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.     

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.     

Occurrence of Phosphonosphingolipids in Bdellovibrio bacteriovorus Strain UKi2

The major phospholipids of two strains of Bdellovibrio bacteriovorus were characterized. Both strain UKi1, which is obligately saprophytic, and strain UKi2, which is facultatively parasitic, contained phosphatidylethanolamine and phosphatidylglycerol as their major glycerophosphatides. A branched, 15-carbon fatty acid is the major component of these alkali-labile lipids. Absent from UKi1 but present in UKi2 were three alkali-stable lipids (compounds 8, 9, and 11) which appear to be phosphosphingolipids. After acid hydrolysis, both compound 8 and 9 yield the identical phosphorus-containing substance that is water soluble, dipolar ionic, and ninhydrin positive. This substance appears to contain a C-P bond since P(i) could not be released from this substance by treatment with alkaline phosphatase or by very harsh mineral acid treatment. Based on chromatographic comparisons, this phosphonate appears to be a novel lipid constituent. Upon degradation, compound 8 yields 1 mol of dihydroxy long-chain base and compound 9 yields 1 mol of a trihydroxy long-chain base. These bases appear to have a 17-carbon, possibly branched, structure based on gas-liquid chromatography retention times. Degradation of both sphingolipids yields a mixture of hydroxy fatty acids, the major component being a branched, 15-carbon hydroxy acid.     

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.     

Respiration of Bdellovibrio bacteriovorus Strain 109J and Its Energy Substrates for Intraperiplasmic Growth

Measurements of oxidation rates, respiratory quotients (RQ), and release of (14)CO(2) from uniformly labeled substrates showed that glutamate, alpha-ketoglutarate, and synthetic and natural amino acid mixtures are oxidized by suspensions of Bdellovibrio bacteriovorus strain 109J. The oxidation of these substrates largely suppress the endogenous respiration of the Bdellovibrio cells and may or may not cause a small increase, 20 to 50%, in their rate of oxygen consumption. The failure of respired substrates to increase markedly the respiration rate of the Bdellovibrio cells over the endogenous value is discussed. Carbon from these substrates is incorporated into the Bdellovibrio cells during oxidation. Acetate is also oxidized, but its oxidation inhibits endogenous respiration by only about 40% and no acetate is assimilated. The RQ of the Bdellovibrio cells changes from a value characteristic of endogenous respiration to that characteristic of the oxidation of glutamate or of a balanced amino mixture very shortly after the attack of the Bdellovibrio cells on their prey, and the latter RQ is maintained during intraperiplasmic growth. Glutamate, or a mixture of amino acids in the external environment, contributes to the carbon dioxide produced by the Bdellovibrio cells growing intraperiplasmically. It is concluded from these data that amino acids, derived from the breakdown of the protein of the prey, serve as a major energy source during intraperiplasmic growth of B. bacteriovorus 108J. Insofar as they were tested, B. bacteriovorus strains 109D and A. 3. 12 were similar in respiration to strain 109J.     

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.     

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 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.     

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 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.     

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。     

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.     

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.