Abstract
The utilization of available substrates, the metabolic potential and the growth rates of bacteria can play significant roles in their pathogenicity. This study concentrates on Mycoplasma 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 of M. 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 pdhB mutant recovered its normal growth and colony morphology. Interestingly, the pdhB mutant 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.
Highlights
Mycoplasmas are the simplest self-replicating organisms and have evolved from Gram-positive bacteria through reductive evolution [1, 2]
Transposon mutants of M. agalactiae were checked for in vitro growth defects, and the results demonstrated slower growth of the pdhB mutant compared to the wild type strain PG2
Transposon mutagenesis is a powerful tool to identify genetic factors involved in the virulence of pathogenic bacteria
Summary
Mycoplasmas are the simplest self-replicating organisms and have evolved from Gram-positive bacteria through reductive evolution [1, 2]. As a result of their limited biosynthetic and metabolic capabilities, mycoplasmas rely on infected host cells for their nutrition and lead a parasitic life [3, 4]. As successful pathogens, they have developed mechanisms to invade and survive within host cells. Surface lipoproteins play an important role in the adhesion and invasion of mycoplasmas and in evading the host immune response because of their variable sequence and expression [5, 6]. Efforts have been made to establish genetic tools through the development of oriC-based shuttle vectors and studies have been undertaken to genetically manipulate mycoplasmas using conventional methods, such as transposition and homologous recombination [17]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
