Carsten Matz

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.

Impact of Violacein-Producing Bacteria on Survival and Feeding of Bacterivorous Nanoflagellates

ABSTRACT We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.

Marine Biofilm Bacteria Evade Eukaryotic Predation by Targeted Chemical Defense

Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.

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.

Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae

Bacteria and protozoa coexist in a wide range of biofilm communities of natural, technical and medical importance. Generally, this interaction is characterized by the extensive grazing activity of protozoa on bacterial prey populations. We hypothesized that the close spatial coexistence in biofilms should allow opportunistic pathogenic bacteria to utilize their eukaryote-targeting arsenal to attack and exploit protozoan host cells. Studying cocultures of the environmental pathogen Pseudomonas aeruginosa and the amoeba Acanthamoeba castellanii, we found that P. aeruginosa rapidly colonized and killed biofilm-associated amoebae by a quorum-sensing independent mechanism. Analysis of the amoeba-induced transcriptome indicated the involvement of the P. aeruginosa type III secretion system (T3SS) in this interaction. A comparison of mutants with specific defects in the T3SS demonstrated the use of the secretion apparatus and the effectors ExoU, ExoS and ExoT in the killing process, of which ExoU had the greatest impact. T3SS-mediated virulence towards A. castellanii was found to be controlled by the global regulators RpoN and RpoS and through modulation of cAMP and alginate biosynthesis. Our findings suggest that conserved virulence pathways and specifically the T3SS play a central role in bacteria–protozoa interactions in biofilms and may be instrumental for the environmental persistence and evolution of opportunistic bacterial pathogens.

High Motility Reduces Grazing Mortality of Planktonic Bacteria

ABSTRACT 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.

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

ABSTRACT 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.

Confusing selective feeding with differential digestion in bacterivorous nanoflagellates.

Abstract Food selectivity and the mechanisms of food selection were analyzed by video microscopy for three species (Spumella, Ochromonas, Cafeteria) of interception-feeding heterotrophic nanoflagellates. The fate of individual prey particles, either live bacteria and/or inert particles, was recorded during the different stages of the particle-flagellate-interaction, which included capture, ingestion, digestion, and egestion. The experiments revealed species-specific differences and new insights into the underlying mechanisms of particle selection by bacterivorous flagellates. When beads and bacteria were offered simultaneously, both particles were ingested unselectively at similar rates. However, the chrysomonads Spumella and Ochromonas egested the inert beads after a vacuole passage time of only 2–3 min, which resulted in an increasing proportion of bacteria in the food vacuoles. Vacuole passage time for starved flagellates was significantly longer compared to that of exponential-phase flagellates for Spumella and Ochromonas. The bicosoecid Cafeteria stored all ingested particles, beads as well as bacteria, in food vacuoles for more then 30 min. Therefore “selective digestion” is one main mechanism responsible for differential processing of prey particles. This selection mechanism may explain some discrepancies of former experiments using inert particles as bacterial surrogates for measuring bacterivory.

Acquired Type III Secretion System Determines Environmental Fitness of Epidemic Vibrio parahaemolyticus in the Interaction with Bacterivorous Protists

Genome analyses of marine microbial communities have revealed the widespread occurrence of genomic islands (GIs), many of which encode for protein secretion machineries described in the context of bacteria-eukaryote interactions. Yet experimental support for the specific roles of such GIs in aquatic community interactions remains scarce. Here, we test for the contribution of type III secretion systems (T3SS) to the environmental fitness of epidemic Vibrio parahaemolyticus. Comparisons of V. parahaemolyticus wild types and T3SS-defective mutants demonstrate that the T3SS encoded on genome island VPaI-7 (T3SS-2) promotes survival of V. parahaemolyticus in the interaction with diverse protist taxa. Enhanced persistence was found to be due to T3SS-2 mediated cytotoxicity and facultative parasitism of V. parahaemolyticus on coexisting protists. Growth in the presence of bacterivorous protists and the T3SS-2 genotype showed a strong correlation across environmental and clinical isolates of V. parahaemolyticus. Short-term microcosm experiments provide evidence that protistan hosts facilitate the invasion of T3SS-2 positive V. parahaemolyticus into a coastal plankton community, and that water temperature and productivity further promote enhanced survival of T3SS-2 positive V. parahaemolyticus. This study is the first to describe the fitness advantage of GI-encoded functions in a microbial food web, which may provide a mechanistic explanation for the global spread and the seasonal dynamics of V. parahaemolyticus pathotypes, including the pandemic serotype cluster O3:K6, in aquatic environments.

Phenotypic variation in Pseudomonas sp CM10 determines microcolony formation and survival under protozoan grazing

Abstract We investigated the survival mechanism of the bacterium Pseudomonas sp. CM10 in the presence of a flagellate predator. The bacterium had been isolated from a continuous culture containing bacterivorous nanoflagellates. On agar plates, we found intraclonal dimorphism of Pseudomonas sp. CM10 colonies at high frequencies: The primary mucoid colony type generated a secondary non-mucoid form. Unlike the repeated generation of non-mucoid colonies from mucoid clones, we did not observe the occurrence of mucoid forms in non-mucoid populations. In semicontinuous and batch cultures, we investigated the ability of the two morphs to survive predation by the bacterivorous flagellate Ochromonas sp. under conditions of growth and starvation. In predator-free cultures, populations of both variants were unicellular but differed in some phenotypic characteristics such as cell motility and hydrophobicity. Grazing treatments revealed that the non-mucoid morph was reduced severely whereas the primary mucoid type survived due to the formation of inert suspended microcolonies stabilized by an extracellular matrix. Effectiveness and competitive trade-offs of microcolony formation were revealed by a competition experiment with the bacterium Pseudomonas putida MM1: Pseudomonas sp. CM10 was displaced in predator-free cultures but outgrew the defenseless and monomorphic competitor under flagellate grazing pressure. We conclude that intraclonal polymorphism may regulate the ability of Pseudomonas sp. CM10 to survive in situations of severe protistan grazing. The formation of inert microcolonies, however, is suggested to be detrimental to rapid growth and dispersal.