scholarly journals Sinorhizobium meliloti Mutants Lacking Phosphotransferase System Enzyme HPr or EIIA Are Altered in Diverse Processes, Including Carbon Metabolism, Cobalt Requirements, and Succinoglycan Production

2008 ◽  
Vol 190 (8) ◽  
pp. 2947-2956 ◽  
Author(s):  
Catalina Arango Pinedo ◽  
Ryan M. Bringhurst ◽  
Daniel J. Gage
Keyword(s):  
Carbon Metabolism ◽  
Sinorhizobium Meliloti ◽  
Carbon Sources ◽  
Deletion Mutants ◽  
Symbiotic Relationship ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Carbon Utilization ◽  
Carbon Regulation ◽  
Extreme Sensitivity

ABSTRACT Sinorhizobium meliloti is a member of the Alphaproteobacteria that fixes nitrogen when it is in a symbiotic relationship. Genes for an incomplete phosphotransferase system (PTS) have been found in the genome of S. meliloti. The genes present code for Hpr and ManX (an EIIAMan-type enzyme). HPr and EIIA regulate carbon utilization in other bacteria. hpr and manX in-frame deletion mutants exhibited altered carbon metabolism and other phenotypes. Loss of HPr resulted in partial relief of succinate-mediated catabolite repression, extreme sensitivity to cobalt limitation, rapid die-off during stationary phase, and altered succinoglycan production. Loss of ManX decreased expression of melA-agp and lac, the operons needed for utilization of α- and β-galactosides, slowed growth on diverse carbon sources, and enhanced accumulation of high-molecular-weight succinoglycan. A strain with both hpr and manX deletions exhibited phenotypes similar to those of the strain with a single hpr deletion. Despite these strong phenotypes, deletion mutants exhibited wild-type nodulation and nitrogen fixation when they were inoculated onto Medicago sativa. The results show that HPr and ManX (EIIAMan) are involved in more than carbon regulation in S. meliloti and suggest that the phenotypes observed occur due to activity of HPr or one of its phosphorylated forms.

scholarly journals Characterization of a Two-Component Regulatory System That Regulates Succinate-Mediated Catabolite Repression in Sinorhizobium meliloti

2010 ◽  
Vol 192 (21) ◽  
pp. 5725-5735 ◽  
Author(s):  
Preston P. Garcia ◽  
Ryan M. Bringhurst ◽  
Catalina Arango Pinedo ◽  
Daniel J. Gage
Keyword(s):  
Catabolite Repression ◽  
Sinorhizobium Meliloti ◽  
Response Regulator ◽  
Energy State ◽  
Carbon Sources ◽  
Regulatory System ◽  
Dicarboxylic Acids ◽  
Deletion Mutants ◽  
State Monitoring ◽  
Secondary Carbon

ABSTRACT When they are available, Sinorhizobium meliloti utilizes C4-dicarboxylic acids as preferred carbon sources for growth while suppressing the utilization of some secondary carbon sources such as α- and β-galactosides. The phenomenon of using succinate as the sole carbon source in the presence of secondary carbon sources is termed succinate-mediated catabolite repression (SMCR). Genetic screening identified the gene sma0113 as needed for strong SMCR when S. meliloti was grown in succinate plus lactose, maltose, or raffinose. sma0113 and the gene immediately downstream, sma0114, encode the proteins Sma0113, an HWE histidine kinase with five PAS domains, and Sma0114, a CheY-like response regulator lacking a DNA-binding domain. sma0113 in-frame deletion mutants show a relief of catabolite repression compared to the wild type. sma0114 in-frame deletion mutants overproduce polyhydroxybutyrate (PHB), and this overproduction requires sma0113. Sma0113 may use its five PAS domains for redox level or energy state monitoring and use that information to regulate catabolite repression and related responses.

scholarly journals Effects of Homologous Phosphoenolpyruvate-Carbohydrate Phosphotransferase System Proteins on Carbohydrate Uptake and Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha H16

2011 ◽  
Vol 77 (11) ◽  
pp. 3582-3590 ◽  
Author(s):  
Chlud Kaddor ◽  
Alexander Steinbüchel
Keyword(s):  
Ralstonia Eutropha ◽  
Insertion Site ◽  
Carbon Sources ◽  
Sugar Transport ◽  
Dry Weight ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Homologous Proteins ◽  
Content Type ◽  
Negative Effect

ABSTRACTSeven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome ofRalstonia eutrophaH16 byin silicoanalysis. Except theN-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACBand H16 ΔnagFECmutants exhibited no growth when cultivated on fructose andN-acetylglucosamine, respectively. Furthermore, a transposon mutant with aptsM-ptsHinsertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mobinsertion.ptsM,ptsH, andptsIsingle, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384,frcACB, ornagFECgenes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of theptsMHIdeletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins inR. eutrophaH16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism.

scholarly journals Quorum Sensing Controls Exopolysaccharide Production in Sinorhizobium meliloti

2003 ◽  
Vol 185 (1) ◽  
pp. 325-331 ◽  
Author(s):  
Melanie M. Marketon ◽  
Sarah A. Glenn ◽  
Anatol Eberhard ◽  
Juan E. González
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Sinorhizobium Meliloti ◽  
Wild Type Strain ◽  
Homoserine Lactone ◽  
Symbiotic Relationship ◽  
Wild Type ◽  
Sensing System ◽  
Quorum Sensing System

ABSTRACT Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C16:1-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.

scholarly journals Roles of Phosphatidylethanolamine and of Its Several Biosynthetic Pathways inSaccharomyces cerevisiae

2001 ◽  
Vol 12 (4) ◽  
pp. 997-1007 ◽  
Author(s):  
Ruth Birner ◽  
Maria Bürgermeister ◽  
Roger Schneiter ◽  
Günther Daum
Keyword(s):  
Biochemical Analysis ◽  
Membrane Vesicle ◽  
Carbon Sources ◽  
Carboxypeptidase Y ◽  
Deletion Mutants ◽  
Wild Type ◽  
Inner Mitochondrial Membrane ◽  
Minimal Level ◽  
Delayed Maturation

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. Inpsd1Δ strains or cho1Δ strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

scholarly journals Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes

2015 ◽  
Vol 112 (51) ◽  
pp. 15630-15635 ◽  
Author(s):  
Cheng-Heng Liao ◽  
Lili Yao ◽  
Ya Xu ◽  
Wei-Bing Liu ◽  
Ying Zhou ◽  
...  
Keyword(s):  
Nitrogen Metabolism ◽  
Carbon Metabolism ◽  
Nitrogen Availability ◽  
Carbon Sources ◽  
Culture Conditions ◽  
Saccharopolyspora Erythraea ◽  
Phosphotransferase System ◽  
Potential Applications ◽  
Regulation Of Transport ◽  
Severe Growth

The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.

scholarly journals Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from Lactobacillus casei Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion

2000 ◽  
Vol 182 (9) ◽  
pp. 2582-2590 ◽  
Author(s):  
Valérie Dossonnet ◽  
Vicente Monedero ◽  
Monique Zagorec ◽  
Anne Galinier ◽  
Gaspar Pérez-Martínez ◽  
...  
Keyword(s):  
Catabolite Repression ◽  
Type Strain ◽  
Lactobacillus Casei ◽  
Wild Type Strain ◽  
Carbon Sources ◽  
Lag Phase ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Inducer Exclusion ◽  
In The Wild

ABSTRACT We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion ofmethyl β-d-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative inptsH mutants altered at Ser-46 of HPr and thehprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.

scholarly journals Autoregulation of Sinorhizobium meliloti exoR gene expression

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2092-2101 ◽  
Author(s):  
Hai-Yang Lu ◽  
Hai-Ping Cheng
Keyword(s):  
Sinorhizobium Meliloti ◽  
Carbon Sources ◽  
Regulatory System ◽  
Leguminous Plant ◽  
Wild Type ◽  
Plant Host ◽  
Active Sensor ◽  
Bacterial Exopolysaccharide ◽  
Membrane Bound ◽  
And Control

The successful nitrogen-fixing symbiosis between the Gram-negative soil bacterium Sinorhizobium meliloti and its leguminous plant host alfalfa (Medicago sativa) requires the bacterial exopolysaccharide succinoglycan. Succinoglycan and flagellum production, along with the ability to metabolize more than 20 different carbon sources and control the expression of a large number of S. meliloti genes, is regulated by the ExoR–ExoS/ChvI signalling pathway. The ExoR protein interacts with and suppresses the sensing activities of ExoS, the membrane-bound sensor of the ExoS/ChvI two-component regulatory system. Here we show that exoR expression is clearly upregulated in the absence of any functional ExoR protein. This upregulation was suppressed by the presence of the wild-type ExoR protein but not by a mutated ExoR protein lacking signal peptide. The levels of exoR expression could be directly modified in real time by changing the levels of total ExoR protein. The expression of exoR was also upregulated by the constitutively active sensor mutation exoS96, and blocked by two single mutations, exoS* and exoSsupA , in the ExoS sensing domain. Presence of the wild-type ExoS protein further elevated the levels of exoR expression in the absence of functional ExoR protein, and reversed the effects of exoS96, exoS* and exoSsupA mutations. Altogether, these data suggest that ExoR protein autoregulates exoR expression through the ExoS/ChvI system, allowing S. meliloti cells to maintain the levels of exoR expression based on the amount of total ExoR protein.

scholarly journals Mutations in Alternative Carbon Utilization Pathways in Candida albicans Attenuate Virulence and Confer Pleiotropic Phenotypes

2006 ◽  
Vol 6 (2) ◽  
pp. 280-290 ◽  
Author(s):  
Melissa A. Ramírez ◽  
Michael C. Lorenz
Keyword(s):  
Candida Albicans ◽  
Carbon Metabolism ◽  
Innate Immune System ◽  
Glyoxylate Cycle ◽  
Transcriptional Profiling ◽  
Carbon Sources ◽  
Innate Immune ◽  
Carbon Utilization ◽  
The Glyoxylate Cycle

ABSTRACT The interaction between Candida albicans and cells of the innate immune system is a key determinant of disease progression. Transcriptional profiling has revealed that C. albicans has a complex response to phagocytosis, much of which is similar to carbon starvation. This suggests that nutrient limitation is a significant stress in vivo, and we have shown that glyoxylate cycle mutants are less virulent in mice. To examine whether other aspects of carbon metabolism are important in vivo during an infection, we have constructed strains lacking FOX2 and FBP1, which encode key components of fatty acid β-oxidation and gluconeogenesis, respectively. As expected, fox2Δ mutants failed to utilize several fatty acids as carbon sources. Surprisingly, however, these mutants also failed to grow in the presence of several other carbon sources, whose assimilation is independent of β-oxidation, including ethanol and citric acid. Mutants lacking the glyoxylate enzyme ICL1 also had more severe carbon utilization phenotypes than were expected. These results suggest that the regulation of alternative carbon metabolism in C. albicans is significantly different from that in other fungi. In vivo, fox2Δ mutants show a moderate but significant reduction in virulence in a mouse model of disseminated candidiasis, while disruption of the glyoxylate cycle or gluconeogenesis confers a severe attenuation in this model. These data indicate that C. albicans often encounters carbon-poor conditions during growth in the host and that the ability to efficiently utilize multiple nonfermentable carbon sources is a virulence determinant. Consistent with this in vivo requirement, C. albicans uniquely regulates carbon metabolism in a more integrated manner than in Saccharomyces cerevisiae, such that defects in one part of the machinery have wider impacts than expected. These aspects of alternative carbon metabolism may then be useful as targets for therapeutic intervention.

Central carbon metabolism of Leishmania parasites

Parasitology ◽  
2010 ◽  
Vol 137 (9) ◽  
pp. 1303-1313 ◽  
Author(s):  
ELEANOR C. SAUNDERS ◽  
DAVID P. DE SOUZA ◽  
THOMAS NADERER ◽  
MARIJKE F. SERNEE ◽  
JULIE E. RALTON ◽  
...  
Keyword(s):  
Carbon Metabolism ◽  
Drug Targets ◽  
Carbon Sources ◽  
Central Carbon Metabolism ◽  
Mammalian Host ◽  
Insect Vector ◽  
Carbon Utilization ◽  
Central Carbon ◽  
Host Infection

SUMMARYLeishmania spp. are sandfly-transmitted protozoa parasites that cause a spectrum of diseases in humans. Many enzymes involved in Leishmania central carbon metabolism differ from their equivalents in the mammalian host and are potential drug targets. In this review we summarize recent advances in our understanding of Leishmania central carbon metabolism, focusing on pathways of carbon utilization that are required for growth and pathogenesis in the mammalian host. While Leishmania central carbon metabolism shares many features in common with other pathogenic trypanosomatids, significant differences are also apparent. Leishmania parasites are also unusual in constitutively expressing most core metabolic pathways throughout their life cycle, a feature that may allow these parasites to exploit a range of different carbon sources (primarily sugars and amino acids) rapidly in both the insect vector and vertebrate host. Indeed, recent gene deletion studies suggest that mammal-infective stages are dependent on multiple carbon sources in vivo. The application of metabolomic approaches, outlined here, are likely to be important in defining aspects of central carbon metabolism that are essential at different stages of mammalian host infection.

scholarly journals AniA Regulates Reserve Polymer Accumulation and Global Protein Expression in Rhizobium etli

2002 ◽  
Vol 184 (8) ◽  
pp. 2287-2295 ◽  
Author(s):  
Sergio Encarnación ◽  
María del Carmen Vargas ◽  
Michael F. Dunn ◽  
Araceli Dávalos ◽  
Guillermo Mendoza ◽  
...  
Keyword(s):  
Protein Expression ◽  
Gene Product ◽  
Sinorhizobium Meliloti ◽  
Carbon Sources ◽  
Proteome Analysis ◽  
Extracellular Polysaccharide ◽  
Oxidative Capacity ◽  
Nucleotide Sequencing ◽  
Rhizobium Etli ◽  
Wild Type

ABSTRACT Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::ΩSmr/Spr mutant CAR1, which is unable to synthesize poly-β-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes β-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.

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