Mycobacterium aviumGenes Associated with the Ability To Form a Biofilm

Abstract

Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression >2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm.Mycobacterium avium complex is widely distributed in the environment, such as in water and soil, and is a chief component of many natural aquatic biofilms (8). M. avium is also known to cause chronic pulmonary infection in patients with predisposing lung disease, such as previous tuberculosis and chronic obstructive pulmonary disease (28). Urban water systems contain organisms of the M. avium complex in biofilm or a biofilm-like structure, and individuals can potentially be exposed to the bacterium, either by inhalation of aerosol particles or ingestion of contaminated water. Studies have established an association between M. avium in urban water and the development of disseminated disease in individuals with AIDS (36).Mycobacterium smegmatis, as well as M. avium, has been shown to produce a biofilm or a biofilm-like structure (6, 19). The outermost layers of the M. smegmatis and M. avium cell walls contain glycopeptidolipid (GPL), whereas the outermost layer of M. tuberculosis is made of phenolic glycolipids, dimycocerosate, and lipo-oligosaccharides (24). Recent studies suggest that the M. smegmatis biofilm is associated with a GPL present on the cell wall, and indirect evidence indicates a similar role in M. avium (6). Aspects of biofilm formation have begun to be examined with M. smegmatis. Transposon inactivation of the GPL gene clusters in M. smegmatis decreased the production of biofilm, and the deletion of the genes tmtp and mps revealed their involvement in biofilm formation upon seeding of the bacterium on polyvinyl chloride (PVC) plates (19,26). The tmtp gene is highly conserved between M. smegmatis and M. avium, ...

Glutamine synthetase encoded by glnA-1 is necessary for cell wall resistance and pathogenicity of Mycobacterium bovis

“…The biofilm formed by M. tuberculosis is known to contain abundant free mycolic acid (Ojha et al, 2005(Ojha et al, , 2008; at this stage we do not know whether mycolic acid synthesis or its content in the cell wall of the mutant is affected. Other genetic loci identified in biofilm formation in mycobacteria are linked to enzymes of the tricarboxylic acid (TCA) cycle, glycolipid biosynthesis (Yamazaki et al, 2006) and extracellular GroEL1 (Ojha et al, 2005). Moreover, how GS influences biofilm formation in M. bovis needs further investigation.…”

AMycobacterium tuberculosisMutant Lacking thegroELHomologuecpn60.1Is Viable but Fails To Induce an Inflammatory Response in Animal Models of Infection

“…Also, it contains three cpn60 genes. Analysis of a transposon mutant library of Mycobacterium avium revealed a number of genes (not including cpn60.1) that impair biofilm formation (42). The virtually identical Cpn60.2 proteins from M. tuberculosis and M. leprae have very different anti-inflammatory actions in mice (30,31), and in general, different Cpn60 proteins exhibit a bewildering variety of activities (4,32).…”

TheMycobacterium aviumsubsp.paratuberculosisMAP3464 Gene Encodes an Oxidoreductase Involved in Invasion of Bovine Epithelial Cells through the Activation of Host Cell Cdc42

“…To identify genes associated with invasion, a transposon library was constructed by transformation of M. avium subsp. paratuberculosis strain 19698 competent cells with the temperature-sensitive plasmid pTNGJC-KAN, a plasmid containing the transposon Tn5367 harboring a kanamycin-resistant gene, as previously described (27). Briefly, M. avium subsp.…”

“…Quantitative fluorogenic amplification of cDNA was performed using an iCycler real-time detection system and SYBR green technology (Bio-Rad, Hercules, CA) as previously described (27). Briefly, PCRs were carried out in 50-l reaction mixtures consisting of 25 l of IQ SYBR green supermix, 1 l of each primer (10 M), 1 l of cDNA, and 23 l of water.…”