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Structural and biophysical characterization of molecular mechanisms of disease-causing pathogens, such as Mycobacterium tuberculosis, often requires recombinant expression of large amounts highly pure protein. For the production of mycobacterial proteins, overexpression in the fast-growing and non-pathogenic species Mycobacterium smegmatis has several benefits over the standard Escherichia coli expression strains. However, unlike for E. coli, the range of expression vectors currently available is limited. Here we describe the development of the pMy vector series, a set of expression plasmids for recombinant production of single proteins and protein complexes in M. smegmatis. By incorporating an alternative selection marker, we show that these plasmids can also be used for co-expression studies. All vectors in the pMy vector series are available in the Addgene repository (www.addgene.com)., (© 2020 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD + -dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD + degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD + phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Type VII secretion systems (ESX) are responsible for transport of multiple proteins in mycobacteria. How different ESX systems achieve specific secretion of cognate substrates remains elusive. In the ESX systems, the cytoplasmic chaperone EspG forms complexes with heterodimeric PE-PPE substrates that are secreted from the cells or remain associated with the cell surface. Here we report the crystal structure of the EspG 1 chaperone from the ESX-1 system determined using a fusion strategy with T4 lysozyme. EspG 1 adopts a quasi 2-fold symmetric structure that consists of a central β-sheet and two α-helical bundles. In addition, we describe the structures of EspG 3 chaperones from four different crystal forms. Alternate conformations of the putative PE-PPE binding site are revealed by comparison of the available EspG 3 structures. Analysis of EspG 1 , EspG 3 , and EspG 5 chaperones using small-angle X-ray scattering reveals that EspG 1 and EspG 3 chaperones form dimers in solution, which we observed in several of our crystal forms. Finally, we propose a model of the ESX-3 specific EspG 3 -PE5-PPE4 complex based on the small-angle X-ray scattering analysis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)