Lynn E. Connolly

The sigmaE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of sigmaE

The extracytoplasmic stress response in Escherichia coli is controlled by the alternative sigma factor, σE. σE activity is uniquely induced by the accumulation of outer membrane protein precursors in the periplasmic space, and leads to the increased production of several proteins, including the periplasmic protease DegP, that are thought to be required for maintaining cellular integrity under stress conditions. Genetic and biochemical experiments show that σE activity is under the control of three genes, rseABC (for regulator of sigma E), encoded immediately downstream of the sigma factor. Deletion of rseA leads to a 25‐fold induction of σE activity. RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits σE‐directed transcription in vitroindicating that RseA acts as an anti‐sigma factor. Deletion of rseB leads to a slight induction of σE, indicating that RseB is also a negative regulator of σE. RseB is a periplasmic protein and was found to co‐purify with the periplasmic domain of RseA, indicating that RseB probably exerts negative activity on σE through RseA. Deletion of rseC, in contrast, has no effect on σE activity under steady‐state conditions. Under induction conditions, strains lacking RseB and/or C show wild‐type induction of σE activity, indicating either the presence of multiple pathways regulating σE activity, or the ability of RseA alone to both sense and transmit information to σE.

Comprehensive Functional Analysis of Mycobacterium tuberculosis Toxin-Antitoxin Systems: Implications for Pathogenesis, Stress Responses, and Evolution

Toxin-antitoxin (TA) systems, stress-responsive genetic elements ubiquitous in microbial genomes, are unusually abundant in the major human pathogen Mycobacterium tuberculosis. Why M. tuberculosis has so many TA systems and what role they play in the unique biology of the pathogen is unknown. To address these questions, we have taken a comprehensive approach to identify and functionally characterize all the TA systems encoded in the M. tuberculosis genome. Here we show that 88 putative TA system candidates are present in M. tuberculosis, considerably more than previously thought. Comparative genomic analysis revealed that the vast majority of these systems are conserved in the M. tuberculosis complex (MTBC), but largely absent from other mycobacteria, including close relatives of M. tuberculosis. We found that many of the M. tuberculosis TA systems are located within discernable genomic islands and were thus likely acquired recently via horizontal gene transfer. We discovered a novel TA system located in the core genome that is conserved across the genus, suggesting that it may fulfill a role common to all mycobacteria. By expressing each of the putative TA systems in M. smegmatis, we demonstrate that 30 encode a functional toxin and its cognate antitoxin. We show that the toxins of the largest family of TA systems, VapBC, act by inhibiting translation via mRNA cleavage. Expression profiling demonstrated that four systems are specifically activated during stresses likely encountered in vivo, including hypoxia and phagocytosis by macrophages. The expansion and maintenance of TA genes in the MTBC, coupled with the finding that a subset is transcriptionally activated by stress, suggests that TA systems are important for M. tuberculosis pathogenesis.

Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

Aminoglycosides: An Overview

Aminoglycosides are natural or semisynthetic antibiotics derived from actinomycetes. They were among the first antibiotics to be introduced for routine clinical use and several examples have been approved for use in humans. They found widespread use as first-line agents in the early days of antimicrobial chemotherapy, but were eventually replaced in the 1980s with cephalosporins, carbapenems, and fluoroquinolones. Aminoglycosides synergize with a variety of other antibacterial classes, which, in combination with the continued increase in the rise of multidrug-resistant bacteria and the potential to improve the safety and efficacy of the class through optimized dosing regimens, has led to a renewed interest in these broad-spectrum and rapidly bactericidal antibacterials.

CarD Tricks and Magic Spots: Mechanisms of Stringent Control in Mycobacteria

Global reprogramming of bacterial gene expression in response to nutritional stress, the stringent response, is well studied in E. coli. Now Stallings et al. report that Mycobacterium tuberculosis employs a different strategy involving the general transcription factor CarD for growth control and persistence in response to stresses encountered during infection.