Disruption of the pdhB Pyruvate Dehydrogenase Gene Affects Colony Morphology, In Vitro Growth and Cell Invasiveness of Mycoplasma agalactiae

The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates onMycoplasma agalactiae, which causes significant economic losses through its contribution to contagious agalactia in small ruminants by as yet unknown mechanisms. This lack of knowledge is primarily due to its fastidious growth requirements and the scarcity of genetic tools available for its manipulation and analysis. Transposon mutagenesis ofM. agalactiae type strain PG2 resulted in several disruptions throughout the genome. A mutant defective in growth in vitro was found to have a transposon insertion in the pdhB gene, which encodes a component of the pyruvate dehydrogenase complex. This growth difference was quite significant during the actively dividing logarithmic phase but a gradual recovery was observed as the cells approached stationary phase. The mutant also exhibited a different and smaller colony morphology compared to the wild type strain PG2. For complementation, pdhAB was cloned downstream of a strong vpma promoter and upstream of a lacZ reporter gene in a newly constructed complementation vector. When transformed with this vector the pdhBmutant recovered its normal growth and colony morphology. Interestingly, the pdhBmutant also had significantly reduced invasiveness in HeLa cells, as revealed by double immunofluorescence staining. This deficiency was recovered in the complemented strain, which had invasiveness comparable to that of PG2. Taken together, these data indicate that pyruvate dehydrogenase might be an important player in infection with and colonization by M. agalactiae. PLOS ONE | DOI:10.1371/journal.pone.0119706 March 23, 2015 1 / 13 OPEN ACCESS Citation: Hegde S, Rosengarten R, ChopraDewasthaly R (2015) Disruption of the pdhB Pyruvate Dehydrogenase Gene Affects Colony Morphology, In Vitro Growth and Cell Invasiveness of Mycoplasma agalactiae. PLoS ONE 10(3): e0119706. doi:10.1371/ journal.pone.0119706 Academic Editor: Alain Charbit, Université Paris Descartes, FRANCE Received: September 9, 2014 Accepted: January 14, 2015 Published: March 23, 2015 Copyright: © 2015 Hegde et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grant P21796B20 (to RCD, Joachim Spergser and RR) of the Austrian Science Fund (FWF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.