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.     

Direct and indirect effects of protist predation on population size structure of a bacterial strain with high phenotypic plasticity

We studied the impact of grazing and substrate supply on the size structure of a freshwater bacterial strain (Flectobacillus sp.) which showed pronounced morphological plasticity. The cell length varied from 2 to >40 microm and encompassed rods, curved cells, and long filaments. Without grazers and with a sufficient substrate supply, bacteria grew mainly in the form of medium-sized rods (4 to 7 microm), with a smaller proportion (<10%) of filamentous forms. Grazing experiments with the bacterivorous flagellate Ochromonas sp. showed that freely suspended cells of <7 microm were highly vulnerable to grazers, whereas filamentous cells were resistant to grazing and became enriched during predation. A comparison of long-term growth in carbon-limited chemostats with and without grazers revealed that strikingly different bacterial populations developed: treatments with flagellates were composed of >80% filamentous cells. These attained a biomass comparable to that of populations in chemostats without grazers, which were composed of medium-sized rods and c-shaped cells. Carbon starvation resulted in a fast decrease in cell length and a shift towards small rods, which were highly vulnerable to grazing. Dialysis bag experiments in combination with continuous cultivation revealed that filament formation was significantly enhanced even without direct contact of bacteria with bacterivores and was thus probably stimulated by grazer excretory products.     

Strain-specific differences in the grazing sensitivities of closely related ultramicrobacteria affiliated with the Polynucleobacter cluster

Ultramicrobacteria (cell volume < 0.1 microm(3)) are the numerically dominant organisms in the plankton of marine and freshwater habitats. Flagellates and other protists are assumed to be the most important predators of these ultramicrobacteria as well as of larger planktonic bacteria. However, due to controversial observations conducted previously, it is not clear as to whether fractions of the ultramicrobacteria are resistant to flagellate predation. Furthermore, it is not known if closely related bacteria vary significantly in their sensitivity to flagellate predation. We investigated the sensitivity of ultramicrobacteria affiliated with the cosmopolitan Polynucleobacter cluster to grazing by Spumella-like nanoflagellates. Laboratory grazing experiments with four closely related (> or =99.6% 16S rRNA gene sequence similarity) bacteria and three closely related (100% 18S rRNA gene sequence similarity) flagellates were performed. In comparison to larger bacteria, predation on the ultramicrobacterial Polynucleobacter strains was weak, and the growth of the predating flagellates was slow. Specific clearance rates ranged between 0.14 x 10(5) and 2.8 x 10(5) units of predator size h(-1). Feeding rates strongly depended on the flagellate and bacterial strain (P < 0.001). Grazing mortality rates of the three flagellate strains investigated varied for the same prey strain by up to almost fourfold. We conclude that (i) ultramicrobacteria affiliated with the Polynucleobacter cluster are not protected from grazing, (ii) strain-specific variations in grazing sensitivity even between closely related bacteria are high, and (iii) strain-specific differences in predator-prey interaction could be an important factor in the evolution and maintenance of microbial microdiversity.     

Interaction of Nutrient Limitation and Protozoan Grazing Determines the Phenotypic Structure of a Bacterial Community

We examined the impact of nutrient conditions (carbon and phosphorus limitation) and grazing by protozoans on the phenotypic community structure of freshwater bacteria in continuous culture systems. Lakewater bacteria were grown on mineral medium, which was supplemented with glucose and amino acids and adjusted by different phosphorus concentrations to achieve either carbon or phosphorus limitation. Each nutrient treatment was inoculated with the same bacterial community and consisted of a nongrazing and a grazing treatment, to which the heterotrophic nanoflagellates Spumella sp. and Ochromonas sp. were added. We found that nutrient conditions alone resulted in differences in the phenotypic structure of the bacterial community: small and motile bacteria dominated under C limitation while large, elongated, and capsulated bacteria were characteristic for P limitation. The genotypic community composition as measured by T-RFLP (terminal restriction fragment length polymorphism) was not severely influenced by the two nutrient treatments. In the presence of flagellate predators, grazing-resistant bacteria developed under both nutrient conditions, but with different survival mechanisms: highly motile bacteria prevailed under C limitation, whereas the P-limited grazing treatment was dominated by filamentous forms. T-RFLP analysis revealed only moderate changes in bacterial community composition due to grazing, which were most pronounced under P limitation. Analysis by video microscopy revealed that high swimming speed is an efficient nonmorphological survival mechanism for bacteria to reduce the capture success of the flagellate predator. The rejection of optimal-sized, nonmotile bacteria under P limitation suggests the importance of other nonmorphological, surface-located cell properties. Our results illustrate that the realized mechanisms of grazing resistance are linked to the actual limitation conditions, and that the combined effects of nutrient limitation and grazing are major determinants of bacterial community structure.     

Effect of gamma-ray irradiation on Escherichia coli motility

The effects of ionizing radiation on bacteria are generally evaluated from the dose-dependent survival ratio, which is determined by colony-forming ability and mutation rate. The mutagenic damage to cellular DNA induced by radiation has been extensively investigated; however, the effects of irradiation on the cellular machinery in situ remain unclear. In the present work, we irradiated Escherichia coli cells in liquid media with gamma rays from ⁶⁰Co (in doses up to 8 kGy). The swimming speeds of the cells were measured using a microscope. We found that the swimming speed was unaltered in cells irradiated with a lethal dose of gamma rays. However, the fraction of motile cells decreased in a dose-dependent manner. Similar results were observed when protein synthesis was inhibited by treatment with kanamycin. Evaluation of bacterial swimming speed and the motile fraction after irradiation revealed that some E. coli cells without the potential of cell growth and division remained motile for several hours after irradiation.     

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%.     

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.     

Size-selective grazing of coastal bacterioplankton by natural assemblages of pigmented flagellates,colorless flagellates,and ciliates

Fluorescently-labelled bacteria (FLB) were used to study the feeding strategies of a natural assemblage of estuarine protozoans and to examine whether the protozoan grazing could account for the in situ size structure of the bacterioplankton. The FLB, DTAF-stained enterococci, ranging in volume from 0.01 to 0.30 × 10^–1 µm³, were added to a natural planktonic assemblage at a density of 5.5% of the natural bacterioplankton. Initial densities (individuals ml^–1) were as follows: total natural bacteria, 2.2 × 10⁶; FLB, 1.2 × 10⁵; pigmented flagellates, 300; colorless flagellates, 250; and ciliates, 30. FLB consumption rates were determined by examining the contents of protozoan food vacuoles, and the long-term effect of grazing (over a period of 100 hours) was determined by monitoring the decline in the FLB density in experimental vessels. The average consumption rates of FLB by pigmented flagellates were similar to those by flagellates that lacked chloroplasts (0.9 and 0.6 FLB protozoan^–1 hour^–1, respectively). The ciliates consumed bacteria at an average rate that was 17-fold higher (per cell) than flagellates, and they displayed a greater preference for larger bacteria than did the flagellates. FLB of the mid-size classes (0.025–0.100 µm³) were heavily grazed by the entire protozoan assemblage; the smallest (<0.025 µm³) and the largest (>0.100 µm³) FLB escaped protozoan grazing. This had a profound effect on the resulting size distribution of FLB. At the end of a 100-hour incubation, the percentage of mid-size FLB (0.025 to 0.100 µm³) decreased 2.0–2.2-fold, while the percentage of the smallest and the largest FLB increased 2.0–2.5-fold. Resultant densities of FLB were consistent with initial clearance rates determined for the protozoan groups. The grazing rates of protozoans on FLB were species-specific; whereas some species consumed FLB, others did not demonstrate bacterivory. The results suggest that protozoan grazing has a major effect on the size distribution of coastal bacterioplankton. By selectively feeding on a particular size-class of bacteria, planktonic ciliates may consume 15–90% day^–1 of the standing stock of largest size classes of bacterioplankton. Thus, ciliates, which were present in low abundance in the field, could not balance the production of the entire bacterial community, but they may strongly influence the portion of the bacterial community represented by the largest bacterial class. The direct effect of flagellates (e.g., grazing) was limited to smaller bacteria.Offprint requests to: M. P. Shiaris.     

Motility and survival ofEuglena ignobilis as affected by different factors

Euglena ignobilis cells in natural puddle water of pH 7.8, when kept at 21 +/- 2 degrees C and under continuous light (intensity of approximately 30 micromol m(-2) s(-1)) in a culture chamber, decreased their speed of movement from > or = 78000 microm/min (after a 12-h cultivation), to 850-1300 microm/min after 18 h. Simultaneously initiated were changes in morphology from the usual elongated motile forms to round motile ones by curving and contraction. Water stress (2 and 4 % agarized puddle water, puddle water with 0.2-1 mol/L NaCl), temperature shock (< or = 10 degrees C, > or = 30 degrees C), darkness and low-light intensity, UV exposure (0.96-2.88 kJ/m2), pH extremes (< or = 6.5 and > or = 10), presence of 'heavy' metals (1-100 ppm Fe, Cu, Zn, Co, Ni, Hg) or organic substances in puddle water (25-1000 ppm 2,4-D, captan, urea, DDT, thiourea), all these factors rapidly (after 5 to 30 min) decreased the speed of the elongated motile form to < or = 300 microm/min, and induced all morphological changes leading to formation of round motile and round nonmotile forms. These features in the alga (i.e. sudden speed reduction and morphological changes from elongate motile to round motile form) may thus be suggested to be used in assessing water quality.     

Impact of fluorochrome stains used to study bacterial transport in shallow aquifers on motility and chemotaxis of Pseudomonas species

One of the most common methods of tracking movement of bacteria in groundwater environments involves a priori fluorescent staining. A major concern in using these stains to label bacteria in subsurface injection-and-recovery studies is the effect they may have on the bacterium's transport properties. Previous studies investigated the impact of fluorophores on bacterial surface properties (e.g. zeta potential). However, no previous study has looked at the impact of fluorescent staining on swimming speed and chemotaxis. It was found that DAPI lowered the mean population swimming speed of Pseudomonas putida F1 by 46% and Pseudomonas stutzeri by 55%. DAPI also inhibited the chemotaxis in both strains. The swimming speeds of P. putida F1 and P. stutzeri were diminished slightly by CFDA/SE, but not to a statistically significant extent. CFDA/SE had no effect on chemotaxis of either strain to acetate. SYBR(?) Gold had no effect on swimming speed or the chemotactic response to acetate for either strain. This research indicates that although DAPI may not affect sorption to grain surfaces, it adversely affects other potentially important transport properties such as swimming and chemotaxis. Consequently, bacterial transport studies conducted using DAPI are biased to nonchemotactic conditions and do not appear to be suitable for monitoring the effect of chemotaxis on bacterial transport in shallow aquifers.     

Measuring microzooplankton grazing on planktonic marine bacteria by its impact on bacterial production

Grazing on planktonic bacteria by microzooplankton was estimated by separating bacteria from the larger plankton with 1m pore Nuclepore filtration and measuring changes in bacteria in filtered and unfiltered samples over 24 hours. In the absence of grazers, bacteria increased linearly. The regression coefficient of linear increase was used to estimatein situ bacterial production. When grazers were present, the changes in bacteria concentration usually took the form of a linear decline, and grazing was estimated by subtracting the regression coefficient of the unfiltered sample from that of the 1m filtrate. Results from the Essex estuary-coastal system of northern Massachusetts show grazing and production at rates that indicate a daily turnover of the standing crop of bacteria, with highest values in mid-estuarine waters. Experiments on the size distribution of grazing showed that microzooplankton from 1–3m were responsible for most of the observed decrease in bacteria. It was suggested that the basic pattern of linear increase of the bacteria in the absence of grazing reflects density-dependent limitation by substrate present at the outset of the incubation and is indicative of a population that has been maintained around the mid-point of the logistic growth curve by grazing.     

Predation as a shaping force for the phenotypic and genotypic composition of planktonic bacteria

Predation is a major mortality factor of planktonic bacteria and an important shaping force for the phenotypic and taxonomic structure of bacterial communities. In this paper we: (1) summarise current knowledge on bacterial phenotypic properties which affect their vulnerability towards grazers, and (2) review experimental evidence demonstrating that this phenotypic heterogeneity results in shifts of bacterial community composition during enhanced protist grazing pressure. Size-structured interactions are especially important in planktonic systems and bacterial cell size influences the mortality rate and the type of grazer to which bacteria are most susceptible. When protists are the major bacterivores, both very small and large bacterial cells gain some size refuge. Recent studies have revealed that also various non-morphological traits such as motility, physicochemical surface characters and toxicity affect bacterial vulnerability and protist feeding success. These properties are effective at different stages during the feeding process of interception feeding flagellates (encounter, capture, ingestion, digestion). Grazing-resistant bacteria in natural communities can account for a substantial portion of the total bacterial biomass at least in more productive aquatic systems. In field and laboratory experiments it has been demonstrated that increased protozoan grazing results in shifts in the phenotypic and genotypic composition of the bacterial assemblage. The importance of this shaping force for the bacterial community structure depends, however, on the overall food web structure, especially on the composition of the metazooplankton. Whereas the structuring impact of bacterial grazers is well documented, relatively little is known about how grazing-mediated changes in bacterial communities influence microbially mediated processes and biogeochemically important transformations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Rate of Bacterial Mortality in Aquatic Environments

A method is proposed which provides a minimum estimate of the rate of bacterial mortality in growing natural populations of planktonic bacteria. This estimate is given by the rate of decrease of radioactivity from the DNA of a [³H]thymidine-labeled natural assemblage of bacteria after all added thymidine has been exhausted from the medium. Results obtained from river water, estuarine water, and seawater show overall bacterial mortality rates in the range 0.010 to 0.030 h⁻¹, in good agreement with the range of growth rates measured in the same environments. Use of selective filtration through Nuclepore filters (pore size, 2 μm) allowed us to determine the contribution of microzooplankton grazing to overall bacterial mortality. Grazing rates estimated by this method ranged from 0 to 0.02 h⁻¹.     

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.     

Swimming Speed of Larval Snail Does Not Correlate with Size and Ciliary Beat Frequency

Many marine invertebrates have planktonic larvae with cilia used for both propulsion and capturing of food particles. Hence, changes in ciliary activity have implications for larval nutrition and ability to navigate the water column, which in turn affect survival and dispersal. Using high-speed high-resolution microvideography, we examined the relationship between swimming speed, velar arrangements, and ciliary beat frequency of freely swimming veliger larvae of the gastropod Crepidula fornicata over the course of larval development. Average swimming speed was greatest 6 days post hatching, suggesting a reduction in swimming speed towards settlement. At a given age, veliger larvae have highly variable speeds (0.8–4 body lengths s⁻¹) that are independent of shell size. Contrary to the hypothesis that an increase in ciliary beat frequency increases work done, and therefore speed, there was no significant correlation between swimming speed and ciliary beat frequency. Instead, there are significant correlations between swimming speed and visible area of the velar lobe, and distance between centroids of velum and larval shell. These observations suggest an alternative hypothesis that, instead of modifying ciliary beat frequency, larval C. fornicata modify swimming through adjustment of velum extension or orientation. The ability to adjust velum position could influence particle capture efficiency and fluid disturbance and help promote survival in the plankton.     

A role for copper in protozoan grazing – two billion years selecting for bacterial copper resistance

The Great Oxidation Event resulted in integration of soft metals in a wide range of biochemical processes including, in our opinion, killing of bacteria by protozoa. Compared to pressure from anthropologic copper contamination, little is known on impacts of protozoan predation on maintenance of copper resistance determinants in bacteria. To evaluate the role of copper and other soft metals in predatory mechanisms of protozoa, we examined survival of bacteria mutated in different transition metal efflux or uptake systems in the social amoeba Dictyostelium discoideum. Our data demonstrated a strong correlation between the presence of copper/zinc efflux as well as iron/manganese uptake, and bacterial survival in amoebae. The growth of protozoa, in turn, was dependent on bacterial copper sensitivity. The phagocytosis of bacteria induced upregulation of Dictyostelium genes encoding the copper uptake transporter p80 and a triad of Cu(I)‐translocating P_IB‐type ATPases. Accumulated Cu(I) in Dictyostelium was monitored using a copper biosensor bacterial strain. Altogether, our data demonstrate that Cu(I) is ultimately involved in protozoan predation of bacteria, supporting our hypothesis that protozoan grazing selected for the presence of copper resistance determinants for about two billion years.     

Direct and Indirect Effects of Protist Predation on Population Size Structure of a Bacterial Strain with High Phenotypic Plasticity

We studied the impact of grazing and substrate supply on the size structure of a freshwater bacterial strain (Flectobacillus sp.) which showed pronounced morphological plasticity. The cell length varied from 2 to >40 μm and encompassed rods, curved cells, and long filaments. Without grazers and with a sufficient substrate supply, bacteria grew mainly in the form of medium-sized rods (4 to 7 μm), with a smaller proportion (<10%) of filamentous forms. Grazing experiments with the bacterivorous flagellate Ochromonas sp. showed that freely suspended cells of <7 μm were highly vulnerable to grazers, whereas filamentous cells were resistant to grazing and became enriched during predation. A comparison of long-term growth in carbon-limited chemostats with and without grazers revealed that strikingly different bacterial populations developed: treatments with flagellates were composed of >80% filamentous cells. These attained a biomass comparable to that of populations in chemostats without grazers, which were composed of medium-sized rods and c-shaped cells. Carbon starvation resulted in a fast decrease in cell length and a shift towards small rods, which were highly vulnerable to grazing. Dialysis bag experiments in combination with continuous cultivation revealed that filament formation was significantly enhanced even without direct contact of bacteria with bacterivores and was thus probably stimulated by grazer excretory products.     

Long Lag Times and High Velocities in the Motility of Natural Assemblages of Marine Bacteria

The motility characteristics of natural assemblages of coastal marine bacteria were examined. Initially, less than 10% of the bacteria were motile. A single addition of tryptic soy broth caused an increase in the motile fraction of cells but only after 7 to 12 h. Motility peaked at 15 to 30 h, when more than 80% of cells were motile. These results support the proposal that energy limits motility in the marine environment. Cell speeds changed more than an order of magnitude on timescales of milliseconds and hours. The maximum community speed was 144 (mu)m s(sup-1), and the maximum individual burst velocity was 407 (mu)m s(sup-1). In uniform medium, speed was an inverse function of tryptic soy broth concentration, declining linearly over 0.001 to 1.0%. In media where concentration gradients existed, the mean speed was a function of position in a spatial gradient, changing from 69 to 144 (mu)m s(sup-1) over as little as 15 to 30 (mu)m. The results suggest that marine bacteria are capable of previously undescribed quick shifts in speed that may permit the bacteria to rapidly detect and keep up with positional changes in small nutrient sources. These high speeds and quick shifts may reflect the requirements for useful motility in a turbulent ocean.     

Dynamics of Snake-like Swarming Behavior of Vibrio alginolyticus

Swarming represents a special case of bacterial behavior where motile bacteria migrate rapidly and collectively on surfaces. Swarming and swimming motility of bacteria has been studied well for rigid, self-propelled rods. In this study we report a strain of Vibrio alginolyticus, a species that exhibits similar collective motility but a fundamentally different cell morphology with highly flexible snake-like swarming cells. Investigating swarming dynamics requires high-resolution imaging of single cells with coverage over a large area: thousands of square microns. Researchers previously have employed various methods of motion analysis but largely for rod-like bacteria. We employ temporal variance analysis of a short time-lapse microscopic image series to capture the motion dynamics of swarming Vibrio alginolyticus at cellular resolution over hundreds of microns. Temporal variance is a simple and broadly applicable method for analyzing bacterial swarming behavior in two and three dimensions with both high-resolution and wide-spatial coverage. This study provides detailed insights into the swarming architecture and dynamics of Vibrio alginolyticus isolate B522 on carrageenan agar that may lay the foundation for swarming studies of snake-like, nonrod-shaped motile cell types.     

Morphological and compositional changes in a planktonic bacterial community in response to enhanced protozoan grazing

We analyzed changes in bacterioplankton morphology and composition during enhanced protozoan grazing by image analysis and fluorescent in situ hybridization with group-specific rRNA-targeted oligonucleotide probes. Enclosure experiments were conducted in a small, fishless freshwater pond which was dominated by the cladoceran Daphnia magna. The removal of metazooplankton enhanced protozoan grazing pressure and triggered a microbial succession from fast-growing small bacteria to larger grazing-resistant morphotypes. These were mainly different types of filamentous bacteria which correlated in biomass with the population development of heterotrophic nanoflagellates (HNF). Small bacterial rods and cocci, which showed increased proportion after removal of Daphnia and doubling times of 6 to 11 h, belonged nearly exclusively to the beta subdivision of the class Proteobacteria and the Cytophaga-Flavobacterium cluster. The majority of this newly produced bacterial biomass was rapidly consumed by HNF. In contrast, the proportion of bacteria belonging to the gamma and alpha subdivisions of the Proteobacteria increased throughout the experiment. The alpha subdivision consisted mainly of rods that were 3 to 6 microm in length, which probably exceeded the size range of bacteria edible by protozoa. Initially, these organisms accounted for less than 1% of total bacteria, but after 72 h they became the predominant group of the bacterial assemblage. Other types of grazing-resistant, filamentous bacteria were also found within the beta subdivision of Proteobacteria and the Cytophaga-Flavobacterium cluster. We conclude that the predation regimen is a major structuring force for the bacterial community composition in this system. Protozoan grazing resulted in shifts of the morphological as well as the taxonomic composition of the bacterial assemblage. Grazing-resistant filamentous bacteria can develop within different phylogenetic groups of bacteria, and formerly underepresented taxa might become a dominant group when protozoan predation is the major selective pressure.