Characterization of a gene responsible for the Na⁺/H⁺ antiporter system of alkalophilic Bacillus species strain C‐125

Abstract

An alkali-sensitive mutant, 38154, of the alkalophilic Bacillus sp. strain C-125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1-4). By subcloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutant's corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly-393 to Arg of the putative ORF1 product, which was deduced to be an 804-amino-acid polypeptide with a molecular weight of 89,070. The N-terminal part of the putative ORF1 product showed amino acid similarity to those of the chain-5 products of eukaryotic NADH quinone oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (delta psi)-driven Na+/H+ antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutant's alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+ antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+ antiporter system is important in the alkalophily of alkalophilic microorganisms.

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4

“…The putative antiporter gene from Bacil-lus sp. strain C-125 is a good candidate for a major role in alkaliphily, but it has yet to be shown to confer Na ϩ /H ϩ antiport activity when expressed alone in a homologous or heterologous deletion strain (16). Such a capacity has been shown for the nhaC gene from B. firmus OF4 (17), but nothing is yet known about the physiological role of this gene.…”

“…strain C-125 (16). Many of these already have obvious homologs in other genera (11,28,29,32,39), and more such homologs are emerging from genome sequencing projects.…”

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity, Sodium Exclusion, Individual Mrp Protein Levels, or Mrp Complex Formation

“…The coupled Na ϩ efflux activity of the antiporters supports Na ϩ resistance and produces an inwardly directed gradient of Na ϩ that drives solute transport and flagellar rotation in alkaliphilic bacteria (6 -9). Although extremely alkaliphilic Bacillus species contain multiple cation/proton antiporters (1,10), the Mrp-type antiporter has a dominant and essential role in alkaliphily (11,12). Mrp antiporters are also broadly distributed among non-alkaliphilic Gram-positive and Gram-negative bacteria and play roles in diverse physiological processes that may depend upon pH and Na ϩ homeostasis (2).…”

Transport of Na+ and K+ by an antiporter-related subunit from the Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae

“…Of the seven hydrophobic subunits comprising the membrane domain of NDH I, NuoL, NuoM and NuoN show sequence homology to subunit A from multicomponent cation/proton antiporters (Hamamoto et al 1994;Hägerhäll 2002, 2003). This evolutionary relationship makes NuoL a prime candidate for a Na + (or proton) channel through NDH I.…”