Osmoregulation in symbiosis-independent mutants of Bdellovibrio bacteriovorus.

Bdellovibrios capable of axenic growth grow in a cell-free medium at a rate considerably lower than that attainable in a two-membered culture with Escherichia coli. The axenic growth rate may be improved either by adjustment of the osmosity of the medium or by the addition of low concentrations of spermine.     

Symbiosis-independent and symbiosis-incompetent mutants of Bdellovibrio bacteriovorus 109J.

Symbiosis-independent (Sin) mutants were isolated from the symbiosis-dependent and symbiosis-competent (Sdcomp+) Bdellovibrio bacteriovorus 109J. Independently isolated Sin mutants were examined for their symbiosis competence and most were found to be comp+. Bdellovibrios comp- were selected from the Sincomp+ mutants. The Sincomp+ bdellovibrios are always at a selective disadvantage, either against Sincomp- bdellovibrios (in organic medium) or against Sdcomp+ bdellovibrios (in buffer with Escherichia coli cells).     

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.     

Chemotaxis in Bdellovibrio bacteriovorus

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

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.     

Isolation and characterization of temperature-sensitive mutants of host-dependent Bdellovibrio bacteriovorus 109D

A variety of temperature-sensitive mutants of host-dependent Bdellovibrio bacteriovorus 109D were selected after ethyl methane sulfonate mutagenesis. Mutants that demonstrated plaque-forming ability reversion frequencies of 10(-8) to 10(-9) were chosen for further study. Representatives of these mutants were then characterized by phase-contrast and electron microscopy, temperature-shifted one-step growth experiments, attachment kinetics, and macromolecular capabilities. Representative mutants demonstrate various types of blockage corresponding to the previously described morphological stages of Bdellovibrio predatious life cycle, i.e., attachment blockage (109D153), penetration blockage (109D3 and 109D48), and blockage of intracellular growth (109D4 and 109D152). The time of release from temperature repression for the mutant classes was found to correspond to the apparent morphological stage of blockage via temperature-shifted, one-step growth experiments. Mutants characterized as exhibiting blockage in the penetration or intracellular stages of the infection cycle exhibited, at the permissive and nonpermissive temperatures, kinetics of attachment to Escherichia coli WP2 similar to those of the wild type. One mutant, 109D153, exhibited depressed attachment at the restrictive temperature even though the Bdellovibrio cells were motile. The extent of 38.5 C attachment of 109D153 to E. coli is at the same level as that of wild-type 109D to Bacillus subtilis, a gram-positive, non-host organism. Subsequent detachments were revealed in the wild-type 109D-B. subtilis or mutant 109D153-Escherichia coli (38.5 C) cultures. These studies reveal a biphasic attachment phenomenon in the early interaction of Bdellovibrio with its host. It appears that, at the restrictive temperature, 109D153 is capable only of the initial, nonspecific type of attachment.     

Analysis of phenotypic diversity among host-independent mutants of Bdellovibrio bacteriovorus 109J

Host-independent (H-I) mutants of the obligate bacterial parasite Bdellovibrio bacteriovorus were isolated from wild-type strain 109J. Seven H-I mutants differed in morphological features such as cell length (2-30 microm) and shape (short or long spirals or rod-like), plaque size, and pigmentation (from almost colorless to bright orange). The mutants exhibited widely different growth capabilities in rich medium, with biomass doubling times and final biomass varying by a factor of two or more. Growth was always enhanced by the addition of host cell extract or divalent cations to the growth medium, but the effect varied widely between the mutants. Analysis of the hit region, mutations in which were previously proposed to be associated with the H-I phenotype, revealed that changes in the nucleotide sequence in this region occurred only in three of the seven mutants.     

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.     

Membrane-associated, energy-linked reactions in Bdellovibrio bacteriovorus.

Disrupted cells of Bdellovibrio bacteriovorus exhibited adenosine triphosphatase activity, 60 to 80% of which was in the soluble fraction. Dicyclohexylcarbodiimide did not inhibit the adenosine triphosphatase activity in membrane particles. The particles did not show energy-linked transhydrogenase activity. The activity of non-energy-linked transhydrogenase as well as the rate of oxygen consumption were higher in membrane particles of the host-independent strain than in the host-dependent strains. The uptake of amino acid uptake was inhibited by cyanide and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Valinomycin, in the presence of K+, did not inhibit the uptake, and only partial inhibition was exerted by arsenate and dicyclohexylarbodiimide. Sulfhydryl reagents inhibited amino acid uptake.     

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).     

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.     

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.     

Chemotaxis of Bdellovibrio bacteriovorus toward pure compounds.

Positive chemotaxis by Bdellovibrio bacteriovorus strain UKi2 was measured for 139 compounds. Twenty-one compounds were attractants; sensitive attraction was elicited by acetate, propionate, thioacetate, malonate, cis-oxalacetate, D-glucose-6-phosphate, acetyl coenzyme A, ammonium ion, barium ion, manganous ion, and potassium ion. Several of the attractants for B. bacteriovorus strain UKi2 also were attractants to strains 6-5-S and 114; however, strains 109D and 109J were not attracted by the compounds tested. Of 33 compounds tested, 8 were repellents for B. bacteriovorus strain UKi2: n-caproate, alanine, isoleucine, leucine, phenylalanine, tyrosine, cobaltous chloride, and hydronium ion. None of the organic repellents for strain UKi2 elicited repulson from strains 114 or 109D. However, all three strains of Bdellovibrio show aerotaxis. Several compounds were tested for their effects on viability and predacious growth of B. bacteriovorus strain UKi2. No simple correlation was found between attraction or repulsion and benefit or harm to bdellovibrios. The data are consistent with the view that in nature, the greatest survival value of chemotaxis for bdellovibros may be in aerotaxis, attraction to certain inorganic ions and acetate, and repulsion by hydronium ion.     

Chemotaxis toward amino acids by Bdellovibrio bacteriovorus.

Chemotaxis toward amino acids by Bdellovibrio bacteriovorous strain UKi2 was studied by the capillary technique of Adler (J. Gen. Microbiol. 74:77-91, 1973). Chemotaxis was shown to be optimal when the capillaries were incubated at between 15 and 40 degrees C for 30 min; the optimal pH was between 7.0 and 8.2. The chemotactic response was proportional to the density of the suspension of bdellovibrios up to a density of 10(8) cells/ml. B. bacteriovorus was attracted to L-asparagine, L-cysteine, L-glutamine, glycine, L-histidine, L-lysine, and L-threonine. The possible roles of chemotaxis in the life of B. bacteriovorus are discussed.     

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.