Construction Of The Insertion-inactivated Mutant Of RocG In Bacillus Subtilis

Many researchers have studied the metabolic networks of Bacillus subtilis,a model organism for gram-positive bacteria. Among them, the ammoniametabolic pathway has important practical significance. The natto, which isfermented by Bacillus natto, has a high nutritional value and healthy function.However, Bacillus natto will produce a large amount of ammonia during themetabolic process, which has a negatively influence on its flavor.Glutamate dehydrogenase (GDH) is a key enzyme in the ammoniametabolic pathway of B. subtilis, it plays an important role in keeping thebalance between carbon and nitrogen metabolism. There are two genesencoding GDH in B. subtilis, gudB and rocG. For B. subtilis168, the gudBencodes an inactive enzyme. and the rocG gene is the only gene whichencodes the active GDH. When the rocG gene is knocked-out, it results in theappearance of mutations with gudB1replaced to gudB, which can encode anactive GDH (gudB1-GDH). The roles of GDH in metabolic and regulatorymechanism have been studied, but all the researches were limited torocG-GDH， not on gudB1-GDH. In order to study the function and regulatory mechanism of gudB1-GDH,rocG homologous recombination sequence was amplified by PCR using theDNA of B. subtilis168as the template, and two restrict enzyme sites of HindIII and BamH I were introduced. Then, the rocG homologous recombinationsequence was connected with pMUTin4, an insertion-inactivated plasmidnamed as pMUTin4-rocG was constructed successfully. The plasmid wastransformed into B. subtilis168, and an insertion-inactivated mutant of rocG(BS-△rocG) was produced. The growth rate of the mutant was similar to thewild type strain. However, its GDH activity was4.641U/mg protein, higherthan the wild type strain, whose activity was3.042U/mg protein. It suggestedthat the gudB1mutation occurred during the growth period of BS-△rocG.Therefore, BS-△rocG can be used for further study on the function andregulatory mechanism of gudB1-GDH in the metabolism network of B.subtilis.In the meanwhile, we tried to get a rocG gene mutant of Bacillus natto,which may produce low amount of ammonia and thus improve the flavor ofnatto. Using the DNA of Bacillus natto (CGMCC No.2801) as the template,the rocG homologous recombination sequence was amplified and connectedwith pMUTin4. In this way, an insertion-inactivated plasmid named aspMUTin4-rocG1was constructed successfully. We hope to get an insertionalrocG inactivated mutant of Bacillus natto later by improving thetransformation methods.