## 3 Mycobacterium Ecology: The Underlying Resilience Biology
### Biofilm Formation and Quorum-Sensing
In addition to the production of these toxins, vesicles also protect mycobacterial cells from the antimycobacterial activity of antituberculous compounds [94]. Hsp18, a heat-shock chaperone, is hypothesized to play an important role in biofilm formation, namely in the cell adherence and attachment stages to surfaces [95]. In _M. smegmatis_, mycolic acids are important cell wall components involved in pellicle biofilm, the type of biofilm formed in solid-air interfaces (Figure 3) [96]. The mycolic acids produced in late pellicle biofilms are shorter (C\({}_{56}\)-C\({}_{68}\)) than the ones produced during the planktonic lifestyle (C\({}_{70}\)-C\({}_{90}\)), being this mechanism regulated by the chaperone GroEL1 [96]. Moreover, free mycolic acids were also detected in the extracellular matrix as the result of the hydrolysis of trehalose dimycolate by serine esterase [96]. Furthermore, the _rpoZ_ gene is also important in sliding motility and biofilm formation, since, compared to the wild type, deletion mutants have reduced levels of short-chain mycolic acids in the cell wall and absence of these in the extracellular matrix [97]. Monomeromycolyl diacylglycerol (MMDAG) and mycolate ester waxes present in the mycobacterial cell surface are important to form normal biofilms. Deletion mutants of the _mmpL11_ gene, which encodes a transporter of these lipids, leads to a delay in biofilm formation [98]. Additionally, Lsr2 (histone-like protein) is also of crucial importance to the biofilm phenotype, since transposon insertion mutants in this gene exhibited deficiency in MMDAG, compromising biofilm formation through increasing sliding motility and by reducing cell surface hydrophobicity [99] which is essential for cell-to-cell interactions. In 2017, [PERSON] and colleagues published a model of biofilm formation for _M. smegmatis_ in which the planktonic cells start the aggregation processes by the overproduction of Lsr2 that further triggers the upregulation of GroEL1 and GroEL1-dependent free mycolate syntheses [100]. After this step, the iron sequestration pathways are induced, promoting biofilm maturation [100]. Additionally, _hadC_ deletion mutants possess a defected dehydratase activity of fatty acid synthase type II (FAS-II), delaying, and attenuating pellicle biofilm formation [53]. The GroEL1 chaperone modulates the synthesis of mycolates specifically during biofilm formation and physically associates with KasA, a key component of the type II fatty acid synthase (FAS II) involved in mycolic acid synthesis [101]. The same occurs with mutants of the mammalian cell entry (_mcc_) 1 operon, responsible for free mycolic acids accumulation on the mycobacterial cell wall [101]. Furthermore, _Rx0024_ gene that encodes a putative peptidoglycanamidase induces biofilm formation by increasing cell hydrophobicity, contrary to the _glmM_ gene that produces a phosphoglucosamine mutase, inhibiting biofilm formation [101]. Thus, the mycobacterial cell wall composition greatly affects the mycobacterial capacity to correctly form biofilms. [PERSON] et al. (2015) showed that biofilm formation is hampered by the absence of genes expressing the PknG, L13, and RenU proteins [102]. These proteins are part of the redox homeostatic system of mycobacteria (RHOCS) that detects the increase of NADH inside the mycobacterial cells resulting from redox stress situations, leading to the phosphorylation of the L13 protein by the PknG, increasing the association of L13 with the RenU protein, triggering the hydrolysis of NADH, then culminating in the redox homeostasis balance of mycobacterial cells [102]. In addition to this redox homeostatic system, thiol reductive stress is also implied in biofilm formation, with _mshC_ and _mscR_ genes (mycothiol biosynthesis and mycothiol dependent metabolism of nitrosothiols, respectively) being necessary to pellicle biofilm formation [103]. Thus, the metabolic status of mycobacterial cells regulates biofilm formation. [PERSON] et al. (2007) performed a transcriptional analysis of _M.