Biodiversity and Phylogeny of Planktic Cyanobacteria in Temperate Freshwater Lakes

Currently, the classification used for cyanobacteria is based mainly on morphology. In many cases the classification is known to be incongruent with the phylogeny of cyanobacteria. The evaluation of this classification is complicated by the fact that numerous strains are only described morphologically and have not been isolated. Moreover, the phenotype of many cyanobacterial strains alters during prolonged laboratory cultivation. In this thesis, cyanobacterial strains were isolated from lakes (mainly Lake Tuusulanjärvi) and both morphology and phylogeny of the isolates were investigated. The cyanobacterial community composition in Lake Tuusulanjärvi was followed for two years in order to relate the success of cyanobacterial phenotypes and genotypes to environmental conditions. In addition, molecular biological methods were compared with traditional microscopic enumeration and their ability and usefulness in describing the cyanobacterial diversity was evaluated. The Anabaena, Aphanizomenon, and Trichormus strains were genetically heterogeneous and polyphyletic. The phylogenetic relationships of the heterocytous cyanobacteria were not congruent with their classification. In contrast to heterocytous cyanobacteria, the phylogenetic relationships of the Snowella and Woronichinia strains, which had not been studied before this thesis, reflected the morphology of strains and followed their current classification. The Snowella strains formed a monophyletic cluster, which was most closely related to the Woronichinia strain. In addition, a new cluster of thin, filamentous cyanobacterial strains identified as Limnothrix redekei was revealed. This cluster was not closely related to any other known cyanobacteria. The cyanobacterial community composition in Lake Tuusulanjärvi was studied with molecular methods [denaturant gradient gel electrophoresis (DGGE) and cloning of the 16S rRNA gene], through enumerations of cyanobacteria under microscope, and by strain isolations. Microcystis, Anabaena/Aphanizomenon, and Synechococcus were the major groups in the cyanobacterial community in Lake Tuusulanjärvi during the two-year monitoring period. These groups showed seasonal succession, and their success was related to different environmental conditions. The major groups of the cyanobacterial community were detected by all used methods. However, cloning gave higher estimates than microscopy for the proportions of heterocytous cyanobacteria and Synechococcus. The differences were probably caused by the high 16S rRNA gene copy numbers in heterotrophic cyanobacteria and by problems in the identification and detection of unicellular cyanobacteria.Syanobakteerit (sinilevät) ovat fotosyntesoivia bakteereita, joiden muodostamat kukinnat ovat yleisiä loppukesäisin. Useat kukintoja muodostavista syanobakteereista, kuten osa Anabaena-, Microcystis- ja Planktothrix-sukujen kannoista, tuottavat myrkkyjä. Nämä myrkylliset kukinnat haittaavat vesien virkistyskäyttöä sekä aiheuttavat terveysriskin ihmisille ja eläimille. Syanobakteerien tunnistus ja ekologinen tutkimus pohjautuvat niiden luokitteluun, joka nykyisellään perustuu lähinnä morfologiaan. Tämä luokittelu on monin osin ristiriidassa niiden geneettisen sukulaisuuden (fylogenian) kanssa. Luokittelun uudelleen arviointia on vaikeuttanut kantojen vähäisyys, tietämättömyys useiden lajien geneettisistä sukulaisuussuhteista ja kantojen morfologian muuttuminen laboratoriokasvatuksessa. Tutkimusta varten eristettiin lukuisia syanobakteerikantoja, joiden geneettiset sukulaisuussuhteet ja morfologia selvitettiin. Ympäristötekijöiden vaikutusta lajien ja genotyyppien runsastumiseen tutkittiin ja samalla vertailtiin eri menetelmien kykyä kuvata syanobakteeriyhteisön diversiteettiä. Tutkimuksessa havaittiin että Anabaena-, Aphanizomenon-, Trichormus- ja Limnothrix redekei-kantojen geneettiset sukulaisuussuhteet eivät vastaa niiden nykyistä luokittelua. Anabaena-, Aphanizomenon-, ja Trichormus- sukujen kannat, jotka muodostivat geneettisesti hajanaisia ryhmiä, voitaisiin luokitella jopa useampaankin sukuun. Sen sijaan Snowella- ja Woronichinia-kantojen sukulaisuussuhteet, joita ei ole aikaisemmin tutkittu, vastasivat niiden morfologiaa ja nykyistä luokittelua. Tuusulanjärven syanobakteeripopulaatio muodostui kolmesta pääryhmästä, Anabaena/Aphanizomenon-, Microcystis- ja Synechococcus-populaatiosta. Nämä ryhmät runsastuivat kesän eri aikoina ja erilaisissa ympäristöolosuhteissa. Elokuun loppupuolella lämpötilan, auringonsäteilyn ja ravinnepitoisuuksien laskiessa Anabaena/Aphanizomenon-populaatio syrjäytti Microcystis-populaation, joka oli runsaimmillaan heinä-elokuussa. Vertailemalla eri menetelmin saatuja tuloksia havaittiin, että vaikka kaikki kolme menetelmää (DGGE, 16S rRNA geenin kloonaus, mikroskopointi) havaitsivat pääryhmät, niin kloonaamalla Anabaena/Aphanizomenon ja Synechococcus-suvun osuudet arvioitiin suuremmiksi kuin mikroskopoimalla. Käyttämällä useita erilaisia menetelmiä saatiin monipuolisempi kuva syanobakteerien diversiteetistä. Uusia tutkimustuloksia syanobakteerien sukulaisuussuhteista voidaan käyttää niiden luokittelun uudistamisessa. Tietoa syanobakteerilajien runsastumiseen johtavista ympäristötekijöistä sekä syanobakteeripopulaatioiden tutkimusmenetelmistä voidaan hyödyntää muun muassa järvien kunnostuksessa ja monitorointimenetelmien kehittämisessä

Deep sequencing of blood and gut T-cell receptor beta-chains reveals gluten-induced immune signatures in celiac disease

Celiac disease (CD) patients mount an abnormal immune response to gluten. T-cell receptor (TCR) repertoires directed to some immunodominant gluten peptides have previously been described, but the global immune response to in vivo gluten exposure in CD has not been systematically investigated yet. Here, we characterized signatures associated with gluten directed immune activity and identified gluten-induced T-cell clonotypes from total blood and gut TCR repertoires in an unbiased manner using immunosequencing. CD patient total TCR repertoires showed increased overlap and substantially altered TRBV-gene usage in both blood and gut samples, and increased diversity in the gut during gluten exposure. Using differential abundance analysis, we identified gluten-induced clonotypes in each patient that were composed of a large private and an important public component. Hierarchical clustering of public clonotypes associated with dietary gluten exposure identified subsets of highly similar clonotypes, the most proliferative of which showing significant enrichment for the motif ASS[LF] R[SW][TD][DT][TE][QA][YF] in PBMC repertoires. These results show that CD-associated clonotypes can be identified and that common gluten associated immune response features can be characterized in vivo from total repertoires, with potential use in disease stratification and monitoring.Peer reviewe

Faecal Microbiota Composition in Adults Is Associated with the FUT2 Gene Determining the Secretor Status

Peer reviewe

Secretor Genotype (FUT2 gene) Is Strongly Associated with the Composition of Bifidobacteria in the Human Intestine

Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa

Quantitative Real-Time PCR Detection of Toxic Nodularia Cyanobacteria in the Baltic Sea

A specific quantitative real-time PCR (qPCR) method was developed for the quantification of hepatotoxin nodularin-producing Nodularia, one of the main bloom-forming cyanobacteria in the Baltic Sea. Specific PCR primers were designed for subunit F of the nodularin synthetase gene (ndaF), which encodes the NdaF subunit of the nodularin synthetase gene complex needed for nodularin production. The qPCR method was applied to water samples (a total of 120 samples) collected from the Baltic Sea in July 2004. As few as 30 ndaF gene copies ml(−1) of seawater could be detected, and thus, the method was very sensitive. The ndaF gene copy numbers and nodularin concentrations were shown to correlate in the Baltic seawater, indicating the constant production of nodularin by Nodularia. This qPCR method for the ndaF gene can be used for detailed studies of Nodularia blooms and their formation. ndaF gene copies and nodularin were detected mostly in the surface water but also in deeper water layers (down to 30 m). Toxic Nodularia blooms are not only horizontally but also vertically widely distributed, and thus, the Baltic fauna is extensively exposed to nodularin

Detection of Microcystin-Producing Cyanobacteria in Finnish Lakes with Genus-Specific Microcystin Synthetase Gene E (mcyE) PCR and Associations with Environmental Factors

We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation

Isolation of bifidobacteria for blood group secretor status targeted personalised nutrition

Background: Currently, there is a constant need to find microbial products for maintaining or even improving host microbiota balance that could be targeted to a selected consumer group. Blood group secretor status, determining the ABO status, could be used to stratify the consumer group. Objective: We have applied a validated upper intestinal tract model (TIM-1) and culturing methods to screen potential probiotic bacteria from faeces of blood secretor and non-secretor individuals. Design: Faecal samples from healthy volunteers were pooled to age- and sex-matched secretor and non-secretor pools. Faecal pools were run through separate TIM-1 simulations, and bacteria were cultivated from samples taken at different stages of simulations for characterisation. Results: Microbes in secretor pool survived the transit through TIM-1 system better than microbes of non-secretor pool, especially bifidobacteria and anaerobes were highly affected. The differences in numbers of bifidobacteria and lactobacilli isolates after plate cultivations and further the number of distinct RAPD-genotypes was clearly lower in non-secretor pool than in secretor pool. Conclusions: In the present study, we showed that microbiota of secretor and non-secretor individuals tolerate gastrointestinal conditions differently and that a combination of gastrointestinal simulations and cultivation methods proved to be a promising tool for isolating potentially probiotic bacteria

Tandem Mass Spectrometry in Resolving Complex Gut Microbiota Functions

This chapter reviews the technological development of high-resolution tandem mass spectrometry (MS) and its capability in analysis of complex microbial systems, particularly in the human gastrointestinal (GI) tract, and presents a few relevant examples of the metaproteomic approach. Protein MS became possible due to the rather simultaneous inventions of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) and is now adopted as a widely used tool in identification as well as characterization of microbiota. These innovations lead to the fast development of proteomics and raised tremendous expectations for the clinical application of proteomic research. The chapter also reviews on studies addressing all the proteins in fecal samples are provided. It describes a more targeted approach that focuses on surface proteins found in intestinal microbiomes. The chapter then focuses on peptide spectral matching (PSM) and de novo sequencing.</p

In Vitro Screen of Lactobacilli Strains for Gastrointestinal and Vaginal Benefits

Traditional probiotics comprise mainly lactic acid bacteria that are safe for human use, tolerate acid and bile, and adhere to the epithelial lining and mucosal surfaces. In this study, one hundred commercial and non-commercial strains that were isolated from human feces or vaginal samples were tested with regards to overall growth in culture media, tolerance to acid and bile, hydrogen peroxide (H2O2) production, and adhesion to vaginal epithelial cells (VECs) and to blood group antigens. As a result, various of the tested lactobacilli strains were determined to be suitable for gastrointestinal or vaginal applications. Commercial strains grew better than the newly isolated strains, but tolerance to acid was a common property among all tested strains. Tolerance to bile varied considerably between the strains. Resistance to bile and acid correlated well, as did VEC adhesion and H2O2 production, but H2O2 production was not associated with resistance to bile or acid. Except for L. iners strains, vaginal isolates had better overall VEC adhesion and higher H2O2 production. Species- and strain-specific differences were evident for all parameters. Rank-ordered clustering with nine clusters was used to identify strains that were suitable for gastrointestinal or vaginal health, demonstrating that the categorization of strains for targeted health indications is possible based on the parameters that were measured in this study