Phylogenetic Relationships and Coaggregation Ability of Freshwater Biofilm Bacteria

Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.     

Coaggregation between Aquatic Bacteria Is Mediated by Specific-Growth-Phase-Dependent Lectin-Saccharide Interactions

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.     

Impact of Water Column Acidification on Protozoan Bacterivory at the Lake Sediment-Water Interface

Although the impact of acidification on planktonic grazer food webs has been extensively studied, little is known about microbial food webs either in the water column or in the sediments. Protozoon-bacterium interactions were investigated in a chronically acidified (acid mine drainage) portion of a lake in Virginia. We determined the distribution, abundance, apparent specific grazing rate, and growth rate of protozoa over a pH range of 3.6 to 6.5. Protozoan abundance was lower at the most acidified site, while abundance, in general, was high compared with other systems. Specific grazing rates were uncorrelated with pH and ranged between 0.02 and 0.23 h^-1, values similar to those in unacidified systems. The protozoan community from an acidified station was not better adapted (P = 0.95) to low-pH conditions than a community from an unacidified site (multivariate analysis of variance on growth rates for each community incubated at pHs 4, 5, and 6). Both communities had significantly lower (P < 0.05) growth rates at pHs 4 and 5 than at pH 6. Reduced protozoan growth rates coupled with high grazing rates and relatively higher bacterial yields (ratio of bacterial-protozoan standing stock) at low pH indicate reduced net protozoan growth efficiency and a metabolic cost of acidification to the protozoan community. However, the presence of an abundant, neutrophilic protozoan community and high bacterial grazing rates indicates that acidification of Lake Anna has not inhibited the bacterium-protozoon link of the sediment microbial food web.     

Grazing Pressure by a Bacterivorous Flagellate Reverses the Relative Abundance of Comamonas acidovorans PX54 and Vibrio Strain CB5 in Chemostat Cocultures

The response of the bacterial strains Comamonas acidovorans PX54 (β subclass of the class Proteobacteria) and Vibrio strain CB5 (γ subclass of the class Proteobacteria) to grazing by the bacterivorous flagellate Ochromonas sp. was examined in one-stage chemostat experiments under conditions of low growth rates with a complex carbon source. The two bacterial strains were cultured together; they were cultured without flagellates in the first phase of the experiments and in the presence of the flagellates in the second phase. Monoclonal and polyclonal antibodies were used to determine the numbers and sizes of C. acidovorans PX54 and Vibrio strain CB5 cells. The flagellates caused strong changes in total bacterial cell numbers, in the relative abundances of the individual bacterial strains, and in bacterial cell size distribution. Vibrio strain CB5 dominated the total bacterial cell numbers during the flagellate-free phase of the experiments with a relative abundance of 93%, but this declined to 33% after inoculation with the flagellate. In contrast to Vibrio strain CB5, C. acidovorans PX54 responded to grazing with a strong expansion of cell length distribution toward large, filamentous cells. These changes in cell morphology resulted in a high percentage of inedible cells in the C. acidovorans PX54 population but not in the Vibrio strain CB5 population, which caused the observed change in the relative abundances of the strains. Batch culture experiments without the flagellate demonstrated that the elongation of C. acidovorans PX54 cells was dependent on their growth rate. This indicates that the occurrence of filamentous C. acidovorans PX54 cells is not a direct response to chemical stimuli released by the flagellates but rather a response to increased growth rates due to flagellate grazing.Predator-prey interactions of coexisting, free-living aquatic bacteria and bacterivorous protozoa have coevolved for more than a billion years (28). This enormous time span and the short generation times of both groups of microorganisms should have resulted in a high degree of evolutionary adaptation on both sides. Bacteria may have developed defense strategies to prevent themselves from being ingested (preingestional strategies) or digested (postingestional strategies) by their protozoan predators, which, expectedly, adaptated to circumvent the bacterial defense mechanisms. Information about the strategies involved in these predator-prey interactions is scarce. Recently, Jürgens and Güde (20) reviewed the strategies of bacteria and stressed the lack of knowledge in this field.Studies on size-selective ingestion (grazing) of bacterivorous protozoa (6, 10, 25) indicate that very small and large bacteria are partly or totally protected from protozoan grazing (12, 20). This finding is supported by field and experimental observations showing the occurrence and persistence of large bacterial filaments and aggregates during times of high grazing pressure (11, 21, 29, 41). The experimental evidence for protection and the increasing number of reports on the presence of filamentous bacteria in freshwater ecosystems (12, 13, 19, 35, 39, 41) indicate that this bacterial morphotype exhibits an ecologically significant defense strategy against protozoan grazing. It is not known to which species these protected forms belong. Additionally, it is unclear if the filamentous bacteria grow permanently with these, with respect to grazing, advantageous morphological properties or if they express these characteristic features only under strong grazing pressure.In a recent study, Pernthaler et al. (30) demonstrated that a slow-growing bacterial community reacted to the addition of bacterivorous flagellates within 1 day: one group produced filamentous, grazing-resistant forms, and another group of bacteria reacted with a massive growth rate increase. Similarly, Jürgens et al. (21) observed in enclosure studies, after experimentally increasing the protozoan grazing pressure, that there was a rapid and strong change in the morphological structure of the bacterial community. After 3 days, mainly filamentous and other inedible bacterial cells dominated the bacterial biomass, with a prevalence of 80 to 90%.Different mechanisms are conceivable for such changes in the morphological structure of bacterial communities. First, nonfilamentous, edible strains may simply be replaced after some time by inedible, permanently filamentous strains. In situations with bacterial generation times longer than 1 day and undetectably low abundances of filamentous cells (30), such an indirect selection mechanism can hardly cause visible changes in community structure within 24 h. But the possibility cannot be ruled out that this mechanism is of relevance in natural ecosystems. Second, medium-size, edible cells may become elongated and thus form filaments. This type of response to strong protistan grazing might be controlled by two different mechanisms: (i) elongation of the cells due to grazing-mediated changes in bacterial growth conditions (indirect induction of filament formation) or (ii) direct induction of morphological changes by chemical stimuli. Such chemical stimuli might be produced and released by the protozoan predators (predator kairomone) or produced by the prey bacteria and set free by the predators during digestion. The second type of stimuli would act as an alarm substance. It is not known if selection or one of the induction mechanisms triggers the observed reactions of bacterial communities. Pernthaler et al. (30) speculated that a chemical stimulus caused the observed changes in their experiments, since they found an immediate response upon addition of a flagellate grazer.Detailed information on the interactions of bacteria with protozoan grazers and the resulting bacterial defense strategies are necessary for a comprehensive understanding of a number of important issues in microbial ecology. This includes questions about the influence of protozoa on (i) the bacterial species composition of natural communities, (ii) the regulation of bacterial production and mineralization in aquatic systems, and (iii) the survival and behavior of allochthonous bacteria such as pathogenic members of the family Enterobacteriaceae or genetically engineered microorganisms in the environment.In this study, we used a model system to investigate the interactions of two bacterial strains with the bacterivorous nanoflagellate Ochromonas sp. The bacterium Vibrio sp. strain CB5 originated from the pelagic zone of Lake Constance (southern Germany) and was isolated from a chemostat inoculated with a water sample from that lake (14). The other strain, Comamonas acidovorans PX54, represents a member of a phylogenetic group which is abundant in Lake Plußsee (located near Plön, northern Germany) and in other lakes in the same area (9).In this study, we investigated mechanisms that control the observed changes in the composition of the model community and investigated possible defense strategies of pelagic bacteria against protozoan grazing.     

A new method for evaluating defense of microalgae against protozoan grazing

Body-size spectrum has proved to be a highly informative indicator to summarize the functional structure of a community at taxon-free resolution. In this study, an approach based on body-size spectrum of protozoan communities was used to detect the defense of microalgae against protozoan grazing. The biofilm-dwelling protozoan communities were used as a test predator system, and two algal species, Chlorella sp. and Nannochloropsis oceanica, were employed as test microalgae. A nine-day bioassay test was carried out by exposing biofilm-dwelling protozoan communities to a gradient of concentrations 10⁰ (control), 10⁴, 10⁵, 10⁶, and 10⁷ cell ml⁻¹ of both microalgae, respectively. Results showed that both algal species represented strong defense effects on the test predator system at different levels of concentration. The body-size distinctness of the protozoan assemblages showed a sharp decrease at high concentration level more than 10⁶ cell ml⁻¹ in both algal treatments. Based on the paired body-size distinctness indices of the protozoa, ellipse tests demonstrated that the body-size spectrum showed an increasing trend of departure from the expected pattern with increasing concentrations of both test algae. Thus, it is suggested that the body-size spectrum of protozoa may be used as a useful indicator to identify the defense of microalgae against protozoan grazing.     

Predation on Multiple Trophic Levels Shapes the Evolution of Pathogen Virulence

The pathogen virulence is traditionally thought to co-evolve as a result of reciprocal selection with its host organism. In natural communities, pathogens and hosts are typically embedded within a web of interactions with other species, which could affect indirectly the pathogen virulence and host immunity through trade-offs. Here we show that selection by predation can affect both pathogen virulence and host immune defence. Exposing opportunistic bacterial pathogen Serratia marcescens to predation by protozoan Tetrahymena thermophila decreased its virulence when measured as host moth Parasemia plantaginis survival. This was probably because the bacterial anti-predatory traits were traded off with bacterial virulence factors, such as motility or resource use efficiency. However, the host survival depended also on its allocation to warning signal that is used against avian predation. When infected with most virulent ancestral bacterial strain, host larvae with a small warning signal survived better than those with an effective large signal. This suggests that larval immune defence could be traded off with effective defence against bird predators. However, the signal size had no effect on larval survival when less virulent control or evolved strains were used for infection suggesting that anti-predatory defence against avian predators, might be less constrained when the invading pathogen is rather low in virulence. Our results demonstrate that predation can be important indirect driver of the evolution of both pathogen virulence and host immunity in communities with multiple species interactions. Thus, the pathogen virulence should be viewed as a result of both past evolutionary history, and current ecological interactions.     

From Grazing Resistance to Pathogenesis: The Coincidental Evolution of Virulence Factors

To many pathogenic bacteria, human hosts are an evolutionary dead end. This begs the question what evolutionary forces have shaped their virulence traits. Why are these bacteria so virulent? The coincidental evolution hypothesis suggests that such virulence factors result from adaptation to other ecological niches. In particular, virulence traits in bacteria might result from selective pressure exerted by protozoan predator. Thus, grazing resistance may be an evolutionarily exaptation for bacterial pathogenicity. This hypothesis was tested by subjecting a well characterized collection of 31 Escherichia coli strains (human commensal or extra-intestinal pathogenic) to grazing by the social haploid amoeba Dictyostelium discoideum. We then assessed how resistance to grazing correlates with some bacterial traits, such as the presence of virulence genes. Whatever the relative population size (bacteria/amoeba) for a non-pathogenic bacteria strain, D. discoideum was able to phagocytise, digest and grow. In contrast, a pathogenic bacterium strain killed D. discoideum above a certain bacteria/amoeba population size. A plating assay was then carried out using the E. coli collection faced to the grazing of D. discoideum. E. coli strains carrying virulence genes such as iroN, irp2, fyuA involved in iron uptake, belonging to the B2 phylogenetic group and being virulent in a mouse model of septicaemia were resistant to the grazing from D. discoideum. Experimental proof of the key role of the irp gene in the grazing resistance was evidenced with a mutant strain lacking this gene. Such determinant of virulence may well be originally selected and (or) further maintained for their role in natural habitat: resistance to digestion by free-living protozoa, rather than for virulence per se.     

High motility reduces grazing mortality of planktonic bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 microm s(-1) as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 microm s(-1)) and a highly motile strain (>45 microm s(-1)) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 microm(3). Grazing mortality was lowest for cells of >0.5 microm(3) and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (< or =0.1 microm(3), >50 microm s(-1)) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.     

High Motility Reduces Grazing Mortality of Planktonic Bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 μm s⁻¹ as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 μm s⁻¹) and a highly motile strain (>45 μm s⁻¹) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 μm³. Grazing mortality was lowest for cells of >0.5 μm³ and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (≤0.1 μm³, >50 μm s⁻¹) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.     

Microbial community dynamics in Mediterranean nutrient-enriched seawater mesocosms: changes in abundances, activity and composition

Quantitative and qualitative changes in bacterial communities from the Mediterranean Sea were compared in duplicate batch mesocosms with or without addition of inorganic nutrients. Methods including traditional microbial ecology techniques, molecular biology and flow cytometry were combined to determine abundances, production, cell size, activity, culturability and taxonomic diversity of bacterial cells. Addition of nutrients and confinement resulted in an increase of bacterial densities which were rapidly controlled by protozoan grazing. Changes in bacterial activity and morphology were observed during the growth phase of bacteria and under grazing pressure. The proportion of medium-size and culturable cells increased during the growth phase. These cells were preferentially consumed by grazers resulting in a strong limitation of bacterial production. As a consequence of the grazing pressure, large cells were produced and contributed to the remaining bacterial productivity after grazing. Grazing had an effect on the taxonomic composition of bacterial communities by preferentially eliminating gamma-Proteobacteria, alpha-Proteobacteria were preserved. It seems that some species from the genera Ruegeria and Cytophaga may have developed defence strategies to escape predation.     

Active Bacterial Populations and Grazing Impact Revealed by an In Situ Experiment in a Shallow Aquifer

To elucidate bacterial population dynamics in an aquifer, we attempted to reveal the impact of protozoan grazing on bacterial productivity and community structure by an in situ incubation experiment using a diffusion chamber. The abundance and vertical distribution of bacteria and protozoa in the aquifer were revealed using wells that were drilled in a sedimentary rock system in Itako, Ibaraki, Japan. The water column in the wells possessed aerobic and anaerobic layers. Active bacterial populations under the grazing pressure of protozoa were revealed through in situ incubation with grazer eliminating experiment by the filtration. On August 19, 2003, the total number of bacteria (TDC) decreased from 1.5 × 10⁶ cells ml^{? 1} at 2.2 m depth to 3.0 × 10⁵ cells ml^{? 1} at 10 m depth. The relative contribution of the domain Bacteria to TDC ranged between 63% and 84%. Protozoa existed at a density of 4.2 × 10⁴ to 1.9 × 10⁵ cells ml^{? 1} in both aerobic and microaerobic conditions. A grazing elimination experiment in situ for 6 days brought about clearly different bacterial community profiles between the 2.2 m and 10 m samples. The bacterial composition of the initial community was predominantly β- and γ -proteobacteria at 2.2 m, while at 10 m β-, α - and γ -proteobacteria represented 56%, 26% and 13% of the community, respectively. The distribution of bacterial abundance, community composition and growth rates in the subsurface were influenced by grazing as well as by geochemical factors (dissolved oxygen and concentrations of organic carbon, methane and sulfate). Results of the in situ incubation experiment suggested that protozoan grazing contributes significantly to bacterial population dynamics.     

Effect of Protozoa on Bacterial Degradation of an Aromatic Compound

Prototrophic and growth factor-requiring strains of Alcaligenes spp. were used to study the effect of a protozoan, Tetrahymena pyriformis, on the degradation of p-aminobenzoate. The protozoan inhibited activity of the prototrophic bacterium by reducing its population size. For the growth factor-requiring strain of Alcaligenes, T. pyriformis provided the required growth factors so that the predator permitted the bacteria to grow and to continue p-aminobenzoate degradation. T. pyriformis inhibited bacterial activity when the amino acid supply was in excess, but activity of the auxotrophic strain of Alcaligenes was stimulated by the protozoan when the amino acid supply was limiting, although the bacterial population size was reduced by the protozoan.     

Changes amid constancy: Flower and leaf microbiomes along land use gradients and between bioregions

Microbial communities inhabiting above-ground parts of plants affect their host's development, fitness and function. Although studies on plant-associated microbes are of growing interest, environmental drivers of flower microbiomes in particular are poorly characterized. In this study, we investigated flower and leaf epiphytic bacterial microbiomes of Ranunculus acris and Trifolium pratense using metabarcoding of 16S ribosomal DNA in three German bioregions and along land-use intensity gradients. Our data suggests that the structures of bacterial communities clearly differed between plant species and tissue types. Also, floral bacterial communities of R. acris showed higher variability in comparison to T. pratense. Bacteria usually associated with pollinators were found solely in flower samples, while bacteria usually associated with the rhizosphere were only present in high abundances on leaves. We identified Pseudomonadaceae, Enterobacteriaceae and Sphingomonadaceae as the most abundant taxa on flowers, while Sphingomonadaceae, Methylobacteriaceae and Cytophagaceae dominated bacterial communities on leaves. We found that bacterial communities did not differ between long-distant regions. However, there was a turnover within each bioregion between short-distant locations. High land use intensity caused phylogenetically less diverse and more homogenous bacterial communities with an exception of T. pratense flowers. This was associated with a loss of rare bacterial families. Intensification of mowing affected the bacterial communities associated with leaves of T. pratense and fertilization led to more homogenous flower and leaf communities of R. acris, while grazing had no effects on the bacterial community composition. However, dominant taxa were not affected by land use intensification. Despite that, we identified indicator taxa for regularly disturbed environments in flower microbiomes. In conclusion, our study contributes to the knowledge about microbial community structures of the phyllosphere and extends the understanding of their community dynamics with respect to biogeographical separation and anthropogenic changes of the environment.     

Identification of Ciliate Grazers of Autotrophic Bacteria in Ammonia-Oxidizing Activated Sludge by RNA Stable Isotope Probing

It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that ¹³C-labeled protozoan RNA can be retrieved from activated sludge amended with ¹³C-labeled protozoa or ¹³C-labeled Escherichia coli cells by using an RNA stable isotope probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate Epistylis galea dominated the acquisition of carbon from bacteria with access to CO₂ under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.Protozoa are the main consumers of bacteria in the environment, and as such they play a major role in controlling bacterial biomass (33) and regulating nutrient recycling (14). Therefore, being able to study the flow of carbon in food webs involving bacteria and protozoa can provide an insight into how protozoan grazing affects bacterial function in a wide range of systems.Protozoan grazing has the potential to alter the genotypic and phenotypic composition of bacterial communities (15, 17, 18, 35). Perhaps as a result of this, protozoan grazing has also been found to have an effect on the function of activated sludge systems, including nitrogen removal processes. Studies using eukaryotic inhibitors to remove grazers from activated sludge have reported a wide range of effects following reduced grazing pressure. These effects include an increase in turbidity or planktonic cell densities, and either no effect (21, 32), higher nitrification rates (16, 20), or lower (29, 30) rates of nitrification in the absence of grazers. These conflicting reports on the effect of predation on nitrification might be due to protozoa displaying feeding preferences or to the indirect ways in which protozoan grazing can affect bacterial processes. For example, it has been shown that the release of substances, such as vitamins and nucleotides, secreted by protozoa as metabolic by-products, can act as growth factors, which enhance bacterial activity, including nitrification (31). In addition, the presence of some types of grazers, particularly ciliates, has been shown to be closely related to a decrease in biochemical oxygen demand (27), and it has been reported that ciliates can alter water flux and help redistribute nutrients in flocs (8), which in turn might have an impact on nitrification rates.Although the data presented in the literature suggest that protozoan grazing is an important factor for activated sludge processes, there are still many questions as to whether its effects are directly linked to predation and possibly feeding preferences. In the present study we sought to identify protozoa assimilating carbon from autotrophic bacteria under ammonia-oxidizing conditions and acetate-consuming bacteria under denitrifying conditions using RNA-stable isotope probing (RNA-SIP).Since it was first developed, RNA-SIP has been successfully used to identify functional groups of bacteria responsible for different processes, including denitrification and benzene and phenol degradation (9, 10, 19, 25, 26). In these studies, the flow of carbon was tracked from soluble labeled substrates into the bacteria consuming it. However, recent studies have shown that it is possible to use this technique to track the flow of carbon across more than one trophic level, unraveling some of the interactions observed in microbial food webs (11, 13, 23, 24). These studies exemplify the broad range of questions that can be addressed with SIP and have begun to define the boundaries beyond which SIP has limited utility.     

Protozoan grazing and its impact upon population dynamics in biofilm communities

AIMS: To determine the impact of protozoan grazing on the population dynamics of a multispecies bacterial biofilm community. METHODS AND RESULTS: Grazing by Acanthamoeba castellanii and the ciliate Colpoda maupasi upon biofilm and planktonic communities, composed of Klebsiella pneumoniae, Pseudomonas fluorescens and Staphylococcus epidermidis was investigated. Biofilms were formed using glass coverslips, held in a carousel device, as substrata for biofilm formation or in glass flow cells. The predatory effects of the amoeba were generally confined to the biofilm, where grazing rates corresponded to losses from the biofilm equivalent to ca 30,000 biofilm cells cm(-2) h(-1), with the amoeba becoming an integral part of the community. C. maupasi reduced the thickness of mature multispecies biofilms at steady-state from 500 to <200 microm. CONCLUSIONS: We report that the presence of the protozoa A. castellanii and C. maupasi markedly influence population dynamics within defined biofilm communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study dispels the popular opinion that biofilms are protected against predation by protozoa. A. castellanii clearly has the capacity to graze mixed biofilm communities and to become integrally associated with them, whereas the ciliate C. maupasi reduced biofilm thickness by up to 60%.     

Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin

The mucosal surfaces of wild and farmed aquatic vertebrates face the threat of many aquatic pathogens, including fungi. These surfaces are colonized by diverse symbiotic bacterial communities that may contribute to fight infection. Whereas the gut microbiome of teleosts has been extensively studied using pyrosequencing, this tool has rarely been employed to study the compositions of the bacterial communities present on other teleost mucosal surfaces. Here we provide a topographical map of the mucosal microbiome of an aquatic vertebrate, the rainbow trout (Oncorhynchus mykiss). Using 16S rRNA pyrosequencing, we revealed novel bacterial diversity at each of the five body sites sampled and showed that body site is a strong predictor of community composition. The skin exhibited the highest diversity, followed by the olfactory organ, gills, and gut. Flectobacillus was highly represented within skin and gill communities. Principal coordinate analysis and plots revealed clustering of external sites apart from internal sites. A highly diverse community was present within the epithelium, as demonstrated by confocal microscopy and pyrosequencing. Using in vitro assays, we demonstrated that two Arthrobacter sp. skin isolates, a Psychrobacter sp. strain, and a combined skin aerobic bacterial sample inhibit the growth of Saprolegnia australis and Mucor hiemalis, two important aquatic fungal pathogens. These results underscore the importance of symbiotic bacterial communities of fish and their potential role for the control of aquatic fungal diseases.     

Effects of Hydrophobic and Electrostatic Cell Surface Properties of Bacteria on Feeding Rates of Heterotrophic Nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.     

Impact of protozoan grazing on bacterial community structure in soil microcosms

The influence of grazing by a mixed assemblage of soil protozoa (seven flagellates and one amoeba) on bacterial community structure was studied in soil microcosms amended with a particulate resource (sterile wheat roots) or a soluble resource (a solution of various organic compounds). Sterilized soil was reinoculated with mixed soil bacteria (obtained by filtering and dilution) or with bacteria and protozoa. Denaturing gradient gel electrophoresis (DGGE) of PCR amplifications of 16S rRNA gene fragments, as well as community level physiological profiling (Biolog plates), suggested that the mixed protozoan community had significant effects on the bacterial community structure. Excising and sequencing of bands from the DGGE gels indicated that high-G+C gram-positive bacteria closely related to Arthrobacter spp. were favored by grazing, whereas the excised bands that decreased in intensity were related to gram-negative bacteria. The percentages of intensity found in bands related to high G+C gram positives increased from 4.5 and 12.6% in the ungrazed microcosms amended with roots and nutrient solution, respectively, to 19.3 and 32.9% in the grazed microcosms. Protozoa reduced the average bacterial cell size in microcosms amended with nutrient solution but not in the treatment amended with roots. Hence, size-selective feeding may explain some but not all of the changes in bacterial community structure. Five different protozoan isolates (Acanthamoeba sp., two species of Cercomonas, Thaumatomonas sp., and Spumella sp.) had different effects on the bacterial communities. This suggests that the composition of protozoan communities is important for the effect of protozoan grazing on bacterial communities.     

Prey-Specific Growth Responses of Freshwater Flagellate Communities Induced by Morphologically Distinct Bacteria from the Genus Limnohabitans

Because their large growth potential is counterbalanced with grazing by heterotrophic nanoflagellates (HNF), bacteria of the genus Limnohabitans, which are common in many freshwater habitats, represent a valuable model for examining bacterial carbon flow to the grazer food chain. We conducted experiments with natural HNF communities taken from two distinct habitats, the meso-eutrophic Římov Reservoir and the oligo-mesotrophic Lake Cep (South Bohemia). HNF communities from each habitat at distinct seasonal phases, a late April algal bloom and a late May clear water phase, were each fed 3 Limnohabitans strains of differing cell sizes. Water samples were prefiltered (5 μm) to release natural HNF communities from zooplankton control and then amended with the Limnohabitans strains L. planktonicus II-D5 (medium sized, rod shaped), Limnohabitans sp. strain T6-5 (thin, long, curved rod), and Limnohabitans sp. strain 2KL-3 (large solenoid). Using temporal sampling and prey treatment, we determined HNF growth parameters such as doubling time, growth efficiency, and length of lag phase prior starting to exponential growth. All three Limnohabitans strains supported HNF growth but in significant prey-, site-, and season-dependent fashions. For instance, addition of the moderately large T6-5 strain yielded very rapid HNF growth with a short lag phase. In contrast, the curved morphology and larger cell size of strain 2KL-3 made this prey somewhat protected against grazing by smaller HNF, resulting in slower HNF growth and longer lag phases. These trends were particularly pronounced during the late May clear-water phase, which was dominated by smaller HNF cells. This may indicate a longer “adaptation time” for the flagellate communities toward the large prey size offered.     

Effects of hydrophobic and electrostatic cell surface properties of bacteria on feeding rates of heterotrophic nanoflagellates

The influence of cell surface hydrophobicity and electrostatic charge of bacteria on grazing rates of three common species of interception-feeding nanoflagellates was examined. The hydrophobicity of bacteria isolated from freshwater plankton was assessed by using two different methods (bacterial adhesion to hydrocarbon and hydrophobic interaction chromatography). The electrostatic charge of the cell surface (measured as zeta potential) was analyzed by microelectrophoresis. Bacterial ingestion rates were determined by enumerating bacteria in food vacuoles by immunofluorescence labelling via strain-specific antibodies. Feeding rates varied about twofold for each flagellate species but showed no significant dependence on prey hydrophobicity or surface charge. Further evidence was provided by an experiment involving flagellate grazing on complex bacterial communities in a two-stage continuous culture system. The hydrophobicity values of bacteria that survived protozoan grazing were variable, but the bacteria did not tend to become more hydrophilic. We concluded that variability in bacterial cell hydrophobicity and variability in surface charge do not severely affect uptake rates of suspended bacteria or food selection by interception-feeding flagellates.