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.     

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.     

Intracellular development of 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.     

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.     

Artificial immobilization of the two-membered bacterial system Bdellovibrio bacteriovorus 109D--Escherichia coli B in polyacrylamide gel

The interaction of a parasite with a host was studied in the two-membered bacterial system, Bdellovibrio bacteriovorus 109D and Escherichia coli B, immobilised in polyacrylamide gel (PAAG). The parasite localised inside the host cells was found to be more resistant to the toxic action of PAAG components than free B. bacteriovorus. The latter lost its mobility and was inactivated in the matrix of the carrier whereas the intracellular parasite had a normal cycle of development in the periplasm of the infected cells. The dynamics of B. bacteriovorus and E. coli incidence in the liquid phase and in PAAG granules was studied while the immobilised system was incubated. The interaction in the immobilised system could be intensified by growing more bacterial host cells in PAAG particles. The immobilisation was shown to favour the survival of the parasite and the host in the two-membered system.     

Early host damage in the infection cycle of Bdellovibrio bacteriovorus

The effects of bdellovibrio infection on host permeability and respiration were investigated by measuring respiration rates and the rate of o-nitrophenyl-beta-d-galactopyranoside hydrolysis during the course of single infection cycles of Bdellovibrio bacteriovorus strain 109 growing on Escherichia coli ML 35 (lac i(-)z(+)y(-)). The data show that among the very early consequences of parasite attack on the host are an increase in permeability and a general disruption of respiratory activity of the host, and it is suggested that both phenomena stem from early damage to host membrane. The rapid onset of damage after inception of the cycle and the failure of streptomycin to prevent the damage indicate that complete penetration of the parasite into the host is not a requirement for the observed effects. The data also show that bdellovibrio does not use host energy-generating mechanisms for its growth and suggest that the parasites may have a search mechanism that permits them, to some degree, to distinguish between infected and uninfected hosts.     

Bdellovibrio bacteriovorus gen. et sp. n., a predatory,ectoparasitic, and bacteriolytic microorganism

Bdellovibrio bacteriovorus, gen. et sp. n., a predatory and ectoparasitic microorganism with lytic activity against susceptible bacteria, is described, as are techniques for isolation and cultivation. These unusual bacteria cause reactions that are similar in their outward manifestations to bacteriophage-induced lysis. Upon plating a mixture of host bacteria and parasites, confluent lysis or single plaque formation occurs, just as in titration experiments with bacteriophage. However, the parasite plaques develop more slowly than phage plaques. Lysis of host bacteria in liquid culture is accompanied by a decrease in optical density; actually, a population of infected host bacteria is replaced by a population of the tiny parasite.Individual cells of the presently known strains ofBdellovibrio bacteriovorus are typically about 0.3 µ in width and, thus, are considerably narrower than ordinary bacteria. Therefore, they can pass Millipore filters of 0.45 µ pore size diameter. Their shape is often vibrio-like. They possess one unusually thick polar flagellum of about 50 mµ diameter, and they show a distinctive type of motility.The interaction betweenBdellovibrio and the attacked host bacterium can be followed in the phase-contrast microscope; it is characterized by a physical attack of the highly motile parasite, attachment to the bacterial cell surface, and lysis of the host cell.It has not yet been possible to cultivateBdellovibrio in its parasitic form on any artificial substrate. All parasitic strains require living host cells for their propagation. However, saprophytic mutants can be selected from a population of the parasite. These saprophytic derivatives are unable to lyse living bacteria as does the wild-type parasite. On the basis of morphological and physiological properties, a saprophyte strain which has been examined in some detail shows no close relationship to any of the already known categories of bacteria.A study of the kinetics of growth ofBdellovibrio in mixed culture with a susceptible host has disclosed that the number of parasites produced is not proportional to the number of host bacteria killed during the same period. After the majority of the host cells has been destroyed, there is still a considerable increase in parasites, indicating that they grow at the expense of material released from the lysed bacteria. Under the conditions of this trial, the generation time is about 100 minutes.All presently known isolates ofBdellovibrio possess lytic activity only against gram-negative bacteria. The individual strains, however, show certain differences in their host activity spectra; some have a restricted host range, while others are able to attack a broad spectrum of host bacteria.     

Bdellovibrio bacteriovorus strains produce a novel major outer membrane protein during predacious growth in the periplasm of prey bacteria

Bdellovibrio bacteriovorus is a predatory bacterium that is capable of invading a number of gram-negative bacteria. The life cycle of this predator can be divided into a nonreproductive phase outside the prey bacteria and a multiplication phase in their periplasm. It was suggested that during the reproduction phase, B. bacteriovorus reutilizes unmodified components of the prey's cell wall. We therefore examined the outer membranes of B. bacteriovorus strains HD100 (DSM 50701) and HD114 (DSM 50705) by using Escherichia coli, Yersinia enterocolitica, and Pseudomonas putida as prey organisms. The combined sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses revealed novel and innate major outer membrane proteins (OMPs) of B. bacteriovorus strains. An incorporation of prey-derived proteins into the cell wall of B. bacteriovorus was not observed. The corresponding genes of the B. bacteriovorus strains were elucidated by a reverse-genetics approach, and a leader peptide was deduced from the gene sequence and confirmed by Edman degradation. The host-independent mutant strain B. bacteriovorus HI100 (DSM 12732) growing in the absence of prey organisms possesses an OMP similar to the major OMPs of the host-dependent strains. The similarity of the primary structure of the OMPs produced by the three Bdellovibrio strains is between 67 and 89%. The leader peptides of all OMPs have a length of 20 amino acids and are highly conserved. The molecular sizes of the mature proteins range from 34.9 to 37.6 kDa. Secondary-structure predictions indicate preferential alpha-helices and little beta-barrel structures.     

Ultrastructure and Cell Division of a Facultatively Parasitic Strain of Bdellovibrio bacteriovorus

Some aspects of cell development and division of Bdellovibrio bacteriovorus strain UKi2 were examined by use of electron microscopic techniques. Under saprophytic and parasitic conditions of growth, the comma-shaped cells enlarge, elongate, and form helical filaments. The mechanism of division appears to consist of an asymmetrical constriction of the filamentous cell by the cytoplasmic membrane, accompanied by a breakdown of the outer layers of the cell wall in the division region. During regeneration of the cell wall, the flagellum and flagellar sheath are formed. The development of the flagellum of the daughter cell is initiated prior to separation of the newly formed cells from the filament. Observations of B. bacteriovorus UKi2 grown under saprophytic and parasitic conditions indicate that development and ultrastructure are similar in both modes of growth.     

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.     

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。     

A conjugation procedure for Bdellovibrio bacteriovorus and its use to identify DNA sequences that enhance the plaque-forming ability of a spontaneous host-independent mutant.

Wild-type bdellovibrios are obligate intraperiplasmic parasites of other gram-negative bacteria. However, spontaneous mutants that can be cultured in the absence of host cells occur at a frequency of 10(-6) to 10(-7). Such host-independent (H-I) mutants generally display diminished intraperiplasmic-growth capabilities and form plaques that are smaller and more turbid than those formed by wild-type strains on lawns of host cells. An analysis of the gene(s) responsible for the H-I phenotype should provide significant insight into the nature of Bdellovibrio host dependence. Toward this end, a conjugation procedure to transfer both IncQ and IncP vectors from Escherichia coli to Bdellovibrio bacteriovorus was developed. It was found that IncQ-type plasmids were capable of autonomous replication in B. bacteriovorus, while IncP derivatives were not. However, IncP plasmids could be maintained in B. bacteriovorus via homologous recombination through cloned B. bacteriovorus DNA sequences. It was also found that genomic libraries of wild-type B. bacteriovorus 109J DNA constructed in the IncP cosmid pVK100 were stably maintained in E. coli; those constructed in the IncQ cosmid pBM33 were unstable. Finally, we used the conjugation procedure and the B. bacteriovorus libraries to identify a 5.6-kb BamHI fragment of wild-type B. bacteriovorus DNA that significantly enhanced the plaque-forming ability of an H-I mutant, B. bacteriovorus BB5.     

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.     

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.     

Chemotaxis in Bdellovibrio bacteriovorus

Chemotaxis toward yeast extract is demonstrated in obligately and facultatively parasitic strains of Bdellovibrio bacteriovorus.     

Facultatively Parasitic Strain of Bdellovibrio bacteriovorus

A strain of Bdellovibrio bacteriovorus (designated strain UKi2) was isolated which was capable of growing either saprophytically in host-free medium or endoparasitically in Escherichia coli B/r. It was quantitatively determined that each bdellovibrio could develop in solid medium to produce a colony, and 65% of the cells in a late exponential-phase culture were capable of inducing E. coli B/r spheroplasts. A photomicrographic sequence of single E. coli spheroplasts containing bdellovibrios demonstrated that parasitically derived B. bacteriovorus UKi2 could develop saprophytically after release from the host cells. Strain UKi2 appears to be morphologically quite similar to previously described obligately parasitic bdellovibrios; biochemical data on this strain suggests its close relationship to some of the previously described host-independent strains of Bdellovibrio.     

Comprehensive analysis of transport proteins encoded within the genome of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a bacterial parasite with an unusual lifestyle. It grows and reproduces in the periplasm of a host prey bacterium. The complete genome sequence of B. bacteriovorus has recently been reported. We have reanalyzed the transport proteins encoded within the B. bacteriovorus genome according to the current content of the Transporter Classification Database. A comprehensive analysis is given on the types and numbers of transport systems that B. bacteriovorus has. In this regard, the potential protein secretory capabilities of at least four types of inner-membrane secretion systems and five types of outer-membrane secretion systems are described. Surprisingly, B. bacteriovorus has a disproportionate percentage of cytoplasmic membrane channels and outer-membrane porins. It has far more TonB/ExbBD-type systems and MotAB-type systems for energizing outer-membrane transport and motility than does Escherichia coli. Analysis of probable substrate specificities of its transporters provides clues to its metabolic preferences. Interesting examples of gene fusions and of potentially overlapping genes are also noted. Our analyses provide a comprehensive, detailed appreciation of the transport capabilities of B. bacteriovorus. They should serve as a guide for functional experimental analyses.     

Lysis of Sphaerotilus natans Swarm Cells by Bdellovibrio bacteriovorus

Six strains of Sphaerotilus natans (smooth form) were lysed by five parasitic strains of Bdellovibrio bacteriovorus. The possible use of Bdellovibrio to control the proliferation of S. natans in the environment was hypothesized.     

Kinetics of deoxyribonucleic acid destruction and synthesis during growth of Bdellovibrio bacteriovorus strain 109D on pseudomonas putida and escherichia coli

During the growth of Bdellovibrio bacteriovorus on Pseudomonas putida or Escherichia coli in either 10(-3)m tris(hydroxymethyl)aminomethane or in dilute nutrient broth, the host deoxyribonucleic acid (DNA) was rapidly degraded, and by 30 to 60 min after the initiation of the bdellovibrio development cycle essentially all host DNA became nonbandable in CsCl gradients. At this stage the host DNA degradation products were nondiffusable, and there was no appreciable pool of low-molecular-weight (cold acid soluble) DNA fragments in the cells or in the suspending medium. Bdellovibrio DNA synthesis occurred only after degradation of host DNA to a nonbandable form was complete. The synthesis occurred in a continuous fashion with P. putida as the host and in two separate periods with E. coli as host. By using E. coli containing a (3)H-thymidine label, it was shown that 73%, on the average, of the thymine residues of host DNA were incorporated into bdellovibrio DNA when E. coli was the only source of nutrient. In the presence of dilute nutrient broth, the host cells still served as the major source of precursors for bdellovibrio DNA synthesis, with only 20% of the precursors arising from the exogenous nutrients. The data indicate an efficient and controlled utilization of host DNA by the bdellovibrio. The host DNA is apparently degraded early in the developmental cycle to oligonucleotides of intermediate molecular weight from which the biosynthetic monomers are generated only as they become needed for bdellovibrio DNA synthesis.