Selection Of Bacillus Subtilis Halotolerant Mutant Resistant To Proline-Feedback Inhibition And Cloning,Expression Of ProBA Gene

NTG was used to make chemical mutation for Bacillus subtilis 93151. An enhanced osmotolerant mutant was obtained, which could grow in minimal medium containing 14% NaCl(w/v) and was not subject to proline-mediated feedback repression. The content of the intracellular free proline from the mutant increased rapidly with the rising of NaCl concentration, which showed that the high osmotolerant ability of the mutant is relative to the intracellular free proline.According to the sequence of proE gene from wild Bacillus subtilis 93151, two primers were designed and synthesized for PCR. Using TaKaRa LA PCR?in vitro Clonging Kit, a 2.3kb DNA fragment from the mutant was amplified and identified. Sequence analysis indicated that the fragment was composed of a complete proE gene and a partial proA gene. Compared with the wild-type strain, three bases within the proE gene changed. One of the mutations was substitution of an A for a T at nt position 781, leading to a change of a Ser to a Thr at amino acid residue 261 of the deduced protein product, while the other two, T777G and T1081C, were silent mutations. The conserved domain of ProB was analyzed by ncbi internet, it showed that T781A mutation was out of the conserved domains. Possibly the mutation leads to conformation changes and result in a loss of the enzyme's allosteric properties and increase the ability of osmotolerance of the mutant.Sequence analysis of pro A showed that pro A and proB overlapped by 4 nt, and there was a SD sequence at nt 14 upstream of the start codon of pro A. The deduced amino acid of pro A gene shared a high similarity with that of Bacillus subtilis 168(77%). This organization of pro A and proE from Bacillus subtilis 93151 was first reported in Bacillus subtilis.The recombinant vector pBE2-MproB(harbouring proE gene from mutant Bacillus subtilis 93151) and pBE2-WproB (harbouring proE gene from wild-type ) were constructed to identity the function of proE gene from the wild-type and mutant Bacillus subtilis 93151. Transforming the vectors into theproline auxotrophy E.coli\.\252(proB~), E. colil. 1252/pBE2-MproB and E. colil. 1252/ pBE2-WproB were screened, and then grown in minimal medium without proline at 37. The result showed that they both could functionally complement the proline auxotrophy E.coli\.\252 in minimal medium with 0%, 0.2%NaCl. However, E.coli 1.1252/pBE2-MproB could grow into large colonies after 30 hours, but E.coli 1.1252/pBE2-WproB cultured small colonies until 48 hours. Furthermore, E.coli 1.1252/pBE2-MproB could survive in minimal medium with 1% NaCl and E.coli 1.12527 pBE2-WproB not, and the osmotolerant ability of 1.1252/pBE2-MpraB was five-fold that of 1.12527 pBE2-WproB. It was showed that the osmotolerant ability increasing of E.coli 1.1252 by theproE gene from the mutant is more remarkable than from the wild-type strain.