Spying on microbial communities, cell by cell
Within any community of organisms, gene expression is heterogeneous, which can manifest in genetically identical individuals having a different ...phenotype. One has to look at individuals in context and analyze patterns in both space and time to see the full picture. Aiming to fill a gap in current methods, Dar
et al
. developed a transcriptome-imaging method named parallel sequential fluorescence in situ hybridization (par-seqFISH). They applied this technique to the opportunistic pathogen
Pseudomonas aeruginosa
, focusing on biofilms where growth conditions can change at microscopic scale. Development of these communities, as revealed by mRNA composition, were followed in space and time. The results revealed a heterogeneous phenotypic landscape, with oxygen availability shaping the metabolism at a spatial scale of microns within a single contiguous biofilm segment. This tool should be applicable to complex microbial communities in the environment and the human microbiome. —MAF
Spatially resolved, parallel transcriptome imaging reveals functional heterogeneity in bacterial cultures and biofilms.
INTRODUCTION
Microbial populations display heterogeneous gene expression profiles that result in phenotypic differences between individual bacteria. This diversity can allow populations to survive under uncertain and fluctuating conditions such as sudden antibiotic exposure, divide costly functions across different subpopulations, and enable interactions between different phenotypes. In addition to the temporal phenotypic heterogeneity seen in planktonic cultures, microbial populations and communities often exist in multicellular biofilms that exhibit considerable heterogeneity at the microscale, both in the local physicochemistry that individuals experience and in the species composition in their neighborhoods. Phenotypic and microscale variation represent central features of microbial populations, but the landscape of possible cellular states, their spatiotemporal regulation, and their roles in many biological phenomena are still largely unknown.
RATIONALE
The microscale heterogeneity that defines microbial life can play important roles in community organization and function, including in antibiotic resistance and virulence. However, our understanding of these basic features has been limited by our ability to capture this heterogeneity at the relevant spatiotemporal scales. Overcoming these limitations could lead to new insights into the inner workings of microbial assemblages.
RESULTS
We developed par-seqFISH (parallel sequential fluorescence in situ hybridization), a high-throughput method that captures gene expression profiles of individual bacteria while also preserving their physical context within spatially structured environments. We applied this approach to the study of
Pseudomonas aeruginosa
, a model biofilm-forming bacterium and an opportunistic human pathogen. Focusing on a set of 105 marker genes representing key aspects of
P. aeruginosa
physiology and virulence, we explored the transcriptional profiles of >600,000 bacteria across dozens of growth conditions. We uncovered a diverse set of metabolic- and virulence-related cellular states and quantified their temporal dynamics during population growth. In addition to recording gene expression, we also demonstrated that par-seqFISH captures cell biological parameters such as cell size and can be further integrated with specific dyes to measure features such as chromosome copy in the same cells. Applying par-seqFISH to developing
P. aeruginosa
biofilms, we exposed the biogeographic context of cellular states, providing new insights into the spatial expression of various genes. These included, among other things, mutually exclusive expression patterns of flagella and type IV pili genes and a localized induction of pyocins, which are involved in kin selection and extracellular DNA release. Looking more closely, we found that pyocin-encoding transcripts strongly localized to the bacterial cell poles. In early biofilms, we identified extensive microscale phenotypic structuring in which anaerobic metabolic processes such as denitrification appeared to locally influence the microenvironment through byproduct production. In more mature biofilms, we detected larger-scale partitions into zones of differential metabolic activities and virulence factor biosynthesis.
CONCLUSION
Transcriptome imaging using par-seqFISH captures the microscale phenotypic variation of free-living and sessile bacterial populations. We report extensive heterogeneity in growing
P. aeruginosa
populations and demonstrate that individual multicellular biofilms can contain coexisting but separated subpopulations with distinct physiological activities. This multiplexed and spatially resolved method offers a generalizable tool for studying bacterial populations in space and time directly in their native contexts. Future applications in natural and clinical samples will provide insights into the conditions experienced by microbes in complex environments and the coordinated physiological responses that emerge in turn and reshape them.
Transcriptome imaging using par-seqFISH reveals the dynamics and spatial organization of transcriptional programs inA
P. aeruginosa
A populations at single-cell resolution.
Transcriptional states of individual bacterial cells were identified using clustering analysis (left). The detected cellular states are depicted in different colors. Cell metabolic states can be directly mapped to their native biofilm context to identify emerging microenvironment dynamics (right).
Capturing the heterogeneous phenotypes of microbial populations at relevant spatiotemporal scales is highly challenging. Here, we present par-seqFISH (parallel sequential fluorescence in situ hybridization), a transcriptome-imaging approach that records gene expression and spatial context within microscale assemblies at a single-cell and molecule resolution. We applied this approach to the opportunistic pathogen
Pseudomonas aeruginosa
, analyzing about 600,000 individuals across dozens of conditions in planktonic and biofilm cultures. We identified numerous metabolic- and virulence-related transcriptional states that emerged dynamically during planktonic growth, as well as highly spatially resolved metabolic heterogeneity in sessile populations. Our data reveal that distinct physiological states can coexist within the same biofilm just several micrometers away, underscoring the importance of the microenvironment. Our results illustrate the complex dynamics of microbial populations and present a new way of studying them at high resolution.
Verteporfin photodynamic therapy (vPDT) is a selective vaso-occlusive treatment that targets choroidal vascular abnormalities. It was initially developed to treat neovascular age-related macular ...degeneration using the 'standard' vPDT protocol (verteporfin 6 mg/m(2), vPDT laser fluence 50 J/cm(2)). vPDT therapy has subsequently evolved as an important treatment modality for a range of other chorioretinal conditions including choroidal haemangioma, central serous chorioretinopathy, polypoidal choroidal vasculopathy, and peripapillary choroidal neovascularisation. Various 'safety-enhanced' vPDT protocols have been devised to optimise treatment outcomes, typically using reduced dose verteporfin (verteporfin 3 mg/m(2)) or reduced fluence vPDT (vPDT laser fluence 25 J/cm(2)). This paper reviews the current role of vPDT therapy in the treatment of chorioretinal conditions.
Descriptions of the changeable, striking colors associated with secreted natural products date back well over a century. These molecules can serve as extracellular electron shuttles (EESs) that ...permit microbes to access substrates at a distance. In this review, we argue that the colorful world of EESs has been too long neglected. Rather than simply serving as a diagnostic attribute of a particular microbial strain, redox-active natural products likely play fundamental, underappreciated roles in the biology of their producers, particularly those that inhabit biofilms. Here, we describe the chemical diversity and potential distribution of EES producers and users, discuss the costs associated with their biosynthesis, and critically evaluate strategies for their economical usage. We hope this review will inspire efforts to identify and explore the importance of EES cycling by a wide range of microorganisms so that their contributions to shaping microbial communities can be better assessed and exploited.
Summary
Pseudomonas aeruginosa, an opportunistic pathogen, produces redox‐active pigments called phenazines. Pyocyanin (PYO, the blue phenazine) plays an important role during biofilm development. ...Paradoxically, PYO auto‐poisoning can stimulate cell death and release of extracellular DNA (eDNA), yet PYO can also promote survival within biofilms when cells are oxidant‐limited. Here, we identify the environmental and physiological conditions in planktonic culture that promote PYO‐mediated cell death. We demonstrate that PYO auto‐poisoning is enhanced when cells are starved for carbon. In the presence of PYO, cells activate a set of genes involved in energy‐dependent defenses, including: (i) the oxidative stress response, (ii) RND efflux systems and (iii) iron‐sulfur cluster biogenesis factors. P. aeruginosa can avoid PYO poisoning when reduced carbon is available, but blockage of adenosine triphosphate (ATP) synthesis either through carbon limitation or direct inhibition of the F0F1‐ATP synthase triggers death and eDNA release. Finally, even though PYO is toxic to the majority of the population when cells are nutrient limited, a subset of cells is intrinsically PYO resistant. The effect of PYO on the producer population thus appears to be dynamic, playing dramatically different yet predictable roles throughout distinct stages of growth, helping rationalize its multifaceted contributions to biofilm development.
The redox active pigment, pyocyanin (PYO), made by Pseudomonas aeruginosa, is a double‐edged sword, whose physiological impact changes dramatically yet predictably. Under nutrient‐replete conditions, PYO is produced and can help cells achieve redox homeostasis in the absence of alternative electron acceptors. However, when cells deplete carbon or other nutrients, PYO becomes toxic and poisons the majority of the population. A small, PYO‐insensitive population persists and multiplies when nutrients again become available.
Secondary metabolites profoundly affect microbial physiology, metabolism and stress responses. Increasing evidence suggests that these molecules can modulate microbial susceptibility to commonly used ...antibiotics; however, secondary metabolites are typically excluded from standard antimicrobial susceptibility assays. This may in part account for why infections by diverse opportunistic bacteria that produce secondary metabolites often exhibit discrepancies between clinical antimicrobial susceptibility testing results and clinical treatment outcomes. In this Review, we explore which types of secondary metabolite alter antimicrobial susceptibility, as well as how and why this phenomenon occurs. We discuss examples of molecules that opportunistic and enteric pathogens either generate themselves or are exposed to from their neighbours, and the nuanced impacts these molecules can have on tolerance and resistance to certain antibiotics.
Most bacteria spend the majority of their time in prolonged states of very low metabolic activity and little or no growth, in which electron donors, electron acceptors and/or nutrients are limited, ...but cells are poised to undergo rapid division cycles when resources become available. These non-growing states are far less studied than other growth states, which leaves many questions regarding basic bacterial physiology unanswered. In this Review, we discuss findings from a small but diverse set of systems that have been used to investigate how growth-arrested bacteria adjust metabolism, regulate transcription and translation, and maintain their chromosomes. We highlight major questions that remain to be addressed, and suggest that progress in answering them will be aided by recent methodological advances and by dialectic between environmental and molecular microbiology perspectives.
Lipid research represents a frontier for microbiology, as showcased by hopanoid lipids. Hopanoids, which resemble sterols and are found in the membranes of diverse bacteria, have left an extensive ...molecular fossil record. They were first discovered by petroleum geologists. Today, hopanoid-producing bacteria remain abundant in various ecosystems, such as the rhizosphere. Recently, great progress has been made in our understanding of hopanoid biosynthesis, facilitated in part by technical advances in lipid identification and quantification. A variety of genetically tractable, hopanoid-producing bacteria have been cultured, and tools to manipulate hopanoid biosynthesis and detect hopanoids are improving. However, we still have much to learn regarding how hopanoid production is regulated, how hopanoids act biophysically and biochemically, and how their production affects bacterial interactions with other organisms, such as plants. The study of hopanoids thus offers rich opportunities for discovery.
Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and ...archaea, their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15–19 nt RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22–24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid-derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids.
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•RsAgo associates with 15–19 nt RNA and 22–24 nt DNA molecules in vivo•RsAgo-associated small RNAs correspond to the majority of cellular transcripts•Small DNAs are complementary to the small RNAs and enriched in foreign sequences•RsAgo degrades plasmid DNA and represses expression of plasmid-encoded genes
Summary
While many studies have explored the growth of Pseudomonas aeruginosa, comparatively few have focused on its survival. Previously, we reported that endogenous phenazines support the anaerobic ...survival of P. aeruginosa, yet the physiological mechanism underpinning survival was unknown. Here, we demonstrate that phenazine redox cycling enables P. aeruginosa to oxidize glucose and pyruvate into acetate, which promotes survival by coupling acetate and ATP synthesis through the activity of acetate kinase. By measuring intracellular NAD(H) and ATP concentrations, we show that survival is correlated with ATP synthesis, which is tightly coupled to redox homeostasis during pyruvate fermentation but not during arginine fermentation. We also show that ATP hydrolysis is required to generate a proton‐motive force using the ATP synthase complex during fermentation. Together, our results suggest that phenazines enable maintenance of the proton‐motive force by promoting redox homeostasis and ATP synthesis. This work demonstrates the more general principle that extracellular redox‐active molecules, such as phenazines, can broaden the metabolic versatility of microorganisms by facilitating energy generation.
Secondary metabolites are important facilitators of plant-microbe interactions in the rhizosphere, contributing to communication, competition, and nutrient acquisition. However, at first glance, the ...rhizosphere seems full of metabolites with overlapping functions, and we have a limited understanding of basic principles governing metabolite use. Increasing access to the essential nutrient iron is one important, but seemingly redundant role performed by both plant and microbial Redox-Active Metabolites (RAMs). We used coumarins, RAMs made by the model plant
, and phenazines, RAMs made by soil-dwelling pseudomonads, to ask whether plant and microbial RAMs might each have distinct functions under different environmental conditions. We show that variations in oxygen and pH lead to predictable differences in the capacity of coumarins vs phenazines to increase the growth of iron-limited pseudomonads and that these effects depend on whether pseudomonads are grown on glucose, succinate, or pyruvate: carbon sources commonly found in root exudates. Our results are explained by the chemical reactivities of these metabolites and the redox state of phenazines as altered by microbial metabolism. This work shows that variations in the chemical microenvironment can profoundly affect secondary metabolite function and suggests plants may tune the utility of microbial secondary metabolites by altering the carbon released in root exudates. Together, these findings suggest that RAM diversity may be less overwhelming when viewed through a chemical ecological lens: Distinct molecules can be expected to be more or less important to certain ecosystem functions, such as iron acquisition, depending on the local chemical microenvironments in which they reside.
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