Elimination of PknL and MSMEG_4242 in alters the character of the outer cell envelope and selects for mutations in Lsr2.

Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of and adjacent regulator 2 in rough colony mc155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated genes due to mutations and different IS insertions. When complemented with , the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS transposase TnpA and , which encodes a protein similar to PKS5. When was deleted, smooth mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough del mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of changes colony morphology from rough to smooth, augments sliding motility and increases expression of and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and regulate lipid components of the outer cell envelope and their absence selects for inactivation A regulatory, phosphorylation cascade model is proposed.