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Background Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens. Methods In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd -I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd , bo ′, or aa 3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects. Results The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd -I activity with median inhibitory concentration (IC50) values of 47 ± 28.9 µg/mL (158 ± 97.2 µM) and 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), respectively. Quinestrol inhibited growth of an E. coli " bd -I only" strain with an IC50 of 0.06 ± 0.02 µg/mL (0.2 ± 0.07 µM). Growth of an S. aureus " bd only" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43 µg/mL (6.0 ± 1.2 µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli. Conclusions Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus. [ABSTRACT FROM AUTHOR]

In this study, we identify a novel two-component system in Acinetobacter baumannii (herein named AmsSR for regulator of alternative metabolic systems) only present in select gammaproteobacterial and betaproteobacterial species. Bioinformatic analysis revealed that the histidine kinase, AmsS, contains 14 predicted N-terminal transmembrane domains and harbors a hybrid histidine kinase arrangement in its C-terminus. Transcriptional analysis revealed the proton ionophore CCCP selectively induces PamsSR expression. Disruption of amsSR resulted in decreased intracellular pH and increased depolarization of cytoplasmic membranes. Transcriptome profiling revealed a major reordering of metabolic circuits upon amsR disruption, with energy generation pathways typically used by bacteria growing in limited oxygen being favored. Interestingly, we observed enhanced growth rates for mutant strains in the presence of glucose, which led to overproduction of pyruvate. To mitigate the toxic effects of carbon overflow, we noted acetate overproduction in amsSR-null strains, resulting from a hyperactive Pta-AckA pathway. Additionally, due to altered expression of key metabolic genes, amsSR mutants favor an incomplete TCA cycle, relying heavily on an overactive glyoxylate shunt. This metabolic reordering overproduces NADH, which is not oxidized by the ETC; components of which were significantly downregulated upon amsSR disruption. As a result, the mutants almost exclusively rely on substrate phosphorylation for ATP production, and consequently display reduced oxygen consumption in the presence of glucose. Collectively, our data suggests that disruption of amsSR affects the function of the aerobic respiratory chain, impacting the energy status of the cell, which in turn upregulates alternative metabolic and energy generation pathways. [ABSTRACT FROM AUTHOR]

Summary: Clostridioides difficile is a Gram‐positive, spore‐forming, toxin‐producing anaerobe that can cause nosocomial antibiotic‐associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR‐AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics. [ABSTRACT FROM AUTHOR]

The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." Staphylococcus aureus growth is inhibited by calprotectin in vitro , and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP , which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals., Importance: Staphylococcus aureus is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during S. aureus infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which S. aureus combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of S. aureus to survive in a metal-limited environment., Competing Interests: The authors declare no conflict of interest.

SSR42 is the longest noncoding RNA in the S. aureus cell and the second-most abundant transcript in the stationary phase transcriptome, second only to RNAIII. It is highly conserved across strains and exhibits pronounced stability in stationary phase, however the mechanism behind its regulatory role has yet to be fully elucidated. Herein, we used transcriptomic and proteomic approaches to probe the role of SSR42, revealing that it is a powerful, novel activator of the primary leukocidin LukAB. SSR42 is required for cytotoxicity towards, and escape from within, human neutrophils, and also mediates survival within human blood. We show that SSR42 wields this role via derepression by the peroxide repressor PerR in response to the presence of human neutrophils and governs lukAB induction in this niche. Importantly, this regulation is driven by direct RNA-RNA interaction, as we show binding of the 5' UTR of the lukAB transcript with the 3' end of SSR42, which ultimately modulates transcript stability as well as translational activity. Finally, we demonstrate that this behavior is absolutely required for full virulence of S. aureus in murine models of both pneumonia and sepsis. Collectively, we present SSR42 as a pleiotropic regulatory RNA that acts as a nexus between environmental sensing and the regulation of pathogenesis, responding to environmental stimuli and host immune factors to bolster cytotoxic behavior and facilitate infection in S. aureus ., Importance: S. aureus is a master pathogen due to its formidable collection of virulence factors. These are tightly controlled by a diverse group of regulators that titrate their abundance to adapt to unique infectious niches. The role of regulatory RNAs in stress adaptation and pathogenesis is becoming increasingly more relevant in S. aureus . In this study, we provide the most comprehensive global analysis to date of just such a factor, SSR42. Specifically, we uncover that SSR42 is required for mediating cytotoxicity - one of the pillars of infection - in response to phagocytosis by human neutrophils. We find that SSR42 is induced by components of the host immune system and facilitates downstream activation of cytotoxic factors via RNA-RNA interactions. This illustrates that SSR42 forms a pivotal link between sensing the external environment and mediating resistance to oxidative stress while promoting virulence, solidifying it as a major global regulator in S. aureus .

Laboratory-viable cultivars of previously uncultured bacteria further taxonomic understanding. Despite many years of modern microbiological investigations, the vast majority of bacterial taxonomy remains uncharacterized. While many attempts have been made to decrease this knowledge gap, culture-based approaches parse away at the unknown and are critical for improvement of both culturing techniques and computational prediction efficacy. To this end of providing culture-based approaches, we present a multi-faceted approach to recovering marine environmental bacteria. We employ combinations of nutritional availability, inoculation techniques, and incubation parameters in our recovery of marine sediment-associated bacteria from the Gulf of Mexico and Antarctica. The recovered biodiversity spans several taxa, with 16S-ITS-23S rRNA gene-based identification of multiple isolates belonging to rarer genera increasingly undergoing phylogenetic rearrangements. Our modifications to traditional culturing techniques have not only recovered rarer taxa, but also resulted in the recovery of biotechnologically promising bacteria. Together, we propose our stepwise combinations of recovery parameters as a viable approach to decreasing the bacterial knowledge gap.

With the post-antibiotic era rapidly approaching, naturally-sourced antimicrobial peptides (AMPs) are a prime resource for restocking our antibiotic medicine cupboard. The efficiency of identification requires high-throughput screens that can identify bioactive peptides present within abundant natural-products chemical-space. While there are multiple amenable and high sensitivity colorimetric-based screening approaches available, resazurin-based assays are cost-effective, peptide compatible, and expedient, allowing one to screen a multitude of AMPs in a high-throughput fashion. Herein, we provide a detailed protocol for the optimization and use of resazurin assays for AMP testing, providing key experimental insight, and highlight pitfalls to be avoided., (Copyright © 2022 Elsevier Inc. All rights reserved.)