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Translesion synthesis by translesion polymerases is a conserved mechanism of DNA damage tolerance. In bacteria, DinB enzymes are the widely distributed promutagenic translesion polymerases. The role of DinBs in mycobacterial mutagenesis was unclear until recent studies revealed a role for mycobacterial DinB1 in substitution and frameshift mutagenesis, overlapping with that of translesion polymerase DnaE2. Mycobacterium smegmatis encodes two additional DinBs (DinB2 and DinB3) and Mycobacterium tuberculosis encodes DinB2, but the roles of these polymerases in mycobacterial damage tolerance and mutagenesis is unknown. The biochemical properties of DinB2, including facile utilization of ribonucleotides and 8-oxo-guanine, suggest that DinB2 could be a promutagenic polymerase. Here, we examine the effects of DinB2 and DinB3 overexpression in mycobacterial cells. We demonstrate that DinB2 can drive diverse substitution mutations conferring antibiotic resistance. DinB2 induces frameshift mutations in homopolymeric sequences, both in vitro and in vivo. DinB2 switches from less to more mutagenic in the presence of manganese in vitro. This study indicates that DinB2 may contribute to mycobacterial mutagenesis and antibiotic resistance acquisition in combination with DinB1 and DnaE2.

Bacterial pathogens that infect phagocytic cells must deploy mechanisms that sense and neutralize host microbicidal effectors. For Mycobacterium tuberculosis, the causative agent of tuberculosis, these mechanisms allow the bacterium to rapidly adapt from aerosol transmission to initial growth in the lung alveolar macrophage. Here, we identify a branched signaling circuit in M. tuberculosis that controls growth in the lung through integrated direct sensing of copper ions and nitric oxide by coupled activity of the Rip1 intramembrane protease and the PdtaS/R two-component system. This circuit uses a two-signal mechanism to inactivate the PdtaS/PdtaR two-component system, which constitutively represses virulence gene expression. Cu and NO inhibit the PdtaS sensor kinase through a dicysteine motif in the N-terminal GAF domain. The NO arm of the pathway is further controlled by sequestration of the PdtaR RNA binding response regulator by an NO-induced small RNA, controlled by the Rip1 intramembrane protease. This coupled Rip1/PdtaS/PdtaR circuit controls NO resistance and acute lung infection in mice by relieving PdtaS/R-mediated repression of isonitrile chalkophore biosynthesis. These studies identify an integrated mechanism by which M. tuberculosis senses and resists macrophage chemical effectors to achieve pathogenesis

RbpA and CarD are essential transcription regulators in mycobacteria. Mechanistic analyses of promoter open complex (RPo) formation establish that RbpA and CarD cooperatively stimulate formation of an intermediate (RP2) leading to RPo; formation of RP2 is likely a bottleneck step at the majority of mycobacterial promoters. Once RPo forms, CarD also disfavors its isomerization back to RP2. We determined a 2.76 Å-resolution crystal structure of a mycobacterial transcription initiation complex (TIC) with RbpA as well as a CarD/RbpA/TIC model. Both CarD and RbpA bind near the upstream edge of the −10 element where they likely facilitate DNA bending and impede transcription bubble collapse. In vivo studies demonstrate the essential role of RbpA, show the effects of RbpA truncations on transcription and cell physiology, and indicate additional functions for RbpA not evident in vitro. This work provides a framework to understand the control of mycobacterial transcription by RbpA and CarD. DOI: http://dx.doi.org/10.7554/eLife.22520.001

Defense against infection by Mycobacterium tuberculosis (Mtb) is mediated by CD4 T cells. CCR2+ inflammatory monocytes (IMs) have been implicated in Mtb-specific CD4 T cell responses but their in vivo contribution remains unresolved. Herein, we show that transient ablation of IMs during infection prevents Mtb delivery to pulmonary lymph nodes, reducing CD4 T cell responses. Transfer of MHC class II-expressing IMs to MHC class II-deficient, monocyte-depleted recipients, while restoring Mtb transport to mLNs, does not enable Mtb-specific CD4 T cell priming. On the other hand, transfer of MHC class II-deficient IMs corrects CD4 T cell priming in monocyte-depleted, MHC class II-expressing mice. Specific depletion of classical DCs does not reduce Mtb delivery to pulmonary lymph nodes but markedly reduces CD4 T cell priming. Thus, although IMs acquire characteristics of DCs while delivering Mtb to lymph nodes, cDCs but not moDCs induce proliferation of Mtb-specific CD4 T cells. DOI: http://dx.doi.org/10.7554/eLife.01086.001, eLife digest Tuberculosis is a disease that kills more than one million people every year. It is caused by mycobacteria, notably Mycobacterium tuberculosis, and the World Health Organization estimates that about one third of the world’s population has latent tuberculosis, although only one person in 10 goes on to develop an active infection. Understanding why some individuals develop active infections, whereas most do not, could help with the development of a vaccine to prevent tuberculosis and/or new treatments for the disease. Disappointing results from vaccine trials and the emergence of drug-resistant strains of tuberculosis have increased the need for more research into the interactions between mycobacteria and the human immune system. Tuberculosis is spread when an infected person coughs or sneezes and someone else inhales the mycobacteria spread by the first person. When M. tuberculosis first enters the human respiratory tract, the innate immune system tries to identify and destroy cells that have been infected. However, if this initial response is not effective, the M. tuberculosis can persist in the lungs and trigger the adaptive immune response. This involves CD4 T cells working to eliminate the infection, but our understanding of the adaptive immune response is not complete. Samstein et al. probed the role that immune cells known as inflammatory monocytes play in the adaptive immune response. Previous research has suggested that inflammatory monocytes may develop into dendritic cells that directly prime the CD4 T cells to respond when the lung has been infected. However, Samstein et al. demonstrate that the inflammatory monocytes carry M. tuberculosis from the lungs of infected mice to the draining lymph nodes during the second week of infection. These monocytes develop many of the characteristics of dendritic cells, but they do not activate the CD4 T cells. Samstein et al. show that dendritic cells, contrary to previous evidence, are not necessary for the transport of the M. tuberculosis from the lungs to the draining lymph nodes. Without the dendritic cells, however, fewer CD4 T cell are primed in the lymph nodes. Samstein et al. suggest that the inflammatory monocytes play a crucial role by transporting the live bacteria to the lymph nodes. And once in the lymph nodes, the monocytes transfer invading antigens to dendritic cells to initiate the production of the CD4 T cells to lead the fight against the infection. DOI: http://dx.doi.org/10.7554/eLife.01086.002