New barcode method reveals hidden functions in tuberculosis bacteria

Mycobacterium tuberculosis (Mtb) is a human bacterial pathogen that establishes chronic infection in the lung, and although its genome was sequenced nearly 25 years ago, many genes remain mysterious. Large-scale efforts to tie genes to functions have been slowed by the limited throughput of traditional transposon sequencing in mycobacteria. To tackle that problem, the team led with Sarah A. Stanley as corresponding author created a new resource: a pooled random barcode transposon-site sequencing (RB-TnSeq) library in Mycobacterium tuberculosis. Each transposon in this library contains a unique twenty-nucleotide barcode, which lets researchers rapidly track mutations and run many genetic tests without the laborious protocols required by standard bacterial TnSeq screens. By building this RB-TnSeq resource, the researchers set out to screen Mtb under many different growth and stress conditions so that the function of uncharacterized genes could be inferred from how mutants fare when the bacterium faces changes in nutrients, stressors, or antibiotics.

Using the RB-TnSeq library, the team ran 95 screens across an array of carbon sources, nitrogen sources, stressors, and antibiotics. The barcode approach enabled high-throughput genetic screening and rapid readout of which gene disruptions caused sensitivity or advantage under each condition. A major focus was the PE/PPE genes, a mycobacterial gene family with long-elusive roles; the screens uncovered 187 novel phenotypes across 37 genes in this family. From these data the authors propose a pathway for lactate utilization in which the ESX-5 type VII secretion system exports PPE3, and PPE3 in turn facilitates import of D- and L-lactate into the bacterial cell. They also identify a candidate D-lactate dehydrogenase that may carry out this metabolic step. Additional findings include that the proton-pumping NADH dehydrogenase Nuo is required for utilization of propionate, and characterization of a novel mutant that confers resistance to the new tuberculosis antibiotic pretomanid.

These results demonstrate how RB-TnSeq can illuminate the functions of genes that were previously uncharacterized in Mtb and reveal new aspects of bacterial metabolism and drug resistance. By tying specific genes and systems to the ability to use particular nutrients such as lactate and propionate, the work highlights metabolic flexibility in Mtb and suggests concrete molecular players—PPE3, a candidate D-lactate dehydrogenase, and Nuo—that deserve focused follow-up. The discovery of 187 novel PE/PPE phenotypes expands understanding of a large gene family that has been difficult to study, while the identification of a mutant linked to pretomanid resistance provides an immediate genetic clue about how Mtb can evade this antibiotic. Overall, the RB-TnSeq dataset will serve as a foundation for further experiments, inspiring new hypotheses and helping researchers prioritize which uncharacterized genes to study next to better understand Mtb biology and responses to stress.