Genome Sequence And Mutational Analysis Of Genes Related To Salt-tolerance Of A Novel Salt-resistant Species Mesorhizobium Alhagi CCNWXJ12-2~T

Mesorhizobium alhagi strain CCNWXJ12-2Tis a novel species of soil-dwelling andnitrogen-fixing bacteria that has a nitrogen-fixing symbiotic relationship with Alhagisparsifolia. This symbiosis successfully survived in the desert region in northwestern Chinaand is important in wind prevention and sand-fixation. This strain showed a high resistance tosalt （0.8M NaCl）, alkali （pH12）, temperature （4℃⁶0℃） and drought （30%PEG6000simulated drought condition）. Whole genome analysis combined with transposon mutagenesistechnology was performed to reveal the osmotic regulation network and molecularmechanisms of this strain.In order to identify genes contributed to salt resistance, strain CCNWXJ12-2Twassubjected to random mutagenesis with the transposon Tn5-pRL27. A total of15,000transposants were screened for inability to grow on the medium supplemented with400mMNaCl after5to6days of incubation. Four of the putative salt-sensitive mutants were obtained.The four salt-sensitive mutants had stable but different sensitive traits to NaCl, especially themutant Mam4. The growth of all mutants was reduced to half when compared to M. alhagiCCNWXJ12-2T（CK） and there was no growth when NaCl concentration increased to0.4mol/L. In addition, the four salt-sensitive mutants were also sensitive to KCl, indicating thatthe interrupted genes had distinctive functions.Plasmid rescue was used to clone the flanking sequence of transposon. Tn5were insertedinto different genes in four mutants （Mam1、Mam2、Mam3and Mam4）, named xj₉52,xj₃794, xj₁521and xj₂74respectively. The locations of interrupted genes were identifiedand physical map of interrupted genes with flanking genes was drawn. In mutant Mam1, theTn5interrupted gene xj₉52encoding a hypothetical protein MAXJ12₀4699. Conserveddomains of the hypothetical protein showed that it is a putatived drug/metabolie transporterwith two EamA domains. In mutant Mam2, xj₃794gene encoding protein MAXJ12₁7498homologous to5,10-methylenetetrahydrofolate reductase （MTHFR） was interrupted by Tn5.In mutant Mam3, the Tn5interrupted xj₁521gene encoding a hypothetical outer membranesecretion protein MAXJ12₀0972. Conserved domains analysis of the protein MAXJ12₀0972showed that it may be a RTX toxin and related Ca2+-binding protein contains12predicted hemolysin-type calcium-binding repeat subordinate to hemolysinCabind family.In mutant Mam4, the Tn5interrupted xj₂74encoding a hypothetical proteinMAXJ12₀1324.This hypothetical protein has28%identity with ExoH and locats in a clusterof genes involved in transport and biosynthesis of extracellular polysaccharides.To verify the salt-sensitive phenotype of mutants, the mutants were complemented viatransformation pBBR1-MCS5with a complete copy of the genes from wide-type. The vivocomplementation assays confirmed that the phenotype observed in each mutant was caused bygenes inactivation by the Tn5insertion. The complemented mutants could restore salttolerance phenotype under0.4M NaCl partially. This result indicated that these four geneswere really related to salt tolerance of M. alhagi CCNWXJ12-2T, and played an importantrole in rhizobial osmotic regulation and stress protection.The genome of M. alhagi strain CCNWXJ12-2Twas sequenced on the Illumina HiSeq2000platform by BGI-Shenzhen, China. The genome was assembled using SOAP de novo.The de novo genes prediction was carried out by Glimmer3.0.7,408open reading frames（ORFs） were predicted with average GC content62.65%. Among all the predicted genes,1,034genes were supposed to code hypothetical or unknown proteins according to BLASTcomparisons against several protein sequences and protein family databases such as KEGG,COG, SwissProt, TrEMBL and NR. RNAmmer and tRNAscan-SE were used to predictrRNAs and tRNA. At least one copy of5S,16S and23S rRNAs were identified in thegenome.In conclusion, three new genes encoding a hypothetical protein MAXJ12₀4699, outermembrane secretion protein MAXJ12₀0972, a hypothetical protein MAXJ12₀1324and a5,10-methylenetetrahydrofolate reductase were identified. Sequence analysis, functionprediction, subcellular localization and extracellular polysaccharide analysis were performedto illustrate the functions of these salt tolerance-related genes.