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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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Growth and ATPase activity of Mmm T1/44 and the Δ619 mutant.A. Growth of Mmm T1/44 (▪) and Δ619 (□) in Hayflick medium at 37°C. B. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□). The figure shows representative results of five independent experiments. C. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□) transformed with the control plasmid pMYSO1, and Δ619 complemented with the MSC_0619 (α-like) and MSC_0618 (β-like) proteins, generated from the plasmid pCC1 (▴).
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pone-0038793-g008: Growth and ATPase activity of Mmm T1/44 and the Δ619 mutant.A. Growth of Mmm T1/44 (▪) and Δ619 (□) in Hayflick medium at 37°C. B. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□). The figure shows representative results of five independent experiments. C. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□) transformed with the control plasmid pMYSO1, and Δ619 complemented with the MSC_0619 (α-like) and MSC_0618 (β-like) proteins, generated from the plasmid pCC1 (▴).

Mentions: A previously obtained library of mutants generated by random insertion of the gentamycin resistance transposon Tn4001 in Mmm T1/44 [32] was screened for insertion in genes belonging to the Type 3 cluster. A mutant carrying Tn4001 inserted at position 307 of the MSC_0619 gene (Figure 7A) was identified by PCR as described in the Materials and Methods. The insertion of the 3.7 kbp transposon leaded to a predicted truncated protein of 108 amino acids whereas the complete protein includes 505 amino acids. Comparative growth studies were performed for preliminary characterization of the mutant phenotype. The T1/44 and Δ619 strains were grown in pyruvate-supplemented Hayflick medium at 37°C and the optical density at 640 nm was recorded over a period of 60 h. The time courses for growth were very similar, except that the cell density observed in stationary growth phase was repeatedly slightly lower for the mutant strain (Figure 8A). Both strains were able to acidify the culture medium, the pH decreasing from an initial value of 7.4 to 5.4 for T1/44, and to 5.8 for the mutant strain at t = 30 h.


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)

Growth and ATPase activity of Mmm T1/44 and the Δ619 mutant.A. Growth of Mmm T1/44 (▪) and Δ619 (□) in Hayflick medium at 37°C. B. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□). The figure shows representative results of five independent experiments. C. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□) transformed with the control plasmid pMYSO1, and Δ619 complemented with the MSC_0619 (α-like) and MSC_0618 (β-like) proteins, generated from the plasmid pCC1 (▴).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369863&req=5

pone-0038793-g008: Growth and ATPase activity of Mmm T1/44 and the Δ619 mutant.A. Growth of Mmm T1/44 (▪) and Δ619 (□) in Hayflick medium at 37°C. B. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□). The figure shows representative results of five independent experiments. C. Rate of release of Pi from ATP in the presence of membrane preparations from T1/44 (▪) and Δ619 (□) transformed with the control plasmid pMYSO1, and Δ619 complemented with the MSC_0619 (α-like) and MSC_0618 (β-like) proteins, generated from the plasmid pCC1 (▴).
Mentions: A previously obtained library of mutants generated by random insertion of the gentamycin resistance transposon Tn4001 in Mmm T1/44 [32] was screened for insertion in genes belonging to the Type 3 cluster. A mutant carrying Tn4001 inserted at position 307 of the MSC_0619 gene (Figure 7A) was identified by PCR as described in the Materials and Methods. The insertion of the 3.7 kbp transposon leaded to a predicted truncated protein of 108 amino acids whereas the complete protein includes 505 amino acids. Comparative growth studies were performed for preliminary characterization of the mutant phenotype. The T1/44 and Δ619 strains were grown in pyruvate-supplemented Hayflick medium at 37°C and the optical density at 640 nm was recorded over a period of 60 h. The time courses for growth were very similar, except that the cell density observed in stationary growth phase was repeatedly slightly lower for the mutant strain (Figure 8A). Both strains were able to acidify the culture medium, the pH decreasing from an initial value of 7.4 to 5.4 for T1/44, and to 5.8 for the mutant strain at t = 30 h.

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