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Specific evolution of F1-like ATPases in mycoplasmas.

Béven L, Charenton C, Dautant A, Bouyssou G, Labroussaa F, Sköllermo A, Persson A, Blanchard A, Sirand-Pugnet P - PLoS ONE (2012)

Bottom Line: Phylogenomic studies identified two types of F(1)-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features.Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media.Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions.

View Article: PubMed Central - PubMed

Affiliation: University Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, Villenave d'Ornon, France.

ABSTRACT
F(1)F(0) ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F(1)F(0) ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, β, γ and ε subunits of F(1) ATPases and could form an F(1)-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F(1)-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F(1)-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F(1)-like structure is associated with a hypothetical X(0) sector located in the membrane of mycoplasma cells.

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Operon structure and expression of the genes of the Type 3 cluster in Mmm.A. RT-PCR experiments were carried out on intergenic regions to demonstrate the co-transcription of the MSC_0618 to MSC_0627 genes. The region of the genome region surrounding the Type 3 cluster in Mmm is shown. Gene mnemonics and numbers are shown for the Type 3 cluster. The site of transposon insertion in the MSC_0619 disrupted mutant (Δ619) is indicated by an arrow. Expected sizes of the putative transcripts are indicated. Amplification products of the expected sizes were obtained with primers binding within and upstream from the cluster (+) but not downstream from the cluster (−). B. Immunodetection of proteins from the Type 3 cluster of T1/44 and Δ619. A control for membrane protein detection was included, in the form of an antibody against a membrane protein, LppQ (anti-LppQ serum kindly supplied by Prof. J. Frey). C. Nano-LC-MS/MS detection of Type 3 ATPase proteins. Numbers of scans and distinct peptides detected are indicated. D. Evaluation of the sensitivity of Protein 5 and β-like subunit to trypsin degradation. Intact and lysed cells of Mmm T1/44 were incubated with (+) or without (−) trypsin enzyme coated on beads for six hours. Protection against hydrolysis was assessed by immunodetection with antibodies raised against Protein 5 (MSC_0620) and β-like subunit (MSC_0618).
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pone-0038793-g007: Operon structure and expression of the genes of the Type 3 cluster in Mmm.A. RT-PCR experiments were carried out on intergenic regions to demonstrate the co-transcription of the MSC_0618 to MSC_0627 genes. The region of the genome region surrounding the Type 3 cluster in Mmm is shown. Gene mnemonics and numbers are shown for the Type 3 cluster. The site of transposon insertion in the MSC_0619 disrupted mutant (Δ619) is indicated by an arrow. Expected sizes of the putative transcripts are indicated. Amplification products of the expected sizes were obtained with primers binding within and upstream from the cluster (+) but not downstream from the cluster (−). B. Immunodetection of proteins from the Type 3 cluster of T1/44 and Δ619. A control for membrane protein detection was included, in the form of an antibody against a membrane protein, LppQ (anti-LppQ serum kindly supplied by Prof. J. Frey). C. Nano-LC-MS/MS detection of Type 3 ATPase proteins. Numbers of scans and distinct peptides detected are indicated. D. Evaluation of the sensitivity of Protein 5 and β-like subunit to trypsin degradation. Intact and lysed cells of Mmm T1/44 were incubated with (+) or without (−) trypsin enzyme coated on beads for six hours. Protection against hydrolysis was assessed by immunodetection with antibodies raised against Protein 5 (MSC_0620) and β-like subunit (MSC_0618).

Mentions: Standard RT-PCR assays were then undertaken to demonstrate the production of transcripts overlapping the various CDS regions within the cluster in Mmm. Based on the DOOR predictions, we carried out nine RT-PCR assays with the primers listed in Table S1. An overview of the binding sites of the primers is provided in Figure 7A. Products of the expected size were obtained for eight out of nine RT-PCRs, confirming linked transcription of the open reading frames in the Type 3 gene cluster in Mmm. No product was detected when the MSC_0617-MSC_0618 intergenic region was amplified. These results suggest that the genes of the Type 3 cluster and at least three upstream genes constitute an operon expressed in Mmm.


Specific evolution of F1-like ATPases in mycoplasmas.

Béven L, Charenton C, Dautant A, Bouyssou G, Labroussaa F, Sköllermo A, Persson A, Blanchard A, Sirand-Pugnet P - PLoS ONE (2012)

Operon structure and expression of the genes of the Type 3 cluster in Mmm.A. RT-PCR experiments were carried out on intergenic regions to demonstrate the co-transcription of the MSC_0618 to MSC_0627 genes. The region of the genome region surrounding the Type 3 cluster in Mmm is shown. Gene mnemonics and numbers are shown for the Type 3 cluster. The site of transposon insertion in the MSC_0619 disrupted mutant (Δ619) is indicated by an arrow. Expected sizes of the putative transcripts are indicated. Amplification products of the expected sizes were obtained with primers binding within and upstream from the cluster (+) but not downstream from the cluster (−). B. Immunodetection of proteins from the Type 3 cluster of T1/44 and Δ619. A control for membrane protein detection was included, in the form of an antibody against a membrane protein, LppQ (anti-LppQ serum kindly supplied by Prof. J. Frey). C. Nano-LC-MS/MS detection of Type 3 ATPase proteins. Numbers of scans and distinct peptides detected are indicated. D. Evaluation of the sensitivity of Protein 5 and β-like subunit to trypsin degradation. Intact and lysed cells of Mmm T1/44 were incubated with (+) or without (−) trypsin enzyme coated on beads for six hours. Protection against hydrolysis was assessed by immunodetection with antibodies raised against Protein 5 (MSC_0620) and β-like subunit (MSC_0618).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369863&req=5

pone-0038793-g007: Operon structure and expression of the genes of the Type 3 cluster in Mmm.A. RT-PCR experiments were carried out on intergenic regions to demonstrate the co-transcription of the MSC_0618 to MSC_0627 genes. The region of the genome region surrounding the Type 3 cluster in Mmm is shown. Gene mnemonics and numbers are shown for the Type 3 cluster. The site of transposon insertion in the MSC_0619 disrupted mutant (Δ619) is indicated by an arrow. Expected sizes of the putative transcripts are indicated. Amplification products of the expected sizes were obtained with primers binding within and upstream from the cluster (+) but not downstream from the cluster (−). B. Immunodetection of proteins from the Type 3 cluster of T1/44 and Δ619. A control for membrane protein detection was included, in the form of an antibody against a membrane protein, LppQ (anti-LppQ serum kindly supplied by Prof. J. Frey). C. Nano-LC-MS/MS detection of Type 3 ATPase proteins. Numbers of scans and distinct peptides detected are indicated. D. Evaluation of the sensitivity of Protein 5 and β-like subunit to trypsin degradation. Intact and lysed cells of Mmm T1/44 were incubated with (+) or without (−) trypsin enzyme coated on beads for six hours. Protection against hydrolysis was assessed by immunodetection with antibodies raised against Protein 5 (MSC_0620) and β-like subunit (MSC_0618).
Mentions: Standard RT-PCR assays were then undertaken to demonstrate the production of transcripts overlapping the various CDS regions within the cluster in Mmm. Based on the DOOR predictions, we carried out nine RT-PCR assays with the primers listed in Table S1. An overview of the binding sites of the primers is provided in Figure 7A. Products of the expected size were obtained for eight out of nine RT-PCRs, confirming linked transcription of the open reading frames in the Type 3 gene cluster in Mmm. No product was detected when the MSC_0617-MSC_0618 intergenic region was amplified. These results suggest that the genes of the Type 3 cluster and at least three upstream genes constitute an operon expressed in Mmm.

Bottom Line: Phylogenomic studies identified two types of F(1)-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features.Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media.Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions.

View Article: PubMed Central - PubMed

Affiliation: University Bordeaux, UMR 1332 de Biologie du Fruit et Pathologie, Villenave d'Ornon, France.

ABSTRACT
F(1)F(0) ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F(1)F(0) ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, β, γ and ε subunits of F(1) ATPases and could form an F(1)-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F(1)-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F(1)-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F(1)-like structure is associated with a hypothetical X(0) sector located in the membrane of mycoplasma cells.

Show MeSH
Related in: MedlinePlus