Screening Of Saline-alkaline Tolerant Genes From Halomonas Zhaodongensis And Analysis On Saline-alkaline Tolerance Of DUF1538 And DUF2062 Family Members

Increasing attentions have been paid to the halophilic bacteria by many researchers in microbiology,ecology and soil because of their unique halo-tolerant and alkaline-resistant genetic mechanisms and physiological characteristics.Halophilic bacteria is a category of important microbial resources with the significance of basic research on halo-tolerance mechanism and the discovery of new halo-alkaline tolerant genes,which can provide important theoretical direction and important gene resources for the development of gene-engineered microbial fertilizers and the transgenic crops with high-capacity of halo-alkaline tolerance.Halomonas zhaodongensis NEAU-ST10-25 T is a Gram-negative moderately halophilic bacterium which was isolated from Zhaodong,Heilongjiang Province,which can grow in S-G medium containing 0-15% Na Cl(optimum 3%).In this study,Escherichia coli KNabc lacking three major Na~+(Li~+)/H~+ antiporters(?nha A,?nha B,?cha A)was used to obtain more unknown halo-alkaline-tolerant genes and identify their protein functions by screening in a gene library of genomic DNA from strain NEAU-ST10-25 T and functional complementation assay through selection in E.coli KNabc.In this study,a gene library of genomic DNA from strain NEAU-ST10-25 T containing the potential halo-alkaline-tolerant genes was constructed and screened by selection in E.coli KNabc on the LBK medium plate containing 0.2 M Na Cl plus 50 ?g·mL-1 ampicillin.We obtained 21 positive clones enabling E.coli KNabc to grow on the LBK medium plate containing 0.2 M Na Cl.According to the digestion and sequencing analysis,two genes designated ump AB were found to encode a pair unknown membrane protein with unknown function belonging to DUF1538 family(domain of unknown function with No.2062 family)and one separate gene designated duf2062 found to encode a protein with unknown function belongin to DUF2062 family(domain of unknown function with No.2062 family),respectively.The total length of the ump AB genes is 1580 bp,consisting of two open reading frames(ORF).The stop codon(TAA)of Hz_Ump A overlaps at the site of base "A" with the initial codon(ATG)of Hz_Ump B.The corresponding proteins contain 254 and 271 amino acid residues,respectively.Transmembrane segment prediction showed that they all have seven transmembrane segments.Protein algnment showed that there are 47 highly conserved amino acid residues between their 12 closestly homologues with them,including 36 hydrophobic amino acids,6 positively charged amino acids,5 polar amino acids.A careful protein alignment at the NCBI website showed that there is no identity between either Hz_Ump A or Hz_Ump B and all known specific Na~+(Li~+)/H~+ antiporters,proteins with Na~+(Li~+)/H~+ antiport activity,or even any subunit of multi-gene Na~+/H~+ antiporters.Phylogenetic analysis confirms that Hz_Ump A and Hz_Ump B should belong to DUF1538 family,which are significantly distant with the above-mentioned proteins with Na~+/H~+ antiport activity.The sole co-expression of ump AB genes could confer E.coli KNabc the tolerance to 0.4 M Na Cl and 30 m M Li Cl,and an alkaline p H resistance at 8.0.Compared with the negative control,single Hz_Ump A or Hz_Ump B could confer E.coli KNabc the tolerance to 0-0.15 M,especially in the alkaline conditions,also ump B was better than ump A.Also,a significant difference is that the longer Hz_Ump B contains a hydrophilic C terminus in contrast to the shorter Hz_Ump A.That suggests that the hydrophilic C terminus of Hz_Ump B may be quite important for the entire function of Hz_Ump AB as a Na~+/H~+ antiporter,since the sole Hz_Ump B exhibits the better Na Cl tolerance and higher alkaline p H resistance,in comparison with the sole Hz_Ump A.At the same time,its Na~+(Li~+,K~+)/H~+ antiporter activity can be detected in the reversed membrane by fluorescence dequenching method,and the activity showed a p H-dependent profile with the optimum p H at 9.0.The K0.5 values for three monovalent cations were 0.26 ± 0.06 mM,0.46 ± 0.09 m M and 0.41 ± 0.06 m M,respectively.In other words,Hz_Ump AB had the best affinity for Na~+,followed by K~+ and Li~+,respectively.In order to further analyze the cell localization of Hz_Ump AB and determination of them as a hetero-dimer,we constructed the co-expression vector p ETDuet-1-ump AB,and then prepared the samples for total protein extract,membrane protein fractions and cytoplasmic protein fractions of the cells by ultracentrifugation.The protein bands(about 25 KDa)of Hz_Ump A and Hz_Ump B were detected only in the total protein extract and membrane protein fractions by Western Blot.This reveals that Hz_Ump A and Hz_Ump B indeed localize in the cytoplasmic membranes.Hz_Ump B co-immunoprecipitated by Hz_Ump A indicates that Hz_Ump AB forms a hetero-dimer.Na~+(Li~+)/H~+ antiport assays confirm that Hz_Ump AB is likely to function as a p H-dependent Na~+(Li~+)/H~+ antiporter.Radchenko et al.showed that one mutant E.coli strain TO114(the different designation with the same genotype as KNabc)lacking three major Na~+(Li~+)/H~+ antiporters(Nha A,Nha B and Cha A)may be used for the identification of K~+/H~+ antiporter candidate genes.K~+/H~+ antiport assay by using Na-free KCl reveals that Hz_Ump AB is also likely to function as a p H-dependent K~+/H~+ antiporter.Therefore,we propose that Hz_Ump AB should function as a two-component p H-dependent Na~+(Li~+,K~+)/H~+ antiporter.The full length of the duf2062 gene designated Hz_DUF2062,was 552 bp.The encoded protein consists of 183 amino acid residues.Functional complementation assay revealed that the gene duf2062 allowed the salt-sensitive mutant KNabc to grow in the medium containing 0.3 M Na Cl or 5 m M Li Cl,and an alkaline p H resistance.Transmembrane segment prediction analysis showed that Hz_DUF2062 had three transmembrane segments.Protein alignment showed that Hz_DUF2062 belongs to DUF2062(domain of unknown function with No.2062)family.Also,54 amino acid residues exists commonly between Hz_DUF2062 and its homologues,including 28 hydrophobic amino acids,10 positively-charged amino acids and 13 polar amino acids.Phylogenetic analysis confirmed that Hz_DUF2062 and other proteins of the same family were clustered on a branch of 91% of a cluster of bootstrap values,which is significantly distant with known Na~+/H~+ antiporter or protein with Na~+/H~+ antiport activity.This indicates that Hz_DUF2062 does not belong to those Na~+/H~+ antiporter or salt-tolerant proteins that have been identified.The activity of Na~+(Li~+)/H~+ antiport activity,as well as relatively K~+/H~+ antiport activity,was detected by fluorescence dequenching method at a wide range of p H 6.5 to 9.5 with the optimum p H at 8.5.The K0.5 values for the three ions were 0.2 ± 0.13 m M,0.22 ± 0.1 m M and 0.43 ± 0.08 m M,respectively.In other words,Hz_Ump AB has the best affinity for Na~+,followed by Li~+ and K~+.Taken together,this is the report on the protein functional analysis of Hz_Ump AB and Hz_DUF2062 families as representatives of DUF1538 family and DUF2063 family.The results presented in this thesis trigger the understanding the function of DUF1538 protein pairs and DUF2062 family protein,and broaden the knowledge of novel Na~+/H~+ antiporters,especially for the ones from the moderately halophilic and alkaliphilic bacterium.Moreover,the discovery of Hz_Ump AB also gives us a new understanding of the structural classification of Na~+/H~+ antiporter.In addition to the forms as a homodimer or a hetero-polymer,Na~+/H~+ antiporters can also fucntion as a hetero-dimer just like Hz_Ump AB.