Conversations with a Killer Bacterium: Identifying new targets of Quorum Sensing sRNAs in Vibrio cholera

A presentation in the Undergraduate Research Kaleidoscope: Library East Commons Performance Space, February 9, 2009Runtime: 06:42 minutes (Edited)Runtime: 06:43 minutes (Original)Bacteria communicate with each other by releasing signal molecules into the environment that travel between cells. When the molecules accumulate, surface receptors detect the signals and trigger pathways that alter gene expression on a population-wide scale. This process, called quorum sensing (QS), allows bacterial groups to behave like multicellular organisms. In the human pathogen Vibrio cholerae, QS synchronizes the production of multiple small RNAs (sRNAs) that do not encode proteins, but instead bind to and alter messenger RNA targets, including one that controls essential virulence traits. We used a new computer algorithm, TargetRNA, to verify the known sRNA-controlled targets and also to predict several new candidates. I will genetically engineer a fusion of each candidate to a bioluminescent “reporter” gene, visually monitor responses to QS signals, and define the mechanism of each sRNA/mRNA binding interaction. These studies will clarify how chemical information is processed and converted into behavioral changes in this deadly microbe.Brian K. Hamme

Characterizing a novel direct target of the quorum-sensing controlled small RNAs in V. cholerae

n/aUndergraduat

High-resolution time series reveals differential behaviors of closely-related microbes in coastal communities

Coastal plankton, primarily composed of heterotrophic bacteria, eukaryotic microalgae, and other small eukaryotes, have an outsized impact on global biogeochemical cycles. Understanding the forces that affect community assembly and dynamics through time is therefore important for our understanding of these cycles. From careful analyses of the genetics and behaviors of isolates we know that very closely related microbes can vary in their potential for growth, defense from predation, and ability to compete and cooperate for resources. However in surveys of the environment, most studies have focused on lower-resolution groupings of taxa, so little is known about how these differences between closely related microbes play out in the wild. In this thesis, I show that when viewed at high temporal, spatial, and genetic resolution, the coastal plankton community is highly dynamic. Making use of a 93-day time series collected from Nahant, Massachusetts, I analyze amplicons at single-nucleotide resolution. First, for the eukaryotic community I show that despite apparent stability at higher taxonomic levels, there is rapid turnover of the community at the sequence level, and that for sequences one nucleotide apart there is evidence for distinct ecologies. Second, in the bacterial community, I use a much more resolved genetic marker library to show that even when sequences emerge from the same species there is evidence for distinct dynamics during the time series. Taken together, these observations demonstrate a seemingly fractal diversity in the coastal ocean plankton, where the further one zooms in the more distinctions one can make between organisms. This dizzying diversity across temporal, spatial, and evolutionary scales may have previously unappreciated impacts on our understanding of these communities.Ph.D

Methanogen Productivity and Microbial Community Composition Varies With Iron Oxide Mineralogy

Quantifying the flux of methane from terrestrial environments remains challenging, owing to considerable spatial and temporal variability in emissions. Amongst a myriad of factors, variation in the composition of electron acceptors, including metal (oxyhydr)oxides, may impart controls on methane emission. The purpose of this research is to understand how iron (oxyhydr)oxide minerals with varied physicochemical properties influence microbial methane production and subsequent microbial community development. Incubation experiments, using lake sediment as an inoculum and acetate as a carbon source, were used to understand the influence of one poorly crystalline iron oxide mineral, ferrihydrite, and two well-crystalline minerals, hematite and goethite, on methane production. Iron speciation, headspace methane, and 16S-rRNA sequencing microbial community data were measured over time. Substantial iron reduction only occurred in the presence of ferrihydrite while hematite and goethite had little effect on methane production throughout the incubations. In ferrihydrite experiments the time taken to reach the maximum methane production rate was slower than under other conditions, but methane production, eventually occurred in the presence of ferrihydrite. We suggest that this is due to ferrihydrite transformation into more stable minerals like magnetite and goethite or surface passivation by Fe(II). While all experimental conditions enriched for Methanosarcina, only the presence of ferrihydrite enriched for iron reducing bacteria Geobacter. Additionally, the presence of ferrihydrite continued to influence microbial community development after the onset of methanogenesis, with the dissimilarity between communities growing in ferrihydrite compared to no-Fe-added controls increasing over time. This work improves our understanding of how the presence of different iron oxides influences microbial community composition and methane production in soils and sediments

Epilithic Bacterial Assemblages on Subtidal Rocky Reefs: Variation Among Alternative Habitats at Ambient and Enhanced Nutrient Levels

Abstract Temperate rocky reefs often support mosaics of alternative habitats such as macroalgal forests, algal turfs and sea urchin barrens. Although the composition of epilithic microbial biofilms (EMBs) is recognized as a major determinant of macroalgal recruitment, their role in regulating the stability of alternative habitats on temperate rocky reefs remains unexplored. On shallow rocky reefs of the Island of Capraia (NW Mediterranean), we compared EMB structure among canopy stands formed by the fucoid Ericaria brachycarpa, algal turfs, and urchin barrens under ambient versus experimentally enhanced nutrient levels. The three habitats shared a core microbial community consisting of 21.6 and 25.3% of total ASVs under ambient and enhanced nutrient conditions, respectively. Although Gammaproteobacteria, Alphaproteobacteria and Flavobacteriia were the most abundant classes across habitats, multivariate analyses at the ASV level showed marked differences in EMB composition among habitats. Enhancing nutrient level had no significant effect on EMBs, although it increased their similarity between macroalgal canopy and turf habitats. At both ambient and enriched nutrient levels, ASVs mostly belonging to Proteobacteria and Bacteroidetes were more abundant in EMBs from macroalgal canopies than barrens. In contrast, ASVs belonging to the phylum of Proteobacteria and, in particular, to the families of Rhodobacteraceae and Flavobacteriaceae at ambient nutrient levels and of Rhodobacteraceae and Bacteriovoracaceae at enhanced nutrient levels were more abundant in turf than canopy habitats. Our results show that primary surfaces from alternative habitats that form mosaics on shallow rocky reefs in oligotrophic areas host distinct microbial communities that are, to some extent, resistant to moderate nutrient enhancement. Understanding the role of EMBs in generating reinforcing feedback under different nutrient loading regimes appears crucial to advance our understanding of the mechanisms underpinning the stability of habitats alternative to macroalgal forests as well as their role in regulating reverse shifts

Viruses of the Nahant Collection, characterization of 251 marine Vibrionaceae viruses

Viruses are highly discriminating in their interactions with host cells and are thought to play a major role in maintaining diversity of environmental microbes. However, large-scale ecological and genomic studies of co-occurring virus-host pairs, required to characterize the mechanistic and genomic foundations of virus-host interactions, are lacking. Here, we present the largest dataset of cultivated and sequenced co-occurring virus-host pairs that captures ecologically representative fine-scale diversity. Using the ubiquitous and ecologically diverse marine Vibrionaceae as a host platform, we isolate and sequence 251 dsDNA viruses and their hosts from three time points within a 93-day time-series study. The virus collection includes representatives of the three Caudovirales tailed virus morphotypes, a novel family of nontailed viruses, and the smallest (10,046 bp) and largest (348,911 bp) Vibrio virus genomes described. We provide general characterization and annotation of the viruses and describe read-mapping protocols to standardize genome presentation. The rich ecological and genomic contextualization of hosts and viruses make the Nahant Collection a unique platform for high-resolution studies of environmental virus-host infection networks.National Science Foundation (U.S.) (OCE 1435993

Virfam annotation of viruses of the Nahant Collection.

Virfam annotation of the tailed viruses of the Nahant Collection -provides predicted morphotype and assignment to categories based on head-neck-tail modules

Rapid evolutionary turnover of mobile genetic elements drives bacterial resistance to phages

Although it is generally accepted that phages drive bacterial evolution, how these dynamics play out in the wild remains poorly understood. We found that susceptibility to viral killing in marine Vibrio is mediated by large and highly diverse mobile genetic elements. These phage defense elements display exceedingly fast evolutionary turnover, resulting in differential phage susceptibility among clonal bacterial strains while phage receptors remain invariant. Protection is cumulative, and a single bacterial genome can harbor 6 to 12 defense elements, accounting for more than 90% of the flexible genome among close relatives. The rapid turnover of these elements decouples phage resistance from other genomic features. Thus, resistance to phages in the wild follows evolutionary trajectories alternative to those predicted from laboratory-based evolutionary experiments