[Cloning and sequencing of the proBA gene from the selected mutant resistant to proline analogue from Bacillus subtilis].
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. A 2.3 kb DNA fragment from the mutant was amplified using PCR method. Sequence analysis indicated that three bases changed within the proB gene, compared with the wild-type strain. 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 other two were silent mutations. The recombinant vector pBE2-proB could functionally complement the proline auxotrophy E. coli 1.1252. Sequence analysis of proA showed that proA and proB overlapped by 4 nt, and there was a SD sequence at nt 14 upstream of the start codon of proA. The deduced amino acid of proA gene shared a high similarity with that of Bacillus subtilis 168 (77%).
Miao
Li-Xia
LX
Department of Technology, College of Life Science, Wuhan University, Wuhan 430072, China.
Cao
Jun-Wei
JW
Liu
Rui-Jie
RJ
Wang
You-Liang
YL
Zeng
Yong-Hui
YH
D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D015247:Deoxyribonuclease HindIII / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D008969:Molecular Sequence Data; D009154:Mutation; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D011392:Proline / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology
D004269:DNA, Bacterial; D012965:Sodium Chloride; D011392:Proline; D000445:Aldehyde Oxidoreductases; D050698:Glutamate-5-Semialdehyde Dehydrogenase; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase; D015246:Deoxyribonuclease EcoRI; D015247:Deoxyribonuclease HindIII