A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa
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Shah, Megha↗
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An Autoinducer Analogue Reveals an Alternative Mode of Ligand Binding for the LasR Quorum-Sensing Receptor
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99 GENERAL AND MISCELLANEOUS↗
Mechanism underlying autoinducer recognition in the Vibrio cholerae DPO-VqmA quorum-sensing pathway
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99 GENERAL AND MISCELLANEOUS↗
Discovery of Signaling Small Molecules (e.g. quorum sensing molecules) from the Microbiome
Microbial communities are shaped through the interactions between their microbial members and the environment (microbe-microbe and host-microbe interactions). Signal transduction pathways in the microbiome are often modulated through the small molecule products of microbial biosynthetic gene clusters (BGCs). Advances in 16S rRNA profiling and shotgun metagenomics have revolutionized our understanding about the microbial composition of various communities and their BGCs. Environmental metagenomes contain thousands of BGCs with uncharacterized small molecule products that potentially play roles in signal transduction. The overarching aim of this proposal was to develop computational techniques for discovering these small molecules and characterizing their bioactivity.
59 BASIC BIOLOGICAL SCIENCES↗
Solutions Network Formulation Report. NASA's Potential Contributions in Remote Quorum Sensing and the Management of Harmful Algal Blooms
This candidate solution proposes to use the night-imaging capabilities of the HSTC from SAC-C and of the HSC from SAC-D/Aquarius to detect bioluminescent events associated with HABs (harmful algal blooms). Once detected, this information could be fed to the NOAA CSCOR (Center for Sponsored Coastal Ocean Research) Harmful Algal Bloom Event Response Program, which acts quickly to fund the mobilization of research teams and to engage local agencies in a response. The HSC/HSTC data can serve as input to the HABSOS decision support system to provide information on location, extent, and duration of HAB events. Society will benefit from improved protection of the health of humans beings, aquatic ecosystems, and coastal economies. This work supports coastal management, public health, and homeland security applications.
Fletcher, Rose↗
Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication
Abstract Background Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication. Results We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with the soxRS oxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulon soxRS, was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture. Conclusions Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.
Chun, Kayla↗
The Exometabolome of Xylella fastidiosa in Contact with Paraburkholderia phytofirmans Supernatant Reveals Changes in Nicotinamide, Amino Acids, Biotin, and Plant Hormones
Microbial competition within plant tissues affects invading pathogens’ fitness. Metabolomics is a great tool for studying their biochemical interactions by identifying accumulated metabolites. Xylella fastidiosa, a Gram-negative bacterium causing Pierce’s disease (PD) in grapevines, secretes various virulence factors including cell wall-degrading enzymes, adhesion proteins, and quorum-sensing molecules. These factors, along with outer membrane vesicles, contribute to its pathogenicity. Previous studies demonstrated that co-inoculating X. fastidiosa with the Paraburkholderia phytofirmans strain PsJN suppressed PD symptoms. Here, we further investigated the interaction between the phytopathogen and the endophyte by analyzing the exometabolome of wild-type X. fastidiosa and a diffusible signaling factor (DSF) mutant lacking quorum sensing, cultivated with 20% P. phytofirmans spent media. Liquid chromatography–mass spectrometry (LC-MS) and the Method for Metabolite Annotation and Gene Integration (MAGI) were used to detect and map metabolites to genomes, revealing a total of 121 metabolites, of which 25 were further investigated. These metabolites potentially relate to host adaptation, virulence, and pathogenicity. Notably, this study presents the first comprehensive profile of X. fastidiosa in the presence of a P. phytofirmans spent media. The results highlight that P. phytofirmans and the absence of functional quorum sensing affect the ratios of glutamine to glutamate (Gln:Glu) in X. fastidiosa. Additionally, two compounds with plant metabolism and growth properties, 2-aminoisobutyric acid and gibberellic acid, were downregulated when X. fastidiosa interacted with P. phytofirmans. These findings suggest that P. phytofirmans-mediated disease suppression involves modulation of the exometabolome of X. fastidiosa, impacting plant immunity.
60 APPLIED LIFE SCIENCES↗
Improving growth of Cupriavidus necator H16 on formate using adaptive laboratory evolution-informed engineering
Conversion of CO 2 to value-added products presents an opportunity to reduce GHG emissions while generating revenue. Formate, which can be generated by the electrochemical reduction of CO 2 , has been proposed as a promising intermediate compound for microbial upgrading. Here we present progress towards improving the soil bacterium Cupriavidus necator H16, which is capable of growing on formate as its sole source of carbon and energy using the Calvin-Benson-Bassham (CBB) cycle, as a host for formate utilization. Using adaptive laboratory evolution, we generated several isolates that exhibited faster growth rates on formate. The genomes of these isolates were sequenced, and resulting mutations were systematically reintroduced by metabolic engineering, to identify those that improved growth. Further, the metabolic impact of several mutations was investigated further using RNA-seq transcriptomics. We found that deletion of a transcriptional regulator implicated in quorum sensing, PhcA, reduced expression of several operons and led to improved growth on formate. Growth was also improved by deleting large genomic regions present on the extrachromosomal megaplasmid pHG1, particularly two hydrogenase operons and the megaplasmid CBB operon, one of two copies present in the genome. Based on these findings, we generated a rationally engineered ..delta..phcA and megaplasmid-deficient strain that exhibited a 24% faster maximum growth rate on formate. Moreover, this strain achieved a 7% growth rate improvement on succinate and a 19% increase on fructose, demonstrating the broad utility of microbial genome reduction. This strain has the potential to serve as an improved microbial chassis for biological conversion of formate to value-added products.
59 BASIC BIOLOGICAL SCIENCES↗
Scrambling Signal Modularity in Bottom-up Assembled Synthetic Pseudomonas Consortia Reveals Robust Information Transfer
There is immense potential in crafting synthetic microbial communities for application in human health, agriculture, the environment, and even biomanufacturing where an appropriately constructed consortium can be assembled with tremendous biosynthetic or degradative capabilities. In many of these cases, bacterial signaling serves as a form of intercellular information transfer that guides the collective’s behavior. Such communication is complex, as many signals, signal disruptors, microbial species, physical barriers, and spatiotemporal constraints may be involved. Here, in this work, we demonstrate that a multisignal pathway for molecular information transfer within a consortium of several Pseudomonas spp. can be scrambled (genetically and organizationally) while the original message is still effectively conveyed. Assembled from the bottom up, we have employed two types of signaling molecules (i) a redox active secondary metabolite (rhizospheric signal, phloroglucinol), and (ii) a bacterial quorum sensing signal (3-oxo-C12 acylhomoserine lactone, AI-1). These signals can be intraconverted and acted upon by designated community members. We show how the order in which the signals are received, transduced, and subsequently transmitted can be rearranged with minimal impact on the intended outcome. In the consortial context, we found this messaging structure can be remarkably robust. Inspired by rhizospheric molecular signaling mechanisms, this work provides a conceptual framework for designing signaling and information transfer processes within assembled communities.
Biological and medical sciences↗
Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis
To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by “programming” cell lysis. Here, in this paper, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon of Escherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including “monoculture” and “transmitter-receiver” models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the “collective” attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.
59 BASIC BIOLOGICAL SCIENCES↗
Quorum-driven microbial consortium for Bioplastic production from agro-waste
Microbial consortia have high relevance in natural environments. Here we present the production of polyhydroxyalkanoates (PHA) from agro-industrial residues by a synthetic interkingdom consortium formed by the saprotrophic fungus Ophiostoma piceae CECT 20146, which encodes a wide range of lignocellulolytic enzymes, and a natural PHA producer, Pseudomonas putida KT2440. Two agro-industrial residues were utilized: Brewer's Spent Grain (BSG) as a carbon/nitrogen source and biofilm scaffold and waste cooking oil (WCO) as a carbon source for PHA synthesis. Through biochemistry, microscopy, and omics analyses, it is shown that P. putida accumulates up to 40.2% of intracellular PHA when the quorum sensing molecule, farnesol (naturally produced by O. piceae) is added, thanks to the increased proliferation of P. putida cells. An interactive Shiny application has also been developed for an easy visualization and comprehension of all the transcriptomics and metabolomics data: https://jgf-bioinformatics.shinyapps.io/Visualization_app/. These results support the increased PHA production of the consortium by an induction of gene phaG, which redirects intermediaries of the fatty acid biosynthesis to PHA precursors, and the repression of the PHA depolymerase phaZ in P. putida. The trophic interaction between microorganisms seems to rely on the citric acid produced by O. piceae and the glycerol liberated from WCO, which can both be consumed by P. putida. Bioreactor scale-up experiments allowed a 3.3-fold increase in the PHA concentration in the consortium (6.7 g·L-1) without pretreatment or sterilization of the substrates, laying the groundwork for the implementation of an industrial consolidated bioprocess (CBP).
Bacteria↗
Strategies to achieve high productivity, high conversion, and high yield in yeast fermentation of algal biomass hydrolysate
The conversion of carbohydrates in biomass via fermentation is an important component of an overall strategy to decarbonize the production of fuels and chemicals. Owing to the cost and resources required to produce biomass hydrolysates, the economic and environmental sustainability of these fermentation processes requires that they operate with high yields, sugar conversion, and productivity. Immobilized-cell technology in a continuous bioprocess can achieve significantly higher volumetric productivities than is possible from standard batch fermentation using free cells. Here, we demonstrate approaches for improvement of ethanol yield from algal hydrolysates and a mock hydrolysate medium. Saccharomyces cerevisiae was immobilized in alginate and incorporated into a two-column immobilized cell reactor system. Furthermore, the yeast quorum-sensing molecule, 2-phenylethanol, was added to improve ethanol yield by restricting growth and diverting sugar to ethanol. The bioreactor system could achieve high ethanol volumetric productivity (>20 g/ Lreactor ·h) and high glucose conversion (>99%) in mock hydrolysate, while the addition of 0.2% 2-phenylethanol resulted in 4.9% higher ethanol yield. With an algal hydrolysate of <10 g/L sugar, the ethanol volumetric productivity reached 9.8 g/ Lreactor ·h, and the addition of 0.2% 2-phenylethanol increased the ethanol yield by up to 7.4%. These results demonstrate the feasibility of novel strategies to achieve sustainability goals in biomass conversions.
59 BASIC BIOLOGICAL SCIENCES↗
Coral mucus rapidly induces chemokinesis and genome-wide transcriptional shifts toward early pathogenesis in a bacterial coral pathogen
Elevated seawater temperatures have contributed to the rise of coral disease mediated by bacterial pathogens, such as the globally distributed Vibrio coralliilyticus, which utilizes coral mucus as a chemical cue to locate stressed corals. However, the physiological events in the pathogens that follow their entry into the coral host environment remain unknown. Here, we present simultaneous measurements of the behavioral and transcriptional responses of V. coralliilyticus BAA-450 incubated in coral mucus. Video microscopy revealed a strong and rapid chemokinetic behavioral response by the pathogen, characterized by a two-fold increase in average swimming speed within 6 min of coral mucus exposure. RNA sequencing showed that this bacterial behavior was accompanied by an equally rapid differential expression of 53% of the genes in the V. coralliilyticus genome. Specifically, transcript abundance 10 min after mucus exposure showed upregulation of genes involved in quorum sensing, biofilm formation, and nutrient metabolism, and downregulation of flagella synthesis and chemotaxis genes. After 60 min, we observed upregulation of genes associated with virulence, including zinc metalloproteases responsible for causing coral tissue damage and algal symbiont photoinactivation, and secretion systems that may export toxins. Together, our results suggest that V. coralliilyticus employs a suite of behavioral and transcriptional responses to rapidly shift into a distinct infection mode within minutes of exposure to the coral microenvironment.
54 ENVIRONMENTAL SCIENCES↗
Machine learning uncovers independently regulated modules in the Bacillus subtilis transcriptome
The transcriptional regulatory network (TRN) of Bacillus subtilis coordinates cellular functions of fundamental interest, including metabolism, biofilm formation, and sporulation. Here, we use unsupervised machine learning to modularize the transcriptome and quantitatively describe regulatory activity under diverse conditions, creating an unbiased summary of gene expression. We obtain 83 independently modulated gene sets that explain most of the variance in expression and demonstrate that 76% of them represent the effects of known regulators. The TRN structure and its condition-dependent activity uncover putative or recently discovered roles for at least five regulons, such as a relationship between histidine utilization and quorum sensing. The TRN also facilitates quantification of population-level sporulation states. As this TRN covers the majority of the transcriptome and concisely characterizes the global expression state, it could inform research on nearly every aspect of transcriptional regulation in B. subtilis.
59 BASIC BIOLOGICAL SCIENCES↗
Redox-enabled electronic interrogation and feedback control of hierarchical and networked biological systems
Abstract Microelectronic devices can directly communicate with biology, as electronic information can be transmitted via redox reactions within biological systems. By engineering biology’s native redox networks, we enable electronic interrogation and control of biological systems at several hierarchical levels: proteins, cells, and cell consortia. First, electro-biofabrication facilitates on-device biological component assembly. Then, electrode-actuated redox data transmission and redox-linked synthetic biology allows programming of enzyme activity and closed-loop electrogenetic control of cellular function. Specifically, horseradish peroxidase is assembled onto interdigitated electrodes where electrode-generated hydrogen peroxide controls its activity. E. coli ’s stress response regulon, oxyRS , is rewired to enable algorithm-based feedback control of gene expression, including an eCRISPR module that switches cell-cell quorum sensing communication from one autoinducer to another—creating an electronically controlled ‘bilingual’ cell. Then, these disparate redox-guided devices are wirelessly connected, enabling real-time communication and user-based control. We suggest these methodologies will help us to better understand and develop sophisticated control for biology.
59 BASIC BIOLOGICAL SCIENCES↗
A global soil plasmidome resource unveils functional and ecological roles of plasmids in soil microbiomes
Plasmids play significant roles in microbial adaptation to ecosystems, yet their dynamics remain poorly understood due to identification challenges. We present the Global Soil Plasmidome Resource (GSPR), a comprehensive dataset of 98,728 plasmid sequences amassed from 6860 terrestrial microbial communities and isolates. We explore this resource through various computational approaches, including phylogenetic diversity analysis, host prediction, and extensive functional annotation, to understand the contribution of plasmids to the genetic and functional diversity in soil, correlating these findings with sample type, as well as the soil habitat they were retrieved from. Our analysis reveals insights into plasmid-encoded functions such as effector modules, quorum sensing, and stress resistance, which may contribute to their persistence and microbial adaptation in soil. Furthermore, CRISPR analysis suggests a prevalent role of these elements related to intra-plasmid competition. By contrasting plasmids from cultivated and uncultivated organisms, we identify important functions that expand existing knowledge of plasmid roles in these habitats. This study represents a notable step forward in elucidating plasmid diversity and function within soil microbiomes and establishes a foundational framework for exploring their roles in natural environments.
Fiamenghi, Mateus B↗
Quantitative dissection of Agrobacterium T-DNA expression in single plant cells reveals density-dependent synergy and antagonism
Agrobacterium pathogenesis, which involves transferring T-DNA into plant cells, is the cornerstone of plant genetic engineering. As the applications that rely on Agrobacterium increase in sophistication, it becomes critical to achieve a quantitative and predictive understanding of T-DNA expression at the level of single plant cells. Here we examine if a classic Poisson model of interactions between pathogens and host cells holds true for Agrobacterium infecting Nicotiana benthamiana. Systematically challenging this model revealed antagonistic and synergistic density-dependent interactions between bacteria that do not require quorum sensing. Using various approaches, we studied the molecular basis of these interactions. To overcome the engineering constraints imposed by antagonism, we created a dual binary vector system termed ‘BiBi’, which can improve the efficiency of a reconstituted complex metabolic pathway in a predictive fashion. Our findings illustrate how combining theoretical models with quantitative experiments can reveal new principles of bacterial pathogenesis, impacting both fundamental and applied plant biology.
Alamos, Simon↗