Karel Horňák

Alga-Derived Substrates Select for Distinct Betaproteobacterial Lineages and Contribute to Niche Separation in Limnohabitans Strains

ABSTRACT We examined the proportions of major Betaproteobacteria subgroups within bacterial communities in diverse nonaxenic, monospecific cultures of algae or cyanobacteria: four species of cryptophyta (genera Cryptomonas and Rhodomonas), four species of chlorophyta (genera Pediastrum, Staurastrum, and Chlamydomonas), and two species of cyanobacteria (genera Dolichospermum and Aphanizomenon). In the cryptophyta cultures, Betaproteobacteria represented 48 to 71% of total bacteria, the genus Limnohabitans represented 18 to 26%, and the Polynucleobacter B subcluster represented 5 to 16%. In the taxonomically diverse chlorophyta group, the genus Limnohabitans accounted for 7 to 45% of total bacteria. In contrast, cyanobacterial cultures contained significantly lower proportions of the Limnohabitans bacteria (1 to 3% of the total) than the cryptophyta and chlorophyta cultures. Notably, largely absent in all of the cultures was Polynucleobacter necessarius (Polynucleobacter C subcluster). Subsequently, we examined the growth of Limnohabitans strains in the presence of different algae or their extracellular products (EPP). Two strains, affiliated with Limnohabitans planktonicus and Limnohabitans parvus, were separately inoculated into axenic cultures of three algal species growing in an inorganic medium: Cryptomonas sp., Chlamydomonas noctigama, and Pediastrum boryanum. The Limnohabitans strains cocultured with these algae or inoculated into their EPP consistently showed (i) pronounced population growth compared to the control without the algae or EPP and (ii) stronger growth stimulation of L. planktonicus than of L. parvus. Overall, growth responses of the Limnohabitans strains cultured with algae were highly species specific, which suggests a pronounced niche separation between two closely related Limnohabitans species likely mediated by different abilities to utilize the substrates produced by different algal species.

Top-down and bottom-up induced shifts in bacterial abundance, production and community composition in an experimentally divided humic lake

We examined in situ abundance and activities of the major bacterial groups in the two most distinct compartments of experimentally divided Lake Grosse Fuchskuhle (Germany). The selected south-west (SW) and north-east basin (NE) differ substantially in their major chemical and biological parameters that potentially influence the dynamics and composition of microbial communities. Water from the basins were incubated in dialysis bags, which allowed for a relatively free exchange of nutrients, limiting solutes and low molecular organic matter but fully prevented exchange of organisms. To investigate the effect of top-down and bottom-up manipulations three size fractions of water samples were produced: (i) unfiltered, (ii) pre-filtered through 5.0 microm pore size membranes to remove large particles, as well as grazers and (iii) pre-filtered through 0.8 microm filters to remove all potential bacterivores. One set of dialysis bags was either incubated in acidic SW (rich in humic matter) or in almost neutral NE basin whereas a second set was transferred from the SW to the NE basin and vice versa. Our study revealed pronounced differences in growth rates among the major bacterial groups in relation to the treatments. Members of the Betaproteobacteria, in particular of the subgroup targeted by the BETA2-870 probe, were highly abundant in both basins, and most of them belonged to the Polynucleobacter necessesarius subcluster PnecC. Their specific growth rates surprisingly increased in all treatments when being transplanted into the acidic SW basin, indicating that pH and humic substances greatly affected growth of this particular group in the lake. In contrast, members of the Sphingobacteria/Flavobacteria group of the Bacteroidetes (both basins) as well as Actinobacteria (SW basin) were less abundant, especially in the presence of flagellates (< 5.0 microm treatments). However, because of their extremely low initial numbers, grazing of heterotrophic nanoflagellates mostly controlled only a small part of the bacterial production (< or = 12%).

Assessing Niche Separation among Coexisting Limnohabitans Strains through Interactions with a Competitor, Viruses, and a Bacterivore

ABSTRACT We investigated potential niche separation in two closely related (99.1% 16S rRNA gene sequence similarity) syntopic bacterial strains affiliated with the R-BT065 cluster, which represents a subgroup of the genus Limnohabitans. The two strains, designated B4 and D5, were isolated concurrently from a freshwater reservoir. Differences between the strains were examined through monitoring interactions with a bacterial competitor, Flectobacillus sp. (FL), and virus- and predator-induced mortality. Batch-type cocultures, designated B4+FL and D5+FL, were initiated with a similar biomass ratio among the strains. The proportion of each cell type present in the cocultures was monitored based on clear differences in cell sizes. Following exponential growth for 28 h, the cocultures were amended by the addition of two different concentrations of live or heat-inactivated viruses concentrated from the reservoir. Half of virus-amended treatments were inoculated immediately with an axenic flagellate predator, Poterioochromonas sp. The presence of the predator, of live viruses, and of competition between the strains significantly affected their population dynamics in the experimentally manipulated treatments. While strains B4 and FL appeared vulnerable to environmental viruses, strain D5 did not. Predator-induced mortality had the greatest impact on FL, followed by that on D5 and then B4. The virus-vulnerable B4 strain had smaller cells and lower biomass yield, but it was less subject to grazing. In contrast, the seemingly virus-resistant D5, with slightly larger grazing-vulnerable cells, was competitive with FL. Overall, our data suggest contrasting ecophysiological capabilities and partial niche separation in two coexisting Limnohabitans strains.

Aggregate formation in a freshwater bacterial strain induced by growth state and conspecific chemical cues.

We investigated the induction of aggregate formation in the freshwater bacterium Sphingobium sp. strain Z007 by growth state and protistan grazing. Dialysis bag batch culture experiments were conducted in which these bacteria were grown spatially separated from bacteria or from co-cultures of bacteria and predators. In pure cultures of Sphingobium sp. strain Z007, the concentrations of single cells and aggregates inside and outside the dialysis membranes developed in a similar manner over 3 days of incubation, and the proportions of aggregates were highest during the exponential growth phase. Cell production of Sphingobium sp. strain Z007 was enhanced in the presence of another isolate, Limnohabitans planktonicus, from an abundant freshwater lineage (R-BT065) outside the bags, and even more so if that strain was additionally grazed upon by the bacterivorous flagellate Poterioochromonas sp. However, the ratios of single cells to aggregates of Sphingobium sp. strain Z007 were not affected in either case. By contrast, the feeding of flagellates on Sphingobium sp. strain Z007 outside the dialysis bags led to significantly higher proportions of aggregates inside the bags. This was not paralleled by an increase in growth rates, and all cultures were in a comparable growth state at the end of the experiment. We conclude that two mechanisms, growth state and the possible release of infochemicals by the predator, may induce aggregate formation of Sphingobium sp. strain Z007. Moreover, these infochemicals only appeared to be generated by predation on cells from the same species.

Differential freshwater flagellate community response to bacterial food quality with a focus on Limnohabitans bacteria.

Different bacterial strains can have different value as food for heterotrophic nanoflagellates (HNF), thus modulating HNF growth and community composition. We examined the influence of prey food quality using four Limnohabitans strains, one Polynucleobacter strain and one freshwater actinobacterial strain on growth (growth rate, length of lag phase and growth efficiency) and community composition of a natural HNF community from a freshwater reservoir. Pyrosequencing of eukaryotic small subunit rRNA amplicons was used to assess time-course changes in HNF community composition. All four Limnohabitans strains and the Polynucleobacter strain yielded significant HNF community growth while the actinobacterial strain did not although it was detected in HNF food vacuoles. Notably, even within the Limnohabitans strains we found significant prey-related differences in HNF growth parameters, which could not be related only to size of the bacterial prey. Sequence data characterizing the HNF communities showed also that different bacterial prey items induced highly significant differences in community composition of flagellates. Generally, Stramenopiles dominated the communities and phylotypes closely related to Pedospumella (Chrysophyceae) were most abundant bacterivorous flagellates rapidly reacting to addition of the bacterial prey of high food quality.

Every coin has a back side: invasion by Limnohabitans planktonicus promotes the maintenance of species diversity in bacterial communities.

One of the earliest challenges for ecologists has been to study the impact of invasive species on microbial communities. Although bacteria are fundamental in biological processes, current knowledge on invasion effects by aquatic non-pathogenic bacteria is still limited. Using pure cultures of diverse planktonic bacteria as model organisms at two different carbon concentration levels, we tested the response of an assembled community to the invasion by Limnohabitans planktonicus, an opportunistic bacterium, successful in freshwaters. The invader, introduced at the early stationary growth phase of the resident community, caused a strong decrement of the abundance of the dominant species. This was due to competition for nutrients and a potential allelopathic interaction. Simultaneously, resident species formerly unable to successfully compete within the community, thus potentially exposed to competitive exclusion, increased their abundances. The overall result of the invasion was preservation of species diversity, the higher the lower was the substrate content available. Our study provides new insights into bacterial invasions, offering an alternative interpretation of invasions for community ecology.

Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 106 cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 104 cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 106 bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.

Suboptimal light conditions negatively affect the heterotrophy of Planktothrix rubescens but are beneficial for accompanying Limnohabitans spp.

We examined the effect of light on the heterotrophic activity of the filamentous cyanobacterium Planktothrix rubescens and on its relationship with the accompanying bacteria. In situ leucine uptake by bacteria and cyanobacteria was determined in a subalpine mesotrophic lake, and natural assemblages from the zone of maximal P. rubescens abundances were incubated for 2 days at contrasting light regimes (ambient, 100× increased, dark). Planktothrix rubescens from the photic zone of the lake incorporated substantially more leucine, but some heterotrophic activity was maintained in filaments from the hypolimnion. Exposure of cyanobacteria to increased irradiance or darkness resulted in significantly lower leucine incorporation than at ambient light conditions. Highest abundances and leucine uptake of Betaproteobacteria from the genus Limnohabitans were found in the accompanying microflora at suboptimal irradiance levels for P. rubescens or in dark incubations. Therefore, two Limnohabitans strains (representing different species) were co-cultured with axenic P. rubescens at different light conditions. The abundances and leucine incorporation rates of both strains most strongly increased at elevated irradiance levels, in parallel to a decrease of photosynthetic pigment fluorescence and the fragmentation of cyanobacterial filaments. Our results suggest that Limnohabitans spp. in lakes might profit from the presence of physiologically stressed P. rubescens.

A novel ion-exclusion chromatography-mass spectrometry method to measure concentrations and cycling rates of carbohydrates and amino sugars in freshwaters

The concentrations of free neutral carbohydrates and amino sugars were determined in freshwater samples of distinct matrix complexity, including meso-, eu- and dystrophic lakes and ponds, using high-performance ion-exclusion chromatography (HPIEC) coupled to mass spectrometry (MS). In contrast to other methods, our approach allowed the quantification of free neutral carbohydrates and amino sugars at low nM concentrations without derivatization, de-salting or pre-concentration. New sample preparation procedures were applied prior to injection employing syringe and hollow fiber filtration. Analytes were separated on a strong cation exchange resin under 100% aqueous conditions using 0.1% formic acid as a mobile phase. To minimize background noise in MS, analytes were detected in a multiple reaction monitoring scan mode with double ion filtering. Detection limits of carbohydrates and amino sugars ranged between 0.2 and 2nM at a signal-to-noise ratio >5. Error ranged between 1 and 12% at 0.5-500nM levels. Using a stable isotope dilution approach, both the utilization and recycling of glucose in Lake Zurich was observed. In contrast, N-acetyl-glucosamine was equally rapidly consumed but there was no visible de novo production. The simple and rapid sample preparation makes our protocol suitable for routine analyses of organic compounds in freshwater samples. Application of stable isotope tracers along with accurate measures of carbohydrate and amino sugar concentrations enables novel insights into the compound in situ dynamics.

High-throughput determination of dissolved free amino acids in unconcentrated freshwater by ion-pairing liquid chromatography and mass spectrometry.

We developed a procedure for the direct determination of dissolved free amino acids (DFAAs) in freshwater samples employing ion-pairing liquid chromatography and mass spectrometry. Our approach allowed accurate quantification of subnanomolar concentrations of DFAAs without prior concentration, derivatization or sample clean-up steps, achieving a throughput of three samples per hour. DFAAs were separated on a C-18 resin using tridecafluoroheptanoic acid as an ion-pairing agent controlling the overall retention. The relative standard deviation of DFAA measurements was <10% in samples from the mesotrophic Lake Zurich (Switzerland), and across concentrations of 0.5-500nM. Recoveries of DFAAs ranged from 94 to 102% within the range of 0.2-10nM. The limits of quantification for individual DFAAs varied between 50pM to 2nM (median, 0.5nM). The new method was employed to compare the spatial variability of DFAA concentrations in samples obtained by two devices. Epilimnetic samples of different size (ml, l) were collected at various spatial scales (cm, m, km) with a traditional 5l Friedinger sampler and with a custom-made multi-syringe sampling apparatus. Concentrations of total DFAAs ranged from 30 to 330nM. Alanine, serine, glutamic acid, arginine and glycine constituted 65% of the total pool, while methionine and tryptophan occurred at sub-nM concentrations only. Concentrations of individual DFAAs varied spatially over 2 orders of magnitude. Their spatial distribution was positively skewed, as characterized by rare peaks, most strongly so for glutamate, glycine and asparagine. The composition of DFAAs significantly differed at all examined spatial scales, and this could be mainly attributed to alanine, aspartic acid, and glycine. Our new method equals or outperforms existing ones in terms of sensitivity and reproducibility, while its procedural simplicity renders it superior for the high-throughput analysis of freshwater samples.