Abstract Edible mushrooms have been reported to have antimicrobial properties and other health benefits. This study aims to test the antimicrobial activities of several edible mushrooms from markets and test if co-culturing them with bacteria could induce stronger anti-bacterial properties. Commercial mushrooms, Hericium erinaceus (lion’s mane), Pleurotus ostreatus (oyster mushroom), Lentinula edodes (Shiitake) and Agaricus bisporus (button mushroom), were grown from strictly controlled/sterile substrates. Ethanol and water extracts from the mushrooms were prepared and tested against the bacteria Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus , and Bacillus subtilis , and the fungus Candida albicans for antimicrobial activities. Shiitake water extract (SWE) showed strong antibacterial effects against all tested bacterial species, inhibitory effects on their biofilms, and antifungal activity. The antimicrobials in SWE seem to damage the cell wall and cell membrane of the bacteria, prefer weak acidic conditions, and are heat labile. Some antimicrobials are likely proteins and polysaccharides. In contrast, 3 other mushrooms displayed only weak antimicrobial effects. The fast-growing lion’s mane and oyster mushroom were co-cultured with different bacteria. The co-cultivation promoted the fruiting body development of lion’s mane. Co-culturing with S. aureus increased the anti-bacterial effects of lion’s mane against S. aureus , E. coli and particularly B. subtilis . Co-culturing the oyster mushroom with bacteria, especially B. subtilis and P. aeruginosa , boosted the mushroom’s growth. All tested bacteria, especially S. aureus , increased oyster mushroom’s anti-bacterial effect against E. coli and B. subtilis . The findings indicate that mushroom-bacteria co-culturing could have benefits both agriculturally and medicinally.

The antagonistic biofilms formed by probiotic Bacilli may significantly mitigate persistent strains of Candida albicans, which are often involved in severe oral, vulvovaginal or systemic infections in humans. Here, we report on a spatiotemporal antagonistic activity mediated through pulcherriminic acid (PA) production by biofilm-forming B. subtilis, which is subsequently transported to the extracellular environment and binds ferric iron to form red-coloured pigment pulcherrimin. We show that the pulcherrimin building-up is targeted towards the C. albicans macrocolony via B. subtilis biofilm branching and successive PA relay. Furthermore, biofilm-forming B. subtilis cells demonstrate robust hyphal colonization that results subsequent eradication of C. albicans. Besides, extracted pulcherrimin mitigates C. albicans biofilm formation and yeast-to-hyphae (Y-H) transition. We further find that the mode of hyphal colonization could be regulated via SpoA-SinI pathway, while pulcherrimin relay is connected to surfactin production machinery. We assume therefore that the pulcherrimin relay for iron hijacking, in parallel to the direct hyphal colonization by biofilm-forming Bacilli, may provide a promising platform for developing therapeutic concepts to overcome antibiotic persistence in pathogenic yeasts.

Despite being one of the driest and harshest deserts on Earth, the Atacama Desert is home to a variety of bacterial life. Microorganisms that reside here may have developed adaptations to help them survive this unique environment. In this study, we used bioinformatic and genetic methods to assess the abundance of phyla that are present in this environment and what types of adaptations individual bacteria have obtained. To assess bacterial diversity, we used 16S rRNA sequencing on soil samples and determined the relative composition of different phyla and archaea at sixteen locations. A selection of eight cultivatable organisms which produce pigments were subjected to whole genome sequencing (WGS). Using these sequences, we screened for stress-tolerance capabilities including pigment production pathways, biofilm-related genes, antibiotic production, and genome stability. We found that all strains we sequenced are predicted to produce bioactive compounds. We also found that the pigments that these bacteria produce have antioxidant, iron and ion chelating, and/or antibiotic properties. This characterization allows us to assess adaptive strategies of bacteria which is important in the fields of agriculture, biotechnology and health. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACT 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.

The Atacama Desert is home to bacteria that use biofilms as a means of protecting themselves against the harsh environment. To inform research regarding this survival mechanism, we cultured and sequenced the genomes of three Bacillus sp. isolates from Atacama Desert soil.

Phenazines are highly prevalent, natural bioactive substances secreted by microbes. However, their mode of action and potential involvement in shaping microbiomes remain elusive. Here we performed a comprehensive analysis of over 1.35 million bacterial genomes to identify phenazine-producing bacteria distributed across 193 species in 34 families. Analysis of rhizosphere microbiome and public rhizosphere metagenomic datasets revealed that phenazines could shape the microbial community by inhibiting Gram-positive bacteria, which was verified by pairwise interaction assays using Phenazine-1-carboxamide (PCN)-producing Pseudomonas chlororaphis. PCN induced DNA damage in Bacillus subtilis, a model Gram-positive target, where it directly bound to the bacterial topoisomerase IV, inhibiting its decatenation activity and leading to cell death. A two-species consortium of phenazine-producing Pseudomonas and resistant B. subtilis exhibited superior synergistic activity in preventing Fusarium crown rot in wheat plants. This work advances our understanding of a prevalent microbial interaction and its potential for biocontrol.

Janthinobacterium is a genus of Gram-negative environmental bacteria that survive extreme conditions by forming biofilms and producing pigments. Janthinobacterium sp. LS2A, an extremophile isolated from soil in the Chilean Patagonia, contains seven known biosynthetic gene clusters, including the purple pigment violacein, which may aid in its survival in harsh environments.

In Bacillus subtilis, biofilm exopolysaccharide (EPS) is made by a 15-gene operon (epsA-O) that assembles, exports, and polymerizes EPS subunits. EpsA, encoded by the first gene in the operon, has multiple functions, including acting as a regulatory protein working in conjunction with the cytoplasmic tyrosine kinase EpsB. EpsA is a transmembrane protein that has an extracellular signal-sensing domain. Published work shows that purified EPS is able to interact with the EpsA signal sensing domain, suggesting a feedback regulation during EPS biosynthesis. In this study, we propose that oligosaccharide analogs, like aminoglycoside antibiotics, may inhibit biofilm formation by interrupting this pathway. Methods In this study, we constructed Bacillus subtilis strains that are resistant to different antibiotics, including kanamycin, neomycin, ampicillin, erythromycin, and spectinomycin, by adding resistance cassettes at the amyE site of the B. subtilis genome. We performed biofilm growth assays of wild-type and antibiotic resistant B. subtilis in the presence of their respective antibiotic types in biofilm inducing medium. To investigate the EPS produced by biofilms with and without antibiotics, we used light scattering to determine the lengths of the EPS chains produced by biofilms grown from kanamycin-resistant cells in the presence and absence of kanamycin. We also performed a sulfuric acid assay to quantify the amount of EPS produced by the strain in the same conditions. A growth curve comparing the growth of this strain was also performed to ensure that using kanamycin in the kanamycin resistant culture wasn't impacting growth. Results In our biofilm growth assays, biofilms that were grown in kanamycin and neomycin were defective and didn't form whereas biofilms grown in other antibiotics grew similar to wild-type biofilm. To further investigate why this may have happened, the EPS produced by kanamycin-resistant B. subtilis in the presence and absence of kanamycin was analyzed. Compared to wild-type, the amount of EPS produced by the kanamycin-resistant culture in the presence of kanamycin was significantly less than in the same strain without kanamycin. Additionally, the lengths of the EPS chains were significantly reduced. In shaking conditions, the growth curves for the strain with and without the presence of kanamycin were the same. Conclusion In our preliminary studies, we show that aminoglycoside antibiotics that exhibit a sugar-like structure, kanamycin and neomycin, inhibit biofilm formation even if the strain is resistant to the respective antibiotic. This inhibition was specific to aminoglycosides, not other antibiotic types. After further investigation of the EPS molecules produced by strains exposed to kanamycin by light scattering and sulfuric acid assays, it was determined that the presence of kanamycin greatly reduced EPS production and the EPS chains produced were of shorter length. We hypothesize that the oligosaccharide-like aminoglycosides are able to interfere with EPS polymerization, without impacting overall cell growth, as showed by a growth curve. This work reveals a potential second mode of action of aminoglycoside antibiotics by showing their ability to inhibit both cell growth, acting inside of the cell, and biofilm formation, acting outside of the cell on EPS biosynthesis. This work was supported by the National Science Foundation and the NSF Graduate Research Fellowship Program.

Summary Eukaryotes have evolved sophisticated post‐translational modifications to regulate protein function and numerous biological processes, including ubiquitination controlled by the coordinated action of ubiquitin‐conjugating enzymes and deubiquitinating enzymes (Dubs). However, the function of deubiquitination in pathogenic fungi is largely unknown. Here, the distribution of Dubs in the fungal kingdom was surveyed and their functions were systematically characterized using the phytopathogen Fusarium graminearum as the model species, which causes devastating diseases of all cereal species world‐wide. Our findings demonstrate that Dubs are critical for fungal development and virulence, especially the ubiquitin‐specific protease 15 (Ubp15). Global ubiquitome analysis and subsequent experiments identified three important substrates of Ubp15, including the autophagy‐related protein Atg8, the mitogen‐activated protein kinase Gpmk1, and the mycotoxin deoxynivalenol (DON) biosynthetic protein Tri4. Ubp15 regulates the deubiquitination of the Atg8, thereby impacting its subcellular localization and the autophagy process. Moreover, Ubp15 also modulates the deubiquitination of Gpmk1 and Tri4. This modulation subsequently influences their protein stabilities and further affects the formation of penetration structures and the biosynthetic process of DON, respectively. Collectively, our findings reveal a previously unknown regulatory pathway of a deubiquitinating enzyme for fungal virulence and highlight the potential of Ubp15 as a target for combating fungal diseases.

Currently, almost all known regulators involved in bacterial phosphorus metabolism are proteins. In this study, we identified a conserved new small regulatory RNA (sRNA), named PhoS, encoded in the 3’ untranslated region (UTR) of the phoPR genes in Bacillus velezensis and B. subtilis. Expression of phoS is strongly induced upon phosphorus scarcity and stimulated by the transcription factor PhoP. Conversely, PhoS positively regulates PhoP translation by binding to the ribosome binding site (RBS) of phoP mRNA. PhoS can promote Bacillus biofilm formation through, at least in part, enhancing the expression of the matrix-related genes, such as the eps genes and the tapA-sipW-tasA operon. The positive regulation of phoP expression by PhoS contributes to the promoting effect of PhoS on biofilm formation. sRNAs regulating biofilm formation have rarely been reported in gram-positive Bacillus species. Here we highlight the significance of sRNAs involved in two important biological processes: phosphate metabolism and biofilm formation.

The Atacama Desert, the driest, with the highest radiation, and one of the most ancient deserts in the world, is a hostile environment for life. We have a collection of 74 unique bacterial isolates after cultivation and confirmation by 16S rRNA gene sequencing. Pigmentation, biofilm formation, antimicrobial production against Escherichia coli MG1655 and Staphylococcus aureus HG003, and antibiotic resistance were assessed on these isolates. We found that approximately a third of the colonies produced pigments, 80% of isolates formed biofilms, many isolates produce growth inhibiting activities against E. coli and/or S. aureus, and many were resistant to antibiotics. The functional characterization of these isolates gives us insight into the adaptive bacterial strategies in harsh environments and enables us to learn about their possible use in agriculture, healthcare, or biotechnology.

The cell wall is a foundational structure in many bacterial cells, functioning to give the cell its shape and mediate the stress from the pressure of turgor. In gram-positive bacteria, such as Bacillus subtilis, the cell wall is decorated with cross-linked peptidoglycan, proteins, and anionic polymers known as teichoic and teichuronic acids. In B. subtilis, it is known that teichoic acids abundantly decorate the cell wall, whereas teichuronic acids are only present in phosphate-limiting conditions. Overall, these anionic polymers have functions that are important for cell function. Firstly, they provide an overall negative charge to the cell wall, allowing them to act as a reservoir for cations. They've also been shown to regulate autolysin activity, scaffold cell-surface proteins, and serve as receptors for the binding of phages. In this project, we hypothesize that teichuronic acid has a potential novel role in biofilm formation and development. To study this, we created a knock-out of the operon responsible for the biosynthesis of teichuronic acid, that which we observed that there's a delay in biofilm formation and an earlier biofilm disassembly compared to the wild-type. We also observed that eliminating teichuronic acid led to an impaired efficacy of bacteria colonizing the root of plants and a significantly lesser sporulation efficiency. Finally, we observe that teichuronic acid is involved in the regulation of pulcherrimin production in B. subtilis, which is a biofilm-associated pigment. Based on our preliminary results, we propose a potential novel function of teichuronic acid, an under-characterized anionic polymer, in biofilm formation and development and the regulation of pulcherrimin in B. subtilis. This project was provided substantial support by the Office of Undergraduate Research and Fellowships (URF) at Northeastern University.

AbstractThe Atacama Desert is home to bacteria that use biofilms as a means of protecting themselves against the harsh environment. We cultured and sequenced the genomes of threeBacillus sp. isolates from the soil of the Atacama Desert. This information will inform research in survival mechanisms of eubacteria in the Atacama Desert.

Bacillus velezensis FZB42 is a plant growth-promoting rhizobacterium (PGPR) and a model microorganism for biofilm studies. Biofilms are required for the colonization and promotion of plant growth in the rhizosphere. However, little is known about how the final stage of the biofilm life cycle is regulated, when cells regain their motility and escape the mature biofilm to spread and colonize new niches. In this study, the non-annotated gene ccdC was found to be involved in the process of biofilm dispersion. We found that the ccdC-deficient strain maintained a wrinkled state at the late stage of biofilm formation in the liquid—gas interface culture, and the bottom solution showed a clear state, indicating that no bacterial cells actively escaped, which was further evidenced by the formation of a cellular ring (biofilm pellicle) located on top of the preformed biofilm. It can be concluded that dispersal, a biofilm property that relies on motility proficiency, is also positively affected by the unannotated gene ccdC. Furthermore, we found that the level of cyclic diguanylate (c-di-GMP) in the ccdC-deficient strain was significantly greater than that in the wild-type strain, suggesting that B. velezensis exhibits a similar mechanism by regulating the level of c-di-GMP, the master regulator of biofilm formation, dispersal, and cell motility, which controls the fitness of biofilms in Pseudomonas aeruginosain. In this study, we investigated the mechanism regulating biofilm dispersion in PGPR.

Infections caused by Acinetobacter baumannii, a Gram-negative opportunistic pathogen, are difficult to eradicate due to the bacterium's propensity to quickly gain antibiotic resistances and form biofilms, a protective bacterial multicellular community. The A. baumannii DNA damage response (DDR) mediates the antibiotic resistance acquisition and regulates RecA in an atypical fashion; both RecALow and RecAHigh cell types are formed in response to DNA damage. The findings of this study demonstrate that the levels of RecA can influence formation and dispersal of biofilms. RecA loss results in surface attachment and prominent biofilms, while elevated RecA leads to diminished attachment and dispersal. These findings suggest that the challenge to treat A. baumannii infections may be explained by the induction of the DDR, common during infection, as well as the delicate balance between maintaining biofilms in low RecA cells and promoting mutagenesis and dispersal in high RecA cells. This study underscores the importance of understanding the fundamental biology of bacteria to develop more effective treatments for infections.

Pulcherriminic acid (PA) is a cyclic-L-leu-L-leu di-peptide produced by Bacillus subtilis during biofilm formation. When secreted, PA strongly chelates extracellular iron and forms a reddish- brown pigment, pulcherrimin. Production of pulcherriminic acid and formation of pulcherrimin modulate iron homeostasis in B. subtilis . Pulcherriminic acid also functions as an antioxidant to protect cells from increasing oxidative stress during biofilm formation. We previously showed that PA is involved in gene regulation, differentially regulating hundreds of genes in B. subtilis . One of the strongly upregulated genes by PA is yhjH , encoding a putative MarR-type transcription repressor. In this study, we characterized the regulation of the yhjH gene by PA, by PchR, a known transcription repressor for PA biosynthesis, and by YhjH itself. We also found that high expression of yhjH triggers rapid cell lysis in B. subtilis . Results from RNA-seq suggest that YhjH differentially regulates about 180 genes, among which there is a significant number of prophage genes. Lastly, we propose that YhjH be re-named as PcdR, for “pulcherriminic acid cell death regulator”. ### Competing Interest Statement The authors have declared no competing interest.

Pulcherrimin is an iron-binding reddish pigment produced by various bacterial and yeast species. In the soil bacterium Bacillus subtilis, this pigment is synthesized intracellularly as the colorless pulcherriminic acid by using two molecules of tRNA-charged leucine as the substrate; pulcherriminic acid molecules are then secreted and bind to ferric iron extracellularly to form the red-colored pigment pulcherrimin. The biological importance of pulcherrimin is not well understood. A previous study showed that secretion of pulcherrimin caused iron depletion in the surroundings and growth arrest on cells located at the edge of a B. subtilis colony biofilm. In this study, we identified that pulcherrimin is primarily produced under biofilm conditions and provides protection to cells in the biofilm against oxidative stress. We presented molecular evidence on how pulcherrimin lowers the level of reactive oxygen species (ROS) and alleviates oxidative stress and DNA damage caused by ROS accumulation in a mature biofilm. We also performed global transcriptome profiling to identify differentially expressed genes in the pulcherrimin-deficient mutant compared with the wild type, and further characterized the regulation of genes by pulcherrimin that are related to iron homeostasis, DNA damage response (DDR), and oxidative stress response. Based on our findings, we propose pulcherrimin as an important antioxidant that modulates B. subtilis biofilm development.