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.     

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.     

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.     

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.     

Molecular Heterogeneity of the Bdellovibrios: Evidence of Two New Species

A systematic examination of a variety of isolates of the bacterial endoparasite Bdellovibrio has revealed extensive molecular diversity. The quantity of deoxyribonucleic acid (DNA) polynucleotide homology ranges from more than 90% among the isolates with DNA containing 50 to 51% guanine plus cytosine (GC) to undetectable levels between the 43% GC and 51% GC isolates. The two isolates with low GC-containing DNA (H-I Bdellovibrio A3.12 and UKi2) have only 16% DNA homology. H-I Bdellovibrio A3.12 and 109 have barely detectable ribosomal ribonucleic acid (rRNA) homology, whereas the homology approaches 100% among all the high GC isolates tested. Cases of high DNA/DNA and DNA/rRNA homologies are reflected in low dissimilarities of enzyme migration patterns in starch gel electrophoresis. The dissimilarities exhibited among the high GC Bdellovibrio isolates are as low as those previously reported for different Escherichia coli strains. The zymograms of H-I Bdellovibrio A3.12 and UKi2 are completely different from each other as well as from all other bdellovibrios (100% dissimilarity). Genome sizes determined for the representative isolates demonstrate three size ranges which coincide with group differences based on the above measurements. Enzyme assays reveal that all isolates possess a tricarboxylic acid cycle and most contain an alanine and glutamic dehydrogenase. We conclude that the use of bacterial endoparasitism as a defining trait has resulted in a molecularly diverse collection of isolates. It is recommended that the specific epitaph bacteriovorus be used only for the type specimen (Bdellovibrio 100 of Stolp and Starr, 1963) and for other related 50 to 51% GC isolates. The heterogeneity of the group warrants two new species. We designate Bdellovibrio A3.12 as the nomenclatural type of B. starrii sp. n. and Bdellovibrio UKi2 as the nomenclatural type of B. stolpii sp. n.     

Verification of the protein in the outer membrane of Bdellovibrio bacteriovorus as the OmpF protein of its Escherichia coli prey.

Two research groups showed that several Bdellovibrio strains incorporated into their outer membranes intact OmpF porin proteins derived from their Escherichia coli prey. These results could not be reproduced by another group using Bdellovibrio bacteriovorus 109J. They showed that a major protein appearing in the Bdellovibrio Triton X-100-insoluble outer membrane was coded for by the bdellovibrios. We reconciled these results by examining the strain used by this group and by reviving a freeze-dried culture of strain 109J which had been stored for almost 9 years. B. bacteriovorus 109J failed to acquire substantial amounts of the OmpF protein from E. coli ML35, and a protein coded for by the bdellovibrios was expressed in its place. However, B. bacteriovorus 109J incorporated the OmpF protein from rough K-12 strains of E. coli, and the revived 9-year-old culture of B. bacteriovorus 109J incorporated more of the OmpF protein from the smooth E. coli ML35 than did its contemporary counterpart. The protein isolated from the outer membrane of the bdellovibrios was identified as the OmpF protein of E. coli by its protease peptide profile on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western blot analysis. This confirmed that bdellovibrios relocalize outer membrane proteins from their prey, but relocalization may be an unstable trait which can be influenced by the prey.     

A novel assay to monitor predator-prey interactions for Bdellovibrio bacteriovorus 109 J reveals a role for methyl-accepting chemotaxis proteins in predation

Bdellovibrio bacteriovorus are Gram-negative bacteria that prey upon other Gram-negative bacteria, including some pathogens, in a wide variety of habitats including soil, sewage, marine and estuarine environments. In order to facilitate studies on predation by this organism, we have developed a method that assays killing of luminescent Escherichia coli by B. bacteriovorus. Moreover, we have used this assay to compare predation of cells by derivatives of B. bacteriovorus containing targeted mutations in genes we have identified. Two genes are described; one, mcp2, encoding a methyl-accepting chemotaxis protein (MCP) and the other, an mviN homologue. Bdellovibrio bacteriovorus mcp2::aphII were less efficient predators on luminescent E. coli than B. bacteriovorus containing a randomly inserted aphII gene via TnphoA transposition. These and other chemotaxis experiments implicated at least a minor role for chemotaxis in predation by B. bacteriovorus. They also open the way for further studies on Bdellovibrio ecology, genomics and predator-prey interactions. The results further confirm that Bdellovibrio uses a chemotaxis system in order to sense, and respond to, changes in its environment, including prey.     

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.     

Penicillin-binding proteins of bdellovibrios.

We examined the predacious gram-negative bacterium Bdellovibrio bacteriovorous 109J and free-living strains 109J-A1 and 109J-KA1 derived therefrom for penicillin-binding proteins (PBPs). We compared their PBPs with those of the host bacterium, Escherichia coli, and with those of a facultatively predacious bdellovibrio, B. stolpii UKi2, grown axenically. The multiple PBPs of the 109J strains and of UKi2 differed from each other and from those of E. coli, which suggests that screening for PBPs may be a convenient way to determine to what extent the bdellovibrios may represent a diverse group of organisms. A method for labeling furazlocillin and cefaperizone with iodine-125 is also described.     

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.     

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.     

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.     

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.     

Effect of paracrystalline protein surface layers on predation by Bdellovibrio bacteriovorus.

We determined that paracrystalline protein surface arrays (S layers) protected gram-negative eubacteria from predation by Bdellovibrio bacteriovorus. Aquaspirillum serpens VHA and MW5 and Aquaspirillum sinuosum were resistant to predation by B. bacteriovorus 6-5-S when fully covered by their S layers. The S layer of Aeromonas salmonicida A449 protected the cells from predication by B. bacteriovorus 109J. A predacious, plaque-forming vibrio that lysed an S-layer- variant of Caulobacter crescentus but was not predacious on the parental strain which possessed an S layer was isolated from raw sewage. Since S layers are stable components of many bacterial surfaces in nature, they can provide this protective function in both aquatic and terrestrial habitats where Bdellovibrio spp. are found.     

Nonidentity of Bdellovibrio bacteriovorus Strains 109D and 109J

Two strains of Bdellovibrio bacteriovorus, both designated as 109 in the literature, differ. They should be referred to as 109D and 109J to avoid further confusion.     

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.     

Attempts to grow bdellovibrios micurgically-injected into animal cells

Incubation in buffer of Bdellovibrio bacteriovorus 109J, B. stolpii UKi2, or B. starrii A3.12 with washed eucaryotic animal cells (mouse liver, hamster kidney, or bovine mammary gland) resulted in neither attachment nor growth of the bdellovibrios. When cells of these bdellovibrio strains were incubated with erythrocyte suspensions (bovine or rabbit) a very low level of bdellovibrio attachment and penetration occurred, but no growth could be detected. Using micurgical procedures, bdellovibrios were injected into the perivetelline space or the cytoplasm of rabbit ova. After 18–24h incubation, neither a significant loss nor increase of injected, intracellular bdellovibrios was observed. Limited axenic growth of bdellovibrios (109J or UKi2) occurred in media containing rabbit ova extracts and dilute nutrient broth. It is concluded that eucaryotic rabbit ova do not provide a suitable environment for intracellular bdellovibrio growth.     

Penicillin-induced formation of osmotically stable spheroplasts in nongrowing Bdellovibrio bacteriovorus

Bdellovibrio peptidoglycan is of typical gram-negative composition. The molar ratios of alanine:glutamic acid:diaminopimelic acid:muramic acid:glucosamine were about 2:1:1:1:1. Nascent, nongrowing Bdellovibrio bacteriovorus 109J were converted from highly motile vibrios to highly motile spheres when shaken in dilute buffer plus penicillin, cephalothin, bacitracin, or D-cycloserine. The spherical forms contained essentially no sedimentable peptidoglycan; i.e., they were spheroplasts. Spheroplasts induced by penicillin, D-cycloserine, and lysozyme were stable in dilute buffer and did not lyse when subjected to osmotic shock. Normal Bdellovibrio suspended in buffer turned over their peptidoglycan at a rate of approximately 30% h during the initial 120 min of starvation. Chloramphenicol and sodium azide strongly inhibited Bdellovibrio peptidoglycan turnover and the induction of spheroplasts by penicillin. The data indicate that nongrowing B. bacteriovorus are sensitive to penicillin and other antibiotics affecting cell walls because of their high rate of peptidoglycan turnover. It is also concluded that an intact peptidoglycan layer is required for maintaining cell shape, but is not required for osmotic stability of B. bacteriovorus.     

Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.     

Isolation and Preliminary Characterization of Bacteriophages for Bdellovibrio bacteriovorus

Ten bacteriophages that attack and lyse saprophytic strains of Bdellovibrio bacteriovorus were isolated. Morphological, serological, and host-range studies revealed that there were four different bdellovibrio phages present among the isolates. One of the phages lysed a strain of B. bacteriovorus that requires the presence of a suitable bacterial host for growth. The phage attached to the bdellovibrio cells in the absence of the bacterial host cells; lysis occurred only in the presence of host cells. The 19 saprophytic bdellovibrio strains employed in the phage host-range studies were grouped on the basis of their susceptibility to phage lysis.