Bdellovibrio bacteriovorus HD100 guards against Pseudomonas tolaasii brown-blotch lesions on the surface of post-harvest Agaricus bisporus supermarket mushrooms.

BACKGROUND: Pseudomonas tolaasii is a problematic pathogen of cultured mushrooms, forming dark brown 'blotches' on mushroom surfaces and causing spoilage during crop growth and post-harvest . Treating P. tolaasii infection is difficult, as other, commensal bacterial species such as Pseudomonas putida are necessary for mushroom growth, so treatments must be relatively specific. RESULTS: We have found that P. tolaasii is susceptible to predation in vitro by the δ-proteobacterium Bdellovibrio bacteriovorus. This effect also occurred in funga, where B. bacteriovorus was administered to post-harvest mushroom caps before and after administration of the P. tolaasii pathogen. A significant, visible improvement in blotch appearance, after incubation, was observed on administration of Bdellovibrio. A significant reduction in viable P. tolaasii cell numbers, recovered from the mushroom tissue, was detected. This was accompanied by a more marked reduction in blotch severity on Bdellovibrio administration. We found that there was in some cases an accompanying overgrowth of presumed-commensal, non-Pseudomonas bacteria on post-harvest mushroom caps after Bdellovibrio-treatment. These bacteria were identified (by 16SrRNA gene sequencing) as Enterobacter species, which were seemingly resistant to predation. We visualised predatory interactions occuring between B. bacteriovorus and P. tolaasii on the post-harvest mushroom cap surface by Scanning Electron Microscopy, seeing predatory invasion of P. tolaasii by B. bacteriovorus in funga. This anti-P. tolaasii effect worked well in post-harvest supermarket mushrooms, thus Bdellovibrio was not affected by any pre-treatment of mushrooms for commercial/consumer purposes. CONCLUSIONS: The soil-dwelling B. bacteriovorus HD100 preys upon and kills P. tolaasii, on mushroom surfaces, and could therefore be applied to prevent spoilage in post-harvest situations where mushrooms are stored and packaged for sale.

Spiral architecture of the nucleoid in Bdellovibrio bacteriovorus.

We present a cryo-electron tomographic analysis of the three-dimensional architecture of a strain of the Gram-negative bacterium Bdellovibrio bacteriovorus in which endogenous MreB2 was replaced with monomeric teal fluorescent protein (mTFP)-labeled MreB2. In contrast to wild-type Bdellovibrio cells that predominantly displayed a compact nucleoid region, cells expressing mTFP-labeled MreB2 displayed a twisted spiral organization of the nucleoid. The more open structure of the MreB2-mTFP nucleoids enabled clear in situ visualization of ribosomes decorating the periphery of the nucleoid. Ribosomes also bordered the edges of more compact nucleoids from both wild-type cells and mutant cells. Surprisingly, MreB2-mTFP localized to the interface between the spiral nucleoid and the cytoplasm, suggesting an intimate connection between nucleoid architecture and MreB arrangement. Further, in contrast to wild-type cells, where a single tight chemoreceptor cluster localizes close to the single polar flagellum, MreB2-mTFP cells often displayed extended chemoreceptor arrays present at one or both poles and displayed multiple or inaccurately positioned flagella. Our findings provide direct structural evidence for spiral organization of the bacterial nucleoid and suggest a possible role for MreB in regulation of nucleoid architecture and localization of the chemotaxis apparatus.

Predation of human pathogens by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus.

AIMS: The focus of this study was to evaluate the potential use of the predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus to control the pathogens associated with human infection. METHODS AND RESULTS: By coculturing B. bacteriovorus 109J and M. aeruginosavorus ARL-13 with selected pathogens, we have demonstrated that predatory bacteria are able to attack bacteria from the genus Acinetobacter, Aeromonas, Bordetella, Burkholderia, Citrobacter, Enterobacter, Escherichia, Klebsiella, Listonella, Morganella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio and Yersinia. Predation was measured in single and multispecies microbial cultures as well as on monolayer and multilayer preformed biofilms. Additional experiments aimed at assessing the optimal predation characteristics of M. aeruginosavorus demonstrated that the predator is able to prey at temperatures of 25-37°C but is unable to prey under oxygen-limiting conditions. In addition, an increase in M. aeruginosavorus ARL-13 prey range was also observed. CONCLUSIONS: Bdellovibrio bacteriovorus and M. aeruginosavorus have an ability to prey and reduce many of the multidrug-resistant pathogens associated with human infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Infectious complications caused by micro-organisms that have become resistant to drug therapy are an increasing problem in medicine, with more infections becoming difficult to treat using traditional antimicrobial agents. The work presented here highlights the potential use of predatory bacteria as a biological-based agent for eradicating multidrug-resistant bacteria, with the hope of paving the way for future studies in animal models.

Shadowing the actions of a predator: backlit fluorescent microscopy reveals synchronous nonbinary septation of predatory Bdellovibrio inside prey and exit through discrete bdelloplast pores.

The Bdellovibrio are miniature "living antibiotic" predatory bacteria which invade, reseal, and digest other larger Gram-negative bacteria, including pathogens. Nutrients for the replication of Bdellovibrio bacteria come entirely from the digestion of the single invaded bacterium, now called a bdelloplast, which is bound by the original prey outer membrane. Bdellovibrio bacteria are efficient digesters of prey cells, yielding on average 4 to 6 progeny from digestion of a single prey cell of a genome size similar to that of the Bdellovibrio cell itself. The developmental intrabacterial cycle of Bdellovibrio is largely unknown and has never been visualized "live." Using the latest motorized xy stage with a very defined z-axis control and engineered periplasmically fluorescent prey allows, for the first time, accurate return and visualization without prey bleaching of developing Bdellovibrio cells using solely the inner resources of a prey cell over several hours. We show that Bdellovibrio bacteria do not follow the familiar pattern of bacterial cell division by binary fission. Instead, they septate synchronously to produce both odd and even numbers of progeny, even when two separate Bdellovibrio cells have invaded and develop within a single prey bacterium, producing two different amounts of progeny. Evolution of this novel septation pattern, allowing odd progeny yields, allows optimal use of the finite prey cell resources to produce maximal replicated, predatory bacteria. When replication is complete, Bdellovibrio cells exit the exhausted prey and are seen leaving via discrete pores rather than by breakdown of the entire outer membrane of the prey.

Three-dimensional imaging of the highly bent architecture of Bdellovibrio bacteriovorus by using cryo-electron tomography.

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.

[Ultrastructural basis of interactions between prokaryotes and eukaryotes in different symbiotic models]. / Basi ultrastrutturali delle interazioni tra procarioti ed eucarioti in differenti modelli simbiotici.

This paper reviews the Author's contribution to the knowledge of the ultrastructural basis of the prokaryote-eukaryote interactions in different models assessed by an ultrastructural approach. In agreement with the hypothesis of the origin of eukaryotic cells, which are chimeras of several prokaryotes with different morpho-functional specializations, symbiosis had major consequence for evolution of life. In Arthropods, one of the most successful lifestyles, the presence of endosymbiotic prokaryotes, plays an important role in their metabolism. In some cases, genome integration has occurred in the endosymbiotic relationships with the host, proving that intracellular symbiosis is not merely a nutritional supplement. Intracellular symbiotic bacteria are also described in nematodes. In particular, the presence of intracellular Wolbachia in filariae, even if its function is not yet completely known, influences positively the reproductive biology and the survival of the host, as proved by antibiotic treatment against this bacterium. The ultrastructural images reported in this review were obtained using different species of cockroaches, termites, ticks and filarial nematodes. The traditional methods of transmission (TEM), scansion (SEM) and immuno electron microscopy were used. In addition, also freeze-fracture and deep-etching techniques were employed. The cockroaches and the primitive termite Mastotermes darwiniensis host symbiotic bacteria in the ovary and in specialized cells (bacteriocytes) of the fat body. These bacteria have the typical cell boundary profile of gram-negative bacteria and are enveloped in a vacuolar membrane produced by the host cell. Molecular sequence data of 16S rDNA of endosymbionts of five species of cockroaches and M. darwiniensis indicate that they are members of the Flavobacteria-bacteroides group and that the infection occurred in an ancestor common to cockroaches and termites probably after the end of the Paleozoic (250 Ma BP). The symbiotic bacteria are transmitted transovarially and, during embryogenesis, they are integrated into the morphogenetic processes. In particular, we were able to demonstrate that the origin of the bacteriocyte should be looked for in the cells of the haemocyte line (embryonic plasmatocytes). The eggs are infected by the bacteria emerging from the bacteriocytes of the ovaric fat body and, at the end of the vitellogenesis, they are actively phagocytized by the egg membrane. In filarial nematodes, intracellular bacteria belonging to the genus Wolbachia have been described: they have evolved an obligatory mutualistic association with their host. In fact, antibiotic treatments lead to the clearance of bacteria and this loss produces a negative impact on reproduction and survival of the filarial host. We evidenced, by TEM, the degenerative events occurring during the embriogenesis of Brugia pahangi and Dirofilaria immitis after tetracycline treatment. The data suggest that the Wolbachia play a direct role in worm metabolism. Finally, a new additional model of the prokaryote-eukaryote interaction has been described: we have recently discovered a new intracellular alpha-proteobacterium, named Iric ES1, which resides in the ovarian tissues of the tick Ixodes ricinus. The intriguing characteristic of this bacterium is its ability to invade and consume the ovaric mitochondria. From an evolutionary perspective, it is interesting to note that Iric ES1 enters mitochondria in a similar way to that employed by the "predatory" bacterium Bdellovibrio bacteriovorus.

[Intracellular membrane structures of Bdellovibrio bacteriovorus during development in the bacterial host cell]. / Vnutrikletochnye membrannye struktury Bdellovibrio bacteriovorus pri razvitii v kletke baktteri-khoziaina

Electron microscope investigation of intracellular membrane structures of Bd. bacteriovorous during its intracellular growth and development has been carried out. Increased amount of membrane structures has been stated. Along with simply organized invaginations of plasmalemma, complicated membrane structures resembling mesosomes of grampositive bacteria are observed. Localization of these structures testifies to their involvement in the synthesis of exotoxins and exoenzymes.

Sharing of prey: coinfection of a bacterium by a virus and a prokaryotic predator.

UNLABELLED: Rarely, if ever, has a single bacterial cell been confirmed to simultaneously host two fundamentally different predators. Two such predators are viruses and the predatory prokaryotes known as Bdellovibrio and like organisms. Viruses or bacteriophage are particles requiring prey cells in an active metabolic state to complete their life cycle. The Bdellovibrio and like organisms, unlike viruses, are bacteria that can efficiently infect and grow in prey which are in stationary phase. In this study, electron microscopic examination revealed an unprecedented coinfection by the two agents of Vibrio vulnificus, introducing a new bacterial predation paradigm. Rather than the viruses and Bdellovibrio and like organisms competing for a single prey cell, both can survive in the same cell and successfully reproduce themselves. This is an especially valuable mechanism when the prey is in short supply, and the survival of the predators may be at stake. IMPORTANCE: This article describes the coinfection of a prokaryotic prey or host cell by both a bacteriophage (phage) and the predatory bacterium of the group Bdellovibrio and like organisms (BALOs). Such coinfection has not been previously reported and therefore introduces a new paradigm for predation of bacteria. This finding invites new studies on the interactions of BALOs, phage, and prey in predation. Predation is an important mechanism in nature for helping to keep bacterial populations in check and also plays a major role in the cycling of nutrients through the microbial loop. How dual infection by phage and BALOs imposes on these and other functions of predation is fertile ground for future studies and serves as a keystone reference on bacterial predation and mortality.

Roles of multiple flagellins in flagellar formation and flagellar growth post bdelloplast lysis in Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus cells have a single polar flagellum whose helical pitch and diameter characteristically change near the midpoint, resulting in a tapered wave. There are six flagellin genes in the genome: fliC1 to fliC6. Accordingly, the flagellar filament is composed of several similar flagellin species. We have used knockout mutants of each gene and analyzed the mutational effects on the filament length and on the composition and localization of each flagellin species in the filament by electron microscopy and one- and two-dimensional polyacrylamide gel electrophoresis. The location and amounts of flagellins in a filament were determined to be as follows: a small amount of FliC3 at the proximal end, followed by a large amount of FliC5, a large amount of FliC1, a small amount of FliC2 in this order, and a large amount of FliC6 at the distal end. FliC4 was present at a low level, but the location was not determined. Filament lengths of newly born progeny cells increased during prolonged incubation in nutrient-deficient buffer. The newly formed part of the elongated filament was composed of mainly FliC6. Reverse transcription PCR analysis of flagellar gene expression over 5 days in buffer showed that fliC gene expression tailed off over 5 days in the wild-type cells, but in the fliC5 mutant, expression of the fliC2, fliC4, and fliC6 genes was elevated on day 5, suggesting that they may be expressed to compensate for the absence of a major component, FliC5.

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.

Predatory mechanisms of Bdellovibrio and like organisms.

Bdellovibrio and like organisms (BALOs) are predatory, Gram-negative delta-proteobacteria with a complex developmental lifecycle. In the free-living attack phase they are highly motile and seek out prey bacteria that they invade. The ensuing intracellular growth and replication is characterized by the development of a long filament that septates into individual cells that differentiate further into the flagellated attack-phase bacterium. The prey bacterium is lysed and motile predators are released. BALOs have recently been considered to have potential as living antibiotics. The idea of using predatory bacteria as therapeutic agents to combat pathogenic Gram-negative bacteria is intriguing, as they can prey upon human pathogenic bacteria including Salmonella, Pseudomonas and Escherichia coli. However, our current knowledge of the amazing biology of these prokaryotes that cause the systematic destruction of Gram-negative bacteria is still rather limited. More has to be learned about their predatory lifestyle before their application as therapeutic agents will become feasible.

Predation by Bdellovibrio bacteriovorus HD100 requires type IV pili.

Early electron microscopy and more recent studies in our laboratory of Bdellovibrio bacteriovorus cells indicated the presence of narrow fibers at the nonflagellar pole of this unusual predatory bacterium. Analysis of the B. bacteriovorus HD100 genome showed a complete set of genes potentially encoding type IV pili and an incomplete gene set for Flp pili; therefore, the role of type IV pili in the predatory life cycle of B. bacteriovorus HD100 was investigated. Alignment of the predicted PilA protein with known type IV pilins showed the characteristic conserved N terminus common to type IVa pilins. The pilA gene, encoding the type IV pilus fiber protein, was insertionally inactivated in multiple Bdellovibrio replicate cultures, and the effect upon the expression of other pilus genes was monitored by reverse transcriptase PCR. Interruption of pilA in replicate isolates abolished Bdellovibrio predatory capability in liquid prey cultures and on immobilized yellow fluorescent protein-labeled prey, but the mutants could be cultured prey independently. Expression patterns of pil genes involved in the formation of type IV pili were profiled across the predatory life cycle from attack phase predatory Bdellovibrio throughout the intraperiplasmic bdelloplast stages to prey lysis and in prey-independent growth. Taken together, the data show that type IV pili play a critical role in Bdellovibrio predation.

Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus.

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.

Transcriptional activity of the host-interaction locus and a putative pilin gene of Bdellovibrio bacteriovorus in the predatory life cycle.

Bdellovibrio bacteriovorus is a predatory bacterium that grows and replicates within the periplasm of a large variety of Gram-negative bacteria. So far, the host-interaction locus (hit locus) is the only genetic locus that is implicated in the obligate predatory lifestyle. Sequence analysis revealed that upstream of the hit locus, the genomic regions of the two obligate predatory B. bacteriovorus-type strains HD100 and HD114 encode genes for pilus formation. As pili might be involved in the invasion process, the transcriptional activity of the hit locus and of a putative pilin gene (flp1) of the pilus cluster were studied in synchronized cultures of B. bacteriovorus with Escherichia coli K-12 as prey bacteria. Stages of the life cycle were monitored with scanning electronic microscopy and transcriptional analyses were performed by quantitative reverse transcription polymerase chain reaction. Our study revealed an increased expression level of the putative hit and flp1 genes in the attack phase of B. bacteriovorus, whereas the transcriptional activity significantly decreased during the intracellular replication phase.

Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus.

We determined that paracrystalline protein surface arrays (S layers) protected gram-negative eubacteria from predation by Bdellovibrio bacteriovorus. Aquaspirillum serpens VHA and MW5 and Aquaspirillum sinuosum were resistant to predation by B. bacteriovorus 6-5-S when fully covered by their S layers. The S layer of Aeromonas salmonicida A449 protected the cells from predication by B. bacteriovorus 109J. A predacious, plaque-forming vibrio that lysed an S-layer- variant of Caulobacter crescentus but was not predacious on the parental strain which possessed an S layer was isolated from raw sewage. Since S layers are stable components of many bacterial surfaces in nature, they can provide this protective function in both aquatic and terrestrial habitats where Bdellovibrio spp. are found.

Interactions between Bdellovibrio and its host cell.

The bdellovibrios are extremely small bacteria with the unique property of being parasites of other (gram-negative) bacteria. In the presence of viable and susceptible bacteria a Bdellovibrio cell physically 'attacks' an individual host cell, attaches to its surface, penetrates the cell wall, and multiples within the periplasmic (intramural) space of its prey. The invading Bdellovibrio and its progeny degrade and consume the cellular constituents of the invaded host bacterium. This process finally results in complete lysis of the host cell and release of the Bdellovibrio progeny. From a population of parasitic bdellovibrios, derivatives can be selected that grow on complex nutrient media. Currently, none of the different nutritional types can be propagated in a fully defined synthetic medium. By degradation of the cellular constituents of the host the Bdellovibrio cell in its periplasmic space has available all the monomeric subunits needed to synthesis of the macromolecules. Peculiarities of Bdellovibrio metabolism with respect to uptake of preformed molecules and energy efficiency are discussed.

Ultrastructural changes during encystment and germination of Bdellovibrio sp.

Under proper conditions, Bdellovibrio sp. strain W cells develop into bdellocysts in appropriate prey bacteria. After attachment and penetration of the prey cell, the encysting bdellovibrio began to accumulate inclusion material and increase in size, and was surrounded by an outer layer of amorphous electrondense material. The cytoplasm of the encysting cell appeared more electron dense, and nuclear areas appeared more compact. During germination of bdellocysts, the outer wall was uniformly broken down the inclusion material changed shape and affinity for the heavy metal stain, and the nuclear areas expanded. As the outer wall was dissolved, outgrowth began with the elongation of the germinant as it emerged from the prey ghost as an actively motile cell.

[Ultrastructural characteristics of Bdellovibrio chlorellavorus]. / Osobennosti ul'trastruktury Bdellovibrio chlorellavorus.

Intracellular and extracellular polar lamellar systems were detected in Bdellovibrio chlorellavorus, the obligate parasite of Chlorella. They were observed in negatively contrasted preparations and thin sections. These systems are considered as an essential character of the bacterium, being connected with its parasitic behaviour.

Waveform analysis and structure of flagella and basal complexes from Bdellovibrio bacteriovorus 109J.

The structure of sheathed flagella from Bdellovibrio bacteriovorus was investigated. The first three periods of these flagella were characterized by progressively smaller wavelengths and amplitudes in periods more distal to the cell. The damped appearance was due to a single nonrandom transition between two helical structures within each filament. The intersection of the two helices, one of which was a threefold-reduced miniature of the other, occurred at a fixed distance along the filament and resulted in a shift in the flagellar axis. Flagella increased in length as the cells aged and assumed a constant miniature waveform at their distal ends. The core filament was the principal determinant of flagellar morphology. It was composed of 28,000- and 29,500-dalton polypeptides. The 28,000-dalton subunits were located in the cell-proximal segment of the filament, and the 29,500-dalton subunits were located in the more distal region. The heteromorphous appearance of bdellovibrio flagella arose from the sequential assembly of these subunits. The basal complex associated with core filaments was examined because of its potential involvement in sheath formation. Bdellovibrio basal organelles were generally similar to those of other gram-negative species, but appeared to lack a disk analogous to the outer membrane-associated L ring which is a normal component of gram-negative basal complexes.

[Intracellular development of Bdellovibrio bacteriovorus]. / Vnutrikletochnoe razvitie Bdellovibrio bacteriovorus.

The intracellular growth of Bdellovibrio bacteriovorus, a bacterial parasite, was studied by a light-optical method using time-lapse cinemicrography. The organism was found to be capable of growth in the periplasmic space of filamentous cells of the host bacterium Pseudomonas fluorescens without any contact with the cytoplasmic membrane. Several B. bacteriovorus cells could grow simultaneously in the bdelloplasm.