Katrin Zwirglmaier

Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization-RING-FISH

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 104−105 copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (101−103 copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method – a halo‐like, ring‐shaped concentration of fluorescence in the cell periphery – we coined the term RING‐FISH (recognition of individual genes) to differentiate it from conventional FISH.

Seasonal and spatial patterns of microbial diversity along a trophic gradient in the interconnected lakes of the Osterseen Lake District, Bavaria

The Osterseen Lake District in Bavaria consists of 19 small interconnected lakes that exhibit a pronounced trophic gradient from eutrophic to oligotrophic. It therefore presents a unique model system to address ecological questions regarding niche adaptation and Baas Beckings long standing hypothesis of “everything is everywhere, but the environment selects.” Here, we present the first assessment of the microbial diversity in these lakes. We sampled the lakes in August and December and used 454 pyrosequencing of 16S rRNA amplicons to analyze the microbial diversity. The diversity patterns between lakes and seasons were compared and the bacterial community composition was correlated with key chemical and physical parameters. Distinct patterns of bacterial diversity only emerged at the level of individual OTUs (operational taxonomic units), but not at the level of the major bacterial phyla. This emphasizes the high functional and physiological diversity among bacterial species within a phylum and calls for analysis of biodiversity at the level of OTUs in order to understand fine-scale biogeography. We were able to identify a number of cosmopolitan OTUs as well as specialist OTUs that were restricted to certain lakes or seasons, suggesting adaptation to specific ecological niches.

Subtraction hybridization in microplates: an improved method to generate strain-specific PCR primers.

An improved subtraction hybridization technique was developed and evaluated. The hybridization is performed in a microplate with the subtractor-DNA immobilized in the plate while the probe-DNA is in solution. After hybridization the probe-specific DNA can easily be removed from the microwell and submitted to further analysis. This new technique has been successfully applied to generate several strain-specific PCR-primers for Lactococcus lactis subsp. lactis, Pediococcus spec., Saccharomyces spec. and Listeria monocytogenes.

In Situ Functional Gene Analysis: Recognition of Individual Genes by Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) using specific probes certainly is one of the most commonly applied molecular techniques in microbial ecology. Monitoring of community composition and dynamics can be combined with localization and identification of individual cells in situ. However, the resolution power of the method is limited by the need for high target numbers per cell. Apart from standard targets (ribosomal RNAs), mRNAs could be used successfully for in situ visualization in some cases. A new promising variant of in situ hybridization could be established that should provide access to any low copy number nucleic acid targets, such as chromosomal genes. The recognition of individual genes by FISH (RING-FISH) technology is based on target visualization mediated by polynucleotide probe network formation. The specificity of the approach is provided by intracellular probe-target hybridization. This initial hybridization apparently acts as a focal point for inter-probe hybridization within and mainly in the periphery of the cells. Probe-conferred fluorescence typically appears halo-like around the cells. RING-FISH can be used in combination with conventional oligonucleotide FISH. Thus, genetic potential can be assigned to in situ identified cells.

Influence of temperature, mixing, and addition of microcystin‐LR on microcystin gene expression in Microcystis aeruginosa

Cyanobacteria, such as the toxin producer Microcystis aeruginosa, are predicted to be favored by global warming both directly, through elevated water temperatures, and indirectly, through factors such as prolonged stratification of waterbodies. M. aeruginosa is able to produce the hepatotoxin microcystin, which causes great concern in freshwater management worldwide. However, little is known about the expression of microcystin synthesis genes in response to climate change‐related factors. In this study, a new RT‐qPCR assay employing four reference genes (GAPDH, gltA, rpoC1, and rpoD) was developed to assess the expression of two target genes (the microcystin synthesis genes mcyB and mcyD). This assay was used to investigate changes in mcyB and mcyD expression in response to selected environmental factors associated with global warming. A 10°C rise in temperature significantly increased mcyB expression, but not mcyD expression. Neither mixing nor the addition of microcystin‐LR (10 μg L−1 or 60 μg L−1) significantly altered mcyB and mcyD expression. The expression levels of mcyB and mcyD were correlated but not identical.

Synechococcus diversity along a trophic gradient in the Osterseen Lake District, Bavaria

Picocyanobacteria are important primary producers in freshwater; however, there is still a knowledge gap regarding their diversity at the strain level. For this reason, the microbial diversity of four lakes with different trophic states was investigated by sequencing of the 16S rRNA gene using universal primers. The study was performed in selected lakes of the Osterseen Lake District, Germany, from 2012 to 2014 (Lake Schiffhuettensee: eutrophic; Lake Ostersee: meso-oligotrophic; Lake Groebensee: oligotrophic; Lake Lustsee: oligotrophic). It was determined that the bacterial community of each of these lakes was characterized by one or more specific phyla. Within the autotrophic plankton, the picocyanobacterium Synechococcus sp. dominated oligotrophic habitats, whereas eukaryotic algae prevailed in eutrophic lakes. The study focused on the occurrence of cyanobacteria, specifically the genus Synechococcus. Genetic analysis of the 16S rRNA gene revealed an extendend diversity of freshwater Synechococcus. The occurrence of the identified operational taxonomic units of Synechococcus did not correlate with the trophic state of their habitat, suggesting that the current, underestimated diversity of picocyanobacteria deserves increased consideration in assessments of microbial and freshwater biodiversity.

Temporal Dynamics of the Microbial Community Composition with a Focus on Toxic Cyanobacteria and Toxin Presence during Harmful Algal Blooms in Two South German Lakes

Bacterioplankton plays an essential role in aquatic ecosystems, and cyanobacteria are an influential part of the microbiome in many water bodies. In freshwaters used for recreational activities or drinking water, toxic cyanobacteria cause concerns due to the risk of intoxication with cyanotoxins, such as microcystins. In this study, we aimed to unmask relationships between toxicity, cyanobacterial community composition, and environmental factors. At the same time, we assessed the correlation of a genetic marker with microcystin concentration and aimed to identify the main microcystin producer. We used Illumina MiSeq sequencing to study the bacterioplankton in two recreational lakes in South Germany. We quantified a microcystin biosynthesis gene (mcyB) using qPCR and linked this information with microcystin concentration to assess toxicity. Microcystin biosynthesis gene (mcyE)-clone libraries were used to determine the origin of microcystin biosynthesis genes. Bloom toxicity did not alter the bacterial community composition, which was highly dynamic at the lowest taxonomic level for some phyla such as Cyanobacteria. At the OTU level, we found distinctly different degrees of temporal variation between major bacteria phyla. Cyanobacteria and Bacteroidetes showed drastic temporal changes in their community compositions, while the composition of Actinobacteria remained rather stable in both lakes. The bacterial community composition of Alpha- and Beta-proteobacteria remained stable over time in Lake Klostersee, but it showed temporal variations in Lake Bergknappweiher. The presence of potential microcystin degraders and potential algicidal bacteria amongst prevalent Bacteroidetes and Alphaproteobacteria implied a role of those co-occurring heterotrophic bacteria in cyanobacterial bloom dynamics. Comparison of both lakes studied revealed a large shared microbiome, which was shaped toward the lake specific community composition by environmental factors. Microcystin variants detected were microcystin-LR, -RR, and -YR. The maximum microcystin concentrations measured was 6.7 μg/L, a value still acceptable for recreational waters but not drinking water. Microcystin concentration correlated positively with total phosphorus and mcyB copy number. We identified low abundant Microcystis sp. as the only microcystin producer in both lakes. Therefore, risk assessment efforts need to take into account the fact that non-dominant species may cause toxicity of the blooms observed.

Spatio-temporal distribution pattern of the picocyanobacterium Synechococcus in lakes of different trophic states: a comparison of flow cytometry and sequencing approaches

In this study, we aimed to investigate the spatio-temporal distribution patterns of the picocyanobacterium Synechococcus, an important contributor to primary production in many freshwater lakes. Our study sites were four lakes with different trophic states within the Osterseen Lake District in Southern Germany. Flow cytometry counts and next-generation sequencing were used from April to October 2015 to analyse the occurrence of Synechococcus and heterotrophic prokaryotes in relation to physical and chemical habitat properties. Synechococcus was identified as the main representative of the autotrophic picoplankton, but cell counts varied widely. The Synechococcus taxa identified by flow cytometry were confirmed by sequencing data, but the comparison of the flow cytometry counts and sequence data revealed discrepancies for cells in the exponential phase. The eutrophic Lake Schiffhuettensee was dominated by algae and had the highest abundance of heterotrophic prokaryotes. The presence of distinct operational taxonomic units of Synechococcus varied seasonally and was lake-specific, indicating local niche adaptation. Our study sheds light on the ecology of these important primary producers in freshwater systems. Furthermore, the discrepancy observed with the direct comparison of the widely used methods of next-generation sequencing and flow cytometry should serve as a caveat for future data analysis.

Influence of cyanobacteria, mixotrophic flagellates, and virioplankton size fraction on transcription of microcystin synthesis genes in the toxic cyanobacterium Microcystis aeruginosa

Toxic cyanobacteria such as Microcystis aeruginosa are a worldwide concern in freshwater reservoirs. Problems associated with their mass occurrence are predicted to increase in the future due to global warming. The hepatotoxic secondary metabolite microcystin is of particular concern in this context. This study aimed to determine whether co‐occurring microorganisms influence the expression of microcystin biosynthesis genes. To this end, we performed cocultivation experiments and measured mcyB and mcyD transcripts in M. aeruginosa using RT‐qPCR. We utilized representatives from three different plankton groups: the picocyanobacterium Synechococcus elongatus, the unicellular flagellate grazer Ochromonas danica, and virioplankton from two different lakes. The presence of S. elongatus significantly increased mcyB and mcyD transcription in M. aeruginosa. Cocultivation with the mixotrophic chrysophyte O. danica did not increase the transcription of mcyB and mcyD; in fact, mcyD transcripts decreased significantly. The virioplankton size fraction of environmental water samples induced a significant increase in mcyB and mcyD transcription when obtained from lakes with cyanobacterial blooms. Our results show that co‐occurring microorganisms influence the expression of microcystin biosynthesis genes in M. aeruginosa.