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BMC Bioinformatics

BioMed Central

Open Access

Proceedings

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 Falquet*1
Address: 1Swiss Institute of Bioinformatics, Génopode-UNIL, 1015 Lausanne, Switzerland, 2Microsynth AG, Schützenstrasse 15, P.O. Box, 9436
Balgach, Switzerland, 3Institute for Veterinary Bacteriology, University of Bern, Länggass-Strasse 122, 3012 Bern, Switzerland and 4Wellcome Trust
Genome Campus, Hinxton, Cambs CB10 1SA, UK
Email: Sandra P Calderon-Copete - Sandra.Calderon@unil.ch; George Wigger - georges.wigger@microsynth.ch;
Christof Wunderlin - Christof.Wunderlin@microsynth.ch; Tobias Schmidheini - t.schmidheini@microsynth.ch;
Joachim Frey - joachim.frey@vbi.unibe.ch; Michael A Quail - mq1@sanger.ac.uk; Laurent Falquet* - Laurent.Falquet@isb-sib.ch
* Corresponding author

from European Molecular Biology Network (EMBnet) Conference 2008: 20th Anniversary Celebration
Martina Franca, Italy. 18–20 September 2008
Published: 16 June 2009
BMC Bioinformatics 2009, 10(Suppl 6):S7

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

<supplement> <title> <p>European Molecular Biology Network (EMBnet) Conference 2008: 20th Anniversary Celebration. Leading applications and technologies in bioinformatics</p> </title> <editor>Erik Bongcam-Rudloff, Domenica D'Elia, Andreas Gisel, Sophia Kossida, Kimmo Mattila and Lubos Klucar</editor> <note>Proceedings</note> <url>http://www.biomedcentral.com/content/pdf/1471-2105-10-S6-info.pdf</url> </supplement>

This article is available from: http://www.biomedcentral.com/1471-2105/10/S6/S7
© 2009 Calderon-Copete et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

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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 socalled 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

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(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 30Gauge 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 cutoff 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.

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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-Sep2008: 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 homologybased 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

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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.
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).

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).

Table 1: Functional assignment criteria

Source

Known Protein

Blastp (Mycoplasma DB)

Evalue < e-20

Blastp (Swissprot DB)
HAMAP families
Interpro domains

Putative

Evalue between
>e-20 and <e-4
Evalue < e-20
Evalue between
>e-20 and <e-4
Confident protein family match
No confident protein family match
If no HAMAP family match. Support evidence from at least one InterPro
member database

Hypothetical
Evalue
> e-4 or No match
Evalue
> e-4 or No match
N/A
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).

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Table 2: Results obtained for M. hyopneumoniae 232 annotation

All genes

ncRNA

727
773
704
23
647
57
69

Total of M. hyopneumoniae 232 known genes
Total annotated genes using pipeline
Total correct predictions *
Total known genes not predicted *
Total correct gene annotations
Total predicted genes incorrectly or differently annotated
Total predicted genes in additionally annotated by pipeline

CDS
691
741
672
19
618
54
69

36
32
32
4
29
3
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

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 noncoding 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.
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 con-

sensus 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.
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 [28-30]. 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

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

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Table 4: Summary of M. conjunctivae genome features

Genome Features
Length (bp)
G + C content (mole%)

846,214
29.0%

Functionally assigned protein CDSs
Putative protein CDSs
Hypothetical protein CDSs
23S rRNA
16S rRNA
5S rRNA
Bacterial RNase P class B
TPP riboswitch (THI element)
tmRNA
Bacterial signal recognition particle RNA
Transfer RNA genes

Figure 1
Genome map
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.

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-3phosphate 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].

344
39
316
1
1
1
1
1
1
1
28

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.

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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
metabolic process
transport
proteolysis
tRNA aminoacylation for protein translation
DNA repair
DNA replication
carbohydrate metabolic process
phosphoenolpyruvate-dependent sugar phosphotransferase system
regulation of transcription
DNA modification
glycolysis
ATP synthesis coupled proton transport
DNA methylation
DNA recombination
DNA integration
electron transport
biosynthetic process
nucleoside metabolic process
Protein folding

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 IS1138b) 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
HpaI 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.

68
32
31
18
15
13
12
11
11
10
9
9
8
7
7
6
6
5
5
5

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.

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.
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.

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NC_007295/2317-2674
NC_007332/1991-2348
NC_006360/2462-2821
M_conj/1970-2271
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NC_007295/2317-2674
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NC_006360/2462-2821
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AATTTTTCGCCCGTCGGCTGTTGACATTCGCGCGCGGAATCCGTGTGTTTTTAAATGTTT
AATTTTTCGCCCGTCGGCTGTTGACATTCGCGCGCGGAATCCGTGTGTTTTTAAATGTTT
AATTTTTCGCCCGTCGGCTGTTGACATTCGCGCGCGGAATCCGTGTGTTTTTAAATGTTT
AACTTTTCTACCATCTGCTGTTTGCATGCGCGCTCTAAATCCATGTGTTTTAATGTGTTT
10
20
30
40
50
60
<-I K R G D A T S M R A R F G H T K L H K
<-I K R G D A T S M R A R F G H T K L H K
<-I K R G D A T S M R A R F G H T K L H K
<-V K R G D A T Q M R A R F G H T K I H K
TCTTTTATTTGGTTGATAAGTTCGCTTCATTTT-TTCTCCTTTTTTT--GCTTAAATTTA
TCTTTTATTTGGTTGATAAGTTCGCTTCATTTT-TTCTCCTTTTTTT--GCTTAAATTTA
TCTTTTATTTGGTTGATAAGTTCGCTTCATTTT-TTCTCCTTTTTTTTTGCTTAAATTTA
TAATTTATTTGGTTGATAAGTTCGTTTCATACTATTCTCCTTTTTCT--------ATT-70
80
90
100
110
120
R K N P Q Y T R K M . . . . . . . . . .
R K N P Q Y T R K M . . . . . . . . . .
R K N P Q Y T R K M . . . . . . . . . .
L K N P Q Y T R K M . . . . . . . . . .
AGTTTTAGAAGTTATTTTAAGGGATTTTAAAAAAATATTGTTAAAATTTAAAACCAAAAT
AGTTTTAGAAGTTATTTTAAGGTATTTTAAAAAAATATTGTTAAAATTTAAAACCAAAAT
AGTTTTAGAAGTTATTTTAAGGTATTTTAAAAAAATATTGTTAAAATTTAAAACCAAAAT
--TTTTATATGGTACTC-AACAATTATGATTAAAGTAATTTACAAATTTTACCACAAATT
130
140
150
160
170
180
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
CAACTTTATTAAATGTGCTAAATAAAGTTGATAAAATGTTTGCAAAAACATTTTTGTTGT
CAACTTTATTAAATGTGCTAAATAAAGTTGATAAAATGTTTGCAAAAACATTTTTGTTGT
CAACTTTATTAAATGTGCTAAATAAAGTTGATAAAATGTTTGCAAAAACATTTTTGTTGT
AAGATATATTTAAT--------TAAAATGGAT----TATTT--------AGCTTTATAAT
190
200
210
220
230
240
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . .
TTTAAACAAAACAAATTGATTTAAAAATTATACTACAAAATTAAAGGAAAATTTATAAAA
TTTAAACAAAACAAATTGATTTAAAAATTATACTACAAAATTAAAGGAAAATTTATAAAA
TTTAAACAAAACAAATTGATTTAAAAATTATACTACAAAATTAAAGGAAAATTTATAAAA
TTCAATTA-------------------TTATAATACCCAAAAAAGGTAGGA---ATAAA250
260
270
280
290
300
. . . . . . . . . . . . . . . . . . . M
. . . . . . . . . . . . . . . . . . . M
. . . . . . . . . . . . . . . . . . . M
. . . . . . . . . . . . . . . . . . . .
TGCAAACAAATAAAAATAATTTAAAGGTTAGAACACAGCAAATTAGACAACAAATTGAAA
TGCAAACAAATAAAAATAATTTAAAGGTTAGAACACAGCAAATTAGGCAACAAATTGAAA
TGCAAACAAATAAAAATAATTTAAAGGTTAGAACACAGCAAATTAGGCAACAAATTGAAA
---AATTGAATAAAACAGATTTAGAGATTAAAACAATCAATCTAAAACAATCTCTTGAAG
310
320
330
340
350
360
Q T N K N N L K V R T Q Q I R Q Q I E->
Q T N K N N L K V R T Q Q I R Q Q I E->
Q T N K N N L K V R T Q Q I R Q Q I E->
. M N K T D L E I K T I N L K Q S L E->

Figure 2
Origin of replication
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).

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Table 6: List of M. conjunctivae transposases detected.

gene

Product

start

MCJ_00022
MCJ_00059
MCJ_00099
MCJ_00207
MCJ_00208
MCJ_00215
MCJ_00399
MCJ_00428
MCJ_00553
MCJ_00635

IS1138
?
IS1138
IS1138
IS1138
ISMag1
IS1138
IS1138
ISMHp1
IS1634AM

23818
61624
97032
192412
192717
198177
430830
474034
639264
742041

end

strand
24999
61728
98213
192660
193088
199265
432011
474138
639500
743714

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

+
+
+
+
+

length

Condition of the sequence

1181
104
1181
248
371
1088
1181
104
236
1673

Complete
Partial
Complete
Same transposase split in two ORFs
Complete
Complete
Partial
Partial
Complete

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.

Table 7: Genome comparison. Functional classification of proteins of 5 sequenced mycoplasma genomes

Functional categories

Translation, ribosomal
structure and biogenesis
Transcription
DNA replication,
recombination and repair
Posttranslational
modification, protein
turnover
Energy production and
conversion
Carbohydrate transport
and metabolism
Amino acid transport and
metabolism
Nucleotide transport and
metabolism
Coenzyme transport and
metabolism
Lipid transport and
metabolism
Inorganic ion transport
and metabolism
Other
No known function
Total CDSs

Mycoplasma.
hyopneumoniae 232
(892 kb)

Mycoplasma pulmonis Mycoplasma genitalium Mycoplasma mobile
(964 kb)

(816 kb)

(777 kb)

Mycoplasma
conjunctivae
(846 kb)

100

108

108

108

90

22
75

27
118

16
49

23
72

16
55

20

24

19

22

15

25

29

20

29

25

57

68

32

50

30

25

27

17

24

20

19

22

20

18

17

9

12

12

17

8

6

10

9

9

7

14

18

17

15

5

118
201
691

124
195
782

87
78
484

107
139
633

95
316
699

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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
CU179680
CP001047
CP000123
AE015450
L43967
AE017332
AE017244
AE017243
AE017308
BX293980
BA000026
U00089
AL445566
AE017245
FM864216

Species name

Genome Size
877,438
820,453
1,010,023
996,422
580,076
892,758
920,079
897,405
777,079
1,211,703
1,358,633
816,394
963,879
799,476
846,214

Mycoplasma agalactiae PG2 chromosome
Mycoplasma arthritidis 158L3-1
Mycoplasma capricolum subsp. capricolum ATCC 27343
Mycoplasma gallisepticum R
Mycoplasma genitalium G37
Mycoplasma hyopneumoniae 232
Mycoplasma hyopneumoniae 7448
Mycoplasma hyopneumoniae J
Mycoplasma mobile 163 K
Mycoplasma mycoides subsp. mycoides SC str. PG1
Mycoplasma penetrans HF-2
Mycoplasma pneumoniae M129
Mycoplasma pulmonis UAB CTIP
Mycoplasma synoviae 53
Mycoplasma conjunctivae

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.

Competing interests

References

2.
3.

4.
5.

6.

The authors declare that they have no competing interests.

Authors' contributions
JF proposed and supported the project. SPCC created the
pipeline and did the analysis. MAQ did the genomic
library. TS and MAQ sequenced the clones. TS, GW and
CW did the assembly. SPCC and LF wrote the manuscript.

7.

8.

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.

29.7%
30.7%
23.8%
31.5%
31.7%
28.6%
28.5%
28.5%
25%
24%
25.7%
40%
26.6%
28.5%
29%

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.

1.

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.

G+C content

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