Transport Mechanism Of Ectoine-like Substances In Virgibacillus Halodenitrificans PDB-F2 With Complete Genome Sequencing And Analyzing

Virgibacillus halodenitrificans strain PDB-F2,a moderate halophilic bacterium which could efficiently degrade phenol under highly salty conditions,can be a good candidate used to enhance the biological treatment of high-salt organic wastewater.In usual,moderate halophilic bacteria are used to accumulate compatible solutes to tolerant the salt when they encounter the external osmotic pressure changes.Up to now,the mechanism studies of V.halodenitrificans PDB-F2 using compatible solutes to resist the salt are still very limited.To study the salt-tolerant mechanism of the compatible solutes in this strain,especially the typical compatible solutes,are important and useful to explain the salt-tolerant mechanism of this bacterium and guide the application of this stain in wastewater treatment as well as provide basic research data for salt-tolerant mechanism of compatible solutes in microorganism.In this study,the osmoprotective effects of different compatible solutes on the strain under high-salt conditions were explored,and the results showed that the osmoprotective effects were hydroxyectoine>ectoine>proline>trehalose>glutamic acid>betaine.Then the 1H-NMR was used to detect the kinds of compatible solutes accumulated in cells with exogenous compatible solutes provided,and according to the results,it was showed that any single compatible solute couldn't provide the necessary osomtic pressure to the cells under high salinity surroundings,and the strain would synthesize varieties of compatible solutes to resist high salt,but ectoine-like substances were the main compatibilie solutes which PDF2 accumulated.The synthesis genes and transport genes related to the above compatible solutes were identified by complete genome sequencing and analyzing.The structural analysis of the transport gene ehuABCD of ectoine-like substances was carried out,and the results suggested that ehuC,ehuD,ehuA were arranged together in the same direction,which was separated from ehuB by 156 bp,and the translation orientation of genes was ehuB-ehuC-ehuD-ehuA.The functional complementation experiments showed that ehuABCD and ehuE genes(which was another transport gene of ectoine-like substances and confirmed by whole genome sequencing analysis)were indeed transporters of ectoine-like substances.The apparent transport processes and transporters expression of ectoine-like substances were investigated by high performance liquid chromatography(HPLC)and real-time fluorescence quantitative PCR.When the hyperosomtic shock happened from 8%(w/v)NaCl to 12%(w/v)NaCl,the results showed that the maximum transport rate of hydroxyectoine was 7.41 ± 0.37 mg/g cell dry weight · min,which was approximate 4.5 folds of the maximum transport rate of ectoine(1.65 ± 0.01 mg/g cell dry weight · min).The changes of ectoine transport rates were consistent with those of transcription of ehuABCD,which increased first and then decreased.It was also found that salinity had a significant inhibition effect on the transport of ectoine because the maximum transport rate of ectoine under hypoosmotic shock was approximate 20-fold than that of hyperosomtic shock.And the transport processes of ectoine were controlled at the levels of transport activity and the transcription of the transport genes.