Divergent gene expression responses in two Baltic Sea heterotrophic model bacteria to dinoflagellate dissolved organic matter.

Phytoplankton release massive amounts of dissolved organic matter (DOM) into the water column during recurring blooms in coastal waters and inland seas. The released DOM encompasses a complex mixture of both known and unknown compounds, and is a rich nutrient source for heterotrophic bacteria. The metabolic activity of bacteria during and after phytoplankton blooms can hence be expected to reflect the characteristics of the released DOM. We therefore investigated if bacterioplankton could be used as "living sensors" of phytoplankton DOM quantity and/or quality, by applying gene expression analyses to identify bacterial metabolisms induced by DOM. We used transcriptional analysis of two Baltic Sea bacterial isolates (Polaribacter sp. BAL334 [Flavobacteriia] and Brevundimonas sp. BAL450 [Alphaproteobacteria]) growing with DOM from axenic cultures of the dinoflagellate Prorocentrum minimum. We observed pronounced differences between the two bacteria both in growth and the expressed metabolic pathways in cultures exposed to dinoflagellate DOM compared with controls. Differences in metabolic responses between the two isolates were caused both by differences in gene repertoire between them (e.g. in the SEED categories for membrane transport, motility and photoheterotrophy) and the regulation of expression (e.g. fatty acid metabolism), emphasizing the importance of separating the responses of different taxa in analyses of community sequence data. Similarities between the bacteria included substantially increased expression of genes for Ton and Tol transport systems in both isolates, which are commonly associated with uptake of complex organic molecules. Polaribacter sp. BAL334 showed stronger metabolic responses to DOM harvested from exponential than stationary phase dinoflagellates (128 compared to 26 differentially expressed genes), whereas Brevundimonas sp. BAL450 responded more to the DOM from stationary than exponential phase dinoflagellates (33 compared to 6 differentially expressed genes). These findings suggest that shifts in bacterial metabolisms during different phases of phytoplankton blooms can be detected in individual bacterial species and can provide insights into their involvement in DOM transformations.

Deciphering Chemical Mediators Regulating Specialized Metabolism in a Symbiotic Cyanobacterium.

Genomes of cyanobacteria feature a variety of cryptic biosynthetic pathways for complex natural products, but the peculiarities limiting the discovery and exploitation of the metabolic dark matter are not well understood. Here we describe the discovery of two cell density-dependent chemical mediators, nostoclide and nostovalerolactone, in the symbiotic model strain Nostoc punctiforme, and demonstrate their pronounced impact on the regulation of specialized metabolism. Through transcriptional, bioinformatic and labeling studies we assigned two adjacent biosynthetic gene clusters to the biosynthesis of the two polyketide mediators. Our findings provide insight into the orchestration of specialized metabolite production and give lessons for the genomic mining and high-titer production of cyanobacterial bioactive compounds.

Effects of allochthonous dissolved organic matter input on microbial composition and nitrogen-cycling genes at two contrasting estuarine sites.

Heterotrophic bacteria are important drivers of nitrogen (N) cycling and the processing of dissolved organic matter (DOM). Projected increases in precipitation will potentially cause increased loads of riverine DOM to the Baltic Sea and likely affect the composition and function of bacterioplankton communities. To investigate this, the effects of riverine DOM from two different catchment areas (agricultural and forest) on natural bacterioplankton assemblages from two contrasting sites in the Baltic Sea were examined. Two microcosm experiments were carried out, where the community composition (16S rRNA gene sequencing), the composition of a suite of N-cycling genes (metagenomics) and the abundance and transcription of ammonia monooxygenase (amoA) genes involved in nitrification (quantitative PCR) were investigated. The river water treatments evoked a significant response in bacterial growth, but the effects on overall community composition and the representation of N-cycling genes were limited. Instead, treatment effects were reflected in the prevalence of specific taxonomic families, specific N-related functions and in the transcription of amoA genes. The study suggests that bacterioplankton responses to changes in the DOM pool are constrained to part of the bacterial community, whereas most taxa remain relatively unaffected.

Insight into the genome and brackish water adaptation strategies of toxic and bloom-forming Baltic Sea Dolichospermum sp. UHCC 0315.

The Baltic Sea is a shallow basin of brackish water in which the spatial salinity gradient is one of the most important factors contributing to species distribution. The Baltic Sea is infamous for its annual cyanobacterial blooms comprised of Nodularia spumigena, Aphanizomenon spp., and Dolichospermum spp. that cause harm, especially for recreational users. To broaden our knowledge of the cyanobacterial adaptation strategies for brackish water environments, we sequenced the entire genome of Dolichospermum sp. UHCC 0315, a species occurring not only in freshwater environments but also in brackish water. Comparative genomics analyses revealed a close association with Dolichospermum sp. UHCC 0090 isolated from a lake in Finland. The genome closure of Dolichospermum sp. UHCC 0315 unraveled a mixture of two subtypes in the original culture, and subtypes exhibited distinct buoyancy phenotypes. Salinity less than 3 g L-1 NaCl enabled proper growth of Dolichospermum sp. UHCC 0315, whereas growth was arrested at moderate salinity (6 g L-1 NaCl). The concentrations of toxins, microcystins, increased at moderate salinity, whereas RNA sequencing data implied that Dolichospermum remodeled its primary metabolism in unfavorable high salinity. Based on our results, the predicted salinity decrease in the Baltic Sea may favor toxic blooms of Dolichospermum spp.

Coupling biogeochemical process rates and metagenomic blueprints of coastal bacterial assemblages in the context of environmental change.

Bacteria are major drivers of biogeochemical nutrient cycles and energy fluxes in marine environments, yet how bacterial communities respond to environmental change is not well known. Metagenomes allow examination of genetic responses of the entire microbial community to environmental change. However, it is challenging to link metagenomes directly to biogeochemical process rates. Here, we investigate metagenomic responses in natural bacterioplankton communities to simulated environmental stressors in the Baltic Sea, including increased river water input, increased nutrient concentration, and reduced oxygen level. This allowed us to identify informative prokaryotic gene markers, responding to environmental perturbation. Our results demonstrate that metagenomic and metabolic changes in bacterial communities in response to environmental stressors are influenced both by the initial community composition and by the biogeochemical factors shaping the functional response. Furthermore, the different sources of dissolved organic matter (DOM) had the largest impact on metagenomic blueprint. Most prominently, changes in DOM loads influenced specific transporter types reflecting the substrate availability and DOC assimilation and consumption pathways. The results provide new knowledge for developing models of ecosystem structure and biogeochemical cycling in future climate change scenarios and advance our exploration of the potential use of marine microorganisms as markers for environmental conditions.

Comparative Genomics of the Baltic Sea Toxic Cyanobacteria Nodularia spumigena UHCC 0039 and Its Response to Varying Salinity.

Salinity is an important abiotic factor controlling the distribution and abundance of Nodularia spumigena, the dominating diazotrophic and toxic phototroph, in the brackish water cyanobacterial blooms of the Baltic Sea. To expand the available genomic information for brackish water cyanobacteria, we sequenced the isolate Nodularia spumigena UHCC 0039 using an Illumina-SMRT hybrid sequencing approach, revealing a chromosome of 5,294,286 base pairs (bp) and a single plasmid of 92,326 bp. Comparative genomics in Nostocales showed pronounced genetic similarity among Nodularia spumigena strains evidencing their short evolutionary history. The studied Baltic Sea strains share similar sets of CRISPR-Cas cassettes and a higher number of insertion sequence (IS) elements compared to Nodularia spumigena CENA596 isolated from a shrimp production pond in Brazil. Nodularia spumigena UHCC 0039 proliferated similarly at three tested salinities, whereas the lack of salt inhibited its growth and triggered transcriptome remodeling, including the up-regulation of five sigma factors and the down-regulation of two other sigma factors, one of which is specific for strain UHCC 0039. Down-regulated genes additionally included a large genetic region for the synthesis of two yet unidentified natural products. Our results indicate a remarkable plasticity of the Nodularia salinity acclimation, and thus salinity strongly impacts the intensity and distribution of cyanobacterial blooms in the Baltic Sea.

Strains of the toxic and bloom-forming Nodularia spumigena (cyanobacteria) can degrade methylphosphonate and release methane.

Nodularia spumigena is a nitrogen-fixing cyanobacterium that forms toxic blooms in the Baltic Sea each summer and the availability of phosphorous is an important factor limiting the formation of these blooms. Bioinformatic analysis identified a phosphonate degrading (phn) gene cluster in the genome of N. spumigena suggesting that this bacterium may use phosphonates as a phosphorus source. Our results show that strains of N. spumigena could grow in medium containing methylphosphonic acid (MPn) as the sole source of phosphorous and released methane when growing in medium containing MPn. We analyzed the total transcriptomes of N. spumigena UHCC 0039 grown using MPn and compared them with cultures growing in Pi-replete medium. The phnJ, phosphonate lyase gene, was upregulated when MPn was the sole source of phosphorus, suggesting that the expression of this gene could be used to indicate the presence of bioavailable phosphonates. Otherwise, growth on MPn resulted in only a minor reconstruction of the transcriptome and enabled good growth. However, N. spumigena strains were not able to utilize any of the anthropogenic phosphonates tested. The phosphonate utilizing pathway may offer N. spumigena a competitive advantage in the Pi-limited cyanobacterial blooms of the Baltic Sea.