The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets.

SUMMARY To understand the adaptation of Mycobacterium tuberculosis to the intracellular environment, we used comprehensive metabolite profiling to identify the biochemical pathways utilized during growth on cholesterol, a critical carbon source during chronic infection. Metabolic alterations observed during cholesterol catabolism centered on propionyl-CoA and pyruvate pools. Consequently, growth on this substrate required the transcriptional induction of the propionyl-CoA assimilating methylcitrate cycle (MCC) enzymes, via the Rv1129c regulatory protein. We show that both Rv1129c and the MCC enzymes are required for intracellular growth in macrophages, and the growth defect of MCC mutants is largely attributable to the degradation of host-derived cholesterol. Together, these observations define a coordinated transcriptional and metabolic adaptation that is required for scavenging carbon during intracellular growth.

Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks.

Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway.

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