title: Phosphoenolpyruvate phosphotransferase system and N-acetylglucosamine metabolism in Bacillus sphaericus.
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.
authors.affiliation: Laboratorio de Microbiología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (1428), Argentina.
authors.forename: Alejandro F
authors.initials: AF
authors.lastname: Alice
authors.affiliation: Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain.
authors.forename: Gaspar
authors.initials: G
authors.lastname: Pérez-Martínez
authors.affiliation: Laboratorio de Microbiología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (1428), Argentina.
authors.forename: Carmen
authors.initials: C
authors.lastname: Sánchez-Rivas
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001407:Bacillus / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics / Q000378:metabolism*; D017852:Phosphotransferases (Nitrogenous Group Acceptor) / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr; D017852:Phosphotransferases (Nitrogenous Group Acceptor); C022360:phosphoenolpyruvate-protein phosphotransferase; D000117:Acetylglucosamine