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.     

Factors affecting the intracellular parasitic growth of Bdellovibrio bacteriovorus developing within Escherichia coli

A procedure for one-step growth experiments on Bdellovibrio bacteriovorus growing parasitically in Escherichia coli B was developed. The resulting one-step growth curves showed that, under defined conditions at 30 C, each singly infected E. coli host cell, on the average, gave rise to 5.7 Bdellovibrio cells. This value was confirmed by single-burst experiments and by microscopic observations. In the temperature range of 25 to 38 C, the average burst size and the duration of the latent period were inversely proportional to the temperature. The effect of hydrogen ion concentration on the one-step growth kinetics in this system indicated a broad pH optimum, ranging from neutrality to slightly alkaline pH values. After Bdellovibrio cells and host cells were mixed, there was always a delay (the so-called "lag phase") before the parasite titer increased in terms of plaque-forming units. Phase-contrast microscopic observations indicated that this delay stems in part from the polyphasic nature of the Bdellovibrio life cycle. We propose the following five terms to make explicit the sequence of events in this life cycle: "attachment," "penetration," "elongation," "fragmentation," and "burst." Nutritional experiments revealed that Bdellovibrio obtains a major fraction of its cellular components from host-cell material. Infection of E. coli by Bdellovibrio without added Mg(++) or Ca(++) (0.003 m Mg(++), 0.002 m Ca(++)) resulted in partial or total lysis of the host cell soon after infection. Protoplast integrity was necessary for the normal completion of the intracellular growth phase of Bdellovibrio in E. coli; normal development of the parasite took place only in the presence of Mg(++) or Ca(++).     

Development of Bdellophage VL-1 in Parasitic and Saprophytic Bdellovibrios

Ultrastructure was correlated with growth kinetics of bdellophage VL-1 infecting host-dependent ("parasitic") Bdellovibrio bacteriovorus 109J in its Escherichia coli B host (the three-membered system), as well as in the host-independent ("saprophytic") derivative of the Bdellovibrio. Electron microscope observations showed the arrested growth of the phage-infected bdellovibrios, polar localization of the phage progeny, and stages in their release. Present evidence indicates that bdellophage DNA is derived from both the Bdellovibrio and its host cell.     

Parasitic interaction of Bdellovibrio bacteriovorus with other bacteria

Starr, Mortimer P. (University of California, Davis), and Nancy L. Baigent. Parasitic interaction of Bdellovibrio bacteriovorus with other bacteria. J. Bacteriol. 91:2006-2017. 1966.-The interactions of the predatory parasite, Bdellovibrio bacteriovorus, with Erwinia amylovora, Pseudomonas tabaci, and P. phaseolicola were examined by means of phase-contrast and electron microscopy. Attachment of the bdellovibrio to the host cell is apparently initially reversible; detachment occurs infrequently in the later stages. Formation of a pore in the host cell wall is followed by disorganization of the host nucleus and of the murein layer of the host cell wall. Short host cells become totally spheroplasted; the longer rods of Pseudomonas usually are partially spheroplasted. The parasite completely invades the host cell, and the cell contents of the host are digested. Bdellovibrios living as parasites inside the host increase considerably in size in comparison with those which have been living away from the host for a time. When the host protoplast is entirely lysed, the parasites leave the disintegrating "ghosted" cell envelope, and are ready to reinitiate the parasitic cycle. The time taken for a mature Bdellovibrio cell to complete the parasitic cycle may vary depending on the length of time the parasite has been away from its hosts.     

Interaction of Bdellovibrio bacteriovorus and Host Bacteria II. Intracellular Growth and Development of Bdellovibrio bacteriovorus in Liquid Cultures

The intracellular life cycle of Bdellovibrio bacteriovorus 109 growing on Escherichia coli in a dilute nutrient medium exhibits a period of constant infective titer while the parasite grows and elongates inside the host cell. This period is terminated after 2 to 4 hr, and the number of the plaque-forming units in the culture rises rapidly to as much as six times the initial titer. The growth pattern of Bdellovibrio is similar with actively growing or resting host cells, or with host cells killed by ultraviolet irradiation or by heating at 70 C. The yield of B. bacteriovorus strain 109 in two-membered cultures with E. coli B depends on the host concentration and may reach 7.5 x 10(10) cells per ml. Penicillin, which has no effect on the attachment and penetration of Bdellovibrio, inhibits its multiplication.     

Penetration of Bdellovibrio bacteriovorus into Host Cells

Electron microscopy reveals that, in Bdellovibrio infection, after the formation of a passage pore in the host cell wall, the differentiated parasite penetration pole is associated with the host protoplast. This firm contact persists throughout the parasite penetration and after this process is completed. In penetrated hosts this contact is also apparent by phase microscopy. The association between the walls of the parasite and the host at the passage pore, on the other hand, is transient. Bdellovibrio do not penetrate hosts whose protoplast and cell walls are separated by plasmolysis, or in which the membrane-wall relationship is affected by low turgor pressure. It is concluded, therefore, that for penetration to occur it is essential that the host protoplast be within reach of the parasite, so that a firm contact can be established between them. A penetration mechanism is proposed that is effected by forces generated by fluxes of water and solutes due to structural changes in the infected host envelope. These forces cause a differential expansion of the host protoplast and cell wall and their separation from each other around the entry site, while the parasite remains firmly anchored to the host protoplast. Consequently, the parasite ends up enclosed in the expanded host periplasm. The actual entry, therefore, is a passive act of the parasite.     

Interaction of Bdellovibrio bacteriovorus and Host Bacteria I. Kinetic Studies of Attachment and Invasion of Escherichia coli B by Bdellovibrio bacteriovorus

Quantitative methods were developed for the study of the early stages in the interaction of Bdellovibrio bacteriovorus and host bacteria. Attachment measurements were based on the differential filtration of host and parasite. Invasion was measured by estimation of radioactively labeled Bdellovibrio cells remaining attached to the host cells after mechanical agitation. The kinetics of attachment and the final number of Bdellovibrio cells attached were dependent on the multiplicity of the parasite, the composition and pH of the medium, and the incubation temperature. Inhibitors of Bdellovibrio motility, including chelating agents, NaN(3), and low pH, all inhibited attachment, as did anaerobiosis. Ultraviolet-killed host cells retained their competence for attachment of Bdellovibrio cells, whereas heat-killed cells lost it. Invasion was selectively inhibited by inhibitors of protein synthesis, such as streptomycin, puromycin, and chloramphenicol. These antibiotics had no effect on attachment.     

蛭弧菌的分离及其生长条件和裂解能力的研究

噬菌蛭弧菌具有裂解病原菌、净化水体的功效,从海洋环境中分离到4株Bh04系列蛭弧菌,对其生长条件进行了测定,同时研究了它们对61株菌株的裂解能力。结果表明,在1%～3%的盐度范围内,蛭弧菌均可生长,最适盐度为3%;在15～30℃温度条件下蛭弧菌也可生长,但最适培养温度为20-25℃;只有在使用活的宿主菌的培养条件下,蛭弧菌才能生长。4株蛭弧菌分别可裂解21、24、40、43株菌,各占总试验菌数(61)的34.4%、39.3%、65.6%和70.5%。4株蛭弧菌一起,则可裂解55菌株,占总试验菌株的90.2%。研究结果揭示了蛭弧菌在消除海洋环境中有害细菌方面的潜在应用价值。     

噬菌蛭弧菌对鱼类常见致病菌裂解作用的研究

调查了北京地区２５份水样,其中２４份检出噬菌蛭弧菌。本次试验选用４株鱼类主要致病菌为宿主菌,检出的蛭弧菌对上述４种细菌的裂解范围有所不同。其中嗜水气单胞菌可被全部检出的蛭弧菌裂解（２４／２４）,其他３株菌仅部分被裂解,依次为肠型点状气单胞菌（１７／２４）,荧光假单胞菌（９／２４）,鳗弧菌（７／２４）。本次试验直接从水样中检出６株对４种宿主菌均有裂解作用的蛭弧菌,为进一步利用蛭弧菌防治鱼类常见细菌性疾病提供了可用资料。     

Importance of Bdellovibrio in regulating microbial cenoses and self-purification processes in domestic sewage

The bacterial parasite Bdellovibrio was directly proved to be involved in the regulation of microbial cenoses and in the self-purification of domestic waste waters. The incidence of heterotrophs, Gram-negative bacteria, E. coli and Bdellovibrio was followed up in dynamics in the microecological system of waste waters for ten days. In control experiments, bdellovibrions were removed using pteridine as a vibriostatic agent. In the absence of bdellovibrions, the cell number of the studied microorganisms did not increase after reaching a stationary level. In the control, the total incidence of heterotrophs decreased 1355 times, that of Gram-negative bacteria fell down 527 times, and that of E. coli cells dropped 3419 times due to the interaction between the host bacteria and Bdellovibrio. The variations in the number of interacting cells were characteristic of a two-component parasite-host system.     

The incidence of Bdellovibrio spp. in man-made water systems: coexistence with legionellas

Bdellovibrios have been isolated from surface waters but there are no reports of its occurrence in mains water supplies. One hundred and thirty five water samples from 81 sources were examined for the presence of Bdellovibrio bacteriovorus and Legionella spp. Bdellovibrios were isolated by a double-layer agar technique with two strains of Legionella pneumophila serogroup 1 as the host organisms. Bdellovibrio spp. were isolated from 57.8% and Legionella spp. from 9.5% of the samples. The two species occurred together in 4.4% of samples. The incidence of Bdellovibrio spp. and its occurrence with legionellas in man-made water systems is discussed.     

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.     

Further taxonomic characterization of the genus Bdellovibrio.

Cultures of Bdellovibrio isolated from different geographic locations have been studied in terms of deoxyribonucleic acid analysis (% G + C, genome size, and DNA hybridization), cytochrome spectrum, and host range. Isolates of the genus exhibit a broad range of % G + C ranging from 37 to 51% and the genome sizes extend from 1300 x 10(6) to 1700 x 10(6) daltons. DNA hybridization continues to reveal a high level of genetic heterogeneity. Bdellovibrio 3294 exhibits 32% relative reassociation to Bdellovibrio W, 37% to Bdellovibrio stolpii Uki2, and an undetectible level to Bdellovibrio starrii A3.12 Bdellovibrio W is 23% related to B. starri A3.12 and 28.5% to B. stolpii Uki2. For the first time differential absorption techniques have revealed peaks of cytochrome b. The analysis of the cytochrome spectrum seems to be limited as a taxonomic tool since most of the recent isolates studied share a common cytochrome spectrum. Host-range studies have been found to be dependent on the experimental conditions, and with the exception of one isolate (B. starrii A3.12) the taxonomic significance of such techniques must be taken with caution.     

The incidence of Bdellovibrio spp. in man-made water systems: coexistence with legionellas

Bdellovibrios have been isolated from surface waters but there are no reports of its occurrence in mains water supplies. One hundred and thirty five water samples from 81 sources were examined for the presence of Bdellovibrio bacteriovorus and Legion-ella spp. Bdellovibrics were isolated by a double-layer agar technique with two strains of Legionella pneumophila serogroup 1 as the host organisms. Bdellovibrio spp. were isolated from 57·8% and Legionella spp. from 9·5% of the samples. The two species occurred together in 4·4% of samples. The incidence of Bdellovibrio spp. and its occurrence with legionellas in man-made water systems is discussed.     

Susceptibility of biofilms to Bdellovibrio bacteriovorus attack

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of Bdellovibrio as low as 10(2) PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of B. bacteriovorus to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh Bdellovibrio; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by Bdellovibrio but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments B. bacteriovorus was able to decrease the biomass of both E. coli and Pseudomonas fluorescens biofilms as determined by phase-contrast and epifluorescence microscopy.     

Molecular parasitism in the Escherichia coli-Bdellovibrio bacteriovorus system: translocation of the matrix protein from the host to the parasite outer membrane

During the intracellular maturation in Escherichia coli of the parasite Bdellovibrio bacteriovorus the outer membrane, major protein I of E. coli (i.e., the matrix protein) becomes associated with the outer membrane of the emerging parasite cells. The binding properties of this protein with the outer membrane of the host and of the parasite are identical. An analogous phenomenon also occurs during Bdellovibrio parasitism on Klebsiella pneumoniae and on Salmonella typhimurium. Possible roles for this scavenging action of Bdellovibrio, and similar phenomena in other parasitic systems, are discussed.     

Role of carbohydrate-protein recognition in the process of Bdellovibrio attaching to bacterial host cells

The authors discuss the role of carbohydrates in the surface layer of the cell wall in the process of Bdellovibrio attachment to host-bacteria cells. The paper presents the results of inhibitory analysis using sugars conducted with two model systems as well as the data about the effectiveness of the interaction between parasite cells and host cells after the modification of their surface polysaccharide layer with concanavalin A and sodium periodate.     

Predatory prokaryotes: an emerging research opportunity

Predatory prokaryotes have evolved a unique strategy of obtaining energy and biosynthetic materials from their surroundings: acquiring them from other living bacterial cells. These types of microbes have been found in a diverse variety of environments, and may play an important role in modulating microbial population structure and dynamics, as has been hypothesized for marine viruses and possibly protists. Only one genus of predatory bacterium, Bdellovibrio, has been extensively described and studied, though several other examples have been reported in the literature. In this review, the four basic strategies used by currently described predatory prokaryotes will be discussed: "wolfpack" group predation, epibiotic attachment, direct cytoplasmic invasion, and periplasmic invasion. Special adaptations to each approach will be considered, and compared overall to the genetic and biochemical characteristics of symbiotic or pathogenic prokaryotes living within eukaryotic cells. Two specific examples of predatory microbes, Bdellovibrio and Ensifer, will be described in terms of predation strategy, association with host cells, and host range. The prospects for bringing to bear the tools of molecular microbial genetics to the study of predatory prokaryotes will be explored, using current research with Bdellovibrio and Ensifer as examples.     

Taxonomic studies of predatory bdellovibrios based on 16S rRNA analysis, ribotyping and the hit locus and characterization of isolates from the gut of animals

The aim of our study was to obtain data for the molecular characterization of bdellovibrio bacteria, which were recently split into the genus Bdellovibrio and the newly designated genus Bacteriovorax. We determined the 16S rDNA sequences of five reference strains and performed a phylogenetic analysis including published 16S rRNA sequences of bdellovibrios. A comparison of the secondary structure showed significant differences in two regions of the 16S rRNAs of the species Bdellovibrio bacteriovorus, Bacteriovorax starrii, and Bacteriovorax stolpii. In addition, ribotyping techniques gave specific hybridization patterns and revealed that two rRNA operons are present in the investigated strains. A hybridization probe derived from the genetic locus hit, associated with the host independent (HI) phenotype of B. bacteriovorus, was found to be specific for this species. Sequence comparison of the hit locus revealed few base pair changes between host independent (HI) and host dependent (HD) strains. Ribotyping and hybridization experiments using the hit probe were applied to characterize bdellovibrio strains isolated from the gut of animals and humans and one isolate from sewage.     

Peculiarities of the fatty acid composition ofBdellovibrio

The fatty acid composition of twelve Bdellovibrio strains isolated upon the growth on bacteria of various taxonomic groups was studied. A dependence of the lipid composition of bdellovibrios on that of bacteria they were parasitizing on was shown. Data pointing to the selective incorporation of fatty acids of host bacteria by bdellovibrios were obtained. Bdellovibrio membranes were shown to contain monounsatured fatty acids with different positions of double bonds indicating that there are at least two alternative mechanisms of synthesis of these acids in the parasites.