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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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Genome circle ofAcholeplasma oculistrain 19L and a summary of the comparative analyses. Circular patterns (from outside to inside): 1 (outer circle), scale in base pairs of the chromosome; 2 (blue), six contigs obtained from the initial SBS paired-end read assembly; 3 (red), 28 differences identified by comparing the results of the Illumina and PacBio sequencing results; 4 (black), predicted proteins encoded on the forward and reverse strands; 5 (green) tRNA genes and (grey) rRNA operons; 6 (violet), predicted unique proteins for A. oculi in comparison to other Acholeplasmataceae species; 7 (orange), orthologous proteins of A. oculi and A. laidlawii; 8 (light orange), orthologous proteins of A. oculi and A. brassicae; 9 (yellow), orthologous proteins of A. oculi and A. palmae; 10-14 (green), orthologous proteins of A. oculi and ‘Ca. P. asteris’ strain OY-M, AY-WB, ‘Ca. P. australiense’ strain rp-A, NZSb11 or ‘Ca. P. mali’ strain AT, respectively; 15 (olive and purple), cumulative G + C skew.
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Fig1: Genome circle ofAcholeplasma oculistrain 19L and a summary of the comparative analyses. Circular patterns (from outside to inside): 1 (outer circle), scale in base pairs of the chromosome; 2 (blue), six contigs obtained from the initial SBS paired-end read assembly; 3 (red), 28 differences identified by comparing the results of the Illumina and PacBio sequencing results; 4 (black), predicted proteins encoded on the forward and reverse strands; 5 (green) tRNA genes and (grey) rRNA operons; 6 (violet), predicted unique proteins for A. oculi in comparison to other Acholeplasmataceae species; 7 (orange), orthologous proteins of A. oculi and A. laidlawii; 8 (light orange), orthologous proteins of A. oculi and A. brassicae; 9 (yellow), orthologous proteins of A. oculi and A. palmae; 10-14 (green), orthologous proteins of A. oculi and ‘Ca. P. asteris’ strain OY-M, AY-WB, ‘Ca. P. australiense’ strain rp-A, NZSb11 or ‘Ca. P. mali’ strain AT, respectively; 15 (olive and purple), cumulative G + C skew.

Mentions: The SBS and SMRT approaches enabled the efficient reconstruction of the complete genome sequence in independent experiments. SBS sequencing provided 1,095,574 paired-end quality passed reads with an average length of 101 nt (total read length of 110,652,974 nt). De novo assembly of the SBS-derived reads alone led to the incorporation of 964,613 reads (88%) into six large contigs (513260 bp, 244477 bp, 109253 bp, 547590 bp, 106516 bp and 52461 bp in size), showing a 64-fold read coverage on average (Table 1, Figure 1) and reaching a total contig length of 1,573,557 bp. In turn, the mapping of SBS reads on the finished genome sequence revealed no uncovered regions. Gap regions derived from the SBS de novo assembly cover repetitive sequences of high similarity. In detail, two gaps (4,748 bp and 4,898 bp in size) include the rRNA operon regions (99% sequence identity), two gaps (1,661 bp each) include two transposases (100% sequence identity), one gap (186 bp) borders Acholeplasma phage L2 (>92% sequence identity) and the smallest gap (176 bp) is located close to a heavy metal translocating P-type ATPase (92% sequence identity). Gaps derived from the assembly of SBS reads were closed by primer-walking (Sanger sequencing), resulting in a complete circular chromosomal sequence.Table 1


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)

Genome circle ofAcholeplasma oculistrain 19L and a summary of the comparative analyses. Circular patterns (from outside to inside): 1 (outer circle), scale in base pairs of the chromosome; 2 (blue), six contigs obtained from the initial SBS paired-end read assembly; 3 (red), 28 differences identified by comparing the results of the Illumina and PacBio sequencing results; 4 (black), predicted proteins encoded on the forward and reverse strands; 5 (green) tRNA genes and (grey) rRNA operons; 6 (violet), predicted unique proteins for A. oculi in comparison to other Acholeplasmataceae species; 7 (orange), orthologous proteins of A. oculi and A. laidlawii; 8 (light orange), orthologous proteins of A. oculi and A. brassicae; 9 (yellow), orthologous proteins of A. oculi and A. palmae; 10-14 (green), orthologous proteins of A. oculi and ‘Ca. P. asteris’ strain OY-M, AY-WB, ‘Ca. P. australiense’ strain rp-A, NZSb11 or ‘Ca. P. mali’ strain AT, respectively; 15 (olive and purple), cumulative G + C skew.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Genome circle ofAcholeplasma oculistrain 19L and a summary of the comparative analyses. Circular patterns (from outside to inside): 1 (outer circle), scale in base pairs of the chromosome; 2 (blue), six contigs obtained from the initial SBS paired-end read assembly; 3 (red), 28 differences identified by comparing the results of the Illumina and PacBio sequencing results; 4 (black), predicted proteins encoded on the forward and reverse strands; 5 (green) tRNA genes and (grey) rRNA operons; 6 (violet), predicted unique proteins for A. oculi in comparison to other Acholeplasmataceae species; 7 (orange), orthologous proteins of A. oculi and A. laidlawii; 8 (light orange), orthologous proteins of A. oculi and A. brassicae; 9 (yellow), orthologous proteins of A. oculi and A. palmae; 10-14 (green), orthologous proteins of A. oculi and ‘Ca. P. asteris’ strain OY-M, AY-WB, ‘Ca. P. australiense’ strain rp-A, NZSb11 or ‘Ca. P. mali’ strain AT, respectively; 15 (olive and purple), cumulative G + C skew.
Mentions: The SBS and SMRT approaches enabled the efficient reconstruction of the complete genome sequence in independent experiments. SBS sequencing provided 1,095,574 paired-end quality passed reads with an average length of 101 nt (total read length of 110,652,974 nt). De novo assembly of the SBS-derived reads alone led to the incorporation of 964,613 reads (88%) into six large contigs (513260 bp, 244477 bp, 109253 bp, 547590 bp, 106516 bp and 52461 bp in size), showing a 64-fold read coverage on average (Table 1, Figure 1) and reaching a total contig length of 1,573,557 bp. In turn, the mapping of SBS reads on the finished genome sequence revealed no uncovered regions. Gap regions derived from the SBS de novo assembly cover repetitive sequences of high similarity. In detail, two gaps (4,748 bp and 4,898 bp in size) include the rRNA operon regions (99% sequence identity), two gaps (1,661 bp each) include two transposases (100% sequence identity), one gap (186 bp) borders Acholeplasma phage L2 (>92% sequence identity) and the smallest gap (176 bp) is located close to a heavy metal translocating P-type ATPase (92% sequence identity). Gaps derived from the assembly of SBS reads were closed by primer-walking (Sanger sequencing), resulting in a complete circular chromosomal sequence.Table 1

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