The
            <i>Bacillus subtilis ywjI</i>
            (
            <i>glpX</i>
            ) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme

Jules, Matthieu; Chat, Ludovic Le; Aymerich, Stéphane; Coq, Dominique Le

doi:10.1128/jb.01783-08

Cited by 19 publications

(25 citation statements)

References 25 publications

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“…Strain and Growth Conditions-All physiological and flux analysis experiments were performed with wild-type B. subtilis BSB168 trpϩ (20). For each growth experiment, frozen glycerol stocks were used to inoculate 5 ml of Luria-Bertani (LB) medium.…”

Section: Methodsmentioning

confidence: 99%

“…Expression Studies-Green fluorescent protein (GFP) reporter strains of glpF or dctP transcription were obtained by insertion of either a fragment containing the entire glpF regulatory region (from Ϫ271 to ϩ 130 relative to the transcription start) or the 3Ј end region of the dctP gene (from ϩ866 to ϩ1466 relative to the translation start) into the pBaSysBioII plasmid upstream of the promoter-less gfpmut3 gene (23) and then inserting the resulting plasmids into BSB168 strain (20) chromosome by single crossing-over recombination.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Kleijn

Buescher²,

Chat³

et al. 2010

Journal of Biological Chemistry

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103

110

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Commonly glucose is considered to be the only preferred substrate in Bacillus subtilis whose presence represses utilization of other alternative substrates. Because recent data indicate that malate might be an exception, we quantify here the carbon source utilization hierarchy. Based on physiology and transcriptional data during co-utilization experiments with eight carbon substrates, we demonstrate that malate is a second preferred carbon source for B. subtilis, which is rapidly co-utilized with glucose and strongly represses the uptake of alternative substrates. From the different hierarchy and degree of catabolite repression exerted by glucose and malate, we conclude that both substrates might act through different molecular mechanisms. To obtain a quantitative and functional network view of how malate is (co)metabolized, we developed a novel approach to metabolic flux analysis that avoids error-prone, intuitive, and ad hoc decisions on 13 C rearrangements. In particular, we developed a rigorous approach for deriving reaction reversibilities by combining in vivo intracellular metabolite concentrations with a thermodynamic feasibility analysis. The thus-obtained analytical model of metabolism was then used for network-wide isotopologue balancing to estimate the intracellular fluxes. These 13 C-flux data revealed an extraordinarily high malate influx that is primarily catabolized via the gluconeogenic reactions and toward overflow metabolism. Furthermore, a considerable NADPH-producing malic enzyme flux is required to supply the biosynthetically required NADPH in the presence of malate. Co-utilization of glucose and malate resulted in a synergistic decrease of the respiratory tricarboxylic acid cycle flux.Typically, microbes prefer a single carbon source whose presence represses utilization of alternative substrates. The classical example is the diauxic shift of Escherichia coli with subsequent growth on first glucose and then lactose (1). This widespread phenomenon, referred to as carbon catabolite repression, has been intensively studied in many chemoorganotrophic microbes, and in the vast majority of documented cases, the preferred carbon source is glucose (2, 3). Apart from repressing the co-utilization of alternative substrates, preferred carbon sources are normally associated with a high specific growth rate and substantial secretion of overflow metabolites such as ethanol, lactate, or acetate. For the Grampositive model bacterium Bacillus subtilis, glucose has long been recognized to be preferred, but recent data indicated that glucose might not be the only preferred carbon source (4, 5).According to the current model of the global carbon catabolite repression mechanism in B. subtilis, glucose-induced catabolite repression is mediated by the HPr kinase-catalyzed phosphorylation of HPr at its Ser-46 residue (2). This effect is propagated throughout the cell by the pleiotropic transcription factor CcpA (6), whose binding to the target sites for catabolite repression is controlled by the presence of HPr(Ser...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Kleijn

Buescher²,

Chat³

et al. 2010

Journal of Biological Chemistry

Self Cite

103

110

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show abstract

“…B. subtilis 168 and a phoPR null mutant (AH024, Howell et al, 2003) were used for transcriptome analysis. The B. subtilis tryptophan prototrophic BSB168 (Jules et al, 2009) and AH024 (DphoPR) were transformed using standard procedures (Anagnostopoulos & Spizizen, 1961). For transcriptome analysis and LCA experiments, B. subtilis strains were grown in a low-phosphate defined medium (LPDM) or in a high-phosphate defined medium (HPDM) (Müller et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

et al. 2011

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The high phosphate content of Bacillus subtilis cell walls dictates that cell wall metabolism is an important feature of the PhoPR-mediated phosphate limitation response. Here we report the expression profiles of cell-envelope-associated and PhoPR regulon genes, determined by live cell array and transcriptome analysis, in exponentially growing and phosphate-limited B. subtilis cells. Control by the WalRK two-component system confers a unique expression profile and high level of promoter activity on the genes of its regulon with yocH and cwlO expression differing both qualitatively and quantitatively from all other autolysin-encoding genes examined. The activity of the PhoPR two-component system is restricted to the phosphate-limited state, being rapidly induced in response to the cognate stimulus, and can be sustained for an extended phosphate limitation period. Constituent promoters of the PhoPR regulon show heterogeneous induction profiles and very high promoter activities. Phosphate-limited cells also show elevated expression of the actin-like protein MreBH and reduced expression of the WapA cell wall protein and WprA cell wall protease indicating that cell wall metabolism in this state is distinct from that of exponentially growing and stationary-phase cells. The PhoPR response is very rapidly deactivated upon removal of the phosphate limitation stimulus with concomitant increased expression of cell wall metabolic genes. Moreover expression of genes encoding enzymes involved in sulphur metabolism is significantly altered in the phosphate-limited state with distinct perturbations being observed in wild-type 168 and AH024 (DphoPR) cells.

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“…While FBPase II enzymes from E. coli (52), M. tuberculosis (50), Synechocystis sp. PCC6803 (25), B. subtilis (33), and C. glutamicum (42) have been biochemically characterized, the characteristics of a promiscuous FBPase/SBPase from a nonphotosynthetic bacterium lacking the Calvin cycle have not yet been determined. …”

Section: Bioinformatic Analysis and Phylogeny Of The Fbpases Glpxmentioning

confidence: 99%

“…Class I, II, and III FBPases are found primarily in bacteria, class IV is found primarily in archaea, and class V is found primarily in thermophiles (21,23). Some microorganisms possess more than one FBPase, mostly combinations of class I and II FBPases, as in E. coli (19), or class II and III FBPases, as found in B. subtilis (26,33). FBPases show a very close functional and structural relationship to SBPases (EC 3.1.3.37) (34).…”

mentioning

confidence: 99%

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

Stolzenberger¹,

Lindner

Persicke

et al. 2013

J Bacteriol

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The genome of the facultative ribulose monophosphate (RuMP) cycle methylotroph Bacillus methanolicus encodes two bisphosphatases (GlpX), one on the chromosome (GlpX C ) and one on plasmid pBM19 (GlpX P ), which is required for methylotrophy. Both enzymes were purified from recombinant Escherichia coli and were shown to be active as fructose 1,6-bisphosphatases (FBPases). The FBPase-negative Corynebacterium glutamicum ⌬fbp mutant could be phenotypically complemented with glpX C and glpX P from B. methanolicus. GlpX P and GlpX C share similar functional properties, as they were found here to be active as homotetramers in vitro, activated by Mn 2؉ ions and inhibited by Li ؉ , but differed in terms of the kinetic parameters. GlpX C showed a much higher catalytic efficiency and a lower K m for fructose 1,6-bisphosphate (86.3 s ؊1 mM ؊1 and 14 ؎ 0.5 M, respectively) than GlpX P (8.8 s ؊1 mM ؊1 and 440 ؎ 7.6 M, respectively), indicating that GlpX C is the major FBPase of B. methanolicus. Both enzymes were tested for activity as sedoheptulose 1,7-bisphosphatase (SBPase), since a SBPase variant of the ribulose monophosphate cycle has been proposed for B. methanolicus. The substrate for the SBPase reaction, sedoheptulose 1,7-bisphosphate, could be synthesized in vitro by using both fructose 1,6-bisphosphate aldolase proteins from B. methanolicus. Evidence for activity as an SBPase could be obtained for GlpX P but not for GlpX C . Based on these in vitro data, GlpX P is a promiscuous SBPase/FBPase and might function in the RuMP cycle of B. methanolicus.

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The Bacillus subtilis ywjI ( glpX ) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme

Cited by 19 publications

References 25 publications

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

Characterization of Fructose 1,6-Bisphosphatase and Sedoheptulose 1,7-Bisphosphatase from the Facultative Ribulose Monophosphate Cycle Methylotroph Bacillus methanolicus

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