Among the few regulatory proteins encoded by Mycoplasma pneumoniae is HPr kinase/phosphatase (HPrK/P), the key regulator of carbon metabolism in low-GC Gram-positive bacteria. The corresponding gene, hprK, and the gene encoding the target protein HPr, ptsH, were overexpressed. In vitro analysis of the purified proteins confirmed ATP-dependent phosphorylation of HPr by HPrK/P. In contrast to HPrK/P of Bacillus subtilis, which is by default a phosphatase and needs high ATP concentrations for kinase activity, the M. pneumoniae enzyme exhibits kinase activity at very low ATP concentrations and depends on P i for phosphatase activity. This inverted control of enzymic activity may result from the adaptation to very different ecological niches. While the standard activities of HPrK/P from M. pneumoniae and other Grampositive bacteria differ, they are both modulated by the concentration of ATP, P i and glycolytic intermediates. Site-directed mutagenesis of a potential ATPbinding site and of the HPrK/P signature sequence resulted in four different activity classes : (i) inactive proteins, (ii) enzymes with reduced kinase and phosphatase activities, (iii) enzymes that had lost phosphatase, but not kinase activity, and (iv) enzymes that exhibited increased phosphatase activity.
The Bacillus subtilis two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (pgi,pfk, fbaA, and pykA) is not affected by the glucose supply, there is an activation of the glycolytic gap gene and the pgk operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the ccpAmutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by theccpA mutation. Our data suggest (i) that ccpAmutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.
The Bacillus subtilis sacY and sacT genes encode antiterminator proteins, similar to the Escherichia coli bglG gene product and required for transcription of sucrose metabolism genes. A Tn10 insertion into bglP (formerly sytA) has been previously identified as restoring sucrose utilization to a strain with deletions of both sacY and sacT. The nucleotide sequence of bglP showed a high degree of homology with the E. coli bglF gene (BglF is a -glucoside permease of the phosphotransferase system and also acts as a negative regulator of the BglG antiterminator). Complementation studies of an E. coli strain with a deletion of the bgl operon showed that BglP was a functional -glucoside permease. In B. subtilis, bglP complemented in trans both the bglP::Tn10 original insertion and a phenotypically similar bglP deletion. Disruption of licT abolished the suppressor phenotype in a bglP mutant. LicT is a recently identified third B. subtilis antiterminator of the BglG/SacY family. These observations indicated that BglP was also a negative regulator of LicT. Both LicT and BglP seem to be involved in the induction by -glucosides of an operon containing at least two genes, bglP itself and bglH, encoding a phospho--glucosidase. Other -glucoside genes homologous to bglP and bglH have been recently described in B. subtilis. Thus, B. subtilis possesses several sets of -glucoside genes, like E. coli, but these genes do not appear to be cryptic.The products of the Bacillus subtilis sacPA operon mediate transport and hydrolysis of sucrose. Mutants affected in sacPA, or in the sacT gene which encodes an antiterminator protein (AT) involved in sacPA induction by sucrose, are impaired for utilization of this sugar (8,12). This defect is not absolute because B. subtilis can also assimilate sucrose through an extracellular pathway involving the exoenzyme levansucrase encoded by sacB (35). This gene is also induced by sucrose through an antitermination mechanism involving SacY, a second AT structurally similar to SacT, but fully activated only in the presence of a high sucrose concentration (Ͼ1%) (7). When activated, SacY can partially replace SacT for induction of sacPA (36); this appears to be due to the structural similarity of the two ATs and of their targets within the sacPA and sacB leader regions (4). The model for induction by sucrose of sacB (7) is similar to that proposed for induction by -glucosides of the Escherichia coli bgl operon (19, 25): an enzyme II bgl (BglF), a permease of the phosphotransferase system (PTS) specific for the inducer, inactivates the AT (BglG) by phosphorylating it, and in the presence of the inducer, its permeation and phosphorylation lead to the concomitant dephosphorylation of BglF and then to that of BglG. The activated (dephosphorylated) AT binds to a specific imperfect palindromic RNA target sequence (RAT sequence), which overlaps the 5Ј part of a regulatory terminator located in the leader region of the regulated gene. This binding stabilizes the RAT secondary structure and therefore preven...
To better understand cellular life, it is essential to decipher the contribution of individual components and their interactions. Minimal genomes are an important tool to investigate these interactions. Here, we provide a database of 105 fully annotated genomes of a series of strains with sequential deletion steps of the industrially relevant model bacterium Bacillus subtilis starting with the laboratory wild type strain B. subtilis 168 and ending with B. subtilis PG38, which lacks approximately 40% of the original genome. The annotation is supported by sequencing of key intermediate strains as well as integration of literature knowledge for the annotation of the deletion scars and their potential effects. The strain compendium presented here represents a comprehensive genome library of the entire MiniBacillus project. This resource will facilitate the more effective application of the different strains in basic science as well as in biotechnology.
The lic operon of Bacillus subtilis is required for the transport and degradation of oligomeric β-glucosides, which are produced by extracellular enzymes on substrates such as lichenan or barley glucan. The licoperon is transcribed from a ςA-dependent promoter and is inducible by lichenan, lichenan hydrolysate, and cellobiose. Induction of the operon requires a DNA sequence with dyad symmetry located immediately upstream of the licBCAH promoter. Expression of the lic operon is positively controlled by the LicR regulator protein, which contains two potential helix-turn-helix motifs, two phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domains (PRDs), and a domain similar to PTS enzyme IIA (EIIA). The activity of LicR is stimulated by modification (probably phosphorylation) of both PRD-I and PRD-II by the general PTS components and is negatively regulated by modification (probably phosphorylation) of its EIIA domain by the specific EIILic in the absence of oligomeric β-glucosides. This was shown by the analysis oflicR mutants affected in potential phosphorylation sites. Moreover, the lic operon is subject to carbon catabolite repression (CCR). CCR takes place via a CcpA-dependent mechanism and a CcpA-independent mechanism in which the general PTS enzyme HPr is involved.
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