Cloning And NaCl Stress Responses Of Genes For The Uptake Of Glycine Betaine From Virgibacillus Halodenitrificans PDB-F2

The gram-positive moderately halophilic bacterium,Virgibacillus halodenitrificans PDB-F2,is able to degrade phenol in high-salinity environment,which copes with external salinity fluctuation by biosynthesis or uptake of compatible solutes.Studies on salt tolerance mechanism of transport compatible solutes in V.halodenitrificans PDB-F2 were rare.This study mainly investigated the salt tolerance mechanism of transport glycine betaine by PDB-F2,which enriched the theoretical study of the salt tolerance mechanism of the moderate halophilic bacteria PDB-F2,the application of PDB-F2 in the treatment of high-salt organic wastewater,and was of great significance to use synthetic biology to enhance the salt tolerance of common degrading bacteria.In this study,firstly,the uptake of glycine betaine by V.halodenitrificans PDB-F2 under high salt conditions was determined by nuclear magnetic resonance(NMR).Compared with biosynthesis of ectoine,PDB-F2 preferentially transported glycine betaine from outside to resist osmotic stress,and exogenously added glycine betaine inhibited the intracellular synthesis of ectoine in PDB-F2.The results of the protection effect of exogenous glycine betaine on PDB-F2 showed that glycine betaine significantly increased the salt-tolerance of PDB-F2 at 6%-15%NaCl,and the maximum tolerated salinity of PDB-F2 was 15%NaCl.The E.coli MKH13 is a mutant strain,and contains deletions of compatible solutes biosynthesis gene and transport genes,which can not grow at 4%NaCl condition.We complemented E.coli MKH13 with betL,opuDl,opuD2,opud3,which predicted from the complete genome sequence of V.halodenitrificans PDB-F2.Functional complementation of E.coli MKH13 showed that all four transporters were able to transport glycine betaine.And the affinity of four transporters for glycine betaine was BetL>OpuD1>OpuD3>OpuD2.Real-time fluorescence quantitative PCR was used to investigated transcriptional levels of genes of betL.opuD1,opuD2,and opuD3 in V.halodenitrificans PDB-F2.The results showed that transcriptional level and intracellular glycine betaine concentration increased with increasing salinity,and the highest transcriptional level of betL,opuDl,opuD3 and a maximum of intracellular glycine betaine concentration were all at 12%NaCl,which indicated that the amount of glycine betaine transported by V.halodenitrificans PDB-F2 was dependent on the degree of salt stress imposed by the external environment.The transcription was induced by NaCl,while glycine betaine had no effect on gene transcription.But it affected the transcriptional levels of genes,and the maximal induction of betL,opuDl,opuD2 and opuD3 genes transcription depends on high salt plus glycine betaine.The transcription of betL,opuD1,opuD2 and opuD3 were specificity for glycine betaine.The uptake of glycine betaine induced by osmosis stress in V.halodenitrificans PDB-F2 was not only dependent on transcription of genes induced by salt stress,but was related to transporter activity stimulated by salt stress.