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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.

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Transcriptional portrait of M. bovis BCG during biofilm production shows genes differentially expressed during intercellular aggregation and substrate attachment