Whole genome sequence of mycoplasma mycoides subsp. Mycoides LC : application to the development of new diagnostic tools and heterologous gene expression

Abstract

Mycoplasmas are the smallest bacteria without a cell wall derived from Gram positive bacteria. A group of mycoplasma known as the “Mycoplasma mycoides cluster” is composed of five subspecies and an unassigned group of strains known for their pathogenicity in ruminant hosts. Phylogenetically, this cluster is found to be closely related to species of mycoplasma plant pathogens. Mycoplasmas have a reduced genome size of about 1 Mbp, characterized by a low GC content of about 25 %. Among members of the Mycoplasma mycoides cluster, Mycoplasma mycoides subsp. mycoides large colony biotype (MmmLC) is one of the agents responsible for contagious agalactia in goats. This organism is closely related to Mycoplasma mycoides subsp. mycoides small colony biotype, the causative agent of contagious bovine pleuropneumonia (CBPP), for which the whole genome sequence is available. Because of the close relationship of these two species we have decided to sequence the genome of an MmmLC strain for comparative genomics. Before whole genome sequencing and assembly, we have estimated the genome size of MmmLC using pulse field gel electrophoresis (PFGE). Data generated from this initial study have permitted us to verify the genome assembly by comparing in-silico profiles. In addition the preliminary analysis included DNA hybridization tests to verify the presence of duplicated genes in MmmLC as that of the genome of MmmSC. Comparative genomics made from the available whole genome sequence data of species within the M. mycoides cluster has permitted the identification of target genes, which were used for the development of specific PCR tests. The target genes chosen included genes of the “arginine deiminase operon”, in most bacteria genes of this operon code for enzymes involved in the degradation of arginine to produce energy. The number of these genes as well as their organization within the operon found to vary between members of the M. mycoides cluster. From this operon arcD has been used to develop a specific PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP), and arcB has been used for the development of specific PCR for the identification of M. putrefaciens, another causative agent of the contagious agalactia syndrome. The glk gene, flanking the operon on the 3' end, was found to be highly conserved among all members of the M. mycoides cluster and was used for the design of specific primers able to detect all members of M. mycoides cluster. Furthermore, annotation of the genome sequence of MmmLC allowed the discovery of two new insertion sequence elements. One of these two insertion sequence elements was found in higher copy in the genome and belongs to the IS3 family. This insertion sequence was not described in any other mycoplasma species or bacteria, was given a new name: ISMmy2. It was also found in some species of the M. mycoides cluster, but not in all the strains under these species. Interestingly, a non-functional vestige of ISMmy2 was also found in the MmmSC genome. Copies of this ISMmy2 were also found in species closely related to the M. mycoides cluster. Finally, we have tried to evaluate the capacity of MmmLC to be transformed and to express a heterologous gene with the ultimate aim to create a multivalent vaccine. For this aim we have chosen the H-gene of peste des petits ruminant virus of veterinary health importance