Electron Microscopic Observations on the Penetration of Bdellovibrio bacteriovorus into Gram-negative Bacterial Hosts

The progressive stages in Bdellovibrio bacteriovorus penetration into two strains of Escherichia coli were examined by use of electron microscopic techniques. The initial change observed in the ultrastructure of the host following parasitic attack was the swelling of the cell envelope at the site of attachment. The Bdellovibrio then appeared to pierce the center of this swelling, forming a pore in the outer wall layers of the host. The edges of this entry pore constricted the Bdellovibrio throughout its penetration into the host cell. Although partial disruption of the cytoplasmic membrane was always apparent, the parasite did not appear to actively penetrate through this barrier. An attempt is made to correlate the fine structural changes involved in penetration with the physiological data that have accumulated to date.     

Host receptor sites involved in the attachment of Bdellovibrio bacteriovorus and Bdellovibrio stolpii

Abstract The location of lipoteichoic acid and 3 cell wall-associated protein antigens in fresh isolates of Streptococcus mutans (serotype c ) and in variants derived from these parental strains by repeated subculture in vitro has been examined by electrophoretic and dot-immunobinding techniques. Following subculture there was a marked shift in the distribution of all four antigens from the cell wall to the extracellular culture supernatants. It is therefore apparent that that the decreased hydrophobicity and the impaired ability of the subcultured variants to colonise in vivo, as observed by others, cannot be correlated with changes in a single surface molecule.     

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.     

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.     

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.     

Chemotaxis in Bdellovibrio bacteriovorus

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

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.     

Energy metabolism of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus, strain Bd. 109 Sa, generates ATP mainly by oxidative phosphorylation during electron transport. During exponential growth the ATP pool is constant (9 nmoles/100 μg N) indicating that energy-producing and energy-consuming reactions are well balanced. The ratio of substrate respiration/endogenous respiration is approx. 2.5/1. Energy charge is constant both in endogenous and substrate respiration at values of 0.62 to 0.64. During endogenous respiration (starvation) the ATP pool oscillates at regular intervals. ATP over-production is started after the ATP pool has decreased to a minimum level of 6 nmoles/100 μg N. The alternating over- and under-production of ATP is interpreted as a special regulation which enables the organism to make economic use of its own cellular materials. Addition of substrate (glutamate) to starving cells does not influence the type of ATP pool oscillation as observed in endogenous respiration. The parasitic strain Bd. 109 Pa exhibits the same periodicity of ATP overproduction as does its saprophytic derivative, Bd. 109 Sa. Decrease of viability during starvation is paralleled by a decrease of the ATP pool.     

Energy metabolism of Bdellovibrio bacteriovorus

The P/O ratio of Bdellovibrio bacteriovorus, strain Bd 109 Sa, was evaluated by two different methods based on the determination of energy-rich phosphate bonds and either NADH oxidation or oxygen-uptake. P/O values calculated on the basis of NADH oxidation were up to 6, which has to be regarded as being overestimated. P/O values calculated from energy-rich phosphate bonds and oxygen uptake were around 2. The P/O values determined for Escherichia coli B were similar. The loss of phosphorylation efficiency at one site is discussed.The ATP pool turnover rate of Bdellovibrio was 8/min during endogenous respiration and 24/min during substrate respiration. The corresponding values in Escherichia coli B were 3/min and 38/min.This study was performed at the University of Hamburg (Institut für Allgemeine Botanik, Abteilung Mikrobiologic).     

Periplasmic enzymes in Bdellovibrio bacteriovorus and Bdellovibrio stolpii.

When cells of either Bdellovibrio bacteriovorus 109J or Bdellovibrio stolpii UKi2 were subjected to osmotic shock by treatment with sucrose-EDTA and MgCl2 solutions, only trace amounts of proteins or enzyme activities were released into the shock fluid. In contrast, when nongrowing cells were converted to motile, osmotically stable, peptidoglycan-free spheroplasts by penicillin treatment, numerous proteins were released into the suspending fluid. For both species, this suspending fluid contained substantial levels of 5'-nucleotidase, purine phosphorylase, and deoxyribose-phosphate aldolase. Penicillin treatment also released aminoendopeptidase N from B. bacteriovorus, but not from B. stolpii. Penicillin treatment did not cause release of cytoplasmic enzymes such as malate dehydrogenase. The data indicated that bdellovibrios possess periplasmic enzymes or peripheral enzymes associated with the cell wall complex. During intraperiplasmic bdellovibrio growth, periplasmic and cytoplasmic enzymes of the Escherichia coli substrate cell were not released upon formation of the spherical bdelloplast during bdellovibrio penetration. Most of the E. coli enzymes were retained within the bdelloplast until later in the growth cycle, when they became inactivated or released into the suspending buffer or both.     

Rapid isolation of host-independent Bdellovibrio bacteriovorus

A new method of isolating host-independent Bdellovibrio bacteriovorus has been developed. Filtered suspensions of host-dependent cells are dropped in small volumes onto 0.2 μm membranes laid on rich media agar. Significant growth is observed within 1–2 days; these cells were confirmed to be B. bacteriovorus using microscopic observations and PCR.     

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.     

Effect of light on Bdellovibrio bacteriovorus.

Bdellovibrio underwent photooxidation by visible light in the presence of exogenous photosensitizer and by near-ultraviolet light (325 to 400 nm) in its absence. The colorless, host-dependent wild type was more sensitive to the lethal effect of light than was its pigmented, facultative parasitic mutant. The latter's ability to form colonies was much more sensitive to light than was its plaque-forming capability. The biosynthesis of the mutant pigment was inhibited by diphenylamine, though this inhibition did not result in additional sensitivity to photokilling.     

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 10² 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.     

Isolation of a Bacteriophage for Bdellovibrio bacteriovorus

A phage infective for Bdellovibrio bacteriovorus was isolated. Electron microscopy revealed that it is tail-less, has a hexagonal appearance and two distinct capsomere layers, and is 60 to 70 nm in size. The nucleic acid appears to be single-stranded deoxyribonucleic acid. This is the first report of the isolation of a phage infective for B. bacteriovorus.     

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.     

Differential Predation by Bdellovibrio bacteriovorus 109J

Bdellovibrio bacteriovorus is a predatory bacterium that can replicate only inside Gram-negative bacteria. We incubated B. bacteriovorus 109J in a mixture of two prey cells present in equal numbers and enumerated prey cells after 3 h of predation. In multiple prey pairings, B. bacteriovorus preferentially lysed on one prey over the other. When prey were individually incubated with B. bacteriovorus, they were preyed on with different efficiencies. Three prey had only 5–8% of cells remaining after Bdellovibrio predation and the other three prey had 37–43% of cells remaining. Timing of attachment of B. bacteriovorus to prey cells also varied with Bdellovibrio attachment to more preferred prey occurring the fastest. These results suggest that B. bacteriovorus 109J does not randomly infect prey cells but infects and kills some prey more readily than others.     

Chemotaxis by Bdellovibrio bacteriovorus toward prey.

A chemotaxis assay system that uses a modified Boyden chamber was characterized and used for measurements of chemotaxis by Bdellovibrio bacteriovorus strain UKi2 toward several bacterial species. Bacteria tested included both susceptible and nonsusceptible cells (Escherichia coli, Pseudomonas fluorescens, Bacillus megaterium, and B. bacteriovorus strains UKi2 and D). None was attractive to bdellovibrios when present at densities below 10(7) cells per ml. Chemotaxis toward E. coli was studied most extensively; under conditions that minimized effects of osmotic shock to the cells, E. coli and exudates from E. coli at densities as high as 10(8) cells per ml failed to elicit a chemotactic response. Cell-free filtrates from mixed cultures of bdellovibrios and E. coli neither attracted nor repelled bdellovibrios. The data indicate that bdellovibrios do not use chemotaxis to locate prey cells.     

噬菌蛭弧菌对嗜水气单胞菌裂解作用的研究

本文报道了8株噬菌蛭弧菌对河水中分离的9株嗜水气单胞菌的裂解作用。结果发现,噬菌蛭弧菌Bd32的裂解率为77.78％;Bd83、Bd98的裂解率为88.89％,其余5株噬菌蛭弧菌对9株嗜水气单胞菌全部裂解。