Genes found essential in other mycoplasmas are dispensable in Mycoplasma bovis

Abstract

Mycoplasmas are regarded to be useful models for studying the minimum genetic complement required for independent survival of an organism. Mycoplasma bovis is a globally distributed pathogen causing pneumonia, mastitis, arthritis, otitis media and reproductive tract disease, and genome sequences of three strains, the type strain PG45 and two strains isolated in China, have been published. In this study, several Tn4001 based transposon constructs were generated and used to create a M. bovis PG45 insertional mutant library. Direct genome sequencing of 319 independent insertions detected disruptions in 129 genes in M. bovis, 48 of which had homologues in Mycoplasma mycoides subspecies mycoides SC and 99 of which had homologues in Mycoplasma agalactiae. Sixteen genes found to be essential in previous studies on other mycoplasma species were found to be dispensable. Five of these genes have previously been predicted to be part of the core set of 153 essential genes in mycoplasmas. Thus this study has extended the list of non-essential genes of mycoplasmas from that previously generated by studies in other species.

Figure 1. Location of 319 transposon integration sites in the M. bovis genome.

The distribution of the transposon insertion sites indicates that insertions were randomly distributed.

Figure 2. A fragment containing an inverted repeat (IR, black bar), the promoter (p), the signal sequence(s) and an FRT site (grey bar) was ligated to a fragment containing an FRT site, the reporter gene (phoA) and an IR using the EcoRI and XhoI cleavage sites in a pUC57 backbone.

Tn4001 with one or both insertion sequences was amplified and inserted in between the FRT sites of the construct to generate pTn4001single (a) and pTn4001complete (b), respectively. The construct pMiniTn4001-gent (c) was developed by amplifying and inserting the gentamicin resistance gene (aacA-aphD) between the two FRT sites of the construct, then the transposase gene (tnp) was amplified and inserted outside of the transposable element (IR, black bar). To generate the plasmid pMiniTn4001-tet (d), a fragment containing the IR, the promoter (p), the signal (s) and an FRT site was ligated to a fragment containing an FRT site, the reporter gene (phoA) and an IR in the pUC57 plasmid backbone. FRT sites have a unique XbaI cleavage site, so ligation of the fragments produced a construct with a single FRT site. The tnp gene was amplified and ligated into the plasmid outside the transposing element, then the tetM resistance gene with its own promoter and terminator was ligated within the construct.

Figure 3. PCR-based screening approach to identify transposon insertions in gene targets.

The insertion of the transposable element in a particular gene can occur in two possible orientations. PCR reaction using a primer pair, one based on the 39-bp IR sequence (uppercase) of the transposon and other one being either the forward (in this figure) or reverse primer flanking the gene of interest (GOI) would generate a single PCR product in the event of gene disruption. The relative position of the transposon insertion within the GOI is estimated based on the size of the PCR fragment including the region of binding of forward or reverse primer and primer based on IR region of transposon.