Halotolerant cyanobacterium Aphanothece halophytica contains an Na+-dependent F1F0-ATP synthase with a potential role in salt-stress tolerance

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H(+)-ATP synthase, Aphanothece halophytica contains a putative F(1)F(0)-type Na(+)-ATP synthase (ApNa(+)-ATPase) operon (ApNa(+)-atp). The operon consists of nine genes organized in the order of putative subunits β, ε, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa(+)-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa(+)-ATPase exhibited Na(+)-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na(+) ion protected the inhibition of ApNa(+)-ATPase by N,N'-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na(+)-loaded inverted membrane vesicles. Expression of the ApNa(+)-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na(+)-dependent F(1)F(0)-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.     

Translational coupling varying in efficiency between different pairs of genes in the central region of the atp operon of Escherichia coli

A series of atp::lacZ fusions has been constructed for use in a study of translational coupling in the central region of the Escherichia coli atp operon. Five genes, atpE, atpF, atpH, atpA and atpG, were shown to be translationally coupled to various degrees of tightness. A new lac promoter vector, compatible with the atp::lacZ fusion vectors, was used to express individual atp genes in the same hosts as the fusion genes. The H(+)-ATPase subunits thus synthesized exercised no significant trans-regulation on the expression of the atp::lacZ fusions, indicating that the coupling is primarily cis. The mechanism of this coupling was investigated using in vitro mutagenesis. At least in the case of the pair atpHA, coupling seems to involve facilitated binding of fresh ribosomes to the atpA translational initiation regions.     

The Na(+)-F(1)F(0)-ATPase operon from Acetobacterium woodii. Operon structure and presence of multiple copies of atpE which encode proteolipids of 8- and 18-kda

Eight genes (atpI, atpB, atpE(1), atpE(2), atpE(3), atpF, atpH, and atpA) upstream of and contiguous with the previously described genes atpG, atpD, and atpC were cloned from chromosomal DNA of Acetobacterium woodii. Northern blot analysis revealed that the eleven atp genes are transcribed as a polycistronic message. The atp operon encodes the Na(+)-F(1)F(0)-ATPase of A. woodii, as evident from a comparison of the biochemically derived N termini of the subunits with the amino acid sequences deduced from the DNA sequences. The molecular analysis revealed that all of the F(1)F(0)-encoding genes from Escherichia coli have homologs in the Na(+)-F(1)F(0)-ATPase operon from A. woodii, despite the fact that only six subunits were found in previous preparations of the enzyme from A. woodii. These results unequivocally prove that the Na(+)-ATPase from A. woodii is an enzyme of the F(1)F(0) class. Most interestingly, the gene encoding the proteolipid underwent quadruplication. Two gene copies (atpE(2) and atpE(3)) encode identical 8-kDa proteolipids. Two additional gene copies were fused to form the atpE(1) gene. Heterologous expression experiments as well as immunolabeling studies with native membranes revealed that atpE(1) encodes a duplicated 18-kDa proteolipid. This is the first demonstration of multiplication and fusion of proteolipid-encoding genes in F(1)F(0)-ATPase operons. Furthermore, AtpE(1) is the first duplicated proteolipid ever found to be encoded by an F(1)F(0)-ATPase operon.     

Complete DNA sequence of the atp operon of the sodium-dependent F1Fo ATP synthase from Ilyobacter tartaricus and identification of the encoded subunits

The atp operon of Ilyobacter tartaricus, strain DSM 2382, was completely sequenced using conventional and inverse polymerase chain reaction (i-PCR) techniques. It contains nine open reading frames that were attributed to eight structural genes of the F(1)F(o) ATP synthase and the atpI gene, which is not part of the enzyme complex. The initiation codons of all atp genes, except that of atpB coding for the a subunit, were identified by the corresponding N-terminal amino acid sequence. The hydrophobic a subunit was identified by MALDI mass spectrometry. The atp genes of I. tartaricus are arranged in one operon with the sequence atpIBEFHAGDC comprising 6,992 base pairs with a GC content of 38.1%. The F(1)F(o) ATP synthase of I. tartaricus has a calculated molecular mass of 510 kDa and includes 4,810 amino acids. The gene sequences and products reveal significant identities to atp genes of other Na(+)-translocating F(1)F(o) ATP synthases, especially in the F(o) subunits a and c which are directly involved in ion translocation.     

Mutant residues suppressing rho(0)-lethality in Kluyveromyces lactis occur at contact sites between subunits of F(1)-ATPase

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.     

Gene structure of Enterococcus hirae (Streptococcus faecalis) F1F0-ATPase, which functions as a regulator of cytoplasmic pH.

Enterococcus hirae (formerly Streptococcus faecalis) ATCC 9790 has an F1F0-ATPase which functions as a regulator of the cytoplasmic pH but does not synthesize ATP. We isolated four clones which contained genes for c, b, delta, and alpha subunits of this enzyme but not for other subunit genes. It was revealed that two specific regions (upstream of the c-subunit gene and downstream of the gamma-subunit gene) were lost at a specific site in the clones we isolated, suggesting that these regions were unstable in Escherichia coli. The deleted regions were amplified by polymerase chain reaction, and the nucleotide sequences of these regions were determined. The results showed that eight genes for a, c, b, delta, alpha, gamma, beta, and epsilon subunits were present in this order. Northern (RNA) blot analysis showed that these eight genes were transcribed to one mRNA. The i gene was not found in the upper region of the a-subunit gene. Instead of the i gene, this operon contained a long untranslated region (240 bp) whose G + C content was only 30%. There was no typical promoter sequence such as was proposed for E. coli, suggesting that the promoter structure of this species is different from that of E. coli. Deduced amino acid sequences suggested that E. hirae H(+)-ATPase is a typical F1F0-type ATPase but that its gene structure is not identical to that of other bacterial F1F0-ATPases.     

Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants.

We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase. The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species. The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms. We constructed two B. subtilis strains from which different parts of the atp operon were deleted. These B. subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source. ATP was synthesized in these strains only by substrate-level phosphorylation. The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio. In the absence of oxidative phosphorylation, B. subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate). The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes. It therefore appears that B. subtilis and E. coli respond in similar ways to the absence of oxidative phosphorylation.     

The F1-ATPase inhibitor Inh1 (IF1) affects suppression of mtDNA loss-lethality in Kluyveromyces lactis

Loss of mtDNA by the petite-negative yeast Kluyveromyces lactis is lethal (rho(o)-lethality). However, mutations in the alpha, beta and gamma subunits of F(1)-ATPase can suppress lethality by increasing intramitochondrial hydrolysis of ATP. Increased hydrolysis of ATP can also occur on inactivation of Inh1, the natural inhibitor of F(1)-ATPase. However, not all strains of K. lactis show suppression of rho(o)-lethality on inactivation of INH1. Genetic analysis indicates that one or more alleles of modifying factors are required for suppression. Papillae showing enhanced resistance to ethidium bromide (EB) in INH1 disruptants have mutations in the alpha, beta and gamma subunits of F(1)-ATPase. Increased growth of double mutants on EB has been investigated by disruption of INH1 in previously characterized atp suppressor mutants. Inactivation of Inh1, with one exception, results in better growth on EB and increased F(1)-ATPase activity, indicating that suppression of rho(o)-lethality is not due to atp mutations preventing Inh1 from interacting with the F(1)-complex. By contrast, in suppressor mutants altered in Arg435 of the beta subunit, disruption of INH1 did not change the kinetic properties of F(1)-ATPase or alter growth on EB. Consequently, Arg435 appears to be required for interaction of Inh1 with the beta subunit. In a previous study, a mex1-1 allele was found to enhance mgi(atp) expression. In accord with results from double mutants, it has been found that mex1-1 is a frameshift mutation in INH1 causing inactivation of Inh1p.     

Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3.

The lantibiotic nisin is produced by several strains of Lactococcus lactis. The complete gene cluster for nisin biosynthesis in L. lactis 6F3 comprises 15 kb of DNA. As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK. Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK. Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified. One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L. lactis 6F3 have now been identified. Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins. Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin. However, in comparison with the wild-type strain, these mutants were more sensitive to nisin. This indicates that besides nisI the newly identified genes are also involved in immunity to nisin. The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.     

Identification of subunits a, b, and c1 from Acetobacterium woodii Na+-F1F0-ATPase. Subunits c1, c2, AND c3 constitute a mixed c-oligomer

The Na(+)-F(1)F(0)-ATPase operon of Acetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunit a and b, a gene encoding a 16-kDa proteolipid (subunit c(1)), and two genes encoding 8-kDa proteolipids (subunits c(2) and c(3)). Because subunits a, b, and c(1) were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a, b, and c(1) are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunits a, c(2), c(3), b, delta, alpha, gamma, beta, and epsilon. Biochemical and immunological analyses revealed that subunits c(1), c(2), and c(3) are all part of the c-oligomer, the first of a F(1)F(0)-ATPase that contains 8- and 16-kDa proteolipids.