Gary R. LeCleir

Master recyclers: features and functions of bacteria associated with phytoplankton blooms.

Marine phytoplankton blooms are annual spring events that sustain active and diverse bloom-associated bacterial populations. Blooms vary considerably in terms of eukaryotic species composition and environmental conditions, but a limited number of heterotrophic bacterial lineages — primarily members of the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria — dominate these communities. In this Review, we discuss the central role that these bacteria have in transforming phytoplankton-derived organic matter and thus in biogeochemical nutrient cycling. On the basis of selected field and laboratory-based studies of flavobacteria and roseobacters, distinct metabolic strategies are emerging for these archetypal phytoplankton-associated taxa, which provide insights into the underlying mechanisms that dictate their behaviours during blooms.

Analysis of microbial gene transcripts in environmental samples.

ABSTRACT We analyzed gene expression in marine and freshwater bacterioplankton communities by the direct retrieval and analysis of microbial transcripts. Environmental mRNA, obtained from total RNA by subtractive hybridization of rRNA, was reverse transcribed, amplified with random primers, and cloned. Approximately 400 clones were analyzed, of which ∼80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both Bacteria (mainly α- and γ-Proteobacteria) and Archaea (mainly Euryarchaeota). Many transcripts could be linked to environmentally important processes such as sulfur oxidation (soxA), assimilation of C1 compounds (fdh1B), and acquisition of nitrogen via polyamine degradation (aphA). Environmental transcriptomics is a means of exploring functional gene expression within natural microbial communities without bias toward known sequences, and provides a new approach for obtaining community-specific variants of key functional genes.

Niche of harmful alga Aureococcus anophagefferens revealed through ecogenomics.

Harmful algal blooms (HABs) cause significant economic and ecological damage worldwide. Despite considerable efforts, a comprehensive understanding of the factors that promote these blooms has been lacking, because the biochemical pathways that facilitate their dominance relative to other phytoplankton within specific environments have not been identified. Here, biogeochemical measurements showed that the harmful alga Aureococcus anophagefferens outcompeted co-occurring phytoplankton in estuaries with elevated levels of dissolved organic matter and turbidity and low levels of dissolved inorganic nitrogen. We subsequently sequenced the genome of A. anophagefferens and compared its gene complement with those of six competing phytoplankton species identified through metaproteomics. Using an ecogenomic approach, we specifically focused on gene sets that may facilitate dominance within the environmental conditions present during blooms. A. anophagefferens possesses a larger genome (56 Mbp) and has more genes involved in light harvesting, organic carbon and nitrogen use, and encoding selenium- and metal-requiring enzymes than competing phytoplankton. Genes for the synthesis of microbial deterrents likely permit the proliferation of this species, with reduced mortality losses during blooms. Collectively, these findings suggest that anthropogenic activities resulting in elevated levels of turbidity, organic matter, and metals have opened a niche within coastal ecosystems that ideally suits the unique genetic capacity of A. anophagefferens and thus, has facilitated the proliferation of this and potentially other HABs.

Chitinase Gene Sequences Retrieved from Diverse Aquatic Habitats Reveal Environment-Specific Distributions

ABSTRACT Chitin is an abundant biopolymer whose degradation is mediated primarily by bacterial chitinases. We developed a degenerate PCR primer set to amplify a ∼900-bp fragment of family 18, group I chitinase genes and used it to retrieve these gene fragments from environmental samples. Clone libraries of presumptive chitinase genes were created for nine water and six sediment samples from 10 aquatic environments including freshwater and saline lakes, estuarine water and sediments, and the central Arctic Ocean. Putative chitinase sequences were also retrieved from the Sargasso Sea metagenome sequence database. We were unable to obtain PCR product with these primers from an alkaline, hypersaline lake (Mono Lake, California). In total, 108 partial chitinase gene sequences were analyzed, with a minimum of 5 and a maximum of 13 chitinase sequences obtained from each library. All chitinase sequences were novel compared to previously identified sequences. Intralibrary sequence diversity was low, while we found significant differences between libraries from different water column samples and between water column and sediment samples. However, identical sequences were retrieved from samples collected at widely distributed locations that did not necessarily represent similar environments, suggesting homogeneity of chitinoclastic communities between some environments.

Composition of the gut microbiota modulates the severity of malaria.

Significance Plasmodium infections cause >200 million cases of malaria and ∼1 million deaths annually. Although these infections result in disease states that range from asymptomatic to life-threatening, factors that contribute to disease severity remain poorly defined. This report demonstrates that the assemblage of microbes in the gut can modulate the severity of malaria. Mice from different vendors with differences in their gut microbiome showed significant differences in pathology after infection with Plasmodium. Among the bacterial populations that were different between “resistant” and “susceptible” mice were Lactobacillus and Bifidobacterium, and treatment of mice with Lactobacillus and Bifidobacterium resulted in decreased Plasmodium burden. These results identify both a previously unidentified risk factor for severe malaria and a potential new avenue of treatment. Plasmodium infections result in clinical presentations that range from asymptomatic to severe malaria, resulting in ∼1 million deaths annually. Despite this toll on humanity, the factors that determine disease severity remain poorly understood. Here, we show that the gut microbiota of mice influences the pathogenesis of malaria. Genetically similar mice from different commercial vendors, which exhibited differences in their gut bacterial community, had significant differences in parasite burden and mortality after infection with multiple Plasmodium species. Germfree mice that received cecal content transplants from “resistant” or “susceptible” mice had low and high parasite burdens, respectively, demonstrating the gut microbiota shaped the severity of malaria. Among differences in the gut flora were increased abundances of Lactobacillus and Bifidobacterium in resistant mice. Susceptible mice treated with antibiotics followed by yogurt made from these bacterial genera displayed a decreased parasite burden. Consistent with differences in parasite burden, resistant mice exhibited an elevated humoral immune response compared with susceptible mice. Collectively, these results identify the composition of the gut microbiota as a previously unidentified risk factor for severe malaria and modulation of the gut microbiota (e.g., probiotics) as a potential treatment to decrease parasite burden.

Phage infection of an environmentally relevant marine bacterium alters host metabolism and lysate composition

Viruses contribute to the mortality of marine microbes, consequentially altering biological species composition and system biogeochemistry. Although it is well established that host cells provide metabolic resources for virus replication, the extent to which infection reshapes host metabolism at a global level and the effect of this alteration on the cellular material released following viral lysis is less understood. To address this knowledge gap, the growth dynamics, metabolism and extracellular lysate of roseophage-infected Sulfitobacter sp. 2047 was studied using a variety of techniques, including liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based metabolomics. Quantitative estimates of the total amount of carbon and nitrogen sequestered into particulate biomass indicate that phage infection redirects ∼75% of nutrients into virions. Intracellular concentrations for 82 metabolites were measured at seven time points over the infection cycle. By the end of this period, 71% of the detected metabolites were significantly elevated in infected populations, and stable isotope-based flux measurements showed that these cells had elevated metabolic activity. In contrast to simple hypothetical models that assume that extracellular compounds increase because of lysis, a profile of metabolites from infected cultures showed that >70% of the 56 quantified compounds had decreased concentrations in the lysate relative to uninfected controls, suggesting that these small, labile nutrients were being utilized by surviving cells. These results indicate that virus-infected cells are physiologically distinct from their uninfected counterparts, which has implications for microbial community ecology and biogeochemistry.

Temporal changes in particle-associated microbial communities after interception by nonlethal sediment traps.

Using marine sediment traps (named RESPIRE for REspiration of Sinking Particles In the subsuRface ocEan) designed to collect sinking particles and associated microbial communities in situ, we collected and incubated marine aggregates/particles in the southern Pacific Ocean from separate phytoplankton bloom events in situ. We determined the phylogenetic affiliation for the microorganisms growing on aggregates by pyrosequencing partial 16S rRNA gene amplicons. Water column samples were also collected and sequenced for comparison between sinking-particle-associated and planktonic bacterial communities. Statistically significant differences were found between the water column and sediment trap bacteria. Relative abundances of Pelagibacter sp. and multiple members of the Flavobacteria, Actinobacteria, and α-Proteobacteria were elevated in water column samples, while trap samples contained members of the Roseobacter clade of α-Proteobacteria in high relative abundances. Our findings indicated that rapid changes - within 24 h of collection - occurred to the microbial community associated with aggregates from either bloom type. There was a little change in the bacterial assemblage after the initial 24-h incubation period. The most abundant early colonizer was a Sulfitobacter sp. This study provides further evidence that Roseobacters are rapid colonizers of marine aggregates and that colonization can occur on short timescales. This study further demonstrates that particle origin may be insignificant regarding the heterotrophic bacterial population that degrades them.

Viral and bacterial abundance and production in the Western Pacific Ocean and the relation to other oceanic realms

We completed a transect through the Western Pacific Warm Pool to examine how environmental variables may influence viral and bacterial abundance and production rates in this globally important oceanic region. Of the variables analyzed, viral abundance and production had the most significant relationship to bacterial cell abundance: viral parameters were not significantly correlated to the measured environmental variables, including temperature. Bacterial production rates were significantly correlated to temperature in open ocean waters, but not in waters close to land masses. Analyses of 16S rRNA gene by pyrosequencing indicated only minor changes in eubacterial community structure across the transect, with α-proteobacteria dominating all sampled populations. Diversity within the prokaryotic community did not correlate directly with viral abundance or activity. Comparisons to two other ocean-scale transects (> 8000 km of open ocean in total) in the Atlantic Ocean indicated that correlations between viral and bacterial abundance and production relative to environmental variables are regime dependent. In particular, correlations to temperature showed remarkable differences across the three transects. Collectively, our observations suggest that seemingly similar oceanic regions may have very different microbial community responses to environmental variables. Our observations and analyses demonstrate that ocean-scale generalizations may not apply in the case of viral ecology.

Examining the impact of acetylene on N-fixation and the active sediment microbial community

Here we examined the impact of a commonly employed method used to measure nitrogen fixation, the acetylene reduction assay (ARA), on a marine sediment community. Historically, the ARA technique has been broadly employed for its ease of use, in spite of numerous known artifacts. To gauge the severity of these effects in a natural environment, we employed high-throughput 16S rRNA gene sequencing to detect differences in acetylene-treated sediments vs. non-treated control sediments after a 7 h incubation. Within this short time period, significant differences were seen across all activity of microbes identified in the sediment, implying that the changes induced by acetylene occur quickly. The results have important implications for our understanding of marine nitrogen budgets. Moreover, because the ARA technique has been widely used in terrestrial and freshwater habitats, these results may be applicable to other ecosystems.

Identifying the source of unknown microcystin genes and predicting microcystin variants by comparing genes within uncultured cyanobacterial cells

ABSTRACT While multiple phylogenetic markers have been used in the culture-independent study of microcystin-producing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (>6 kb) encompassing two genes involved in microcystin biosynthesis (mcyA2 and mcyB1) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel mcyA genotype previously termed Microcystis-like that is pervasive in the Laurentian Great Lakes and they predict the microcystin variant(s) that it produces.