Orr H. Shapiro

A bacterial pathogen uses dimethylsulfoniopropionate as a cue to target heat-stressed corals.

Diseases are an emerging threat to ocean ecosystems. Coral reefs, in particular, are experiencing a worldwide decline because of disease and bleaching, which have been exacerbated by rising seawater temperatures. Yet, the ecological mechanisms behind most coral diseases remain unidentified. Here, we demonstrate that a coral pathogen, Vibrio coralliilyticus, uses chemotaxis and chemokinesis to target the mucus of its coral host, Pocillopora damicornis. A primary driver of this response is the host metabolite dimethylsulfoniopropionate (DMSP), a key element in the global sulfur cycle and a potent foraging cue throughout the marine food web. Coral mucus is rich in DMSP, and we found that DMSP alone elicits chemotactic responses of comparable intensity to whole mucus. Furthermore, in heat-stressed coral fragments, DMSP concentrations increased fivefold and the pathogen’s chemotactic response was correspondingly enhanced. Intriguingly, despite being a rich source of carbon and sulfur, DMSP is not metabolized by the pathogen, suggesting that it is used purely as an infochemical for host location. These results reveal a new role for DMSP in coral disease, demonstrate the importance of chemical signaling and swimming behavior in the recruitment of pathogens to corals and highlight the impact of increased seawater temperatures on disease pathways.

Advantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity

ABSTRACT To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively to Proteobacteria (95.8%). Sequences belonging to Firmicutes (42.6%) and Thermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, while Proteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.

Bacteriophage predation regulates microbial abundance and diversity in a full-scale bioreactor treating industrial wastewater

Changes in the microbial community composition of a full-scale membrane bioreactor treating industrial wastewater were studied over a period of 462 days using a series of 16S rRNA gene clone libraries. Frequent changes in the relative abundance of specific taxonomic groups were observed, which could not be explained by changes in the reactors conditions or wastewater composition. Phage activity was proposed to drive some of the observed changes. Bacterial hosts were isolated from a biomass sample obtained towards the end of the study period, and specific phage counts were carried out for some of the isolated hosts using stored frozen biomass samples as the phage inocula. Plaque-forming unit concentrations were shown to change frequently over the study period, in correlation with changes in the relative abundance of taxonomic groups closely related by 16S rRNA gene sequence to the isolated strains. Quantitative PCR was used to verify changes in the abundance of a taxonomic group closely related to one of the isolated hosts, showing good agreement with the changes in relative abundance in the clone libraries of that group. The emerging pattern was consistent with the ‘killing the winner’ hypothesis, although alternative interaction mechanisms could not be ruled out. This is the first time that phage–host interactions in a complex microbial community are demonstrated over an extended period, and possibly the first in situ demonstration of ‘killing the winner’ stochastic behavior.

Vortical ciliary flows actively enhance mass transport in reef corals

Significance The fitness of corals and their ability to form large reefs hinge on their capacity to exchange oxygen and nutrients with their environment. Lacking gills or other ventilating organs, corals have been commonly assumed to depend entirely on ambient flow to overcome the mass transport limitations associated with molecular diffusion. Here, we show that corals are not enslaved to ambient flow but instead, can actively enhance mass transport by producing intense vortical flows with their epidermal cilia. By vigorously stirring the water immediately adjacent to their surface, this active process allows corals to increase mass transport and thus, can be a fundamental survival mechanism in regions or at times of weak ambient flow. The exchange of nutrients and dissolved gasses between corals and their environment is a critical determinant of the growth of coral colonies and the productivity of coral reefs. To date, this exchange has been assumed to be limited by molecular diffusion through an unstirred boundary layer extending 1–2 mm from the coral surface, with corals relying solely on external flow to overcome this limitation. Here, we present direct microscopic evidence that, instead, corals can actively enhance mass transport through strong vortical flows driven by motile epidermal cilia covering their entire surface. Ciliary beating produces quasi-steady arrays of counterrotating vortices that vigorously stir a layer of water extending up to 2 mm from the coral surface. We show that, under low ambient flow velocities, these vortices, rather than molecular diffusion, control the exchange of nutrients and oxygen between the coral and its environment, enhancing mass transfer rates by up to 400%. This ability of corals to stir their boundary layer changes the way that we perceive the microenvironment of coral surfaces, revealing an active mechanism complementing the passive enhancement of transport by ambient flow. These findings extend our understanding of mass transport processes in reef corals and may shed new light on the evolutionary success of corals and coral reefs.

Bacteriophage ecology in environmental biotechnology processes

Heterotrophic bacteria are an integral part of any environmental biotechnology process (EBP). Therefore, factors controlling bacterial abundance, activity, and community composition are central to the understanding of such processes. Among these factors, top-down control by bacteriophage predation has so far received very limited attention. With over 10(8) particles per ml, phage appear to be the most numerous biological entities in EBP. Phage populations in EBP appear to be highly dynamic and to correlate with the population dynamics of their hosts and genomic evidence suggests bacteria evolve to avoid phage predation. Clearly, there is much to learn regarding bacteriophage in EBP before we can truly understand the microbial ecology of these globally important systems.

Multicellular photo‐magnetotactic bacteria

Multicellular magnetotactic bacteria (MMB) are unique microorganisms typically comprised of 10-40 bacterial cells arranged around a central acellular compartment. Their life cycle has no known unicellular stage and division occurs by separation of a single MMB aggregate into two identical offspring. In this study, South-seeking multicellular magnetotactic bacteria (ssMMB) were enriched from a New England salt marsh. When exposed to light, ssMMB reversed their magnetotactic behaviour to become North-seeking. The exposure time needed to generate the reversal response varied with light wavelength and intensity. Extensive exposure to light appeared to be lethal. This is the first report of a Northern hemisphere MMB displaying South-seeking behaviour and the first time a MMB is found to exhibit photo-magnetotaxis. We suggest that this mechanism enables ssMMB to optimize their location with regard to chemical gradients and light intensities, and propose a model to explain the peculiar balance between photo- and magnetotaxis.

Substitution by Inosine at the 3′-Ultimate and Penultimate Positions of 16S rRNA Gene Universal Primers

Universal 16S rRNA gene primers (8F and 518R) bearing inosine substitutions at either the 3′-ultimate or the 3′-ultimate and penultimate base positions were exploited for the first time to study the bacterial community associated with coral polymicrobial Black Band Disease (BBD). Inosine-modified universal primer pairs display some shifting in the composition of 16S rRNA gene libraries, as well as expanding the observed diversity of a BBD bacterial community at the family/class level. Possible explanations for the observed shifts are discussed. These results thus point to the need for adopting multiple approaches in designing 16S rRNA universal primers for PCR amplification and subsequent construction of 16S rRNA gene libraries or pyrosequencing in the exploration of complex microbial communities.

Application of a unique miniature MBR for screening the biodegradation of brominated flame retardants

Abstract A unique miniature membrane bioreactor (mMBR) was designed and applied to examine the biodegradability of two complex organic compounds belonging to a family of brominated flame retardants (BFR) under continuous culture conditions using a bacterial consortium. BFRs are a widely used group of anthropogenic environmental contaminants. Many of these compounds are toxic, persistent, have limited biodegradability, and tend to bioaccumulate in the environment. Their widespread production and use combined with the inappropriate treatment and disposal of industrial wastewater have caused myriad global health and environmental concerns. Dibromoneopentyl glycol (DBNPG) and tribromoneopentyl alcohol (TBNPA) are aliphatic BFRs, classified as recalcitrant compounds, having half-lives of more than 100 years. Following successful debromination and complete biodegradation of the two target compounds in the mMBR, we used molecular and bioinformatic techniques to track changes in bacterial community composition du...

Microscale tracking of coral disease reveals timeline of infection and heterogeneity of polyp fate.

Coral disease is often studied at scales ranging from single colonies to the entire reef. This is particularly true for studies following disease progression through time. To gain a mechanistic understanding of key steps underlying infection dynamics, it is necessary to study disease progression, and host-pathogen interactions, at relevant microbial scales. Here we provide a dynamic view of the interaction between the model coral pathogen Vibrio coralliilyticus and its coral host Pocillopora damicornis at unprecedented spatial and temporal scales. This view is achieved using a novel microfluidics-based system specifically designed to allow microscopic study of coral infection in-vivo under controlled environmental conditions. Analysis of exudates continuously collected at the system’s outflow, allows a detailed biochemical and microbial analyses coupled to the microscopic observations of the disease progression. The resulting multilayered dataset provides the most detailed description of a coral infection to-date, revealing distinct pathogenic processes as well as the defensive behavior of the coral host. We provide evidence that infection in this system occurs following ingestion of the pathogen, and may then progress through the gastrovascular system. We further show infection may spread when pathogens colonize lesions in the host tissue. Copious spewing of pathogen-laden mucus from the polyp mouths results in effective expulsion of the pathogen from the gastrovascular system, possibly serving as a first line of defense. A secondary defense mechanism entails the severing of calicoblastic connective tissues resulting in the controlled isolation of diseased polyps, or the survival of individual polyps within infected colonies. Further investigations of coral-pathogen interactions at these scales will help to elucidate the complex interactions underlying coral disease, as we as the versatile adaptive response of the coral ecosystems to fluctuating environments.

Phenotypic variability in chloroplast redox state predicts cell fate in a marine diatom

Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in diverse aquatic ecosystems. They are subjected to a wide range of environmental cues, including abiotic stressors and biotic interactions with associated bacteria, viruses and grazers. However, the cellular strategies that underline their ecological success and their rapid acclimation to fluctuating conditions are still underexplored. This study investigates heterogeneity within diatom populations in response to oxidative stress, which mediates a wide range of environmental stress conditions. We combined flow cytometry and microfluidics system for live-imaging microscopy to measure redox dynamics at the single-cell level. Using the redox-sensitive sensor roGFP we measured in vivo organelle-specific oxidation patterns in the model diatom Phaeodactylum tricornutum. Chloroplast targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to oxidative stress, revealing two distinct subpopulations. Remarkably, the “oxidized” subpopulation was sensitive to the stress and subsequently died, while the “reduced” subpopulation was resilient to it and recovered. We further characterized an early phase of “pre-commitment” to cell death following oxidative stress, after which cell death was irreversibly activated in the “oxidized” cells, even upon removal of the stress. Oxidation of the chloroplast glutathione pool preceded commitment to cell death, and was used as a novel predictor of cell fate. We propose that phenotypic variability within diatom populations can provide an ecological strategy to cope with rapid environmental fluctuations in the marine ecosystem.Abstract Diatoms are photosynthetic microorganisms of great ecological and biogeochemical importance, forming vast blooms in diverse aquatic ecosystems. Current understanding of phytoplankton acclimation to stress is based on population-level analysis, masking cell-to-cell variability. Here we investigated heterogeneity within Phaeodactylum tricornutum populations in response to oxidative stress, which is induced by environmental stress conditions. We combined flow cytometry and a microfluidics system for live imaging to measure redox dynamics at the single-cell level using the roGFP sensor. Chloroplast-targeted roGFP exhibited a light-dependent, bi-stable oxidation pattern in response to H2O2, revealing distinct subpopulations of sensitive oxidized cells and resilient reduced cells. Subpopulation proportions depended on growth phase, linking the bi-stable phenotype to proliferation. Oxidation of chloroplast-targeted roGFP preceded commitment to cell death and was used as a novel cell fate predictor. We propose that light-dependent metabolic heterogeneity results in differential stress responses that regulate cell fate within diatom populations.