Predation on Prokaryotes in the Water Column and Its Ecological Implications

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PREDATION ON PROKARYOTES IN THE WATER COLUMN AND ITS ECOLOGICAL IMPLICATIONS

Jakob Pernthaler Abstract | The oxic realms of freshwater and marine environments are zones of high prokaryotic mortality. Lysis by viruses and predation by ciliated and flagellated protists result in the consumption of microbial biomass at approximately the same rate as it is produced. Protist predation can favour or suppress particular bacterial species, and the successful microbial groups in the water column are those that survive this selective grazing pressure. In turn, aquatic bacteria have developed various antipredator strategies that range from simply ‘outrunning’ protists to the production of highly effective cytotoxins. This ancient predator–prey system can be regarded as an evolutionary precursor of many other interactions between prokaryotic and eukaryotic organisms.

PELAGIC HABITAT The size of a population of organisms in the environ- the rate of viral infection of aquatic bacteria seems to The parts of a lake, river and ment is determined by the balance between their specific be higher during periods of intense protistan grazing8, ocean that make up the water cell growth and mortality rates. Microorganisms are no however protists themselves can also be targets for viral column. exception to this ecological truism. Bacteria and archaea attack11,12. 1 OLIGOTROPHIC in PELAGIC habitats can grow in OLIGOTROPHIC conditions Theoretical models and empirical surveys indicate An aquatic environment that has and can survive extended periods of starvation2. It is that protistan predation might often be most influential low levels of nutrient and algal therefore unlikely that they die predominantly from a in limiting the total abundance and biomass of the BAC photosynthetic production (for lack of resources. The main sources of microbial mortal- TERIOPLANKTON, whereas viruses are considered to more example, high mountain lakes). ity in the water column are currently considered to be profoundly affect prokaryotic community diversity3,5,13. PHAGOTROPHY viral-mediated lysis and grazing by PHAGOTROPHIC pro- The unequal effects of these two mortality sources on The uptake of particles by tists3. Viruses and protists have been deemed the most microbial assemblages are probably due to the OMNIVORY eukaryotic cells. important cause of microbial death by both environ- of HETEROTROPHIC protists on the one hand (they can mental virologists and protozoologists, respectively 4,5, feed on a range of different prokaryotic species) and BACTERIOPLANKTON Bacteria that inhabit the water sometimes in conspicuous agreement with their respec- the typically narrow host range of viruses on the other. column of lakes and oceans, tive research focuses. It is also possible that autolysis (for Moreover, viral-associated mortality is highly density either freely suspended or example, owing to ultraviolet (UV) radiation damage)6 is dependent — viruses will primarily affect the largest, attached to particles. a source of mortality in some habitats and, furthermore, most rapidly growing bacterial populations in mixed Max-Planck-Institute for genes that are involved in programmed cell death are assemblages (known as the ‘killing the winners’ phe- Marine Microbiology, present in numerous prokaryotic species7. nomenon)3. By contrast, protistan predation can act Celsiusstrasse 1, The interplay between viruses and protists in the on less abundant bacteria in mixed assemblages. This D-28359 Bremen, Germany. control of aquatic prokaryotes is still poorly under- is owing to selective foraging — although many protists e-mail: [email protected] stood. There are indications that virus-induced mor- are omnivorous, some important groups, particularly doi:10.1038/nrmicro1180 tality is more substantial when bacterial productivity flagellates, might nevertheless show a strong preference Published online June 10 2005 is enhanced8 or in anoxic conditions9,10. Sometimes, for particular prey species14–16 or morphotypes17,18.

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Phylogenetic affiliations of predators and prey Most organic carbon is produced and consumed in Predators. The identification of the BACTERIVOROUS these microbial food webs, which themselves are NANOFLAGELLATES that form large populations in situ is embedded in the ‘classic’ grazing food chain (algae– often difficult, owing to the paucity of distinct morpho- zooplankton–fish). logical features, the destruction of their delicate forms The ‘microbial loop’ concept40 refers to the by fixation procedures and the selectivity of culturing production of dissolved organic material (DOM) approaches19,20. Accordingly, only a few ecological stud- in aquatic food webs during the flux of particulate ies have reported the population dynamics of individual matter towards larger organisms, and the reincor- taxonomic groups and their relative contributions to poration of this DOM by heterotrophic bacteria and total bacterivory 20–22. archaea. Bacterivorous nanoflagellates and, to a lesser Heterokont taxa (that is, those with unequal extent, small ciliates ‘close’ this loop by forming a flagellae, mainly chrysomonads and bicosoecids) con- bridge between the DOM-consuming microorgan- stitute 20%–50% of heterotrophic nanoflagellate (HNF) isms of the picoplankton, which have cell lengths biomass in both marine and freshwater pelagic com- <1–2 mm, and those planktonic organisms (such as munities, followed by choanoflagellates (5–40%) and crustacean zooplankton, rotifers, larger ciliates and kathablepharids (10–>25%)23. Members of the genus dinoflagellates) that can only feed on cells >3–5 mm Spumella seem to be particularly abundant in lakes21. in diameter41. Protists, moreover, can induce ‘short- Numerous phylogenetic lineages of novel unicellular circuits’ in the microbial loop22 by either releasing eukaryotes have been described recently in the oceans DOM42 in a process that is graphically described as 43 OMNIVORY by comparative sequence analysis of 18S ribosomal ‘sloppy feeding’ , by competing for DOM with bac- Ability of animals to feed on RNA genes24. At least two of these lineages comprise teria44 and by directly feeding on DETRITAL PARTICLES45 different types of prey. small (2–3 mm) phagotrophic organisms that can or viruses46. Furthermore, protists also feed directly seasonally form a substantial fraction of all HNFs in on bacterial PRIMARY PRODUCERS from the AUTOTROPHIC HETEROTROPHY 25 22,47,48 The acquisition of metabolic coastal waters . The important ciliated bacterivores picoplankton , that is, on free-living unicellular energy by consumption of are small oligotrichs, peritrichs and scuticociliates in cyanobacteria that are mainly affiliated to the genera particulate or dissolved organic freshwaters26, and aloricate (naked) oligotrichous forms Synechococcus and Prochlorococcus, and on small matter. in marine environments27. eukaryotic algae49. In the EUPHOTIC ZONE of the world’s

BACTERIVOROUS oceans, at an ecosystem level, this protistan HERBIVORY NANOFLAGELLATES Prey. Over the past 15 years, cultivation-independent might be even more important than the consumption Small, flagellated protists that molecular approaches have provided a first insight of heterotrophic bacteria50. range in size from 3 to 15 mm into the taxonomic composition of the heterotrophic Protistan grazing on bacteria is also an important and that can feed on bacteria. prokaryotic assemblages in the water column of lakes mechanism of NUTRIENT REGENERATION, in particular, of 28,29 PICOPLANKTON and oceans . Members of the SAR11 clade of the nitrogen and phosphorus. These two elements (as well Organisms suspended in the α-proteobacteria are probably among the most com- as iron and silicium) limit the growth of prokaryotic water column that are less than mon prokaryotes in marine plankton30. These bacte- and eukaryotic autotrophs in many aquatic systems51. 2 mm in size. ria form >50% of the total number of bacteria in the Aquatic bacteria are better competitors for phos-

DETRITAL PARTICLES surface waters of the Sargasso Sea. A large fraction of phorus than eukaryotic algae at low ambient nutrient 52 Dead organic material prokaryotes in the deeper waters of the open oceans concentrations . Therefore, bacteria can incorporate suspended in the water column. are related to Crenarchaeota of ‘marine group I’31. The the nutrients that are required for the proliferation α-proteobacteria that are affiliated to Roseobacter of primary producers in their biomass. However, PRIMARY PRODUCERS Organisms that are the original species are another common component of coastal bacteria also depend on the release of photosyntheti- 32 source of organic material in an and offshore microbial assemblages , and they season- cally fixed organic carbon that is overproduced dur- ecosystem — plants, algae or ally constitute 10–25% of marine PICOPLANKTON33. The ing phytoplankton growth40. Consequently, the high chemosynthetic microorganisms. γ-proteobacteria that are related to the NOR5 and SAR86 bacterial affinity for nutrients has a negative effect on lineages make up another 5–10% of bacteria in coastal their main source of organic carbon. This ‘lose–lose’ AUTOTROPHY 33 The acquisition of metabolic waters . Such habitats, moreover, host numerous bac- aspect of bacterial and algal coexistence is counter- energy from the fixation of teria that are affiliated to the Cytophaga-Flavobacterium- balanced by the activity of the bacterivorous protists. inorganic carbon, for example, Flexibacter lineage of the Bacteroidetes34. Other clades Compared with eukaryotes, prokaryotes have a higher by photo- or chemosynthesis. from this large phylogenetic group are typically encoun- nitrogen and phosphorus concentration per volume tered in freshwaters29,35. Several lineages of β-proteo- of biomass, owing to their higher ratio of proteins and EUPHOTIC ZONE 29,36,37 38 53 Upper realms of the oceans that bacteria and Gram-positive Actinobacteria are nucleic acids to total cell mass . Protists that graze are penetrated by sufficient common in the water column of lakes and rivers29,36,37. on picoplankton cells release into the environment amounts of light for the growth By contrast, α- and γ-proteobacteria are usually rare. excess nutrients that are not required for growth, for of photosynthetic organisms. Both marine and freshwater systems also contain many example, ammonium54 or dissolved amino acids55. 39 HERBIVORY small unicellular cyanobacteria . This nutrient recycling can stimulate the proliferation 56 57 The consumption of plants. of both primary producers and other bacteria . Bacterivores in aquatic microbial food webs The abundance of HNFs in different pelagic NUTRIENT REGENERATION The microbial food webs in the water column of lakes, habitats varies greatly, but typically ranges from 100 Processes by which nutrients –1 that are bound in organismic rivers and oceans are composed of viruses, chemo- to 10,000 cells ml in the plankton of lakes, rivers 23 biomass are retransformed into organotrophic prokaryotes and cyanobacteria, as well and marine surface waters . It has been claimed that their inorganic form. as autotrophic and heterotrophic unicellular eukaryotes. the typical ratio of HNFs to heterotrophic bacteria is

538 | JULY 2005 | VOLUME 3 www.nature.com/reviews/micro estuaries. lowland lakes and coastal Examples include shallow sources. other or production matter from photosynthetic organic dissolved of availability Aquatic systems with high mortality owing to starvation. by alack of resources and organisms ismainly determined abundancethe or biomass of inwhich scenario Ecological predation. by mortality owing to organisms ismainly determined abundancethe or biomass of inwhich scenario Ecological NATURE REVIEWS EUTROPHIC CONTROL BOTTOMUP TOPDOWN CONTROL |

MICROBIOLOGY plankton plankton resources. waterin the column should mainly be limited by growth rate of at least th top-down control of prokaryotic the assemblages framework inthe web: of understood food the be This might sound counter-intuitive at but first, must competition for nutrients inmore productive waters. trophic systems, whereas growth their islimited by controlled by protistan predation oligo- inhighly models microbial food webs system productivity inmarineand both freshwater carbon and nutrients might related be to overall eco- tems than inmore productive regions of oceans the in nutrient-poor, higher tend tobe oligotrophic sys- that abundance the ratios of HNFsto prokaryotes organic matter sediments tothe and rapid the of produced, export newly particulate bon extremely low ambient levels of labile organic car- of HNFsand prey. bacterial their By contrast, the a consequence of similar the ranges of growth rates and colleagues 45 years ago years 45 colleagues and macroscopic (terrestrial) world asoutlined by Hairston Is world the of aquatic microorganisms amirror of the ‘Top-down’ or‘bottom-up’control? approximately 1:1,000 (FIG. 1) been disputed or or chlorophyll in numbers ismuch lessthan, for example, that of planktonic organisms, and annual the fluctuation grazing mortality compared with the biomass of other prokaryotes more to seems be tightly controlled by aquatic heterotrophic of biomass the level, munity from that those limit prokaryotic growth. At acom- late standing bacterial stocks must distinguished be which hypothesis iscorrect, and factors the that regu- plankton. freshwater and concentrations of prokaryotic cellsfound inmarine mortality, and protists at typical might the starved be microbial standing stocks might controlled be by grazers should limited be by predation and nutrients, and biomass the of protistan their column should limited be by competition for carbon webs, biomass the food of prokaryotes water inthe predators. If concept this applies to aquatic microbial whereas abundance the of herbivores iscontrolled by of resources (for example, light), not by animal grazing, plantbecause biomass iscontrolled by availability the paper, the authors argue that ‘‘the world is green’’ waters ciliated protists productive highest to invery seems be is unknown. Therelative importance of grazing by heterotrophic flagellates (>15mm)to total bacterivory ally by afactor of 100.Thecontribution of larger ‘BOTTOMUP’ CONTROL There are indications that issueof the pico- Currently, there seems to be no consensus about

66 or growth-limiting nutrients inaquatic systems, . Other empirical. Other observations 26,60,61 69 also predict that bacteria are more tightly ‘TOPDOWN’ CONTROL . a or microzooplankton or 59 . HNFbiomass can change season- 63,68 by the availability of organic organic of availability the by REF. 58 . Comparative analyses show e freely-suspended bacteria bacteria e freely-suspended 62 ? In this classic ecology ecology ? In classic this by protistan grazing , but estimate has this 67 , indicate that the 64,65 5 and theoretical theoretical and 63 . This might be . Thismight be . Alternatively, 65

b rates are unrelated, predator control isassumed. By contrast,ifbacterialabundanceandspecificgrowth bacterial-specific growth ratesandabundanceispredicted. (resource limitation),anegativecorrelation between approaches thecarryingcapacityofenvironment By contrast, more the nutrient-rich controlled. bottom-up is it prey, is, of that availability protistan predators themselves islimited by low the in nutrient-poor habitats implies that growth the of proposed byWright andCoffin conditions. a importance ofpredation inoligotrophic andeutrophic Figure 1| might be a featuremight be only of marine waters. In freshwater between system productivity and community control control. predation thereby releasing prokaryote the community from control abundance the of bacterivorous the HNFs, — larger protists, zooplankton — that and larvae fish can sustain aricher community of top predators from HNFs andtheirbacterialprey. Adaptedwithpermission reflectswhich, inturn, thedegree ofuncouplingbetween theoretical maximumandthemeasured concentration, lines reflects thedegree ofuncouplingbetweenthe different environments. Thedistancebetweenthesetwo actual observedabundanceofHNFs(red dashedline)in maximum abundanceofHNFs(blacksolidline)andthe in oligotrophic systems.Thegraphshowsthetheoretical resource-limited ineutrophic systemsandpredation-limited prey decreases. Bacteriaare therefore regarded as rates), andtherefore thecouplingbetweenpredator and derived from energeticcalculationsandmeasured grazing habitat increases (compared with theoretical predictions steeply thanpredator abundanceasthetrophic stateofthe observation thatbacterialabundancetendstorisemore b a

|Theempiricalmodelproposed byGasol Log (HNF cell numbers) Bacterial cell numbers However, such an unambiguous relationship However, unambiguous an such lgtohcEutrophic Oligotrophic REF. 65 Two modelsthataddress therelative ©(2002)Springer. Carrying capacity |Thedensity-dependentlogisticmodel maximum Theoretical by predation Regulation Numbers observed Time Log (bacterialcellnumbers) by resources Regulation VOLUME 3 147 . Ifbacterialabundance EUTROPHIC REVIEWS uncoupling Degree of Log (specific Log (specific | JULY 2005 68

reflects the growth rate) growth rate) Cell numbers Cell numbers systems systems |

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systems, the pattern seems to be substantially modified process, the food vacuole is transported towards the by other parameters, which include the influx of posterior end of the protist and undigested remains organic material from rivers or terrestrial sources70 are subsequently expelled by exocytosis83. and cascading effects from higher levels of the food Owing to the specific mechanisms of particle web (that is, the feeding modes of the dominant uptake and handling, protistan predators cannot zooplankton species)71. feed on all sizes of bacteria with equal efficiency. Furthermore, hydrodynamic considerations also Protists attached to particles indicate that the rates of encounter between flagellates Productive and unproductive habitats in aquatic and bacteria are minimal at bacterial cell diameters of environments are not separated by large geographi- ~0.5 mm84. Consequently, microbial cells that range cal distances. A significant proportion of microbial in length from 1 to 3 mm are preferentially consumed activity in the pelagic zone occurs on, or near, parti- by many planktonic HNFs85,86 and bacterivorous cles such as senescent algae or macroscopic organic ciliates18, whereas smaller or larger cells benefit from aggregates >0.3–0.5 mm in diameter that are formed reduced loss rates87,88. by the flocculation of phytoplankton and detritus The processing of microorganisms that have (known as marine snow72 or lake snow73). Both the been engulfed in food vacuoles depends not only on CHEMOSPHERE and particle surfaces are preferential temperature89 but also on the bacterial species. Such sites of colonization by mobile microbial species differential digestion is an important mechanism that that are guided by CHEMOTAXIS74. However, suspended is responsible for the apparently selective grazing organic aggregates offer no refuge from protistan pressure of HNFs on mixed bacterial assemblages. For predation75. HNFs are also found at elevated con- example, it seems to be much more time-consuming centrations on and around such aggregates, where to digest a Gram-positive than a Gram-negative bacte- they form communities that can differ from those rial cell90. As a consequence of the various aspects of in the ambient water76. The colonization of marine feeding, the growth efficiencies of protists can depend snow particles by HNFs occurs in hours to days of strongly on the available bacterial prey species and its aggregate formation77, and contact is favoured by nutritional value14. both random mobility and chemotaxis78. Attached However, finicality might not always be an optimal protists might control the abundance of attached foraging strategy for a suspension-feeding omnivore. bacteria and, hence, influence the rate of aggregate Active selection might only be advantageous at high degradation and the export of organic matter from particle concentration, that is, if the formation of the water column to the sediments. new food vacuoles is the rate-limiting step in feed- The attachment of HNFs or ciliates to suspended ing. At low bacterial concentrations, the selectivity of particles or large algae79,80 can also offer other advan- interception-feeding flagellates markedly decreases, tages. For hydrodynamic reasons, protists are more as it is energetically more effective to feed on all efficient in collecting suspended bacteria if they are particles that can be morphologically ingested and attached to a surface81,82. Larger particles might more- then to attempt to digest them91. This is the case, for over represent a refuge from zooplankton predation. example, in oligotrophic waters: assuming a random Considering the spatial heterogeneity of bacterial distribution, 1,000,000 bacteria ml–1 occupy only ~0.1 productivity, alternation between the attached and millionth part of this volume. Accordingly, protists free-living lifestyle in response to prey availability 82 need to collect prey with astonishing efficiency, and seems to be highly advantageous for bacterivorous an individual protist can clear the particles from at protists in the water column of aquatic systems. Such least 105 times its own body volume of water every CHEMOSPHERE a dual lifestyle is indeed adopted by many pelagic hour92,93. Moreover, as for many animals, uptake selec- Zone of elevated concentration HNFs. tivity can depend on the nutritional state of the grazer: of organic molecules that whereas a population of satiated flagellates readily diffuse from the surface of a suspended particle. Feeding behaviour discriminated between bacteria and similarly sized To understand how aquatic bacterivorous protists latex beads, a starved population of the same species CHEMOTAXIS shape the composition of microbial assemblages, it is did not94. Protists might encounter such a situation Ability of microorganisms to crucial to study their feeding strategies. FILTER FEEDING in aquatic habitats that are rich in inedible mineral follow a chemical gradient. is particularly advantageous for attached protists81, particles95, for example, from terrestrial influx.

FILTER FEEDING but this strategy is also adopted by free-living marine Diverging reaction norms between clones seems to Feeding mode that filters and freshwater flagellates (for example, choanoflagel- be an inherited trait of protistan populations. There can particles from the water by lates) and small ciliates26. Bacterivory by INTERCEPTION be significant interspecific and intraspecific variability means of a sieving structure. FEEDING is a multi-step process that is as complex as in grazing rates and feeding-related parameters such as Usually the prey is very small 96 compared with the predator. the feeding habits of multicellular predators. First, the growth efficiency . Moreover, protistan feeding can be prey is drawn to the flagellate by a feeding current that highly variable over the entire cell cycle81,82. For exam- INTERCEPTION FEEDING is induced by the beat of one flagellum. It is then cap- ple, individual flagellates can cease to ingest bacteria The capture of individual tured between the flagella and brought into contact during cell division. Therefore, it is debatable whether bacteria or particles by direct random contact with a protistan with a sensitive area of the cell surface. A phagocytotic the levels of protistan bacterivory found in short-term cell. Usually the sizes of the vesicle is formed at this morphologically inconspicu- measurements can be extrapolated to periods of days predator and prey are similar. ous spot and the prey is ingested. During the digestive or more.

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Cell wall structure, During the past decade, an increasing number of surface potential Size reduction bacteria have been isolated from phylogenetic lineages that also occur in high densities in the water column of lakes and oceans33,106,107. Many of these isolates are so-called ULTRAMICROBACTERIA. Although these bacteria might not be entirely protected from flagellate predation, they are nevertheless close to the lower size limits Motility patterns of ingestibility87. Therefore, the loss rates of ultramicrobacteria are substantially reduced compared with other bacterial isolates. Currently, there are no con- vincing alternative suggestions why such a small cell Morphology, filamentation size should be advantageous for microbial growth and survival in aquatic habitats. Some bacterial strains show considerable morphological plasticity at different growth conditions. For example, some species can form large thread-like morphotypes at enhanced growth rates103. Filament formation in subpopulations can also occur at sub- 108 Toxin release optimal growth conditions or during starvation . Exopolymer formation Some bacterial species in freshwater habitats are Communication of a permanently filamentous morphotype35,109. Figure 2 | Phenotypic properties of aquatic bacteria that might provide protection from Filamentous bacteria either exceed the size range of predation by heterotrophic protists. The different strategies are discussed in the text. cells that most bacterivorous protists can feed on, or filaments are grazed at substantially lower rates than smaller bacteria88,110,111. Filament formation is Mixotrophy therefore regarded as an efficient means of protection Bacterivorous PHOTOTROPHS occur in various freshwater from protistan predation (but thread-like morpho- and marine habitats97. The term ‘MIXOTROPHY’ refers to types might nevertheless be vulnerable to grazing a broad array of ecological strategies that range from by larger zooplankton71). It is conceivable that such a predominantly phototrophic to an almost exclu- morphological shifts could be triggered by external sively heterotrophic lifestyle. The metabolic costs of chemical cues that might even be released by the forming and maintaining a phagotrophic apparatus predator itself. in addition to the photosynthetic machinery seem to Aquatic bacteria produce a wide range of extra- be rather low (<10% of total metabolic expenditure) cellular polymeric substances (EPS) that comprise and are easily matched by the potential benefits98. polysaccharides, proteins, nucleic acids, lipids and Mixotrophy is an efficient means of acquiring limit- other biological macromolecules112. EPS secretion ing nutrients in oligotrophic pelagic environments99, significantly enhanced bacterial survival on expo- and might also provide a further source of organic sure to intense HNF grazing in experiments with carbon at light-limiting growth conditions100. If the EPS-deficient mutants113. EPS-producing planktonic higher affinity of prokaryotes for some inorganic bacteria typically develop subpopulations of single PHOTOTROPHS Organisms that fix inorganic nutrients is considered, phagotrophy in phototrophic cells and microcolonies that are embedded in an 114 carbon using light energy. eukaryotes is understood conceptually as a strategy EPS matrix , and the larger microcolonies are proof ‘eating your competitor’101. In some systems such tected from flagellate predation owing to their size. MIXOTROPHS as nutrient-poor, high mountain lakes, mixotrophic The shift to the colonial cell type may be a passive Organisms that are part nanoflagellates can even be the dominant primary consequence of selective feeding on single cells114, autotrophic and part heterotrophic, for example, producer species. Such mixotrophs severely affect but microcolony formation can also be specifically carnivorous plants. the productivity of the prokaryotic assemblages, and induced in the presence of predators by cell–cell they create a scenario for bacteria of commensalism communication QUORUM SENSING113. ULTRAMICROBACTERIA and predation that has been poetically described as Protection from protistan grazing might more- Bacteria that maintain cell 102 volumes of <0.1 mm3 even ‘neither with nor without you’ . over be mediated by properties of the bacterial cell during exponential growth on wall. Gram-positive enteric bacteria are consumed at rich media. Microbial antipredator strategies significantly lower rates by protists than are Gram- Several phenotypic features of aquatic bacteria have negative strains115. The consumption of naturally QUORUM SENSING been interpreted as adaptations to escape protistan occurring Gram-positive freshwater Actinobacteria is Bacterial communication system based on the secretion grazing pressure (FIG. 2). Some of these features, such selectively avoided by HNFs, as deduced from analysis 116 and detection of a quorum, as exopolymer secretion and filament formation, could of protistan food vacuoles . An ultramicrobacterial which is a substance that be the consequence of physiological conditions that are Gram-positive isolate from a lake was found to be com- increases with population unrelated to predation, such as growth rate103. Others, pletely protected from grazing107. Bacterial cell surface density and that induces 104 87 expression of specific genes in such as high-speed motility , cell miniaturization and charge and hydrophobicity have also been suggested 105 117,118 the population above a toxin production , seem to have specifically evolved in as parameters that might influence grazing losses . threshold concentration. response to grazing mortality. This has been explained on physicochemical grounds

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Some bacteria can even poison their predators in a Edible size range concerted manner: Chromobacterium violaceum spe- A cifically induced the eukaryotic APOPTOTIC RESPONSE after being ingested by a flagellate105. The constitutive production of the toxic agent, violacein, was only observed in strains that were capable of quorum sensing. Toxin B secretion related to quorum-sensing abilities has also C been reported for mature biofilms of Pseudomonas aeruginosa113. D E Community-level effects of predation Cell size The current conceptual model of the impact of predation on the morphological structure of bacterioplank- Protistan grazing ton implies that flagellate grazing eliminates mainly medium-sized cells and shifts the size-structure of bacterial assemblages towards smaller and/or larger 121 D cells (FIG. 3). Blooms of protist-inedible filamen- E tous or microcolony-forming bacteria are typically encountered in the spring communities of productive B C lakes35,122,123 and in acidified lakes with high levels of grazing by mixotrophic flagellates124. Similar pheno- Biomass Biomass typic changes in the bacterioplankton can be induced experimentally by food-web manipulation125. The

A growth of resistant bacteria sometimes completely compensates for the overall losses in bacterial biomass Cell size owing to grazing mortality126,127. Figure 3 | The effect of predation on microbial The small average cell size of aquatic bacteria is community structure. The effects on biomass size usually thought to be a result of starvation2 or protection distributions of individual populations (depicted in different against size-selective protistan feeding. This might not colours) are shown. Bacterial species that are in the size necessarily be a contradiction. One of the less obvious range that can be readily ingested by protists (A, B and C) are consequences of HNF grazing is its negative influence disproportionally reduced by grazing (shown in the upper panel), unless they can change their morphotype (B, C) as on the activity of bacterial assemblages. Starvation of shown in the bottom panel. Populations outside the edible aquatic bacteria often results in a marked decrease in cell size range (D, E) will be favoured by grazing. Combined from size, and non-growing bacteria from many culturable concepts by several authors127,134,136. genera tend to be smaller than growing cells2. HNFs feed on these small starving cells at lower rates85,120. They seem to control the overall productivity of bacterioplankton as the effect of molecular interaction forces on the by specifically feeding on the larger, most rapidly grow- probabilities of contact between predator and prey118. ing64 or dividing128 cells in the assemblage (for example, Hydrophobicity is viewed as important in regulating cells with high nucleic acid content129) rather than by vulnerability to phagocytosis by mammalian leuko- unselectively cropping bacterial standing stocks. cytes119, but at present, it is unclear if such surface prop- The formation of grazing-resistant filamentous erties have a role as grazing protection mechanisms morphotypes in some freshwater bacteria has been for aquatic bacteria. A more hydrophobic strain of the described as a side effect of enhanced growth rates103. marine cyanobacterium Prochlorococcus showed sig- Therefore, the response of bacterioplankton assem- nificantly higher vulnerability to predation than a simi- blages to HNF predation might also be related to the lar-sized, but more hydrophilic, strain118. By contrast, supply or qualitative composition of dissolved organic the feeding rates of three freshwater flagellate species substrates130. There are even indications that different on various strains of heterotrophic bacteria were not growth-limiting factors might favour the development significantly dependent on either prey hydrophobicity of specific types of grazing-resistant bacteria in mixed or cell-surface potential117. assemblages. When freshwater bacteria in phospho- Bacterial motility seems to be a two-sided coin. rus-limited, continuous culture were exposed to HNF On the one hand, the probability of random contacts grazing, large filamentous cells were dominant. If the between predators and prey increases with bacterial same experimental assemblages were limited by organic swimming, and motile bacteria can therefore be con- carbon, small, highly motile bacterial morphotypes sumed at higher rates by HNFs120. On the other hand, prevailed that could escape from predation131. Such the capture efficiency of rapidly swimming bacteria observations invite speculation as to whether the rare- APOPTOTIC RESPONSE (>25 mm s–1) by flagellates is decreased compared ness of filamentous bacteria in marine picoplankton Phenotypic changes that occur during programmed cell death with less rapid swimmers and some bacteria may reach — compared with freshwater picoplankton — might in eukaryotic cells, for example, an ‘escape velocity’ that is high enough to effectively be related to a different interplay of resource limitation cell shrinkage. protect them from flagellate grazing104. and predation in the two types of habitat.

542 | JULY 2005 | VOLUME 3 www.nature.com/reviews/micro extended periods of starvation. extended periods optimal and that can survive proliferate ifconditions are microorganisms that rapidly of strategy Growth ofspecies acommunity. number of individuals ineach Balance of the respective present inacommunity. Number that of are species NATURE REVIEWS FEAST OR FAMINE EVENNESS COMMUNITY SPECIES RICHNESS |

MICROBIOLOGY both inlakesboth and ocean inthe groups densities of inhigh ultramicrobacteria occur natural aquatic microbial assemblages: phylogenetic a direct imprint on taxonomic the composition of might related be to size-selective protistan grazing into species bacterial strains with different cell sizes cate that microdiversification the of freshwater asingle indirectly indi- experiments nevertheless Laboratory composition webs can induce profound incommunity shifts lation of predator densities innatural microbial food assemblagesto bacterial experimental or manipu- the grazers protistan of addition The predation. protistan assemblagesbacterial can be indicate that community the of structure pelagic wordfinal on subject. the there are legitimate doubts isthe this asto whether tic assumption of totally unselective protistan feeding, However, analyses asthese were on unrealis- the based to directly investigate predation whether the affects Alteromonas species incoastalmarinewaters.Rare bacteria,suchas predation cancounterbalancethegrowth ofinvasivebacterial column. Datacompiledfrom grazing probably accountsfortheirrarityinthebacterialwater flagellate population.Thevulnerabilityofsuchbacteriato disproportionately reduced during the regrowth ofthe larger thanotherplanktonicbacteriaandare therefore in dilutioncultures). Thesespeciestypicallyform cellsthatare assemblages atartificiallyreduced grazingpressure (suchas assemblages freshwater many in bacterioplankton the of fraction protected from grazing, constitute aprominent positive Actinobacteria, which are allegedly better for bacterioplankton community composition have deemedprotistan predation of minor importance theoretical of models microbial interactions food-web of rabbits from plots of chalk grassland dominance of rare plant after species fencing-out the Tansley and Adamson invasion the described and discoveries of in1925, early the twentieth century: community isone of fundamental the ecological The impact of predators on diversity the of prey the Do protists affect diversity? Figure 4| SPECIES RICHNESS Alteromonas spp. Experimental investigationsExperimental and studies field both For practical reasons, it iscurrently not possible

(% of all bacteria) 10 15 0 5 0 ‘Killing thewinner’bypredation. Alteromonas spp., rapidlyovergrow theoriginalmicrobial 38 37,127,133 . 1 innatural microbial assemblages. . Moreover, bacterivores the leave Day ofexperiment spp. 2 REF. 16 flagellates Heterotrophic shaped substantially by 3 . 30,36 , and Gram- the 4 132 Protistan . By contrast, 5 2 3 0 1 4 5

3,69 Heterotrophic flagellates 87

. . (x103 cells ml–1) Vibrio protist-inedible species bacterial typic successions microbial inthe assemblages towards a bloom of HNFs,followed by taxonomic and pheno- top-predator from aeutrophic pond rapidly induced of total bacterioplankton biomass inalake mentous bacterial species transiently constituted >40% ofDuring intense periods HNFgrazing, asingle fila- bacteria. freshwater of assemblages experimental in is empirical for evidence asignificant relationship to bottom-up control, or viceversa. Although there changes are induced by from suddenshifts top-down community rapid that and mortality, or productivity encountered different at very levels of total microbial be can composition taxonomic stable apparently an can cause substantial changes inbacterioplankton There ismore that predation evidence direct mortality occur when grazing when pressureoccur isrelieved of genus the photypes) are suppressed by filter-feeding zooplankton protists (aswell asalgaeand filamentous mor- bacterial during spring, the ‘clear-water’ phase, bacterivorous ate lakes, isanatural this and recurrent phenomenon: sity probably show a high degreethat different bacterivorous co-occurring species of functional diver- metabolically active bacteria that pursue a‘ large, of blooms planktonic suppress can plankton Sea β example, with similarspecies morphological characteristics, for trol free-living the subpopulations of related pathogenic sible that protistan selective predation might con- also (FIG. 5) tor (or species both) were to grazing by exposed aflagellate or aciliate preda- shown microbial inexperimental assemblages that diversitythe assemblages. of bacterial Thishasbeen composition of HNFcommunities might influence be partly predator specific, and that the taxonomic terial adaptations that protect against grazing might predator species some against effective only are that strategies follow single bacterial species as individual microorganisms HNF community might not allow dominance the of a unicellular cyanobacteria abundance of groups, some bacterial for example, of overall bacterial mortality rate to growth rate sition might deduced be from changes ratios inthe magnitude community of inbacterial shifts compo- poorly understood. It has been suggested that the structure of aquatic microbial assemblages are still factors inshaping and stabilizing community the species tinct blooms of an ultramicrobacterial Gram-positive COMMUNITY EVENNESS COMMUNITY FAMINE -proteobacteria Shifts in bacterial phylogenetic inbacterial Shifts composition can also The complex feeding strategies of protists indicate Protistan grazing can selectively reduce relative the The interactions of top-down and bottom-up 138 . There are indications that of benefits the bac- and and . Thisconcept predicts that communities with ’ growth strategy (for example, 133 Vibrio cholerae and aflock-forming Pseudoalteromonas Daphnia 139 37 . Some HNF species in coastal North incoastal HNFspecies . Some . . High HNFpredation induced dis- 133 71 . It argued hasbeen that adiverse . Experimental removal. Experimental of this 137 . VOLUME 3 48 or specific freshwater or specific species species Pseudomonas 136 . (FIG. 4) REVIEWS | JULY 2005 135 Alteromonas . In temper- 35 16 . . It ispos- strain FEAST OR |

543 140 134 ,

544 . Schut,F., Prins,R.A.&Gottschal,J.C.Oligotrophy and 1. probes. targeted hybridization with rRNA- fluorescence sequencing of rRNA genes and environmental samples by identification of protists in Cultivation-independent surrogate particles. feeding rates through uptake of conditions,field for example, of physiological properties under Determination of protistan REVIEWS . Thingstad,T. F. Elementsofatheoryforthemechanisms 3. Morita,R.Y. in 2. APPROACHES MOLECULAR BIOLOGICAL APPROACHES ECOPHYSIOLOGICAL viruses. bacterial production between protistan grazers and First systematicattempttomodelthepartitioning of Oceanogr. of lyticbacterialvirusesinaquaticsystems. controlling abundance,diversity, andbiogeochemicalrole York, 1997). Lifestyle Starvation-Survival Ecol. pelagic marinebacteria:factsandfiction. | JULY 2005 12 , 177–202(1997). in situ in 45 , 1320–1328(2000). Bacteria inOligotrophicEnvironments:

| VOLUME 3 , 529(ChapmanHall,New without prior cultivation ological activities of prokaryotic defined taxa have available become to physi- determine the biogeochemical processes. For example, members can magnitude the influence and stability of specific predation prokaryoticselective species on particular approaches will eventually allow us to assess whether lation or field inthe inmixed cultures nomic community manipu- after experimental shifts between growth-to-mortality-rate ratios and taxo- (‘competition specialists’). rapidly tofavourablechangesingrowth conditions predation losses(‘defencespecialists’)ortorespond most species are favoured thatare ableeithertominimize control. Dependingonthedirection ofsuchshifts,bacterial triggered byrapidshiftsfrom ‘top-down’to‘bottom-up’ species compositionofthemicrobial assemblageare bacterial production andprotistan bacterivory. Changesin productivity ifthere isanapproximate balancebetween microbial specieswillexistatdifferent levelsofmicrobial by Simek community-level growth andloss. changes inmicrobial speciescomposition to Figure 5| by acombination of ing behaviour of aquatic protists addressed can be bial ecology. Thecommunity composition and feed- by rapid the advance of field micro- of inthe methods structure of aquatic prokaryotes iscurrently favoured natural microbial assemblages. investigatedto be in verified can be this also whether BIOLOGICAL Grazing pressure The study of predator on community the effects Aquat. Microb. Limnol. (top-down control) Defence specialists A simpleconceptualmodelthatrelates et. al. approaches 140 , predicts thatstablecommunitiesof . Strom, S.L.in 5. . Bidle, K.D.&Falkowski,P. G.Cell deathinplanktonic, 7. Sommaruga,R.,Obernosterer, G.J.&Psenner, I.,Herndl, R. 6. Fuhrman,J.A.&Noble,R.T. andprotists Viruses cause 4. Bacterial productivity ECOPHYSIOLOGICAL 643–655 (2004). photosynthetic microorganisms. Appl. Environ.Microbiol. incorporation byfreshwater andmarine bacterioplankton. Inhibitory effect ofsolarradiationonthymidineandleucine 2000). &Son,NewYork,Kirchman, D.L.)351–386(J.Wiley Oceanogr. similar bacterialmortalityincoastalseawaters. 25,116,141 Stable community 142 . Acombination of these . Moreover, techniques (bottom-up control) Competition specialists 40 , 1236–1242(1995). Microbial EcologyoftheOceans This model,proposed and and

63 140 , 4178–4184(1997). , it remains MOLECULAR Nature Rev. Microbiol. in situ in

Limnol. (ed. photosynthesis. evolution asprofoundly as,for example, oxygenic protists by microbial eukaryotic hasshaped terivory summary, it isconceivable that invention the of bac- mechanisms that promote pathogenicity. bacterial In against protistan grazing to elaborate, the specific tations that free-living bacteria use for protection require only asmallstep to progress from adap- the of the the of protists or leukocytes unicellular cells,which can aquatic eukaryotic be nisms and toxins effective by against phagocytosis have developed various pre-ingestionmecha- defence sent ahabitat, patch, afood or Microorganisms both. a eukaryote athreat, might pose but might repre- also evolutionary ‘arms race’. From perspective, abacterial environment. than for direct the secretion of into exoenzymes the engulfed prokaryotic predators inside cells eukaryotic economical for controlled and flexible processing of digestive These processesesses. are probably more lutionary development digestive proc- of specific organic matter incellvacuoles allows for evo- the The uptake and subsequent trapping of particulate removal by protistan predation. aconsequence could be ofpurposes selective their microbialcally modified) strains for bioremediation (for release ofunsuccessful specific example, geneti- It is,moreover, conceivable that conspicuously the closely relatedclosely topathogens endosymbionts of amoebae are phylogenetically related lifestyles, for example, typical the bacterial sis between the prokaryotic the between and worlds eukaryotic Predation could one be of oldest the interactions Evolution ofmicrobial predator–prey systems in eukaryotes phenomenamental ecological suchasphototrophy by circumventing digestion, giving to funda- rise evolved means to control take vacuole of food the be relatedbe to structure the of microbial the web food tion rates ofmight some pathogenic species bacterial applied motivations. As outlined above, elimina- the tor–prey interactions stimulated might be also by more web. Furthermore, investigations of microbial preda- abundance iscontrolled of bacteria these by food the marine water column turnoverthe of organic sulphur compounds inthe 2 146 However, prokaryotes are ‘senior the players’ inthe , . Symbiosis and parasitism are evolutionarily Roseobacter Roseobacter . Weinbauer, M.G.,Brettar, I.&Höfle,M.G.Lysogeny 9. Weinbauer, M.G.,Christaki,U.,Nedoma, A.& 8. 0 Weinbauer, M.G.&Höfle,Size-specific 10. Oceanogr. surface, deep,andanoxicmarinewaters. and virus-inducedmortalityofbacterioplankton in (2003). eutrophic reservoir. enrichment andgrazingonviralproduction inameso- Šimek, K.Comparingtheeffects ofresource (1998). communities. mortality oflakebacterioplanktonbynatural virus 145 and other forms of endosymbio- clade are important mediators of 48 143 , 1457–1465(2003). Aquat. Microb.Ecol.

105,119 , but it isunknown how the www.nature.com/reviews/micro Aquat. Microb.Ecol. 146 . Some bacteria have bacteria . Some . It therefore to seems

15 , 103–113

31 Limnol. , 137–144 144 137 . . REVIEWS

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39. Yabuuchi, E. et al. Burkholderia uboniae sp. nov., 44. Fraser, C., Hanage, W. P. & Spratt, B. G. Neutral Ghent, Belgium, 25 October 2004, and has the intention to L-arabinose-assimilating but different from microepidemic evolution of bacterial pathogens? stir up the interdisciplinary debate on the prokaryotic species. Burkholderia thailandensis and Burkholderia Proc. Natl Acad. Sci. USA 102, 1968–1973 (2005). We thank all participants as well as D. Mazel, G. Manfio and vietnamiensis. Microbiol. Immunol. 44, 307–317 45. Cohan, F. M. Does recombination constrain neutral T. Iida for their contributions. T.C., P.V. and J.S. are indebted (2000). divergence among bacterial taxa. Evolution 49, 164–175 to the Fund for Scientific Research — Flanders (Belgium) for 40. Cohan, F. M. Concepts of Bacterial Biodiversity for the Age (1995). a position as postdoctoral fellow and research funding, of Genomics. in Microbial Genomes (eds Fraser, C. M., 46. Keim, P. et al. Anthrax molecular epidemiology and respectively. F.M.C. acknowledges funding from the National Read, T. D. & Nelson, K. E.) Ch. 11, 175–194 (Humana forensics: using the appropriate marker for different Science Foundation. Press, Totowa, 2004). evolutionary scales. Infect. Genet. Evol. 4, 205–213 (2004). 41. Lawrence, J. G. Gene transfer in bacteria: speciation 47. Dunlap, P. V. and Ast, J. C. Genomic and Competing interests statement without species. Theor. Popul. Biol. 61, 449–460 phylogenetic characterization of luminous bacteria The authors declare no competing financial interests. (2002). symbiotic with the deep-sea fish Chlorophthalmus 42. Palys, T., Nakamura, L. K. & Cohan, F. M. Discovery and albatrossis (Aulopiformes: Chlorophthalmidae). Appl. classification of ecological diversity in the bacterial world: Environ. Microbiol. 71, 930–939 (2005). Online links the role of DNA sequence data. Int. J. Syst. Bacteriol. 47, 1145–1156 (1997). Acknowledgements FURTHER INFORMATION 43. Papke, R. T., Ramsing, N. B., Bateson, M. M. & Ward, D. This paper is the outcome of a workshop entitled ‘The Get into the debate on the prokaryotic species at: M. Geographical isolation in hot spring cyanobacteria. prokaryotic species: genome plasticity, microevolution and http://bioinformatics.psb.ugent.be/publications/prokspec Environ. Microbiol. 5, 650–659 (2003). taxonomy’, organized by F. Thompson and J. Swings in Access to this interactive links box is free online.

CORRIGENDUM PREDATION ON PROKARYOTES IN THE WATER COLUMN AND ITS ECOLOGICAL IMPLICATIONS Jakob Pernthaler Nature Reviews Microbiology 3, 537–546 (2005), doi:10.1038/nrmicro1180 In the above article, the following passage of text on page 542 might have been misleading: ‘Some bacteria can even poison their predators in a concerted manner: Chromobacterium violaceum specifically induced the eukaryotic APOPTOTIC RESPONSE after being ingested by a flagellate105. The constitutive production of the toxic agent, violacein, was only observed in strains that were capable of quorum sensing.’ It should have read: ‘Some bacteria can even poison their predators in a concerted manner: Chromobacterium violaceum caused rapid cell death after being ingested by a flagellate105. The toxic agent violacein is known to induce the APOPTOTIC RESPONSE in eukaryotic cells105a. Its constitutive production was only observed in strains that were capable of quorum sensing.’ The author apologizes to readers for any confusion caused.

105a. Melo, P. S., Justo, G. Z., de Azevedo, M. B., Duran, N. & Haun, M. Violacein and its β-cyclodextrin complexes induce apoptosis and differentiation in HL60 cells. Toxicology 186, 217–225 (2003).

ERRATUM Also in this article, a global formatting problem unfortunately resulted in all units in µm being mistakenly changed to mm. We wish to apologize to the author, and to readers, for any confusion caused.