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PubMed2025

The SinR·SlrR Heteromer Attenuates Transcription of a Long Operon of Flagellar Genes in Bacillus subtilis.

During growth, Bacillus subtilis differentiates into subpopulations of motile individuals and non-motile chains, associated with dispersal and biofilm formation, respectively. The two cell types are dictated by the activity of the alternative sigma factor SigD encoded as the penultimate gene of the 27-kb long fla/che flagellar operon. The frequency of SigD-ON motile cells is increased by the heteromeric transcription factor SwrA·DegU that activates the fla/che promoter. Conversely, the frequency of motile cells is decreased by the heteromeric transcription factor SinR·SlrR, but the mechanism and location of inhibition is poorly understood. Here, using ChIP-Seq analysis, we determine the binding sites of the SinR·SlrR heteromer on the genome. We identified two sites within the fla/che operon that were necessary and sufficient to attenuate transcript abundance by causing premature termination upstream of the gene that encodes SigD. Thus, cell motility and the transition to biofilm formation depend on the expression of a long operon governed by two opposing heteromeric transcription factors that operate at two different stages of the transcription cycle. More broadly, our study serves as a model for transcription factors that control transcriptional elongation and the regulation of long operons in bacteria.

Bacillus subtilis↗
PubMed2025

Autoregulation of the Master Regulator Spo0A Controls Cell-Fate Decisions in Bacillus subtilis.

Spo0A in Bacillus subtilis is activated by phosphorylation (Spo0A~P) upon starvation and differentially controls a set of genes involved in biofilm formation and sporulation. The spo0A gene is transcribed by two distinct promoters, a σA-recognized upstream promoter Pv during growth, and a σH-recognized downstream promoter Ps during starvation, and appears to be autoregulated by four Spo0A~P binding sites (0A1-4 boxes) localized between two promoters. However, the autoregulatory mechanisms and their impact on differentiation remain elusive. Here, we determined the relative affinity of Spo0A~P for each 0A box and dissected each promoter in combination with the systematic 0A box mutations. The data revealed that (1) the Pv and Ps promoters are on and off, respectively, under nutrient-rich conditions without Spo0A~P, (2) the Ps promoter is activated by first 0A3 and then 0A1 during early starvation with low Spo0A~P, (3) during later starvation with high Spo0A~P, the Pv promoter is repressed by first 0A1 and then 0A2 and 0A4, and (4) during prolonged starvation, both promoters are silenced by all 0A boxes with very high Spo0A~P. Our results indicate that the autoregulation of spo0A is one of the key determinants to achieve a developmental increase in Spo0A~P, leading to a temporal window for entry into biofilm formation or sporulation.

Bacillus subtilis↗
PubMed2025

A novel regulation on the developmental checkpoint protein Sda that controls sporulation and biofilm formation in Bacillus subtilis.

UNLABELLED: Biofilm formation by Bacillus subtilis is triggered by an unusually simple environmental sensing mechanism. Certain serine codons, the four TCN codons (N for A, T, C, or G), in the gene for the biofilm repressor SinR caused lowered SinR translation and subsequent biofilm induction during transition from exponential to stationary growth. Global ribosome profiling showed that ribosomes pause when translating the four UCN (U for T on the mRNA) serine codons on mRNA, but not the two AGC/AGU serine codons. We proposed a serine codon hierarchy (AGC/AGT vs TCN) in that genes enriched in the TCN serine codons may experience reduced translation efficiency when serine is limited. In this study, we designed an algorithm to score all protein-coding genes in B. subtilis NCIB3610 based on the serine codon hierarchy. We generated a short list of 50 genes that could be subject to regulation by this novel mechanism. We further investigated one such gene from the list, sda, which encodes a developmental checkpoint protein regulating both sporulation and biofilm formation. We showed that synonymously switching the TCN serine codons to AGC in sda led to delayed biofilm formation and sporulation. This engineered strain also outgrew strains with other synonymously substituted sda alleles (TCN) in competition assays for biofilm formation and sporulation. Finally, we showed that the AGC serine codon substitutions in sda elevated the Sda protein levels. This serine codon hierarchy-based novel signaling mechanism could be exploited by bacteria in adapting to stationary phase and regulating important biological processes. IMPORTANCE: Genome-wide ribosome profiling in Bacillus subtilis shows that under serine limitation, ribosomes pause on the four TCN (N for A, C, G, and T), but not AGC/AGT serine codons, during translation at a global scale. This serine codon hierarchy (AGC/T vs TCN) differentially influences the translation efficiency of genes enriched in certain serine codons. In this study, we designed an algorithm to score all 4,000+ genes in the B. subtilis genome and generated a list of 50 genes that could be subject to this novel serine codon hierarchy-mediated regulation. We further investigated one such gene, sda, encoding a developmental checkpoint protein. We show that sda and cell developments controlled by Sda are also regulated by this novel mechanism.

Bacillus subtilis↗
PubMed2025

Identification of sporulation genes in Bacillus anthracis highlights similarities and significant differences with Bacillus subtilis.

The molecular basis of endospore formation in the model gram-positive bacterium Bacillus subtilis has been investigated for over half a century. Here, using high throughput and classical genetic approaches, we performed a comparative analysis of sporulation in the human pathogen Bacillus anthracis. A transposon-sequencing screen identified >150 genes required for B. anthracis sporulation. As anticipated, many of the genes that are critical for sporulation in B. subtilis were also required for B. anthracis sporulation. However, we identified >50 genes that are important for sporulation in B. anthracis but not in B. subtilis, and 22 B. anthracis sporulation genes that are absent from the B. subtilis genome. To validate the hits from our screen, we generated an ordered transposon-mutant library using Knockout Sudoku. Cytological analysis of a subset of the canonical sporulation-defective mutants revealed similar but not identical phenotypes in the pathogen compared to the model. We investigated several of the newly identified sporulation genes, with an in-depth analysis of one, ORF 04167, renamed ipdA. Sporulating cells lacking ipdA are blocked in the morphological process of engulfment, generating septal bulges. An AlphaFold-Multimer screen and a classical genetic enrichment revealed that IpdA is a secreted inhibitor of the polysaccharide deacetylase PdaN. Our data support a model in which induction of IpdA at the onset of sporulation inhibits deacetylation of the cell wall peptidoglycan (PG), enabling the sporulation-specific PG hydrolases to catalyze engulfment. Altogether, our studies reveal that B. subtilis is an excellent model for endospore formation in B. anthracis, while underscoring the importance of direct analysis in B. anthracis. The suite of tools that we have generated will catalyze the molecular dissection of sporulation and other cell biological processes in this important human pathogen.

Bacillus anthracis↗
PubMed2026

Insights into the mechanism of enhanced tetramethylpyrazine production in dehulled adlay fermented by Bacillus subtilis BJ3-2.

Tetramethylpyrazine (TTMP) is a vital bioactive alkaloid and characteristic flavor compound in fermented foods. Our previous study found that fermentation of adlay by Bacillus subtilis BJ3-2 efficiently accumulates TTMP, whereas the underlying high-yield mechanism remains unclear. This study investigated the fermentation characteristics, gene transcription and protein expression of B. subtilis BJ3-2 in dehulled adlay (BDA) and soybean (BSB), respectively, and elucidated the mechanism responsible for high-yield TTMP production. The results showed that glutamate, leucine and phenylalanine were major free amino acids in BDA. The TTMP yield in BDA at 48 h (6.11 mg/g dry weight) was 360-fold higher than that in BSB. Transcriptomic and proteomic analysis demonstrated that compared with the soybean substrate, dehulled adlay substrate significantly up-regulated the expression of alsSD and ilvBH genes and their encoding proteins in B. subtilis BJ3-2, which were involved in C5-branched dibasic acid metabolism, 2-oxocarboxylic acid metabolism, and valine, leucine and isoleucine biosynthesis. Meanwhile, acetoin degradation was inhibited by down-regulating acetoin dehydrogenase complex (acoABCL) in citrate cycle, glycolysis/gluconeogenesis and carbon metabolism. Additionally, nitrogen metabolism pathway was transcriptionally enhanced to guarantee sufficient ammonium supply. Notably, protein-protein interaction and molecular docking analyses revealed that acetohydroxyacid synthase (ilvBH) interacted tightly with α-acetolactate decarboxylase (alsD), potentially forming a metabolic channel for acetoin synthesis. In conclusion, the efficient synthesis of TTMP in BDA was primarily attributed to the high synthesis and low degradation of acetoin, and the moderate synthesis of ammonium/ammonia. This study provided a theoretical basis for the targeted and efficient biosynthesis of TTMP.

Bacillus subtilis↗
PubMed2026

Genome-informed qPCR tracking revealed preferential persistence of Bacillus subtilis BS9 in the broiler chicken gastrointestinal tract.

This study aimed to develop a strain-specific quantitative PCR (qPCR) assay for Bacillus subtilis BS9 and characterize its persistence and spatial distribution in the broiler chicken gastrointestinal tract. Whole-genome sequencing and comparative genomic analysis identified a unique 110-bp sequence within a strain-specific genomic island, which was used to design a highly specific qPCR assay with excellent efficiency and sensitivity. In a 14-day in vivo trial, broiler chicks receiving daily oral doses of BS9 were analyzed using both culture-based methods and the newly developed qPCR. The assay was applied qualitatively, presence or absence, to detect BS9 in intestinal samples. BS9 was detected exclusively in the duodenum, jejunum, and cecum, with no presence in the gizzard or ileum. These findings demonstrate that BS9 exhibits region-specific persistence in the gut, likely reflecting adaptation to distinct physiological niches, which may contribute to its probiotic mechanisms.IMPORTANCEThis work provides the first detailed account of B. subtilis BS9's spatial persistence in poultry, revealing preferential adherence to specific intestinal regions. The strain-specific qPCR assay developed here offers a precise, culture-independent tool for tracking BS9 in complex gut environments. These insights into the genetic basis and tissue tropism of BS9 persistence advance our understanding of probiotic-host interactions and establish a framework for characterizing novel probiotic strains.

Bacillus subtilis↗
PubMed2026

Complete genome sequence of Bacillus subtilis strain S-LA1, a potential plant probiotic endophyte from the medicinal plant Leucas aspera.

Bacillus subtilis strain S-LA1 is an endophytic bacterium isolated from Leucas aspera roots that harbors a 4.2 Mbp genome predicted to encode several traits for nutrient acquisition, plant growth promotion, and plant probiotic efficacy. Genomic characterization underscores its potential as a microbial resource supporting sustainable agriculture and crop disease management strategies.

Bacillus↗
PubMed2026

Bacillus subtilis RNase HII Is Inefficient at Processing Guanosine Monophosphate and Damaged Ribonucleotides.

During one round of DNA replication, nearly 2000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphates (dNMPs). Given their high rate of insertion, genomic DNA could contain rNMPs that are damaged or mismatched. Here, we test the activity of Bacillus subtilis and Escherichia coli RNase HII on canonical, mismatched, and damaged rNMPs. We show that E. coli RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, B. subtilis RNase HII efficiently incises rAMP, rCMP, and rUMP but is inefficient at processing rGMP in both a canonical and mismatched base pair. We test damaged ribonucleotides and find that B. subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that B. subtilis RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.

Bacillus subtilis↗
PubMed2025

Bacillus subtilis RNase HII is inefficient at processing guanosine monophosphate and damaged ribonucleotides.

During one round of DNA replication, nearly 2,000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphate (dNMP). Given their high rate of insertion, genomic DNA would contain rNMPs that are damaged or mismatched. Here, we tested the activity of Bacillus subtilis and Escherichia coli RNase HII on all four canonical, mismatched, and damaged rNMPs. We show that E. coli RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, B. subtilis RNase HII efficiently incised rAMP, rCMP, and rUMP, but was inefficient at processing rGMP in both a canonical and mismatched base pair. We tested damaged ribonucleotides and found that B. subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that B. subtilis RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.

Bacillus subtilis↗
PubMed2026

Engineering Bacillus Subtilis for Efficient Biosynthesis of Riboflavin: Current Knowledge and Future Perspectives.

Riboflavin is an essential water-soluble vitamin that serves as a precursor for the biosynthesis of the flavin cofactors FMN and FAD, which play pivotal roles in numerous redox and energy metabolism reactions. With the growing global demand for sustainable vitamin production, microbial fermentation has become an attractive alternative to chemical synthesis due to its environmental and economic advantages. Among microbial hosts, Bacillus subtilis has emerged as a leading cell factory for riboflavin production owing to its GRAS status, well-characterized genetics, and efficient protein secretion system. This review provides a comprehensive overview of recent advances in metabolic engineering strategies to enhance riboflavin biosynthesis in B. subtilis. Key topics include strengthening biosynthetic and precursor pathways, relieving feedback inhibition, balancing metabolic flux and cell growth, employing adaptive laboratory evolution, and utilizing omics-guided optimization and 13C metabolic flux analysis. Moreover, the integration of synthetic biology tools such as riboswitch engineering, regulatory element design, and high-throughput screening has significantly accelerated strain improvement. Despite remarkable progress, challenges remain in achieving precise regulatory control, optimizing multi-gene expression, and enhancing genome integration efficiency. Future research combining multi-omics data, synthetic regulatory design, and machine learning-driven predictive modeling is expected to further advance the development of intelligent B. subtilis cell factories. However, the practical implementation of these systems remains constrained by the metabolic burden of overproduction and the lack of universal regulatory models that can predict strain performance across varying industrial scales.

Bacillus subtilis↗
PubMed2026

Bacillus subtilis isolated from medicinal plants rhizosphere effectively controls Cercospora leaf spot and improves plant growth in mung bean (Vigna radiata).

BACKGROUND: Mung bean is an important leguminous crop, which is reported to face devastating yield losses of up to 70% due to Cercospora leaf spot (CLS) disease. Traditional methods, such as the application of agrochemicals and fungicides, have been used to control CLS, but their intensive use has toxic effects on edible crops. METHODS: To find out a sustainable alternative, this study characterizes a strain, Bacillus subtilis Medicinal_04, isolated from Cannabis sativa rhizosphere and explores its role as an eco-friendly biofungicide and biostimulant. The species level identification of the isolate was confirmed by Average Nucleotide Identity (ANIb) and a digital DNA-DNA hybridization (dDDH). The antagonistic efficacy of B. subtilis Medicinal_04 against Cercospora canescens was evaluated in vitro as well as in planta assays. RESULTS: ANIb of 97.80% and a dDDH score of 85.90% against the reference B. subtilis str. 168. confirmed this isolate as B. subtilis. The in-vitro results showed that B. subtilis robustly inhibited C. canescens growth by 81.5%, strongly correlated with positive chitinolytic activity and a diverse genomic array of secondary metabolite biosynthetic gene clusters. The in planta results demonstrated that B. subtilis seed priming reduced disease incidence by 80 and 71.4%, while foliar application resulted in reductions of 90 and 85.7% for NM-51 and NM-20-21 varieties, respectively. Furthermore, fungicide application successfully reduced disease, however it caused noticeable phytotoxic reductions in root-shoot architecture and chlorophyll content. In contrast, biological interventions completely bypassed these trade-offs as B. subtilis application displayed improved root-shoot length, pod number, and chlorophyll content, while simultaneously enhancing antioxidative enzyme activities (SOD, POD, and CAT) and PR-1 gene expression. CONCLUSION: These findings demonstrate that B. subtilis Medicinal_04 has the potential to serve as a multifunctional biocontrol agent and is capable of securing high-level disease suppression and optimizing plant productivity, offering a valuable toolkit for climate-smart, sustainable agriculture.

Bacillus subtilis↗
PubMed2026

Structural characterization and predicted biosynthetic pathway of the polysaccharide component of bioflocculant from starch-degrading Bacillus subtilis ZHX3.

Polysaccharides-based bioflocculant is a promising eco-friendly alternative to conventional flocculants, yet their application is limited by high production cost. Understanding the biosynthetic pathway is essential for targeted strain improvement. In this study, we characterized polysaccharides structure of bioflocculant MBF-ZHX3 from Bacillus subtilis ZHX3 and predicted its biosynthetic pathway via genomic analysis combined with quantitative real-time PCR (qPCR). Two purified polysaccharide fractions, PS1-1 (5982 Da) and PS2-1 (17,577 Da), were obtained. Both were mainly composed of glucose, with a backbone of →4)-α-D-Glcp-(1 → and α-D-Glcp-(1 → branches attached at O-6. Whole-genome sequencing revealed a circular chromosome of 4,122,369 bp and two plasmids. Functional annotation showed high carbohydrate metabolism activity, with 284 genes (9.52%) and 264 genes (11.28%) assigned to carbohydrate metabolism in the COG and KEGG database, respectively. A complete eps gene cluster consisting of 15 open reading frames was identified. qPCR showed that key genes involved in substrate uptake (ptsG, malP, mdxEFG-msmX) and nucleotide sugar synthesis (pgcA, gtaB) were significantly upregulated. The priming glycosyltransferase (GT) epsL and the primary GT epsF were upregulated, along with the flippase epsK, polymerase epsG, and chain-length regulators epsA and epsB. Based on these findings, we propose a putative biosynthetic pathway for the polysaccharide component of MBF-ZHX3, and identify epsL, epsF, and epsG as prioritized targets for future genetic engineering. This work provides an integrated structural-genomic-transcriptomic framework that can guide rational strain improvement to enhance bioflocculant production.

Polysaccharides structure↗
PubMed2024

Engineering a probiotic Bacillus subtilis for acetaldehyde removal: A hag locus integration to robustly express acetaldehyde dehydrogenase.

We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the hag locus, a σD-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter, following a deletion of the gene encoding a post-translational regulator of σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics.

Acetaldehyde↗
PubMed2026

Genome mining and metabolomics unveil new napyradiomycin antibiotics from Streptomyces sp. 0H2M.

Napyradiomycins are a family of meroterpenoid natural products known for their promising antibiotic activities. In this study, four new napyradiomycins derivatives were identified, SF2415B4 (1), SF2415B5 (2), SF2415B6 (3), and SF2415B7 (4) from Streptomyces sp. 0H2M, alongside a known molecule, A80915A (5) through the synergy between genome mining and metabolomics analysis. Their structures were elucidated through a combination of spectroscopic and spectrometric analyses, including HRMS-ESI, NMR, and DP4+. Genome sequencing identified a putative biosynthetic gene cluster, and subsequent analyses revealed a distinct biosynthetic pathway with an unprecedented tailoring mechanism mediated by novel hydroxylases and halogenases. Biological assays demonstrated significant activity against Bacillus subtilis, Bacillus cereus and methicillin-resistant Staphylococcus aureus due to perturbation of cell membrane integrity, and minimum inhibitory concentration (MIC) values ranged from 0.24 to 30.7 μM. Additionally, in vitro cytotoxicity experiments indicated that compounds 2-5 very mildly inhibited the viability of human non-small cell lung cancer (NSCLC) cell line A549 in a concentration-dependent manner, with IC50 values of 16.7, 39.1, 65.0, and 32.8 μM, respectively. Moreover, they were shown to induce apoptosis and autophagy in A549 cells, evidenced by increased levels of cleaved PARP, decreased expression of anti-apoptotic proteins (Bcl-2, Bcl-xL, and Survivin), and accumulation of LC3-II. These findings offer new insights into the natural product chemistry in Streptomyces and the pharmacology of napyradiomycin class antibiotics.

Streptomyces↗
PubMed2026

Draft genome sequences of four bacterial isolates from the Indian Fort Nature Preserve in Geneseo, NY.

We report the genome sequences of four bacterial strains, Bacillus subtilis, Bacillus pumilus, Pseudomonas kermanshahensis, and Kocuria rhizophila, isolated from soil or plant material from Geneseo, NY. Bacterial strains were selected based on preliminary, qualitative screening for antimicrobial production via zones of clearing and/or inhibition against lab strains.

antimicrobial production↗
PubMed2026

Revealing Functional Traits of Insect Pest Suppressive Rhizobacterial Strains Through Comparative Genomics.

Root inoculation with rhizobacteria is an emerging strategy to enhance plant resistance to aphid herbivory, yet the microbial functional traits underpinning these responses remain poorly characterised. Here, we present a comparative genomic analysis of five rhizobacteria (Acidovorax radicis N35, Bacillus subtilis B171, Bacillus velezensis FZB42, Rhizobium radiobacter F4 and Pseudomonas simiae WCS417r) that suppress aphids when inoculated onto barley. As expected, functional variation largely reflected phylogenetic relatedness; however, candidate traits implicated in modulation of plant immune defences were conserved across all strains, including biosynthesis of 2,3-butanediol, riboflavin and salicylic acid. Additional shared functions, linked to plant defence signalling, included phytoene and squalene biosynthesis (absent in P. simiae) and N-acyl homoserine lactone quorum sensing (absent in Bacillus spp.). Strain-specific traits were also identified, including surfactin production in Bacillus spp. and hydrogen cyanide biosynthesis in A. radicis and P. simiae. Comparison with a broader collection of rhizobacteria revealed that many putative plant-beneficial functions identified were widely conserved, including among closely related phytopathogens. This extensive functional overlap suggests aphid suppression cannot be explained solely by presence or absence of broad functional traits, but rather by specific trait combinations, regulatory differences, or context-dependent expression. This highlights the need for genome-informed approaches for bioinoculant discovery.

Animals↗
PubMed2025

Exploring biosynthetic potential of the endophytic Penicillium turbatum BLH34 using whole-genome sequence analysis and molecular networking.

An in-depth genomic and metabolomic investigation was conducted on the endophytic fungus Penicillium turbatum BLH34, isolated from Macleaya cordata. Hybrid sequencing (Illumina-Nanopore) generated a high-quality 27.9 Mb genome (GC 48.6%) encoding 9798 proteins, with functional annotation linking 5350 genes to the NCBI non-redundant database and 3404 to KEGG pathways. AntiSMASH analysis uncovered 35 biosynthetic gene clusters (BGCs), 23 of which lacked homology to known pathways, highlighting BLH34's potential for novel metabolite discovery. Molecular networking (GNPS) and LC-MS/MS identified 19 specialised metabolites, including antimicrobial polyketides. Bioassays demonstrated potent inhibition against Staphylococcus aureus (36 mm), Bacillus subtilis (28 mm) and Escherichia coli (24 mm), underscoring its pharmaceutical relevance.

Penicillium↗