borinquense DSM 11551 and Aphanothece halophytica PCC 6803 Their

borinquense DSM 11551 and Aphanothece halophytica PCC 6803. Their amino acid sequences are aligned in Fig. 3. The amino acid sequence deduced from the ORF, designated as M-Nha (Na+/H+ antiporter from metagenomic library), MK-2206 cost consisted of 523 amino acid residues with a calculated molecular weight of 58 147 Da and a pI of 5.50. The most abundant amino acid residues of this protein were Leu (75/523), followed by Ile (48/523), Val (46/523), Ala (38/523) and Gly (37/239). The least abundant residue was Cys (two

residues) and Trp (five residues). Among the 523 amino acid residues, only 89 residues were charged, indicating that M-Nha is of low polarity. This is consistent with the belief that the Na+/H+ antiporter is an integral membrane protein. Although the dense alignment surface approach revealed that the M-NhaP contained 11 peaks (Fig. 4), the probability for the 10th peak was only around 20% when its transmembrane segment (TMS) was analyzed using tmhmm computer program (data not shown). The sosui analysis further confirmed this result of total 10 peaks in M-NhaP released by tmhmm (Fig. 5). Thus it was Selleckchem Trametinib likely that the M-Nhap only contained 10, not 11, transmembrane domains. The conserved domain analysis against CDD suggested that M-NhaP is a cpa1 Na+/H+ antiporter from bacteria, which was classified as a model that may span more than one domain and had not been assigned to any domain superfamily yet. Furthermore, CDD also showed

that M-Nha had significant similarity to NhaP type Na+/H+ and K+/H+ antiporter with a unique C-terminal domain in the Na+/H+ exchanger family. A similar result was also obtained Decitabine cost when it was analyzed by interproscan. Gene ontology delineation indicated that M-Nha was integrated to membrane (GO: 0016021) and exchanged Na+ for H+ in an electroneutral manner. The effects of NaCl concentration on the growth of transformant

cell E. coli KNabc/pM-Nha, which harbored the recombinant Na+-resistant plasmid pM-Nha, and E. coli KNabc/pUC18, which contained only empty pUC18 vector, were evaluated. The E. coli KNabc/pM-Nha strains can grow well in LBK medium containing 0.2 M NaCl and can even survive in the presence of 0.25 M NaCl, whereas cells of E. coli KNabc/pUC18 do not (Fig. 6). To test the effect of pH on cell growth, E. coli KNabc/pUC18 and KNabc/pM-Nha were grown in minimal medium as described above but at different pH values from 7 to 8.5. The results were similar to that influenced by NaCl, with a greatly reduced growth of E. coli KNabc/pUC18 under alkaline conditions, especially at pH above 8.0, compared with that below neutral pH. However, only a certain growth reduction range was observed for E. coli KNabc/pM-Nha harboring nha gene in alkaline medium (Fig. 6). This result indicated that the protein encoded by m-nha gene offered the antiporter-negative mutant E. coli KNabc cells not only resistance to Na+, but also the ability to grow under alkaline conditions.

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