atpC Gene Research Articles

Clinical optochin resistant Streptococcus pneumoniae and Streptococcus pseudopneumoniae strains in Tunisia.

Streptococcus pneumoniae can be responsible for severe human infections. Optochin resistance has been a potential cause of misidentification of pneumococcus and other members of the mitis group. Hence, rapid and easy optochin resistant (Optr) S. pneumoniae identification is essential. Atypical pneumococci were characterized using optochin susceptibility, bile solubility based on spectrophotometric reading, serotyping, pulsed field gel electrophoresis (PFGE), 16S rRNA sequencing and PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802 and Spn9828. Optical density values for the bile solubility test suggest the identification of four Optr S. pneumoniae and one Streptococcus pseudopneumoniae. All Optr pneumococci harbored cpsA, lytA, ply, Spn9802, Spn9828 and pspA genes. Only ply, spn9802 and Spn9828 genes were detected in S. pseudopneumoniae. The 16S rRNA sequencing differentiates between these two species. Optr S. pneumoniae strains belonged to different genotypes and serotypes (14, 19A, 3 and 9V). Three Optr S. pneumoniae isolates were typed as pspA family 2, while one belonged to pspA family 1. Sequencing of the atpA and atpC gene of the Optr variants revealed three mutations in the ATPase a-subunit (L99I, M23V and V52I) and one mutation in ATPase c-subunit (V48I). Our data indicate that bile OD-values provides an accurate, fast and easy method to discriminate between Optr S. pneumoniae and other Streptococcus mitis group. Moreover molecular techniques, confirming the bile test, can be used in order to prevent these atypical pneumococci and alert clinical microbiologists of the presence of these strains in the community.

Description of optochin-resistant Streptococcus pneumoniae due to an uncommon mutation in the atpA gene and comparison with previously identified atpC mutants from Brazil

Optochin susceptibility testing is a major assay used for presumptive identification of Streptococcus pneumoniae. Still, atypical optochin-resistant (Optr) pneumococci have been reported and this phenotype has been attributed to nucleotide substitutions in the genes coding for the F0F1ATPase. While substitutions in the atpC gene (c-subunit of ATPase) are more common and better characterized, data on mutations in the atpA (a-subunit) are still limited. We have characterized five Optr isolates presenting alterations in the atpA (Trp206Cys in four isolates and Trp206Ser in one isolate), constituting the first report of such mutations in Brazil. Most of the Optr isolates consisted of heterogeneous populations. Except for Opt MICs and the nucleotide changes in the atpA gene, Optr and Opts subpopulations originating from the same culture had identical characteristics. In addition, we compared phenotypic and genetic characteristics of these atpA mutants with those of atpC mutants previously identified in Brazil. No structural alterations were detected among predicted proteins, regardless of mutations in the coding gene, suggesting that, despite the occurrence of mutations, protein structures tend to be highly conserved, ensuring their functionalities. Phylogenetic analysis revealed that atypical Optr strains are true pneumococci and Opt resistance does not represent any apparent selective advantage for clinical isolates.

Description of a novel mutation in the atpC gene in optochin-resistant Streptococcus pneumoniae strains isolates from Tunisia

Identification of Streptococcus pneumoniae among other α-haemolytic streptococci is based on phenotypic or genotypic characteristics such as colony morphology, bile solubility and optochin susceptibility. This study reports three optochin-resistant S. pneumoniae strains isolated from immunocompromised patients in Tunisia. The three isolates were positive for the bile solubility test. Biochemical identification with API® 20 Strep was not discriminatory for two strains. The three strains had different serotypes (6C, 19F and 23F) and three different sequence types (ST386, ST320 and ST326). Sequencing of the atpA and atpC genes for each strain showed only modification in atpC. The mutations Met13→Val or Val48→Ile were observed in two strains. However, in the third strain a novel type of mutation (Val15→Ile) was identified.

Rates and Mechanisms of Resistance Development inMycobacterium tuberculosisto a Novel Diarylquinoline ATP Synthase Inhibitor

R207910 (also known as TMC207) is an investigational drug currently in clinical studies for the treatment of multidrug-resistant (MDR) tuberculosis. It has a high degree of antimycobacterial activity and is equally effective against drug-susceptible and MDR Mycobacterium tuberculosis isolates. In the present study, we characterized the development of resistance to R207910 in vitro. Ninety-seven independent R207910-resistant mutants were selected from seven different clinical isolates of M. tuberculosis (three drug-susceptible and four MDR isolates) at 10x, 30x, and 100x the MIC. At a concentration of 0.3 mg/liter (10x the MIC), the mutation rates ranged from 4.7 x 10(-7) to 8.9 x 10(-9) mutations per cell per division, and at 1.0 mg/liter (30x the MIC) the mutation rate ranged from 3.9 x 10(-8) to 2.4 x 10(-9). No resistant mutants were obtained at 3 mg/liter (100x the MIC). The level of resistance ranged from 0.12 to 3.84 mg/liter for the mutants identified; these concentrations represent 4- to 128-fold increases in the MICs. For 53 of the resistant mutants, the atpE gene, which encodes a transmembrane and oligomeric C subunit of the ATP synthase and which was previously shown to be involved in resistance, was sequenced. For 15/53 mutants, five different point mutations resulting in five different amino acid substitutions were identified in the atpE gene. For 38/53 mutants, no atpE mutations were found and sequencing of the complete F0 ATP synthase operon (atpB, atpE, and atpF genes) and the F1 ATP synthase operon (atpH, atpA, atpG, atpD, and atpC genes) from three mutants revealed no mutations, indicating other, alternative resistance mechanisms. Competition assays showed no measurable reduction in the fitness of the mutants compared to that of the isogenic wild types.

P189 The appearance of atpC gene from clinical isolates of Streptococcus pneumoniae in Tehran, Iran

P189 The appearance of atpC gene from clinical isolates of Streptococcus pneumoniae in Tehran, Iran

Characterization of in vitro-generated and clinical optochin-resistant strains of Streptococcus pneumoniae isolated from Argentina.

Optochin susceptibility is a key test used for pneumococcal diagnosis, but optochin-resistant (Opt(r)) pneumococci have been reported in the last 2 decades. In this work, we characterized eight Opt(r) clinical strains which presented a new mutation, G47V, a predominant A49S mutation (recently reported in Brazil) and A49T. These mutations were found in the c subunit of the F(0)F(1) ATPase encoded by the atpC gene, and W206C was found in the a subunit encoded by the atpA gene. The Opt(r) clinical isolates were analyzed by BOX PCR, multilocus sequence typing, and serotype and antimicrobial resistance profiles, and they showed no epidemiological relationship. To characterize the Opt(r) mutations that could emerge among clinical strains, we studied a pool of spontaneous Opt(r) colonies obtained in vitro from the virulent D39 strain. We compared the atpAC mutations of these Opt(r) pneumococci (with or without passage through C57BL/6 mice) with those described in the clinical isolates. This analysis revealed three new mutations, G47V and L26M in the c subunit and L184S in the a subunit. Most of the mutations identified in the laboratory-generated Opt(r) strains were also found in clinical strains, with the exception of the L26M and L184S mutations, and we suppose that both mutations could emerge among invasive strains in the future. Considering that atpAC are essential genes, we propose that all spontaneous mutations that confer in vitro optochin resistance would not present severe physiological alterations in S. pneumoniae and may be carried by circulating pneumococcal strains.

Diversity of mutations in the atpC gene coding for the c Subunit of F0F1 ATPase in clinical isolates of optochin-resistant Streptococcus pneumoniae from Brazil.

We report the characteristics of four optochin-resistant (Opt(r)) Streptococcus pneumoniae isolates from Brazil. All four Opt(r) isolates presented mutations in the nucleotide sequence coding for the c subunit of F(0)F(1) ATPase. Two isolates showed mutations in codons 23 (leading to the deduced amino acid substitution isoleucine instead of alanine) and 49 (serine instead of alanine, a novel type of mutation detected at this position), respectively. Two additional novel mutations, both located in codon 45, were detected in the other two isolates, corresponding to leucine or valine (instead of phenylalanine). The data indicate that three previously unrecognized alterations were detected in the atpC gene of S. pneumoniae and that Opt resistance among Brazilian pneumococcal isolates is not related to a specific pneumococcal serotype, antimicrobial-resistance profile, or clonal group.

Biochemical characteristics of Escherichia coli ATP synthase with insulin peptide a fused to the globular part of the γ-subunit

The codon 5383-5385 (CCG) in the atpC gene of the unc operon of Escherichia coli cells was replaced with the sequence encoding peptide A of human insulin. The foreign protein fused to the middle part of the gamma-subunit of ATP synthase affects neither biosynthesis of the chimeric polypeptide nor the integration of the EF(0) x F(1) enzyme into the membranes of the E. coli cells. The inserted peptide A does not inhibit the process of oxidative phosphorylation. The ATPase activity of the mutant EF(0) x F(1) enzyme was equal to that of the wild-type enzyme and was regulated by modifiers in the similar way, suggesting that the space in the stalk area of F(0)/F(1) interaction is enough for the introduction of an additional oligopeptide without changing catalytic properties of the ATP synthase.

A novel mutation in the α-helix 1 of the C subunit of the F 1/F 0 ATPase responsible for optochin resistance of a Streptococcus pneumoniae clinical isolate

Previously reported mutations involved in optochin resistance of Streptococcus pneumoniae clinical isolates changed residues 48, 49 or 50, in the transmembrane α-helix 2 of the F 1/F 0 ATPase subunit. We report here an unusual mutation which changes the sequence of the transmembrane α-helix 1 of the AtpC subunit. This mutation involves a Gly to Ser substitution resulting from a G to A transition at codon 14 of the atpC gene.

The Assembly of the CAAT-box Binding Complex at a Photosynthesis Gene Promoter Is Regulated by Light, Cytokinin, and the Stage of the Plastids

A functionally important region in the promoter of the spinach photosynthesis gene AtpC, which encodes the subunit gamma of the chloroplast ATP synthase, is located immediately upstream of the CAAT-box. A single nucleotide exchange in this region (AAAATTCAAT --> AAGATCAAT) uncouples the expression of an AtpC promoter::uidA gene fusion from the regulation by light, cytokinin, and functional plastids and results in a high constitutive expression of the reporter gene. By screening an Arabidopsis thaliana expression library with a double-stranded wild-type oligonucleotide from this promoter region, we have isolated cDNAs from Arabidopsis libraries that code for plant homologs of the CAAT-box binding factor (CBF)-C. Binding occurs only in the presence of nuclear extracts, consistent with reports from metazoa CBFs that the subunits A and B in addition to C are required for the formation of the CBF-DNA complex. At least eight genes with homologies to CBF-C are present in the Arabidopsis genome; one of them exhibits striking similarities to the gene for the human global transcriptional repressor Drap1. In gel mobility shift assays, low binding activity of CBF to the wild-type AtpC promoter sequence was observed with nuclear extracts from tissue with low AtpC expression levels, i.e. extracts from etiolated and photobleached seedlings, whereas high binding activity was detectable with extracts from tissues with high AtpC expression levels, i.e. extracts from light-grown seedlings and etiolated seedlings treated with cytokinin. Binding to the mutant sequence, which directs constitutive high level uidA expression in vivo, is significantly stronger than to the wild-type sequence. The data are consistent with the idea that the assembly of CBF at the AtpC promoter is regulated in response to light and cytokinin and that the low level of expression in etiolated and photobleached material is caused by an inhibitory effect. The structure/function relationships of the Arabidopsis CBFs are discussed in relation to their regulatory function in AtpC gene expression.

Plastid translation is required for the expression of nuclear photosynthesis genes in the dark and in roots of the pea lip1 mutant

The expression of nuclear photosynthesis genes in pea seedlings requires both light and a postulated signal produced by developing plastids. The requirement for the plastid signal for the accumulation of transcripts of Lhcb1, RbcS, PetE, and AtpC genes was investigated in the pea mutant lip1, which shows light-independent photomorphogenic development. Lincomycin and erythromycin, inhibitors of plastid translation, decreased the accumulation of transcripts of nuclear photosynthesis genes in shoots of light-grown wild-type and lip1 seedlings, indicating that the plastid signal is required in the lip1 mutant. Treatment with lincomycin or erythromycin also reduced the accumulation of transcripts in shoots of dark-grown lip1 seedlings, indicating that light is not an obligate requirement for the synthesis or activity of the plastid signal. Lincomycin had a similar effect on the accumulation of Lhcb1 transcripts in dark-grown cop1-4 seedlings of Arabidopsis. Accumulation of transcripts of nuclear photosynthesis genes was also observed in roots of light-grown lip1 seedlings, and this accumulation, which was associated with the development of chloroplasts, was again dependent on plastid translation. The plastid signal therefore regulates the expression of nuclear photosynthesis genes in the dark and in roots of the lip1 mutant.

Molecular bases of three characteristic phenotypes of pneumococcus: optochin-sensitivity, coumarin-sensitivity, and quinolone-resistance.

Streptococcus pneumoniae is uniquely sensitive to amino alcohol antimalarials in the erythro configuration, such as optochin, quinine, and quinidine. The protein responsible for the optochin (quinine)-sensitive (Opts, Qins) phenotype of pneumococcus is the proteolipid c subunit of the FzeroF1 H(+)-ATPase. OptR/QinR isolates arose by point mutations in the atpC gene and produce different amino acid changes in one of the two transmembrane alpha-helices of the c subunit. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opts) and M222 (an OptR strain produced by interspecies recombination between pneumococcus and S. oralis), and S. oralis (OptR) revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin, quinine, and related compounds specifically inhibited the membrane-bound ATPase activity. Equivalent differences between Opts/Qins and OptR/QinR strains, both in growth inhibition and in membrane ATPase resistance, were found. Pneumococci also show a characteristic sensitivity to coumarin drugs, and a relatively high level of resistance to most quinolones. We have cloned and sequenced the gyrB gene, and characterized novobiocin resistant mutants. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates. This residue position is equivalent to Val-120 of Escherichia coli ryGB, a residue that lies inside the ATP-binding domain but is not involved in novobiocin binding in E. coli, as revealed by crystallographic data. In addition, the genes encoding the ParC and ParE subunits of topoisomerase IV, together with the region encoding amino acids 46 to 172 (residue numbers as in E. coli) of the pneumococcal ryGA subunit, were characterized in respect to fluoroquinolone resistance. The gyrA gene maps to a physical location distant from the gyrB and parEC loci on the chromosome. Ciprofloxacin-resistant (CpR) clinical isolates had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level CpR), or in both resistance-determining regions of ParC and GyrA (high-level CpR). Mutations were found in residue positions equivalent to Ser-83 and Asp-87 of the E. coli GyrA subunit. Transformation experiments demonstrated that topoisomerase IV is the primary target of ciprofloxacin, DNA gyrase being a secondary one.

The spinach AtpC and AtpD genes contain elements for light-regulated, plastid-dependent and organ-specific expression in the vicinity of the transcription start sites.

Run-on assays with isolated nuclei demonstrate that the transcription rates of AtpC and AtpD (gene products: the CF1 subunits gamma and delta of the chloroplast ATP synthase) are comparable in spinach seedlings. However, chimeric GUS gene fusions with 5'-flanking regions of the AtpC gene direct an approximately 10-fold lower GUS level in transgenic tobacco compared with equivalent fragments from the AtpD gene. Both promoters contain sequences in the vicinity of the respective TATA boxes, which are sufficient to direct light-regulated, plastid-dependent and organ-specific expression of the GUS gene. In contrast, the upstream regions of both promoters differ the higher GUS level directed by the AtpD promoter is caused by enhancer-like elements located upstream of the region involved in the regulated expression, while nucleotides upstream of -73 in the AtpC promoter contribute relatively little to the promoter activity. 5'-Deletion analyses and site-directed mutagenesis studies indicated that the -73/-48 bp AtpC region contains cis-elements crucial for this regulated expression. If five nucleotides within this region (-59/-55) are exchanged, the GUS gene is constitutively expressed and the activity in etiolated seedlings, in seedlings with photobleached plastids and in roots increases to the level detectable in green cotyledons. It is concluded that signal transduction pathways from different regulators converge prior to gene regulation and that these five nucleotides are part of a cis-element which functions as a repressor in darkness, in tissues with impaired plastids and in roots.

Nucleotide Sequence for the Genomic DNA Encoding the Chloroplast ATP Synthase γ-Subunit Gene (atpC) from Chlamydomonas reinhardtii

The ATP synthase complex (CFo-CF1) in chloroplast thylakoid membranes couples the chemical potential energy in the protonmotive force to the synthesis of ATP. A unique feature of the chloroplast enzyme is that the catalytic activity is partially regulated by the oxidation state of the CF, ysubunit (2, 4). The a-subunit contains two cysteine residues that are speculated to form an intrapeptide disulfide bridge (5). The oxidation of the vicinal sulfhydryls together with the reduction of the ensuing disulfide is believed to act as a redox regulatory switch (2. 4). The eukaryotic green alga Chlamvl,,dornonas reinhardtli can grow exclusively on acetate, which allows one to isolate conditionally lethal mutant strains defective in photosynthesis. This makes C. reinhardtii an ideal model system for the application of both classical and modern molecular genetics. Although the cDNA for the C. reinhardtii CF, --subunit gene has been cloned and sequenced (6), in order to identify mutations in the nuclear gene (atpC), it was necessary to obtain the genomic DNA sequence (7). Furthermore, the genomic atpC gene can be used to rescue atpC mutants and to generate and tag other types of mutations in C reinhardtil. The atpC gene was obtained from genomic DNA by using DNA primers corresponding to the 5' and 3' ends of the cDNA and polymerase chain reaction technology to amplify the intervening sequence (Table I). The nucleotide and deduced amino acid sequences are shown in Figure 1. The atpC gene contains four introns. The first intron, only 85 bases long, resides very close to the transit peptide/mature peptide cleavage site. It is interesting to note that the third intron in the C. reinhardtil gene corresponds exactly to the position of the second intron in the spinach -y-subunit genomic DNA (3). All four introns are located in the 5' portion of the gene while the sequence encoding the putative regulatory cysteines are located in the 3' portion. This is in contrast to the spinach sequence in which the DNA encoding the two cysteines are located between the two introns (3). The exon-intron boundaries for the first two introns and the 5' junction of the third intron coincide with the AG:GT motif which is the consensus splice junction in plants (1). However, the 3' junction for the third intron and both junctions for the fourth intron do not have the consensus sequence. The four introns do not have any significant sequence identity with each other and have approximately the same G-C content as the coding region.

Isolation and Characterization of a Chlamydomonas reinhardtii Mutant Lacking the 7-Subunit of Chloroplast Coupling Factor 1 (CF1)

Several nonphotoautotrophic mutants of Chlamydomonas reinhardtii were generated by transforming strain nit1-305 (cw 15) with exogenous DNA. An enrichment for potential photophosphorylation mutants was performed on medium containing arsenate, and acetate-requiring auxotrophs were then identified by replica plating. Strains containing a potential mutation in the nuclear DNA encoding the chloroplast coupling factor 1 (CF1) gamma-subunit (the atpC gene) were first identified serologically with a monospecific antiserum directed against the CF1 gamma-subunit polypeptide. Of several mutants isolated, one, designated T1-54, was characterized at the protein, DNA, and RNA levels. Mutant strain T1-54 lacks anti-CF1 gamma-subunit cross-reacting material, exhibits polymorphism at the atpC locus compared with the parental strain, and lacks the mRNA transcript for the CF1 gamma-subunit. The data are consistent with there being an insertion of exogenous DNA, a deletion of DNA, or both at the 5' end of the gene encoding the CF1 gamma-subunit.

Isolation and characterization of a Chlamydomonas reinhardtii mutant lacking the gamma-subunit of chloroplast coupling factor 1 (CF1).

Several nonphotoautotrophic mutants of Chlamydomonas reinhardtii were generated by transforming strain nit1-305 (cw 15) with exogenous DNA. An enrichment for potential photophosphorylation mutants was performed on medium containing arsenate, and acetate-requiring auxotrophs were then identified by replica plating. Strains containing a potential mutation in the nuclear DNA encoding the chloroplast coupling factor 1 (CF1) gamma-subunit (the atpC gene) were first identified serologically with a monospecific antiserum directed against the CF1 gamma-subunit polypeptide. Of several mutants isolated, one, designated T1-54, was characterized at the protein, DNA, and RNA levels. Mutant strain T1-54 lacks anti-CF1 gamma-subunit cross-reacting material, exhibits polymorphism at the atpC locus compared with the parental strain, and lacks the mRNA transcript for the CF1 gamma-subunit. The data are consistent with there being an insertion of exogenous DNA, a deletion of DNA, or both at the 5' end of the gene encoding the CF1 gamma-subunit.

The promoters of the atp operon of Escherichia coli K12.

The nucleotide sequence has been determined of a 900 bp segment of chromosomal DNA located between 2.6 and 3.5 kb left of the origin of replication, oriC. This segment, which overlaps with the known sequence of the atp operon coding for the eight subunits of the Escherichia coli K12 ATP synthase, contains two coding sequences with the same polarity (counterclockwise) as the atp genes: One of these, designated atpI, which codes for the N-terminal part of a 14 kD polypeptide, is located in front (upstream) of the atpB gene (the first structural gene in the atp operon), the other one codes for the C-terminal part of the gidB gene. The 606 bp segment located between the gidB and the atpI genes contains no coding sequences. By employing the nuclease S1 mapping technique, we have determined a promoter, designated atpIp, for the atp operon located in front of the atpI gene; two additional, weak transcription starts were located within the atpI gene. No transcription start sites were detected up to 1,000 bp upstream of the atpIp promoter, neither were any transcription start sites detected within the cluster of the eight structural atp genes. The atp operon transcription terminates at a site approximately 50 bp downstream from the atpC gene.