The actinomyceteCorynebacterium glutamicumgrows as rod-shaped cells by zonal peptidoglycan synthesis at the cell poles. In this bacterium, experimental depletion of the polar DivIVA protein (DivIVA_Cg) resulted in the inhibition of polar growth; consequently, these cells exhibited a coccoid morphology. This result demonstrated that DivIVA is required for cell elongation and the acquisition of a rod shape. DivIVA fromStreptomycesorMycobacteriumlocalized to the cell poles of DivIVA_Cg-depletedC. glutamicumand restored polar peptidoglycan synthesis, in contrast to DivIVA proteins fromBacillus subtilisorStreptococcus pneumoniae, which localized at the septum ofC. glutamicum. This confirmed that DivIVAs from actinomycetes are involved in polarized cell growth. DivIVA_Cglocalized at the septum after cell wall synthesis had started and the nucleoids had already segregated, suggesting that inC. glutamicumDivIVA is not involved in cell division or chromosome segregation.

Corynebacterium glutamicum is able to grow in media containing up to 12 mM arsenite and 500 mM arsenate and is one of the most arsenic-resistant microorganisms described to date. Two operons ( ars1 and ars2 ) involved in arsenate and arsenite resistance have been identified in the complete genome sequence of Corynebacterium glutamicum . The operons ars1 and ars2 are located some distance from each other in the bacterial chromosome, but they are both composed of genes encoding a regulatory protein ( arsR ), an arsenite permease ( arsB ), and an arsenate reductase ( arsC ); operon ars1 contains an additional arsenate reductase gene ( arsC1′ ) located immediately downstream from arsC1 . Additional arsenite permease and arsenate reductase genes ( arsB3 and arsC4 ) scattered on the chromosome were also identified. The involvement of ars operons in arsenic resistance in C. glutamicum was confirmed by gene disruption experiments of the three arsenite permease genes present in its genome. Wild-type and arsB3 insertional mutant C. glutamicum strains were able to grow with up to 12 mM arsenite, whereas arsB1 and arsB2 C. glutamicum insertional mutants were resistant to 4 mM and 9 mM arsenite, respectively. The double arsB1-arsB2 insertional mutant was resistant to only 0.4 mM arsenite and 10 mM arsenate. Gene amplification assays of operons ars1 and ars2 in C. glutamicum revealed that the recombinant strains containing the ars1 operon were resistant to up to 60 mM arsenite, this being one of the highest levels of bacterial resistance to arsenite so far described, whereas recombinant strains containing operon ars2 were resistant to only 20 mM arsenite. Northern blot and reverse transcription-PCR analysis confirmed the presence of transcripts for all the ars genes, the expression of arsB3 and arsC4 being constitutive, and the expression of arsR1 , arsB1 , arsC1 , arsC1′ , arsR2 , arsB2 , and arsC2 being inducible by arsenite.

Staphylococcus aureus escapes from immune recognition by invading a wide range of human cells. Once the pathogen becomes intracellular, the most important last resort antibiotics are not effective. Therefore, novel anti-infective therapies against intracellular S. aureus are urgently needed. Here, we have studied the physiological changes induced in the host cells by S. aureus during its intracellular proliferation. This is important, because the pathogen exploits the host cell’s metabolism for its own proliferation. We find that S. aureus severely depletes glucose and amino acid pools, which leads to increased breakdown of glutamine by the host cell in an attempt to meet its own metabolic needs. All of these metabolic changes activate autophagy in the host cell for nutrient scavenging and energy generation. The metabolic activation of autophagy could be used by the pathogen to sustain its own intracellular survival, making it an attractive target for novel anti-infectives.

Antimicrobial resistance is becoming one of the most important human health issues. Accordingly, the research focused on finding new antibiotherapeutic strategies is again becoming a priority for governments and major funding bodies. The development of treatments based on the generation of oxidative stress with the aim to disrupt the redox defenses of bacterial pathogens is an important strategy that has gained interest in recent years. This approach is allowing the identification of antimicrobials with repurposing potential that could be part of combinatorial chemotherapies designed to treat infections caused by recalcitrant bacterial pathogens. In addition, there have been important advances in the identification of novel plant and bacterial secondary metabolites that may generate oxidative stress as part of their antibacterial mechanism of action. Here, we revised the current status of this emerging field, focusing in particular on novel oxidative stress-generating compounds with the potential to treat infections caused by intracellular bacterial pathogens.

Corynebacterium glutamicum contains four serine/threonine protein kinases (STPKs) named PknA, PknB, PknG, and PknL. Here we present the first biochemical and comparative analysis of all four C. glutamicum STPKs and investigate their potential role in cell shape control and peptidoglycan synthesis during cell division. In vitro assays demonstrated that, except for PknG, all STPKs exhibited autokinase activity. We provide evidence that activation of PknG is part of a phosphorylation cascade mechanism that relies on PknA activity. Following phosphorylation by PknA, PknG could transphosphorylate its specific substrate OdhI in vitro. A mass spectrometry profiling approach was also used to identify the phosphoresidues in all four STPKs. The results indicate that the nature, number, and localization of the phosphoacceptors varies from one kinase to the other. Disruption of either pknL or pknG in C. glutamicum resulted in viable mutants presenting a typical cell morphology and growth rate. In contrast, we failed to obtain null mutants of pknA or pknB, supporting the notion that these genes are essential. Conditional mutants of pknA or pknB were therefore created, leading to partial depletion of PknA or PknB. This resulted in elongated cells, indicative of a cell division defect. Moreover, overexpression of PknA or PknB in C. glutamicum resulted in a lack of apical growth and therefore a coccoid-like morphology. These findings indicate that pknA and pknB are key players in signal transduction pathways for the regulation of the cell shape and both are essential for sustaining corynebacterial growth.

Summary: To survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol‐dependent reductase (mycoredoxin‐1) with a CGYC catalytic motif. With mycoredoxin‐1 and mycothiol deletion strains of Mycobacterium smegmatis, we show that mycoredoxin‐1 and mycothiol are involved in the protection against oxidative stress. Mycoredoxin‐1 acts as an oxidoreductase exclusively linked to the mycothiol electron transfer pathway and it can reduce S‐mycothiolated mixed disulphides. Moreover, we solved the solution structures of oxidized and reduced mycoredoxin‐1, revealing a thioredoxin fold with a putative mycothiol‐binding site. With HSQC snapshots during electron transport, we visualize the reduction of oxidized mycoredoxin‐1 as a function of time and find that mycoredoxin‐1 gets S‐mycothiolated on its N‐terminal nucleophilic cysteine. Mycoredoxin‐1 has a redox potential of −218 mV and hydrogen bonding with neighbouring residues lowers the pK_a of its N‐terminal nucleophilic cysteine. Determination of the oxidized and reduced structures of mycoredoxin‐1, better understanding of mycothiol‐dependent reactions in general, will likely give new insights in how M. tuberculosis survives oxidative stress in human macrophages.