RbpA and CarD are essential transcription regulators in mycobacteria. Mechanisticanalyses 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 À10 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.

SummarypH-responsive transcription factors of the Rim101/ PacC family govern virulence in many fungal pathogens. These family members control expression of target genes with diverse functions in growth, morphology and environmental adaptation, so the mechanistic relationship between Rim101/PacC and infection is unclear. We have focused on Rim101 from Candida albicans, which we find to be required for virulence in an oropharyngeal candidiasis model. Rim101 affects the yeast-hypha morphological transition, a major virulence requirement in disseminated infection models. However, virulence in the oropharyngeal candidiasis model is independent of the yeast-hypha transition because it is unaffected by an nrg1 mutation, which prevents formation of yeast cells. Here we have identified Rim101 target genes in an nrg1D/D mutant background and surveyed function using an overexpression-rescue approach. Increased expression of Rim101 target genes ALS3, CHT2, PGA7/RBT6, SKN1 or ZRT1 can partially restore pathogenic interaction of a rim101D/D mutant with oral epithelial cells. Four of these five genes govern cell wall structure. Our results indicate that Rim101-dependent cell wall alteration contributes to C. albicans pathogenic interactions with oral epithelial cells, independently of cell morphology.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death. Synthesis of lipids critical for Mtb’s cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4’-phosphopantetheine (Ppt) from coenzyme A to diverse acyl carrier proteins (ACPs). We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss of function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor co-crystal structure may aid further development of anti-mycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

Live-cell imaging by light microscopy has demonstrated that all cells are spatially and temporally organized. Quantitative, computational image analysis is an important part of cellular imaging, providing both enriched information about individual cell properties and the ability to analyze large datasets. However, such studies are often limited by the small size and variable shape of objects of interest. Here, we address two outstanding problems in bacterial cell division by developing a generally applicable, standardized, and modular software suite termed Projected System of Internal Coordinates from Interpolated Contours (PSICIC) that solves common problems in image quantitation. PSICIC implements interpolated-contour analysis for accurate and precise determination of cell borders and automatically generates internal coordinate systems that are superimposable regardless of cell geometry. We have used PSICIC to establish that the cell-fate determinant, SpoIIE, is asymmetrically localized during Bacillus subtilis sporulation, thereby demonstrating the ability of PSICIC to discern protein localization features at sub-pixel scales. We also used PSICIC to examine the accuracy of cell division in Esherichia coli and found a new role for the Min system in regulating division-site placement throughout the cell length, but only prior to the initiation of cell constriction. These results extend our understanding of the regulation of both asymmetry and accuracy in bacterial division while demonstrating the general applicability of PSICIC as a computational approach for quantitative, high-throughput analysis of cellular images.

During host infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential in Mycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets. However, their molecular partners in protein reactivation have not been characterized in mycobacteria. Here, we reconstituted the activities of the Mtb ClpB/DnaK bichaperone system with the cofactors DnaJ1, DnaJ2, and GrpE and the small heat shock protein Hsp20. We found that DnaJ1 and DnaJ2 activate the ATPase activity of DnaK differently. A point mutation in the highly conserved HPD motif of the DnaJ proteins abrogates their ability to activate DnaK, although the DnaJ2 mutant still binds to DnaK. The purified Mtb ClpB/DnaK system reactivated a heat-denatured model substrate, but the DnaJ HPD mutants inhibited the reaction. Finally, either DnaJ1 or DnaJ2 is required for mycobacterial viability, as is the DnaK-activating activity of a DnaJ protein. These studies lay the groundwork for strategies to target essential chaperone–protein interactions in Mtb, the leading cause of death from a bacterial infection.