Phosphoenolpyruvate phosphotransferase system and N-acetylglucosamine metabolism in Bacillus sphaericus.
(2003)
Journal - Microbiology (Reading, England) (England )
Abstract :
Bacillus sphaericus, a bacterium of biotechnological interest due to its ability to produce mosquitocidal toxins, is unable to use sugars as carbon source. However, ptsHI genes encoding HPr and EI proteins belonging to a PTS were cloned, sequenced and characterized. Both HPr and EI proteins were fully functional for phosphoenolpyruvate-dependent transphosphorylation in complementation assays using extracts from Staphylococcus aureus mutants for one of these proteins. HPr(His(6)) was purified from wild-type and a Ser46/Gln mutant of B. sphaericus, and used for in vitro phosphorylation experiments using extracts from either B. sphaericus or Bacillus subtilis as kinase source. The results showed that both phosphorylated forms, P-Ser46-HPr and P-His15-HPr, could be obtained. The findings also proved indirectly the existence of an HPr kinase activity in B. sphaericus. The genetic structure of these ptsHI genes has some unusual features, as they are co-transcribed with genes encoding metabolic enzymes related to N-acetylglucosamine (GlcNAc) catabolism (nagA, nagB and an undetermined orf2). In fact, this bacterium was able to utilize this amino sugar as carbon and energy source, but a ptsH null mutant had lost this characteristic. Investigation of GlcNAc uptake and streptozotocin inhibition in both a wild-type and a ptsH null mutant strain led to the proposal that GlcNAc is transported and phosphorylated by an EII(Nag) element of the PTS, as yet uncharacterized. In addition, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase activities were determined; both were induced in the presence of GlcNAc. These results, together with the authors' recent findings of the presence of a phosphofructokinase activity, are strongly indicative of a glycolytic pathway in B. sphaericus. They also open new possibilities for genetic improvements in industrial applications.
| ISSN : | 1350-0872 |
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| Mesh Heading : | Acetylglucosamine Adenosine Triphosphate Bacillus Base Sequence Biological Transport, Active Cloning, Molecular DNA, Bacterial Genes, Bacterial Genetic Complementation Test Molecular Sequence Data Multigene Family Mutagenesis, Site-Directed Phosphoenolpyruvate Phosphoenolpyruvate Sugar Phosphotransferase System Phosphotransferases (Nitrogenous Group Acceptor) Reverse Transcriptase Polymerase Chain Reaction Staphylococcus aureus Transcription, Genetic metabolism genetics genetics metabolism genetics genetics metabolism genetics metabolism |
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| Mesh Heading Relevant : | Bacterial Proteins metabolism metabolism metabolism |
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Existence of a true phosphofructokinase in Bacillus sphaericus: cloning and sequencing of the pfk gene.
(2002)
Journal - Applied and environmental microbiology (United States )
Abstract :
Some strains of Bacillus sphaericus are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the pfk gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes.
| ISSN : | 0099-2240 |
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| Mesh Heading : | Amino Acid Sequence Bacillus Base Sequence Cloning, Molecular Genetic Complementation Test Molecular Sequence Data Phosphofructokinases Polymerase Chain Reaction genetics chemistry metabolism |
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| Mesh Heading Relevant : | enzymology genetics |
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DNA supercoiling and osmoresistance in Bacillus subtilis 168.
(1998)
Journal - Current microbiology (UNITED STATES )
Abstract :
The importance of the DNA structure for the expression of the osmotic response (osmotolerance) was investigated in Bacillus subtilis 168. Plasmid pUB110 DNA was used as a reporter of the chromosomal DNA topology, and analyses were performed in chloroquine agarose gels. Plasmidic DNA obtained from cultures in Schaeffer medium (D) taken in those periods in which B. subtilis is able to express osmotolerance (early stationary phase or from germinating spores) or from adapted cultures to hyperosmotic medium (DN) presented a higher level of negative supercoiling than DNA samples from vegetative cultures, normally refractory to induction of osmotolerance. The involvement of the DNA gyrase was investigated through the sensitivity to novobiocin, an antibiotic inhibitor of its activity and the behavior of a gyrB1 mutant strain (RG1). In the wild-type strain, the addition of a sublethal concentration of novobiocin (0.5 microg/ml) to the hyperosmotic medium relaxed DNA and inhibited growth. Moreover, already growing cultures in DN medium and later submitted to the same antibiotic presented a relaxed DNA and stopped growing. The RG1 mutant strain submitted to similar novobiocin treatments displayed normal growth in DN novobiocin medium. These results pointed to the requirement of a highly negative supercoiled DNA structure involving the gyrase activity in osmotic response.
| ISSN : | 0343-8651 |
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| Mesh Heading : | Anti-Bacterial Agents Bacillus subtilis Chloroquine Culture Media DNA Gyrase DNA Topoisomerases, Type II DNA, Bacterial DNA, Superhelical Genes, Reporter Novobiocin Plasmids Spores, Bacterial pharmacology genetics metabolism genetics metabolism physiology chemistry drug effects metabolism drug effects pharmacology genetics physiology |
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| Mesh Heading Relevant : | Osmotic Pressure metabolism physiology metabolism physiology |
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Existence of a True Phosphofructokinase in Bacillus sphaericus: Cloning and Sequencing of the pfk Gene
(2002)
Journal - Applied and Environmental Microbiology
Abstract :
Some strains of Bacillus sphaericus are entomopathogenic to mosquito larvae, which transmit diseases, such as filariasis and malaria, affecting millions of people worldwide. This species is unable to use hexoses and pentoses as unique carbon sources, which was proposed to be due to the lack of glycolytic enzymes, such as 6-phosphofructokinase (PFK). In this study, PFK activity was detected and the pfk gene was cloned and sequenced. Furthermore, this gene was shown to be present in strains belonging to all the homology groups of this heterogeneous species, in which PFK activity was also detected. A careful sequence analysis revealed the conservation of different catalytic and regulatory residues, as well as the enzyme's phylogenetic affiliation with the family of allosteric ATP-PFK enzymes.