Differential Predation by Bdellovibrio bacteriovorus 109J

Bdellovibrio bacteriovorus is a predatory bacterium that can replicate only inside Gram-negative bacteria. We incubated B. bacteriovorus 109J in a mixture of two prey cells present in equal numbers and enumerated prey cells after 3 h of predation. In multiple prey pairings, B. bacteriovorus preferentially lysed on one prey over the other. When prey were individually incubated with B. bacteriovorus, they were preyed on with different efficiencies. Three prey had only 5–8% of cells remaining after Bdellovibrio predation and the other three prey had 37–43% of cells remaining. Timing of attachment of B. bacteriovorus to prey cells also varied with Bdellovibrio attachment to more preferred prey occurring the fastest. These results suggest that B. bacteriovorus 109J does not randomly infect prey cells but infects and kills some prey more readily than others.     

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.     

Uptake of intact nucleoside monophosphates by Bdellovibrio bacteriovorus 109J

The degraded nucleic acids and ribosomes of its prey cell provide Bdellovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 microM in attack phase cells and 35 microM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated.     

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).     

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.     

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.     

An ATP transport system in the intracellular bacterium, Bdellovibrio bacteriovorus 109J.

The intracellularly growing bacterium Bdellovibrio bacteriovorus 109J transports intact ATP by a specific, energy-requiring process. ATP transport does not involve either an ADP-ATP or an AMP-ATP exchange mechanism but, instead, has characteristics of an active transport permease. Kinetically distinct systems for ATP transport are expressed by the two developmental stages of the bdellovibrio life cycle.     

Change in the surface hydrophobicity of substrate cells during bdelloplast formation by Bdellovibrio bacteriovorus 109J.

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J, the substrate cell surface becomes more hydrophobic. This was shown (i) by comparing the sensitivity to hydrophobic antibiotics of wild-type and lipopolysaccharide mutant strains of Salmonella typhimurium to that of the bdellovibrio growing on these strains and (ii) by measuring the binding efficiency of these strains, Escherichia coli, and their derived bdelloplasts to octyl Sepharose. The kinetics of increase in surface hydrophobicity was similar to the kinetics of the conversion of the substrate cell peptidoglycan to a lysozyme-resistant form (M. Thomashow and S. Rittenberg, J. Bacteriol. 135:1008-1014, 1978), and hydrophobicity reached a maximum at about 60 min in a synchronous culture. The change in hydrophobicity was inhibited by chloramphenicol, suggesting that bdellovibrio protein synthesis was required. Control experiments revealed that the free-swimming bdellovibrio had a more hydrophobic surface than the deep rough mutants of S. typhimurium.     

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.     

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.     

Waveform analysis and structure of flagella and basal complexes from Bdellovibrio bacteriovorus 109J.

The structure of sheathed flagella from Bdellovibrio bacteriovorus was investigated. The first three periods of these flagella were characterized by progressively smaller wavelengths and amplitudes in periods more distal to the cell. The damped appearance was due to a single nonrandom transition between two helical structures within each filament. The intersection of the two helices, one of which was a threefold-reduced miniature of the other, occurred at a fixed distance along the filament and resulted in a shift in the flagellar axis. Flagella increased in length as the cells aged and assumed a constant miniature waveform at their distal ends. The core filament was the principal determinant of flagellar morphology. It was composed of 28,000- and 29,500-dalton polypeptides. The 28,000-dalton subunits were located in the cell-proximal segment of the filament, and the 29,500-dalton subunits were located in the more distal region. The heteromorphous appearance of bdellovibrio flagella arose from the sequential assembly of these subunits. The basal complex associated with core filaments was examined because of its potential involvement in sheath formation. Bdellovibrio basal organelles were generally similar to those of other gram-negative species, but appeared to lack a disk analogous to the outer membrane-associated L ring which is a normal component of gram-negative basal complexes.     

Artificial immobilization of the two-membered bacterial system Bdellovibrio bacteriovorus 109D--Escherichia coli B in polyacrylamide gel

The interaction of a parasite with a host was studied in the two-membered bacterial system, Bdellovibrio bacteriovorus 109D and Escherichia coli B, immobilised in polyacrylamide gel (PAAG). The parasite localised inside the host cells was found to be more resistant to the toxic action of PAAG components than free B. bacteriovorus. The latter lost its mobility and was inactivated in the matrix of the carrier whereas the intracellular parasite had a normal cycle of development in the periplasm of the infected cells. The dynamics of B. bacteriovorus and E. coli incidence in the liquid phase and in PAAG granules was studied while the immobilised system was incubated. The interaction in the immobilised system could be intensified by growing more bacterial host cells in PAAG particles. The immobilisation was shown to favour the survival of the parasite and the host in the two-membered system.     

Investigations into the polymorphism of rat tail tendon fibrils using atomic force microscopy

Collagen type I displays a typical banding periodicity of 67 nm when visualized by atomic force or transmission electron microscopy imaging. We have investigated collagen fibers extracted from rat tail tendons using atomic force microscopy, under different ionic and pH conditions. The majority of the fibers reproduce the typical wavy structure with 67 nm spacing and a height difference between the peak and the grooves of at least 5 nm. However, we were also able to individuate two other banding patterns with 23+/-2 nm and 210+/-15 nm periodicities. The small pattern showed height differences of about 2 nm, whereas the large pattern seems to be a superposition of the 67 nm periodicity showing height differences of about 20 nm. Furthermore, we could show that at pH values of 3 and below the fibril structure gets dissolved whereas high concentrations of NaCl and CaCl(2) could prevent this effect.     

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.     

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.     

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.     

Early host damage in the infection cycle of Bdellovibrio bacteriovorus

The effects of bdellovibrio infection on host permeability and respiration were investigated by measuring respiration rates and the rate of o-nitrophenyl-beta-d-galactopyranoside hydrolysis during the course of single infection cycles of Bdellovibrio bacteriovorus strain 109 growing on Escherichia coli ML 35 (lac i(-)z(+)y(-)). The data show that among the very early consequences of parasite attack on the host are an increase in permeability and a general disruption of respiratory activity of the host, and it is suggested that both phenomena stem from early damage to host membrane. The rapid onset of damage after inception of the cycle and the failure of streptomycin to prevent the damage indicate that complete penetration of the parasite into the host is not a requirement for the observed effects. The data also show that bdellovibrio does not use host energy-generating mechanisms for its growth and suggest that the parasites may have a search mechanism that permits them, to some degree, to distinguish between infected and uninfected hosts.     

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.     

Humicola lanuginosa lipase hydrolysis of mono-oleoyl-rac-glycerol at the lipid-water interface observed by atomic force microscopy

A new type of planar lipid substrate for Humicola lanuginosa lipase (HLL) has been prepared by depositing a monolayer of 1-mono-oleoyl-rac-glycerol (MOG) on top of a monolayer of dipalmitoyl-phosphatidylcholine (DPPC) on mica by the Langmuir-Blodgett (LB) technique. The bilayer was subsequently exposed to HLL in a liquid cell of an atomic force microscope (AFM) allowing the time course of the lipolytic degradation to be observed. By analysing a series of AFM images, we find that enzymes are preferentially activated at the edge of nano-scale defects present in the bilayer prior to enzyme injection, while defect-free areas of the substrate are surprisingly stable towards enzyme degradation. The initial rate of hydrolysis is found to be proportional to the perimeter length, P, of the initial nano-scale defects as well as the bulk enzyme concentration, c(HLL); d(lipid)/dt=k P c(HLL). We estimate the specific rate of MOG hydrolysis by HLL to be 2.5x10(4) MOG molecules/(minute x molecule of HLL).     

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.