Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was ...shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M. smegmatis. Here, by means of RNA-Seq, we monitored the early steps of biofilm production in M. bovis BCG, to distinguish intercellular aggregation from attachment to a surface. Genes encoding for the transcriptional regulators dosR and BCG0114 (Rv0081) were significantly regulated and responded differently to intercellular aggregation and surface attachment. Moreover, a M. tuberculosis H37Rv deletion mutant in the Rv3134c-dosS-dosR regulon, formed less biofilm than wild type M. tuberculosis, a phenotype reverted upon reintroduction of this operon into the mutant. Combining RT-qPCR with microbiological assays (colony and surface pellicle morphologies, biofilm quantification, Ziehl-Neelsen staining, growth curve and replication of planktonic cells), we found that BCG0642c affected biofilm production and replication of planktonic BCG, whereas ethR affected only phenotypes linked to planktonic cells despite its downregulation at the intercellular aggregation step. Our results provide evidence for a stage-dependent expression of genes that contribute to biofilm production in slow-growing mycobacteria.
Biofilm formed in vitro by mycobacteria has been associated with increased antibiotic tolerance as compared with planktonic cells. Cellulose has been identified as a component of DTT-exposed biofilms ...formed by M. tuberculosis. The celA1 gene of M. tuberculosis encodes a cellulase, which could affect the formation of biofilm by slow-growing mycobacteria. In this work, the celA1 gene of M. tuberculosis was cloned into the integrative pMV361 plasmid and then transformed into M. bovis BCG Pasteur to produce BCG:celA1, to have celA1 expressed from the strong promoter hsp60. We compared planktonic and biofilm growth, possible presence of CelA1 in whole protein extracts, quantitated biofilm, presence of monosaccharides, and bacillary burden in lungs after aerosol infection in BALB/c mice. Differences in the appearance of the surface pellicle and of the biofilm attached to the substrate were observed. In biofilms, we observed a significant decrease of glucosamine in BCG:celA1 compared with BCG:pMV361. Finally, BCG:celA1 had lower viable bacteria than the BCG:pMV361 strain after 24 h and 3 weeks post-infection, but no difference was found at 9 weeks post-infection.
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