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Complete genome determination and analysis of Acholeplasma oculi strain 19L, highlighting the loss of basic genetic features in the Acholeplasmataceae.

Siewert C, Hess WR, Duduk B, Huettel B, Reinhardt R, Büttner C, Kube M - BMC Genomics (2014)

Bottom Line: Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence.Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi.The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.

View Article: PubMed Central - PubMed

Affiliation: Humboldt-Universität zu Berlin, Faculty of Life Science, Thaer-Institute, Division Phytomedicine, Lentzeallee 55/57, 14195 Berlin, Germany. Michael.Kube@agrar.hu-berlin.de.

ABSTRACT

Background: Acholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and 'Candidatus Phytoplasma'. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing.

Results: The A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120 bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F1FO-type Na+ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins.

Conclusions: The application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.

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Composition of theAcholeplasmaand ‘Ca. Phytoplasma’ core-genomes as predicted by PanOCT. The total number of proteins inferred from the respective core genome is given in the middle (uncoloured part).
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Fig6: Composition of theAcholeplasmaand ‘Ca. Phytoplasma’ core-genomes as predicted by PanOCT. The total number of proteins inferred from the respective core genome is given in the middle (uncoloured part).

Mentions: Only a basic set of proteins is shared between A. oculi and the five complete phytoplasma genomes. The 293 to 310 predicted shared proteins (27% to 59%) are consistent with previously calculated numbers for other acholeplasmas [7, 8] (Figure 3). The second highest number of unique proteins (440) is predicted for A. palmae (Figure 4), which is the closest known relative of the phytoplasmas [7] (Figure 2) and is also supported by the highest number of predicted shared proteins with the five completely sequenced phytoplasmas (Additional file 1). In second position among the acholeplasmas, A. oculi shares many of its proteins with the phytoplasmas, supported by its phylogenetic assignment and the received PanOCT results. This analysis is also supported by A. oculi and A. laidlawii, which share the highest number of proteins amongst the acholeplasmas (Figure 3). The phytoplasmas’ genome reduction process is reflected by the low number of 294 proteins assigned to the shared core within the pan-genome (2,077 proteins in total; Figure 5). Phytoplasma genomes are characterised by extensive gene losses, transposon-mediated gene duplication [12] and horizontal gene-integration events [15]. Comparing the pan-genomes of acholeplasmas and phytoplasmas, Venn analysis highlights basic differences in the overall gene content. Complete Acholeplasma and ‘Ca. Phytoplasma’ genomes collectively encode 402 and 14 predicted unique proteins, respectively (Figure 6, Additional file 2). The 14 unique genes, which are common to the genus ‘Ca. Phytoplasma’, encode nine hypothetical proteins and five proteins with known functions. Two of the hypothetical proteins contain a sequence-variable mosaic (SVM) motif [16] and comprise SAP05 (AYWB_032), which is described as a putative effector protein [17] inducing the formation of smooth young rosette leaves that lack serrations along the leaf margin [18], and SAP30 (AYWB_402), which is similar to SAP11 containing an eukaryotic nuclear localisation signal [19, 20]. This group of unique genes also includes two phytoplasma proteins involved in a suggested alternative pathway in the carbohydrate metabolism of phytoplasmas [7, 13, 21]. The malate/Na + symporter (MleP) provides a carbon source which undergoes oxidative decarboxylation by malate dehydrogenase (SfcA), thereby providing pyruvate processed by the dehydrogenase multienzyme complex and providing acetyl coenzyme A. The phosphotransacetylation of acetyl-CoA performed by the PduL-like protein provides acetyl-phosphate, which is processed via acetate kinase (AckA) and results in the formation of ATP and acetate. A. oculi does not encode MleP, SfcA and the phosphate acetyltransferase (Pta), which is common in mycoplasmas, though it is suggested that PduL fulfills this function in Acholeplasmataceae[7, 21] including A. oculi. However, the alternative energy-yielding pathway of phytoplasmas utilising malate is clearly not encoded in the analysed acholeplasma genomes.Figure 5


Complete genome determination and analysis of Acholeplasma oculi strain 19L, highlighting the loss of basic genetic features in the Acholeplasmataceae.

Siewert C, Hess WR, Duduk B, Huettel B, Reinhardt R, Büttner C, Kube M - BMC Genomics (2014)

Composition of theAcholeplasmaand ‘Ca. Phytoplasma’ core-genomes as predicted by PanOCT. The total number of proteins inferred from the respective core genome is given in the middle (uncoloured part).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4221730&req=5

Fig6: Composition of theAcholeplasmaand ‘Ca. Phytoplasma’ core-genomes as predicted by PanOCT. The total number of proteins inferred from the respective core genome is given in the middle (uncoloured part).
Mentions: Only a basic set of proteins is shared between A. oculi and the five complete phytoplasma genomes. The 293 to 310 predicted shared proteins (27% to 59%) are consistent with previously calculated numbers for other acholeplasmas [7, 8] (Figure 3). The second highest number of unique proteins (440) is predicted for A. palmae (Figure 4), which is the closest known relative of the phytoplasmas [7] (Figure 2) and is also supported by the highest number of predicted shared proteins with the five completely sequenced phytoplasmas (Additional file 1). In second position among the acholeplasmas, A. oculi shares many of its proteins with the phytoplasmas, supported by its phylogenetic assignment and the received PanOCT results. This analysis is also supported by A. oculi and A. laidlawii, which share the highest number of proteins amongst the acholeplasmas (Figure 3). The phytoplasmas’ genome reduction process is reflected by the low number of 294 proteins assigned to the shared core within the pan-genome (2,077 proteins in total; Figure 5). Phytoplasma genomes are characterised by extensive gene losses, transposon-mediated gene duplication [12] and horizontal gene-integration events [15]. Comparing the pan-genomes of acholeplasmas and phytoplasmas, Venn analysis highlights basic differences in the overall gene content. Complete Acholeplasma and ‘Ca. Phytoplasma’ genomes collectively encode 402 and 14 predicted unique proteins, respectively (Figure 6, Additional file 2). The 14 unique genes, which are common to the genus ‘Ca. Phytoplasma’, encode nine hypothetical proteins and five proteins with known functions. Two of the hypothetical proteins contain a sequence-variable mosaic (SVM) motif [16] and comprise SAP05 (AYWB_032), which is described as a putative effector protein [17] inducing the formation of smooth young rosette leaves that lack serrations along the leaf margin [18], and SAP30 (AYWB_402), which is similar to SAP11 containing an eukaryotic nuclear localisation signal [19, 20]. This group of unique genes also includes two phytoplasma proteins involved in a suggested alternative pathway in the carbohydrate metabolism of phytoplasmas [7, 13, 21]. The malate/Na + symporter (MleP) provides a carbon source which undergoes oxidative decarboxylation by malate dehydrogenase (SfcA), thereby providing pyruvate processed by the dehydrogenase multienzyme complex and providing acetyl coenzyme A. The phosphotransacetylation of acetyl-CoA performed by the PduL-like protein provides acetyl-phosphate, which is processed via acetate kinase (AckA) and results in the formation of ATP and acetate. A. oculi does not encode MleP, SfcA and the phosphate acetyltransferase (Pta), which is common in mycoplasmas, though it is suggested that PduL fulfills this function in Acholeplasmataceae[7, 21] including A. oculi. However, the alternative energy-yielding pathway of phytoplasmas utilising malate is clearly not encoded in the analysed acholeplasma genomes.Figure 5

Bottom Line: Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence.Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi.The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.

View Article: PubMed Central - PubMed

Affiliation: Humboldt-Universität zu Berlin, Faculty of Life Science, Thaer-Institute, Division Phytomedicine, Lentzeallee 55/57, 14195 Berlin, Germany. Michael.Kube@agrar.hu-berlin.de.

ABSTRACT

Background: Acholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and 'Candidatus Phytoplasma'. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing.

Results: The A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120 bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F1FO-type Na+ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins.

Conclusions: The application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.

Show MeSH
Related in: MedlinePlus