Deeply branching Bacillota species exhibit atypical Gram-negative staining.

The Gram staining method differentiates bacteria based on their cell envelope structure, with the monoderm and diderm cell envelope types traditionally being synonymous with Gram-positive and Gram-negative stain results, respectively. Monoderms have a single phospholipid membrane surrounded by a thick layer of peptidoglycan, while diderms have a lipopolysaccharide outer membrane exterior to a thin peptidoglycan layer. The Bacillota (formerly Firmicutes) phylum has members with both cell wall types, and recent phylogenetic analyses have shown that monoderm Bacillota evolved from diderm ancestors on multiple occasions. Here, we compiled Gram staining and ultrastructural data for Bacillota species with complete genomes to further investigate the evolution of Gram-positive and Gram-negative cell wall types. The results indicate that many deeply branching lineages at the root of Bacillota phylum stain Gram-negative but do not harbor genes for outer membrane protein or lipopolysaccharide biosynthesis. Phylogenetic reconstructions suggest that several deeply branching Bacillota species have retained a thin peptidoglycan layer in their cell walls, which was inherited from a diderm ancestor. Taxa with this atypical Gram-negative-staining cell wall structure include the thermophilic anaerobe Symbiobacterium thermophilum and members of the Desulfotomaculia, Syntrophamonadia, Desulfitobacteriia, Thermosediminibacteria, and Thermoanaerobacteria. Using Gram-staining results as a proxy for cell wall thickness, our analysis indicates that several independent peptidoglycan thickening events may have occurred in the evolution of the Gram-positive cell envelope. IMPORTANCE: In this study, we examined the evolution of bacterial cell envelopes, specifically focusing on Gram-positive and Gram-negative cell wall types in the Bacillota phylum. Our results indicate that certain bacteria can stain Gram-negative despite having a monoderm cell wall structure, thus challenging the conventional interpretation of Gram-staining results. Our observations also question the assumption that Gram-negative staining is always indicative of a diderm structure. These findings have broader implications for understanding how and when cell walls thicken during the evolution of bacterial cell envelopes.

Novel Insights from Clinical Practice Autologous Blood Patch Pleurodesis and Endobronchial Valves for Management of Persistent Air Leaks in Two Cases of Tuberculosis.

INTRODUCTION: Pulmonary infections, such as tuberculosis, can result in numerous pleural complications including empyemas, pneumothoraces with broncho-pleural fistulas, and persistent air leak (PAL). While definitive surgical interventions are often initially considered, management of these complications can be particularly challenging if a patient has an active infection and is not a surgical candidate. CASE PRESENTATION: Autologous blood patch pleurodesis and endobronchial valve placement have both been described in remedying PALs effectively and safely. PALs due to broncho-pleural fistulas in active pulmonary disease are rare, and we present two such cases that were managed with autologous blood patch pleurodesis and endobronchial valves. CONCLUSION: The two cases presented illustrate the complexities of PAL management and discuss the treatment options that can be applied to individual patients.

Extracellular organic disulfide reduction by Shewanella oneidensis MR-1.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

Metal-binding amino acid ligands commonly found in metalloproteins differentially fractionate copper isotopes.

Copper (Cu) is a cofactor in numerous key proteins and, thus, an essential element for life. In biological systems, Cu isotope abundances shift with metabolic and homeostatic state. However, the mechanisms underpinning these isotopic shifts remain poorly understood, hampering use of Cu isotopes as biomarkers. Computational predictions suggest that isotope fractionation occurs when proteins bind Cu, with the magnitude of this effect dependent on the identity and arrangement of the coordinating amino acids. This study sought to constrain equilibrium isotope fractionation values for Cu bound by common amino acids at protein metal-binding sites. Free and bound metal ions were separated via Donnan dialysis using a cation-permeable membrane. Isotope ratios of pre- and post-dialysis solutions were measured by MC-ICP-MS following purification. Sulfur ligands (cysteine) preferentially bound the light isotope (63Cu) relative to water (Δ65Cucomplex-free = - 0.48 ± 0.18‰) while oxygen ligands favored the heavy isotope (65Cu; + 0.26 ± 0.04‰ for glutamate and + 0.16 ± 0.10‰ for aspartate). Binding by nitrogen ligands (histidine) imparted no isotope effect (- 0.01 ± 0.04‰). This experimental work unequivocally demonstrates that amino acids differentially fractionate Cu isotopes and supports the hypothesis that metalloprotein biosynthesis affects the distribution of transition metal isotopes in biological systems.