Defining a temporal order of genetic requirements for development of mycobacterial biofilms

Abstract

Summary Most mycobacterial species spontaneously form biofilms, inducing unique growth physiologies and reducing drug sensitivity. Biofilm growth progresses through three genetically programmed stages: substratum attachment, intercellular aggregation and architecture maturation. Growth of Mycobacterium smegmatis biofilms requires multiple factors including a chaperonin (GroEL1) and a nucleoid-associated protein (Lsr2), although how their activities are linked remains unclear. Here we show that Lsr2 participates in intercellular aggregation, but substratum attachment of Lsr2 mutants is unaffected, thereby genetically distinguishing these developmental stages. Further, we identify a suppressor mutation in a glycopeptidolipid synthesis gene (mps) that results in hyperaggregation of cells and fully restores the form and functions of Δlsr2 mutant biofilms. Suppression by the mps mutation is specific to Δlsr2; it does not rescue the maturation-deficient biofilms of a ΔgroEL1 mutant, thereby differentiating the process of aggregation from maturation. Gene expression analysis supports a stepwise process of maturation, highlighted by temporally separated, transient inductions of iron and nitrogen import genes. Furthermore, GroEL1 activity is required for induction of nitrogen, but not iron, import genes. Together, our findings begin to define molecular checkpoints during development of mycobacterial biofilms.

Adaptation of Mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

“…1A). While colonies and pellicles represent different growth models in vitro , the two have overlapping gene expression patterns in M. smegmatis (14). Inclusion of both models in the Tn-seq screen allowed similarities and differences in genetic requirements for Mtb fitness to be determined in these two models of aggregated growth.…”

“…We thus hypothesized that the signal regulating inlps induction is dependent on the specific nature of growth environment in mature pellicle biofilms, which are not created by other growth conditions, including during colony development on agar surfaces and in high-density detergent-free shaken cultures. To address this hypothesis we used a previously described, hyper-aggregating suppressor strain of M. smegmatis that reports gene regulation responsive to cell-surface properties and/or biofilm microenvironments (14). The strain carries a suppressor mutation in the glycopeptidolipid biosynthesis gene ( mps ) in a Δ lsr2 background that rescues the deficiencies in cell-cell adhesion and biofilm formation of the parent Δ lsr2 strain(14).…”

“…Growth and development of biofilms is a multi-stage process that requires dedicated genetic programs expressed in a spatiotemporal order(13–15). Initial stages involving substratum attachment of planktonic cells and aggregated growth are followed by a maturation stage accompanied by synthesis of an extracellular matrix (13, 14). Physical contacts and chemical communications among cells in developing biofilms, as well as physiological adaptation to self-generated gradients of nutrients and oxygen stratify the architecture resulting in phenotypically heterogeneous cells (15–18).…”

The impact of leadered and leaderless gene structures on translation efficiency, transcript stability, and predicted transcription rates inMycobacterium smegmatis

“…Mycobacterium smegmatis strain ΔMSMEG_2952 [63] and its derivatives (Table 1) were grown in Middlebrook 7H9 medium with albumin/dextrose/catalase supplementation (ADC; final concentrations, 5 g/L bovine serum albumin fraction V, 2 g/L dextrose, 0.85 g/L NaCl, and 3 mg/L catalase), 0.2% glycerol, and 0.05% Tween 80. Cultures were shaken at 200 rpm and 37°C to an optical density at 600 nm (OD 600 ) of ∼0.8 at the time of harvest.…”

“…All constructs (pSS303 and derivatives noted in Table 1) were built using NEBuilder HiFi DNA Assembly Master Mix (E2621). Each assembled plasmid was integrated in M. smegmatis ΔMSMEG_2952 [63] at the Giles phage site, and selected with 200 µg/mL hygromycin.…”