Chemical Mediators at the Bacterial-Fungal Interface.

Interactions among microbes are key drivers of evolutionary progress and constantly shape ecological niches. Microorganisms rely on chemical communication to interact with each other and surrounding organisms. They synthesize natural products as signaling molecules, antibiotics, or modulators of cellular processes that may be applied in agriculture and medicine. Whereas major insight has been gained into the principles of intraspecies interaction, much less is known about the molecular basis of interspecies interplay. In this review, we summarize recent progress in the understanding of chemically mediated bacterial-fungal interrelations. We discuss pairwise interactions among defined species and systems involving additional organisms as well as complex interactions among microbial communities encountered in the soil or defined as microbiota of higher organisms. Finally, we give examples of how the growing understanding of microbial interactions has contributed to drug discovery and hypothesize what may be future directions in studying and engineering microbiota for agricultural or medicinal purposes.

Large-Scale Identification of Known and Novel RRNPP Quorum-Sensing Systems by RRNPP_Detector Captures Novel Features of Bacterial, Plasmidic, and Viral Coevolution.

Gram-positive Firmicutes bacteria and their mobile genetic elements (plasmids and bacteriophages) encode peptide-based quorum-sensing systems (QSSs) that orchestrate behavioral transitions as a function of population densities. In their simplest form, termed "RRNPP", these QSSs are composed of two adjacent genes: a communication propeptide and its cognate intracellular receptor. RRNPP QSSs notably regulate social/competitive behaviors such as virulence or biofilm formation in bacteria, conjugation in plasmids, or lysogeny in temperate bacteriophages. However, the genetic diversity and the prevalence of these communication systems, together with the breadth of behaviors they control, remain largely underappreciated. To better assess the impact of density dependency on microbial community dynamics and evolution, we developed the RRNPP_detector software, which predicts known and novel RRNPP QSSs in chromosomes, plasmids, and bacteriophages of Firmicutes. Applying RRNPP_detector against available complete genomes of viruses and Firmicutes, we identified a rich repertoire of RRNPP QSSs from 11 already known subfamilies and 21 novel high-confidence candidate subfamilies distributed across a vast diversity of taxa. The analysis of high-confidence RRNPP subfamilies notably revealed 14 subfamilies shared between chromosomes/plasmids/phages, 181 plasmids and 82 phages encoding multiple communication systems, phage-encoded QSSs predicted to dynamically modulate bacterial behaviors, and 196 candidate biosynthetic gene clusters under density-dependent regulation. Overall, our work enhances the field of quorum-sensing research and reveals novel insights into the coevolution of gram-positive bacteria and their mobile genetic elements.

Review on computer-assisted biosynthetic capacities elucidation to assess metabolic interactions and communication within microbial communities.

Microbial communities thrive through interactions and communication, which are challenging to study as most microorganisms are not cultivable. To address this challenge, researchers focus on the extracellular space where communication events occur. Exometabolomics and interactome analysis provide insights into the molecules involved in communication and the dynamics of their interactions. Advances in sequencing technologies and computational methods enable the reconstruction of taxonomic and functional profiles of microbial communities using high-throughput multi-omics data. Network-based approaches, including community flux balance analysis, aim to model molecular interactions within and between communities. Despite these advances, challenges remain in computer-assisted biosynthetic capacities elucidation, requiring continued innovation and collaboration among diverse scientists. This review provides insights into the current state and future directions of computer-assisted biosynthetic capacities elucidation in studying microbial communities.

Computer-assisted biosynthetic capacities elucidation accelerates our ability to interpret microbial interactions, allowing us to understand better and establish a balance within ecosystems.

Chemotactic Interactions of Scenedesmus sp. and Azospirillum brasilense Investigated by Microfluidic Methods.

The use of algae for industrial, biotechnological, and agricultural purposes is spreading globally. Scenedesmus species can play an essential role in the food industry and agriculture due to their favorable nutrient content and plant-stimulating properties. Previous research and the development of Scenedesmus-based foliar fertilizers raised several questions about the effectiveness of large-scale algal cultivation and the potential effects of algae on associative rhizobacteria. In the microbiological practice applied in agriculture, bacteria from the genus Azospirillum are one of the most studied plant growth-promoting, associative, nitrogen-fixing bacteria. Co-cultivation with Azospirillum species may be a new way of optimizing Scenedesmus culturing, but the functioning of the co-culture system still needs to be fully understood. It is known that Azospirillum brasilense can produce indole-3-acetic acid, which could stimulate algae growth as a plant hormone. However, the effect of microalgae on Azospirillum bacteria is unclear. In this study, we investigated the behavior of Azospirillum brasilense bacteria in the vicinity of Scenedesmus sp. or its supernatant using a microfluidic device consisting of physically separated but chemically coupled microchambers. Following the spatial distribution of bacteria within the device, we detected a positive chemotactic response toward the microalgae culture. To identify the metabolites responsible for this behavior, we tested the chemoeffector potential of citric acid and oxaloacetic acid, which, according to our HPLC analysis, were present in the algae supernatant in 0.074 mg/ml and 0.116 mg/ml concentrations, respectively. We found that oxaloacetic acid acts as a chemoattractant for Azospirillum brasilense.

Effects of potassium-mediated electrical communication inhibition on nitrogen removal in microbial fuel cells.

Potassium ion signaling mediates microbial communication in electroactive biofilms within microbial fuel cells (MFCs), but its role in nitrogen removal remains unclear. This study investigated the impact of inhibiting potassium signaling on nitrogen removal in MFCs using tetraethylammonium chloride (TEA) as an inhibitor. Results demonstrated that 5 mM and 10 mM TEA reduced the maximum power generation of MFCs from 77.95 mW/cm2 to 57.18 mW/cm2 and 48.23 mW/cm2, respectively. Correspondingly, total nitrogen (TN) removal efficiency was decreased from 46.57 ± 1.01% to 35.93 ± 0.63% and 38.97 ± 0.74%, respectively. This decline was attributed to inhibited potassium ion signaling, which compromised the electrochemical performance of the MFC and hindered the nitrogen removal process. The relative abundance of exoelectrogen Geobactor decreased from 15.37% to 5.17% and 8.05%, while the relative abundance of cathodic nitrifying bacteria Nitrosomonas decreased from 17.87% to 4.92% and 3.63% under 5 mM and 10 mM TEA. These findings underscore the crucial role of potassium ion signaling in enhancing the bioelectrochemical nitrogen removal process in MFCs.

Characterizing chemical signaling between engineered "microbial sentinels" in porous microplates.

Living materials combine a material scaffold, that is often porous, with engineered cells that perform sensing, computing, and biosynthetic tasks. Designing such systems is difficult because little is known regarding signaling transport parameters in the material. Here, the development of a porous microplate is presented. Hydrogel barriers between wells have a porosity of 60% and a tortuosity factor of 1.6, allowing molecular diffusion between wells. The permeability of dyes, antibiotics, inducers, and quorum signals between wells were characterized. A "sentinel" strain was constructed by introducing orthogonal sensors into the genome of Escherichia coli MG1655 for IPTG, anhydrotetracycline, L-arabinose, and four quorum signals. The strain's response to inducer diffusion through the wells was quantified up to 14 mm, and quorum and antibacterial signaling were measured over 16 h. Signaling distance is dictated by hydrogel adsorption, quantified using a linear finite element model that yields adsorption coefficients from 0 to 0.1 mol m-3 . Parameters derived herein will aid the design of living materials for pathogen remediation, computation, and self-organizing biofilms.

Effect of different vacuum levels for beef brisket during cold storage: A microbiological and physicochemical analysis.

Effect of packaging at different vacuum levels such as 7.2 Pa (99.99% vacuum), 30 kPa (70.39%), 70 kPa (30.91%), and 101.33 kPa (0%, atmospheric condition) using a specially designed airtight container on physicochemical and microbial properties of beef brisket cuts during cold storage was investigated. Dramatic pH increase was found only in air atmospheric packaging. Higher vacuum level yielded higher water holding capacity and lower volatile basic nitrogen (VBN), 2-thiobarbituric acid (TBA), and growth rate of aerobic bacteria and coliforms, whereas the fatty acid composition showed no difference among various vacuum levels. The highest vacuum level (7.2 Pa) yielded no increases in VBN, TBA, and coliform and the least increase in aerobe counts. For bacterial communities, higher vacuum levels yielded higher proportions of Leuconostoc, Carnobacterium, and lactobacilli belonging to the phylum Firmicutes and lower proportions of Pseudomonas belonging to the phylum Proteobacteria. Predictive curves for bacterial communities showed that just a little oxygen significantly affects the bacterial dominance based on different oxygen dependence of individual bacteria and their logarithmic changes by vacuum level.

GNPS-Guided Discovery of Madurastatin Siderophores from the Termite-Associated Actinomadura sp. RB99.

In this study, we analyzed if Actinomadura sp. RB99 produces siderophores that that could be responsible for the antimicrobial activity observed in co-cultivation studies. Dereplication of high-resolution tandem mass spectrometry (HRMS/MS) and global natural product social molecular networking platform (GNPS) analysis of fungus-bacterium co-cultures resulted in the identification of five madurastatin derivatives (A1, A2, E1, F, and G1), of which were four new derivatives. Chemical structures were unambiguously confirmed by HR-ESI-MS, 1D and 2D NMR experiments, as well as MS/MS data and their absolute structures were elucidated based on Marfey's analysis, DP4+ probability calculation and total synthesis. Structure analysis revealed that madurastatin E1 (2) contained a rare 4-imidazolidinone cyclic moiety and madurastatin A1 (5) was characterized as a Ga3+ -complex. The function of madurastatins as siderophores was evaluated using the fungal pathogen Cryptococcus neoformans as model organism. Based on homology models, we identified the putative NRPS-based gene cluster region of the siderophores in Actinomadura sp. RB99.

Structural and Functional Analysis of Bacterial Sulfonosphingolipids and Rosette-Inducing Factor 2 (RIF-2) by Mass Spectrometry-Guided Isolation and Total Synthesis.

We have analyzed the abundance of bacterial sulfonosphingolipids, including rosette-inducing factors (RIFs), in seven bacterial prey strains by using high-resolution tandem mass spectrometry (HRMS2 ) and molecular networking (MN) within the Global Natural Product Social Molecular Networking (GNPS) web platform. Six sulfonosphingolipids resembling RIFs were isolated and their structures were elucidated based on comparative MS and NMR studies. Here, we also report the first total synthesis of two RIF-2 diastereomers and one congener in 15 and eight synthetic steps, respectively. For the total synthesis of RIF-2 congeners, we employed a decarboxylative cross-coupling reaction to synthesize the necessary branched α-hydroxy fatty acids, and the Garner-aldehyde approach to generate the capnine base carrying three stereogenic centers. Bioactivity studies in the choanoflagellate Salpingoeca rosetta revealed that the rosette inducing activity of RIFs is inhibited dose dependently by the co-occurring sulfonosphingolipid sulfobacins D and F and that activity of RIFs is specific for isolates obtained from Algoriphagus.

Multispecies biofilms in fermentation: Biofilm formation, microbial interactions, and communication.

Food fermentation is driven by microorganisms, which usually coexist as multispecies biofilms. The activities and interactions of functional microorganisms and pathogenic bacteria in biofilms have important implications for the quality and safety of fermented foods. It was verified that the biofilm lifestyle benefited the fitness of microorganisms in harsh environments and intensified the cooperation and competition between biofilm members. This review focuses on multispecies biofilm formation, microbial interactions and communication in biofilms, and the application of multispecies biofilms in food fermentation. Microbial aggregation and adhesion are important steps in the early stage of multispecies biofilm formation. Different biofilm-forming abilities and strategies among microorganisms lead to several types of multispecies biofilm formation. The spatial distribution of multispecies biofilms reflects microbial interactions and biofilm function. Then, we discuss the intrinsic factors and external manifestations of multispecies biofilm system succession. Several typical interspecies cooperation and competition modes and mechanisms of microbial communication were reviewed in this review. The main limitations of the studies included in this review are the relatively small number of studies of biofilms formed by functional microorganisms during fermentation and the lack of direct evidence for the formation process of multispecies biofilms and microbial interactions and communication within biofilms. This review aims to provide the food industry with a sufficient understanding of multispecies biofilms in food fermentation. Practical Application: Meanwhile, it offers a reference value for better controlling and utilizing biofilms during food fermentation process, and the improvement of the yield, quality, and safety of fermented products including Chinese Baijiu, cheeese,kefir, soy sauce, kombucha, and fermented olive.

Setting the Stage: Genes Controlling Mechanosensation and Ca2+ Signaling in Escherichia coli.

Although mechanistic understanding of calcium signaling in bacteria remains inchoate, current evidence clearly links Ca2+ signaling with membrane potential and mechanosensation. Adopting a radically new approach, Luder et al. scanned the Keio collection of Escherichia coli gene knockouts (R. Luder, G. N. Bruni, and J. M. Kralj, J Bacteriol 203:e00509-20, 2021, https://doi.org/10.1128/JB.00509-20) to identify mutations that cause changes in Ca2+ transients. They identify genes associating Ca2+ signaling with outer membrane biogenesis, proton motive force, and, surprisingly, long-term DNA damage. Their work has major implications for electrophysiological communication between bacteria and their environment.

Uncovering potential interspecies signaling factors in plant-derived mixed microbial culture.

Microbes use signaling factors for intraspecies and interspecies communications. While many intraspecies signaling factors have been found and characterized, discovery of factors for interspecies communication is lagging behind. To facilitate the discovery of such factors, we explored the potential of a mixed microbial culture (MMC) derived from wheatgrass, in which heterogeneity of this microbial community might elicit signaling factors for interspecies communication. The stability of Wheatgrass MMC in terms of community structure and metabolic output was first characterized by 16S ribosomal RNA amplicon sequencing and liquid chromatography/mass spectrometry (LC/MS), respectively. In addition, detailed MS analyses led to the identification of 12-hydroxystearic acid (12-HSA) as one of the major metabolites produced by Wheatgrass MMC. Stereochemical analysis revealed that Wheatgrass MMC produces mostly the (R)-isomer, although a small amount of the (S)-isomer was also observed. Furthermore, 12-HSA was found to modulate planktonic growth and biofilm formation of various marine bacterial strains. The current study suggests that naturally derived MMCs could serve as a simple and reproducible platform to discover potential signaling factors for interspecies communication. In addition, the study indicates that hydroxylated long-chain fatty acids, such as 12-HSA, may constitute a new class of interspecies signaling factors.

Animated Guide to Represent a Novel Means of Gut-Brain Axis Communication.

Novel scientific concepts must be made understandable to allow their further development, highlighting the need for better communication of abstract ideas that these discoveries are built upon. This project focused on visually communicating the discovery of microbiome-derived molecules that play a major role in microbiome-gut-brain axis communication through multimedia learning.A 4-min animated video that was segmented and used a combination of 2D and 3D models was created. It communicated the important information about the process of discovering the molecules in mouse models, their production by bacteria and their potential implications for human health. The animation was then provided to a scientific audience, alongside a short-answer survey and a Likert scale, to assess how visual aspects accompanied with narration compare to learning and comprehension of the same content if it is read.The findings are based on the total of 15 participants, 9 of which were exposed to the information via animation (Group A) and 6 who were given information in a form of written narrative (Group B). It was found that Group A scored average M = 15 (out of 25) on the post assessment compared to Group B with an average of M = 7. Higher scores correlated with higher rating on questions about perceived understanding through animated media. Additionally, the animation scored higher on helpfulness in learning abstract ideas, especially having to do with structure and spatial navigation. This indicates that scientific abstract concepts that are likely comprehended are needed in order to make definite conclusions.

Microbial consortia: a critical look at microalgae co-cultures for enhanced biomanufacturing.

Monocultures have been the preferred production route in the bio-industry, where contamination has been a major bottleneck. In nature, microorganisms usually exist as part of organized communities and consortia, gaining benefits from co-habitation, keeping invaders at bay. There is increasing interest in the use of co-cultures to tackle contamination issues, and simultaneously increase productivity and product diversity. The feasibility of extending the natural phenomenon of co-habitation to the biomanufacturing industry in the form of co-cultures requires careful and systematic consideration of several aspects. This article will critically examine and review current work on microbial co-cultures, with the intent of examining the concept and proposing a design pipeline that can be developed in a biomanufacturing context.

Plant root-microbe communication in shaping root microbiomes.

A growing body of research is highlighting the impacts root-associated microbial communities can have on plant health and development. These impacts can include changes in yield quantity and quality, timing of key developmental stages and tolerance of biotic and abiotic stresses. With such a range of effects it is clear that understanding the factors that contribute to a plant-beneficial root microbiome may prove advantageous. Increasing demands for food by a growing human population increases the importance and urgency of understanding how microbiomes may be exploited to increase crop yields and reduce losses caused by disease. In addition, climate change effects may require novel approaches to overcoming abiotic stresses such as drought and salinity as well as new emerging diseases. This review discusses current knowledge on the formation and maintenance of root-associated microbial communities and plant-microbe interactions with a particular emphasis on the effect of microbe-microbe interactions on the shape of microbial communities at the root surface. Further, we discuss the potential for root microbiome modification to benefit agriculture and food production.

Use of sulfur-oxidizing bacteria as recognition elements in hydrogen sulfide biosensing system.

Four sulfur-oxidizing bacteria (Thiobacillus thioparus, Acidithiobacillus thiooxidans PTCC1717, Acidithiobacillus ferrooxidans PTCC1646, and Acidithiobacillus ferrooxidans PTCC1647) were used as biorecognition elements in a hydrogen sulfide biosensing system. All the experiments were performed in 0.1 M phosphate buffer solution containing 1-20 ppm H2S with optimum pH and temperature for each species. Although H2 S was applied to the biosensing system, the dissolved O2 content decreased. Dissolved O2 consumed by cells in both free and immobilized forms was measured using a dissolved oxygen sensor. Free bacterial cells exhibit fast response (<200 Sec). Immobilization of the cells on polyvinyl alcohol was optimized using an analytical software. Immobilized A. ferrooxidans and A. thiooxidans retained more than 50% of activity after 30 days of immobilization. According to the data, A. thiooxidans and A. ferrooxidans are appropriate species for hydrogen sulfide biosensor.

Quorum sensing bacteria improve microbial networks stability and complexity in wastewater treatment plants.

Quorum-sensing bacteria (QSB) are crucial factors for microbial communication, yet their ecological role in wastewater treatment plants (WWTPs) remains unclear. Here, we developed a method to identify QSB by comparing 16S rRNA gene sequences. QSB in 388 activated sludge samples collected from 130 WWTPs across China primarily were identified as rare taxa and conditionally rare taxa. A co-occurrence network shared by all sludge communities revealed that QSB exhibited higher average clustering coefficient (0.46) than non-QSB (0.15). Individual sludge networks demonstrated that quorum sensing microbiomes were positively correlated with network robustness and network complexity, including average clustering coefficient and link density. We confirmed that QSB keystones and QSB nodes have a positive impact on network complexity by influencing network modularity through a structural equation model. Meanwhile, QSB communities directly contributed to maintaining network robustness (r = 0.29, P < 0.05). Hence, QSB play an important role in promoting network complexity and stability. Furthermore, QSB communities were positively associated with the functional composition of activated sludge communities (r = 0.33, P < 0.01), especially the denitrification capacity (r = 0.45, P < 0.001). Overall, we elucidated the ecological significance of QSB and provided support for QS-based regulation of activated sludge microbial communities.

RNA:DNA triplexes: a mechanism for epigenetic communication between hosts and microbes?

Molecular communication between host and microbe is mediated by the transfer of many different classes of macromolecules. Recently, the trafficking of RNA molecules between organisms has gained prominence as an efficient way to manipulate gene expression via RNA interference (RNAi). Here, we posit a new epigenetic control mechanism based on triple helix (triplex) structures comprising nucleic acids from both host and microbe. Indeed, RNA:DNA triplexes are known to regulate gene expression in humans, but it is unknown whether interkingdom triplexes are formed either to manipulate host processes during pathogenesis or as a host defense response. We hypothesize that a fraction of the extracellular RNAs commonly released by microbes (e.g., bacteria, fungi, and protists) and their hosts form triplexes with the genome of the other species, thereby impacting chromatin conformation and gene expression. We invite the field to consider interkingdom triplexes as unexplored weaponry in the arms race between host and microbe.

Amino Acid-Derived Bacterial Metabolites in the Colorectal Luminal Fluid: Effects on Microbial Communication, Metabolism, Physiology, and Growth.

Undigested dietary and endogenous proteins, as well as unabsorbed amino acids, can move from the terminal part of the ileum into the large intestine, where they meet a dense microbial population. Exfoliated cells and mucus released from the large intestine epithelium also supply nitrogenous material to this microbial population. The bacteria in the large intestine luminal fluid release amino acids from the available proteins, and amino acids are then used for bacterial protein synthesis, energy production, and in other various catabolic pathways. The resulting metabolic intermediaries and end products can then accumulate in the colorectal fluid, and their concentrations appear to depend on different parameters, including microbiota composition and metabolic activity, substrate availability, and the capacity of absorptive colonocytes to absorb these metabolites. The aim of the present review is to present how amino acid-derived bacterial metabolites can affect microbial communication between both commensal and pathogenic microorganisms, as well as their metabolism, physiology, and growth.

Hierarchical encapsulation of bacteria in functional hydrogel beads for inter- and intra- species communication.

To sequester prokaryotic cells in a biofilm-like niche, the creation of a pertinent and reliable microenvironment that reflects the heterogeneous nature of biological systems is vital for sustenance. Design of a microenvironment that is conducive for growth and survival of organisms, should account for factors such as mass transport, porosity, stability, elasticity, size, functionality, and biochemical characteristics of the organisms in the confined architecture. In this work we present an artificial long-term confinement model fabricated by natural alginate hydrogels that are structurally stable and can host organisms for over 10 days in physiologically relevant conditions. A unique feature of the confinement platform is the development of stratified habitats wherein bacterial cells can be entrapped in the core as well as in the shell layers, wherein the thickness and the number of shell layers are tunable at fabrication. We show that the hydrogel microenvironment in the beads can host complex subpopulations of organisms similar to that in a biofilm. Dynamic interaction of bacterial colonies encapsulated in different beads or within the core and stratified layers of single beads was demonstrated to show intra- species communication. Inter- species communication between probiotic bacteria and human colorectal carcinoma cells was also demonstrated to highlight a possible bidirectional communication between the organisms in the beads and the environment. STATEMENT OF SIGNIFICANCE: Bacteria confinement in a natural soft hydrogel structure has always been a challenge due to the collapse of hydrogel architectures. Alternative methods have been attempted to encapsulate microorganisms by employing various processes to avoid/minimize rupturing of hydrogel structures. However, most of the past approaches have been unfavorable in balancing cell proliferation and functionality upon confinement. Our study addresses the fundamental gap in knowledge necessary to create favorable and complex 3D biofilm mimics utilizing natural hydrogel for microbial colonization for long-term studies. Our approach represents a cornerstone in the development of 3D functional architectures not only to advance studies in microbial communication, host-microbe interaction but also to address basic and fundamental questions in biology.