Alice Alejandro F, Pérez-Martínez Gaspar, Sánchez-Rivas Carmen
Laboratorio de Microbiología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires (1428), Argentina.
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos, CSIC, Paterna, Valencia, Spain.
Microbiology (Reading). 2003 Jul;149(Pt 7):1687-1698. doi: 10.1099/mic.0.26231-0.
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.
球形芽孢杆菌由于能够产生杀蚊毒素而具有生物技术应用价值，但它不能利用糖类作为碳源。然而，编码属于磷酸烯醇式丙酮酸磷酸转移酶系统（PTS）的HPr和EI蛋白的ptsHI基因被克隆、测序并进行了表征。在使用金黄色葡萄球菌突变体提取物进行的互补试验中，对于其中一种蛋白，HPr和EI蛋白在磷酸烯醇式丙酮酸依赖性转磷酸化反应中均具有完全功能。从球形芽孢杆菌的野生型和Ser46/Gln突变体中纯化出了HPr(His(6))，并将其用于以球形芽孢杆菌或枯草芽孢杆菌提取物作为激酶源的体外磷酸化实验。结果表明，可以获得磷酸化形式的P-Ser46-HPr和P-His15-HPr。这些发现还间接证明了球形芽孢杆菌中存在HPr激酶活性。这些ptsHI基因的遗传结构具有一些不寻常的特征，因为它们与编码与N-乙酰葡糖胺（GlcNAc）分解代谢相关的代谢酶的基因（nagA、nagB和一个未确定的orf2）共同转录。事实上，这种细菌能够利用这种氨基糖作为碳源和能源，但ptsH基因敲除突变体失去了这一特性。对野生型和ptsH基因敲除突变体菌株中GlcNAc摄取和链脲佐菌素抑制作用的研究表明，GlcNAc是由PTS的EII(Nag)元件转运并磷酸化的，该元件尚未被表征。此外，还测定了GlcNAc-6-磷酸脱乙酰酶和GlcN-6-磷酸脱氨酶的活性；二者在GlcNAc存在时均被诱导。这些结果，连同作者最近发现的磷酸果糖激酶活性，强烈表明球形芽孢杆菌中存在糖酵解途径。它们也为工业应用中的基因改良开辟了新的可能性。
