Whole genome sequencing of M. agalactiae
strain 5632 revealed that it contains an additional 95 genes representing an extra-130 kbps when compared to the PG2 type strain. For organisms that have a small genome size such as mycoplasmas, this is a rather significant feature. The additional material is mostly composed of repeated elements, so that our knowledge of the M. agalactiae
pan-genome has been enriched by 39 new genes. A large portion of those, more specifically 23, is present in ICEs or corresponds to IS. Recent mathematical models by Tettelin et al.
] show that the pan-genome of the mollicute Ureaplasma urealyticum
is limited, based on the draft sequences of nine strains. This implies that the sequencing of additional strains might not significantly increase our knowledge of this species unless it is targeting a specific biological question [40
]. Although U. urealyticum
is a human pathogen and has a genome slightly smaller (ca. 750 kbp), the same observation may apply to the M. agalactiae
species as indicated by the low number of new genes discovered in our study. Thus, sequencing additional M. agalactiae
strains might bring little more information on the global coding capacity of this organism.
Overall, data obtained here and elsewhere indicate that about 10% of the 5632 genome is highly dynamic in that large regions corresponding to ICE can excise [16
] and, theoretically, relocate elsewhere or be transferred to a recipient cell during conjugation, if such event is further shown to occur in this species. The two ICE's vestiges, ICE IV and V, represent scars of past ICE insertions followed by a progressive decay. Interestingly, these more resemble the larger ICE vestige of PG2 or the ICE of M. capricolum
than the three entire ICE copies of 5632 suggesting that this strain may have, at one point, hosted two types of ICEs. These data indicate that the circulation of ICEs in some strains might not be such a rare event. The presence of ICE circular forms in 5632 [16
] together with the low number of SNPs detected between the three copies indicate that multiple ICE insertions are recent. The mechanisms underlying ICE insertion, excision and putative transfer in mycoplasmas have yet to be investigated, but recent studies on ICE elements in Gram-positive bacteria suggest that these events can be under the control of sophisticated regulation systems in response to changing environmental conditions such as stress or population density [41
]. The finding of ICE in M. agalactiae
and members of the "mycoides" cluster together with evidence of HGT in between these species further raised the prospect that these simple bacteria could conjugate. So far, a single report has supported the occurrence of conjugation in mycoplasmas by showing the exchange of genetic material in between M. pulmonis
cells via a mechanism resistant to DNAse [42
]. The idea that this phenomenon might be more common among mycoplasmas than first expected is very exciting because, if occurring, it would change the way we see the evolution of these so called "minimal organisms".
Although smaller in size than ICE, IS elements as a whole represent a dynamic potential for the genome because of their copy number. In other bacteria, their contribution to genome plasticity and dynamics is well known [43
]. Here, no major DNA inversion or rearrangement was detected between the two M. agalactiae
genomes that could be associated to IS except for two cases. As previously shown by our group, the first one refers to the duplication in 5632 of the single vpma
cluster of PG2 that has been most likely driven by IS elements and that resulted in 5632 having extended possibilities for surface diversification when compared to PG2 [17
]. The second case refers to a region which organization significantly differs in between PG2 and 5632 (see Figure ) and which contains several IS related elements (i.e. IS, transposases or pseudogenes of transposase). Events underlying rearrangements in this region cannot be exactly retraced but most likely they are ancient and have resulted in duplication of the ptsG
gene in PG2 (MAG3250 and MAG3320). Interestingly, this region, like many others associated with IS, contains several genes or pseudogenes that have undergone HGT suggesting that IS may directly contribute to this phenomenon as suggested for other bacteria [43
]. Finally, we showed that IS insertions may have an impact on gene expression, thus modifying some of the strain properties such as those associated with restriction-modification in 5632.
Compared to the PG2 type strain, 5632 seems better equipped for DNA exchange. Besides harbouring an impressive "mobilome", some of which may be tailored for conjugative transfer, it contains a number of operating RM systems. On one hand, these may act as a barrier to DNA invasion [44
] and explain why 5632 DNA is resistant to several methylase-sensitive restriction enzymes and to DNA transformation (data not shown). On the other hand, while methylated DNA is protected against degradation, it might be more likely accepted by a recipient cell displaying similar RM systems, regardless of the DNA transfer or uptake mechanisms. Indeed, some of the 5632 specific RM systems not present in PG2 have homologs in members of the "mycoides" cluster (Table ). Whether the structure of the M. agalactiae
population is made of a majority of PG2-like strains that are deficient in mobile elements as well as in RM systems with only some strains such as 5632 being more prone to gene exchange with selected partners, is not yet known. Finally, DNA methylases, whether they belong or not to RM systems, could play a number of functions related to fitness or virulence, including the regulation of various physiological processes such as chromosome replication, mismatch repair, transposition, and transcription as described in other bacteria [45
]. They may also be involved in the epigenetic switch of some key factors such as in the Pap of the uropathogenic E. coli
Interestingly, a fairly good portion of the flexible gene pool of M. agalactiae
is dedicated to producing surface proteins, many of which are lipoproteins. Based on in silico
analysis, 5632 contains ca. 100 lipoproteins with at least 56 expressed under laboratory conditions. These include the Vpma family composed of 16 different lipoproteins that are encoded by 23 genes in 5632 and 6 in PG2 and that are phase variable in expression and probably in size [17
]. Phase variation of surface molecules is a common mechanism in mycoplasma species [33
] and is probably a major adaptive strategy for these minimal pathogens. Vpma phase variants are produced at high frequencies and in a reversible manner by site-specific recombination [26
] but comparative proteogenomics conducted here suggest that other variable systems may co-exist. For instance, expression of the P48-like protein as a lipoprotein that is soluble in Triton-X114 may depend on a riboshifting mechanism or on reversible hypermutation in a polyG tract localised at the 5' coding sequence (Figure ). Indeed, data obtained by Lynyansky et al.
] showed that translation of a full length P68 lipoprotein in M. bovis
is associated with the length of a similar polyG tract. The length of this homopolymer varies from 8 to 10 residues when comparing four M. bovis
strains, with nine G allowing translation of a complete P68. Indeed, expression of the two homologs, the M. agalactiae
P48-like and the M. bovis
P68, is most likely phase variable in the two ruminant pathogens. Several other polyG tracts, some containing up to 13 residues, were found in the study that are associated with the 5' end of genes encoding surface lipoproteins suggesting that this may be a common slippage mechanism in M. agalactiae
. Finally, the drp
family involves genes that circulate by HGT between M. agalactiae
and members of the "mycoides" cluster. Based on comparative proteogenomics, 5632 and PG2 have a same size repertoire each composed of a different set with only 2 out of 12 or 13 drp
products being expressed, one common to the two strains and one specific. Whether this reflects a mechanism of phase variation is unlikely, but silent drp
genes may act as a sequence reservoir for the emergence of new Drp expression patterns. Taken together these results suggest that the two M. agalactiae
strains might display very different surface architectures with highly dynamic compositions during clonal propagation.
The strain 5632 was initially chosen because of its particular genetic features, several of which were found in its close relative M. bovis
. This was further confirmed in this study which shows that 5632, unlike PG2, possesses (i) mobile elements such ICE and IS in multiple copies, (ii) a P48-like gene that is expressed, and (iii) two genes related to phage immunity that are also present in M. bovis
]. The ovine/caprine pathogen M. agalactiae
and the cattle pathogen M. bovis
were first classified as the same species and our findings indicate that a continuum of strains might exist in between these two species. The genome sequence of M. bovis
has been achieved (Craig Venter Institute, unpublished data and [31
]) and its analysis may unravel even more common traits as well as some specificities that may explain their respective host-specificity.