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.     

Translocation of an outer membrane protein into prey cytoplasmic membranes by bdellovibrios.

Within minutes of Bdellovibrio bacteriovorus attack on prey cells, such as Escherichia coli, the cytoplasmic membrane of the prey is altered. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified invaded prey cell (bdelloplast) membranes revealed the appearance of a noncytoplasmic membrane protein. This protein is not observed in preparations of noninvaded E. coli membranes and migrates in a manner similar to that of E. coli OmpF. Isoelectric focusing and two-dimensional gel electrophoresis of bdelloplast cytoplasmic membrane preparations also revealed the presence of a protein with electrophoretic properties similar to those of OmpF and the major Bdellovibrio outer membrane proteins. The protein appears in cytoplasmic membrane preparations within minutes of attack and persists throughout most of the intraperiplasmic developmental cycle. The appearance of this protein is consistent with our hypothesis that bdellovibrios translocate a pore protein into the bdelloplast cytoplasmic membrane to kill their prey and to gain access to the cytoplasmic contents for growth.     

Acquisition of Escherichia coli outer membrane proteins by Bdellovibrio sp. strain 109D

The ability of Bdellovibrio sp. to acquire the OmpF major outer membrane protein from its Escherichia coli prey was examined to determine if there were other outer membrane proteins which could or could not be acquired. Growth of bdellovibrios on mutant prey which were defective in the expression of outer membrane proteins revealed that Bdellovibrio sp. could acquire the OmpC protein in the absence of the OmpF protein. However, the OmpA, LamB, and protein 2 proteins could not be found in the Bdellovibrio Triton-insoluble outer membrane. The disappearance of the OmpF and OmpC proteins from the bdelloplast surface was measured, and it was determined that Bdellovibrio sp. exhibited a kinetic and temporal preference for the OmpF protein. Bdellovibrios could be grown on porin-deficient prey, and the progeny bdellovibrios possessed outer membranes with a protein mass deficiency.     

Bdellovibrio possesses a prey-derived OmpF protein in its outer membrane

Bdellovibrio bacteriovorus 109D andBdellovibrio stolpii derive one of their major outer membrane proteins from the outer membrane of their prey. This prey-derived protein corresponds to the OmpF protein ofEscherichia coli. Bdellovibrios cultivated onSalmonella typhimurium prey acquire theSalmonella OmpF protein; this protein is distinguishable electrophoretically from the OmpF protein ofE. coli. Bdellovibrios containing the prey-derived OmpF protein are sensitive to killing by colicin A but not colicin E1, whereas bdellovibrios without this protein are completely resistant to colicin killing.     

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.     

Prey-derived signals regulating duration of the developmental growth phase of Bdellovibrio bacteriovorus.

The filamentous elongation typical of growth-phase cells of the predatory bacterium Bdellovibrio bacteriovorus is mediated by regulatory signals that are derived from the prey cell itself. These signals regulate the differentiation of growth-phase cells into the attack phase and appear to be required for continued filamentous growth by prey-dependent wild-type bdellovibrios and their prey-independent mutant derivatives alike. Using a prey-independent bdellovibrio strain, we have developed an assay for the detection and quantification of the growth-extending signal activity present in extracts of prey cells. This prey-derived regulatory activity was shown to be independent of its nutritional contribution to the bdellovibrios and was found to occur in heat-stable, proteinlike compounds of a variety of native molecular weights within the soluble fraction of extracts from both gram-negative and gram-positive bacteria.     

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.     

Isolation and composition of sheathed flagella from Bdellovibrio bacteriovorus 109J.

A procedure was developed for the purification of sheathed flagella from Bdellovibrio bacteriovorus 109J. Preparations of isolated flagella appeared as filaments 28 nm in diameter, did not vary in sheath content by more than 10% from the mean, and contained 50% protein, 38% phospholipid, and 12% lipopolysaccharide (LPS) by weight. The sheath was readily solubilized by Triton X-100, whether or not EDTA was present, and contained all of the LPS and phospholipid and 30 to 40% of the protein of the intact flagella; sedimentable core filament polypeptides accounted for the remainder. Flagellar LPS was significantly enriched in nonadecenoic acid (19:1) and depleted in beta-hydroxymyristic acid relative to outer membrane LPS from intraperiplasmically grown bdellovibrios. These observations suggest that the sheath is a stable domain distinct from the bulk of the outer membrane. The sheath also contained substantially more phospholipid (57%) and less protein (26%) of a more heterogeneous composition than that of previously described outer membranes. This unusual balance of constituents was predicted to result in a fluid membrane compatible with a model for the generation of motility by rotation of the core filament within a highly flexible sheath.     

Molecular parasitism in the Escherichia coli-Bdellovibrio bacteriovorus system: translocation of the matrix protein from the host to the parasite outer membrane

During the intracellular maturation in Escherichia coli of the parasite Bdellovibrio bacteriovorus the outer membrane, major protein I of E. coli (i.e., the matrix protein) becomes associated with the outer membrane of the emerging parasite cells. The binding properties of this protein with the outer membrane of the host and of the parasite are identical. An analogous phenomenon also occurs during Bdellovibrio parasitism on Klebsiella pneumoniae and on Salmonella typhimurium. Possible roles for this scavenging action of Bdellovibrio, and similar phenomena in other parasitic systems, are discussed.     

Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp. isolated on phytopathogenic bacteria

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 x 10(2) to 6 x 10(3) and 2.8 x 10(2) to 2.3 x 10(4) PFU g(-1). A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.     

A new model for the penetration of prey cells by bdellovibrios.

Bdellovibrio bacteriovorus 109J and most other bdellovibrios cause prey cells to round following penetration. Bdellovibrio sp. strain W does not cause rounding of the prey. Analysis of enzyme activities during the early stages of bdellovibrio attack indicated that strain W differs from most other bdellovibrios in that there is no glycanase activity produced during penetration. Likewise, heat-killed prey were penetrated normally by strain 109J, but the resulting bdelloplast did not become round and no glycanase was detected, indicating that glycanase is not essential for penetration. Peptidoglycan from prey cells penetrated by strain W was sensitive to lysozyme, but these cells were not susceptible to attack and penetration by strain 109J, indicating that peptidoglycan deacetylation is not the primary exclusion mechanism. We propose a model in which it is the peptidase activity of the bdellovibrios which allows them to breach the peptidoglycan of their prey and in which the glycanase activity exhibited by strain 109J and other bdellovibrios is responsible for the rounding of the bdelloplast.     

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.     

Bdellovibrio bacteriovorus synthesizes an OmpF-like outer membrane protein during both axenic and intraperiplasmic growth.

Outer membrane preparations of Bdellovibrio bacteriovorus grown intraperiplasmically on Escherichia coli containing OmpF were prepared by the Triton X-100 procedure of Schnaitman (J. Bacteriol. 108:545-552, 1971). They contained a protein that migrated to almost the same position as E. coli OmpF in sodium dodecyl sulfate-acrylamide gradient gel electrophoresis and to the same position as E. coli OmpF when urea was incorporated into the gel. The mobility of this protein increased relative to that of OmpC in urea-containing gels as does E. coli OmpF. However, the same protein was also produced during axenic growth and during intraperiplasmic growth on prey lacking OmpF. The peptide profile generated by partial proteolysis of this protein showed no homology to that produced from E. coli OmpF. We conclude that B. bacteriovorus synthesizes an OmpF-like protein. Previous claims that the bdellovibrio incorporates an intact E. coli OmpF are not consistent with these observations.     

Investigations into the life cycle of the bacterial predator Bdellovibrio bacteriovorus 109J at an interface by atomic force microscopy

Atomic force microscopy was used to image Bdellovibrio bacteriovorus 109J, a gram-negative bacterial predator that consumes a variety of other gram-negative bacteria. In predator-prey communities grown on filters at hydrated air-solid interfaces, repeated cycles of hunting, invasion, growth, and lysis occurred readily even though the cells were limited to near two-dimensional movement. This system allowed us to image the bacteria directly without extensive preparation or modification, and many of the cells remained alive during imaging. Presented are images of the life cycle in two species of prey organisms, both Escherichia coli (a small prey bacterium that grows two-dimensionally on a surface) and Aquaspirillum serpens (a large prey bacterium that grows three-dimensionally on a surface), including high-resolution images of invaded prey cells called bdelloplasts. We obtained evidence for multiple invasions per prey cell, as well as significant heterogeneity in morphology of bdellovibrios. Mutant host-independent bdellovibrios were observed to have flagella and to excrete a coating that causes the predators to clump together on a surface. Most interestingly, changes in the texture of the cell surface membranes were measured during the course of the invasion cycle. Thus, coupled with our preparation method, atomic force microscopy allowed new observations to be made about Bdellovibrio at an interface. These studies raise important questions about the ways in which bacterial predation at interfaces (air-solid or liquid-solid) may be similar to or different from predation in solution.     

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).     

Glycolytic and tricarboxylic acid cycle enzyme activities during intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli.

Selected enzyme activities were measured in extracts of the total cell pellets obtained at various times during aerobic intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on anaerobically grown Escherichia coli substrate cells. Initially, the glycolytic enzyme activities were associated with the input of E. coli and the tricarboxylic acid cycle enzyme activities with the input of bdellovibrios. During the first 90 min of Bdellovibrio development, the glycolytic activities declined about 25 to 60%, whereas the tricarboxylic acid cycle activities increased about 10%. Between 110 and 180 min, the glycolytic activities decreased to trace levels and tricarboxylic acid cycle activities increased about 50 to 90%. Both bdellovibrio cell extracts and the cell-free growth menstruum (obtained after bdellovibrio growth on E. coli) caused the inactivation of glycolytic enzymes in E. coli extracts.     

Comprehensive analysis of transport proteins encoded within the genome of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a bacterial parasite with an unusual lifestyle. It grows and reproduces in the periplasm of a host prey bacterium. The complete genome sequence of B. bacteriovorus has recently been reported. We have reanalyzed the transport proteins encoded within the B. bacteriovorus genome according to the current content of the Transporter Classification Database. A comprehensive analysis is given on the types and numbers of transport systems that B. bacteriovorus has. In this regard, the potential protein secretory capabilities of at least four types of inner-membrane secretion systems and five types of outer-membrane secretion systems are described. Surprisingly, B. bacteriovorus has a disproportionate percentage of cytoplasmic membrane channels and outer-membrane porins. It has far more TonB/ExbBD-type systems and MotAB-type systems for energizing outer-membrane transport and motility than does Escherichia coli. Analysis of probable substrate specificities of its transporters provides clues to its metabolic preferences. Interesting examples of gene fusions and of potentially overlapping genes are also noted. Our analyses provide a comprehensive, detailed appreciation of the transport capabilities of B. bacteriovorus. They should serve as a guide for functional experimental analyses.     

The obligate predatory Bdellovibrio bacteriovorus possesses a neutral lipid A containing alpha-D-Mannoses that replace phosphate residues: similarities and differences between the lipid As and the lipopolysaccharides of the wild type strain B. bacteriovorus HD100 and its host-independent derivative HI100

Bdellovibrio bacteriovorus are predatory bacteria that penetrate Gram-negative bacteria and grow intraperiplasmically at the expense of the prey. It was suggested that B. bacteriovorus partially degrade and reutilize lipopolysaccharide (LPS) of the host, thus synthesizing an outer membrane containing structural elements of the prey. According to this hypothesis a host-independent mutant should possess a chemically different LPS. Therefore, the lipopolysaccharides of B. bacteriovorus HD100 and its host-independent derivative B. bacteriovorus HI100 were isolated and characterized by SDS-polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. LPS of both strains were identified as smooth-form LPS with different repeating units. The lipid As were isolated after mild acid hydrolysis and their structures were determined by chemical analysis, by mass spectrometric methods, and by NMR spectroscopy. Both lipid As were characterized by an unusual chemical structure, consisting of a beta-(1-->6)-linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six fatty acids that were all hydroxylated. Instead of phosphate groups substituting position O-1 of the reducing and O-4' of the nonreducing end alpha-d-mannopyranose residues were found in these lipid As. Thus, they represent the first lipid As completely missing negatively charged groups. A reduced endotoxic activity as determined by cytokine induction from human macrophages was shown for this novel structure. Only minor differences with respect to fatty acids were detected between the lipid As of the host-dependent wild type strain HD100 and for its host-independent derivative HI100. From the results of the detailed analysis it can be concluded that the wild type strain HD100 synthesizes an innate LPS.     

Development of Bdellophage VL-1 in Parasitic and Saprophytic Bdellovibrios

Ultrastructure was correlated with growth kinetics of bdellophage VL-1 infecting host-dependent ("parasitic") Bdellovibrio bacteriovorus 109J in its Escherichia coli B host (the three-membered system), as well as in the host-independent ("saprophytic") derivative of the Bdellovibrio. Electron microscope observations showed the arrested growth of the phage-infected bdellovibrios, polar localization of the phage progeny, and stages in their release. Present evidence indicates that bdellophage DNA is derived from both the Bdellovibrio and its host cell.     

Fate of predator and prey proteins during growth of Bdellovibrio bacteriovorus on Escherichia coli and Pseudomonas syringae prey

A two-dimensional electrophoretic analysis of protein distribution followed by identification of selected proteins by mass spectrometry was performed on fresh bdellovibrio cultures containing attack phase cells of the predatory bacterium Bdellovibrio bacteriovorus strain 109J-1 and the remains of an Escherichia coli or a Pseudomonas syringae pv. tomato prey. Cleavage of the peptidoglycan-associated outer membrane proteins (OMPs) OmpA in E. coli and OprF in P. syringae occurred in both prey. The tryptic peptides obtained from the cleavage products of OmpA and OprF were all located within the 19-kDa pronase-resistant N-terminal parts of the corresponding proteins. The predator cell fraction was separated from the prey ghosts in fresh bdellovibrio cultures by centrifugation on a Percoll-sucrose cushion. Proteins from each fraction were separated by two-dimensional electrophoresis and identified by mass spectrometric analysis. As no prey OMP could be detected in the predator cell fraction, it was concluded that prey OMPs are not transferred to the predator, as had been suggested previously. However, a protein from the predator was found bound to ghost cell envelopes. This protein may correspond to a protein earlier suggested to be associated with the prey outer or cytoplasmic membranes. Along with recently described polypeptides from B. bacteriovorus strains 100 and 114, it forms a new family of putative outer membrane proteins.