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Nucleic Acids Res. 2001 May 15; 29(10): 2145–2153. | PMCID: PMC55444 |
The complete genome sequence
of the murine respiratory pathogen Mycoplasma pulmonis Isabelle Chambaud,1,2 Roland Heilig,5 Stéphane Ferris,2 Valérie Barbe,5 Delphine Samson,5 Frédérique Galisson,3 Ivan Moszer,4 Kevin Dybvig,6 Henri Wróblewski,7 Alain Viari,8 Eduardo P.C.
Rocha,4,9 and Alain Blanchard1a 1INRA–Université de Bordeaux 2, Institut de Biologie Végétale Moléculaire, 71 avenue Edouard Bourleaux, BP 81, 33883 Villenave D’Ornon Cedex, France, 2Unité d’Oncologie Virale, 3Service d’Informatique Scientifique and 4Unité de Régulation de l’Expression Génétique, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France , 5Genoscope, Centre National de Séquençage, 2 rue Gaston Crémieux, BP 191, 91006 Evry Cedex, France , 6Department of Genomics and Pathology, University of Alabama at Birmingham, 1670 University Boulevard, Volker Hall, Room 418A, Birmingham, AL 35294-0019, USA , 7Université de Rennes 1, UMR CNRS 6026, Campus de Beaulieu, F-35042 Rennes Cedex, France , 8INRIA Rhone-Alpes-Projet HELIX, 655 Avenue de l’Europe, 38330 Montbonnot-Saint Martin, France and 9Atelier de Bioinformatique, 12 Rue Cuvier, 75005 Paris, France Received January 8, 2001; Revised March 19, 2001; Accepted March 19, 2001. Mycoplasma pulmonis is a wall-less eubacterium belonging
to the Mollicutes (trivial name, mycoplasmas) and
responsible for murine respiratory diseases. The genome of strain
UAB CTIP is composed of a single circular 963 879 bp chromosome
with a G + C content of 26.6 mol%, i.e. the lowest
reported among bacteria, Ureaplasma urealyticum apart.
This genome contains 782 putative coding sequences (CDSs) covering
91.4% of its length and a function could be assigned to
486 CDSs whilst 92 matched the gene sequences of hypothetical proteins,
leaving 204 CDSs without significant database match. The genome contains
a single set of rRNA genes and only 29 tRNAs genes. The replication
origin oriC was localized by sequence analysis
and by using the G + C skew method. Sequence polymorphisms
within stretches of repeated nucleotides generate phase-variable
protein antigens whilst a recombinase gene is likely to catalyse
the site-specific DNA inversions in major M.pulmonis surface
antigens. Furthermore, a hemolysin, secreted nucleases and a glyco-protease
are predicted virulence factors. Surprisingly, several of the genes
previously reported to be essential for a self-replicating minimal
cell are missing in the M.pulmonis genome although
this one is larger than the other mycoplasma genomes fully sequenced
until now. Mycoplasmas are prokaryotes that cause slowly progressive, chronic
diseases in human and animals. Mycoplasmal infections in humans
are associated with a variety of respiratory and urogenital diseases,
and those in farm animals are responsible for important economic
losses ( 1– 3).
Because current antibiotic treatments often fail to eradicate mycoplasmas
from the host, better control of these infections is needed. As
for other pathogens, it is expected that the availability of fully
sequenced genomes will help in understanding the physiology and
the pathogenesis mechanisms of these bacteria. This should subsequently
allow the development of new and efficient means for the control
of these pathogens. Mycoplasmas (class Mollicutes) lack a cell
wall but are related to Gram-positive eubacteria from which they
evolved by a drastic reduction of genome size, resulting in the
loss of many biosynthetic abilities. They are, thus, considered
as the best representatives of the concept of a minimal cell ( 4). Three mycoplasma genomes have been
sequenced to date, two from closely related species, Mycoplasma
pneumoniae (Mpn) ( 5) and Mycoplasma genitalium (Mge) ( 6),
and the third from Ureaplasma urealyticum (Uu)
( 7), which belongs to the same phylogenetic
group. Mycoplasma pulmonis, the subject of our work,
is the etiologic agent of murine respiratory mycoplasmosis in rats
and mice. This natural pathogen is considered as the most useful
model for studying mycoplasmal respiratory infections, including those
commonly caused by Mpn in humans. Mpn is a leading cause of respiratory
tract infections, especially in children and young adults. Although
it has been estimated that only 3–10% of Mpn-infected
patients develop pneumonia, up to 30% of cases involving
all age groups may be caused by this bacterium (for review see 8). In accordance with observations made
during Mpn infections, innate immunity provides anti- M.pulmonis defence
of the lungs and humoral immunity plays a role against the dissemination
of the infection ( 9). A key factor
in the ability of mycoplasmas to establish a chronic infection is
their genome flexibility, which allows them to produce a highly variable
mosaic of surface antigens ( 10, 11). One of the better-understood systems
exhibiting this antigenic variation is the one responsible for the
expression of the Vsa lipoproteins in M.pulmonis ( 12). Here we report the complete M.pulmonis genome
sequence. Its analysis revealed hypervariable domains relevant to
pathogenesis and provides clues for deciphering the respective roles
of putative virulence factors. Mycoplasma strain The strain UAB CT of M.pulmonis was kindly provided
by Maureen Davidson (University of Florida, Gainesville, FL). This
strain was passaged twice in a modified PPLO broth medium in our
laboratory and the obtained stock named UAB CTIP. Growth medium
contained per liter: 22.5 g of PPLO broth powder (Difco), 0.2 g
of salmon sperm DNA (Sigma), 200 ml of horse serum, 5 ml of Isovitalex
solution (Becton Dickinson), 10 ml of 25% (w/v)
glucose, 4 ml of 1% (w/v) phenol red and 120 mg
of ampicillin. Genomic DNA was isolated by lysing mycoplasma cells
with SDS and proteinase K followed by phenol–chloroform
extractions. The same DNA preparation was used for the construction
of the different libraries. Construction of genomic libraries A preliminary library, named A (1–2 kb inserts obtained
by partial Sau3AI digestion of genomic DNA and
cloned into pUC18), was constructed for a pilot study. We have subsequently
generated three other libraries of different insert size to favor
the scaffold formation between sequence contigs during the assembly
stage. Library C was a short insert (2–3 kb) library constructed
by partial Sau3AI digestion followed by gel sizing
and ligation to the BamHI site of pUC18. Library
R was made by partial Tsp509I digestion, sizing
of fragments to 5–6 kb and insertion into the EcoRI
site of the pBAM3 vector (derived from pBluescript II SK+;
R.Heilig, unpublished material). Library B was a ‘miniBAC’ library,
constructed by partial Tsp509I digestion of genomic
DNA, sizing >15 kb and ligation to the gel-purified 8.7
kb EcoRI-linearized pBACe3.6 BAC vector. Shotgun sequencing and sequence assembly Cycle sequencing was performed, from both ends of the plasmid
and miniBAC clones using flanking vector sequences as primers. Dye
primer reactions were analyzed on a Li-Cor 4200L (4000 clones from
library R) and dye terminator reactions on an ABI 377 (500, 2000
and 1500 clones, from libraries A, C and B, respectively). The whole
genome sequence assembly was performed using the PHRED and PHRAP
software packages ( 13). A total
of 6018 end sequences from library R (mean length 930 nt; 5.8 genome
equivalents) and 574, 3840 and 1920 reads from libraries A, C and
B, respectively (mean length 600 nt; 4.0 genome equivalents), were
incorporated into contigs. Moreover, the vsa-containing
region, spanning 10–15 kb, revealed a high level of heterogeneity
between the subclones, which hindered the correct sequence assembly
and was attributed to sequence heterogeneity in the M.pulmonis culture used
for the genomic DNA preparation. To circumvent this problem, we
performed restriction digest analysis of all the miniBACs covering
this region to identify an over-represented population of clones
and selected two of them for subsequent subcloning and complete
sequencing. Finally, the five individual assemblies were combined
to form a consensus, which was used to generate the full genomic
sequence. Finishing and validation One cloning gap (39 bp) and several regions with uncertainties were
resolved by sequencing duplicate PCR products. To fullfil the sequence
quality criteria (a minimum of three reads for each position, sequencing
on both strands or using two types of chemistry and a PHRAP quality
value of 40 or more), a total of 1144 finishing reactions, requiring
481 primers, were performed. The assembly was validated by restriction
digest analysis, both on total genomic DNA by pulsed-field gel electrophoresis
using the enzymes EagI, KpnI, NruI and SacII, and on a selection
of overlapping miniBACs and plasmid clones using the enzymes EcoRI, EcoRV, HindIII and BssHII. Identification of CDSs and annotation The annotation was mainly performed using the integrated computer
environment specialized for large-scale sequence annotation, Imagene
( 14). The see text on p.1 were
identified using GLIMMER, software for compositional analysis with
an interpolated Markov model ( 15).
Putative coding sequences (CDSs) extracted from 300 kb of the M.pulmonis genome were used to train the Markov
model. Once trained, the model was applied to the genome using 100 bp
as the cut-off value for the smallest CDSs. A total of 875 CDSs
were detected using this method but some of them were removed because
either they significantly overlapped other CDSs and/or
they were of small size (100–150 bp) with a deduced polypeptide
presenting no detectable similarity with sequences and patterns
in databases. CDSs in intergenic spaces were also searched using
BLASTX2 ( 16). The CDSs were numbered
starting with dnaA as the first gene (MYPU_10010)
and recD as the last (MYPU_7820). Search of homology of the products deduced from these CDSs was
performed using BLASTP2 ( 16)
against SWISS-PROT and SP-TrEMBL. Additional investigations with
CDSs showing no homologs using this first strategy were performed using
FASTA ( 17) and Smith and Waterman
algorithms. Motifs were also searched on translated genes using
the ProfileScan server ( http://www.isrec.isb-sib.ch/software/PFSCAN_form.html) against
the databases PROSITE, Pfam-A and Gribskov ( 18– 20). For putative membrane proteins and ABC
transporters, transmembrane domains were searched using TMpred ( 21). In the comparison of mollicute genomes, two
genes were regarded as homologous if the proteins they encode were
similar both in sequence and in size. For this, we made pair-wise
comparisons of all proteins of all proteome pairs, filtering potential
homologs using a threshold in BlastP of E-value <10 –5 and
in maximal difference of protein lengths of 20%. Subsequently,
we aligned the sequences, using a variant of the classical dynamic
programming algorithm for global alignment, where one counts 0-weight
for gaps at both ends of the largest sequence, using the BLOSUM62
matrix ( 22). Finally, we retained
pairs of proteins presenting similarity >40%.
The set of orthologous genes (supposed to have diverged following
a speciation event) was defined by adding a further criterion of
double best hit, i.e. two genes are defined as orthologous if they
are homologous and if they are the best match of one another in
the respective genomes. For the comparaison of genomes, we also
used the information provided in the KEGG catalog of genes for Mpn,
Mge and Uu ( http://star.scl.genome.ad.jp/kegg/kegg2.html#genes)
( 23). Terminators were searched
using PETRIN ( 24). The tRNAs were
located using the software fastRNA ( 25)
and rRNA by detecting regions of homology with mycoplasmal rRNAs. Analysis
of codon usage through factorial correspondence analysis was done
using software developed at the Atelier de BioInformatique ( 26). The search for close repeats was
done through the use of the KMR algorithm in windows as explained
elsewhere ( 27). Dot-plots were
drawn by using the program dotter from the Sanger Center ( 28). Database submission and web-based access to the
sequences These sequence data have been submitted to the EMBL/GenBank/DDBJ databases
under accession number AL445566. The information of individual CDSs, data retrieval and graphical views
are available on the web ( http://genolist.pasteur.fr/MypuList).
This server, which has an organization identical to that described
for Subtilist, the Bacillus subtilis-dedicated
web server ( 29), also provides
the possibility of performing similarity searches, using FASTA or
BLAST, and motif searches using pattern matching. General features of the genome The general features of the genome are listed in Table 1. The genome size is 963 879 bp and its average
G + C content is 26.6%, which is, along with that
of Uu, the lowest of all bacterial genomes ( 7).
In M.pulmonis, the base composition is rather homogeneous
along the entire chromosome (Fig. A), with regions
corresponding to rRNA and vsa genes showing the lowest
A + T values. The average A + T content of the
coding regions is 73% with 85.3% at the third
position of the codon. An analysis of the codon usage among the
782 identified CDSs using factorial correspondence analysis ( 26) indicated that the code is homogeneously
used among all the CDSs, the only exception being the vsa genes
and a few other lipoprotein genes (data not shown). This result
suggests that there was, if any, a low level of horizontal transfer
during the recent evolution of this mollicute. Like all species
belonging to the genus Mycoplasma, M.pulmonis reads
UGA as a tryptophan codon instead of a stop codon. In fact, the
UGA codon is strongly preferred to UGG (91.2 versus 8.8%).
There is also a strong preference for the usage of the UAA stop
codon (86.2%) as compared with 13.8% for UAG,
the other stop codon. This bias confirms the association between
the use of these two stop codons and the G + C% of
the entire genome which was recently reported for different bacterial
species ( 31). The use of only
two stop codons is in accordance with the finding of a single peptide
chain release factor-encoding gene ( prfA), capable
of recognizing both UAA and UAG. | Table 1. General features of the M.pulmonis genome |
The M.pulmonis genome encodes a single set
of rRNA genes. In contrast to Mpn, Mge and Uu, the three genes are
not organized as an operon: the 16S rRNA and 23S rRNA genes are
adjacent, without intervening tRNA genes, which seems to be common
to all mollicutes, the phytoplasmas excepted. The 5S rRNA is located
350 kb from the two other rRNA genes, such a separation having previously
been described for two other mycoplasmas, Mycoplasma
hyopneumoniae and Mycoplasma flocculare ( 32). The set of 29 tRNA genes with specificity
for all amino acids was identified in the M.pulmonis genome
and currently represents the smallest set of tRNAs among the sequenced
bacterial genomes. Likewise, the four sequenced mollicute genomes
share a single gene for a sigma factor, the one encoding σ70. With the software Petrin, that uses Escherichia
coli default parameters, we were able to identify 281 ρ-independent terminators
in the genome of M.pulmonis. Although these terminators
were detected in the genome of Mpn ( 5),
they have not been reported in the other sequenced mycoplasma genomes and
they have been predicted not to exist in such low G + C genomes
( 33). Origin of replication The origin of replication of the M.pulmonis chromosome
has not been experimentally identified. However, as described for other
bacterial genomes, but not for the Mpn and Mge genomes, the tandem
arrangement of dnaA and dnaN genes was
identified. However, the two other genes, gyrA and gyrB, commonly found in the vicinity of the origin
of replication, are located elsewhere on the genome. We have designated
as base 1 of the M.pulmonis genome the first non-coding
base upstream dnaA. We identified five putative
DnaA boxes (consensus sequence 5′-TTATCCACA-3′), three being upstream dnaA and
the two other downstream of this gene. This organization was not
found in the Mpn, Mge and Uu genomes ( 5– 7), but is reminiscent of that described
for other mollicutes including Spiroplasma citri ( 34) and Mycoplasma capricolum ( 35). It is, therefore, possible that this
divergence from the typical eubacterial oriC is
restricted to the phylogenetic cluster of mycoplasma species that
includes Mpn, Mge and Uu. The analysis of the G + C skew, defined as [(G – C)/(G + C)] ( 36), on the M.pulmonis genome
provided additional evidence that the origin of replication was
properly located (Fig. B). There was indeed
a marked inversion of G + C skew upstream of dnaA.
This bias is stronger than in Mpe and Mge but weaker when compared
with other bacterial genomes ( 37).
On both sides of the putative origin of replication, there is also
a strong bias in the polarity of the genes transcribed on the two
DNA strands. Comparison with other sequenced mollicute genomes The M.pulmonis genome is currently the largest
completely sequenced genome from a mollicute. Consequently, the number
of CDSs identified is larger than for the other three mollicutes:
782 as compared with 483, 688 and 605 for Mpn ( 38),
Mge and Uu, respectively (Table 2). This
increased number of genes is not evenly distributed in all the functional categories.
In some categories, including those governing nucleotide metabolism,
intermediary metabolism and protein synthesis and fate, the number
of genes is equivalent in the four mollicute genomes (Table 2). In these categories, these genomes appear
to approach, to a similar level, the concept of a minimal cell.
Furthermore, and quite surprisingly, in contrast to Mpn, Mge and
Uu which share the same set of genes for transcription, M.pulmonis has
adopted in this category a still more minimalist organization. Indeed,
the gene rpoE encoding the δ subunit
of RNA polymerase is lacking; this gene has been shown by mutagenesis
experiments to be dispensable in B.subtilis ( 39). The larger number of genes in M.pulmonis as compared
with the other three mollicutes can be mainly attributed to an increased
number of membrane proteins, in particular those devoted to transport.
It is difficult to predict from the sequence analysis the specificity
of ABC (ATPase) transporters ( 40),
56 M.pulmonis CDSs were found to potentially encode
subunits for these transporters as compared with 32 and 33 CDSs
for Mpn and Uu, respectively. Differences were also found in the ‘Energy
metabolism’ category (Table 2) and
are due, in part, to the presence of four CDSs encoding α ( atpA) and β ( atpD)
ATP synthase subunits. A set of atpA- atpD genes
is located within the operon encoding the other six subunits of
the ATP synthase and the three other atpA- atpD tandem
copies are scattered on the chromosome (Fig. ). | Table 2. Functional classification of proteins in the completely
sequenced mollicute genomes |
Of the 179 M.pulmonis CDSs, which do not have
an ortholog in the Mpn, Mge and Uu genomes, 123 encode putative proteins
showing no significant homology in the databases. Additionally,
eight other M.pulmonis CDSs encode conserved hypothetical
proteins with the closest homologs among the polypeptides deduced
from Gram-positive bacterial genomes, as expected from the mycoplasmal
phylogenetic position. One noteworthy exception is Mypu_4730,
which shares homology with a putative Thermotoga maritima polypeptide.
Some other M.pulmonis polypeptides with no ortholog
deduced from the Mpn, Mge and Uu genomes include ribosomal protein
S1 (Mypu_1300), glucokinase (GlcK; Mypu_2280),
5′-nucleosidase precursor (UshA; Mypu_0550)
and signal peptidase I (SipS; Mypu_6300). It should be
noted that re-annotating the Mpn genome suggested that one of the
identified proteases in this organisms (MPN386) could act as a signal
peptidase I ( 38). Mycoplasma
pulmonis also seems to have the ability to metabolize sugars
other than those metabolized by Mpn, Mge and Uu. Indeed, a set of
genes involved in the catabolism of maltodextrins has been identified;
these genes are clustered together in one region of the genome and
encode a maltodextrin ABC transport system (Mypu_6390–6410),
a β- d-phosphate-glucomutase
(PgmB; Mypu_6350), a dextrinase with unknown specificity
(Mypu_6330) and an α-amylase
3 (AmyC, Mypu_6320). This finding suggests that M.pulmonis can
use glycogen as a source of energy and carbon which is in accordance
with reports that all glucose-fermenting mycoplasmas ferment glycogen.
However, these results were based on experiments using media containing
serum or serum fractions, which contain saccharolytic enzymes ( 41). Therefore, the use of complex sugars
by M.pulmonis will have to be experimentally verified.
Interestingly, the closely located Mypu_6220 CDS potentially
encodes a polypeptide that belongs to the LacI
family of transcriptional regulators. This polypeptide, with no
ortholog in Mpn, Mge and Uu, together with the heat-inducible transcription
repressor (Mypu_1420) is the only indication for transcriptional
regulation in this mycoplasma. Among the M.pulmonis CDSs for which an ortholog
was found in Uu but not in Mpn, are CTP synthase (Mypu_6660), cardiolipin
synthetase (Mypu_6650) and a poorly understood enzyme (PslX; Mypu_1620) involved in the phospholipid metabolism.
In addition, six IS-elements including three which are incomplete
were found in the M.pulmonis genome. Although the gene order in Mpn and Mge is highly conserved ( 42), this is the case neither in Uu ( 7) nor in M.pulmonis (data
not shown). The regions of syntheny between the four genomes consist
in most cases of genes encoding products such as ribosomal proteins,
ABC transporters or ATP synthase subunits. Comparison with the set of genes for a minimal
cell The Mge genome is the smallest bacterial genome and as such is
considered as the best representative for the concept of a minimal
cell ( 43). With the assumption
that genes conserved across large phylogenetic distances are likely
to be essential, a minimal set of genes for a living cell was defined
by others ( 4). Among the 256 Mge
genes belonging to this minimal set ( http://www.ncbi.nlm.nih.gov/Complete_Genomes/Minset/),
eight genes do not have an ortholog in the M.pulmonis genome [MG278, guanosine
5′-diphosphate 3′-diphosphate
pyrophosphohydrolase ( spoT); MG453, UTP-glucose-1-phosphate
uridylyltransferase ( gtaB); MG053, phosphomannomutase
( cpsG); MG063, 1-phosphofructokinase ( fruK);
MG393, heat shock protein ( groES); MG392, heat
shock protein ( groEL); MG382, uridine kinase ( udk); MG118, UDP-glucose 4-epimerase ( galE)]. Although
the Mpn and Mge genomes have genes for the synthesis of the heat
shock proteins-chaperonins GroEL and GroES, the absence of these
genes has also been reported for Uu ( 7).
GroEL has also been shown to be dispensable for Mpn in
vitro ( 44). The fact that
the M.pulmonis genome lacks fruK but
has a probably functional fruA gene has to be regarded
in the light of some recently reported data showing that in the mollicute S.citri, fructose catabolism requires fruA but
not fruK ( 45).
Another striking absence in M.pulmonis is that
of SpoT, an enzyme playing a key role in the stringent response. Strong
evidence supports that there is only one relA/spoT product
in Gram-positive bacteria (in contrast to two in Gram-negative bacteria)
that displays activity for both synthesis and degradation of (p)ppGpp
( 46). SpoT was shown by transposon mutagenesis
to be dispensable for Mpn and Mge ( 44).
There are only a few examples of eubacteria, which lack the stringent response,
in particular Helicobacter pylori ( 47)
and Chlamydia trachomatis ( 48). Highly recombinogenic loci in the M.pulmonis genome The vsa locus encodes highly variable surface
lipoprotein antigens. It has been shown in strain KD735-15 that
high-frequency, site-specific DNA inversions serve to regulate the phase-variable
expression of individual vsa genes ( 12).
Only one vsa gene, located in the expression site,
is transcribed at a given time, the other vsa genes
being silent. A model involving DNA strand exchange at conserved vsa recombination sites ( vrs boxes)
has been proposed to mediate these inversions ( 12). The
completed genomic sequence allows for the first time analysis of
the complete vsa locus of the UAB CTIP strain and reveals
seven vsa genes (Fig. ),
with four of them ( vsaA, vsaC, vsaE and vsaF) being previously
described in the strain KD735-15 containing 11 vsa genes
( 12). As expected from such a
highly recombinogenic locus, neither the gene order nor the number
of repeated units (see below) in individual genes are conserved
between strains. The expressed gene in the sequenced locus is the
newly described vsaI gene. At the 5′-end of each vsa gene a vrs box was identified,
an additional vrs box laying within the genes vsaC and vsaI (Fig. ). The dot-plot analysis of this locus highlights
repeated elements within each individual vsa gene;
for example, a 57 bp unit (19 amino acids) is repeated 27 times
in the vsaI gene. There are also common repeats
between different vsa genes such as between vsaF and vsaG (data not shown). Within the vsa locus,
three lipoprotein-encoding CDSs ( lipA, lipB and lipD; Fig. ) were also
identified. Although the deduced sequences share significant similarity
with Vsas, they do not exhibit a repeated C-terminus nor vrs boxes
within their genes. Furthermore, it was predicted that the putative
site-specific recombinase that promotes DNA inversion within this
locus would be a member of the λ integrase family.
Indeed a gene, the product of which potentially encodes this type
of recombinase, was located immediately downstream of the vsa locus
(Fig. ). Experiments are underway to confirm
that this gene is indeed responsible for the variability of cell
surface antigens and also to determine if antigenic variation confers
some protection from the host’s immune system. Previous studies have shown that M.pulmonis strain
KD735-15 contains at least two highly recombinogenic hsd loci
that code for type I restriction and modification systems ( 49). The UAB CTIP genome has two hsd loci
similar to that of KD735-15 and an additional third hsd locus
that is not completely functional (Fig. ).
At the hsd3 locus, the two hsdS are
most likely functional but the hsdR and hsdM have
been disrupted by frameshift mutations and are predicted to be non-functional. The
three loci are not co-located on the chromosome (Fig. ) and, in contrast to the vsa locus,
no CDS encoding a putative recombinase involved in the site-specific
DNA inversions was found in their vicinity or elsewhere on the chromosome. Although
detected in the Mpn and Mge genomes, the hsd systems
do not seem to be active in these species. In addition to these hsd genes, we identified a complex locus of a type
III RM system and CpG methylase genes that were not identified previously
within the genus Mycoplasma. Lipoproteins, polymorphisms and repeats In addition to the expressed vsa product (VsaI),
55 other lipoprotein CDSs (7% of the total number of CDSs),
were identified as compared with 46, 21 and 42 in Mpn, Mge and Uu,
respectively. Very few of these lipoproteins had orthologs in the
databases and a putative function could be attributed to only three
of them: MYPU_1390 and MYPU_6930 as membrane nucleases,
and MYPU_6320 as an amylase. Three other lipoproteins with
unknown function (MYPU_0070, MYPU_3430 and MYPU_5260)
have orthologs in other mycoplasmas. However, several of the lipoproteins
could be classified into highly homologous families. Two of the enzymes
involved in the maturation of bacterial lipoproteins could be deduced
from the genome, the prolipoprotein diacylglycerol transferase ( lgt) and the prolipoprotein signal peptidase ( lsp). A third enzyme, the apolipoprotein:phospholipid N-acyl transferase ( lnt), was
not found, which substantiates the finding that lipoprotein acylation
may not be complete in some mycoplasma species ( 50, 51). However, one cannot rule out that
other types of acylation occur, similar to that recently described
for Borrelia burgdorferi ( 52). The expression of several mycoplasmal lipoprotein genes has
been shown to be phase-variable (for review see 11)
and one of the underlying mechanisms is due to variation in the length
of homopolymeric nucleotide tracts located either in the promoter
or in the coding sequence itself. A stretch of repeated T (T 31)
and A (A 39) nucleotides is located upstream of two M.pulmonis genes encoding the putative lipoproteins MYPU_0190
and MYPU_4780, respectively. These homopolymeric tracts
are highly suggestive of phase-variation in the expression of these
genes because they are hotspots for frameshift mutations generated
by replication slippage. Additional evidence for phase-variation
was provided by polymorphisms in the sequence of these repeats.
The assembly revealed variations in these tracts among sequences
from otherwise identical clones. We verified that this was not due
to sequencing difficulties. For MYPU_0190, T 31 was
found six times and T 29 two times (6×T31/2×T29). The situation was more complicated
for MYPU_4780, with the following polymorphism: 1×A36/2×A39/2×A40/2×A41/2×A44.
The detection of variation in these homopolymeric tracts was favored because
the DNA for generating the sequencing libraries (see Materials and
Methods) was obtained from a M.pulmonis isolate
rather than from a single clone. Should this phase-variation experimentally
be confirmed, this would be the first indication that two different
mechanisms (length variation in a homopolymeric tract and site-specific
inversions in the vsa genes) driving phase-variation
of surface antigens operate in a single mycoplasma cell. Virulence factors Like other mollicutes, this organism contains few recognizable genes
likely to be involved in virulence. Exceptions are a hemolysin (MYPU_1710),
a set of membrane-associated or secreted nucleases (MYPU_6930–6940
and MYPU_1390) and a potentially secreted protease (MYPU_1180).
The hemolytic activity of M.pulmonis was previously
described ( 53). The MYPU_1710
product has no homolog in Mpn or Mge, but in Uu, another mollicute
displaying a hemolytic activity ( 7).
This hemolysin belongs to the hlyA family, members
of which are recognized virulence factors in Serpulina
hyodysenteriae and in mycobacteria ( 54, 55). Mollicutes have limited biosynthetic capacity,
and membrane-associated or secreted proteases and nucleases are
thought to help these microorganisms to acquire metabolic precursors
from the host. The study of the nuclease produced by Mycoplasma
penetrans clearly indicated that mycoplasmal nucleases could
act as virulence factors ( 56, 57). A thiol peroxidase (MYPU_7080)
is also of interest because the production of hydrogen peroxide
has been suggested to be a virulence factor during M.pulmonis infections
( 58). This enzyme was not deduced
from the genomes of other mollicutes. The complete sequence of the M.pulmonis genome
opens a wide range of perspectives for the study of the mollicutes,
and, more generally, of bacterial evolution related to pathogenicity. The
analysis of this genome will be more easily exploited than those
of the preceding mollicutes because of the availability of tools,
transposons and oriC-based replicative plasmids,
to genetically manipulate M.pulmonis ( 59, 60)
(A.Blanchard, unpublished material). Furthermore, the mouse, one
of the natural hosts of M.pulmonis, appears to
be a particularly useful model system to dissect mycoplasma–host
interactions notably owing to the existence of mice strains differing
in their susceptibility to mycoplasmal disease. Mycoplasma
pulmonis belongs to a phylogenetic branch distinct from that
of the other sequenced mycoplasmas. We are aware of at least six
other ongoing mollicute genome projects ( M.mycoides subsp.
mycoides SC, M.hyopneumoniae, M.gallisepticum, M.penetrans, S.citri and S.kunkelii).
From an evolutionary perspective, the availability of the genome
sequence of these mycoplasmal species will make this group of organisms
an ideal target for genome comparisons and studies of microbial
evolution. Still, the most striking feature of the genome of M.pulmonis is
that it reveals an astonishing diversity of strategies related to the
evolution of pathogenicity. It contains genes that can participate
in intra-chromosomal homologous recombination, like in M.pneumoniae and M.genitalium, antigenic variation through slipped-strand
mispairing of repetitive motifs in control regions, as found in H.pylori ( 61)
and Haemophilus influenzae ( 62),
highly variable repetitive proteins, like in Mycobacterium
tuberculosis ( 63), and variable
restriction and modification systems, as proposed for Neisseria
gonorrhoea ( 64, 65). Hence, in spite of its minimalist
character in terms of biochemical potential, M.pulmonis is
an extremely suited model organism to study pathogenesis and co-evolution
of bacteria with the host. This work paves the way for a wide variety of experimental studies.
One obvious experiment is to inactivate the gene(s) potentially
controlling the inversions within the vsa locus
to confirm the contribution of this enzyme in the variability of cell
surface antigens and also to determine whether antigenic variation
confers protection against the host’s immune system. The
fact that a single recombinase gene was found in the genome suggests
that this enzyme catalyzes DNA inversions in both the vsa and hsd loci. We would like to thank J.Weissenbach and L.Montagnier for continuous
support during this program, Paul Scott for carefully reading the
manuscript and Sophie Oztas for technical help during the sequencing
at Genoscope. This work was funded by the Institut Pasteur, Genoscope,
INRA, Région Aquitaine and the Université Victor
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