Permeability of the boundary layers of Bdellovibrio bacteriovorus 109J and its bdelloplasts to small hydrophilic molecules.

Measurements of the sucrose-permeable and -impermeable volumes during Bdellovibrio bacteriovorus attack on Escherichia coli or Pseudomonas putida showed that the volume of the bdelloplast increased over that of the substrate cell. Although the pattern of the increase differed with the two organisms, the volumes reached maximum at about 60 min into the bdellovibrio growth cycle. By this time, the cytoplasmic membranes of the attacked cells were completely permeable to sucrose. The kinetics of increase in sucrosepermeable volumes were similar to the kinetics of attachment and penetration (Varon and Shilo, J. Bacteriol. 95:744-753, 1968). These data show that the original cytoplasmic and periplasmic compartmentalization of the substrate cell ceases to exist with respect to small hydrophilic molecules during bdellovibrio attack. In contrast, the effective pore size of the outer membrane of the substrate cell to small oligosaccharides remains unaltered during bdelloplast formation as was shown by direct measurements of its exclusion limits. The major porin protein of E. coli, OmpF, was recoverable from the bdelloplast outer membrane fraction until the onset of lysis. The Braun lipoprotein was removed from the bdelloplast wall early, and OmpA was lost in the terminal part of the bdellovibrio growth cycle.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: solubilization of Escherichia coli peptidoglycan.

During penetration of Bdellovibrio bacteriovorus into Escherchia coli, two enzymatic activities, a glycanase and a peptidase, rapidly solubilized some 10 to 15% of the E. coli peptidoglycan. The glycanase activity, which solubilizes peptidoglycan amino sugars, came to a sharp halt with completion of the penetration process. Peptidase activity, which cleaves diaminopimelic acid residues from the peptidoglycan, continued, but at a decreasing rate. By 90 min after bdellovibrio attack, some 30% of the initial E. coli diaminopimelic acid residues were solubilized and present in the culture fluid as free diaminopimelic acid. During bdellovibrio penetration some 25% of the lipopolysaccharide glucosamine was also solubilized by an as yet undefined enzymatic activity that yielded products having molecular weights below 2,000. The solubilization of E. coli lipopolysaccharide glucosamine also terminated at completion of bdellovibrio penetration. At the end of bdellovibrio growth, a second period of rapid solubilization of bdelloplast peptidoglycan began which resulted in lysis of the bdelloplast and complete solubilization of the peptidoglycan amino sugars and diaminopimelic acid. The final lytic enzyme(s) was synthesized just before the time of lysis.     

Regulated breakdown of Escherichia coli deoxyribonucleic acid during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J.

During growth of Bdellovibrio bacteriovorus on [2-14C]deoxythymidine-labeled Escherichia coli, approximately 30% of the radioactivity was released to the culture fluid as nucleoside monophosphates and free bases; the remainder was incorporated by the bdellovibrio. By 60 min after bdellovibrio attack, when only 10% of the E. coli deoxyribonucleic acid (DNA) had been solubilized, the substrate cell DNA was degraded to 5 X 10(5)-dalton fragments retained within the bdelloplast. Kinetic studies showed these fragments were formed as the result of sequential accumulation of single- and then double-strand cuts. DNA fragments between 2 X 10(3) and 5 X 10(5) daltons were never observed. Chloramphenicol, added at various times after initiation of bdellovibrio intraperiplasmic growth on normal or on heated E. coli, which have inactivated deoxyribonucleases, inhibited further breakdown and solubilization of substrate cell DNA. Analysis of these intraperiplasmic culture deoxyribonuclease activities showed that bdellovibrio deoxyribonucleases are synthesized while E. coli nucleases are inactivated. It is concluded that continuous and sequential synthesis of bdellovibrio deoxyribonucleases of apparently differing specificities is necessary for complete breakdown and solubilization of substrate cell DNA, and that substrate cell deoxyribonucleases are not involved in any significant way in the degradation process.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus on heat-treated Escherichia coli.

Heat treatment (55 degrees C for 40 min) of cell suspensions in buffer (ca. 3 x 10(9) cells per ml) of Escherichia coli ML35 caused a 4- to 4.5-log loss of cell viability. Similar results were found for several other E. coli strains that were examined. As a result of this heat treatment, 260-nm- and 280-nm-absorbing materials were released into the suspending buffer, along with about 10% of the total cellular radioactivity, when cells uniformly labeled with (14)C were used. In comparison with untreated cells, heat-treated E. coli ML35 cells showed (i) no significant changes in macromolecular composition other than ca. 22% less RNA content, (ii) an increased permeability to o-nitrophenyl-beta-d-galactopyranoside (a compound to which untreated cells are impermeable), (iii) almost complete loss of respiratory potential, and (iv) substantial losses of numerous glycolytic enzyme activities in cell extracts prepared from these cells. Intraperiplasmic development of Bdellovibrio bacteriovorus 109J with heat-treated E. coli ML35 as substrate cells appeared normal when observed microscopically, although bdellovibrio attachment and resultant bdelloplast formation were slightly retarded. No significant changes were observed in cell yields or in the ratios and contents of DNA, RNA, or protein between bdellovibrios harvested from untreated cells and those from heat-treated substrate cells after single-developmental-cycle growth on these cells. The average Y(ATP) values for intraperiplasmic growth on untreated and heat-treated substrate cells were 16.0 and 17.9, respectively. It is concluded that intraperiplasmic bdellovibrio growth on gently heat-treated E. coli substrate cells is very similar to growth on untreated substrate cells, even though the former substrate cells are nonviable and substantially impaired in many metabolic activities.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: N-deacetylation of Escherichia coli peptidoglycan amino sugars.

During intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli, the substrate cell peptidoglycan is extensively modified as it is converted to bdelloplast peptidoglycan. The initially lysozyme-sensitive peptidoglycan of E. coli was rapidly converted to a lysozyme-resistant form. The conversion was due to the N-deacetylation of a large portion of the peptidoglycan amino sugars. Chemically acetylating the isolated peptidoglycan restored its sensitivity to lysozyme digestion. However, approximately half of the products of lysozyme digestion exhibited hydrophobic interactions that were shown not to be due to the presence of protein. This suggests that a molecule capable of hydrophobic interactions, other than protein, becomes linked to the bdelloplast peptidoglycan. The data also suggest that much of the Braun lipoprotein is removed from the E. coli peptidoglycan early during bdellovibrio development.     

Intraperiplasmic growth of Bdellovibrio bacteriovorus 109J: attachment of long-chain fatty acids to escherichia coli peptidoglycan.

During the initial stages of intraperiplasmic growth of Bdellovibrio bacteriovorus on Escherichia coli, the peptidoglycan of the E. coli becomes acylated with long-chain fatty acids, primarily palmitic acid (60%) and oleic acid (20%). The attachment of the fatty acids to the peptidoglycan involves a carboxylic-ester bond, i.e., they were removed by treatment with alkaline hydroxylamine. Their linkage to the peptidoglycan does not involve a protein molecule. When the bdelloplast peptidoglycan was digested with lysozyme, the fatty acid-containing split products behaved as lipopeptidoglycan, i.e., they were extracted into the organic phase of 1-butanol:acetic acid:water (4:15) two-phase system; all of the lysozyme split products generated from normal E. coli peptidoglycan were extracted into the water phase. It is suggested that the function of the acylation reaction is to help stabilize the bdelloplast outer membrane against osmotic forces. In addition, a model is presented to explain how a bdellovibrio penetrates, stabilizes, and lyses a substrate cell.     

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.     

Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E. coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing. This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus. Exogenous thymidine-5'-monophosphate competed effectively with [3H]thymine residues of E. coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all. A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E. coli phosphorus as a phosphorus source for B. bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids. A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E. coli phosphorus by B. bacteriovorus, as did orthophosphate alone. A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E. coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid. These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B. bacteriovorus growing intraperiplasmically. As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth. The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved.     

Ribonucleic acid destruction and synthesis during intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on (2-14C)uracil-labeled Escherichia coli approximately 50% of the radioactivity is incorporated by the bdellovibrio and most of the remainder is released as free nucleic acid bases. Kinetic studies showed that 50 and 30S ribosomal particles and 23 and 16S ribosomal ribonucleic acid (RNA) of E. coli are almost completely degraded by the first 90 min in a 210- to 240-min bdellovibrio developmental cycle. Synthesis of bdellovibrio ribosomal RNA was first detected after 90 min. The specific activity and the ratio of radioactivity in the bases of the synthesized bdellovibrio RNA was essentially the same as those of the substrate E. coli. The total radioactivity of the bdellovibrio deoxyribonucleic acid (DNA) exceeded that in the DNA of the substrate E. coli cell, and the ratio of radioactivity of cytosine to thymine residues differed. Intraperiplasmic growth of B. bacteriovorus in the presence of added nucleoside monophosphates (singly or in combination) significantly decreased the uptake of radioactivity from (2-14C)uracil-labeled E. coli; nucleosides or nucleic acid bases did not. It is concluded that the RNA of the substrate cell, in the form of nucleoside monophosphates, is the major or exclusive precursor of the bdellovirbrio RNA and also serves as a precursor for some of the bdellovibrio DNA.     

Attachment of diaminopimelic acid to bdelloplast peptidoglycan during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J.

An early event in the predatory lifestyle of Bdellovibrio bacteriovorus 109J is the attachment of diaminopimelic acid (DAP) to the peptidoglycan of its prey. Attachment occurs over the first 60 min of the growth cycle and is mediated by an extracellular activity(s) produced by the bdellovibrio. Some 40,000 DAP residues are incorporated into the Escherichia coli bdelloplast wall, amounting to ca. 2 to 3% of the total initial DAP content of its prey cells. Incorporation of DAP occurs when E. coli, Pseudomonas putida, or Spirillum serpens are the prey organisms. The structurally similar compounds lysine, ornithine, citrulline, and 2,4-diaminobutyric acid are not attached. The attachment process is not affected by heat-killing the prey nor by the addition of inhibitors of either energy generation (cyanide, azide, or arsenate), protein or RNA synthesis (chloramphenicol and rifamycin), or de novo synthesis of cell wall (penicillin or vancomycin). Approximately one-third of the incorporated DAP is exchangeable with exogenously added unlabeled DAP, whereas the remaining incorporated DPA is solubilized only during the lysis of the bdelloplast wall. Examination of DAP incorporation at low prey cell densities suggests that bdellovibrios closely couple the incorporation to an independent, enzymatic solubilization of DAP by a peptidase. The data indicate that DAP incorporation is a novel process, representing the second example of the ability of the bdellovibrio to biosynthetically modify the wall of its 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.     

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.     

Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent or very low in cell extracts prepared from heat-treated E. coli cells. It is concluded that during intraperiplasmic growth B. bacteriovorus degrades some 20 to 40% of the ribonucleotides derived from the initial E. coli RNA into the base and ribose-1-phosphate moieties. The ribose-1-phosphate is further metabolized by B. bacteriovorus both for energy production and for biosynthesis, of non-nucleic acid cell material. In addition, the data indicate that during intraperiplasmic growth B. bacteriovorus can metabolize ribose only if this compound is available to it as the ribonucleoside monophosphate.     

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.     

Kinetics of deoxyribonucleic acid destruction and synthesis during growth of Bdellovibrio bacteriovorus strain 109D on pseudomonas putida and escherichia coli

During the growth of Bdellovibrio bacteriovorus on Pseudomonas putida or Escherichia coli in either 10(-3)m tris(hydroxymethyl)aminomethane or in dilute nutrient broth, the host deoxyribonucleic acid (DNA) was rapidly degraded, and by 30 to 60 min after the initiation of the bdellovibrio development cycle essentially all host DNA became nonbandable in CsCl gradients. At this stage the host DNA degradation products were nondiffusable, and there was no appreciable pool of low-molecular-weight (cold acid soluble) DNA fragments in the cells or in the suspending medium. Bdellovibrio DNA synthesis occurred only after degradation of host DNA to a nonbandable form was complete. The synthesis occurred in a continuous fashion with P. putida as the host and in two separate periods with E. coli as host. By using E. coli containing a (3)H-thymidine label, it was shown that 73%, on the average, of the thymine residues of host DNA were incorporated into bdellovibrio DNA when E. coli was the only source of nutrient. In the presence of dilute nutrient broth, the host cells still served as the major source of precursors for bdellovibrio DNA synthesis, with only 20% of the precursors arising from the exogenous nutrients. The data indicate an efficient and controlled utilization of host DNA by the bdellovibrio. The host DNA is apparently degraded early in the developmental cycle to oligonucleotides of intermediate molecular weight from which the biosynthetic monomers are generated only as they become needed for bdellovibrio DNA synthesis.     

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.     

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.     

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.     

Energy efficiency of intraperiplasmic growth of Bdellovibrio bacteriovorus.

The Y-ATP (energy efficiency) of intraperiplasmic growth of Bdellovibrio bacteriovorus was determined from the distribution of radioactivity of the substrate organism ([U-14C]Escherichia coli) btween CO2 and bdellovibrio cells at the end of growth. A "best" Y-ATP value of 18.5 was obtained from single growth cycle experiments and an average value of 25.9 from multicycle experiments. Both values are much higher than the usual value of 10.5 for bacteria growing in rich media. The bases for the unusual energy efficiency for growth of B. bacteriovorus are discussed.     

Taxonomic studies of predatory bdellovibrios based on 16S rRNA analysis, ribotyping and the hit locus and characterization of isolates from the gut of animals

The aim of our study was to obtain data for the molecular characterization of bdellovibrio bacteria, which were recently split into the genus Bdellovibrio and the newly designated genus Bacteriovorax. We determined the 16S rDNA sequences of five reference strains and performed a phylogenetic analysis including published 16S rRNA sequences of bdellovibrios. A comparison of the secondary structure showed significant differences in two regions of the 16S rRNAs of the species Bdellovibrio bacteriovorus, Bacteriovorax starrii, and Bacteriovorax stolpii. In addition, ribotyping techniques gave specific hybridization patterns and revealed that two rRNA operons are present in the investigated strains. A hybridization probe derived from the genetic locus hit, associated with the host independent (HI) phenotype of B. bacteriovorus, was found to be specific for this species. Sequence comparison of the hit locus revealed few base pair changes between host independent (HI) and host dependent (HD) strains. Ribotyping and hybridization experiments using the hit probe were applied to characterize bdellovibrio strains isolated from the gut of animals and humans and one isolate from sewage.