Complete genome determination and analysis of Acholeplasma oculi strain 19L, highlighting the loss of basic genetic features in the Acholeplasmataceae
© Siewert et al.; licensee BioMed Central Ltd. 2014
Received: 4 June 2014
Accepted: 25 September 2014
Published: 24 October 2014
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.
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.
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.
Acholeplasma species comprise bacteria of the family Acholeplasmataceae in the class Mollicutes, characterised by the lack of sterol requirement for growth and thereby separated from Mycoplasmataceae and Spiroplasmataceae. The majority of Acholeplasma spp. are described as saprophytes and commensals. An evident assignment as pathogens is hampered by the fact that several Acholeplasma spp. are distributed ubiquitously. Moreover, no primary pathogen is described for this genus. However, the isolation of strains from diseased animals, and classification as putative animal pathogens, applies to species such as A. axanthum and A. oculi[2, 3]. This assignment of the type strain A. oculi 19L (syn. A. oculusi) was the result of its isolation from goat eyes infected with keratoconjunctivitis and re-infection experiments . However, the assignment of A. oculi to this disease is rare in contrast to several Mycoplasma spp. .
Besides Acholeplasma, the Acholeplasmataceae family also includes the provisory taxon ‘Candidatus Phytoplasma’. Phytoplasmas are associated with several hundred plant diseases – and thus significant economic losses . After insect vector-mediated transmission, phytoplasmas colonise as intracellular obligate parasites the sieve cells of a plant, often resulting in abnormal growth and reduced vitality. No general evidence for pathogenesis by acholeplasmas in colonised insects and plants has been provided to date. However, a recently published study on the A. laidlawii strain PG-8 supports its phytopathogenicity, which can be increased after nanotransformation in ultramicroform cells and might be correlated to extracellular vesicle formation under experimental conditions . Further studies are needed in this respect, but the results may indicate a mechanism shared by both genera. In contrast, experimentally proven effector proteins or membrane proteins involved in phytoplasma-host interaction were not identified in the acholeplasmas . These genetic elements of phytoplasmas might have originated from horizontal gene transfers. Massive gene loss, in combination with duplication events and genome instability, separates the phytoplasmas from the acholeplasmas. The complete genome sequences of eight strains of this family have been published, comprising the acholeplasmas A. laidlawii strain PG-8A , A. brassicae strain O502 and A. palmae strain J233  and the phytoplasmas ‘Ca. P. australiense’ strain rp-A  and NZSb11 , ‘Ca. P. asteris’ strain OY-M  and AY-WB  and ‘Ca. P. mali’ strain AT . In the past, all five phytoplasma strains and A. laidlawii were sequenced by applying the whole genome shotgun approach and using plasmid or fosmid libraries as templates for dye-terminator sequencing (Sanger sequencing). In determining the chromosome sequences of A. brassicae and A. palmae, a combination of Sanger sequencing and next generation sequencing methods (pyrosequencing, 454 Life Sciences/Roche) was applied for the first time to this bacterial family .
Both taxa show characteristic gene losses. In comparison to acholeplasmas, phytoplasmas lack the F1FO ATPase synthetase complex, the cell division protein FtsZ, a wider variety of ABC transporters, the Rnf complex and the membrane protein SecG of the Sec-dependent secretion system. Moreover, acholeplasmas possess a rich repertoire of enzymes involved in carbohydrate metabolism, fatty acids, isoprenoids and partial amino acid metabolism . Because these findings were inferred from the analyses of three acholeplasma and five phytoplasma genome sequences, it remains unclear as to what extent these differences between the two genera can be truly generalised or if the other acholeplasmas might share some of these features of their genetic repertoire with the phytoplasmas. Therefore, we determined the complete genome of A. oculi strain 19L by applying two different strategies based on sequencing by synthesis (SBS, Illumina) and, in a second approach, single molecule real time (SMRT, PacBio) sequencing. The subsequent analyses highlight the efficiency of current sequencing technologies and provide remarkable insights into the evolution of Acholeplasmataceae.
Comparison of assemblies derived from SBS and SMRT sequencing
Results obtained by the de novo assembly of paired-end SBS reads
Consensus length [bp]
Total read count
The SMRT sequencing approach provided 42,300 SMRT reads with a mean read length of 6,747 nt (total read length of 285,414,973 nt). A total of 38,875 reads enabled the gapless reconstruction of the circular chromosome at a size of 1,587,116 bp. A consensus concordance of 99.9991% and 144.1-fold average sequence coverage were reached. Around 8% of the SMRT-derived reads were rejected due to insufficient quality or incomplete read processing during the assembly.
Evaluation of differences by PCR and Sanger sequencing
Sequence 5′- 3′
No. of differences
Position of the conflict
In summary, Sanger sequencing confirmed the SBS-derived sequences for 22 out of 28 differences. The deviating SMRT data at these positions may indicate errors or rare sequence variations within the final chromosome sequence of 1,587,120 bp. Polymorphic sites at six sequence positions of the chromosome are supported by SMRT assembly, deviating SBS reads and Sanger sequencing.
Benchmarks of the genome of A. oculi and its comparison to other Acholeplasmataceae
Overview of the currently completely determined Acholeplasmataceae genomes
G + C [%]
G + C [%]1
Coding percentage1 [%]
G + C [%]
G + C [%]
Furthermore, the PanOCT analysis predicted that phytoplasmas encode unique AAA+ ATPase, thymidylate kinase and a DNA-dependent RNA polymerase sigma 70 factor RpoD (IPR013325, IPR014284, IPR007627, Additional file 2). RpoD exhibits only insignificant BlastP hits to acholeplasmas’ sigma factors (minimal e-value 9e-08, score 47), and no ortholog was predicted via PanOCT. The existence of a phytoplasma-specific sigma factor points towards some peculiarities in their regulatory system. The other two deduced proteins showed similarities in BlastP analysis to some acholeplasma proteins, albeit they differed in small domain structures. For instance, the AAA+ ATPase of phytoplasmas gave a hit to the ATP-dependent zinc metalloprotease FtsH, which also contains the AAA+ domain structure, and the thymidylate kinases of acholeplasmas showed an additional conserved site (predicted by the PROSITE database search, http://prosite.expasy.org/) – contrary to the thymidylate kinases of phytoplasmas. The overall high number of 402 unique proteins for the four acholeplasmas is interpreted with respect to the diverse environments colonised by acholeplasmas.
Particularities of A. oculistrain 19L
A high percentage (55% – 148) of the 271 proteins predicted to be ‘unique’ for A. oculi in the Acholeplasmataceae are annotated as hypothetical proteins (Figure 1). This set of unique proteins contains phage-related proteins similar to the Acholeplasma phage L2 [GenBank: L13696.1] , including the proteins L2_7, L2_9, L2_11 and L2_12, and which are organised in clusters at three different chromosome regions (118,599-126,732 Aocu_01170-01290; 650,878-659,350 Aocu_05790-5880; 1,062,230-1,068,906 Aocu_09710-9810). In addition, two recombinases (Aocu_08890, Aocu_13990), two integrases (Aocu_05790, Aocu_08840) and one resolvase (Aocu_14000) belong to the list of unique proteins. Besides phage-assigned elements, six transposases were identified, including a mutator type (Aocu_01380), IS3/IS911 family protein (Aocu_01570), IS200-like (Aocu_03680), IS204/IS1001/IS1096/IS1165 (Aocu_04230/50) and other transposon-related elements (insertion element subunit, Aocu_03690). Furthermore, a putative complex transposon is associated with a region carrying a high number of unique proteins (134,519-157,461, Aocu_01380-01570). Beside other proteins, it encodes two oligopeptide ABC transporter components (periplasmatic component oppA and ATP-binding protein oppD) and six glycosyl hydrolase family proteins and a periplasmatic binding protein/LacI transcriptional regulator. A second candidate for a complex transposon (423,889-490,302 Aocu_03680-4250) encodes a UDP-galactopyranose mutase (Glf, Aocu_04190) in addition to another Glf (Aocu_04670) outside the proposed complex transposon. These genes set A. oculi apart from other acholeplasmas. The UDP-galactopyranose mutase is involved in the conversion of UDP-galactopyranose (UDP-GALP) into UDP-galactofuranose (UDP-GALF) (IPR004379), which is the precursor to D-galactofuranose and is often found in the lipopolysaccharide O antigens of several Gram-negative bacteria .
A. oculi is also separated from the other three acholeplasmas by the presence of a putative manganese efflux pump (MntP, Aocu_03470), thereby enabling the exportation of manganese ions, which are toxic in higher amounts. The functional relevance of MntP for manganese homoeostasis has been demonstrated for E. coli. The direct comparison of the A. oculi and the E. coli MntP protein shows 31% identical and 56% similar residues. In addition, A. oculi encodes a cadmium resistance transporter (CadD, Aocu_08600) and one amidohydrolase (AmhX, Aocu_08940). In Bacillus subtilis, AmhX enables the cleavage of the amide bond between non-active conjugated amino acids and may mobilise indole-3-acetic acid (IAA) from inactive storage forms in plants besides several other functions  (IPR017439 ). A. oculi was also detected on plant surfaces . Therefore, one may speculate whether A. oculi can stimulate the growth of colonised plant tissue. Hints for such a manipulation of the IAA metabolism of plants have also been obtained for A. palmae and A. brassicae encoding a putative auxin efflux carrier protein , though no experimental studies are available.
A. oculi is separated from the other Acholeplasma spp. by encoding several additional transcriptional regulators such as ubiC (Aocu_00680), gntR (Aocu_00690), Cro/C1 family proteins (Aocu_01770, Aocu_05750, Aocu_08910 and Aocu_13020) and TetR family proteins (Aocu_14450) not assigned to other Cro/C1-type or TetR family proteins in this family. In total, A. oculi encodes 13 Cro/C1 family proteins, nine of which are shared, and four TetR family proteins, one of which is shared by the other acholeplasmas.
Furthermore, A. oculi is separated from other Acholeplasmataceae by encoding the GDP-D-glycero-α-D-manno-heptose biosynthesis pathway providing D-glycero-D-manno-heptose (HddA, GmhA, HddC, GmhB; Aocu_04590-620). This is a precursor of the inner core lipopolysaccharide . These proteins are similar to those found in the pathway that was reconstructed for the Gram-positive bacteria Aneurinibacillus thermoaerophilus strain DSM 10155 (member of Bacillus/Clostridium group) . For acholeplasmas, there is only one report by Mayberry et al.  that A. modicum contains heptose among the glycolipids.
Moreover, A. oculi encodes two additional proteins, thus playing a role in the biosynthesis of the amino acid methionine. MetW (Aocu_08790) synthesises methionine from homoserine (IPR010743 ), which provides an additional pathway to produce methionine needed in the initiation of translation. The diaminopimelate epimerase (DapF, Aocu_08990) belongs to the aspartate pathway (IPR001653), from which the four amino acids lysine, threonine, methionine and isoleucine can be synthesised.
F1FO ATPases and V1VO ATPases are membrane complexes which function either as H+- or Na+-translocators  (Figure 9). The F1FO ATPase consists of two units – the integral membrane protein FO (atpBEF) acting as a proton channel and the peripheral catalytic stalk F1 (atpHAGDC). The V1VO ATPase is built by the integral membrane protein VO (ntpIK) and the peripheral catalytic stalk V1 (ntpECFABD) . The difference between both transporters is that the V-type ATPase only works in one direction by hydrolysing ATP to produce either a proton or a sodium motive force, while additionally the F-type ATPase is able to act in the other direction by allowing the regulation of the cellular ion pool using the proton motive force, which leads to ATP generation .
Following the sequence-based prediction of Dzioba et al. , the classification of the ion translocating profile can be inferred from the alignment of the protein sequences of the subunits AtpE (F-type ATPase) and NtpK (V-type ATPase). Certain conserved binding motifs are represented by the amino acids at specific positions, in order to specify an H+- or a Na+-translocation (Figure 9). As a result, one F1FO-type Na+ ATPase is suggested to be encoded by all acholeplasmas except for A. oculi, and one V-type Na+ ATPase is predicted for all acholeplasmas except for A. brassicae. It remains unclear as to whether the V-type Na+ ATPase of A. palmae is working despite the lack of an atpC subunit, although this species additionally encodes the F1FO-type Na+ ATPase. Moreover, protein sequence alignment leads to the conclusion that all acholeplasmas encode one V-type H+ ATPase. Deductively, all acholeplasmas encode at least one Na+ and one H+ translocator. This finding stands in accordance with the evidence that Acholeplasma laidlawii strain B possesses a (Na+-Mg2+)-ATPase which is capable of actively extruding sodium ions against the concentration gradient . This previously described, but not genetically characterised, cation pump was linked to the characteristically low intracellular sodium level of these bacteria.
Ultimately, the loss of the F1FO-type Na+ ATPase in Acholeplasmataceae, as is the case for A. oculi, may probably be compensated by the V-type Na+ ATPase. The loss of this genetic module in phytoplasmas remains unclear, but it might be interpreted in respect to the adaptation of phytoplasmas in the intracellular environment with constant osmotic conditions. The comparison of both V-type ATPase operons encoded by A. oculi highlights low sequence identities of the involved proteins (24% to 52%) and differences in protein lengths (Figure 9). This leads to the suggestion that the operons did not derive from a duplication event.
Besides F1FO ATPase, the loss of groEL/ES is remarkable. Native protein folding is conducted by molecular chaperones such as GroEL/ES (Hsp60), DnaK (Hsp70), DnaJ, GrpE, SecB and other heat-shock proteins (Hsp) . GroEL complexes (800 kDa) consisting of two stacked heptameric rings exhibit ATPase activity . The smaller GroES (10 kDa), together with ATP, binds to GroEL and forms the GroEL/GroES complex. DnaK prevents off-pathway reactions or stabilises certain folding intermediates. DnaJ and GrpE act as co-helpers for DnaK . GroEL/ES is probably replaced by the trigger factor (TF) and DnaK, which has already been shown by Kerner et al.  for E. coli or by the HrcA protein, which is commonly found as a part of the heat-shock regulation of bacteria . TF/DnaK and HrcA are encoded in all analysed species of the Acholeplasmataceae (Figure 7). Several Mollicutes are known to have lost groEL and groES, such as Mesoplasma florum, Mycoplasma hyopneumoniae, Ureaplasma parvum serovar 3, Ureaplasma urealyticum, Mycoplasma mobile and some further Mycoplasma spp. . It is likely that there are even more Mollicutes lacking these proteins. Saccardo et al.  suggested, based on draft sequences, that there are four phytoplasma strains of the 16SrIII group that probably lack GroEL/ES. The possibility that this genetic feature can be lost within the Mollicutes is supported by experiments with transposon mutagenesis, showing that GroEL is not or only weakly regulated during heat shock for M. genitalium or M. pneumonia, thereby leading to the suggestion that this chaperone is not essential for Mycoplasma spp. in general and may represent an evolutionary remnant. Evolutionary loss could apparently be possible due to either the fact that GroEL is immunogenic, and therefore it would be advantageous to get rid of it by avoiding an immune response in mammals  – a benefit for A. oculi when infecting mammals – or alternatively Mollicutes possess small genomes which encode few proteins; consequently, they own fewer substrate proteins, which have to be correctly folded by GroEL.
This study demonstrated the efficiency of the SMRT approach in the complete de novo determination of bacterial genomes. A. oculi encodes, like other Acholeplasma spp., rich genetic content in comparison to phytoplasmas. The relatively small core genome of phytoplasmas should be interpreted with respect to their intracellular parasitism and their corresponding poor metabolic repertoire. In contrast, acholeplasmas depend on a richer genetic repertoire due to their widespread distribution and colonisation of diverse micro-habitats. However, for the first time, the deduced protein content of A. oculi highlights that the loss of basic genetic elements, including the chaperone GroEL/ES and the F1FO-type Na+ ATPase system, took place in both genera of the Acholeplasmataceae. One could therefore speculate that the common V-type H+ ATPase system in acholeplasmas may regulate the cellular proton pool, and the V-type Na+ ATPase system may compensate for the lack of the F1FO-type Na+ ATPase. The loss of GroEL/ES is interpreted as being not extraordinary for Mollicutes and seems to have occurred several times within this class.
A. oculi strain 19L isolate was kindly provided by Jerry K. Davis (Purdue University School of Veterinary Medicine, West Lafayette, Ind., USA) from the strain collection of the International Organization for Mycoplasmology (IOM). Cells were cultivated in ATCC® Medium 1039 (http://www.atcc.org) supplemented with 0.2% polymyxin B (Roth, Karlsruhe, Germany) and 0.2% penicillin G (Merck, Darmstadt, Germany) at 28°C for about 14 days and collected by centrifugation (20 min, 10,000 rpm, 4°C). The DNA isolation of A. oculi strain 19L for SBS was performed with the DNeasy Blood & Tissue Kit (Qiagen, Hildesheim, Germany) and according to the manufacturer’s instruction. DNA isolation needed for preparing the PacBio 10-kb library high molecular weight genomic DNA was performed according to Moore et al. .
Sequencing and assembly of SBS data
DNA-Seq libraries were prepared from fragmented DNA (COVARIS S2, Woburn, Massachusetts, USA) according to recommendations made by the supplier (TruSeq DNA sample preparation v2 guide, Illumina, San Diego, CA, USA). Libraries were quantified by fluorometry, immobilised and processed onto a flow cell with a cBot followed by sequencing by synthesis by applying TruSeq v3 chemistry on a HiSeq2500 (all components by Illumina).
The de novo assembly of the reads was performed in CLC Genomics Workbench 7.0 (http://www.clcbio.com). The assembly data was exported as a BAM file, indexed using SAMtools  and imported in Gap5 . Gaps were closed by PCR and primer-walking by applying dye-terminator sequencing performed on an ABI 310 capillary sequencer (Life technologies, Carlsbad, CA, USA).
Sequencing and assembly of SMRT data
A 10-kb library was prepared and processed as recommended by Pacific Biosciences (http://www.smrtcommunity.com/SampleNet/Sample-Prep). Library construction and subsequent sequencing were performed using the SMRTbell Template Preparation Reagent Kit 1.0, DNA/Polymerase binding kit P4-C2, MagBead Kit and DNA Sequencing Kit 2.0 (all components supplied by Pacific Biosciences, Menlo Park CA, USA.). The genome was sequenced using PacBio RS II technology (P4-C2 chemistry). Data collected on the PacBio RS II instrument were processed and filtered (SMRT analysis software, version 2.1). All experiments were conducted according to the manufacturers’ instructions on a single SMRT cell. Obtained data were analysed on the SMRT Portal V2.1.1 (http://www.pacb.com/devnet/) by applying the integrated Celera® Assembler. SMRT sequencing and SBS were performed by the Max Planck-Genome-centre Cologne, Germany (http://mpgc.mpipz.mpg.de/home/).
Identification of sequencing differences comparing both sequencing methods
Rare sequencing differences were identified via BlastN (low complexity filter off, word size 7)  by applying the SMRT-derived genome sequence as a reference. In addition, SBS data were mapped onto the SMRT sequence in CLC Genomics Workbench 7.0, and Primer-BLAST  was used for designing oligonucleotide pairs, thus enabling the PCR amplification of conflict regions (Table 2). Sequences of PCR products were determined by applying dye-terminator sequencing.
Annotation of the genome sequence
The oriC region was determined through the cumulative GC-skew calculation of the chromosome sequence in Artemis  and the determination of the DnaA-boxes . The adjusted genome sequence was automatically annotated in RAST  and annotation was manually curated in Artemis by incorporating additional analyses obtained from the InterProScan database , RNAmmer  and tRNAscan-SE . The annotated genome sequence of A. oculi strain 19L, including information on polymorphisms, was deposited [EMBL:LK028559]). Read data for SBS [EMBL:ERX463488] and SMRT [EMBL:ERX470328] were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena/).
Prediction of orthologous proteins within the Acholeplasmataceaefamily
Orthologous proteins of Acholeplasmataceae were calculated by PanOCT  by applying the default parameters and protein data of A. laidlawii strain PG-8A [Genbank:CP000896.1] , A. brassicae strain O502 [GenBank:FO681348.1] , A. palmae strain J233 [GenBank:FO681347] , ‘Ca. P. australiense’ strain rp-A [GenBank:AM422018.1]  and NZSb11 [GenBank:CP002548.1] , ‘Ca. P. asteris’ strain OY-M [GenBank:AP006628.2]  and AY-WB [GenBank:CP000061.1]  and ‘Ca. P. mali’ strain AT [GenBank:CU469464.1] . The results obtained by the software were also used for the prediction of the pan-, dispensable- and core-genome  of each genera and the family .
Phylogenetic analysis of the Acholeplasmataceae
Alignment and the phylogenetic tree were calculated for 64 16S rRNA genes of the Acholeplasmataceae by using the maximum likelihood algorithm implemented in CLC Genomics Workbench 7.0 and by applying the parameter ‘very accurate’ for the alignment with a gap open cost of 10.0 and a gap extension cost of 1.0. The phylogenetic tree was constructed by using a 1,000 bootstrapped maximum likelihood algorithm, whereby the ‘UPGMA’ construction method and the ‘Jukes Cantor’ nucleotide substitution model were chosen. Mycoplasma genitalium strain G37 was added, in order to root the tree.
The German Research Foundation (DFG), through projects KU 2679/2-1 and BU 890/21-1, the German Ministry for Education and Research (BMBF, RNomics no. 01GS0805), the Ministry of Education and Science (Republic of Serbia, Grant No. TR31043) and the German Academic Exchange Service (DAAD/BMBF), through project 56266384, supported this work.
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