Identification of genes involved in Mycoplasma gallisepticum biofilm formation using mini-Tn4001-SGM transposon mutagenesis

Highlights

• A novel transposon vector containing the gentamicin gene was constructed.

• Of 738 mutants obtained, 12 had significantly reduced biofilm formation.

• These genes were associated with cellular processes, translation, metabolism, gene regulation.

• The new transposon vector provided a powerful tool for study of MG pathogenesis.

Abstract

Mycoplasma gallisepticum (MG) is an important pathogen that can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. MG has the ability to form biofilms. The molecular mechanisms underlying MG biofilm formation are complex and poorly understood. To better understand the mechanisms involved in biofilm formation, mini-Tn4001-SGM, a novel transposon vector containing the gentamicin gene was constructed and electroporated into MG strain R_low. Of the 738 mutants obtained, 12 had significantly reduced capacity to form biofilms in a polystyrene microtiter-plate biofilm assay. Ten different genes were identified as disrupted in these mutants using genomic walking from the transposon insertion sites and Southern bolt hybridization with a transposon-based probe. Four genes were associated with cellular processes, especially synthesis of extracellular polysaccharide and several lipoproteins encoded. Other genes were associated with translation, metabolism and gene regulation, and one had unknown function. Seven genes identified in this study have been previously associated with biofilm formation in MG or other bacterial species. The other three have not been previously reported to play a role in biofilm formation in MG. In conclusion, a new transposon vector was shown to be a powerful tool for future studies of MG pathogenesis. This study adds to our understanding of the molecular mechanisms involved in MG biofilm formation and may shed light on the persistence of MG infections.

Introduction

Mycoplasma gallisepticum (MG) is an important poultry pathogen that causes considerable economic losses worldwide (Ron et al., 2015). It is the causative agent of chronic respiratory disease in chickens and turkeys, and reduces hatchability and egg production and quality (Zhang et al., 2015). MG causes a persistent infection in chickens and is present at high levels in respiratory tissues (Chen et al., 2012).

A number of bacterial species is thought to persist in the environment in biofilms. Many bacteria use biofilm formation to survive inhospitable conditions and cause persistent infections (Wang et al., 2012). Bacterial pathogens in biofilms have a unique survival advantage. Our previous studies showed that MG strains can produce biofilms and this might be related to the persistence of MG infections (Chen et al., 2012), although the mechanisms involved in persistence and its relationship to biofilm formation are poorly understood. The limited genomic size of MG (1012800 bp) offers an opportunity to understand the molecular basis of biofilm formation and unravel interactions between the host and this pathogen. Transposons are effective tools that have been widely used to create mutant libraries for identifying essential and biofilm-related genes in many bacteria (Chen et al., 2010).

In this study, the results of that work were used to develop a transposon mutagenesis system for MG. Mini-Tn4001SpGm was constructed and additional genes associated with biofilm formation were identified. This study of biofilm-formation genes in mycoplasmas using transposon mutagenesis could potentially be applied to studying the molecular mechanisms involved in biofilm processes of other mycoplasmas.