Gene Cloning And Salt-tolerance Evaluation Of The Membrane-integral Pyrophosphatase Of Human Gut Microbial Clostridium Methylpentosum

The primeval life or the contemporary organisms suffering with stress conditions appeared to depend on PPi rather than ATP as the main source of energy, with an important PPi-energized escort as the membrane homodimeric pyrophosphatases（PPases） composed of H+-PPase, Na+-PPase and Na+,H+-PPase. The long known H+-PPase with broad distributions has already been given with numerous functional characterizations and applications in constructing stress-resistant organisms particularly of plants, whereas no any correlated reports have pointed to Na+-PPase, Na+,H+-PPase that were uniquely found in prokaryotes several years ago or recently. In this study, we presented the gene cloning and salt-tolerance evaluation for the membrane-integral PPase of the human gut microbe Clostridium methylpentosum（CmPP） that was speculated as a member of Na+,H+-PPase subfamily. The major results were shown as follows:（1） Gene cloning and bioinformatic analysis of CmPP and salt-tolerance evaluation of its recombinant E. coli strainUsing the human gut metagenome as DNA template, the full length of CmPP gene was successfully amplified by two rounds of PCR, which was subcloned into the plasmid pET32a（+） by enzyme digestion to achieve the E. coli expression vector pET（CmPP）. Sequencing data indicated the amplified CmPP gene contained an entire ORF（2100 bp） encoding a single polypeptide of 699 amino acids（with theoretical molecular weight of 72.3kDa and pI value of 6.14）, with the G+C percentage of 59.9 %, relatively balanced base distribution, and no cryptic polyadenylation signals. Topological prediction showed CmPP as a multi-spanning membrane protein consisting of 16 transmembrane helix segments, in accordance to the membrane topological model of those known membrane PPases. Furthermore, phylogenetic tree analysis implicated CmPP as a member of Na+,H+-PPase subfamily, the nearest ortholog of which is the membrane-integral PPase（GenBank Acc. No. ZP₀2078667） of the anaerobiotic Clostridium leptum（coexisted with C. methylpentosum in human gut） that has already been identified.Through the colony dot-plating test and spectrometric analysis of bacterial dynamic growth curve, E. coli strain BL21（DE3） harboring the recombinant vector pET（CmPP） was found to exhibit a remarkably better growth under all appointed salt stresses（0.7 M, 0.8 M, 0.9 M NaCl） than the control strain carrying the empty vector pET32a（+）. This data indicatd the heterologous expression of CmPP could improve the salt tolerance of E. coli.（2） Salt-tolerance evaluation of CmPP recombinant yeast strainThe CmPP gene fragment was excised from the vector pET（CmPP） by enzyme digestion and subcloned into the plasmid pYES2 to create the yeast expression vector pYES2（CmPP）. Through the colony dot-plating test and spectrometric analysis of yeast dynamic growth curve, salt-sensitive Saccharomyces cerevisiae mutant strain ena1- bearing the recombinant vector pYES2（CmPP） was found to demonstrate a notably better growth under all chosen salt stresses（0.2M, 0.25 M, 0.3M NaCl） than the control strain of the empty vector pYES2, implicating the heterologously expressed CmPP could also enhance the salt tolerance of S. cerevisiae.（3） Salt-tolerance analysis of CmPP transgenic tobacco plantsUsing the constructed plasmid pET（CmPP） as DNA template, the CmPP gene fragment was re-amplified, and subcloned into the plasmid pBI121 by enzyme digestion to achieve the plant expression vector pBI121（CmPP） that was further transformed into the Agrobacterium tumefaciens strain LBA4404. Through the leaf disc transformation by Agrobacterium infection and multiple PCR identification, the positive transgenic tobacco plants of CmPP gene were obtained.An extensive salt-tolerance analysis was selectively assigned to the F1 offspring plants of three CmPP transgenic tobacco lines（No. 9, 11, 12） with a dominant performance in the preliminary salt-tolerance test. Our results indicated all CmPP transgenic lines had a better growth status and a lower degree of leaf green-loss than the wild-type upon salt stresses. The dry mass content, total chlorophyll content and root activity declined more severely in the wild-type than CmPP transgenic plants under salt stress conditions. Meanwhile, the MDA content and leakage rate of electrolyte in leaves increased with the concentration of salt in both wild-type and CmPP transgenic plants. However, the values of these two parameters were commonly lower in all three transgenic lines than in wild-type under the same salt stress condition. Taken together, these data explicitly showed that the introduction of heterologous CmPP gene could improve the salt tolerance of the corresponding transgenic tobacco plants.In conclusion, to our knowledge, this study seemed for the first time to convey a functional evaluation on a prokaryotic membrane-bound PPase with the putative Na+-transporting activity. Heterologous expression of CmPP conferred enhanced salt tolerance commonly in bacteria, yeast and tobacco, which was proposed to arise from its Na+-pumping activity that drive Na+ efflux from the cytosol into the extracellular milieu or vacuoles to ameliorate the salt toxicity to cells. Therefore, it could be envisioned that CmPP should have potential uses in genetic engineering to develop transgenic plants especially of agronomical species with increased salt tolerance.