Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

Global diversity of enterococci and description of 18 novel species.

Enterococci are commensal gut microbes of most land animals. They diversified over hundreds of millions of years adapting to evolving hosts and host diets. Of over 60 known enterococcal species, Enterococcus faecalis and E. faecium uniquely emerged in the antibiotic era among leading causes of multidrug resistant hospital-associated infection. The basis for the association of particular enterococcal species with a host is largely unknown. To begin deciphering enterococcal species traits that drive host association, and to assess the pool of Enterococcus-adapted genes from which known facile gene exchangers such as E. faecalis and E. faecium may draw, we collected 886 enterococcal strains from nearly 1,000 specimens representing widely diverse hosts, ecologies and geographies. This provided data on the global occurrence and host associations of known species, identifying 18 new species in the process expanding genus diversity by >25%. The novel species harbor diverse genes associated with toxins, detoxification, and resource acquisition. E. faecalis and E. faecium were isolated from a wide diversity of hosts highlighting their generalist properties, whereas most other species exhibited more restricted distributions indicative of specialized host associations. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades as well as genes associated with range expansion, such as B-vitamin biosynthesis and flagellar motility. Collectively, this work provides an unprecedentedly broad and deep view of the genus Enterococcus, potential threats to human health, and new insights into its evolution.

Bacterial growth in multicellular aggregates leads to the emergence of complex life cycles.

Facultative multicellular behaviors expand the metabolic capacity and physiological resilience of bacteria. Despite their ubiquity in nature, we lack an understanding of how these behaviors emerge from cellular-scale phenomena. Here, we show how the coupling between growth and resource gradient formation leads to the emergence of multicellular lifecycles in a marine bacterium. Under otherwise carbon-limited growth conditions, Vibrio splendidus 12B01 forms clonal multicellular groups to collectively harvest carbon from soluble polymers of the brown-algal polysaccharide alginate. As they grow, groups phenotypically differentiate into two spatially distinct sub-populations: a static "shell" surrounding a motile, carbon-storing "core." Differentiation of these two sub-populations coincides with the formation of a gradient in nitrogen-source availability within clusters. Additionally, we find that populations of cells containing a high proportion of carbon-storing individuals propagate and form new clusters more readily on alginate than do populations with few carbon-storing cells. Together, these results suggest that local metabolic activity and differential partitioning of resources leads to the emergence of reproductive cycles in a facultatively multicellular bacterium.

Historical contingencies and phage induction diversify bacterioplankton communities at the microscale.

In many natural environments, microorganisms decompose microscale resource patches made of complex organic matter. The growth and collapse of populations on these resource patches unfold within spatial ranges of a few hundred micrometers or less, making such microscale ecosystems hotspots of heterotrophic metabolism. Despite the potential importance of patch-level dynamics for the large-scale functioning of heterotrophic microbial communities, we have not yet been able to delineate the ecological processes that control natural populations at the microscale. Here, we address this challenge by characterizing the natural marine communities that assembled on over 1,000 individual microscale particles of chitin, the most abundant marine polysaccharide. Using low-template shotgun metagenomics and imaging, we find significant variation in microscale community composition despite the similarity in initial species pools across replicates. Chitin-degrading taxa that were rare in seawater established large populations on a subset of particles, resulting in a wide range of predicted chitinolytic abilities and biomass at the level of individual particles. We show, through a mathematical model, that this variability can be attributed to stochastic colonization and historical contingencies affecting the tempo of growth on particles. We find evidence that one biological process leading to such noisy growth across particles is differential predation by temperate bacteriophages of chitin-degrading strains, the keystone members of the community. Thus, initial stochasticity in assembly states on individual particles, amplified through ecological interactions, may have significant consequences for the diversity and functionality of systems of microscale patches.

What can we learn from honey bees?

The Western honey bee provides a model system for studying how closely related species of bacteria are able to coexist in a single community.

Regulatory involvement of the PerR and SloR metalloregulators in the Streptococcus mutans oxidative stress response.

Streptococcus mutans is a commensal of the human oral microbiome that can promote dental caries under conditions of dysbiosis. This study investigates metalloregulators and their involvement in the S. mutans oxidative stress response. Oxidative stress in the human mouth can derive from temporal increases in reactive oxygen species (ROS) after meal consumption and from endogenous bacterial ROS-producers that colonize the dentition. We hypothesize that the S. mutans PerR (SMU.593) and SloR (SMU.186) metalloregulatory proteins contribute to the regulation of oxidative stress genes and their products. Expression assays with S. mutans UA159 wild type cultures exposed to H2O2 reveal that H2O2 upregulates perR, and that PerR represses sloR transcription upon binding directly to Fur and PerR consensus sequences within the sloR operator. In addition, the results of Western blot experiments implicate the Clp proteolytic system in SloR degradation under conditions of H2O2-stress. To reveal a potential role for SloR in the H2O2-resistant phenotype of S. mutans GMS802 (a perR-deficient strain), we generated a sloR/perR double knockout mutant, GMS1386, where we observed upregulation of the tpx and dpr antioxidant genes. These results are consistent with GMS802 H2O2 resistance and with a role for PerR as a transcriptional repressor. Cumulatively, these findings support a reciprocal relationship between PerR and SloR during the S. mutans oxidative stress response and begin to elucidate the fitness strategies that evolved to foster S. mutans persistence in the transient environments of the human oral cavity.IMPORTANCEIn 2020, untreated dental caries, especially in the permanent dentition, ranked among the most prevalent infectious diseases worldwide, disproportionately impacting individuals of low socioeconomic status. Untreated caries can lead to systemic health problems and has been associated with extended school and work absences, inappropriate use of emergency departments, and an inability for military forces to deploy. Together with public health policy, research aimed at alleviating S. mutans -induced tooth decay is important because it can improve oral health (and overall health), especially in underserved populations. This research, focused on S. mutans metalloregulatory proteins and their gene targets, is significant because it can promote virulence gene control in an important oral pathogen, and contribute to the development of an anti-caries therapeutic that can reduce tooth decay.

Acidic pH promotes lipopolysaccharide modification and alters colonization in a bacteria-animal mutualism.

Environmental pH can be an important cue for symbiotic bacteria as they colonize their eukaryotic hosts. Using the model mutualism between the marine bacterium Vibrio fischeri and the Hawaiian bobtail squid, we characterized the bacterial transcriptional response to acidic pH experienced during the shift from planktonic to host-associated lifestyles. We found several genes involved in outer membrane structure were differentially expressed based on pH, indicating alterations in membrane physiology as V. fischeri initiates its symbiotic program. Exposure to host-like pH increased the resistance of V. fischeri to the cationic antimicrobial peptide polymixin B, which resembles antibacterial molecules that are produced by the squid to select V. fischeri from the ocean microbiota. Using a forward genetic screen, we identified a homolog of eptA, a predicted phosphoethanolamine transferase, as critical for antimicrobial defense. We used MALDI-MS to verify eptA as an ethanolamine transferase for the lipid-A portion of V. fischeri lipopolysaccharide. We then used a DNA pulldown approach to discover that eptA transcription is activated by the global regulator H-NS. Finally, we revealed that eptA promotes successful squid colonization by V. fischeri, supporting its potential role in initiation of this highly specific symbiosis.

Ambient pH Alters the Protein Content of Outer Membrane Vesicles, Driving Host Development in a Beneficial Symbiosis.

Outer membrane vesicles (OMVs) are continuously produced by Gram-negative bacteria and are increasingly recognized as ubiquitous mediators of bacterial physiology. In particular, OMVs are powerful effectors in interorganismal interactions, driven largely by their molecular contents. These impacts have been studied extensively in bacterial pathogenesis but have not been well documented within the context of mutualism. Here, we examined the proteomic composition of OMVs from the marine bacterium Vibrio fischeri, which forms a specific mutualism with the Hawaiian bobtail squid, Euprymna scolopes We found that V. fischeri upregulates transcription of its major outer membrane protein, OmpU, during growth at an acidic pH, which V. fischeri experiences when it transitions from its environmental reservoir to host tissues. We used comparative genomics and DNA pulldown analyses to search for regulators of ompU and found that differential expression of ompU is governed by the OmpR, H-NS, and ToxR proteins. This transcriptional control combines with nutritional conditions to govern OmpU levels in OMVs. Under a host-encountered acidic pH, V. fischeri OMVs become more potent stimulators of symbiotic host development in an OmpU-dependent manner. Finally, we found that symbiotic development could be stimulated by OMVs containing a homolog of OmpU from the pathogenic species Vibrio cholerae, connecting the role of a well-described virulence factor with a mutualistic element. This work explores the symbiotic effects of OMV variation, identifies regulatory machinery shared between pathogenic and mutualistic bacteria, and provides evidence of the role that OMVs play in animal-bacterium mutualism.IMPORTANCE Beneficial bacteria communicate with their hosts through a variety of means. These communications are often carried out by a combination of molecules that stimulate responses from the host and are necessary for development of the relationship between these organisms. Naturally produced bacterial outer membrane vesicles (OMVs) contain many of those molecules and can stimulate a wide range of responses from recipient organisms. Here, we describe how a marine bacterium, Vibrio fischeri, changes the makeup of its OMVs under conditions that it experiences as it goes from its free-living lifestyle to associating with its natural host, the Hawaiian bobtail squid. This work improves our understanding of how bacteria change their signaling profile as they begin to associate with their beneficial partner animals.

Identification of a Botulinum Neurotoxin-like Toxin in a Commensal Strain of Enterococcus faecium.

Botulinum neurotoxins (BoNTs), produced by various Clostridium strains, are a family of potent bacterial toxins and potential bioterrorism agents. Here we report that an Enterococcus faecium strain isolated from cow feces carries a BoNT-like toxin, designated BoNT/En. It cleaves both VAMP2 and SNAP-25, proteins that mediate synaptic vesicle exocytosis in neurons, at sites distinct from known BoNT cleavage sites on these two proteins. Comparative genomic analysis determines that the E. faecium strain carrying BoNT/En is a commensal type and that the BoNT/En gene is located within a typical BoNT gene cluster on a 206 kb putatively conjugative plasmid. Although the host species targeted by BoNT/En remains to be determined, these findings establish an extended member of BoNTs and demonstrate the capability of E. faecium, a commensal organism ubiquitous in humans and animals and a leading cause of hospital-acquired multi-drug-resistant (MDR) infections, to horizontally acquire, and possibly disseminate, a unique BoNT gene cluster.

Mapping Transposon Insertions in Bacterial Genomes by Arbitrarily Primed PCR.

Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to (1) amplify DNA spanning the transposon insertion junction, and (2) increase the specific yield of transposon insertion junction fragments for sequence analysis. The resulting sequence is mapped to a bacterial genome to identify the site of transposon insertion. In this protocol, AP-PCR as it is routinely used to map sites of transposon insertion within Staphylococcus aureus, is used to illustrate the principle. Guidelines are provided for adapting this protocol for mapping insertions in other bacterial genomes. Mapping transposon insertions using this method is typically achieved in 2 to 3 days if starting from a culture of the transposon insertion mutant. © 2017 by John Wiley & Sons, Inc.

Stress as a Normal Cue in the Symbiotic Environment.

All multicellular hosts form associations with groups of microorganisms. These microbial communities can be taxonomically diverse and dynamic, and their persistence is due to robust, and sometimes coevolved, host-microbe and microbe-microbe interactions. Chemical and physical sources of stress are prominently situated in this molecular exchange, as cues for cellular responses in symbiotic microbes. Stress in the symbiotic environment may arise from three sources: host tissues, microbe-induced immune responses, or other microbes in the host environment. The responses of microbes to these stresses can be general or highly specialized, and collectively may contribute to the stability of the symbiotic system. In this review, we highlight recent work that emphasizes the role of stress as a cue in the symbiotic environment of plants and animals.

A conserved chemical dialog of mutualism: lessons from squid and vibrio.

Microorganisms shape, and are shaped by, their environment. In host-microbe associations, this environment is defined by tissue chemistry, which reflects local and organism-wide physiology, as well as inflammatory status. We review how, in the squid-vibrio mutualism, both partners shape tissue chemistry, revealing common themes governing tissue homeostasis in animal-microbe associations.

A Single Host-Derived Glycan Impacts Key Regulatory Nodes of Symbiont Metabolism in a Coevolved Mutualism.

UNLABELLED: Most animal-microbe mutualistic associations are characterized by nutrient exchange between the partners. When the host provides the nutrients, it can gain the capacity to shape its microbial community, control the stability of the interaction, and promote its health and fitness. Using the bioluminescent squid-vibrio model, we demonstrate how a single host-derived glycan, chitin, regulates the metabolism of Vibrio fischeri at key points in the development and maintenance of the symbiosis. We first characterized the pathways for catabolism of chitin sugars by V. fischeri, demonstrating that the Ccr-dependent phosphoenolpyruvate-pyruvate phosphotransferase system (PTS) prioritizes transport of these sugars in V. fischeri by blocking the uptake of non-PTS carbohydrates, such as glycerol. Next, we found that PTS transport of chitin sugars into the bacterium shifted acetate homeostasis toward a net excretion of acetate and was sufficient to override an activation of the acetate switch by AinS-dependent quorum sensing. Finally, we showed that catabolism of chitin sugars decreases the rate of cell-specific oxygen consumption. Collectively, these three metabolic functions define a physiological shift that favors fermentative growth on chitin sugars and may support optimal symbiont luminescence, the functional basis of the squid-vibrio mutualism. IMPORTANCE: Host-derived glycans have recently emerged as a link between symbiont nutrition and innate immune function. Unfortunately, the locations at which microbes typically access host-derived glycans are inaccessible to experimentation and imaging, and they take place in the context of diverse microbe-microbe interactions, creating a complex symbiotic ecology. Here we describe the metabolic state of a single microbial symbiont in a natural association with its coevolved host and, by doing so, infer key points at which a host-controlled tissue environment might regulate the physiological state of its symbionts. We show that the presence of a regulatory glycan is sufficient to shift symbiont carbohydrate catabolism, acetate homeostasis, and oxygen consumption.

The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation.

Glycans have emerged as critical determinants of immune maturation, microbial nutrition, and host health in diverse symbioses. In this study, we asked how cyclic delivery of a single host-derived glycan contributes to the dynamic stability of the mutualism between the squid Euprymna scolopes and its specific, bioluminescent symbiont, Vibrio fischeri. V. fischeri colonizes the crypts of a host organ that is used for behavioral light production. E. scolopes synthesizes the polymeric glycan chitin in macrophage-like immune cells called hemocytes. We show here that, just before dusk, hemocytes migrate from the vasculature into the symbiotic crypts, where they lyse and release particulate chitin, a behavior that is established only in the mature symbiosis. Diel transcriptional rhythms in both partners further indicate that the chitin is provided and metabolized only at night. A V. fischeri mutant defective in chitin catabolism was able to maintain a normal symbiont population level, but only until the symbiotic organ reached maturity (∼ 4 wk after colonization); this result provided a direct link between chitin utilization and symbiont persistence. Finally, catabolism of chitin by the symbionts was also specifically required for a periodic acidification of the adult crypts each night. This acidification, which increases the level of oxygen available to the symbionts, enhances their capacity to produce bioluminescence at night. We propose that other animal hosts may similarly regulate the activities of epithelium-associated microbial communities through the strategic provision of specific nutrients, whose catabolism modulates conditions like pH or anoxia in their symbionts' habitat.

Non-native acylated homoserine lactones reveal that LuxIR quorum sensing promotes symbiont stability.

Quorum sensing, a group behaviour coordinated by a diffusible pheromone signal and a cognate receptor, is typical of bacteria that form symbioses with plants and animals. LuxIR-type N-acyl L-homoserine (AHL) quorum sensing is common in Gram-negative Proteobacteria, and many members of this group have additional quorum-sensing networks. The bioluminescent symbiont Vibrio fischeri encodes two AHL signal synthases: AinS and LuxI. AinS-dependent quorum sensing converges with LuxI-dependent quorum sensing at the LuxR regulatory element. Both AinS- and LuxI-mediated signalling are required for efficient and persistent colonization of the squid host, Euprymna scolopes. The basis of the mutualism is symbiont bioluminescence, which is regulated by both LuxI- and AinS-dependent quorum sensing, and is essential for maintaining a colonization of the host. Here, we used chemical and genetic approaches to probe the dynamics of LuxI- and AinS-mediated regulation of bioluminescence during symbiosis. We demonstrate that both native AHLs and non-native AHL analogues can be used to non-invasively and specifically modulate induction of symbiotic bioluminescence via LuxI-dependent quorum sensing. Our data suggest that the first day of colonization, during which symbiont bioluminescence is induced by LuxIR, is a critical period that determines the stability of the V. fischeri population once symbiosis is established.

Di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens via modulation of c-di-GMP-dependent pathways.

Dinucleoside tetraphosphates are common constituents of the cell and are thought to play diverse biological roles in organisms ranging from bacteria to humans. In this study we characterized two independent mechanisms by which di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens. Null mutations in apaH, the gene encoding nucleoside tetraphosphate hydrolase, resulted in a marked increase in the cellular level of Ap4A. Concomitant with this increase, Pho regulon activation in low-inorganic-phosphate (P(i)) conditions was severely compromised. As a consequence, an apaH mutant was not sensitive to Pho regulon-dependent inhibition of biofilm formation. In addition, we characterized a Pho-independent role for Ap4A metabolism in regulation of biofilm formation. In P(i)-replete conditions Ap4A metabolism was found to impact expression and localization of LapA, the major adhesin regulating surface commitment by P. fluorescens. Increases in the level of c-di-GMP in the apaH mutant provided a likely explanation for increased localization of LapA to the outer membrane in response to elevated Ap4A concentrations. Increased levels of c-di-GMP in the apaH mutant were associated with increases in the level of GTP, suggesting that elevated levels of Ap4A may promote de novo purine biosynthesis. In support of this suggestion, supplementation with adenine could partially suppress the biofilm and c-di-GMP phenotypes of the apaH mutant. We hypothesize that changes in the substrate (GTP) concentration mediated by altered flux through nucleotide biosynthetic pathways may be a significant point of regulation for c-di-GMP biosynthesis and regulation of biofilm formation.

Conservation of the Pho regulon in Pseudomonas fluorescens Pf0-1.

The Pho regulon integrates the sensing of environmental inorganic phosphate (Pi) availability with coregulation of gene expression, mediating an adaptive response to Pi limitation. Many aspects of the Pho regulon have been addressed in studies of Escherichia coli; however, it is unclear how transferable this knowledge is to other bacterial systems. Here, we report work to discern the conservation of the Pho regulon in Pseudomonas fluorescens Pf0-1. We demonstrate by mutational studies that PhoB/PhoR and the Pst system have conserved functions in the regulation of Pi-induced phosphatase activities, as well as expression of other Pi-regulated genes. A genetic screen was carried out to isolate factors that affect Pho-regulated phosphatase activity. We identified the Pho-regulated phosphatases PhoX and PhoD and present evidence that these enzymes are exported via the Tat system. The phoX and phoD genes were shown to be members of the Pho regulon by reverse transcription-PCR, as well as by functional assessment of putative PhoB binding sites (Pho boxes). Our data also suggested that at least one other non-Tat-secreted Pho-regulated phosphatase exists. From the genetic screen, numerous siderophore mutants that displayed severe defects in Pho-activated phosphatase activity were isolated. Subsequently, iron was shown to be important for modulating the activity of Pho-regulated phosphatases, but it does not regulate this activity at the level of transcription. We also identify and demonstrate a novel role in siderophore production and Pho-regulated phosphatase activity for ApaH, the hydrolase for the nucleotide-signaling molecule AppppA. Finally, numerous mutations in multiple cellular pathways were recovered that may be required for maximal induction of the Pho regulon under Pi-limiting conditions.