# Stringent Response in Mycobacteria: From Biology to Therapeutic Potential
## 6 Stringent Response and Biofilm Formation  
Biofilms are structured communities of bacteria embedded in self-produced polymeric matrices and attached to an abiotic or living surface [95; 96]. Biofilms protect bacteria from antibiotics, host immune system, and other environmental insults; they are a common cause of persistent bacterial infection [95; 97]. NTMs or environmental mycobacteria have been shown to form biofilms. These include _M. chelonae_, _M. avium_, _M. fortuitum_, _M. abscessus_ and _M. smegmatis_[42; 98; 99; 100; 101; 102]. These mycobacterial biofilms have been found in both natural and manufactured settings such as soil, showerheads, hospital water system and medical equipment. NTMs cause skin and soft tissue infections, aseptic meningitis, lymphadenitis, and pulmonary infections [103; 104; 105]. Disseminated and mixed NTM infections have been found in immunocompromised individuals suffering from cystic fibrosis, renal failures, leukemia and organ transplant recipients [106]. The prevalence of _M. abscessus_ in patients with chronic lung infections is also rising steadily [107; 108]. _M. tuberculosis_ has also been shown to form biofilms both in vitro and in vivo [109; 110; 111]. The mycobacterial biofilm matrix comprises extracellular DNA, carbohydrates, lipids and proteins [112; 113]. Biofilm formation, like a stringent response, is a way to survive in harsh or unfavorable environments. Hence, it is likely that stringent response might also regulate biofilm formation in mycobacteria. In the last two decades, the role of (p)ppGpp in the regulation of biofilm formation, particularly in pathogenic bacteria, is becoming well understood. For example, in _Listeria monocytogenes_, deletion of _relA_ impairs biofilm formation and reduces virulence [114]. In _Streptococcus mutans_, _relA_ inactivation causes a reduction in biofilm formation capacity [115]. _Enterococcus faecalis_ lacking (p)ppGpp show diminished capacity to form biofilms [116]. In _Vibrio cholerae_, the inactivation of stringent response results in reduced ability to form biofilms [117]. In the ppGpp null mutant strain of _P. aeruginosa_, the biofilms cells are more sensitive to antibiotics compared with the cells from wild-type biofilms [91]. Given the important role stringent response plays in biofilm formation in several bacterial pathogens, its role in mycobacterial biofilm formation has also been investigated. In _M. smegmatis,_ the stringent response has been shown to control biofilm formation [41] as the \(\Delta rel_{M\text{s}m}\) strain is deficient in biofilm formation, has reduced sliding motility and rough colony morphology. These phenotypes are governed by glycopeptidolipids (GPLs), which are a peculiar class of lipids, found in NTMs and _M. smegmatis,_ and are needed for biofilm formation [118]. The \(\Delta rel_{M\text{s}m}\) strain has reduced levels of GPLs in its cell wall compared to the parental mc\({}^{2}\)155 strain. This is indicative of the fact that the stringent response may regulate biofilm formation and colony morphology in _M. smegmatis_ by regulating the synthesis of GPLs (Figure 2). Moreover, both \(\Delta rel_{M\text{s}h}\) and \(\Delta rel_{M\text{s}m}\) strains also exhibit differential expression of several genes involved in cell envelope biosynthesis [42; 65; 119]. The deletion of small alarmone synthetase, RelZ, also impaired the biofilm formation in _M. smegmatis._ However, the degree of impairment is not as strong as that seen for the \(\Delta rel_{M\text{s}m}\) strain. Moreover, the double knockout \(\Delta rel\Delta relZ\) of _M. smegmatis_ shows the strongest inhibition of biofilm formation [30]. Based on these observations, it seems that \(\text{Rel}_{M\text{s}m}\) is the principal mediator of stringent response, while RelZ has a relatively minor contribution in this process. Thus, the stringent response regulates biofilm in mycobacteria by regulating the expression of genes involved in GPL and other cell wall components.