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Adaptive laboratory evolution of Micrococcus luteus and identification of genes associated with radioresistance through genome-wide association study.

Micrococcus luteus (V017) is a Gram-positive bacterium that was isolated from a sterilization area exposed to 60Co radiation. In this study, we performed an adaptive laboratory evolution experiment with M. luteus, exposing it to 24 continuous cycles of gamma irradiation at four different doses (1.5 kGy, 3.5 kGy, 5.5 kGy, and 7.5 kGy). This led to the creation of four evolved populations with different levels of radioresistance, which were positively correlated with the radiation dose applied. The survival rate of the evolved population that underwent adaptive treatment at the highest dose (7.5 kGy) was 0.69% after exposure to 5.5 kGy, which is about five orders of magnitude higher than that of the original strain V017. Furthermore, 76 evolved strains were selected from these populations, and their genomes were re-sequenced, uncovering a total of 3072 mutations. A genome-wide association study identified 56 single nucleotide polymorphisms (SNPs) significantly associated with radioresistance, linked to 62 candidate genes. Ultimately, 9 genes were selected for functional validation. Inactivating 6 of these genes, including H0H31_RS03855 (SMC family ATPase, SbcC), H0H31_RS04250 (ribonuclease HII), H0H31_RS04570 (endonuclease VIII), H0H31_RS07595 (bifunctional 3'-5' exonuclease/DNA polymerase I), H0H31_RS00170 (serine/threonine phosphatase PPP), and H0H31_RS05860 (CBS-domain-containing protein), significantly increased sensitivity to gamma radiation, underscoring their importance in radioresistance.

Micrococcus luteus

Characterization of Micrococcus luteus Lipidome Containing Novel Lipid Families by Multiple Stage Linear Ion-Trap with High Resolution Mass Spectrometry.

Micrococcus luteus (M. luteus) is a ubiquitous, long-existing Gram-positive bacterium with a distinctive yellow or golden color. It is a model organism for laboratory studies due to its small genome and ease of cultivation. However, only limited knowledge about its constituent lipid structure is known, and its entire lipid profile remains unclear. Here, we applied linear ion trap (LIT) multiple-stage mass spectrometry (MSn) with high resolution for structural characterization of the native lipid extract, along with GC/MS analysis of the acid hydrolysate to reveal the structural details of the entire lipidome, which includes the major phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylinositol (PI), dimannosyl diacylglycerol (DMDG), and minor diacylglycerol (DAG) lipid families. Importantly, we also found two extra lipid families, the new phosphatidyl 1,3-propanediol and the known polyprenyl 1-phosphosate that was not previously reported for M. luteus. We also revealed the unique lipidome simplified by the dominance of branched 15:0-fatty acid substituents (>90% branched 15:0-FA with anteiso-15:0 to iso-15:0 abundance ratio of 4/1), which is in line with the small genome of M. luteus. In addition, the applied LIT MSn mass spectrometry revealed a fragmentation pathway that undergoes internal loss of a glycerol residue specific to DMDG, leading to its structural characterization.

Micrococcus luteus

Integrated computational and experimental benchmarking of Bacillus phage endolysins reveals the relationship between peptidoglycan-fragment recognition descriptors and antibacterial performance.

Protein-based antibacterials such as bacteriophage endolysins offer a targeted therapeutic strategy against Gram-positive pathogens. However, prioritizing the most effective candidates from the large sequence diversity available remains a significant challenge. Here we present a standardized computational-experimental benchmarking framework that evaluates seven phage-derived endolysin variants (E1, E2, E3, E7, E10, E12, and E15) identified from Bacillus genomes. We combined molecular docking and residue-level interaction mapping against muramyl dipeptide (MDP), a minimal conserved peptidoglycan motif, with 1000-ns molecular dynamics simulations, MM/PBSA binding free-energy estimation, and matched functional inhibition assays against Staphylococcus aureus and Micrococcus luteus. Computational analyses revealed generally favorable MDP recognition across variants, albeit with notable differences in contact patterns and complex stability profiles. Experimental screening identified E2 as the most potent antibacterial agent against both species, while E7 and E1 performed strongly in selected computational metrics. Integrated analysis showed only modest correlations between computational descriptors of fragment recognition/stability and observed antibacterial performance. This study establishes a practical comparative benchmarking platform for endolysin candidate prioritization, nominates E2 and E7 as promising candidates for further development, and highlights E1 as a potential structural scaffold for rational engineering, while explicitly demonstrating both the utility and the current limitations of using minimal peptidoglycan fragments as proxies for full cell-wall recognition in lysin benchmarking.

Endopeptidases

The Streptococcus pyogenes mannose phosphotransferase system (Man-PTS) influences antimicrobial activity and niche-specific nasopharyngeal infection.

Streptococcus pyogenes is a human-adapted pathogen that can cause multiple diseases, including pharyngitis and skin infections. Although this bacterium produces many virulence factors, how S. pyogenes competes with the host microbiota is not well understood. Here, we detected antimicrobial activity from S. pyogenes MGAS8232 that prevented the growth of Micrococcus luteus. This activity was produced when cells were grown in 5% CO2 in M17 media supplemented with galactose; however, the addition of alternative sugars coupled with genome sequencing experiments revealed that the antimicrobial phenotype was not related to classical bacteriocins. To further determine genes involved in the production of this activity, a transposon mutant library in S. pyogenes MGAS8232 identified the mannose phosphotransferase system (Man-PTS), a major sugar transporter, as important for the antimicrobial phenotype. Loss-of-function transposon mutants linked to the antimicrobial activity were identified to also be involved in alternative sugar utilization, and additionally, the Man-PTS was further identified from an inadvertent secondary mutation in a bacteriocin operon mutant. Sugar utilization in the Man-PTS mutants demonstrated that galactose, mannose, and N-acetylglucosamine utilization was impaired. RNA-seq experiments in high and low glucose concentrations further characterized the Man-PTS as a glucose transporter; however, transcriptional regulators or virulence factors were not affected with the loss of the Man-PTS. Deletion of Man-PTS demonstrated defects in a mouse model of nasopharyngeal infection but not skin infection. This work suggests that the ability of S. pyogenes to utilize alternative sugars presented by glycans may play a role in acute infection and interactions with the endogenous microbial population existing in the nasopharynx.IMPORTANCEStreptococcus pyogenes is responsible for over 500,000 deaths per year primarily due to invasive infections and post-infection sequelae, although the most common manifestations include pharyngitis and impetigo. S. pyogenes can adapt to its environment through alternative sugar metabolism. Here, we identified an antimicrobial phenotype that was not bacteriocin-related but a by-product of alternative sugar metabolism. The mannose phosphotransferase system was involved in the production of the antimicrobial and was also important for S. pyogenes to utilize alternative sugars and establish nasopharyngeal infection but not skin infection. Overall, this study identified potential strategies used by S. pyogenes for interactions with the endogenous microbiota and further elucidated the importance of sugar metabolism in acute upper respiratory tract infection.

Streptococcus pyogenes

Integrated Optimization, Genomic Characterization, and Functional Evaluation of Biogenic Selenium Nanoparticles from Bacillus licheniformis BLN313: Antibacterial and Anticancer Potential.

Microbial synthesis of selenium nanoparticles (SeNPs) offers a sustainable alternative to chemical routes, but the genetic basis of selenium handling in Bacillus remains poorly defined, which limits rational strain selection. Here, SeNP production, physicochemical characterization, and closed-genome sequencing are combined for Bacillus licheniformis BLN313. Selenite reduction peaked at 500 µg/mL Na2SeO3 (88.8% conversion; 444 ± 27 µg/mL Se0); at higher concentrations, conversion efficiency and viability diverged, indicating that tolerance and reductive capacity are distinct traits. Purified SeNPs were spherical and partially crystalline trigonal Se0 (TEM 190 ± 52 nm; DLS 166 nm, PDI 0.03; zeta potential -20.8 mV), carrying a proteinaceous capping layer confirmed by XPS, EDS, and FTIR and shown by LC-MS to be enriched in cell wall-derived metabolites. The particles were bactericidal against Micrococcus luteus (MIC 62.5 µg/mL) and Klebsiella pneumoniae (MIC 250 µg/mL) and reduced MCF-7 viability (IC50 2.7 µg/mL) while sparing MCF-10A cells. The 4.11 Mb genome (46.3% GC; ANI 99.7%, dDDH 97.8%) encodes SulP and Pit transporters, multiple trxB copies, and sulfur-metabolism and oxidative-stress genes, defining a candidate gene set for selenium uptake, reduction and detoxification. BLN313 thus provides a genetically defined platform for SeNP production in biomedical and environmental applications.

Selenium