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Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis (Bt) are spore-forming members of the Bacillus cereus group. Spores of B. cereus group species are encircled by exosporium, which is composed of an external hair-like nap and a paracrystalline basal layer. Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus group. Herein, we studied the regulation of several exosporium-related genes in Bt. A SigK consensus sequence is present upstream of genes encoding hair-like nap proteins ( bclA and bclB ), basal layer proteins ( bxpA, bxpB, cotB and exsY  ) and inosine hydrolase ( iunH ). Mutation of sigK decreased the transcriptional activities of all these genes, indicating that the transcription of these genes is controlled by SigK. Furthermore, mutation of gerE decreased the transcriptional activities of bclB, bxpB, cotB and iunH but increased the expression of bxpA and GerE binds to the promoters of bclB, bxpB, cotB, bxpA and iunH . These results suggest that GerE directly regulates the transcription of these genes, increasing the expression of bclB, bxpB, cotB and iunH and decreasing that of bxpA . These findings provide insight into the exosporium assembly process at the transcriptional level.

We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.

Background Outdoor malaria transmission is becoming an increasingly important problem in malaria control in Africa. Larval control is a promising intervention as it can target both indoor and outdoor biting mosquitoes. However, the currently available biolarvicide formulations have a short effective duration, and consequently larval control incurs a high operational expense due to the requirement for frequent re-treatment of larval habitats. Formulations of biolarvicides with long-lasting effects is highly desired. A recently developed FourStar® slow-release briquet formulation of Bacillus thuringiensis israelensis and Bacillus sphaericus was evaluated to test its efficacy on malaria vectors. Methods The study evaluated FourStar™ briquets 180-days formulation under semi-natural and natural conditions to test their efficacy in reducing the mosquito population in western Kenya. The semi-natural habitats used the formulation dissolved in rainwater with appropriate concentrations, and second-instar larvae of Anopheles gambiae were introduced and the number of surviving larvae and pupae produced was recorded daily as the outcome. The briquets formulation was then tested in natural habitats for efficacy on pupal productivity reduction in highland and lowland sites in western Kenya. The formulation was finally tested for efficacy in reducing adult mosquito populations in randomized clusters in western Kenya highland. Results In semi-natural conditions, the FourStar™ briquets 180-days formulation completely inhibited mosquito pupal production in the first 3 months, and then reduced pupal productivity by 87–98% (P < 0.001) 4–6 months after application. In natural habitats, during the first 2 months no pupae were detected from any of the treated habitats in highland sites, and Anopheles spp. pupal density was reduced by 60–90% in the next 3–5 months (P < 0.001). In the lowland site, pupal productivity reduction was 100% in the first 3 months, and 75–90% in the next 4–5 months (P < 0.001). The randomized cluster trial found that the application of the briquets formulation reduced mean densities of indoor-biting mosquitoes by 76–82% (P < 0.001) and by 67–75% (P < 0.001) for outdoor-biting mosquitoes. Conclusion This study demonstrated that long-lasting biological larviciding was effective in reducing pupal productivity of larval habitats, and reducing indoor and outdoor resting mosquitoes. The study suggests that long-lasting microbial larviciding may be a promising complementary malaria vector control tool and warrants further large-scale evaluation.

Many strains from the Bacillus subtilis species complex exert strong plant growth‐promoting activities. However, their efficacy in relevant conditions is variable, due in part to their inability to establish a strong interaction with roots in stressful environmental conditions. Adaptative laboratory evolution (ALE) is a powerful tool to generate novel strains with traits of interest. Many Bacillus evolved isolates, stemming from ALE performed with plants, possess a stronger root colonization capacity. An in‐depth analysis of these isolates also allowed the identification of key features influencing the interaction with plant roots. However, many variables can influence the outcome of these assays, and thus, caution should be taken when designing ALE destined to generate better root colonizers. This opinion article reviews the potential of adaptative laboratory evolution assays to develop Bacillus species with higher root‐colonization efficacy.

Strains of Bacillus species, plant growth‐promoting rhizobacteria, have been commercialised as biofertilisers; they are ideal for this because these species form spores that can be stored stably for a long time. However, for these spores to exert their full beneficial effects, they must germinate. The specific germination signals in the rhizosphere, particularly those from plant root exudates, remain largely unknown. Here, we investigated the germination signals from different growth states of cucumber (Cucumis sativus) for spores of Bacillus velezensis SQR9 and Bacillus subtilis NCIB 3610. We identified the corresponding germination receptors and compared them biochemically between the Bacillus species. Larger plants better stimulated spore germination. Five amino acids—L‐isoleucine, L‐ornithine, L‐valine, L‐serine and β‐alanine were—identified as spore germination signals. Combined application of a mixture of these amino acids with bacterial spores markedly enhanced the cucumber growth‐promoting properties of B. velezensis SQR9. The germination receptor for these amino acids was GerA in both Bacillus species. Differences in spore germination efficiency between B. subtilis and B. velezensis may be attributable to variations in the GerA ligand‐recognition sites. Expression of GerA from B. subtilis NCIB 3610 in B. velezensis SQR9 enhanced the spore germination rate of the latter. Our study highlights the pivotal role of amino acids in regulating spore germination of Bacillus and subsequent plant root colonisation, emphasising their potential to enhance the efficacy of Bacillus‐based biofertilisers. Engineering of germination receptors is a promising approach to enhance the spore germination efficiency of biofertiliser strains. Amino acids in cucumber root exudates trigger Bacillus spore germination via GerA, enhancing root colonisation and plant growth. This study reveals key germinators and receptors, advancing targeted biofertiliser applications for improved agricultural sustainability.

Bacillus species have demonstrated beneficial effects on intestinal health, production parameters, and immune function in poultry under both standard and disease-challenged conditions. Previously, we found that several novel ingredients (beta-glucan, vegetable protein hydrolysate, and liquid seaweed extract) demonstrated growth stimulation effects on Bifidobacterium lactis and Lactobacillus plantarum , Here, we extended this approach to examine in vitro synbiotic combinations of five novel marine-derived candidate probiotic Bacillus strains to assess their potential for in ovo applications. Beta-glucan enhanced the growth of all candidate Bacillus probiotic strains compared to a glucose control ( p  ≤ 0.05), suggesting a broad-spectrum modulatory role over a 24-h period, with variable magnitudes of response observed between strains. Species specificity was also observed, with lentinus stimulating the Bacillus pumilus but not the Bacillus altitudinis strains. A seaweed extract consistently stimulated the growth of one of the B. altitudinis strains ( p  ≤ 0.05), which, like all of the strains evaluated here, is seaweed-derived. This suggests potential ecological adaptation in substrate utilization. The shared environmental origin may influence substrate specificity and metabolic complementarity between strains and prebiotic candidates. Both B. altitudinis strains also exhibited enhanced growth at almost all time points ( p  ≤ 0.05) when cultured with vegetable protein hydrolysate. Based on these findings, we evaluated the effect of a potential synbiotic formulation comprising one of the B. altitudinis strains and vegetable protein hydrolysate in chickens, in ovo. The components were administered intra-amniotically at embryonic development day 18.5, utilizing a standard vaccination protocol. The hatchability of the chickens was not affected, thereby demonstrating the established dose as safe and applicable for further investigation. Key points • Shared origin of bioactive compounds may enhance probiotic-prebiotic compatibility in vitro • Protein hydrolysate offers a novel alternative to carbohydrate prebiotics • In ovo delivery of Bacillus-based synbiotic formulations offers potential as an early microbiome programming strategy

Phosphorus (P) use efficiency in alkaline/calcareous soils is only 20% due to precipitation of P 2 O 5 with calcium and magnesium. However, coating Diammonium Phosphate (DAP) with phosphorus solubilizing bacteria (PSB) is more appropriate to increase fertilizer use efficiency. Therefore, with the aim to use inorganic fertilizers more effectively present study was conducted to investigate comparative effect of coated DAP with PSB strains Bacillus subtilis ZE15 (MN003400), Bacillus subtilis ZR3 (MN007185), Bacillus megaterium ZE32 (MN003401) and Bacillus megaterium ZR19 (MN007186) and their extracted metabolites with uncoated DAP under axenic conditions. Gene sequencing was done against various sources of phosphorus to analyze genes responsible for phosphatase activity. Alkaline phosphatase (ALP) gene amplicon of 380bp from all tested strains was showed in 1% w/v gel. Release pattern of P was also improved with coated fertilizer. The results showed that coated phosphatic fertilizer enhanced shoot dry weight by 43 and 46% under bacterial and metabolites coating respectively. Shoot and root length up to 44 and 42% with metabolites coated DAP and 41% with bacterial coated DAP. Physiological attributes also showed significant improvement with coated DAP over conventional. The results supported the application of coated DAP as a useful medium to raise crop yield even at lower application rates i.e., 50 and 75% DAP than non-coated 100% DAP application which advocated this coating technique a promising approach for advancing circular economy and sustainable development in modern agriculture.

ObjectivesTo develop a simple pectin-degrading microorganism screening method.ResultsWe developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample.ConclusionsThe developed method is useful for identifying pectin-degrading microorganisms.

Bacterial nitric oxide synthases (bNOS) are present in many Gram-positive species and have been demonstrated to synthesize NO from arginine in vitro and in vivo. However, the physiological role of bNOS remains largely unknown. We show that NO generated by bNOS increases the resistance of bacteria to a broad spectrum of antibiotics, enabling the bacteria to survive and share habitats with antibiotic-producing microorganisms. NO-mediated resistance is achieved through both the chemical modification of toxic compounds and the alleviation of the oxidative stress imposed by many antibiotics. Our results suggest that the inhibition of NOS activity may increase the effectiveness of antimicrobial therapy.

Abstract The bacterial growth curve graphically represents the development of a bacterial population over time. This analysis is essential to optimize efficiency in research with microorganisms. The research aimed to identify the temperature and time at which bacterial growth reaches the stationary phase and obtain the point of maximum performance. Four species of growth-promoting bacteria were evaluated: Pseudomonas fluorescens, Bacillus aryabhattai, Bacillus subtilis and Azospirillum brasilense, subjected to temperatures of 25 °C, 30 °C and 35 °C. The bacteria were inoculated in a nutrient solution and incubated in a water bath. Samples were taken at four-hour intervals for serial dilution and plating. The colony-forming units (CFUs) were counted every 12 hours. Pseudomonas fluorescens showed maximum growth at 29.9 °C and 52.3 hours. Bacillus aryabhattai reached maximum growth at 25 °C in 48 hours. Bacillus subtilis had maximum growth at 24.1 °C and 47.2 hours. Azospirillum brasilense reached maximum growth at 25.9 °C and 42.5 hours. The results indicate that temperature variations significantly influence the growth of the bacteria analyzed. Based on this research, it is possible to adjust laboratory methodologies to improve the efficiency of microorganism production processes. Resumo A curva de crescimento bacteriano representa graficamente o desenvolvimento de uma população bacteriana ao longo do tempo. Essa análise é essencial para otimizar a eficiência em pesquisas com microrganismos. A pesquisa teve como objetivo identificar a temperatura e o tempo em que o crescimento bacteriano atinge a fase estacionária e obter o ponto de máximo desempenho. Quatro espécies de bactérias promotoras de crescimento foram avaliadas: Pseudomonas fluorescens, Bacillus aryabhattai, Bacillus subtilis e Azospirillum brasilense, submetidas às temperaturas de 25 °C, 30 °C e 35 °C. As bactérias foram inoculadas em solução nutritiva e incubadas em banho-maria. Amostras foram coletadas em intervalos de quatro horas para diluição seriada e plaqueamento. As unidades formadoras de colônias (UFCs) foram contadas a cada 12 horas. Pseudomonas fluorescens apresentou crescimento máximo em 29,9 °C e 52,3 horas. Bacillus aryabhattai atingiu crescimento máximo a 25 °C em 48 horas. Bacillus subtilis apresentou crescimento máximo a 24,1 °C e 47,2 horas. Azospirillum brasilense atingiu crescimento máximo a 25,9 °C e 42,5 horas. Os resultados indicam que variações de temperatura influenciam significativamente o crescimento das bactérias analisadas. Com base nesta pesquisa, é possível ajustar metodologias laboratoriais para melhorar a eficiência dos processos de produção de microrganismos.