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Identification of exopolysaccharide-deficient mutants of Mycoplasma pulmonis
James M Daubenspeck1, Jeffrey R Bolland, Wenyi Luo
1Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Abstract:
The presence of capsular exopolysaccharide (EPS) in Mollicutes has been inferred from electron micrographs for over 50 years without conclusive data to support the production of complex carbohydrates by the organism. Mycoplasma pulmonis binds the lectin Griffonia simplicifolia I (GS-I), which is specific for terminal beta-linked galactose residues. Mutants that failed to produce the EPS bound by GS-I were isolated from a transposon library. All of the mutants had the transposon located in open reading frame MYPU_7410 or MYPU_7420. These overlapping genes are predicted to code for a heterodimeric pair of ABC transporter permeases and may code for part of a new pathway for synthesis of EPS. Analysis by lectin-affinity chromatography in conjunction with gas chromatography demonstrated that the wild-type mycoplasma produced an EPS (EPS-I) composed of equimolar amounts of glucose and galactose that was lacking in the mutants. Phenotypic analysis revealed that the mutants had an increased propensity to form a biofilm on glass surfaces, colonized mouse lung and trachea efficiently, but had a decreased association with the A549 lung cell line. Confounding the interpretation of these results is the observation that the mutants missing EPS-I had an eightfold overproduction of an apparent second EPS (EPS-II) containing N-acetylglucosamine.
Insights
Researchers identified genes MYPU_7410 and MYPU_7420 involved in Mycoplasma pulmonis exopolysaccharide (EPS) production. Mutants lacking EPS-I showed altered biofilm formation and host cell association, suggesting EPS
Area of Science:
- Microbiology
- Molecular Biology
- Carbohydrate Chemistry
Background:
- Capsular exopolysaccharide (EPS) presence in Mollicutes has been suggested for decades but lacked definitive proof.
- Mycoplasma pulmonis exhibits binding to Griffonia simplicifolia I (GS-I) lectin, indicating the presence of terminal beta-linked galactose residues.
Purpose of the Study:
- To conclusively identify the genetic basis for exopolysaccharide (EPS) production in Mycoplasma pulmonis.
- To characterize the composition and function of EPS in Mycoplasma pulmonis.
Main Methods:
- Isolation of transposon mutants deficient in GS-I binding.
- Identification of mutated genes using transposon sequencing.
- Analysis of EPS composition via lectin-affinity chromatography and gas chromatography.
- Phenotypic analysis of mutant strains, including biofilm formation and host cell association.
Main Results:
- Mutants lacking GS-I binding had transposon insertions in overlapping genes MYPU_7410 and MYPU_7420, predicted to encode ABC transporter permeases.
- Wild-type Mycoplasma pulmonis produces an exopolysaccharide (EPS-I) composed of equimolar glucose and galactose, absent in mutants.
- Mutants exhibited increased biofilm formation on glass and efficient colonization of mouse lung and trachea, but decreased association with A549 lung cells.
- Mutants lacking EPS-I showed an eightfold overproduction of a second exopolysaccharide (EPS-II) containing N-acetylglucosamine.
Conclusions:
- Genes MYPU_7410 and MYPU_7420 are crucial for the synthesis of EPS-I in Mycoplasma pulmonis, potentially representing a novel EPS synthesis pathway.
- EPS-I plays a role in modulating biofilm formation and host cell interactions.
- The absence of EPS-I leads to the overproduction of EPS-II, suggesting complex regulatory mechanisms governing exopolysaccharide production.
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