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.     

Biosynthesis of oxygen-detoxifying enzymes in Bdellovibrio stolpii.

Axenically grown Bdellovibrio stolpii (i.e., grown independently of the host) was examined for superoxide dismutase, catalase, and peroxidase activities. Kinetics of enzyme synthesis were determined for aerobically grown cultures and for cultures exposed to 100% oxygen. Enzymatic activities varied with the age of the culture. Normally grown cultures exhibited maximum activity during the first 10 h of growth and again as the stationary phase was approached, beginning at about 48 h. Polyacrylamide gel electropherograms of cell-free extracts revealed that B. stolpii contained one major band (1) and two minor bands (II, III) of superoxide dismutase activity. Each of these enzymes was inactivated by H2O2, indicating that they were iron-containing enzymes. Manganese-containing superoxide dismutase was not detected in B. stolpii. Increased oxygenation did not appreciably stimulate enzyme synthesis, for only superoxide dismutase was induced, reaching maximum activity at 10 h and then rapidly falling to normal levels. Superoxide dismutase appears to be the main enzymatic defense against oxygen toxicity in B. stolpii. Induction of superoxide dismutase with 100% oxygen was manifested as an increase in the intensities of the two minor bands of activity, suggesting that isozyme I is constitutive, whereas isozymes II and III are inducible. The induction of isozymes II and III by 100% oxygen was prevented by an inhibitor of protein biosynthesis, chloramphenicol.     

Chemotaxis in Bdellovibrio bacteriovorus

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

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.     

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.     

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.     

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.     

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.     

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).     

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.     

Elongation and cell division in Bdellovibrio bacteriovorus

Elongation and division of Bdellovibrio bacteriovorus were studied in axenic synchronous cultures. The cells elongate unidirectionally from one end attaining a length of several unit cells, and then divide into the corresponding number of cells. The length of the filament and, consequently, the progeny number, vary within the range of two to several dozen cells, according to the conditions used. A protein and a low molecular weight component are required for normal division.     

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.     

Bdellovibrio bacteriovorus Parasitizing Rhizobium in Western Australia

S ummary . Bdellovibrio bacteriovorus strains which parasitize Rhizobium meliloti, R. trifolii, Agrobacterium tumefaciens and A. radiobacter have been found in Western Australian soils. Rhizobium lupini was not lysed by any strain. Some Bdellovibrio cells pass through 0·22 μm membrane filters, whilst the 0·45 μm filters allow several saprophytic soil bacteria to pass. The strength of the medium is shown to be of great importance for the detection or counting of Bdellovibrio cells. The Bdellovibrio strains are capable of destroying large numbers of rhizobia in the laboratory.     

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.     

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.