Prey Cells Research Articles

A Transcriptional “Scream” Early Response of E. coli Prey to Predatory Invasion by Bdellovibrio

We have transcriptionally profiled the genes differentially expressed in E. coli prey cells when predatorily attacked by Bdellovibrio bacteriovorus just prior to prey cell killing. This is a brief, approximately 20–25 min period when the prey cell is still alive but contains a Bdellovibrio cell in its periplasm or attached to and penetrating its outer membrane. Total RNA was harvested and labelled 15 min after initiating a semi-synchronous infection with an excess of Bdellovibrio preying upon E. coli and hybridised to a macroarray spotted with all predicted ORFs of E. coli. SAM analysis and t-tests were performed on the resulting data and 126 E. coli genes were found to be significantly differentially regulated by the prey upon attack by Bdellovibrio. The results were confirmed by QRT-PCR. Amongst the prey genes upregulated were a variety of general stress response genes, potentially “selfish” genes within or near prophages and transposable elements, and genes responding to damage in the periplasm and osmotic stress. Essentially, the presence of the invading Bdellovibrio and the resulting damage to the prey cell elicited a small “transcriptional scream”, but seemingly no specific defensive mechanism with which to counter the Bdellovibrio attack. This supports other studies which do not find Bdellovibrio resistance responses in prey, and bodes well for its use as a “living antibiotic”.

Predatory Lifestyle of Bdellovibrio bacteriovorus

Bdellovibrio species are naturally predatory, small, motile, Deltaproteobacteria that invade the periplasm of other larger gram-negative bacteria, killing and digesting them. Bdellovibrio grows and divides inside the prey cell, in a structure called a bdelloplast, which then lyses, releasing the Bdellovibrio to prey upon more bacteria. This capability makes Bdellovibrio a potential therapeutic agent, but since its discovery in the 1960s it has not been applied in this way. This review considers what is known postgenomically about Bdellovibrio and its predatory lifestyle, drawing also from what was learned by the excellent microbial physiology work of the early Bdellovibrio researchers. Recent work on the diversity and evolution of predatory bdellovibrios, the role of surface structures in predation, and the ongoing questions about how Bdellovibrio switches between axenic and predatory growth and how its predatory activities may be tempered in the wild, as well as suggestions for future research priorities, are discussed.

Molecular Phylogeny and Description of the Novel Katablepharid Roombia truncata gen. et sp. nov., and Establishment of the Hacrobia Taxon nov

BackgroundPhotosynthetic eukaryotes with a secondary plastid of red algal origin (cryptophytes, haptophytes, stramenopiles, dinoflagellates, and apicomplexans) are hypothesized to share a single origin of plastid acquisition according to Chromalveolate hypothesis. Recent phylogenomic analyses suggest that photosynthetic “chromalveolates” form a large clade with inclusion of several non-photosynthetic protist lineages. Katablepharids are one such non-photosynthetic lineage closely related to cryptophytes. Despite their evolutionary and ecological importance, katablepharids are poorly investigated.Methodology/Principal FindingsHere, we report a newly discovered flagellate, Roombia truncata gen. et sp. nov., that is related to katablepharids, but is morphologically distinct from othermembers of the group in the following ways: (1) two flagella emerge from a papilla-like subapical protrusion, (2) conspicuous ejectisomes are aligned in multiple (5–11) rows, (3) each ejectisome increases in size towards the posterior end of the rows, and (4) upon feeding, a part of cytoplasm elastically stretch to engulf whole prey cell. Molecular phylogenies inferred from Hsp90, SSU rDNA, and LSU rDNA sequences consistently and strongly show R. truncata as the sister lineage to all other katablepharids, including lineages known only from environmental sequence surveys. A close association between katablepharids and cryptophytes was also recovered in most analyses. Katablepharids and cryptophytes are together part of a larger, more inclusive, group that also contains haptophytes, telonemids, centrohelids and perhaps biliphytes. The monophyly of this group is supported by several different molecular phylogenetic datasets and one shared lateral gene transfer; therefore, we formally establish this diverse clade as the “Hacrobia.”Conclusions/SignificanceOur discovery of R. truncata not only expands our knowledge in the less studied flagellate group, but provide a better understanding of phylogenetic relationship and evolutionary view of plastid acquisition/losses of Hacrobia. Being an ancestral to all katablepharids, and readily cultivable, R. truncata is a good candidate for multiple gene analyses that will contribute to future phylogenetic studies of Hacrobia.

Deciphering the hunting strategy of a bacterial wolfpack

Myxococcus xanthus is a common soil bacterium with an intricate multicellular lifestyle that continues to challenge the way in which we conceptualize the capabilities of prokaryotic organisms. Myxococcus xanthus is the preferred laboratory representative from the Myxobacteria, a family of organisms distinguished by their ability to form highly structured biofilms that include tentacle-like packs of surface-gliding cell groups, synchronized rippling waves of oscillating cells and massive spore-filled aggregates that protrude upwards from the substratum to form fruiting bodies. But most of the Myxobacteria are also predators that thrive on the degradation of macromolecules released through the lysis of other microbial cells. The aim of this review is to examine our understanding of the predatory life cycle of M. xanthus. We will examine the multicellular structures formed during contact with prey, and the molecular mechanisms utilized by M. xanthus to detect and destroy prey cells. We will also examine our understanding of microbial predator-prey relationships and the prospects for how bacterial predation mechanisms can be exploited to generate new antimicrobial technologies.

Formation of harmful algal blooms cannot be explained by allelopathic interactions

Many planktonic microalgae produce a range of toxins and may form harmful algal blooms. One hypothesis is that some toxins are allelopathic, suppressing the growth of competitors, and it has been suggested that allelopathy may be one important mechanism causing algal blooms. In a metaanalysis of recent experimental work, we looked for evidence that allelopathy may explain the initiation of algal blooms. With few exceptions, allelopathic effects were only significant at very high cell densities typical of blooms. We conclude that there is no experimental support for allelopathy at prebloom densities, throwing doubts on allelopathy as a mechanism in bloom formation. Most studies tested allelopathy using cell-free manipulations. With simple models we show that cell-free manipulations may underestimate allelopathy at low cell densities if effects are transmitted during cell-cell interactions. However, we suggest that the evolution of allelopathy under field conditions may be unlikely even if based on cell-cell interactions. The spatial dispersion of cells in turbulent flow will make it difficult for an allelopathic cell to receive an exclusive benefit, and a dispersion model shows that dividing cells are rapidly separated constraining clone selection. Instead, we propose that reported allelopathic effects may be nonadaptive side effects of predator-prey or casual parasitic cell-cell interactions.

A New Method for Isolating Host-Independent Variants of Bdellovibrio bacteriovorus Using E. coli Auxotrophs

Bdellovibrios are Gram-negative bacteria that are characterized by predatory behavior. Although Bdellovibrios exhibit an obligatory parasitic life cycle, it is possible to isolate Bdellovibrio variants that no longer require host cells for their growth. In this study, a new method for isolating Bdellovibrio bacteriovorus host-independent (HI) variants was developed. Filtered B. bacteriovorus prey cells were cultured with E. coli diaminopimelic acid (DAP) auxotrophs as host cells. Thereafter, the lysate was plated on DAP minus media, allowing only HI colonies to develop. Using this method, we have isolated numerous HI variants and demonstrated that the emergence of HI variants may be occurring at a higher frequency than was previously suggested.

A fratricidal mechanism is responsible for eDNA release and contributes to biofilm development of Enterococcus faecalis

Extracellular DNA (eDNA), a by-product of cell lysis, was recently established as a critical structural component of the Enterococcus faecalis biofilm matrix. Here, we describe fratricide as the governing principle behind gelatinase (GelE)-mediated cell death and eDNA release. GFP reporter assays confirmed that GBAP (gelatinase biosynthesis-activating pheromone) quorum non-responders (GelE-SprE-) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE+SprE+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, we address a mechanism by which GelE and SprE may modify the cell wall affinity of proteolytically processed AtlA resulting in either a pro- or anti-lytic outcome.

Biofilm formation of Bdellovibrio bacteriovorus host-independent derivatives

Bdellovibrios are Gram-negative predatory bacteria which are ubiquitous to many environmental niches, including natural biofilms. In this study, host-independent (HI) variants of Bdellovibrio bacteriovorus 109J were isolated. Predation assays and genetic analysis confirmed that the selected HI variants are derivatives of B. bacteriovorus. When grown in microtiter plates, HI variants were able to form tenacious biofilms on the surface of the wells. HI biofilm formation on different surfaces, media and temperatures was examined. HI biofilm development was seen on all of the examined surfaces, with the most robust biofilm developing at 22 °C and in media supplemented with yeast extract. Biofilm detachment experiments revealed that the HI cells are firmly attached to the surface of the wells and are not easily removed by physical and chemical treatments. Treating the biofilm with proteinase K and DNase-I caused rapid detachment of the biofilm as well as inhibition of biofilm formation, suggesting that DNA and proteins are major components of the HI biofilm extracellular matrix. Our data suggest that under conditions that might favor the development of HI variants, such as a rich nutrient environment, Bdellovibrio facultative prey cells are capable of attaching to abiotic surfaces and forming biofilms.

Physical Properties of Native Biofilm Cells Explored by Atomic Force Microscopy

Biofilms are complex microbial communities that grow at interfaces. Bacteria in biofilms are phenotypically different than their planktonic (free swimming) relatives; they adapt to the communal, sessile lifestyle by optimizing their motility, adhesion, and metabolism. We used Atomic Force Microscopy (AFM) to directly probe the physical properties of native bacterial cells in simple biofilm communities and demonstrated that widely dissimilar biofilm-colonizing cells all have a high cellular spring constant, indicating that they are quite stiff. However, the lab strain E. coli ML35 that does not form robust biofilms is much less stiff, hinting that stiff bacteria may preferentially colonize surfaces in the early stages of biofilm formation. Adhesive forces between the retracting AFM tip and bacterial cells vary between cell types in terms of the force components, the distance components, and the number of adhesion events, reflecting differences in associated extracellular polymeric substances (EPS), pili, and flagella. Because biofilms are dynamic, robust, and challenging to control or destroy, potential removal agents are of great interest in industrial, medical, and agricultural settings. We also explored the changes that occur in E. coli biofilm cells as they are devoured Bdellovibrio bacteriovorus, a bacterial predator of other bacteria. Invaded prey cells, called bdelloplasts, undergo substantial chemical and physical changes that we probed directly with the AFM tip. Bdelloplasts are significantly shorter than uninvaded E. coli biofilm cells, and prey cells clearly lose elasticity after invasion by B. bacteriovorus predators. On average, the spring constant of uninvaded E. coli cells was three times stiffer than that of bdelloplasts. The retraction portions of the force curves indicate that bdelloplasts adhere to the AFM tip with larger pull-off forces as compared to uninvaded E. coli. Thus, AFM provides provocative information about native biofilms.

Evaluation of the efficiency of metabolism of dinoflagellate phosphorus and carbon by a planktonic ciliate

The trophic transfer of nutrients through the microbial food web is a key top-down control in aquatic ecosystems which is notoriously difficult to evaluate, particularly for planktonic protists. In this study, a sensitive dual-radioactive tracer technique was developed to simultaneously assess the ingestion rate, and carbon- and phosphorus-specific assimilation efficiencies, of the marine planktonic ciliate Strobilidium neptuni feeding on the autotrophic dinoflagellate Heterocapsa triquetra. Dinoflagellate prey were simultaneously 16 h pulse labelled with NaH 14CO 3 and H 3 33PO 4 before being fed to the ciliate, and radioactive labels were traced into ciliate biomass and the experimental medium, as well as being monitored in the prey cells. Rates measured in short-term (10 min) incubations, as commonly used to estimate protist uptake of fluorescently labelled prey, were approximately 6 times higher and 3–6 times more variable than rates measured in longer 3–5 h incubations. The efficiency of accumulation of prey carbon (54±9%) by ciliates was lower than that of prey phosphorus (68±3%) suggesting that the phosphorus to carbon ratio in the ciliates was 1.3 times higher than in the labelled dinoflagellate biomass. Rates of phosphorus accumulation and release were combined to reveal that ciliates consumed 3.2±0.6 dinoflagellates cell −1 h −1. The assessment of carbon tracer release by ciliates was less reliable due to 14CO 2 exchange between the experimental media and air. The study concludes that the dual phosphorus–carbon radioactive tracer labelling of algal prey allowed the quantification of protist herbivory and nutrient remineralisation in laboratory experiments, thereby providing a potential technique for studying planktonic microbial trophic interactions in situ.

Active prey selection in two pelagic copepods feeding on potentially toxic and non-toxic dinoflagellates

Grazing on two red tide dinoflagellates, the potentially toxic Karenia mikimotoi and the non-toxic Gyrodinium instriatum, was examined in two species of marine copepods, Pseudocalanus elongatus and Temora longicornis. Both copepods cleared K. mikimotoi at rates that were a little lower but comparable to those at which they cleared the slightly larger G. instriatum when the two dinoflagellates were offered separately. However, when feeding on mixtures of the two prey species, the clearance rates on K. mikimotoi were substantially reduced in both copepods while their clearances of G. instiatum remained unaltered, suggesting active prey selection. Video observations of individual prey capture and feeding events showed prey rejection frequencies (caught and then released cells) that did not differ between mixed and mono-specific diets. This suggests that the selection between prey cells occurs prior to capture and that it is based on remote characterization of the cells.

A New Method for Isolating Host-Independent Variants of Bdellovibrio bacteriovorus Using E. coli Auxotrophs

Bdellovibrios are Gram-negative bacteria that are characterized by predatory behavior. Although Bdellovibrios exhibit an obligatory parasitic life cycle, it is possible to isolate Bdellovibrio variants that no longer require host cells for their growth. In this study, a new method for isolating Bdellovibrio bacteriovorus host-independent (HI) variants was developed. Filtered B. bacteriovorus prey cells were cultured with E. coli diaminopimelic acid (DAP) auxotrophs as host cells. Thereafter, the lysate was plated on DAP minus media, allowing only HI colonies to develop. Using this method, we have isolated numerous HI variants and demonstrated that the emergence of HI variants may be occurring at a higher frequency than was previously suggested.

Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types

Subcellular selenium (Se) distributions in the oligochaete Tubifex tubifex and in the insect Chironomus riparius did not vary with Se exposure duration, which was consistent with the observations that the duration of prey Se exposure had little influence on either Se assimilation or loss by a predatory insect (the alderfly Sialis velata). However, these two prey types differed in how Se was distributed in their cells. Overall, the predator assimilated a mean of 66% of the Se present in its prey, which was similar to the mean percentage of Se in prey cells (62%) that was theoretically available for uptake (that is, Se in the protein and organelle fractions). Likewise, data for cadmium, nickel and thallium suggest that predictions of trace element transfer between prey and predator are facilitated by considering the subcellular partitioning of these contaminants in prey cells.

Predataxis behavior inMyxococcus xanthus

Spatial organization of cells is important for both multicellular development and tactic responses to a changing environment. We find that the social bacterium, Myxococcus xanthus utilizes a chemotaxis (Che)-like pathway to regulate multicellular rippling during predation of other microbial species. Tracking of GFP-labeled cells indicates directed movement of M. xanthus cells during the formation of rippling wave structures. Quantitative analysis of rippling indicates that ripple wavelength is adaptable and dependent on prey cell availability. Methylation of the receptor, FrzCD is required for this adaptation: a frzF methyltransferase mutant is unable to construct ripples, whereas a frzG methylesterase mutant forms numerous, tightly packed ripples. Both the frzF and frzG mutant strains are defective in directing cell movement through prey colonies. These data indicate that the transition to an organized multicellular state during predation in M. xanthus relies on the tactic behavior of individual cells, mediated by a Che-like signal transduction pathway.

Proteome-Based Comparative Analyses of Growth Stages Reveal New Cell Cycle-Dependent Functions in the Predatory Bacterium Bdellovibrio bacteriovorus

Bdellovibrio and like organisms are obligate predators of bacteria that are ubiquitously found in the environment. Most exhibit a peculiar dimorphic life cycle during which free-swimming attack-phase (AP) cells search for and invade bacterial prey cells. The invader develops in the prey as a filamentous polynucleoid-containing cell that finally splits into progeny cells. Therapeutic and biocontrol applications of Bdellovibrio in human and animal health and plant health, respectively, have been proposed, but more knowledge of this peculiar cell cycle is needed to develop such applications. A proteomic approach was applied to study cell cycle-dependent expression of the Bdellovibrio bacteriovorus proteome in synchronous cultures of a facultative host-independent (HI) strain able to grow in the absence of prey. Results from two-dimensional gel electrophoresis, mass spectrometry, and temporal expression of selected genes in predicted operons were analyzed. In total, about 21% of the in silico predicted proteome was covered. One hundred ninety-six proteins were identified, including 63 hitherto unknown proteins and 140 life stage-dependent spots. Of those, 47 were differentially expressed, including chemotaxis, attachment, growth- and replication-related, cell wall, and regulatory proteins. Novel cell cycle-dependent adhesion, gliding, mechanosensing, signaling, and hydrolytic functions were assigned. The HI model was further studied by comparing HI and wild-type AP cells, revealing that proteins involved in DNA replication and signaling were deregulated in the former. A complementary analysis of the secreted proteome identified 59 polypeptides, including cell contact proteins and hydrolytic enzymes specific to predatory bacteria.

A new mechanism of cell cohesion during interaction between predatory bacteria and their preys

Parasitism of some bacterial species on others, which is based on cell cohesion, has been found in five genera of Gram-negative bacteria: Bdellovibrio [1], Bacteriovorax [2], Micavibrio [3], Ensifer [4], and Cupriavidus [5]. Only Bdellovibrio and Bacteriovorax are intracellular parasites (true predators, according to Gromov and Pavlenko’s definition [6]), whereas members of the genera Micavibrio, Ensifer , and Cupriavidus are ectoparasites. Note that Bdellovibrio, Bacteriovorax , and Micavibrio are obligate parasites, whereas Ensifer and Cupriavidus are facultative ones. However, the diversity of bacterial parasites seems to be poorly studied so far; some unknown species are expected in this physiological and trophic group of prokaryotes. This conclusion is supported by in situ analysis of indigenous forms of microorganisms in natural environment [7‐9]. We recently showed that chemoorganotrophic Gram-negative bacteria of the genus Kaistia (strains NF1 and NF3) isolated from various habitats were facultative parasites on both phototrophic [10] and chemoorganotrophic [11] bacteria; they were typical ectoparasites, whose parasitism was determined by strong adhesion to the prey cells. Note that NF1 and NF3 cells displayed no motility; they lacked flagella, and, hence, the mechanism of their connection and adhesion to prey cells was quite different from that characteristic of the other aforementioned bacterial parasites. The latter, having flagella and displaying motility were capable of searching for and approaching the preys by means of chemotaxis. We studied specific intercellular contacts in the system of an immovable bacterial parasite and a prey (the latter was a parasitesensitive species of chemorganotrophic or phototrophic bacteria). Bacterial parasites of the genus Kaistia (strains NF1 and NF3) proved to have special adhesive structures, the such as the trapping polysaccharide networks, whose filaments carried sticky nodules or granules; cone-shaped protrusions of these bacterial cells mediated anchoring and approaching the prey cells and tight attachment of the parasitic cells to the prey cells. This mechanism of cell interaction substantially differed from those characteristic of other known bacterial parasites, which lacked specific adhesive structures but used chemotactic reactions to interact with their preys.

Quantitative Changes in the Elasticity and Adhesive Properties of Escherichia coli ZK1056 Prey Cells During Predation by Bdellovibrio bacteriovorus 109J

Atomic force microscopy (AFM) was used to explore the changes that occur in Escherichia coli ZK1056 prey cells while they are being consumed by the bacterial predator Bdellovibrio bacteriovorus 109J. Invaded prey cells, called bdelloplasts, undergo substantial chemical and physical changes that can be directly probed by AFM. In this work, we probe the elasticity and adhesive properties of uninvaded prey cells and bdelloplasts in a completely native state in dilute aqueous buffer without chemical fixation. Under these conditions, the rounded bdelloplasts were shown to be shorter than uninvaded prey cells. More interestingly, the extension portions of force curves taken on both kinds of cells clearly demonstrate that bdelloplasts are softer than uninvaded prey cells, reflecting a decrease in bdelloplast elasticity after invasion by Bdellovibrio predators. On average, the spring constant of uninvaded E. coli cells (0.23 +/- 0.02 N/m) was 3 times stiffer than that of the bdelloplast (0.064 +/- 0.001 N/m) when measured in a HEPES-metals buffer. The retraction portions of the force curves indicate that compared to uninvaded E. coli cells bdelloplasts adhere to the AFM tip with much larger pull-off forces but over comparable retraction distances. The strength of these adhesion forces decreases with increasing ionic strength, indicating that there is an electrostatic component to the adhesion events.

Laboratory Maintenance of Bdellovibrio

AbstractBdellovibrio bacteriovorus is a Gram‐negative bacterium that preys upon other Gram‐negative bacteria. It does this by penetrating the outer membrane and peptidoglycan to establish itself in the periplasm where it grows at the expense of the contents of the prey cell before ultimately lysing the prey. Wild‐type Bdellovibrio are prey‐(or host‐) dependent and the protocols described in this unit deal with the techniques required to grow this bacterium on its prey cells. Protocols are also presented to generate and culture host‐independent mutants as well as novel protocols to evaluate Bdellovibrio predation. Curr. Protoc. Microbiol. 9:7B.2.1‐7B.2.13. © 2008 by John Wiley & Sons, Inc.

Feeding mechanism, prey specificity and growth in light and dark of the plastidic dinoflagellate Karlodinium armiger

The food uptake mechanism and prey specificity of the most recently described member of the ichthyotoxic photosynthetic dinoflagellate genus Karlodinium (K. armiger) was studied. K. armiger extracts the contents of prey through an inconspicuous feeding tube (peduncle), but may also ingest whole prey cells. This species is omnivorous, ingesting prey from all major groups of marine protists. K. armiger displays a searching pre-capture behavior with attraction to prey cells and formation of feeding aggregates. In batch cultures, growth rates in the light without food were low (0.01 to 0.10 d -1 ), but when the culture medium was enriched with soil extract, initial growth rate increased (0.19 d -1 ); it further increased (0.60 d -1 ) when fed the cryptophyte Rhodomonas marina in the light (170 µmol photons m 2 s -1 ). R. marina was also ingested in the dark, but did not support posi- tive growth rates and survival. Thus, K. armiger is an omnivorous obligate phototrophic mixotroph which seems to obtain a growth-essential substance or growth factor through phagotrophy.

Ca 2+- and glycoconjugates-dependent prey capture in the heliozoon Actinophrys sol

Exocytosis of extrusomes, secretory granules found in protozoa, is involved in prey capture by the heliozoon Actinophrys sol. Here, we show that extracellular Ca 2+ is necessary for exocytosis and prey capture in A. sol. We found that A. sol could not capture prey cells in a Ca 2+-free solution. L-type Ca 2+ channel blockers and a calmodulin antagonist also inhibited the capture of prey. These results suggest that Ca 2+ influx via L-type Ca 2+ channels plays a crucial role in exocytosis in A. sol. Concanavalin A (Con A) also inhibited prey capture, and the inhibition was relieved by the addition of its hapten sugar, α-mannoside, suggesting that Con A-binding glycoconjugates are implicated in exocytosis of extrusomes and the adhesion of prey cells.