Molecular (PCR-DGGE) versus morphological approach: analysis of taxonomic composition of potentially toxic cyanobacteria in freshwater lakes.

BACKGROUND: The microscopic Utermöhl method is commonly used for the recognition of the presence and taxonomic composition of potentially toxic cyanobacteria and is especially useful for monitoring reservoirs used as drinking water, recreation and fishery resources. However, this method is time-consuming and does not allow potentially toxic and nontoxic cyanobacterial strains to be distinguished. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) of the marker gene ITS and the mcy-gene cluster, and DNA sequencing. We have attempted to calibrate the DGGE-method with a microscopic procedure, using water samples taken in 2011 from four lakes of the Great Mazurian Lakes system. RESULTS: Results showed that the classic microscopic method was much more precise and allowed the classification of the majority of cyanobacterial taxa to the species or genus. Using the molecular approach, most of the sequences could only be assigned to a genus or family. The results of DGGE and microscopic analyses overlapped in the detection of the filamentous cyanobacteria. For coccoid cyanobacteria, we only found two taxa using the molecular method, which represented 17% of the total taxa identified using microscopic observations. The DGGE method allowed the identification of two genera of cyanobacteria (Planktothrix and Microcystis) in the studied samples, which have the potential ability to produce toxins from the microcystins group. CONCLUSIONS: The results confirmed that the molecular approach is useful for the rapid detection and taxonomic distinction of potentially toxic cyanobacteria in lake-water samples, also in very diverse cyanobacterial communities. Such rapid detection is unattainable by other methods. However, with still limited nucleotide sequences deposited in the public databases, this method is currently not sufficient to evaluate the entire taxonomic composition of cyanobacteria in lakes.

Seasonal and spatial diversity of picocyanobacteria community in the Great Mazurian Lakes derived from DGGE analyses of ITS region of rDNA and cpcBAIGS [corrected] markers.

The seasonal and spatial diversity of picocyanobacteria (Pcy) in lakes of the Great Mazurian Lakes (GLM) system was examined by DGGE analysis of molecular markers derived from the 16S-23S internal transcribed spacer (ITS) of the ribosomal operon and the phycocyanin operon (cpcBA-IGS). The study of nine lakes, ranging from mesotrophy to hypereutrophy, demonstrated seasonal variance of Pcy. The richness and Shannon diversity index calculated on the basis of both markers were higher in spring and lower in early and late summer. No statistically significant relationships were found between the markers and trophic status of the studied lakes or Pcy abundance. There were, however, statistically significant relationships between the diversity indices and sampling time. The analysis pointed to a different distribution of the two markers. The ITS marker exhibited more unique sequences in time and space, whereas a greater role for common and ubiquitous sequences was indicated by the cpcBA-IGS data. Examination of the Pcy community structure demonstrated that communities were grouped in highly similar clusters according to sampling season/time rather than to the trophic status of the lake. Our results suggest that time is more important than trophic status in shaping the diversity and structure of Pcy communities. The seasonal changes in picocyanobacteria and differences in diversity and community structures are discussed in the context of well-established ecological hypotheses: the PEG model, intermediate disturbance hypothesis (IDH), and horizontal gene transfer (HGT).

Factors controlling bacteria and protists in selected Mazurian eutrophic lakes (North-Eastern Poland) during spring.

BACKGROUND: The bottom-up (food resources) and top-down (grazing pressure) controls, with other environmental parameters (water temperature, pH) are the main factors regulating the abundance and structure of microbial communities in aquatic ecosystems. It is still not definitively decided which of the two control mechanisms is more important. The significance of bottom-up versus top-down controls may alter with lake productivity and season. In oligo- and/or mesotrophic environments, the bottom-up control is mostly important in regulating bacterial abundances, while in eutrophic systems, the top-down control may be more significant. RESULTS: The abundance of bacteria, heterotrophic (HNF) and autotrophic (ANF) nanoflagellates and ciliates, as well as bacterial production (BP) and metabolically active cells of bacteria (CTC, NuCC, EST) were studied in eutrophic lakes (Mazurian Lake District, Poland) during spring. The studied lakes were characterized by high nanoflagellate (mean 17.36 ± 8.57 × 103 cells ml-1) and ciliate abundances (mean 59.9 ± 22.4 ind. ml-1) that were higher in the euphotic zone than in the bottom waters, with relatively low bacterial densities (4.76 ± 2.08 × 106 cells ml-1) that were lower in the euphotic zone compared to the profundal zone. Oligotrichida (Rimostrombidium spp.), Prostomatida (Urotricha spp.) and Scuticociliatida (Histiobalantium bodamicum) dominated in the euphotic zone, whereas oligotrichs Tintinnidium sp. and prostomatids Urotricha spp. were most numerous in the bottom waters. Among the staining methods used to examine bacterial cellular metabolic activity, the lowest percentage of active cells was recorded with the CTC (1.5-15.4%) and EST (2.7-14.2%) assay in contrast to the NuCC (28.8-97.3%) method. CONCLUSIONS: In the euphotic zone, the bottom-up factors (TP and DOC concentrations) played a more important role than top-down control (grazing by protists) in regulating bacterial numbers and activity. None of the single analyzed factors controlled bacterial abundance in the bottom waters. The results of this study suggest that both control mechanisms, bottom-up and top-down, simultaneously regulated bacterial community and their activity in the profundal zone of the studied lakes during spring. In both lake water layers, food availability (algae, nanoflagellates) was probably the major factor determining ciliate abundance and their composition. In the bottom waters, both groups of protists appeared to be also influenced by oxygen, temperature, and total phosphorus.

Persistence of bacterial proteolytic enzymes in lake ecosystems.

This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2)  = 0.99, n = 17, P ≤ 0.0001, V(max)  = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems.

Effect of organic phosphorus and nitrogen enrichment of mesotrophic lake water on dynamics and diversity of planktonic microbial communities--DNA and protein case studies (mesocosm experiments).

Effects of mesotrophic lake water enrichment with organic phosphorus and nitrogen substrates (DNA and model protein, bovine serum albumin--BSA) on dynamics and diversity of natural microbial communities (bacteria, heterotrophic nanoflagellates, ciliates) were studied in mesocosm experiments. Simultaneous enrichment with DNA and BSA strongly increased the abundance and biomass of all studied groups of microorganisms and induced changes in their morphological and taxonomic structure. The increased participation of large heterotrophic nanoflagellates cells (larger than 10 microm) in their total numbers and shifts in taxonomic and trophic structure of the ciliates, from algivorous to small bacterivorous, species were observed. Grazing caused changes in bacterial size distribution in all enriched mesocosms. Large (10-50 microm) filamentous bacteria significantly contributed to the total bacterial numbers and biomass. Pronounced increase in populations of beta- and gamma-Proteobacteria was found in lake water enriched with organic P and N sources, whereas alpha-Proteobacteria did not change markedly in the studied mesocosms. DNA additions stimulated the rates of bacterial secondary production. BSA shortened the rates of bacterial biomass turnover in lake water. Relatively high and constant (approximately 30%) percentage contribution of active bacteria (MEM+) in two mesocosms enriched with DNA and DNA+BSA suggested the important role of nucleic acids as a source of phosphorus for bacterial growth, activity and production. Numerous and statistically significant correlations between bacteria and protists indicated the direct and selective predator-prey relationship.

Inorganic phosphorus and nitrogen modify composition and diversity of microbial communities in water of mesotrophic lake.

The effects of inorganic nutrients (N, P) enrichment of mesotrophic lake water on changes in bacterial and protistan (heterotrophic nanoflagellates and ciliates) communities compositions were studied in the mesocosm experiment. Phosphorus (PO4(3-)) and nitrogen (NH4+) alone and in combination were added to three types of experimental mesocosms. Mesocosms results suggested that simultaneous addition of P and N stimulated phytoplankton growth and production rates of bacterial biomass its turnover rate. Strong positive correlations between chlorophyll a and bacterial secondary production rates suggested that bacteria were mainly controlled by organic substrates released in course ofphytoplankton photosynthesis. Both nutrients increased distinctly protistan biomass and resulted in the shift in ciliate community composition from algivorous to large omnivorous species. The response of bacterial numbers and biomass to nutrients addition was less evident. However, intensive grazing caused their dynamic changes. Fluorescence in situ hybridization (FISH) revealed only small changes in bacterial taxonomic composition. There was an apparent shift in dominance from Cytophaga-Flavobacterium to the Alphaproteobacteria group in the mesocosm with simultaneous addition of P and N, which positively related to increased abundance of bacterivorous protists. Experiment demonstrated that inorganic N and P nutrients directly influenced the bottom-down control of microbial communities, which had a crucial effect on morphological diversity of bacteria.

The dynamics of protein decomposition in lakes of different trophic status--reflections on the assessment of the real proteolytic activity in situ.

Abstract The aim of this paper is to discuss the methodology of our investigation of the dynamics of protein degradation and the total in situ proteolytic activity in meso/eutrophic, eutrophic, and hypereutrophic freshwater environments. Analysis of the kinetics and rates of enzymatic release of amino acids in water samples preserved with sodium azide allows determination of the concentrations of labile proteins (C(LAB)), and their half-life time (T(1/2)). Moreover, it gives more realistic information on resultant activity in situ (V(T(1/2))) of ecto- and extracellular proteases that are responsible for the biological degradation of these compounds. Although the results provided by the proposed method are generally well correlated with those obtained by classical procedures, they better characterize the dynamics of protein degradation processes, especially in eutrophic or hypereutrophic lakes. In these environments, processes of protein decomposition occur mainly on the particles and depend primarily on a metabolic activity of seston-attached bacteria. The method was tested in three lakes. The different degree of eutrophication of these lakes was clearly demonstrated by the measured real proteolytic pattern and confirmed by conventional trophic state determinants.

Urea and ureolytic activity in lakes of different trophic status.

Urea and uraease (U-ase) activity were determined in water samples taken from the surface layers of 17 lakes of different trophic status. Urea concentrations were inversely correlated with the trophic status of the studied lakes and varied from below the detection limit to 25 micromol l(-1). Maximal potential ureolytic activity (V(max)) ranged from 0.2 to 7.0 micromol l(-1) h(-1). The highest urea concentrations and the lowest U-ase activities were recorded in the spring, whereas the lowest urea concentrations and the highest rates of urea hydrolysis were observed late in summer, during heavy phytoplankton blooms. Since in the majority of the Great Mazurian Lakes microplankton growth was limited by nitrogen supply, urea was an important N source for both auto- and heterotrophic planktonic microorganisms throughout the growth period. U-ase activity was mainly related to the seston. Only up to 25% of total activity could be attributed to free enzymes dissolved in lake water. In epilimnetic water samples the bulk of the ureolytic activity originated from seston-attached bacteria. However, a positive, statistically significant correlation between ureolytic activity and chlorophyll a (Chl(a)) concentrations suggests that phytoplankton may also be responsible for at least a some of the observed ureolytic activity in the highly eutrophic Great Mazurian Lakes.