Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal phosphate-binding protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.

16Organisms are defined by their genomes, yet many distinguishing features of a given organism are 17 encoded by genes that are functionally unannotated. Mycobacterium tuberculosis (Mtb), the leading 18 cause of death due to a single microbe, co-evolved with humans as its only known natural reservoir, yet 19 the factors mediating Mtb's pathogenicity remain incompletely defined. rv3722c is a gene of unknown 20 function predicted to encode a pyridoxal phosphate binding protein and to be essential for in vitro 21 growth of Mtb. Using metabolomic, genetic and structural approaches, we show that Rv3722c is the 22 primary aspartate aminotransferase of Mtb and mediates an essential but underrecognized role in 23 metabolism: nitrogen distribution. Together with the attenuation of Rv3722c-deficient Mtb in 24 macrophages and mice, these results identify aspartate biosynthesis and nitrogen distribution as 25 potential species-selective drug targets in Mtb. 26 survival of Mtb in macrophages and in mice. We further show that this essentiality is due, in part, to a 49 non-redundant role of Asp in the metabolic distribution of assimilated nitrogen. These findings identify 50Rv3722c as an essential metabolic mediator of Asp biosynthesis and Asp-dependent nitrogen 51 metabolism as an essential determinant of Mtb growth and virulence. 52 Results 53Rv3722c is conditionally essential in vitro 54We first sought to confirm the predicted essentiality of Rv3722c. We constructed an Mtb strain in which 55 expression of Rv3722c is regulated by its native promoter, but protein stability is controlled by a 56 tetracycline-repressible protein degradation system (Rv3722c-TetON) 20,21 . An isogenic strain lacking a 57 functional protein degradation system (Rv3722c-Control) served as a control. As predicted, omission of 58 anhydrotetracycline (ATC) resulted in depletion of Rv3722c protein and attenuation of Mtb growth in 59Glu-based Middlebrook 7H9 medium ( Fig 1A-B). This growth defect could be overcome by culturing 60Rv3722c-TetON in the same medium supplemented with casein hydrolysate, which contains a complex 61 mixture of amino acids and small peptides ( Fig 1C), and mitigated by culture on Middlebrook 7H10 agar 62 ( Fig 1D). This conditional rescue made it possible to deplete Rv3722c to levels below the limit of 63 detection ( Fig 1E) before a subsequent experimental challenge. Such pre-depletion completely 64 prevented subsequent growth in unsupplemented 7H9 ( Fig 1F). Rv3722c could also be depleted below 65 the limit of detection without impairing growth by culturing the cells in an Asn-based minimal medium 66 (Sauton's) ( Fig S1). 67Page 5 of 65 68 Figure 1. Rv3722c is conditionally esssential in vitro. A) Growth curve of Rv3722c-proficient and -69 deficient Mtb in 7H9 culture media. Rv3722c-TetOn and Rv3722c-control were cultured in Middlebrook 70 7H9 culture media with or without 500 ng/mL anhydrotetracycline (ATC). Bacterial growth was 71 monitored by optical density at 600 nm. B) Western blot showing the depletion of Rv3722c 7H9 cul...

Comparative sequence analysis has enabled the annotation of millions of genes from organisms across the evolutionary tree. However, this approach has inherently biased the annotation of phylogenetically ubiquitous, rather than species-specific, functions. The ecologically unusual pathogen Mycobacterium tuberculosis (Mtb) has evolved in humans as its sole reservoir and emerged as the leading bacterial cause of death worldwide. However, the physiological factors that define Mtb’s pathogenicity are poorly understood. Here, we report the structure and function of a protein that is required for optimal in vitro fitness and bears homology to two distinct enzymes, Rv0812. Despite diversification of related orthologues into biochemically distinct enzyme families, rv0812 encodes a single active site with aminodeoxychorismate lyase and D–amino acid transaminase activities. The mutual exclusivity of substrate occupancy in this active site mediates coupling between nucleic acid and cell wall biosynthesis, prioritizing PABA over D-Ala/D-Glu biosynthesis. This bifunctionality reveals a novel, enzymatically encoded fail-safe mechanism that may help Mtb and other bacteria couple replication and division.

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