Phototrophic marine benthic microbiomes: the ecophysiology of these biological entities.

Phototrophic biofilms are multispecies, self-sustaining and largely closed microbial ecosystems. They form macroscopic structures such as microbial mats and stromatolites. These sunlight-driven consortia consist of a number of functional groups of microorganisms that recycle the elements internally. Particularly, the sulfur cycle is discussed in more detail as this is fundamental to marine benthic microbial communities and because recently exciting new insights have been obtained. The cycling of elements demands a tight tuning of the various metabolic processes and require cooperation between the different groups of microorganisms. This is likely achieved through cell-to-cell communication and a biological clock. Biofilms may be considered as a macroscopic biological entity with its own physiology. We review the various components of some marine phototrophic biofilms and discuss their roles in the system. The importance of extracellular polymeric substances (EPS) as the matrix for biofilm metabolism and as substrate for biofilm microorganisms is discussed. We particularly assess the importance of extracellular DNA, horizontal gene transfer and viruses for the generation of genetic diversity and innovation, and for rendering resilience to external forcing to these biological entities.

Bioremediation of chromium contaminated water by diatoms with concomitant lipid accumulation for biofuel production.

Hexavalent chromium compounds such as chromate and dichromate, commonly designated as Cr (VI) compounds, are widely used heavy metals in different industries and are considered highly toxic to most life forms. Unfortunately, they have become a major pollutant of groundwater and rivers around dichromate using industries. Bioremediation is widely used to decrease the amount of dichromate in wastewater but requires large amounts of precious fresh water. Here we tested two marine micro-algal species, Phaeodactylum tricornutum strain CCY0033 and Navicula pelliculosa strain CCMP543, for their ability of dichromate bioremediation and concomitantly producing lipids that can serve as biofuel. Dichromate tolerance of the strains was investigated under different growth conditions in order to obtain high biomass yields, high lipid accumulation and high dichromate removal from the medium. Both algal strains grew well and produced high biomass in media containing up to 1 mg of dichromate per liter. Variations in growth conditions revealed that dichromate removal from the medium correlated positively with biomass yield. Dichromate removal using living cells was in the same order of magnitude as with autoclaved dead cells or when using extracted extracellular polymeric substances (EPS). This suggests biosorption of dichromate to cell-associated polymeric substances as the major mechanism of the bioremediation process. For both strains, optimal dichromate removal and lipid production were achieved at a light intensity of 55 µmol m-2s-1 and at a sodium nitrate concentration of 3 mM. The optimal temperature for dichromate removal and lipid production was 23 °C for P. tricornutum and 27 °C for N. pelliculosa. Compared to P. tricornutum strain CCY0033, N. pelliculosa strain CCMP543 produced an overall higher lipid yield under these conditions.

Gregarious cyanobacteria.

Huber and collaborators reported in this issue of Environmental Microbiology about freshwater picocyanobacteria that showed phenotypic plasticity in the sense that they appeared as single cells as well as in aggregates. The authors suggested that aggregation might be an inducible defense as a response to the presence of grazers. This has been described for eukaryotic phytoplankton and for the cyanobacterium Microcystis but thus far not for picocyanobacteria. Although inducible defense as an explanation is an attractive possibility, it is also problematic. Aggregation is common among cyanobacteria and it offers many advantages as compared with a free-living lifestyle. Here these advantages are highlighted and the possibility of inducible defense is critically assessed.

Comparison of gas chromatography/isotope ratio mass spectrometry and liquid chromatography/isotope ratio mass spectrometry for carbon stable-isotope analysis of carbohydrates.

RATIONALE: We compared gas chromatography/isotope ratio mass spectrometry (GC/IRMS) and liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) for the measurement of δ(13)C values in carbohydrates. Contrary to GC/IRMS, no derivatisation is needed for LC/IRMS analysis of carbohydrates. Hence, although LC/IRMS is expected to be more accurate and precise, no direct comparison has been reported. METHODS: GC/IRMS with the aldonitrile penta-acetate (ANPA) derivatisation method was compared with LC/IRMS without derivatisation. A large number of glucose standards and a variety of natural samples were analysed for five neutral carbohydrates at natural abundance as well as at (13)C-enriched levels. Gas chromatography/chemical ionisation mass spectrometry (GC/CIMS) was applied to check for incomplete derivatisation of the carbohydrate, which would impair the accuracy of the GC/IRMS method. RESULTS: The LC/IRMS technique provided excellent precision (±0.08‰ and ±3.1‰ at natural abundance and enrichment levels, respectively) for the glucose standards and this technique proved to be superior to GC/IRMS (±0.62‰ and ±19.8‰ at natural abundance and enrichment levels, respectively). For GC/IRMS measurements the derivatisation correction and the conversion of carbohydrates into CO2 had a considerable effect on the measured δ(13)C values. However, we did not find any significant differences in the accuracy of the two techniques over the full range of natural δ(13)C abundances and (13)C-labelled glucose. The difference in the performance of GC/IRMS and LC/IRMS diminished when the δ(13)C values were measured in natural samples, because the chromatographic performance and background correction became critical factors, particularly for LC/IRMS. The derivatisation of carbohydrates for the GC/IRMS method was complete. CONCLUSIONS: Although both LC/IRMS and GC/IRMS are reliable techniques for compound-specific stable carbon isotope analysis of carbohydrates (provided that derivatisation is complete and the calibration requirements are met), LC/IRMS is the technique of choice. The reasons for this are the improved precision, simpler sample preparation, and straightforward isotopic calibration.

Cyanobacterial lactate oxidases serve as essential partners in N2 fixation and evolved into photorespiratory glycolate oxidases in plants.

Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N(2)-fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N(2)-fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N(2) fixation was more sensitive to O(2) in the Δlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O(2)-scavenging enzyme to protect nitrogenase in extant N(2)-fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.

A versatile method for simultaneous stable carbon isotope analysis of DNA and RNA nucleotides by liquid chromatography/isotope ratio mass spectrometry.

RATIONALE: Liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) is currently the most accurate and precise technique for the measurement of compound-specific stable carbon isotope ratios ((13)C/(12)C) in biological metabolites, at their natural abundance. However, until now this technique could not be applied for the analysis of nucleic acids, the building blocks of the carriers of genetic information in living cells and viruses, DNA and RNA. METHODS: Mixed-mode chromatography (MMC) was applied to obtain the complete separation of nine nucleotides (eight originating from DNA/RNA and one nucleotide (inosine monophosphate) that may serve as an internal standard) in a single run using LC/IRMS. We also developed and validated a method for DNA and RNA extraction and an enzymatic hydrolysis protocol for natural samples, which is compatible with LC/IRMS analysis as it minimizes the carbon blank. The method was used to measure the concentration and stable carbon isotope ratio of DNA and RNA nucleotides in marine sediment and in the common marine macro alga (Ulva sp.) at natural abundance levels as well as for (13)C-enriched samples. RESULTS: The detection limit of the LC/IRMS method varied between 1.0 nmol for most nucleotides and 2.0 nmol for late-eluting compounds. The intraday and interday reproducibility of nucleotide concentration measurements was better than, respectively, 4.1% and 8.9% and for δ(13)C measurements better than, respectively, 0.3‰ and 0.5‰. The obtained nucleic acid concentrations and nucleic acid synthesis rates were in good agreement with values reported in the literature. CONCLUSIONS: This new method gives reproducible results for the concentration and δ(13)C values of nine nucleotides. This solvent-free chromatographic method may also be used for other purposes, such as for instance to determine nucleotide concentrations using spectrophotometric detection. This sensitive method offers a new avenue for the study of DNA and RNA biosynthesis that can be applied in various fields of research.

The morphology and bioactivity of the rice field cyanobacterium Leptolyngbya.

The genus Leptolyngbya comprises filamentous cyanobacteria that are important in rice fields. In the rhizosphere, cyanobacteria produce a variety of secondary metabolites such as auxins that are important in agriculture soil performance. To assess this, Leptolyngbya strain MMG-1, was isolated from the rhizosphere of rice plants and described. For this, the morphology of this strain was studied by light microscopy as well as by confocal laser scanning microscopy. Besides, the ability of this strain to synthesize an auxin-like bioactive com- pound was demonstrated under various culture conditions (different amounts of tryptophan; pH; different alter- nating light:dark periods; duration of the incubation). The auxin-like compound was extracted from the culture of Leptolyngbya strain MMG-1 and identified as indole-3-acetic acid (IAA) by thin layer chromatography (TLC) as well as by high performance liquid chromatography (HPLC). Our results showed that the strain required the precursor L-tryptophan for the synthesis of IAA. Leptolyngbya strain MMG-1 accumulated IAA intracellularly. The IAA secreted by Leptolyngbya strain MMG-1 was significantly correlated with the initial concentration of L-tryptophan in the medium, as well as with the duration of the incubation. The bioactivity of the secreted IAA was determined by its effect on the rooting pattern of Pisum sativum seedlings. The culture supernatant of Leptolyngbya strain MMG-1 stimulated the seedling lateral rooting, while it decreased root length. Hence, rhizospheric Leptolyngbya produced auxin under different conditions and affected the plants rooting pattern.

The marine cyanobacterium Crocosphaera watsonii WH8501 synthesizes the compatible solute trehalose by a laterally acquired OtsAB fusion protein.

Compatible solutes are small organic molecules that are involved in the acclimation to various stresses such as temperature and salinity. Marine or moderate halotolerant cyanobacteria accumulate glucosylglycerol, while cyanobacteria with low salt tolerance (freshwater strains) usually accumulate sucrose or trehalose as the main compatible solutes. The screening of the genome of the marine, unicellular N(2) -fixing cyanobacterium Crocosphaera watsonii WH8501 revealed that instead of genes for glucosylglycerol biosynthesis, a fusion protein for the synthesis of trehalose was found that displayed similarities to trehalose-phosphate-synthase and -phosphatase (OtsAB pathway) from enterobacteria. Accordingly, cells of Crocosphaera showed salt-stimulated expression of the otsAB gene as well as a salt-dependent trehalose accumulation. The biochemical characterization of recombinant full-length OtsAB and truncated OtsB versions revealed that the otsAB gene in Crocosphaera encodes for an active trehalose-phosphate-synthase/phosphatase fusion protein. Genes coding for such proteins were not found in the genomes of other cyanobacteria but were present in many other, non-related marine bacteria, suggesting that otsAB might have been acquired by lateral gene transfer into the Crocosphaera genome.

Effect of salinity on nitrogenase activity and composition of the active diazotrophic community in intertidal microbial mats.

Microbial mats are often found in intertidal areas experiencing a large range of salinities. This study investigated the effect of changing salinities on nitrogenase activity and on the composition of the active diazotrophic community (nifH transcript libraries) of three types of microbial mats situated along a littoral gradient. All three mat types exhibited highest nitrogenase activity at salinities close to ambient seawater or lower. The response to lower or higher salinity was strongest in mats higher up in the littoral zone. Changes in nitrogenase activity as the result of exposure to different salinities were accompanied by changes in the active diazotrophic community. The two stations higher up in the littoral zone showed nifH expression by Cyanobacteria (Oscillatoriales and Chroococcales) and Proteobacteria (Gammaproteobacteria and Deltaproteobacteria). At these stations, a decrease in the relative contribution of Cyanobacteria to the nifH transcript libraries was observed at increasing salinity coinciding with a decrease in nitrogenase activity. The station at the low water mark showed low cyanobacterial contribution to nifH transcript libraries at all salinities but an increase in deltaproteobacterial nifH transcripts under hypersaline conditions. In conclusion, increased salinities caused decreased nitrogenase activity and were accompanied by a lower proportion of cyanobacterial nifH transcripts.

DTAF: an efficient probe to study cyanobacterial-plant interaction using confocal laser scanning microscopy (CLSM).

A variety of microscopic techniques have been utilized to study cyanobacterial associations with plant roots, but confocal laser scanning microscopy (CLSM) is the least used due to the unavailability of a suitable fluorescent dye. Commonly used lectins have problems with their binding ability with root cells and their visualization under CLSM. DTAF (5-(4,6-dichlorotriazinyl) aminofluorescein) is a fluorescent dye that has been widely used for staining various biological samples for fluorescent microscopy. It reacts with polysaccharides and peptides at ordinary conditions. The possible application and efficiency of DTAF for CLSM studies were examined in various aspects of cyanobacterial-plant interactions. Seedlings of Pisum sativum, Vigna rediata and Triticum aestivum were co-cultivated and stained with DTAF as a fluorochrome. Extracellular and intracellular interactions of cyanobacteria and the plant root surface were observed by CLSM. Results were compared with staining by other commonly used lectins. Advantages of the use of DTAF over other stains are its penetration into root tissues and binding with polysaccharides, mainly the cellulose. The staining was smooth, which clearly showed minute details on the cell of surface and root hairs with higher resolution. The emission wavelength for DTAF is 517 nm, which is highly advantageous as cyanobacteria have auto-fluorescence at 665 nm, and both can be simultaneously used in CLSM by visualizing in different channels. This worked efficiently with all three plants used and with filamentous and unicellular cyanobacterial strains. Cyanobacterial presence was not only clearly observed on the root surface, but also inside the root tissue and epidermal cells. The easy protocol and absence of tissue processing make DTAF a useful probe for studies of cyanobacterial associations with plant roots by CLSM.

The non-ribosomal assembly and frequent occurrence of the protease inhibitors spumigins in the bloom-forming cyanobacterium Nodularia spumigena.

Nodularia spumigena is a filamentous nitrogen-fixing cyanobacterium that forms toxic blooms in brackish water bodies worldwide. Spumigins are serine protease inhibitors reported from a single strain of N. spumigena isolated from the Baltic Sea. These linear tetrapeptides contain non-proteinogenic amino acids including a C-terminal alcohol derivative of arginine. However, very little is known about these compounds despite the ecological importance of N. spumigena. We show that spumigins are assembled by two non-ribosomal peptide synthetases encoded in a 21 kb biosynthetic gene cluster. The compact non-ribosomal peptide synthetase features a reductive loading and release mechanism. Our analyses demonstrate that the bulk of spumigins produced by N. spumigena are released as peptide aldehydes in contrast to earlier findings. The main spumigin E variant contains an argininal residue and is a potent trypsin inhibitor. Spumigins were present in all of the N. spumigena strains isolated from the Baltic Sea and comprised up to 1% of the dry weight of the cyanobacterium. Our results demonstrate that bloom-forming N. spumigena strains produce a cocktail of enzyme inhibitors, which may explain in part the ecological success of this cyanobacterium in brackish water bodies worldwide.

Oxygen and the light-dark cycle of nitrogenase activity in two unicellular cyanobacteria.

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well-defined levels of oxygen to which the organisms are exposed, using an online and near real-time acetylene reduction assay combined with sensitive laser-based photoacoustic ethylene detection. The cultures were grown under an alternating 12-12 h light-dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 mumol m(-2) s(-1) provided at the same light-dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5-7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29 degrees C. At 31 degrees C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41 degrees C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.

Temperature excludes N2-fixing heterocystous cyanobacteria in the tropical oceans.

Whereas the non-heterocystous cyanobacteria Trichodesmium spp. are the dominant N2-fixing organisms in the tropical oceans, heterocystous species dominate N2 fixation in freshwater lakes and brackish environments such as the Baltic Sea. So far no satisfactory explanation for the absence of heterocystous cyanobacteria in the pelagic of the tropical oceans has been given, even though heterocysts would seem to represent an ideal strategy for protecting nitrogenase from being inactivated by O2, thereby enabling cyanobacteria to fix N2 and to perform photosynthesis simultaneously. Trichodesmium is capable of N2 fixation, apparently without needing to differentiate heterocysts. Here we show that differences in the temperature dependence of O2 flux, respiration and N2 fixation activity explain how Trichodesmium performs better than heterocystous species at higher temperatures. Our results also explain why Trichodesmium is not successful in temperate or cold seas. The absence of heterocystous cyanobacteria in the pelagic zone of temperate and cold seas, however, requires another explanation.

Adaptive divergence in pigment composition promotes phytoplankton biodiversity.

The dazzling diversity of the phytoplankton has puzzled biologists for decades. The puzzle has been enlarged rather than solved by the progressive discovery of new phototrophic microorganisms in the oceans, including picocyanobacteria, pico-eukaryotes, and bacteriochlorophyll-based and rhodopsin-based phototrophic bacteria. Physiological and genomic studies suggest that natural selection promotes niche differentiation among these phototrophic microorganisms, particularly with respect to their photosynthetic characteristics. We have analysed competition for light between two closely related picocyanobacteria of the Synechococcus group that we isolated from the Baltic Sea. One of these two has a red colour because it contains the pigment phycoerythrin, whereas the other is blue-green because it contains high contents of the pigment phycocyanin. Here we report theory and competition experiments that reveal stable coexistence of the two picocyanobacteria, owing to partitioning of the light spectrum. Further competition experiments with a third marine cyanobacterium, capable of adapting its pigment composition, show that this species persists by investing in the pigment that absorbs the colour not used by its competitors. These results demonstrate the adaptive significance of divergence in pigment composition of phototrophic microorganisms, which allows an efficient utilization of light energy and favours species coexistence.

The ecology of nitrogen fixation in cyanobacterial mats.

All cyanobacterial mats that have been investigated have been proven to be diazotrophic, i.e., use atmospheric dinitrogen (N(2)) as the source of nitrogen. Many cyanobacteria possess the capacity to fix N(2) and different species have evolved various ways to cope with the sensitivity of nitrogenase toward oxygen which is produced by these oxygenic phototrophs. These different strategies give rise to complex patterns of nitrogenase activity in microbial mats. Nitrogenase activity may exhibit complex variations over a day-night cycle but different types of microbial mats may also have their own characteristic patterns. Besides the cyanobacteria, numerous other members of the Bacteria as well as some Archaea are known to be diazotrophic. The complexity of the microbial community and of the observed patterns of nitrogenase activity makes it difficult to understand how the different groups of organisms contribute to N(2) fixation in microbial mats. Cyanobacteria have ample access to energy (sunlight) and reducing equivalents (water) and therefore easily satisfy the demands of nitrogenase. As well, since they also fix CO(2), they are able to synthesize the acceptor molecules for the fixed nitrogen. However, it is also feasible that other diazotrophs in a joint venture with cyanobacteria are responsible for the bulk of the fixed nitrogen. In this review we discuss the importance of cyanobacteria as diazotrophs in microbial mats, their interactions with other potential N(2)-fixing microorganisms, and the factors that control their activities.

Circadian clock-controlled gene expression in co-cultured, mat-forming cyanobacteria.

Natural coastal microbial mat communities are multi-species assemblages that experience fluctuating environmental conditions and are shaped by resource competition as well as by cooperation. Laboratory studies rarely address the natural complexity of microbial communities but are usually limited to homogeneous mono-cultures of key species grown in liquid media. The mat-forming filamentous cyanobacteria Lyngbya aestuarii and Coleofasciculus chthonoplastes were cultured under different conditions to investigate the expression of circadian clock genes and genes that are under their control. The cyanobacteria were grown in liquid medium or on a solid substrate (glass beads) as mono- or as co-cultures under a light-dark regime and subsequently transferred to continuous light. TaqMan-probe based qPCR assays were used to quantify the expression of the circadian clock genes kaiA, kaiB, and kaiC, and of four genes that are under control of the circadian clock: psbA, nifH, ftsZ, and prx. Expression of kaiABC was influenced by co-culturing the cyanobacteria and whether grown in liquid media or on a solid substrate. Free-running (i.e. under continuous light) expression cycle of the circadian clock genes was observed in L. aestuarii but not in C. chthonoplastes. In the former organism, maximum expression of psbA and nifH occurred temporally separated and independent of the light regime, although the peak shifted in time when the culture was transferred to continuous illumination. Although functionally similar, both species of cyanobacteria displayed different 24-h transcriptional patterns in response to the experimental treatments, suggesting that their circadian clocks have adapted to different life strategies adopted by these mat-forming cyanobacteria.

Is the distribution of nitrogen-fixing cyanobacteria in the oceans related to temperature?

Approximately 50% of the global natural fixation of nitrogen occurs in the oceans supporting a considerable part of the new primary production. Virtually all nitrogen fixation in the ocean occurs in the tropics and subtropics where the surface water temperature is 25°C or higher. It is attributed almost exclusively to cyanobacteria. This is remarkable firstly because diazotrophic cyanobacteria are found in other environments irrespective of temperature and secondly because primary production in temperate and cold oceans is generally limited by nitrogen. Cyanobacteria are oxygenic phototrophic organisms that evolved a variety of strategies protecting nitrogenase from oxygen inactivation. Free-living diazotrophic cyanobacteria in the ocean are of the non-heterocystous type, namely the filamentous Trichodesmium and the unicellular groups A-C. I will argue that warm water is a prerequisite for these diazotrophic organisms because of the low-oxygen solubility and high rates of respiration allowing the organism to maintain anoxic conditions in the nitrogen-fixing cell. Heterocystous cyanobacteria are abundant in freshwater and brackish environments in all climatic zones. The heterocyst cell envelope is a tuneable gas diffusion barrier that optimizes the influx of both oxygen and nitrogen, while maintaining anoxic conditions inside the cell. It is not known why heterocystous cyanobacteria are absent from the temperate and cold oceans and seas.

Seasonal development of a coastal microbial mat.

Growth and activity of coastal microbial mats is strongly seasonal. The development of these mats starts in early spring and fully maturate during late summer, where after growth ceases and subsequently the mat deteriorates by erosion and decomposition in winter. Here, the composition of the microbial community of three different mats developing along the tidal gradient of the North Sea beach of the Dutch barrier island Schiermonnikoog was analysed. The 16S ribosomal RNA molecules and the associated gene were sequenced in order to obtain the active (RNA) and resident (DNA) community members, respectively. Proteobacteria, Cyanobacteria, and Bacteroidetes dominated the mats during the whole year but considerable differences among these groups were found along the tidal gradient and seasonally when observed at a finer taxonomic resolution. Richness and diversity increased during the year starting from a pioneering community that is gradually succeeded by a more diverse climax community. The initial pioneers consisted of the cold-adapted photoautotrophic cyanobacterium Nodularia sp. and potential cold adapted members of the alphaproteobacterial Loktanella genus. These pioneers were succeeded by, amongst others, cyanobacteria belonging to the genera Leptolyngbya, Lyngbya, and Phormidium. At the upper littoral (Dune site), which was characterized by an extensive salt marsh vegetation, the mats contained a distinct bacterial community that potentially contribute to or benefit from plant decay. This study reports in detail on the seasonal changes and succession of these coastal microbial mat communities and discusses the potential forces that drive these changes.

Diversity and phylogeny of Baltic Sea picocyanobacteria inferred from their ITS and phycobiliprotein operons.

Picocyanobacteria of the genus Synechococcus span a range of different colours, from red strains rich in phycoerythrin (PE) to green strains rich in phycocyanin (PC). Here, we show that coexistence of red and green picocyanobacteria in the Baltic Sea is widespread. The diversity and phylogeny of red and green picocyanobacteria was analysed using three different genes: 16S rRNA-ITS, the cpeBA operon of the red PE pigment and the cpcBA operon of the green PC pigment. Sequencing of 209 clones showed that Baltic Sea picocyanobacteria exhibit high levels of microdiversity. The partial nucleotide sequences of the cpcBA and cpeBA operons from the clone libraries of the Baltic Sea revealed two distinct phylogenetic clades: one clade containing mainly sequences from cultured PC-rich picocyanobacteria, while the other contains only sequences from cultivated PE-rich strains. A third clade of phycourobilin (PUB) containing strains of PE-rich Synechococcus spp. did not contain sequences from the Baltic Sea clone libraries. These findings differ from previously published phylogenies based on 16S rRNA gene analysis. Our data suggest that, in terms of their pigmentation, Synechococcus spp. represent three different lineages occupying different ecological niches in the underwater light spectrum. Strains from different lineages can coexist in light environments that overlap with their light absorption spectra.