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ABSTRACT: Attempts to identify members of the antiporter complement of the alkali- and saline-adapted soda lake alkaliphile Alkalimonas amylolytica N10 have used screens of DNA libraries in antiporter-deficient Escherichia coli KNabc. Earlier screens used Na(+) or Li(+) for selection but only identified one NhaD-type antiporter whose properties were inconsistent with a robust role in pH homeostasis. Here, new screens using elevated pH for selection identified three other putative antiporter genes that conferred resistance to pH >or=8.5 as well as Na(+) resistance. The three predicted gene products were in the calcium/cation antiporter (CaCA), cation/proton antiporter-2 (CPA2) and cation/proton antiporter-1 (CPA1) families of membrane transporters, and were designated Aa-CaxA, Aa-KefB and Aa-NhaP respectively, reflecting homology within those families. Aa-CaxA conferred the poorest Na(+) resistance and also conferred modest Ca(2+) resistance. Aa-KefB and Aa-NhaP inhibited growth of a K(+) uptake-deficient E. coli mutant (TK2420), suggesting that they catalysed K(+) efflux. For Aa-NhaP, the reversibility of the growth inhibition by high K(+) concentrations depended upon an organic nitrogen source, e.g. glutamine, rather than ammonium. This suggests that as well as K(+) efflux is catalysed by Aa-NhaP. Vesicles of E. coli KNabc expressing Aa-NhaP, which conferred the strongest alkali resistance, exhibited K(+)/H(+) antiport activity in a pH range from 7.5 to 9.5, and with an apparent K(m) for K(+) of 0.5 mM at pH 8.0. The properties of this antiporter are consistent with the possibility that this soda lake alkaliphile uses K(+)( )/H(+) antiport as part of its alkaline pH homeostasis mechanism and part of its capacity to reduce potentially toxic accumulation of cytoplasmic K(+) or respectively, under conditions of high osmolarity or active amino acid catabolism.
Microbiology 08/2007; 153(Pt 7):2168-79. · 3.06 Impact Factor
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ABSTRACT: The growth properties of a new panel of Bacillus subtilis tetL deletion strains and of a derivative set of strains in which tetL is restored to the chromosome support earlier indications that deletion of tetL results in a range of phenotypes that are unrelated to tetracycline resistance. These phenotypes were not reversed by restoration of a tetL gene to its native locus and were hypothesized to result from secondary mutations that arise when multifunctional tetL is deleted. Such genetic changes would temper the alkali sensitivity and Na(+) sensitivity that accompany loss of the monovalent cation/proton activity of TetL. Microarray comparisons of the transcriptomes of wild-type B. subtilis, a tetL deletion strain, and its tetL-restored derivative showed that 37 up-regulated genes and 13 down-regulated genes in the deletion strain did not change back to wild-type expression patterns after tetL was returned to the chromosome. Up-regulation of the citM gene, which encodes a divalent metal ion-coupled citrate transporter, was shown to account for the Co(2+)-sensitive phenotype of tetL mutants. The changes in expression of citM and genes encoding other ion-coupled solute transporters appear to be adaptive to loss of TetL functions in alkali and Na(+) tolerance, because they reduce Na(+)-coupled solute uptake and enhance solute uptake that is coupled to H(+) entry.
Journal of Bacteriology 11/2006; 188(20):7090-100. · 3.83 Impact Factor
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ABSTRACT: In extreme alkaliphiles, Na(+)/H(+) antiporters play a central role in the Na(+) cycle that supports pH homeostasis, Na(+) resistance, solute uptake, and motility. Properties of individual antiporters have only been examined in extremely alkaliphilic soil Bacillus spp., whereas the most alkaline natural habitats usually couple high pH with high salinity. Here, studies were conducted on a Na(+)(Li(+))/H(+) antiporter, NhaD, from the soda lake haloalkaliphile Alkalimonas amylolytica. The activity profile of A. amylolytica NhaD at different pH values and Na(+) concentrations reflects its unique natural habitat. In membrane vesicles from antiporter-deficient Escherichia coli EP432 (DeltanhaA DeltanhaB), the pH optimum for NhaD-dependent Na(+)(Li(+))/H(+) antiport was at least 9.5, the highest pH that could be tested; no activity was observed at pH < or =8.5. NhaD supported low Na(+)/H(+) antiport activity at pH 9.5 that was detectable over a range of Na(+) concentrations from 10 mM to at least 800 mM, with a 600 mM optimum. Although A. amylolytica nhaD was isolated by complementing the Li(+) sensitivity of the triple mutant E. coli strain KNabc (DeltanhaA DeltanhaB DeltachaA), sustained propagation of nhaD-bearing plasmids in this strain resulted in a glycine (Gly(327))-->serine mutation in a putative cytoplasmic loop of the mutant transporter. The altered activity profile of NhaD-G327S appears to be adaptive to the E. coli setting: a much higher activity than wild-type NhaD at Na(+) concentrations up to 200 mM but lower activity at 400 to 600 mM Na(+), with a pH optimum and minimal pH for activity lower than those of wild-type NhaD.
Journal of Bacteriology 11/2005; 187(22):7589-95. · 3.83 Impact Factor
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ABSTRACT: The prokaryotic voltage-gated Na(+) channel, NaChBac, is one of a growing channel superfamily of unknown function. Here we show that Na(V)BP, the NaChBac homologue encoded by ncbA in alkaliphilic Bacillus pseudofirmus OF4, is a voltage-gated Na(+) channel potentiated by alkaline pH. Na(V)BP has roles in motility, chemotaxis, and pH homeostasis at high pH. Reduced motility of bacteria lacking functional Na(V)BP was reversed by restoration of the native channel but not by a mutant Na(V)BP engineered to be Ca(2+)-selective. Motile ncbA mutant cells and wild-type cells treated with a channel inhibitor exhibited behavior opposite to the wild type in response to chemoeffectors. Mutants lacking functional Na(V)BP were also defective in pH homeostasis in response to a sudden alkaline shift in external pH under conditions in which cytoplasmic [Na(+)] is limiting for this crucial process. The defect was exacerbated by mutation of motPS, the motility channel genes. We hypothesize that activation of Na(V)BP at high pH supports diverse physiological processes by a combination of direct and indirect effects on the Na(+) cycle and the chemotaxis system.
Proceedings of the National Academy of Sciences 08/2004; 101(29):10566-71. · 9.68 Impact Factor
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ABSTRACT: The atp operon of alkaliphilic Bacillus pseudofirmus OF4, as in most prokaryotes, contains the eight structural genes for the F-ATPase (ATP synthase), which are preceded by an atpI gene that encodes a membrane protein of unknown function. A tenth gene, atpZ, has been found in this operon, which is upstream of and overlapping with atpI. Most Bacillus species, and some other bacteria, possess atpZ homologues. AtpZ is predicted to be a membrane protein with a hairpin topology, and was detected by Western analyses. Deletion of atpZ, atpI, or atpZI from B. pseudofirmus OF4 led to a requirement for a greatly increased concentration of Mg2+ for growth at pH 7.5. Either atpZ, atpI, or atpZI complemented the similar phenotype of a triple mutant of Salmonella typhimurium (MM281), which is deficient in Mg2+ uptake. atpZ and atpI, separately and together, increased the Mg2+-sensitive 45Ca2+ uptake by vesicles of an Escherichia coli mutant that is defective in Ca2+ and Na+ efflux. We hypothesize that AtpZ and AtpI, as homooligomers, and perhaps as heterooligomers, are Mg2+ transporter, Ca2+ transporter, or channel proteins. Such proteins could provide Mg2+, which is required by ATP synthase, and also support charge compensation, when the enzyme is functioning in the hydrolytic direction; e.g., during cytoplasmic pH regulation.
Proceedings of the National Academy of Sciences 10/2003; 100(18):10213-8. · 9.68 Impact Factor
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ABSTRACT: A putative transport protein (Orf9) of alkaliphilic Bacillus pseudofirmus OF4 belongs to a transporter family (CPA-2) of diverse K+ efflux proteins and cation antiporters. Orf9 greatly increased the concentration of K+ required for growth of a K+ uptake mutant of Escherichia coli. The cytoplasmic K+ content of the cells was reduced, consistent with an efflux mechanism. Orf9-dependent translocation of K+ in E. coli is apparently bidirectional, since ammonium-sensitive uptake of K+ could be shown in K+ -depleted cells. The upstream gene product Orf8 has sequence similarity to a subdomain of KTN proteins that are associated with potassium-translocating channels and transporters; Orf8 modulated the transport capacities of Orf9. No Orf9-dependent K+(Na+)/H+ antiport activity was found in membrane vesicles. Nonpolar deletion mutants in the orf9 locus of the alkaliphile chromosome exhibited no K+ -related phenotype but showed profound phenotypes in medium containing high levels of amine-nitrogen. Their patterns of growth and ammonium content suggested a physiological role for the orf9 locus in bidirectional ammonium transport. Orf9-dependent ammonium uptake was observed in right-side-out membrane vesicles of the alkaliphile wild type and the mutant with an orf8 deletion. Uptake was proton motive force dependent and was inhibited by K+. Orf9 is proposed to be designated AmhT (ammonium homeostasis). Ammonium homeostasis is important in high-amine-nitrogen settings and is particularly crucial at high pH since cytosolic ammonium accumulation interferes with cytoplasmic pH regulation. Endospore formation in amino-acid-rich medium was significantly defective and germination was modestly defective in the orf9 and orf7-orf10 deletion mutants.
Journal of Bacteriology 10/2003; 185(17):5133-47. · 3.83 Impact Factor
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ABSTRACT: Members of the cation diffusion facilitator (CDF) family of membrane transport proteins are found in eukaryotes and prokaryotes. The family encompasses transporters of zinc ions, with cobalt, cadmium and lead ions being additional substrates for some prokaryotic examples. No transport mechanism has previously been established for any CDF protein. It is shown here that the CzcD protein of Bacillus subtilis, a CDF protein, uses an antiporter mechanism, catalysing active efflux of Zn2+ in exchange for K+ and H+. The exchange is probably electroneutral, energized by the transmembrane pH gradient and oppositely oriented gradients of the other cation substrates. The data suggest that Co2+ and Cd2+ are additional cytoplasmic substrates for CzcD. A second product of the same operon that encodes czcD has sequence similarity to oxidoreductases and is here designated CzcO. CzcO modestly enhances the activity of CzcD but is not predicted to be an integral membrane protein and has no antiport activity of its own.
Molecular Microbiology 08/2002; 45(1):145-53. · 5.01 Impact Factor
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ABSTRACT: The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of tet(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two- to threefold increase in tet(L) mRNA stability. Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a B. subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5' end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other B. subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.
Journal of Bacteriology 03/2002; 184(4):889-94. · 3.83 Impact Factor
