BioDeepfuse: a hybrid deep learning approach with integrated feature extraction techniques for enhanced non-coding RNA classification.

The accurate classification of non-coding RNA (ncRNA) sequences is pivotal for advanced non-coding genome annotation and analysis, a fundamental aspect of genomics that facilitates understanding of ncRNA functions and regulatory mechanisms in various biological processes. While traditional machine learning approaches have been employed for distinguishing ncRNA, these often necessitate extensive feature engineering. Recently, deep learning algorithms have provided advancements in ncRNA classification. This study presents BioDeepFuse, a hybrid deep learning framework integrating convolutional neural networks (CNN) or bidirectional long short-term memory (BiLSTM) networks with handcrafted features for enhanced accuracy. This framework employs a combination of k-mer one-hot, k-mer dictionary, and feature extraction techniques for input representation. Extracted features, when embedded into the deep network, enable optimal utilization of spatial and sequential nuances of ncRNA sequences. Using benchmark datasets and real-world RNA samples from bacterial organisms, we evaluated the performance of BioDeepFuse. Results exhibited high accuracy in ncRNA classification, underscoring the robustness of our tool in addressing complex ncRNA sequence data challenges. The effective melding of CNN or BiLSTM with external features heralds promising directions for future research, particularly in refining ncRNA classifiers and deepening insights into ncRNAs in cellular processes and disease manifestations. In addition to its original application in the context of bacterial organisms, the methodologies and techniques integrated into our framework can potentially render BioDeepFuse effective in various and broader domains.

Multiscale spatial segregation analysis in digital images of biofilms.

Quantifying the degree of spatial segregation of two bacterial strains in mixed biofilms is an important topic in microbiology. Spatial segregation is dependent on spatial scale as two strains may appear to be well mixed if observed from a distance, but a closer look can reveal strong separation. Typically, this information is encoded in a digital image that represents the binary system, e.g., a microscopy image of a two species biofilm. To decode spatial segregation information, we have developed quantitative measures for evaluating the degree of the spatial scale-dependent segregation of two bacterial strains in a digital image. The constructed algorithm is based on the new segregation measures and overcomes drawbacks of existing approaches for biofilm segregation analysis. The new approach is implemented in a freely available software and was successfully applied to biofilms of two strains and bacterial suspensions for detection of the different spatial scale-dependent segregation levels.

DegQ is an important policing link between quorum sensing and regulated adaptative traits in Bacillus subtilis.

Quorum sensing (QS) is a widespread bacterial communication system that controls important adaptive traits in a cell density-dependent manner. However, mechanisms by which QS-regulated traits are linked within the cell and mechanisms by which these links affect adaptation are not well understood. In this study, Bacillus subtilis was used as a model bacterium to investigate the link between the ComQXPA QS system, DegQ, surfactin and protease production in planktonic and biofilm cultures. The work tests two alternative hypotheses predicting that hypersensitivity of the QS signal-deficient mutant (comQ::kan) to exogenously added ComX, resulting in increased surfactin production, is linked to an additional genetic locus, or alternatively, to overexpression of the ComX receptor ComP. Results are in agreement with the first hypothesis and show that the P srfAA hypersensitivity of the comQ::kan mutant is linked to a 168 strain-specific mutation in the P degQ region. Hence, the markerless ΔcomQ mutant lacking this mutation is not overresponsive to ComX. Such hyper-responsiveness is specific for the P srfAA and not detected in another ComX-regulated promoter, the P aprE , which is under the positive control by DegQ. Our results suggest that DegQ by exerting differential effect on P srfAA and P aprE acts as a policing mechanism and the intracellular link, which guards the cell from an overinvestment into surfactin production. IMPORTANCE DegQ levels are known to regulate surfactin synthesis and extracellular protease production, and DegQ is under the control of the ComX-dependent QS. DegQ also serves as an important policing link between these QS-regulated processes, preventing overinvestment in these costly processes. This work highlights the importance of DegQ, which acts as the intracellular link between ComX production and the response by regulating extracellular degradative enzyme synthesis and surfactin production.

Nutrient Availability and Biofilm Polysaccharide Shape the Bacillaene-Dependent Antagonism of Bacillus subtilis against Salmonella Typhimurium.

Salmonella enterica is one of the most common foodborne pathogens and, due to the spread of antibiotic resistance, new antimicrobial strategies are urgently needed to control it. In this study, we explored the probiotic potential of Bacillus subtilis PS-216 and elucidated the mechanisms that underlie the interactions between this soil isolate and the model pathogenic strain S. Typhimurium SL1344. The results reveal that B. subtilis PS-216 inhibits the growth and biofilm formation of S. Typhimurium through the production of the pks cluster-dependent polyketide bacillaene. The presence of S. Typhimurium enhanced the activity of the PpksC promoter that controls bacillaene production, suggesting that B. subtilis senses and responds to Salmonella. The level of Salmonella inhibition, overall PpksC activity, and PpksC induction by Salmonella were all higher in nutrient-rich conditions than in nutrient-depleted conditions. Although eliminating the extracellular polysaccharide production of B. subtilis via deletion of the epsA-O operon had no significant effect on inhibitory activity against Salmonella in nutrient-rich conditions, this deletion mutant showed an enhanced antagonism against Salmonella in nutrient-depleted conditions, revealing an intricate relationship between exopolysaccharide production, nutrient availability, and bacillaene synthesis. Overall, this work provides evidence on the regulatory role of nutrient availability, sensing of the competitor, and EpsA-O polysaccharide in the social outcome of bacillaene-dependent competition between B. subtilis and S. Typhimurium. IMPORTANCE Probiotic bacteria represent an alternative for controlling foodborne disease caused by Salmonella enterica, which constitutes a serious concern during food production due to its antibiotic resistance and resilience to environmental stress. Bacillus subtilis is gaining popularity as a probiotic, but its behavior in biofilms with pathogens such as Salmonella remains to be elucidated. Here, we show that the antagonism of B. subtilis is mediated by the polyketide bacillaene and that the production of bacillaene is a highly dynamic trait which depends on environmental factors such as nutrient availability and the presence of competitors. Moreover, the production of extracellular polysaccharides by B. subtilis further alters the influence of these factors. Hence, this work highlights the inhibitory effect of B. subtilis, which is condition-dependent, and the importance of evaluating probiotic strains under conditions relevant to the intended use.

Reduction of hexavalent chromium using bacterial isolates and a microbial community enriched from tannery effluent.

We investigated microbial growth in increasing concentrations of hexavalent chromium (Cr(VI)) and its reduction by a microbial community enriched from tannery effluent and by the bacterial strains isolated from the enriched community. The bacterial growth was monitored by measuring the optical cell density (OD650), while the Cr(VI) concentration in the samples was determined using spectrophotometry and liquid chromatography hyphenated to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). At a Cr(VI) concentration of 100 mg/L, the isolates affiliated with Pseudomonas aeruginosa (P. aeruginosa) reached higher optical cell densities, but were in general less effective for Cr(VI) reduction than the isolates affiliated with Mammaliicoccus sciuri (M. sciuri). All three M. sciuri isolates and only one of the seven P. aeruginosa isolates were able to reduce 50% of the Cr(VI) with an initial concentration of 100 mg/L within 24 h (pH 7.1), while the six isolates affiliated with P. aeruginosa were less effective. Compared to the isolated, individual bacterial strains, the enriched microbial community was better adapted to the elevated Cr(VI) concentrations, but needed a longer time (48 h) to reduce the Cr(VI) with the same efficacy as the most efficient individual isolates. The ability of the enriched microbial community and the isolated bacterial strains to reduce the Cr(VI) highlights their potential for use in the rapid bioremediation of wastewaters contaminated with Cr(VI).

Involvement of Flagellin in Kin Recognition between Bacillus velezensis Strains.

Kin discrimination in nature is an effective way for bacteria to stabilize population cooperation and maintain progeny benefits. However, so far, the research on kin discrimination for Bacillus still has concentrated on "attack and defense" between cells and diffusion-dependent molecular signals of quorum sensing, kin recognition in Bacillus, however, has not been reported. To determine whether flagellar is involve in the kin recognition of Bacillus, we constructed Bacillus velezensis SQR9 assembled with flagellin of its kin and non-kin strains, and performed a swarm boundary assay with SQR9, then analyzed sequence variation of flagellin and other flagellar structural proteins in B. velezensis genus. Our results showed that SQR9 assembled with flagellin of non-kin strains was more likely to form a border phenotype with wild-type strain SQR9 in swarm assay than that of kin strains, and that non-kin strains had greater variation in flagellin than kin strains. In B. velezensis, these variations in flagellin were prevalent and had evolved significantly faster than other flagellar structural proteins. Therefore, we proposed that flagellin is an effective tool partly involved in the kin recognition of B. velezensis strains. IMPORTANCE Kin selection plays an important role in stabilizing population cooperation and maintaining the progeny benefits for bacteria in nature. However, to date, the role of flagellin in kin recognition in Bacillus has not been reported. By using rhizospheric Bacillus velezensis SQR9, we accomplished flagellin region interchange among its related strains, and show that flagellin acts as a mediator to distinguish kin from non-kin in B. velezensis. We demonstrated the polymorphism of flagellin in B. velezensis through alignment analysis of flagellin protein sequences. Therefore, it was proposed that flagellin was likely to be an effective tool for mediating kin recognition in B. velezensis.

Housekeeping gene gyrA, a potential molecular marker for Bacillus ecology study.

Bacillus is a genus of microorganisms (bacteria) and contains many important commercial species used in industry, agriculture and healthcare. Many different Bacilli are relatively well understood at the single-cell level; however, molecular tools that determine the diversity and ecology of Bacillus community are limited, which limits our understanding of how the Bacillus community works. In the present study, we investigated the potential of the housekeeping gene gyrA as a molecular marker for determining the diversity of Bacillus species. The amplification efficiency for Bacillus species diversity could be greatly improved by primer design. Therefore, we designed a novel primer pair gyrA3 that can detect at least 92 Bacillus species and related species. For B. amyloliquefaciens, B. pumilus, and B. megaterium, we observed that the high variability of the gyrA gene allows for more detailed clustering at the subspecies level that cannot be achieved by the 16S rRNA gene. Since gyrA provides better phylogenetic resolution than 16S rRNA and informs on the diversity of the Bacillus community, we propose that the gyrA gene may have broad application prospects in the study of Bacillus ecology.

Kin Discrimination Modifies Strain Distribution, Spatial Segregation, and Incorporation of Extracellular Matrix Polysaccharide Mutants of Bacillus subtilis Strains into Mixed Floating Biofilms.

Microorganisms in nature form multicellular groups called biofilms. In biofilms, bacteria embedded in the extracellular matrix (ECM) interact intensely due to their proximity. Most studies have investigated genetically homogeneous biofilms, leaving a gap in knowledge on genetically heterogeneous biofilms. Recent insights show that a Gram-positive model bacterium, Bacillus subtilis, discriminates between strains of high (kin) and low (nonkin) genetic similarity, reflected in merging (kin) and boundaries (nonkin) between swarms. However, it is unclear how kinship between interacting strains affects their fitness, the genotype assortment, and incorporation of the mutant lacking the main structural ECM polysaccharide (EpsA-O) into floating biofilms (pellicles). We cultivated Bacillus subtilis strains as mixtures of isogenic, kin, and nonkin strain combinations in the biofilm-promoting minimal medium under static conditions, allowing them to form pellicles. We show that in nonkin pellicles, the dominant strain strongly reduced the frequency of the other strain. Segregation of nonkin mixtures in pellicles increased and invasion of nonkin EpsA-O-deficient mutants into pellicles decreased compared to kin and isogenic floating biofilms. Kin and isogenic strains had comparable relative frequencies in pellicles and showed more homogenous cell mixing. Overall, our results emphasize kin discrimination as a social behavior that shapes strain distribution, spatial segregation, and ECM mutant ability to incorporate into genetically heterogenous biofilms of B. subtilis. IMPORTANCE Biofilm communities have beneficial and harmful effects on human societies in natural, medical, and industrial environments. Bacillus subtilis is a biotechnologically important bacterium that serves as a model for studying biofilms. Recent studies have shown that this species engages in kin discriminatory behavior during swarming, which may have implications for community assembly, thus being of fundamental importance. Effects of kin discrimination on fitness, genotype segregation, and success of extracellular matrix (ECM) polysaccharide (EpsA-O) mutant invasion into biofilms are not well understood. We provide evidence that kin discrimination depends on the antagonism of the dominant strain against nonkin by using environmental strains with determined kin types and integrated fluorescent reporters. Moreover, this antagonism has important implications for genotype segregation and for when the bacteria are mixed with ECM producers. The work advances the understanding of kin-discrimination-dependent bacterial sociality in biofilms and its role in the assembly of multicellular groups.

Bacillus subtilis PS-216 Spores Supplemented in Broiler Chicken Drinking Water Reduce Campylobacter jejuni Colonization and Increases Weight Gain.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, or campylobacteriosis, in humans worldwide, and poultry serves as a major source of infection. To reduce the risk associated with C. jejuni transmission via poultry meat, effective interventions during poultry production are needed, and the use of probiotics is a promising approach. In this study, 15 Bacillus subtilis strains were initially screened for their anti-Campylobacter activities. B. subtilis PS-216 strain demonstrated the best anti-Campylobacter activity against 15 C. jejuni isolates when examined using in vitro co-cultures. To evaluate the suitability of B. subtilis PS-216 for probiotic use, its susceptibility to eight clinically important antimicrobials and simulated gastric conditions was investigated. B. subtilis PS-216 was sensitive to all of the tested antibiotics. Although vegetative cells were sensitive to gastric conditions, B. subtilis PS-216 spores were highly resistant. We further evaluated the use of a B. subtilis PS-216 spore preparation (2.5 × 106 CFU/mL water) to prevent and/or reduce C. jejuni colonization in broiler chickens in vivo. Compared to the untreated group, significantly lower Campylobacter counts were detected in caeca of broilers continuously treated with B. subtilis PS-216 spores in their drinking water. Furthermore, broilers continuously treated with B. subtilis PS-216 spores showed improved weight gain, compared to the control group. Together, these results demonstrate the potential of B. subtilis PS-216 for use in poultry to reduce C. jejuni colonization and improve weight gain.

Systems view of Bacillus subtilis pellicle development.

In this study, we link pellicle development at the water-air interface with the vertical distribution and viability of the individual B. subtilis PS-216 cells throughout the water column. Real-time interfacial rheology and time-lapse confocal laser scanning microscopy were combined to correlate mechanical properties with morphological changes (aggregation status, filament formation, pellicle thickness, spore formation) of the growing pellicle. Six key events were identified in B. subtilis pellicle formation that are accompanied by a major change in viscoelastic and morphology behaviour of the pellicle. The results imply that pellicle development is a multifaceted response to a changing environment induced by bacterial growth that causes population redistribution within the model system, reduction of the viable habitat to the water-air interface, cell development, and morphogenesis. The outcome is a build-up of mechanical stress supporting structure that eventually, due to nutrient deprivation, reaches the finite thickness. After prolonged incubation, the formed pellicle collapses, which correlates with the spore releasing process. The pellicle loses the ability to support mechanical stress, which marks the end of the pellicle life cycle and entry of the system into the dormant state.

Annulment of Bacterial Antagonism Improves Plant Beneficial Activity of a Bacillus velezensis Consortium.

Bacillus sp. strains that are beneficial to plants are widely used in commercial biofertilizers and biocontrol agents for sustainable agriculture. Generally, functional Bacillus strains are applied as single-strain communities since the principles of synthetic microbial consortia constructed with Bacillus strains remain largely unclear. Here, we demonstrated that the mutual compatibility directly affects the survival and function of two-member consortia composed of Bacillus velezensis SQR9 and FZB42 in the rhizosphere. A mutation in the global regulator Spo0A of SQR9 markedly reduced the boundary phenotype (appearance of a visible boundary line at the meeting point of two swarms) with wild-type FZB42, and the combined use of the SQR9(△spo0A) mutant and FZB42 improved biofilm formation, root colonization, and the production of secondary metabolites that are beneficial to plants. Furthermore, alleviation of antagonistic interactions of two-member Bacillus consortia improved its beneficial effects to cucumber in a greenhouse experiment. Our results provide evidence that social interactions among bacteria could be an influencing factor for achieving a desired community-level function. IMPORTANCE Bacillus velezensis is one of the most widely applied bacteria in biofertilizers in China and Europe. Additionally, the molecular mechanisms of plant growth promotion and disease suppression by representative model strains are well established, such as B. velezensis SQR9 and FZB42. However, it remains extremely challenging to design efficient consortia based on these model strains. Here, we showed that swarm encounter phenotype is one of the major determinants that affects the performance of two-member Bacillus consortia in vitro and in the rhizosphere. Deletion in global regulatory gene spo0A of SQR9 reduced the strength of boundary formation with FZB42 and resulted in the improved plant growth promotion performance of the dual consortium. This knowledge provides new insights into efficient probiotics consortia design in Bacillus spp.

Bacillus subtilis PS-216 Antagonistic Activities against Campylobacter jejuni NCTC 11168 Are Modulated by Temperature, Oxygen, and Growth Medium.

As the incidence of Campylobacter jejuni and campylobacteriosis grows, so does the need for a better understanding and control of this pathogen. We studied the interactions of C. jejuni NCTC 11168 and a potential probiotic, Bacillus subtilis PS-216, in cocultures at different starting ratios and temperatures (20 °C, 37 °C, 42 °C), under different atmospheres (aerobic, microaerobic), and in different growth media (Mueller-Hinton, chicken litter medium, chicken intestinal-content medium). Under microaerobic conditions, B. subtilis effectively inhibited the growth of C. jejuni at 42 °C (log reduction, 4.19), even when C. jejuni counts surpassed B. subtilis by 1000-fold in the starting inoculum. This inhibition was weaker at 37 °C (log reduction, 1.63), while no impact on CFUs was noted at 20 °C, which is a temperature nonpermissive of C. jejuni growth. Under aerobic conditions, B. subtilis supported C. jejuni survival. B. subtilis PS-216 inhibited the growth of C. jejuni in sterile chicken litter (4.07 log reduction) and in sterile intestinal content (2.26 log reduction). In nonsterile intestinal content, B. subtilis PS-216 was able to grow, to a lesser extent, compared to Mueller-Hinton media, still showing potential as a chicken probiotic that could be integrated into the chicken intestinal microbiota. This study showed the strong influence of environmental parameters on the variability of C. jejuni and B. subtilis interactions. Furthermore, B. subtilis PS-216 antagonism was strongest against C. jejuni NCTC 11168 under conditions that might represent conditions in the chicken environment (42 °C, microaerobic atmosphere, chicken litter medium).

Kin discrimination drives territorial exclusion during Bacillus subtilis swarming and restrains exploitation of surfactin.

Swarming is the collective movement of bacteria across a surface. It requires the production of surfactants (public goods) to overcome surface tension and provides an excellent model to investigate bacterial cooperation. Previously, we correlated swarm interaction phenotypes with kin discrimination between B. subtilis soil isolates, by showing that less related strains form boundaries between swarms and highly related strains merge. However, how kin discrimination affects cooperation and territoriality in swarming bacteria remains little explored. Here we show that the pattern of surface colonization by swarming mixtures is influenced by kin types. Closely related strain mixtures colonize the surface in a mixed swarm, while mixtures of less related strains show competitive exclusion as only one strain colonizes the surface. The outcome of nonkin swarm expansion depends on the initial ratio of the competing strains, indicating positive frequency-dependent competition. We find that addition of surfactin (a public good excreted from cells) can complement the swarming defect of nonkin mutants, whereas close encounters in nonkin mixtures lead to territorial exclusion, which limits the exploitation of surfactin by nonkin nonproducers. The work suggests that kin discrimination driven competitive territorial exclusion may be an important determinant for the success of cooperative surface colonization.

Kin discrimination promotes horizontal gene transfer between unrelated strains in Bacillus subtilis.

Bacillus subtilis is a soil bacterium that is competent for natural transformation. Genetically distinct B. subtilis swarms form a boundary upon encounter, resulting in killing of one of the strains. This process is mediated by a fast-evolving kin discrimination (KD) system consisting of cellular attack and defence mechanisms. Here, we show that these swarm antagonisms promote transformation-mediated horizontal gene transfer between strains of low relatedness. Gene transfer between interacting non-kin strains is largely unidirectional, from killed cells of the donor strain to surviving cells of the recipient strain. It is associated with activation of a stress response mediated by sigma factor SigW in the donor cells, and induction of competence in the recipient strain. More closely related strains, which in theory would experience more efficient recombination due to increased sequence homology, do not upregulate transformation upon encounter. This result indicates that social interactions can override mechanistic barriers to horizontal gene transfer. We hypothesize that KD-mediated competence in response to the encounter of distinct neighbouring strains could maximize the probability of efficient incorporation of novel alleles and genes that have proved to function in a genomically and ecologically similar context.

Surfactin Facilitates Horizontal Gene Transfer in Bacillus subtilis.

Genetic competence for the uptake and integration of extracellular DNA is a key process in horizontal gene transfer (HGT), one of the most powerful forces driving the evolution of bacteria. In several species, development of genetic competence is coupled with cell lysis. Using Bacillus subtilis as a model bacterium, we studied the role of surfactin, a powerful biosurfactant and antimicrobial lipopeptide, in genetic transformation. We showed that surfactin itself promotes cell lysis and DNA release, thereby promoting HGT. These results, therefore, provide evidence for a fundamental mechanism involved in HGT and significantly increase our understanding of the spreading of antibiotic resistance genes and diversification of microbial communities in the environment.

Peptide signaling without feedback in signal production operates as a true quorum sensing communication system in Bacillus subtilis.

Bacterial quorum sensing (QS) is based on signal molecules (SM), which increase in concentration with cell density. At critical SM concentration, a variety of adaptive genes sharply change their expression from basic level to maximum level. In general, this sharp transition, a hallmark of true QS, requires an SM dependent positive feedback loop, where SM enhances its own production. Some communication systems, like the peptide SM-based ComQXPA communication system of Bacillus subtilis, do not have this feedback loop and we do not understand how and if the sharp transition in gene expression is achieved. Based on experiments and mathematical modeling, we observed that the SM peptide ComX encodes the information about cell density, specific cell growth rate, and even oxygen concentration, which ensure power-law increase in SM production. This enables together with the cooperative response to SM (ComX) a sharp transition in gene expression level and this without the SM dependent feedback loop. Due to its ultra-sensitive nature, the ComQXPA can operate at SM concentrations that are 100-1000 times lower than typically found in other QS systems, thereby substantially reducing the total metabolic cost of otherwise expensive ComX peptide.

Identification and Characterization of a Novel Plasmid-Encoded Laccase-Like Multicopper Oxidase from Ochrobactrum sp. BF15 Isolated from an On-Farm Bio-Purification System.

RESEARCH BACKGROUND: In recent decades, laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researchers due to their wide range of biotechnological and industrial applications. Laccases can oxidize a variety of organic and inorganic compounds, making them suitable as biocatalysts in biotechnological processes. Even though the most traditionally used laccases in the industry are of fungal origin, bacterial laccases have shown an enormous potential given their ability to act on several substrates and in multiple conditions. The present study aims to characterize a plasmid-encoded laccase-like multicopper oxidase (LMCO) from Ochrobactrum sp. BF15, a bacterial strain previously isolated from polluted soil. EXPERIMENTAL APPROACH: We used in silico profile hidden Markov models to identify novel laccase-like genes in Ochrobactrum sp. BF15. For laccase characterization, we performed heterologous expression in Escherichia coli, purification and activity measurement on typical laccase substrates. RESULTS AND CONCLUSIONS: Profile hidden Markov models allowed us to identify a novel LMCO, named Lac80. In silico analysis of Lac80 revealed the presence of three conserved copper oxidase domains characteristic of three-domain laccases. We successfully expressed Lac80 heterologously in E. coli, allowing us to purify the protein for further activity evaluation. Of thirteen typical laccase substrates tested, Lac80 showed lower activity on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), pyrocatechol, pyrogallol and vanillic acid, and higher activity on 2,6-dimethoxyphenol. NOVELTY AND SCIENTIFIC CONTRIBUTION: Our results show Lac80 as a promising laccase for use in industrial applications. The present work shows the relevance of bacterial laccases and highlights the importance of environmental plasmids as valuable sources of new genes encoding enzymes with potential use in biotechnological processes.

The ComX Quorum Sensing Peptide of Bacillus subtilis Affects Biofilm Formation Negatively and Sporulation Positively.

Quorum sensing (QS) is often required for the formation of bacterial biofilms and is a popular target of biofilm control strategies. Previous studies implicate the ComQXPA quorum sensing system of Bacillus subtilis as a promoter of biofilm formation. Here, we report that ComX signaling peptide deficient mutants form thicker and more robust pellicle biofilms that contain chains of cells. We confirm that ComX positively affects the transcriptional activity of the PepsA promoter, which controls the synthesis of the major matrix polysaccharide. In contrast, ComX negatively controls the PtapA promoter, which drives the production of TasA, a fibrous matrix protein. Overall, the biomass of the mutant biofilm lacking ComX accumulates more monosaccharide and protein content than the wild type. We conclude that this QS phenotype might be due to extended investment into growth rather than spore development. Consistent with this, the ComX deficient mutant shows a delayed activation of the pre-spore specific promoter, PspoIIQ, and a delayed, more synchronous commitment to sporulation. We conclude that ComX mediated early commitment to sporulation of the wild type slows down biofilm formation and modulates the coexistence of multiple biological states during the early stages of biofilm development.

Influence of Diclofenac on Activated Sludge Bacterial Communities in Fed-Batch Reactors.

RESEARCH BACKGROUND: The occurrence and environmental toxicity of pharmaceuticals have recently attracted increasing attention. Diclofenac is a highly consumed non-steroidal anti-inflammatory drug, which is often detected in wastewaters, but investigations of its influence on bacteria are scarce. EXPERIMENTAL APPROACH: We investigated the influence of this pharmaceutical on bacterial community in activated sludge exposed to increasing concentrations of diclofenac in fed-batch reactors over 41 days. Nitrification activity of the activated sludge was measured and changes in bacterial community structure were followed using culture-independent molecular method (terminal restriction fragment length polymorphism, T-RFLP) and by the cultivation approach. RESULTS AND CONCLUSIONS: Nitrification activity was not detectably influenced by the addition of diclofenac, while the main change of the bacterial community structure was detected only at the end of incubation (after 41 days) when diclofenac was added to artificial wastewater as the only carbon source. Changes in community composition due to enrichment were observed using cultivation approach. However, taxonomic affiliation of isolates did not match taxons identified by T-RFLP community profiling. Isolates obtained from activated sludge used as inoculum belonged to five genera: Comamonas, Arthrobacter, Acinetobacter, Citrobacter and Aeromonas, known for their potential to degrade aromatic compounds. However, only Pseudomonas species were isolated after the last enrichment step on minimal agar plates with diclofenac added as the sole carbon source. NOVELTY AND SCIENTIFIC CONTRIBUTION: Our results suggest that the selected recalcitrant and commonly detected pharmaceutical does not strongly influence the sensitive and important nitrification process of wastewater treatment. Moreover, the isolated strains obtained after enrichment procedure that were able to grow on minimal agar plates with diclofenac added as the only carbon source could serve as potential model bacteria to study bacterial diclofenac degradation.

Antibiotic Bacillomycin D Affects Iron Acquisition and Biofilm Formation in Bacillus velezensis through a Btr-Mediated FeuABC-Dependent Pathway.

Bacillus spp. produce a wide range of secondary metabolites, including antibiotics, which have been well studied for their antibacterial properties but less so as signaling molecules. Previous results indicated that the lipopeptide bacillomycin D is a signal that promotes biofilm development of Bacillus velezensis SQR9. However, the mechanism behind this signaling is still unknown. Here, we show that bacillomycin D promotes biofilm development by promoting the acquisition of iron. Bacillomycin D promotes the transcription of the iron ABC transporter FeuABC by binding to its transcription factor, Btr. These actions increase intracellular iron concentration and activate the KinB-Spo0A-SinI-SinR-dependent synthesis of biofilm matrix components. We demonstrate that this strategy is beneficial for biofilm development and competition with the Pseudomonas fluorescens PF-5. Our results unravel an antibiotic-dependent signaling mechanism that links iron acquisition to biofilm development and ecological competition.