Volume 10 Supplement 6

European Molecular Biology Network (EMBnet) Conference 2008: 20th Anniversary Celebration. Leading applications and technologies in bioinformatics

Open Access

The Mycoplasma conjunctivae genome sequencing, annotation and analysis

  • Sandra P Calderon-Copete1,
  • George Wigger2,
  • Christof Wunderlin2,
  • Tobias Schmidheini2,
  • Joachim Frey3,
  • Michael A Quail4 and
  • Laurent Falquet1Email author
BMC Bioinformatics200910(Suppl 6):S7

DOI: 10.1186/1471-2105-10-S6-S7

Published: 16 June 2009

Abstract

Background

The mollicute Mycoplasma conjunctivae is the etiological agent leading to infectious keratoconjunctivitis (IKC) in domestic sheep and wild caprinae. Although this pathogen is relatively benign for domestic animals treated by antibiotics, it can lead wild animals to blindness and death. This is a major cause of death in the protected species in the Alps (e.g., Capra ibex, Rupicapra rupicapra).

Methods

The genome was sequenced using a combined technique of GS-FLX (454) and Sanger sequencing, and annotated by an automatic pipeline that we designed using several tools interconnected via PERL scripts. The resulting annotations are stored in a MySQL database.

Results

The annotated sequence is deposited in the EMBL database (FM864216) and uploaded into the mollicutes database MolliGen http://cbi.labri.fr/outils/molligen/ allowing for comparative genomics.

Conclusion

We show that our automatic pipeline allows for annotating a complete mycoplasma genome and present several examples of analysis in search for biological targets (e.g., pathogenic proteins).

Background

Mycoplasmas (class Mollicutes) are among the smallest microorganisms capable of self-replication and autonomous life [1]. The genus Mycoplasma includes a large number of highly genomically-reduced species which in nature are associated with hosts either commensally or pathogenically [2]. General features of the class Mollicutes are small genome, lack of cell wall and low GC content.

Indeed, the Mycoplasma species have genomes of 0.6 to 1.3 Mbp. Weisburg et al. (1989) [3] and Woese et al. (1980) [4] revealed that Mycoplasma have evolved from more classical bacteria of the firmicutes taxon by a so-called regressive evolution that resulted in massive genome reduction [5] and minimal metabolic activities. Consequently, they adopted a strict parasitic life style, mainly occurring as extracellular parasites often restricted to a living host, with some species having the ability to invade host cells as described by Sirand-Pugnet et al. (2007) [5], Rosengarten et al. (2000) [6] and Citti et al. (2005) [7]. They have a predilection for the mucosal surfaces, where they successfully compete for nutrients with many other organisms, establishing chronic infections [5]. They do not show specific virulence factor as known in other bacteria, instead they seem to use toxic metabolic intermediates that they secrete and translocate to the host cells as virulence factors [8]. Additionally, due to the lack of cell wall, they are not affected by some antibiotics which target synthesis of cell wall such penicillin or other beta-lactam antibiotics making these organisms particularly interesting in medicine.

Infectious keratoconjunctivitis (IKC)

Mycoplasma conjunctivae is considered as the major etiological agent of Infectious KeratoConjunctivitis (IKC) for both domestic and wild caprinae species. In the European Alps it affects several species such as alpine ibex (Capra ibex ibex), alpine chamois (Rupicapra rupicapra rupicapra), and mouflon (Ovis orientalis musimon), as well as in domestic sheep and goat [9]. In Switzerland, M. conjunctivae is known to be the primary cause of this disease [10].

The implied role of M. conjunctivae is based on the frequent isolation of this organism from inflamed eyes and on limited attempts to induce ocular disease experimentally showing that M. conjunctivae is one agent responsible for epidemic keratoconjunctivitis [11]. Nonetheless, even if the molecular epidemiology has been well described by Belloy et al. (2003) [9], the molecular infection mechanism is still not established and remains a mystery.

Methods

Bacterial strain

M. conjunctivae type strain HRC/581T (NCTC10147) [12] was grown on standard mycoplasma broth medium enriched with 20% horse serum, 2.5% yeast extract and 1% glucose (Axcell Biotechnologies). The cells were harvested by centrifugation at 13,000 × g for 20 min, washed three times in TES buffer (10 mM Tris-HCl, 1 mM EDTA, 0.8% NaCl, pH 7.5), and then re-suspended in TES buffer to a concentration of approximately 109 bacteria/ml. DNA was extracted by the guanidium thiocyanate method [13], extracted 3 times with PCIA (Phenol: CHCl3: Isoamylalcohol = 49.5: 49.5: 1) and 3 times with CIA (CHCl3: Isoamylalcohol = 99: 1), precipitated with 50% isoproanol, washed 2 times with 70% ethanol to remove salt, dried in the air for 15 min and re-suspended in double distilled H2O at a concentration of 500 μg/ml.

Sequencing

Sequencing and assembly of the genome was carried out by Microsynth AG. The quality of the isolated genomic DNA was verified by gel electrophoresis and displayed a pure high molecular weight DNA. The DNA was sheared by passing it several times through a needle, in order to construct two different libraries: a plasmid library and a fosmid library. For the plasmid library (2–12 Kbp inserts), the genomic DNA was passed 30 times through a 30-Gauge needle and sonicated for 10 seconds (sonication strength 3 on a Digital Sonifier 450 from Branson Ultrasonics corp, Danbury, CT, USA). For the fosmid library (32 Kbp inserts), the genomic DNA was passed 10 times through a 23-Gauge needle without sonication.

Small fragments were ligated with a linker, fractionated twice through 0.8% agarose gels. Fractions of 6 different sizes (from 2 to 12 Kbp) were cut out from the gel and cloned into vector pOTW12 (Sanger Institute). Moreover, the large fragments were fractionated using a CHEF-DR II System (BIORAD). Fragments of 32 Kbp were cut out from the gel and ligated into pCC1Fos (Epicentre Biotechnology Inc.).

From the plasmid library 11'300 clones and from the fosmid library 384 clones were end-sequenced on an ABI 3730 capillary sequencer. A second part of the small fragments were sequenced using 454 Life Science FLX technology leading to 263'163 reads that were reduced to 78'498 reads covering 20'569'079 bp after applying a quality cut-off filter (approx. 22× coverage).

Assembly

The assembly was carried out using the SeqMan module of the DNASTAR Lasergene version 7 combining both classical Sanger sequences (ABI3730) and 454 FLX reads. A check was conducted with "amosvalidator" of the AMOS package [14], allowing identifying suspicious regions in the assembly. To help in the assembly process the 384 fosmids paired-end reads were aligned to the final sequence. The reads display a nice spreading at regular intervals except for 2 clones that were absent from the results. The 2 regions were analyzed for the presence of potentially lethal genes for E. coli. The first region contains a homologue of the gene lepA that is known to be lethal when overexpressed in E. coli [15]. This region also contains a restriction enzyme that might cut E. coli genome. The second region contains a transposase and some phage genes. This might explain the toxicity of these two fosmids in E. coli.

Automatic annotation

The automatic annotation pipeline was entirely built locally using available software and linking them with Perl scripts.

Gene prediction was carried out using Glimmer 3.02 [16] and the genetic code specific for Mycoplasma (e.g., UGA encodes a tryptophane). The interpolated context models (ICM) were calculated by self-training on the long ORFs of the contigs. The RNAs were predicted using Infernal with models obtained from RFAM [17], tRNAscan-SE [18], and blastn for 16S and 23S [19].

Predicted coding sequences (CDS) were translated using the EMBOSS package (extractseq, transeq, revseq) [20] and a similarity search was run by blastp against the UniProt/Swiss-Prot knowledgebase (Release 56.2 of 23-Sep-2008: 398181 entries) [21]. The CDS were also scanned against the HAMAP families [22] to identify orthologous protein families. In addition the CDS were searched for potential known domains using InterProScan [23], and for biased compositional regions with SEG [24] and Marcoil [25].

The biological interest of an annotation project is to identify the gene products by designating a descriptive common name for the protein and its function with as much specificity as the evidence supports. We use homology-based annotation transfer to assign the name and associated information of gene product: Gene symbol, EC number if protein is identified as an enzyme and other features.

Homology search is performed by blastp that allows finding the best matches with the highest significant sequence similarity appearing between the putative proteins sequences compared first to a database of known mycoplasma proteins and secondly to proteins from the UniProt/Swiss-Prot knowledgebase. Additionally, matches with HAMAP protein family permits to support homology annotation and raise the confidence level of annotation transfer. Other characterization features like functional domains constitute an additional support evidence of function assignment (Table 1). The results obtained from the various programs are parsed and stored in GFF3 format in a local MySQL database. The EMBL format is produced from the data stored in this database and deposited at the EBI EMBL database, the accession number is FM864216.
Table 1

Functional assignment criteria

Source

Known Protein

Putative

Hypothetical

Blastp (Mycoplasma DB)

Evalue < e-20

Evalue between

Evalue

  

>e-20 and <e-4

> e-4 or No match

Blastp (Swissprot DB)

Evalue < e-20

Evalue between

Evalue

  

>e-20 and <e-4

> e-4 or No match

HAMAP families

Confident protein family match

No confident protein family match

N/A

Interpro domains

If no HAMAP family match. Support evidence from at least one InterPro member database

Support evidence from one or none Interpro member database

Description of the criteria used to assign the genes products into the 3 following categories: Known Protein (known function: significant e-value and supported by confident protein family and functional domains), Putative protein (unclear function: twilight zone e-value but supported by functional domains) and Hypothetical protein (unknown function: non-significant e-value or no match in databases).

In order to assess the confidence of the results provided by our annotation pipeline, we used the known genome of Mycoplasma hyopneumoniae (strain 232) already annotated (NCBI entry AE017332) for comparing pipeline results with those provided at NCBI. The annotation pipeline identifies 741 CDS and 32 non-coding RNAs (ncRNAs) whereas only 691 CDS and 36 ncRNAS are known. The 91.1% of total genes were correctly predicted leaving only 23 genes not found by the predictor programs. Regarding the functional annotation, our pipeline provides 83.7% of correct gene annotations and even in some cases complements the existing one. 7.4% of CDS are wrongly annotated or in a different way. The 69 genes predicted in addition to known genes could be considered as false positives even if they also could represent new potential genes of M. hyopneumoniae genome (Table 2). Using results of M. hyopneumoniae 232 annotation, we evaluated the specificity and sensitivity of the pipeline and obtained a sensitivity of 92% and a specificity of 90% (Table 3).
Table 2

Results obtained for M. hyopneumoniae 232 annotation

 

All genes

CDS

ncRNA

Total of M. hyopneumoniae 232 known genes

727

691

36

Total annotated genes using pipeline

773

741

32

   Total correct predictions *

704

672

32

   Total known genes not predicted *

23

19

4

   Total correct gene annotations

647

618

29

   Total predicted genes incorrectly or differently annotated

57

54

3

   Total predicted genes in additionally annotated by pipeline

69

69

0

* CDS were predicted using Glimmer3 and ncRNAs were predicted using Blastn for 23S and 16S rRNAs, tRNAScan-SE for tRNAs and Infernal for other ncRNAs

Table 3

Evaluation of the sensitivity and the specificity of the pipeline based on re-annotation of the M. hyopneumoniae 232 genome.

Total genes detected

FP

TP

FN

Specificity TP/(TP+FP)

Sensitivity TP/(TP+FN)

773

69

647

57

90%

92%

FP = False Positive, TP = True Positive, FN = False Negative

Results

General genome features

Composition and functional gene assignment, origin of replication

The genome consists of a single chromosome with a size estimated of about 0.9 Mbp. Currently, more than 95% of the genome is available as a single contig, but due to the presence of repeated sequences, we experienced difficulties in assembling the sequences to close the gap. The contig has a size of 846'214 bp with a G+C content of 29% (Figure 1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2105-10-S6-S7/MediaObjects/12859_2009_Article_3302_Fig1_HTML.jpg
Figure 1

Genome map. Mycoplasma conjunctivae genome map generated by GenomeProjector tool and available in http://myconj.vital-it.ch/GenomeProjector/. This map represents from the outer ring in wards, genes on direct strand (pink), genes on complementary strand (yellow), tRNAs (green arrows), rRNAs (pink or orange stripes depending on the strand), GC content (brown lines), GC skew (yellow lines). The replication origin and terminus are predicted from the GC skew shift points and are in a different position than the one we found.

A total of 734 genes have been computationally predicted. We found both 23S and 16S ribosomal RNAs in unique copies located next to each other. The 5S ribosomal RNA is located remotely of 23S and 16S genes. We identified 28 transfer RNAs covering all 20 amino acids. Other non-coding RNAs were found: bacterial RNase P class B, TPP riboswitch (THI element), tmRNA (proteolysis signal) and the bacterial signal recognition particle RNA.

Additionally, 699 genes were predicted as coding sequences for proteins. 49% of those genes have a clear homologue with a known function. 5.6% of those genes were annotated as "putative proteins" because the closest known protein was aligned with a marginal e-value. While the remaining 45% have unknown function, and were named as "hypothetical" proteins. From those hypothetical proteins, 75% matched non-significatively to other proteins and 25% are considered unique for M. conjunctivae because no match was obtained by blastp against both databases. A summary is shown in Table 4 and Table 5.
Table 4

Summary of M. conjunctivae genome features

Genome Features

 

Length (bp)

846,214

G + C content (mole%)

29.0%

   Functionally assigned protein CDSs

344

   Putative protein CDSs

39

   Hypothetical protein CDSs

316

   23S rRNA

1

   16S rRNA

1

   5S rRNA

1

   Bacterial RNase P class B

1

   TPP riboswitch (THI element)

1

   tmRNA

1

   Bacterial signal recognition particle RNA

1

   Transfer RNA genes

28

Table 5

Top 20 biological processes. Relevant information with a biological meaning was searched in priority. We list the top 20 of biological process that are accomplished by the newly annotated genes.

Biological process

Proteins matched

translation

68

metabolic process

32

transport

31

proteolysis

18

tRNA aminoacylation for protein translation

15

DNA repair

13

DNA replication

12

carbohydrate metabolic process

11

phosphoenolpyruvate-dependent sugar phosphotransferase system

11

regulation of transcription

10

DNA modification

9

glycolysis

9

ATP synthesis coupled proton transport

8

DNA methylation

7

DNA recombination

7

DNA integration

6

electron transport

6

biosynthetic process

5

nucleoside metabolic process

5

Protein folding

5

It is important to note that our method (homology based annotation) does not allow to distinguish between close homologues having different functions.

The origin of replication (oriC) was searched by comparing the sequences of 3 strains of M. hyopneumoniae with the sequence of M. conjunctivae in the region of dnaA gene (Figure 2). We attempted to identify several features that have been associated with replication origins in other bacterial species, including mollicutes. Bacterial origins of replication are typically located in the vicinity of the dnaA and dnaN genes. Usually several dnaA-box motifs are found within the intergenic regions around dnaA gene [26]. We searched unsuccessfully for the presence of consensus dnaA-box motifs with the pattern TTATC [CA]A [CA] using fuzznuc of the EMBOSS package [27]. When we used a slightly different, more relaxed dnaA-box consensus motifs TT [AT] [AC] [ACT]A [AC]A, two sequences matching each of these patterns were found between the dnaA and rpmH genes (Figure 2). However, well over 3,000 hits located throughout the rest of the genome were also seen. Therefore, the specificity of the pattern used to try to detect dnaA-box motifs was very low, decreasing our confidence in the significance of the sequences identified.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2105-10-S6-S7/MediaObjects/12859_2009_Article_3302_Fig2_HTML.jpg
Figure 2

Origin of replication. The region between rpmH and dnaA genes for the 3 M. hyopneumoniae strains aligned with M. conjunctivae. The two putative dnaA boxes are shown in black. M. hyopneumoniae J (NC_007295), M. hyopneumoniae 7448 (NC_007332), M. hyopneumoniae 232 (NC_006360), M. conjunctivae (FM864216).

These findings are in contrast to the multiple dnaA-boxes found in the intergenic regions surrounding dnaA in other mollicutes [26]. In addition to the presence of dnaA-box motifs, replication origins can also frequently be identified by looking for biases in strand composition through measures such as the cumulative GC skew [2830]. For M. conjunctivae, we found no significant asymmetries that can be readily detected with GC skew. The lack of a clear bias in M. conjunctivae is similar to that observed for the M. hyopneumoniae [31]. Therefore, the only significant feature of the M. conjunctivae genome that provides any possible indication of the location of the origin of replication is the presence of the dnaA gene. Otherwise, there are no features that allow definitive mapping of the origin to the intergenic region upstream of the dnaA gene, as seen in other bacteria.

Potential pathogenic features

Bacteria have many ways to produce virulence that reside in the ability to adhere, invade and cause damage to host cells. Various strategies of pathogenicity such as cytolysins, toxins and invasins enable other bacteria to produce infection. In Mycoplasma species no such typical primary virulence genes have been found. Mycoplasmas seem rather to use intrinsic metabolic and catabolic functions to cause disease in the affected host and to ensure the microbe's survival. Our efforts to identify genes involved in the pathogenicity of Mycoplasma conjunctivae were concentrated on the one hand, try to find those primary virulence genes, toxins principally, rare in other mycoplasmas. On the other hand, on metabolic pathways that has been proposed by studies carried out in other mycoplasmas [8].

Glycerol pathway

We found using manual blastp queries by an expert, the genes for a glycerol-3-phosphate dehydrogenase (glpO), a glycerol kinase (glpK), a glycerol uptake facilitator protein (glpF) and an ABC transporter system (Sn-glycerol-3-phosphate transport system permease) that are implicated in the glycerol metabolism producing cell damage, inflammation and disease in Mycoplasma mycoides subsp. mycoides Small Colony (SC) [8].

The pathway starts with the assimilation of glycerol by the ABC glycerol transporter (gtsA, gtsB and gtsC). Afterwards, the glycerol is phosphorylated into glycerol-3-phosphate, then oxidized by GlpO in presence of O2 into dihydroxyactone-phosphate (DHAP) producing one molecule of H2O2. H2O2 is released directly inside the host cells by the transmembrane GlpO protein leading to cell death [8]. The absence of any gene having a catalase or dismutase activity favors this hypothesis.

The identification of those genes in Mycoplasma conjunctivae constitutes an important discovery given that a relationship between the glycerol metabolism and cytotoxicity is established in the laboratory[8]. Further work to validate this hypothesis in M. conjunctivae is required and has been started in collaboration with a laboratory of the Institute for Veterinary Bacteriology (University of Bern).

Toxins

Toxins constitute an important type of virulence factors in several bacteria. Thereby, we searched for toxins in M. conjunctivae and we found 3 proteins highly similar with toxins of Treponema hyodysenteriae (Brachyspira hyodysenteriae). Those proteins are Hemolysin A (hlyA), Hemolysin B (hlyB) and Hemolysin C (hlyC). The 3 genes are scattered on the genome.

Those proteins are present in other mycoplasmas, particularly M. hyopneumoniae and M. capricolum, and even if in those species, these toxins are not essential for pathogenicity mechanisms, it can not be excluded that these toxins contribute to the pathogenicity of M. conjunctivae.

IS elements

Insertion sequences (IS) are short DNA elements that function as simple transposable elements by coding for proteins implicated in the transposition activity. Transposase and other regulatory protein are the proteins generally coded by IS elements: The transposase catalyses the enzymatic reaction allowing the IS to move. Regulatory proteins act by enhancing or inhibiting the transposition activity. The coding region in an insertion sequence is usually flanked by inverted repeats [32].

We found several genes coding for complete or partial transposases (Table 6). An IS1138 insertion element has particularly brought our attention. IS1138 elements belong to IS3 family are prevalent in other mycoplasmas and are the only (with IS1138 b) that have been demonstrated directly to undergo autonomous transposition [32, 33]. Interestingly a transposase for one IS1138 insertion elements is followed by homologues of a methylase Hpa I and a type II restriction enzyme HpaI from Haemophilus parainfluenzae forming a restriction-methylation cassette. The hypothesis of a horizontal transfer from H. parainfluenza to M. conjunctivae was formulated. We evaluated the G+C content of this cassette, but we did not observe a higher G+C content inside the cassette compared to the surrounding area. If the G+C content inside the cassette would be different from that of M. conjunctivae (29%) and similar to that of H. parainfluenzae (~41%) it could constitute an evidence of the transfer.
Table 6

List of M. conjunctivae transposases detected.

gene

Product

start

end

strand

length

Condition of the sequence

MCJ_00022

IS1138

23818

24999

-

1181

Complete

MCJ_00059

?

61624

61728

+

104

Partial

MCJ_00099

IS1138

97032

98213

+

1181

Complete

MCJ_00207

IS1138

192412

192660

-

248

Same transposase split in two ORFs

MCJ_00208

IS1138

192717

193088

-

371

 

MCJ_00215

ISMag1

198177

199265

+

1088

Complete

MCJ_00399

IS1138

430830

432011

-

1181

Complete

MCJ_00428

IS1138

474034

474138

+

104

Partial

MCJ_00553

ISMHp1

639264

639500

-

236

Partial

MCJ_00635

IS1634AM

742041

743714

+

1673

Complete

Comparative genomics

The list of proteins was classified and compared to 4 other mycoplasma genomes as shown in Table 7. The main difference with other mycoplasma is an apparently low carbohydrate and transport metabolism that could explain the need for a strong glycerol pathway, as well as the large number of hypothetical proteins probably due to the fully automatic annotation process.
Table 7

Genome comparison. Functional classification of proteins of 5 sequenced mycoplasma genomes

Functional categories

Mycoplasma. hyopneumoniae 232

Mycoplasma pulmonis

Mycoplasma genitalium

Mycoplasma mobile

Mycoplasma conjunctivae

 

(892 kb)

(964 kb)

(816 kb)

(777 kb)

(846 kb)

Translation, ribosomal structure and biogenesis

100

108

108

108

90

Transcription

22

27

16

23

16

DNA replication, recombination and repair

75

118

49

72

55

Posttranslational modification, protein turnover

20

24

19

22

15

Energy production and conversion

25

29

20

29

25

Carbohydrate transport and metabolism

57

68

32

50

30

Amino acid transport and metabolism

25

27

17

24

20

Nucleotide transport and metabolism

19

22

20

18

17

Coenzyme transport and metabolism

9

12

12

17

8

Lipid transport and metabolism

6

10

9

9

7

Inorganic ion transport and metabolism

14

18

17

15

5

Other

118

124

87

107

95

No known function

201

195

78

139

316

Total CDSs

691

782

484

633

699

Discussion

Mycoplasma conjunctivae is the fourteenth genome of a mycoplasma species that has been fully sequenced. Phylogenetically, the closest relative among the sequenced mycoplasmas is M. hyopneumoniae reflected by the high similarity of most of the proteins identified in M. conjunctivae.

The analysis of M. conjunctivae genome features, describes this organism as a typical mycoplasma, with a genome size and a G+C content within the range of other mycoplasma genomes. The comparison of mycoplasma genome sizes demonstrates that the sequence length is variable not only within the same genus but even among strains of the same species as shown in Table 8. Even if we do not know the final size of the genome, we expect a chromosome length of about 900'000 bp, size almost similar with the genome size of M. hyopneumoniae.
Table 8

Genome comparison. Genome size of 15 sequenced genomes of species belonging to Mycoplasma genus http://www.ebi.ac.uk/genomes/bacteria.html, including M. conjunctivae.

EMBL AC

Species name

Genome Size

G+C content

CU179680

Mycoplasma agalactiae PG2 chromosome

877,438

29.7%

CP001047

Mycoplasma arthritidis 158L3-1

820,453

30.7%

CP000123

Mycoplasma capricolum subsp. capricolum ATCC 27343

1,010,023

23.8%

AE015450

Mycoplasma gallisepticum R

996,422

31.5%

L43967

Mycoplasma genitalium G37

580,076

31.7%

AE017332

Mycoplasma hyopneumoniae 232

892,758

28.6%

AE017244

Mycoplasma hyopneumoniae 7448

920,079

28.5%

AE017243

Mycoplasma hyopneumoniae J

897,405

28.5%

AE017308

Mycoplasma mobile 163 K

777,079

25%

BX293980

Mycoplasma mycoides subsp. mycoides SC str. PG1

1,211,703

24%

BA000026

Mycoplasma penetrans HF-2

1,358,633

25.7%

U00089

Mycoplasma pneumoniae M129

816,394

40%

AL445566

Mycoplasma pulmonis UAB CTIP

963,879

26.6%

AE017245

Mycoplasma synoviae 53

799,476

28.5%

FM864216

Mycoplasma conjunctivae

846,214

29%

Globally the mycoplasma genomes have a characteristically low G+C content within the range of 23.8 to 40 mol% (Table 8). The highest G+C content found in M. pneumoniae and the lowest in M. capricolum. Regarding Mycoplasma conjunctivae, G+C content has a typical value of about 29%. The codon usage is similar to that of M. hyopneumoniae and opposite to that of M. capricolum and M. mycoides [31, 34, 35].

The presence of repeats across the genome was the principal difficulty for finishing the genome assembly. Insertion sequence (IS) elements are reported in the majority of mycoplasmas and in M. conjunctivae we found transposases for IS-elements in the genome. Some of those transposases genes are complete sequences and some other are fragmented showing a predicted length of less than 1000 bp. Since those insertion elements are nearly identical they created difficulties for assembling the genome.

The findings highlighted by this project, principally the glycerol pathway, require further experimental confirmation. In particular, the hypothesis for damaging the host cells by the glycerol metabolism need to be confirmed by demonstrating the localization of GlpO in the membrane and the release of H2O2 outside the cell. If this hypothesis can be verified, the possibility to block at any stage the glycerol pathway could constitute a candidate target for controlling the disease.

Conclusion

In conclusion, we created an automatic pipeline to annotate a prokaryotic genome sequence using various tools for the prediction and the identification of the genes. This pipeline is customized for handling sequences of mycoplasma species.

We deposited the Mycoplasma conjunctivae genome fully annotated in the EMBL database (FM864216). Data stored into our local database can be searched and genome can be visualized through our website http://myconj.vital-it.ch. Analysis of annotated genes gives new insights about potential mechanisms of pathogenicity as well as the possibility to go deeper into the knowledge of Mycoplasma conjunctivae and the IKC disease and opens the way to finding methods to prevent M. conjunctivae infections of domestic animals as reservoir for this pathogen and hence prevent IKC in wild animals.

Declarations

Acknowledgements

We thank for support the "Programmes actions intégrées PAI" – Germaine de Staël. We would like to particularly thank Mrs Denise Schmidheini for her kind help in initiating and supporting this sequencing project.

We are grateful to the Vital-IT platform for offering calculation time on their computing cluster, and in particular to Mr Volker Flegel for his help in installing and debugging the necessary software.

This article has been published as part of BMC Bioinformatics Volume 10 Supplement 6, 2009: European Molecular Biology Network (EMBnet) Conference 2008: 20th Anniversary Celebration. Leading applications and technologies in bioinformatics. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/10?issue=S6.

Authors’ Affiliations

(1)
Swiss Institute of Bioinformatics, Génopode-UNIL
(2)
Microsynth AG
(3)
Institute for Veterinary Bacteriology, University of Bern
(4)
Wellcome Trust Genome Campus

References

  1. Pettersson B, Uhlén M, Johansson KE: Phylogeny of some mycoplasmas from ruminants based on 16S rRNA sequences and definition of a new cluster within the hominis group. Int J Syst Bacteriol 1996, 46(4):1093–1098.View ArticlePubMed
  2. Balish MF, Krause DC: Mycoplasmas: A Distinct Cytoskeleton for Wall-Less Bacteria. J Mol Microbiol Biotechnol 2006, 11: 244–255. 10.1159/000094058View ArticlePubMed
  3. Weisburg WG, Tully JG, Rose DL, Petzel JP, Oyaizu H, Yang D, Mandelco L, Sechrest J, Lawrence TG, Van Etten J, Maniloff J, Woese CR: A phylogenetic analysis of the mycoplasmas: Basis for their classification. J Bacteriol 1989, 171: 6455–6467.PubMed CentralPubMed
  4. Woese CR, Maniloff J, Zablen LB: Phylogenetic analysis of the mycoplasmas. Proc Natl Acad Sci USA 1980, 77: 494–498. 10.1073/pnas.77.1.494PubMed CentralView ArticlePubMed
  5. Sirand-Pugnet P, Lartigue C, Marenda M, Jacob D, Barré A, Barbe V, Schenowitz C, Mangenot S, Couloux A, Segurens B, de Daruvar A, Blanchard A, Citti C: Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome. PLoS Genet 2007, 3(5):e75. 10.1371/journal.pgen.0030075PubMed CentralView ArticlePubMed
  6. Rosengarten R, Citti C, Glew M, Lischewski A, Droesse M, Much P, Winner F, Brank M, Spergser J: Hostpathogen interactions in mycoplasma pathogenesis: Virulence and survival strategies of minimalist prokaryotes. Int J Med Microbiol 2000, 290(1):15–25.View ArticlePubMed
  7. Citti C, Browning GF, Rosengarten R: Phenotypic diversity and cell invasion in host subversion by pathogenic mycoplasmas. In Mycoplasmas: pathogenesis, molecular biology, and emerging strategies for control. Edited by: Blanchard A, Browning GF. Horizon Bioscience, Wymondham, Norfolk, United Kingdom; 439–483.
  8. Pilo P, Vilei EM, Peterhans E, Bonvin-Klotz L, Stoffel MH, Dobbelaere D, Frey J: A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony. J Bacteriol 2005, 187(19):6824–6831. 10.1128/JB.187.19.6824-6831.2005PubMed CentralView ArticlePubMed
  9. Belloy L, Janovsky M, Vilei EM, Pilo P, Giacometti M, Frey J: Molecular epidemiology of Mycoplasma conjunctivae in Caprinae: transmission across species in natural outbreaks. Appl Environ Microbiol 2003, 69(4):1913–1919. 10.1128/AEM.69.4.1913-1919.2003PubMed CentralView ArticlePubMed
  10. Belloy L, Vilei EM, Giacometti M, Frey J: Characterization of LppS, an adhesin of Mycoplasma conjunctivae. Microbiology 2003, 149(1):185–193. 10.1099/mic.0.25864-0View ArticlePubMed
  11. Baas EJ, Trotter SL, Franklin RM, Barile MF: Epidemic caprine keratoconjunctivitis: recovery of Mycoplasma conjunctivae and its possible role in pathogenesis. Infect Immun 1977, 18(3):806–815.PubMed CentralPubMed
  12. Barile MF, Del Giudice RA, Tully JG: Isolation and characterization of Mycoplasma conjunctivae sp. n. from sheep and goats with keratoconjunctivitis. Infect Immun 1972, 5: 70–76.PubMed CentralPubMed
  13. Pitcher DG, Saunders NA, Owen RJ: Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 1989, 8: 151–156. 10.1111/j.1472-765X.1989.tb00262.xView Article
  14. Phillippy AM, Schatz MC, Pop M: Genome assembly forensics: finding the elusive mis-assembly. Genome Biol 2008, 9(3):R55. 10.1186/gb-2008-9-3-r55PubMed CentralView ArticlePubMed
  15. March PE, Inouye M: Characterization of the lep operon of Escherichia coli. Identification of the promoter and the gene upstream of the signal peptidase I gene. J Biol Chem 1985, 260(12):7206–7213.PubMed
  16. Delcher AL, Bratke KA, Powers EC, Salzberg SL: Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatic 2007, 23(6):673–679. 10.1093/bioinformatics/btm009View Article
  17. Griffiths-Jones S, Moxon S, Marshall M, Khanna A, Eddy S, Bateman A: Rfam: annotating non-coding RNAs in complete genomes. Nucleic Acids Res 2005, (33 Database):D121-D124.
  18. Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997, 25(5):955–964. 10.1093/nar/25.5.955PubMed CentralView ArticlePubMed
  19. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25(17):3389–3402. 10.1093/nar/25.17.3389PubMed CentralView ArticlePubMed
  20. Rice P, Longden I, Bleasby A: EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics 2000, 16(6):276–277. 10.1016/S0168-9525(00)02024-2View ArticlePubMed
  21. Bairoch A, Apweiler R, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin MJ, Natale DA, O'Donovan C, Redaschi N, Yeh LS: The Universal Protein Resource (UniProt). Nucleic Acids Res 2005, (33 Database):D154-D159.
  22. Lima T, Auchincloss AH, Coudert E, Keller G, Michoud K, Rivoire C, Bulliard V, de Castro E, Lachaize C, Baratin D, Phan I, Bougueleret L, Bairoch A: HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot. Nucleic Acids Res 2009, (37 Database):D471–478. 10.1093/nar/gkn661
  23. Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Das U, Daugherty L, Duquenne L, Finn RD, Gough J, Haft D, Hulo N, Kahn D, Kelly E, Laugraud A, Letunic I, Lonsdale D, Lopez R, Madera M, Maslen J, McAnulla C, McDowall J, Mistry J, Mitchell A, Mulder N, Natale D, Orengo C, Quinn AF, Selengut JD, Sigrist CJ, Thimma M, Thomas PD, Valentin F, Wilson D, Wu CH, Yeats C: InterPro: the integrative protein signature database. Nucleic Acids Res 2009, (37 Database):D211–215. 10.1093/nar/gkn785
  24. Wootton JC, Federhen S: Statistics of local complexity in amino acid sequences and sequence databases. Computers & Chemistry 1993, 17: 149–163. 10.1016/0097-8485(93)85006-XView Article
  25. Delorenzi M, Speed T: An HMM model for coiled-coil domains and a comparison with PSSM based predictions. Bioinformatics 2002, 18(4):617–625. 10.1093/bioinformatics/18.4.617View ArticlePubMed
  26. Cordova CM, Lartigue C, Sirand-Pugnet P, Renaudin J, Cunha RA, Blanchard A: Identification of the origin of replication of the Mycoplasma pulmonis chromosome and its use in oriC replicative plasmids. J Bacteriol 2002, 184(19):5426–5435. 10.1128/JB.184.19.5426-5435.2002PubMed CentralView ArticlePubMed
  27. Fujita MQ, Yoshikawa H, Ogasawara N: Structure of the dnaA and DnaA-box region in the Mycoplasma capricolum chromosome: conservation and variations in the course of evolution. Gene 1992, 110(1):17–23. 10.1016/0378-1119(92)90439-VView ArticlePubMed
  28. Roten CA, Gamba P, Barblan JL, Karamata D: Comparative Genometrics (CG): a database dedicated to biometric comparisons of whole genomes. Nucleic Acids Res 2002, 30(1):142–144. 10.1093/nar/30.1.142PubMed CentralView ArticlePubMed
  29. Mrázek J, Karlin S: Strand compositional asymmetry in bacterial and large viral genomes. Proc Natl Acad Sci USA 1998, 95(7):3720–3725. 10.1073/pnas.95.7.3720PubMed CentralView ArticlePubMed
  30. Rocha EP, Danchin A, Viari A: Universal replication biases in bacteria. Mol Microbiol 1999, 32(1):11–16. 10.1046/j.1365-2958.1999.01334.xView ArticlePubMed
  31. Minion FC, Lefkowitz EJ, Madsen ML, Cleary BJ, Swartzell SM, Mahairas GG: The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol 2004, 186(21):7123–7133. 10.1128/JB.186.21.7123-7133.2004PubMed CentralView ArticlePubMed
  32. Bhugra B, Dybvig K: Identification and characterization of IS a transposable element from Mycoplasma pulmonis that belongs to the IS3 family. Mol Microbiol 1138, 7: 577–584. 10.1111/j.1365-2958.1993.tb01148.xView Article
  33. Dybvig K, Voelker L: Molecular biology of Mycoplasmas. Annu Rev Microbiol 1996, 50: 25–57. 10.1146/annurev.micro.50.1.25View ArticlePubMed
  34. Westberg J, Persson A, Holmberg A, Goesmann A, Lundeberg J, Johansson KE, Pettersson B, Uhlén M: The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res 2004, 14(2):221–227. 10.1101/gr.1673304PubMed CentralView ArticlePubMed
  35. Jaffe JD, Stange-Thomann N, Smith C, DeCaprio D, Fisher S, Butler J, Calvo S, Elkins T, FitzGerald MG, Hafez N, Kodira CD, Major J, Wang S, Wilkinson J, Nicol R, Nusbaum C, Birren B, Berg HC, Church GM: The complete genome and proteome of Mycoplasma mobile. Genome Res 2004, 14(8):1447–1461. 10.1101/gr.2674004PubMed CentralView ArticlePubMed

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