For the detection of E. coli clones exhibiting Na+/H+ antiporter activity, the antiporter-negative mutant strain E. coli KNabc was used as the host for the recombinant plasmids of metagenomic DNA libraries. The
resulting recombinant E. coli strains were screened on selective LBK agar plates containing 5.0 mM Daporinad LiCl. The growth of E. coli KNabc was completely inhibited under this condition due to the toxic effect of Li+ on pyruvate kinase in the absence of an antiporter (Majernik et al., 2001). Thus, only recombinant E. coli strains harboring a gene conferring resistance to Li+ could grow under the conditions used. By functional complementation tests, 10 clone candidates were obtained out of approximately 300 E. coli clones during the initial screening procedure. To confirm that the Li+-resistant phenotype of the clones selected was determined
Dabrafenib order by recombinant plasmids, the plasmids in the clones were isolated and retransformed into E. coli KNabc, and the resulting clones growing in 7.5 mM Li+ medium were screened again on selective plates with high concentrations of NaCl (0.20 and 0.25 M). However, only one recombinant plasmid, designated as pM-Nha, conferred a stable Na+-resistant phenotype on the resulting recombinant E. coli KNabc strains. The hybrid plasmid pM-Nha was sequenced, and it was revealed that pM-Nha carried a common DNA fragment of a putative Na+/H+ antiporter gene. The nucleotide sequence analyses of Na+/H+ antiporter gene revealed that the length of the DNA insert of pM-Nha was 1814 bp, and it contained one intact ORF (1572 bp), a promoter (ATG) and a terminator (TAA) (Fig. 1). A Shine-Dalgarno
(AGGAGG), −10 region (TATTAT) and −35 region (TTGACA) in the downstream and a terminator-like sequence (5′-GCAGGCTGT-3′; 5′-ACAGCCTGC-3′) in the upstream were also found Cobimetinib in the ORF (Fig. 1). A homology search revealed that the protein encoded by ORF had the highest homology of 92%, 86% and 64% identity with the NhaH from Halobacillus dabanensis D-8T (accession no. ABA03152) and Halobacillus aidingensis AD-6T (accession no. ABX57744) and with Nhe2 from Bacillus sp. NRRL B-14911 (accession no. EAR67303), respectively, and a slightly lower similarity (31% and 33% identity) to the Na+ antiporter from Halogeometricum borinquense DSM 11551 (accession no. EEJ57208) or Cyanothece sp. ATCC 29155 (accession no. ACK72385). In terms of the phylogenetic relationship between the Na+/H+ antiporter protein from the metagenomic library constructed in current study and those from other strains reported, the ORF products of these antiporters were clearly divided into two groups (Fig. 2). The M-Nha was closely related to NhaH from the moderately halophilic strains of H. dabanensis D-8T and H. aidingensis AD-6T, and also similar to Nhe2 from Bacillus sp.