP was not a phosphoryl donor to purified EI(Ntr), but it was phosphorylated by ATP in the presence of cell extracts. This phosphorylation was inhibited in the presence of aspartokinase. The findings demonstrate a role for a PTS protein in the transport of a non-sugar solute and suggest an unusual regulatory function for aspartokinase in regulating the phosphorylation state of EI(Ntr).  相似文献   

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14.
Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde-3-phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser-46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low-molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.  相似文献   

15.
16.
A promoter-like mutation, ptsP160, has been identified which drastically reduces expression of the genes specifying two proteins, HPr and enzyme I, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Salmonella typhimurium. This mutation lies between trzA, a gene specifying susceptibility to 1,2,4-triazole, and ptsH, the structural gene for HPr. It leads to a loss of active transport of those sugars that require the PTS for entry into the cell. Pseudorevertants of strains carrying this promoter-like mutation have additional lesions very closely linked to ptsP160 by transduction analysis and are noninducible for HPr and enzyme I above a basal level. Presumably, strains carrying ptsP160 are defective in the normal induction mechanism for HPr and enzyme I, and the pseudorevertants derived from them result from second-site initiation signals within or near this promoter-like element. The induction of HPr and enzyme I above their noninduced levels apparently is not required for transport of at least one PTS sugar, methyl alpha-d-glucopyranoside, since this sugar is taken up by the pseudorevertants at the same rate as by the wild type. The existence of a promoter-like element governing the coordinate inducibility of both HPr and enzyme I suggests that ptsH and ptsI constitute an operon. Wild-type levels of a sugar-specific PTS protein, factor III, are synthesized in response to the crr(+) gene in both a ptsP160 strain and its pseudorevertants; this suggests that the crr(+) gene has its own promoter distinct from ptsP.  相似文献   

17.
The nitrogen-related phosphotransferase system (Ntr-PTS) is a paralogous system working in parallel to the well-known carbohydrate:PTS. In a chain of phosphotransfer reactions, EINtr and NPr (PtsO) deliver phosphoryl groups to the EIIANtr (PtsN) protein. EIIANtr is implicated in important regulatory processes such as the σE-dependent cell envelope stress response and regulation of K+ uptake. Phosphorylation is believed to trigger the output of EIIANtr in these regulations. EIIANtr is encoded within the gene cluster ptsN–yhbJ–ptsO , which is highly conserved in Proteobacteria . In this study, we investigated the phosphorylation of the Escherichia coli EIIANtr protein in vivo by 32P-labeling. We show that EIIANtr is readily phosphorylated in wild-type cells. This phosphorylation occurs at a single site, the histidine 73 in EIIANtr. YhbJ and NPr are dispensable for this phosphorylation. A detailed analysis revealed that both the energy coupling phosphotransferases of the Ntr-PTS as well as the 'sugar'-PTS contribute to the phosphorylation of EIIANtr, suggesting cross talk between both systems.  相似文献   

18.
19.
We here tabulate and describe all currently recognized proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and their homologues encoded within the genomes of sequenced E. coli strains. There are five recognized Enzyme I homologues and six recognized HPr homologues. A nitrogen-metabolic PTS phosphoryl transfer chain encoded within the rpoN and ptsP operons and a tri-domain regulatory PTS protein encoded within the dha (dihydroxyacetone catabolic) operon, probably serve regulatory roles exclusively. In addition to several additional putative regulatory proteins, there are 21 (and possibly 22) recognized Enzyme II complexes. Of the 21 Enzyme II complexes, 7 belong to the fructose (Fru) family, 7 belong to the glucose (Glc) family, and 7 belong to the other PTS permease families. All of these proteins are briefly described, and phylogenetic data for the major families are presented.  相似文献   

20.
The Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS) in prokaryotes mediates the uptake and phosphorylation of its numerous substrates through a phosphoryl transfer chain where a phosphoryl transfer protein, HPr, transfers its phosphoryl group to any of several sugar-specific Enzyme IIA proteins in preparation for sugar transport. A phosphoryl transfer protein of the PTS, NPr, homologous to HPr, functions to regulate nitrogen metabolism and shows virtually no enzymatic cross-reactivity with HPr. Here we describe the genetic engineering of a "chimeric" HPr/NPr protein, termed CPr14 because 14 amino acid residues of the interface were replaced. CPr14 shows decreased activity with most PTS permeases relative to HPr, but increases activity with the broad specificity mannose permease. The results lead to the proposal that HPr is not optimal for most PTS permeases but instead represents a compromise with suboptimal activity for most PTS permeases. The evolutionary implications are discussed.  相似文献   

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