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Terry A. Krulwich - One of the best experts on this subject based on the ideXlab platform.
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the c ring stoichiometry of atp synthase is adapted to cell physiological requirements of alkaliphilic bacillus pseudofirmus of4
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Laura Preiss, Terry A. Krulwich, Oliver J Fackelmayer, David Hicks, Adriana L Klyszejko, O Yildiz, Thomas MeierAbstract:The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.
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mutations in a helix 1 motif of the atp synthase c subunit of bacillus pseudofirmus of4 cause functional deficits and changes in the c ring stability and mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis
Biochemistry, 2011Co-Authors: Jun Liu, Oliver J Fackelmayer, David Hicks, Laura Preiss, Thomas Meier, Eric A Sobie, Terry A. KrulwichAbstract:The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have a GxGxGxG motif. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for nonfermentative growth and, for most of the mutants, on ATP synthesis by ADP- and Pi-loaded membrane vesicles at pH 7.5 and 10.5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. Via SDS–PAGE, most of the mutant c-monomers exhibited increased mobility relative to the wild-type (WT) c-subunit, but among the i...
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three two component transporters with channel like properties have monovalent cation proton antiport activity
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Makoto Fujisawa, Terry A. KrulwichAbstract:Properties of four two-component bacterial transport systems of the cation/proton antiporter-2 (CPA2) family led to suggestions that this CPA2 subset may use a channel rather than an antiport mechanism [see Booth IR, Edwards MD, Gunasekera B, Li C, Miller S (2005) in Bacterial Ion Channels, eds Kubalski A, Martinac B (Am Soc Microbiol, Washington, DC), pp 21–40]. The transporter subset includes the intensively studied glutathione-gated K+ efflux systems from Escherichia coli, KefGB, and KefFC. KefG and KefF are ancillary proteins. They are peripheral membrane proteins that are encoded in operons with the respective transporter proteins, KefB and KefC, and are required for optimal efflux activity. The other two-component CPA2 transporters of the subset are AmhMT, an NH4+ (K+) efflux system from alkaliphilic Bacillus pseudofirmus OF4; and YhaTU, a K+ efflux system from Bacillus subtilis. Here a K+/H+ antiport capacity was demonstrated for YhaTU, AmhMT, and KefFC in membrane vesicles from antiporter-deficient E. coli KNabc. The apparent Km for K+ was in the low mM range. The peripheral protein was required for YhaU- and KefC-dependent antiport, whereas both AmhT and AmhMT exhibited antiport. KefFC had the broadest range of substrates, using Rb+≈K+>Li+>Na+. Glutathione significantly inhibited KefFC-mediated K+/H+ antiport in vesicles. The inhibition was enhanced by NADH, which presumably binds to the KTN/RCK domain of KefC. The antiport mechanism accounts for the H+ uptake involved in KefFC-mediated electrophile resistance in vivo. Because the physiological substrate of AmhMT in the alkaliphile is NH4+, the results also imply that AmhMT catalyzes NH4+/H+ antiport, which would prevent net cytoplasmic H+ loss during NH4+ efflux.
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three putative cation proton antiporters from the soda lake alkaliphile alkalimonas amylolytica n10 complement an alkali sensitive escherichia coli mutant
Microbiology, 2007Co-Authors: Yi Wei, Jun Liu, Terry A. KrulwichAbstract: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 ≥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 Ca2+ 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.
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effects of nonpolar mutations in each of the seven bacillus subtilis mrp genes suggest complex interactions among the gene products in support of na and alkali but not cholate resistance
Journal of Bacteriology, 2000Co-Authors: Arthur A. Guffanti, Wei Wang, Terry A. KrulwichAbstract:The mrp operon was first identified in the genome of Bacillus subtilis as a homologue of a locus that had been found to be centrally important to cytoplasmic pH regulation in alkaliphilic Bacillus halodurans C-125 (3, 16). A point mutation in the first gene of the alkaliphile homologue resulted in loss of Na+/H+ antiporter activity (3). Such antiport is widely used by prokaryotes for alkali and Na+ resistance inasmuch as coupled Na+ exclusion and H+ accumulation can be accomplished via electrogenic exchange of cytoplasmic Na+ for a greater number of H+ (14, 20). The complete mrp operon of B. subtilis is predicted to encode seven hydrophobic gene products (9, 12), as is also posited for homologues from diverse organisms, including alkaliphilic Bacillus pseudofirmus OF4 (14, 15), Rhizobium meliloti (21), Staphylococcus aureus (6), and others annotated in genome databases. Apart from the apparent role of the alkaliphile mrp operons in Na+-dependent pH homeostasis, studies with mutants have suggested that the R. meliloti homologue, pha, may encode a K+/H+ antiporter that is required for symbiotic nitrogen fixation (21) and that B. subtilis mrp (called ntr and sha by other investigators [12, 13]) has multiple functions. First, the B. subtilis mrp locus has been shown to play a role in Na+ resistance and in both Na+- and K+-dependent cytoplasmic pH homeostasis (9, 12). This is consistent with one or more mrp genes encoding an Na+(K+)/H+ antiport activity. Recently, Kosono et al. (13) showed that a B. subtilis mrpA (shaA) mutant fails to sporulate normally and suggested that an early step in sporulation is sensitive to the elevated cytoplasmic Na+ concentration that results from mrp mutations. The second B. subtilis mrp activity, in which the mrpF gene has been implicated, encompasses cholate and Na+ efflux activities, which may be mechanistically coupled. Demonstration of cholate efflux activity has thus far been made only in a mutant with a disruption in mrpF that also lowered expression of mrpG (9), but in the current study, separate mutations in mrpF and mrpG have been examined. Before the discovery of the mrp operon and its homologues, the well-studied examples of Na+/H+ antiporters all involved a single structural gene product (20). Data to date suggest that monovalent cation/H+ antiporter activity requires the first gene of the operon, mrpA, in B. subtilis, but that other genes of the operon are required for some combination of antiporter activity, expression, and assembly (9, 12). That is, MrpA is necessary but not sufficient for Na+/H+ activity. Similarly, Hiramatsu et al. (6) have suggested from studies in which the S. aureus homologue, designated mnh, was expressed in an Na+-sensitive Escherichia coli mutant, that all the genes of the operon may be required for the Na+ resistance conferred in that system. There are recent reports of secondary multidrug transporters with two heterologous protein components (10, 18) but the complexity of the mrp product interactions might be of a much higher order. In addition, the long-recognized sequence similarity of several mrp products to membrane-embedded subunits of energy-coupled NADH dehydrogenase complexes (3, 9) raises the possibility that there is a capacity for electron transport that could provide a primary energy coupling option for mrp functions. In the current study, individual in-frame deletions were made in each of the B. subtilis mrp genes for which no such mutations had been made earlier, i.e., mrpB, -C, -D, -E, -F, and -G. For each of those strains, a version was also made in which an active copy of the disrupted gene was returned to the amyE locus of the chromosome under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. Each mrp gene was similarly introduced into the amyE locus of an mrp null mutant, VKN1, of B. subtilis, and into that of a polar mutant, VK6, that lacks mrpA and expresses mrpB to -G at greatly reduced levels. Resistance and transport studies have supported earlier indications that MrpF is the Na+-cholate efflux protein and further show that MrpF activity is independent of the expression of additional mrp genes. By contrast, MrpA function, which is shown to correlate with a protonophore-sensitive Na+ efflux activity, requires all six other mrp genes. In addition, evidence is presented for a complex regulatory relationship between loss of function of particular mrp genes and expression of the polycistronic mrp mRNA.
David Hicks - One of the best experts on this subject based on the ideXlab platform.
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the c ring stoichiometry of atp synthase is adapted to cell physiological requirements of alkaliphilic bacillus pseudofirmus of4
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Laura Preiss, Terry A. Krulwich, Oliver J Fackelmayer, David Hicks, Adriana L Klyszejko, O Yildiz, Thomas MeierAbstract:The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.
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mutations in a helix 1 motif of the atp synthase c subunit of bacillus pseudofirmus of4 cause functional deficits and changes in the c ring stability and mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis
Biochemistry, 2011Co-Authors: Jun Liu, Oliver J Fackelmayer, David Hicks, Laura Preiss, Thomas Meier, Eric A Sobie, Terry A. KrulwichAbstract:The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have a GxGxGxG motif. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for nonfermentative growth and, for most of the mutants, on ATP synthesis by ADP- and Pi-loaded membrane vesicles at pH 7.5 and 10.5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. Via SDS–PAGE, most of the mutant c-monomers exhibited increased mobility relative to the wild-type (WT) c-subunit, but among the i...
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purification of a cytochrome bd terminal oxidase encoded by the escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from bacillus firmus of4
Journal of Bacteriology, 1996Co-Authors: Michael G Sturr, Terry A. Krulwich, David HicksAbstract:Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and ctyochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3.4-kb region of DNA from alkaliphilic Bacillus firmus OF4. The complementing DNA was predicted to encode five proteins, but neither sequence analysis nor complementation experiments with subclones provided insight into the basis for the complementation. Cytochrome difference spectra of everted membrane vesicles from the transformed strain had characteristics of a cytochrome bd spectrum but with features different from those observed for alkaliphile membranes. To determine the bacterial source and identity of the structural genes for the cytochrome bd in the transformed mutant, the complex was extracted and partially purified. On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa. An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E. coli appC. Consistent with the microsequencing results pMS100 failed to complement a triple mutant of E. coli carrying a deletion in appB as well as in the cyo and cyd loci. The deduced sequence of AppBC had been predicted to be very similar to the sequence of CydAB (J. Dassa et al., Mol. Gen. Genet. 229:341-352, 1991) but this is the first demonstration that the former is indeed a cytochrome bd terminal oxidase. The enzyme catalyzed oxygen uptake coupled to quinol or N,N,N9,N9-tetramethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide. No cross-reactivity to subunit-specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrome bd was detected. Since this is the second cytochrome bd discovered in E. coli, it is proposed that the two complexes be designated cytochrome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded enzyme). In addition, cbdAB is suggested as a more appropriate gene designation for cytochrome bd than either appBC or cyxAB.
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purification of three catalase isozymes from facultatively alkaliphilic bacillus firmus of4
Biochimica et Biophysica Acta, 1995Co-Authors: David HicksAbstract:Cell extracts of facultatively alkaliphilic B. firmus OF4 were assayed for catalase activity and their catalase isozyme content was analyzed on native polyacrylamide gels stained for catalase activity. pH-10.5-grown cells had about twice the specific catalase activity of pH-7.5-grown cells. The higher activity, however, did not confer resistance to exogenous hydrogen peroxide challenge relative to pH-7.5-grown cells and, in fact, the pH-10.5-grown cells were much more sensitive to the challenge. Electrophoresis resolved three catalase isozymes in cell extracts. The isozymes, labeled I–III in order of decreasing electrophoretic mobility, were purified and their Nterminal amino acid sequences were obtained. Isozyme III corresponded to the product of a cloned gene fragment that had been shown to possess substantial sequence similarity to the KatE (HP-II) catalase of E. coli (Quirk, P.G., Krulwich, T.A. and Hicks, D.B. (1993) Biophys. J. 64, 164A) and which had similar biochemical properties to HP-II, i.e., it was a chlorin-containing enzyme expressed only in stationary phase. Isozyme II, a protoheme enzyme, was responsible for the higher activity of alkaline-grown cells and was induced in cells treated with hydrogen peroxide or ascorbate. It showed sequence similarity to katA of Bacillus subtilis (Bol, D. and Yasbin, R. (1991) Gene 109, 31–37). Isozyme I was the only isozyme that exhibited detectable levels of peroxidase activity in addition to catalase activity, resembling a catalase enzyme purified from a different alkaliphile, Bacillus YN-2000 (Yumoto, I., Fukumori, Y. and Yamanaka, T. (1990) J. Biochem. 108, 583–587), to which it showed some sequence similarity.
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evidence for multiple terminal oxidases including cytochrome d in facultatively alkaliphilic bacillus firmus of4
Journal of Bacteriology, 1991Co-Authors: David Hicks, R J Plass, P G QuirkAbstract:The terminal oxidase content of Bacillus firmus OF4, a facultative alkaliphile that grows well over the pH range of 7.5 to 10.5, was studied by difference spectroscopy. Evidence was found for three terminal oxidases under different growth conditions. The growth pH and the stage of growth profoundly affected the expression of one of the oxidases, cytochrome d. The other two oxidases, cytochrome caa3 and cytochrome o, were expressed under all growth conditions tested, although the levels of both, especially cytochrome caa3, were higher at more alkaline pH (P.G. Quirk, A.A. Guffanti, R.J. Plass, S. Clejan, and T.A. Krulwich, Biochim. Biophys. Acta, in press). These latter oxidases were identified in everted membrane vesicles by reduced-versus-oxidized difference spectra (absorption maximum at 600 nm for cytochrome caa3) and CO-reduced-versus-reduced difference spectra (absorption maxima at 574 and 414 nm for cytochrome o). All three terminal oxidases were solubilized from everted membranes and partially purified. The difference spectra of the solubilized, partially purified cytochrome caa3 and cytochrome o complexes were consistent with these assignments. Cytochrome d, which has not been identified in a Bacillus species before, was tentatively assigned on the basis of its absorption maxima at 622 and 630 nm in reduced-versus-oxidized and CO-reduced-versus-reduced difference spectra, respectively, resembling the maxima exhibited by the complex found in Escherichia coli. The B. firmus OF4 cytochrome d was reducible by NADH but not by ascorbate-N,N,N9,N9-tetramethyl-p-phenylenediamine in everted membrane vesicles. Cytochrome d was expressed under two conditions: in cells growing exponentially at pH 7.5 (but not at pH 10.5) and in cells stationary phase at either pH 7.5 or 10.5. Protein immunoblots with antibodies against subunit I of the E. coli cytochrome d complex reacted only with membrane vesicles that contained spectrally identifiable cytochrome d. Additional evidence that this B. firmus OF4 cytochrome is related to the E. coli complex was obtained with a solubilized, partially purified fraction of cytochrome d that also reacted with antibodies against the subunits of the E. coli cytochrome d. Images
Arthur A. Guffanti - One of the best experts on this subject based on the ideXlab platform.
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effects of nonpolar mutations in each of the seven bacillus subtilis mrp genes suggest complex interactions among the gene products in support of na and alkali but not cholate resistance
Journal of Bacteriology, 2000Co-Authors: Arthur A. Guffanti, Wei Wang, Terry A. KrulwichAbstract:The mrp operon was first identified in the genome of Bacillus subtilis as a homologue of a locus that had been found to be centrally important to cytoplasmic pH regulation in alkaliphilic Bacillus halodurans C-125 (3, 16). A point mutation in the first gene of the alkaliphile homologue resulted in loss of Na+/H+ antiporter activity (3). Such antiport is widely used by prokaryotes for alkali and Na+ resistance inasmuch as coupled Na+ exclusion and H+ accumulation can be accomplished via electrogenic exchange of cytoplasmic Na+ for a greater number of H+ (14, 20). The complete mrp operon of B. subtilis is predicted to encode seven hydrophobic gene products (9, 12), as is also posited for homologues from diverse organisms, including alkaliphilic Bacillus pseudofirmus OF4 (14, 15), Rhizobium meliloti (21), Staphylococcus aureus (6), and others annotated in genome databases. Apart from the apparent role of the alkaliphile mrp operons in Na+-dependent pH homeostasis, studies with mutants have suggested that the R. meliloti homologue, pha, may encode a K+/H+ antiporter that is required for symbiotic nitrogen fixation (21) and that B. subtilis mrp (called ntr and sha by other investigators [12, 13]) has multiple functions. First, the B. subtilis mrp locus has been shown to play a role in Na+ resistance and in both Na+- and K+-dependent cytoplasmic pH homeostasis (9, 12). This is consistent with one or more mrp genes encoding an Na+(K+)/H+ antiport activity. Recently, Kosono et al. (13) showed that a B. subtilis mrpA (shaA) mutant fails to sporulate normally and suggested that an early step in sporulation is sensitive to the elevated cytoplasmic Na+ concentration that results from mrp mutations. The second B. subtilis mrp activity, in which the mrpF gene has been implicated, encompasses cholate and Na+ efflux activities, which may be mechanistically coupled. Demonstration of cholate efflux activity has thus far been made only in a mutant with a disruption in mrpF that also lowered expression of mrpG (9), but in the current study, separate mutations in mrpF and mrpG have been examined. Before the discovery of the mrp operon and its homologues, the well-studied examples of Na+/H+ antiporters all involved a single structural gene product (20). Data to date suggest that monovalent cation/H+ antiporter activity requires the first gene of the operon, mrpA, in B. subtilis, but that other genes of the operon are required for some combination of antiporter activity, expression, and assembly (9, 12). That is, MrpA is necessary but not sufficient for Na+/H+ activity. Similarly, Hiramatsu et al. (6) have suggested from studies in which the S. aureus homologue, designated mnh, was expressed in an Na+-sensitive Escherichia coli mutant, that all the genes of the operon may be required for the Na+ resistance conferred in that system. There are recent reports of secondary multidrug transporters with two heterologous protein components (10, 18) but the complexity of the mrp product interactions might be of a much higher order. In addition, the long-recognized sequence similarity of several mrp products to membrane-embedded subunits of energy-coupled NADH dehydrogenase complexes (3, 9) raises the possibility that there is a capacity for electron transport that could provide a primary energy coupling option for mrp functions. In the current study, individual in-frame deletions were made in each of the B. subtilis mrp genes for which no such mutations had been made earlier, i.e., mrpB, -C, -D, -E, -F, and -G. For each of those strains, a version was also made in which an active copy of the disrupted gene was returned to the amyE locus of the chromosome under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. Each mrp gene was similarly introduced into the amyE locus of an mrp null mutant, VKN1, of B. subtilis, and into that of a polar mutant, VK6, that lacks mrpA and expresses mrpB to -G at greatly reduced levels. Resistance and transport studies have supported earlier indications that MrpF is the Na+-cholate efflux protein and further show that MrpF activity is independent of the expression of additional mrp genes. By contrast, MrpA function, which is shown to correlate with a protonophore-sensitive Na+ efflux activity, requires all six other mrp genes. In addition, evidence is presented for a complex regulatory relationship between loss of function of particular mrp genes and expression of the polycistronic mrp mRNA.
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role of the nhac encoded na h antiporter of alkaliphilic bacillus firmus of4
Journal of Bacteriology, 1997Co-Authors: Masahiro Ito, Arthur A. Guffanti, D M Ivey, J Zemsky, Terry A. KrulwichAbstract:Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.
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oxidative phosphorylation by adp p i loaded membrane vesicles of alkaliphilic bacillus firmus of4
Journal of Biological Chemistry, 1994Co-Authors: Arthur A. Guffanti, Terry A. KrulwichAbstract:ATP synthesis in ADP + P(i)-loaded membrane vesicles of the facultative alkaliphile Bacillus firmus OF4 at an external pH of 10.5 did not depend upon the presence of cell wall polymers, e.g. as a proton barrier or sequestration device. Upon energization with ascorbate plus phenazine methosulfate, vesicles at pH(out) = 7.5 generated an electrochemical proton gradient (delta p) of -160 mV, acid and positive out, whereas at pH(out) = 10.5, the delta p was -40 mV, alkaline and positive out. Nonetheless, ATP synthesis was more rapid at the more alkaline pH value, especially in the presence of 200 mM K2SO4, which markedly lowered the surface potential. No synthesis was observed upon abolition of the delta p. Respiration-derived transmembrane potentials (delta psi) energized ATP synthesis much better than an equally large diffusion potential. The diffusion potential failed to energize ATP synthesis above pH 9.5. When delta p, all in the form of a delta psi, was titrated downward at either pH 7.8 or 9.5, ATP synthesis by the latter vesicles was much less adversely affected in the delta p range of -150 to -50 mV, supporting the existence of a sparing, non-chemiosmotic energy component at high pH.
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the cadc gene product of alkaliphilic bacillus firmus of4 partially restores na resistance to an escherichia coli strain lacking an na h antiporter nhaa
Journal of Bacteriology, 1992Co-Authors: D M Ivey, Arthur A. Guffanti, Z Shen, N Kudyan, Terry A. KrulwichAbstract:Abstract A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.
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protonophore resistance and cytochrome expression in mutant strains of the facultative alkaliphile bacillus firmus of4
Biochimica et Biophysica Acta, 1991Co-Authors: P G Quirk, Arthur A. Guffanti, Robert J Plass, Sanda Clejan, Terry A. KrulwichAbstract:Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m -chlorophenylhydrazone (CCCP) to collapse the protonmotive force ( Δ μ ¯ H + ) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa 3 , b, c and o in strain CC2. Strain CC2 also maintained a higher Δ μ ¯ H + than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of Δ μ ¯ H + than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o . Cells of strain CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium , neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of Δ μ ¯ H + . Instead, both resistant alkaliphile strains maintained a higher Δ μ ¯ H + and a correspondingly higher Δ G p than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa 3 -type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk Δ μ ¯ H + .
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role of the nhac encoded na h antiporter of alkaliphilic bacillus firmus of4
Journal of Bacteriology, 1997Co-Authors: Masahiro Ito, Arthur A. Guffanti, D M Ivey, J Zemsky, Terry A. KrulwichAbstract:Application of protoplast transformation and single- and double-crossover mutagenesis protocols to alkaliphilic Bacillus firmus OF4811M (an auxotrophic strain of B. firmus OF4) facilitated the extension of the sequence of the previously cloned nhaC gene, which encodes an Na+/H+ antiporter, and the surrounding region. The nhaC gene is part of a likely 2-gene operon encompassing nhaC and a small gene that was designated nhaS; the operon is preceded by novel direct repeats. The predicted alkaliphile NhaC, based on the extended sequence analysis, would be a membrane protein with 462 amino acid residues and 12 transmembrane segments that is highly homologous to the deduced products of homologous genes of unknown function from Bacillus subtilis and Haemophilus influenzae. The full-length version of nhaC complemented the Na+-sensitive phenotype of an antiporter-deficient mutant strain of Escherichia coli but not the alkali-sensitive growth phenotypes of Na+/H+-deficient mutants of either alkaliphilic B. firmus OF4811M or B. subtilis. Indeed, NhaC has no required role in alkaliphily, inasmuch as the nhaC deletion strain of B. firmus OF4811M, N13, grew well at pH 10.5 at Na+ concentrations equal to or greater than 10 mM. Even at lower Na+ concentrations, N13 exhibited only a modest growth defect at pH 10.5. This was accompanied by a reduced capacity to acidify the cytoplasm relative to the medium compared to the wild-type strain or to N13 complemented by cloned nhaC. The most notable deficiency observed in N13 was its poor growth at pH 7.5 and Na+ concentrations up to 25 mM. During growth at pH 7.5, NhaC is apparently a major component of the relatively high affinity Na+/H+ antiport activity available to extrude the Na+ and to confer some initial protection in the face of a sudden upshift in external pH, i.e., before full induction of additional antiporters. Consistent with the inference that NhaC is a relatively high affinity, electrogenic Na+/H+ antiporter, N13 exhibited a defect in diffusion potential-energized efflux of 22Na+ from right-side-out membrane vesicles from cells that were preloaded with 2 mM Na+ and energized at pH 7.5. When the experiment was conducted with vesicles loaded with 25 mM Na+, comparable efflux was observed in preparations from all the strains.
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the cadc gene product of alkaliphilic bacillus firmus of4 partially restores na resistance to an escherichia coli strain lacking an na h antiporter nhaa
Journal of Bacteriology, 1992Co-Authors: D M Ivey, Arthur A. Guffanti, Z Shen, N Kudyan, Terry A. KrulwichAbstract:Abstract A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes that are apparent homologs of the cadA and cadC genes of cadmium resistance-conferring plasmid pI258 of Staphylococcus aureus. E. coli NM81 transformed with pJB22 had enhanced membrane Na+/H+ antiporter activity that was cold labile and that decreased very rapidly following isolation of everted vesicles. Subclones of pJB22 containing cadC as the only intact gene showed identical complementation patterns in vivo and in vitro. The cadC gene product of S. aureus has been proposed to act as an accessory protein for the Cd2+ efflux ATPase (CadA) (K. P. Yoon and S. Silver, J. Bacteriol. 173:7636-7642, 1991); perhaps the alkaliphile CadC also binds Na+ and enhances antiporter activity by delivering a substrate to an integral membrane antiporter. A 6.0-kb fragment overlapping the pJB22 insert was isolated to complete the sequence of the cadA homolog. A partial sequence of a region approximately 2 kb downstream of the cadA locus shares sequence similarity with plasmids from several gram-positive bacteria. These results suggest that the region of alkaliphile DNA containing the cadCA locus is present on a transposon that could reside on a heretofore-undetected endogenous plasmid.
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molecular cloning and sequencing of a gene from alkaliphilic bacillus firmus of4 that functionally complements an escherichia coli strain carrying a deletion in the nhaa na h antiporter gene
Journal of Biological Chemistry, 1991Co-Authors: D M Ivey, Arthur A Guffanti, J S Bossewitch, Etana Padan, Terry A. KrulwichAbstract:Abstract A gene has been cloned from a DNA library from alkaliphilic Bacillus firmus OF4 that functionally complements a mutant strain of Escherichia coli, NM81, that carries a deletion for one of that strain's Na+/H+ antiporter genes (delta nhaA). The cloned alkaliphile gene restored to NM81 the ability to grow at pH 7.5 in the presence of 0.6 M NaCl and on 100 mM Li+ in the presence of melibiose, and concomitantly led to an increase in the membrane associated Na+/H+ antiport activity. The biologically active alkaliphile DNA was identified as an incomplete open reading frame, the sequence of which would encode a hydrophobic protein. The insert was used to isolate clones containing the complete open reading frame, which would be predicted to encode a protein with a molecular weight of 42,960 and multiple membrane spanning regions. When the open reading frame was expressed under the control of the T7 promoter, the gene product was localized in the membrane. Southern analysis indicated no homology between the alkaliphile gene, which we propose to call nhaC, and the nhaA gene of Escherichia coli, nor with other genes in digests of DNA from E. coli, Bacillus subtilis, or Bacillus alcalophilus. Although there was also no significant similarity between the deduced protein products of the alkaliphile gene and the nhaA gene of E. coli, there was a small region of significant similarity between the deduced alkaliphile gene product and the protein encoded by a human Na+/H+ antiporter gene (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280).
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sequence analysis and functional studies of a chromosomal region of alkaliphilic bacillus firmus of4 encoding an abc type transporter with similarity of sequence and na exclusion capacity to the bacillus subtilis natab transporter
Extremophiles, 1999Co-Authors: Yi Wei, Arthur A Guffanti, Terry A. KrulwichAbstract:A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na+ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na+ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na+/H+ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs.
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ph tolerance in bacillus alkaliphiles versus non alkaliphiles
Novartis Foundation Symposium 221 - Bacterial Responses to Ph, 1999Co-Authors: Terry A. Krulwich, Arthur A Guffanti, Masahiro ItoAbstract:Monovalent cation/proton antiporters that catalyse electrogenic uptake of H+ in exchange for cytoplasmic K+ and/or Na+ are centrally involved in bacterial pH homeostasis under alkaline challenge. Systematic attempts have identified some, but not yet all, of the genes encoding such antiporters that participate in pH homeostasis in the neutrophilic Bacillus subtilis and the extremely alkaliphilic Bacillus firmus OF4. In each organism there are at least three distinct antiporters involved in pH homeostasis. They differ in cation requirement, with pH homeostasis specifically utilizing Na+/H+ antiport in the alkaliphile and using either Na+ or K+/H+ antiport in B. subtilis. Some of the antiporters involved in pH homeostasis are constitutive and are in place to respond to sudden pH shifts, but there is also an inducible component. At least two sets of homologous antiporters (NhaC and Mrp/Pha) function in both alkaliphiles and neutrophiles. An additional antiporter of a different transport protein family, the Gram-positive tetracycline-metal/H+ antiporter, is important in pH homeostasis in B. subtilis but has not yet been shown to be present in any alkaliphile. There are also differences outside of the antiporters themselves that contribute to the greater capacity of the alkaliphiles for pH homeostasis, including cation re-entry capacity and possible surface properties.
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molecular cloning and sequencing of a gene from alkaliphilic bacillus firmus of4 that functionally complements an escherichia coli strain carrying a deletion in the nhaa na h antiporter gene
Journal of Biological Chemistry, 1991Co-Authors: D M Ivey, Arthur A Guffanti, J S Bossewitch, Etana Padan, Terry A. KrulwichAbstract:Abstract A gene has been cloned from a DNA library from alkaliphilic Bacillus firmus OF4 that functionally complements a mutant strain of Escherichia coli, NM81, that carries a deletion for one of that strain's Na+/H+ antiporter genes (delta nhaA). The cloned alkaliphile gene restored to NM81 the ability to grow at pH 7.5 in the presence of 0.6 M NaCl and on 100 mM Li+ in the presence of melibiose, and concomitantly led to an increase in the membrane associated Na+/H+ antiport activity. The biologically active alkaliphile DNA was identified as an incomplete open reading frame, the sequence of which would encode a hydrophobic protein. The insert was used to isolate clones containing the complete open reading frame, which would be predicted to encode a protein with a molecular weight of 42,960 and multiple membrane spanning regions. When the open reading frame was expressed under the control of the T7 promoter, the gene product was localized in the membrane. Southern analysis indicated no homology between the alkaliphile gene, which we propose to call nhaC, and the nhaA gene of Escherichia coli, nor with other genes in digests of DNA from E. coli, Bacillus subtilis, or Bacillus alcalophilus. Although there was also no significant similarity between the deduced protein products of the alkaliphile gene and the nhaA gene of E. coli, there was a small region of significant similarity between the deduced alkaliphile gene product and the protein encoded by a human Na+/H+ antiporter gene (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280).