Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Abstract

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.

Fig. 1

Schematic representation of the plasmids used in this study. The recombinant plasmid pMM20-1 corresponds to the pBluescript KS vector backbone (double line) ligated to the tetM determinant (thick single line) and to a 6.9 kb DNA fragment of M. agalactiae (thin single line) that carries the dnaA gene. Plasmids pMMΔ oriC, pMM21-4 and pMM21-7 were derived from pMM20-1 by total or partial deletion of the insert. Numbers mentioned below the dotted arrows indicate the approximate size of the fragments and bla designates the ampicillin resistance gene. Restriction enzymes: C, ClaI; E, EcoRI; EV, EcoRV; H, HindIII; N: NotI; P, PstI; S, SalI; Sm, SmaI; X, XbaI.

Fig. 2

Free versus integrated oriC plasmids in M. agalactiae transformants. (a) Schematic representation of the integration of oriC plasmids (P: pMM20-1, pMM21-4 or pMM21-7) into genomic DNA of M. agalactiae PG2. The size of the chromosomal ClaI DNA fragment bearing the oriC is designated as G, whereas X and Y correspond, respectively, to the right and left sequences flanking the oriC within the fragment G. A single putative homologous recombination event between the oriC copy carried by any of the three plasmids and the chromosomal oriC region is represented by crossed lines. This crossing over would lead to the integration of the plasmid into the chromosome and would segregate the two oriC copies onto two ClaI fragments GY and GX. Regardless of the plasmid used, the length of the GY fragment that carries the oriC and the tetM and bla markers is constant, while that of the GX fragment varies accordingly with the size of the plasmid inserts. (b) Replication versus integration of the oriC plasmids as observed in Southern blot hybridization using the tetM specific probe. ClaI-digested DNA from non-transformed M. agalactiae (NT) and from three transformants C1, C2 and C3, respectively, obtained using plasmids pMM21-7 (P1), pMM21-4 (P2) and pMM20-1 (P3), was probed with a DIG-labeled tetM specific probe. (c) Localization of the integrated plasmids at the chromosomal oriC locus of M. agalactiae PG2. Southern blot analysis was performed as in (b) except that the DNA was probed with a 1.2 kb DIG-labeled fragment specific to the oriC of M. agalactiae. DNA size standards are indicated in the left margin.

Fig. 3

Stability of the oriC plasmid pMM21-7 in M. agalactiae. Southern blot hybridizations were performed with BamHI digested DNA obtained from three independent pMM21-7 transformants (T1, T2 and T3) which had undergone 6, 16 and 21 additional passages in Aluotto (T1 and T2) or in SP-4 (T3) broth containing tetracycline. The DNA was probed with a DIG-labeled tetM specific probe. BamHI digested DNA corresponding to nontransformed M. agalactiae (NT) and pMM21-7 (P) served as negative and positive controls, respectively.