RefZ and Noc act synthetically to prevent aberrant divisions during Bacillus subtilis sporulation

During sporulation, Bacillus subtilis undergoes an atypical cell division that requires overriding mechanisms which protect chromosomes from damage and ensure inheritance by daughter cells. Instead of assembling between segregated chromosomes at midcell, the FtsZ-ring (Z-ring) coalesces polarly, directing division over one chromosome. The DNA-binding protein RefZ facilitates the timely assembly of polar Z-rings and partially defines the region of chromosome initially captured in the forespore. RefZ binds to motifs (RBMs) located proximal to the origin of replication (oriC). Although refZ and the RBMs are conserved across the Bacillus genus, a refZ deletion mutant sporulates with wildtype efficiency, so the functional significance of RefZ during sporulation remains unclear. To further investigate RefZ function, we performed a candidate-based screen for synthetic sporulation defects by combining ∆refZ with deletions of genes previously implicated in FtsZ regulation and/or chromosome capture. Combining ∆refZ with deletions of ezrA, sepF, parA, or minD did not detectably affect sporulation. In contrast, a ∆refZ ∆noc mutant exhibited a sporulation defect, revealing a genetic interaction between RefZ and Noc. Using reporters of sporulation progression, we determined the ∆refZ ∆noc mutant exhibited sporulation delays after Spo0A activation but prior to late sporulation, with a subset of cells failing to divide polarly or activate the first forespore-specific sigma factor, SigF. The ∆refZ ∆noc mutant also exhibited extensive dysregulation of cell division, producing cells with extra, misplaced, or otherwise aberrant septa. Our results reveal a previously unknown epistatic relationship that suggests refZ and noc contribute synthetically to regulating cell division and supporting spore development.

The Use of Ascophyllum nodosum and Bacillus subtilis C-3102 in the Management of Canine Chronic Inflammatory Enteropathy: A Pilot Study

The aim was to assess the effects of Ascophyllum nodosum (AN) with/without Bacillus subtilis C-3102 as alternative treatments for Chronic Inflammatory Enteropathy (CIE) of dogs. Fourteen CIE patients, which had received the same control (CTR) diet, were enrolled to serially receive three diets: (1) hydrolysed protein (HP) diet; (2) 4.0% AN supplemented HP (HPA) food, (3) HPA diet fortified with 125 billion B. subtilis C-3102 spores/10 kg body weight (HPAB diet). Clinical outcome was assessed by Canine Inflammatory Bowel Disease Activity Index (CIBDAI), whereas gut microbiota compositional variations were investigated via 16S rRNA gene analysis, and faecal fermentation end-products by liquid chromatography. Higher abundances of the Ruminococcaceae and Rikenellaceae families were shown in HPA relative to CTR treatment, with Bacillus genus being differentially abundant on HPAB diet. Concentrations of acetate were higher (p < 0.05) in dogs fed HPA compared to CTR diet, and amounts of isovalerate and isobutyrate were greater (p < 0.05) in HPA compared to HP food. A tendency for higher amounts of faecal butyrate was found for the HPAB treatment (p = 0.06). Comprehensively, while displaying potentially positive effects on faecal fermentations, the tested substances failed to improve CIBDAI scores and microbial richness in CIE dogs.

Characterization of Vegetative Bacillus cereus and Bacillus subtilis Strains Isolated from Processed Cheese Products in an Italian Dairy Plant

Processed cheese is a commercial product characterized by high microbiological stability and extended shelf life obtained through the application of severe heat treatment. However, spore-forming bacteria can survive through thermal processes. Among them, microorganisms belonging to Bacillus genus have been reported. In this study, we examined the microbiological population of the first hours’ production of processed cheeses in an Italian dairy plant during two seasons, between June and October 2020. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify bacteria colonies, allowing the isolation of Bacillus cereus and Bacillussubtilis strains. These results were further confirmed by amplification and sequencing of 16 rRNA bacterial region. A multi-locus sequence type (MLST) analysis was performed to assess the genetic similarity among a selection of isolates. The fourteen B. cereus strains showed two sequence types: ST-32 was observed in only one strain and the ST-371 in the remaining thirteen isolates. On the contrary, all twenty-one B. subtlis strains, included in the study, showed a new allelic profile for the pycA gene, resulting in a new sequence type: ST-249. For B. cereus strains, analysis of toxin genes was performed. All isolates were positive for nheABC, entFM, and cytK, while hblABCD, bceT, and ces were not detected. Moreover, the biofilm-forming ability of B. cereus and B. subtilis strains was assessed, and all selected isolates proved to be biofilm formers (most of them were stronger producers). Considering the genetical similarity between isolates, jointly with the capacity to produce biofilm, the presence of a recurring Bacillus population could be hypothesized.

Bacterial Degradation of Bacteriostatic Polyphenols, Tannins

Tannin degradation by bacteria has not been studied much as tannins are commonly known to be bacteriostatic due to enzyme inhibition, substrate deprivation, and the enzyme activity on the bacterial cell wall. However, about a handful of bacteria have been found to tolerate certain concentrations of tannin. This study focuses on isolating and identifying bacteria from decaying portions of tree bark for tannase production and effective catalysis of ester bond hydrolysis in tannins. Different concentrations of commercial tannic acid were used as the sole carbon source on mineral salt medium (MSM) agar plates, to test the maximum tolerable concentrations (MTCs) by the isolates. Tannin degradation was confirmed by a visual reading method and bacterial tannase activity and the biodegradation percentage were determined. One particular isolate was identified to have 50 g/L MTC of tannin, with a tannase activity of 51.61 U/mL that is optimum after 96 hours of incubation. The 16s rRNA sequencing results showed that the isolate belonged to Bacillus genus and the resulting bacterial strain isolate was found to be a new strain of Bacillus subtilis which was submitted to GenBank under the accession number MH330408.

Development of a probiotic for animals and aquaculture based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains

This article aims to develop a probiotic for animals and aquaculture based on the Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains. The selection of a nutrient medium was conducted for cultivating the inoculum of these microorganisms. Several bacteria fermentations of the Bacillus genus were performed in biological reactors with a capacity of 15 and 250 l. A technology for obtaining a finished probiotic for animals and aquaculture was developed. The results indicate that L-broth is the most optimal nutrient medium for cultivating the studied strains. The cultivation of B. toyonensis B-13249 and B. pumilus B-13250 strains in fermenters revealed that sporulation begins after 4–8 hours of fermentation. In contrast to the vegetative medium, the fermentative medium helped the bacilli develop a higher optical density (the maximum value in the B. pumilus strain – 2.400±0.149), pH value (maximum value in the B. toyonensis strain – 8.483±0.609) and titer (at least 1010 CFU/g). After 20–24 hours of incubation, both strains of bacilli in the fermenter, almost completely pass into endospores, which serve as a signal for the start of biomass centrifugation. This was indicated by the following: from a 15 l fermenter – 83.3±6.1 g of concentrate, from a 250 l fermenter – 499.8±51.4 g. The number of bacilli in a concentrated state was at least 1·1011 CFU/g for both strains. Obtaining a finished preparation required mixing bacterial concentrates with maltodextrin to a titer of at least 1·1010 CFU/g. The number of bacteria in the preparation checked every month during the year, recorded no value less than 1·1010 CFU/g. Thus, L-broth is most favorable for growing the mother culture of the B. toyonensis B-13249 and B. pumilus B-13250 strains, and fermentative nutrient medium – for the cultivation in fermenters. The expiry date of the bacilli-based biological preparation is at least 12 months, during which the drug’s polycomponence, color and consistency are preserved, in addition to the bacteria titer (at least 1·1010 CFU/g) and their viability.

Ocimum basilicum L. associated bacteria with antifungal activity

The presence of pathogens, like fungi, is one of the most important causes of basil crop loss around the world; however, many microorganisms have a crucial role on plant development including protection against pathogenic ones. In the present study, basil associated bacteria were isolated, quantified and preserved. Characterization of isolated bacteria showed 165 Gram positive strains, 152 with bacillary and 13 with coccoid morphology. Later, in vitro antagonism assays were performed, first against Aspergillus spp. and then against Neoscytalidium dimidiatum, Alternaria spp. and Aspergillus spp. Finally, the effect of the isolated bacteria on basil seed germination and first stages of development were carried out. Sampled basil plants, produced around La Paz, Baja California Sur, Mexico, were colonized by known antagonistic bacteria of the Bacillus genus. Bacillus amyloliquefaciens strains were the prevailing species with antifungal activity. Moreover, strains ALMH42, ALMR73 and ALAH91 did not show any deleterious effect on basil seedling development. Biotechnological potential exploration of these isolated strains from healthy basil plants is of great interest for future applications on this and other crops.

Isolation and selection of Bacillus sp. with the potential biosynthesis of protease from fermented soybean products

This study aims to isolate and select strains of Bacillus for protease production from fermented soybean products. To evaluate the ability of protease production, the authors applied the hydrolysis diameter measurement method on Skim milk agar (SMA) and submerged fermentation (SmF). The results showed that 29 bacterial strains were isolated from fermented soybean products in An Giang and Can Tho provinces. Based on the morphological and biochemical characteristics of bacteria, these are defined belong to the Bacillus genus. The results of the protease generation test on SMA helped to select eight strains with a high halo diameter. In SmF with 106 cells/ml, pH 7.2, at 37°C for 48 hours, the ML01 strain gave the highest specific protease activity of 185.92 U/mg. Sequence analysis results of the 16S rRNA gene region of ML01 exhibited a high relationship with the sequence of Bacillus subtilis.

Bioaerosol Emissions during Organic Waste Treatment for Biopolymer Production: A Case Study

Environmentally sustainable methods of waste disposal are a strategic priority. For organic waste management and innovative biological treatments present advantageous opportunities, although organic waste treatment also includes environmental drawbacks, such as bioaerosol production. This study aims to evaluate bioaerosol spread during an innovative experimental treatment. The process consists of two anaerobic steps: acidogenesis, which includes polyhydroxyalkanoate accumulation, followed by methanogenesis. Bioaerosol, PM10, and endotoxin concentrations were measured at three sampling points during different campaigns to evaluate: (1) the background levels, (2) the contamination produced in the pre-treatment stage, and (3) the residual contamination of the outgoing digested sludge. Environmental PM10 seemed to be generally quite contained, while the endotoxin determination was close to 90 EU/m3. Significant microbial concentrations were detected during the loading of the organic fraction of municipal solid waste (fungi > 1300 CFU/m3, Bacillus genus (≈103 CFU/m3), higher Clostridium spp. and opportunistic human pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae), suggesting a significant contamination level. Such results are useful for hazard identification in the risk assessment of innovative processes, as they reveal contaminants potentially harmful to both workers’ health and the environment.

Micellar Antibiotics of Bacillus

Members of the Bacillus genus, particularly the “Bacillus subtilis group”, are known to produce amphipathic lipopeptides with biosurfactant activity. This includes the surfactins, fengycins and iturins that have been associated with antibacterial, antifungal, and anti-viral properties. We have screened a large collection of Bacillus, isolated from human, animal, estuarine water and soil samples and found that the most potent lipopeptide producers are members of the species Bacillus velezensis. B. velezensis lipopeptides exhibited anti-bacterial activity which was localised on the surface of both vegetative cells and spores. Interestingly, lipopeptide micelles (6–10 nm diameter) were detectable in strains exhibiting the highest levels of activity. Micelles were stable (heat and gastric stable) and shown to entrap other antimicrobials produced by the host bacterium (exampled here was the dipeptide antibiotic chlorotetaine). Commercially acquired lipopeptides did not exhibit similar levels of inhibitory activity and we suspect that micelle formation may relate to the particular isomeric forms produced by individual bacteria. Using naturally produced micelle formulations we demonstrated that they could entrap antimicrobial compounds (e.g., clindamycin, vancomycin and resveratrol). Micellar incorporation of antibiotics increased activity. Bacillus is a prolific producer of antimicrobials, and this phenomenon could be exploited naturally to augment antimicrobial activity. From an applied perspective, the ability to readily produce Bacillus micelles and formulate with drugs enables a possible strategy for enhanced drug delivery.