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.     

Membrane Adenosine Triphosphatase of Escherichia coli: Activation by Calcium Ion and Inhibition by Monovalent Cations

Membrane ghost preparations of Escherichia coli K-12 obtained by osmotic lysis of lysozyme-induced spheroplasts were found to possess both Mg(++)- and Ca(++)-activated adenosine 5'-triphosphatase (ATPase, EC 3.6.1.3) activities. Maximal activities of 1.0 to 1.5 mumoles of orthophosphate released per min per mg of protein were obtained at pH 9.0 with a molar Mg(++) to adenosine 5'triphosphate (ATP) ratio of 2:5 and at pH 9.9 with a molar Ca(++) to ATP ratio of 1:5. These ATPase activities were not altered by ouabain, fluoride, N-ethylmaleimide, 2,4-dinitrophenol, cyanide, or dithionite, but were inhibited by low concentrations of azide, p-chloromercuribenzoate, and pentachlorophenol. Mg(++) ATPase was more susceptible to inhibition by azide than was Ca(++) ATPase. Fifty per cent inactivation of both activities was observed when membrane ghost preparations were preincubated at 66 C for 10 min. The Mg(++) and Ca(++) ATPase activities of these preparations were not additive, but did respond independently to inhibition by monovalent cations. Ca(++) ATPase was found to be very sensitive to inhibition by K(+), Na(+), Li(+), Rb(+), and Cs(+); Mg(++) ATPase was relatively insensitive to these ions. One possible interpretation of the results presented in this paper is that the membrane of E. coli possesses an ATPase which is activated by either Mg(++) or Ca(++) and that activation by Ca(++) increases the susceptibility of this enzyme to inhibition by monovalent cations. Increased susceptibility of E. coli membrane ATPase to inhibition by monovalent cations such as Na(+) and K(+) as a consequence of Ca(++) activation could represent a regulatory mechanism.     

Isolation and characterization of temperature-sensitive mutants of host-dependent Bdellovibrio bacteriovorus 109D

A variety of temperature-sensitive mutants of host-dependent Bdellovibrio bacteriovorus 109D were selected after ethyl methane sulfonate mutagenesis. Mutants that demonstrated plaque-forming ability reversion frequencies of 10(-8) to 10(-9) were chosen for further study. Representatives of these mutants were then characterized by phase-contrast and electron microscopy, temperature-shifted one-step growth experiments, attachment kinetics, and macromolecular capabilities. Representative mutants demonstrate various types of blockage corresponding to the previously described morphological stages of Bdellovibrio predatious life cycle, i.e., attachment blockage (109D153), penetration blockage (109D3 and 109D48), and blockage of intracellular growth (109D4 and 109D152). The time of release from temperature repression for the mutant classes was found to correspond to the apparent morphological stage of blockage via temperature-shifted, one-step growth experiments. Mutants characterized as exhibiting blockage in the penetration or intracellular stages of the infection cycle exhibited, at the permissive and nonpermissive temperatures, kinetics of attachment to Escherichia coli WP2 similar to those of the wild type. One mutant, 109D153, exhibited depressed attachment at the restrictive temperature even though the Bdellovibrio cells were motile. The extent of 38.5 C attachment of 109D153 to E. coli is at the same level as that of wild-type 109D to Bacillus subtilis, a gram-positive, non-host organism. Subsequent detachments were revealed in the wild-type 109D-B. subtilis or mutant 109D153-Escherichia coli (38.5 C) cultures. These studies reveal a biphasic attachment phenomenon in the early interaction of Bdellovibrio with its host. It appears that, at the restrictive temperature, 109D153 is capable only of the initial, nonspecific type of attachment.     

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.     

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.     

Importance of Bdellovibrio in regulating microbial cenoses and self-purification processes in domestic sewage

The bacterial parasite Bdellovibrio was directly proved to be involved in the regulation of microbial cenoses and in the self-purification of domestic waste waters. The incidence of heterotrophs, Gram-negative bacteria, E. coli and Bdellovibrio was followed up in dynamics in the microecological system of waste waters for ten days. In control experiments, bdellovibrions were removed using pteridine as a vibriostatic agent. In the absence of bdellovibrions, the cell number of the studied microorganisms did not increase after reaching a stationary level. In the control, the total incidence of heterotrophs decreased 1355 times, that of Gram-negative bacteria fell down 527 times, and that of E. coli cells dropped 3419 times due to the interaction between the host bacteria and Bdellovibrio. The variations in the number of interacting cells were characteristic of a two-component parasite-host system.     

Interaction of Bdellovibrio bacteriovorus and Host Bacteria I. Kinetic Studies of Attachment and Invasion of Escherichia coli B by Bdellovibrio bacteriovorus

Quantitative methods were developed for the study of the early stages in the interaction of Bdellovibrio bacteriovorus and host bacteria. Attachment measurements were based on the differential filtration of host and parasite. Invasion was measured by estimation of radioactively labeled Bdellovibrio cells remaining attached to the host cells after mechanical agitation. The kinetics of attachment and the final number of Bdellovibrio cells attached were dependent on the multiplicity of the parasite, the composition and pH of the medium, and the incubation temperature. Inhibitors of Bdellovibrio motility, including chelating agents, NaN(3), and low pH, all inhibited attachment, as did anaerobiosis. Ultraviolet-killed host cells retained their competence for attachment of Bdellovibrio cells, whereas heat-killed cells lost it. Invasion was selectively inhibited by inhibitors of protein synthesis, such as streptomycin, puromycin, and chloramphenicol. These antibiotics had no effect on attachment.     

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.     

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.     

Parasitic interaction of Bdellovibrio bacteriovorus with other bacteria

Starr, Mortimer P. (University of California, Davis), and Nancy L. Baigent. Parasitic interaction of Bdellovibrio bacteriovorus with other bacteria. J. Bacteriol. 91:2006-2017. 1966.-The interactions of the predatory parasite, Bdellovibrio bacteriovorus, with Erwinia amylovora, Pseudomonas tabaci, and P. phaseolicola were examined by means of phase-contrast and electron microscopy. Attachment of the bdellovibrio to the host cell is apparently initially reversible; detachment occurs infrequently in the later stages. Formation of a pore in the host cell wall is followed by disorganization of the host nucleus and of the murein layer of the host cell wall. Short host cells become totally spheroplasted; the longer rods of Pseudomonas usually are partially spheroplasted. The parasite completely invades the host cell, and the cell contents of the host are digested. Bdellovibrios living as parasites inside the host increase considerably in size in comparison with those which have been living away from the host for a time. When the host protoplast is entirely lysed, the parasites leave the disintegrating "ghosted" cell envelope, and are ready to reinitiate the parasitic cycle. The time taken for a mature Bdellovibrio cell to complete the parasitic cycle may vary depending on the length of time the parasite has been away from its hosts.     

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.     

Escherichia coli K Bacteriophages I. Isolation and Introductory Characterization of Five Escherichia coli K Bacteriophages

A set of five Escherichia coli K phages has been isolated. These phages are adsorbed to and lyse the capsular forms of the host bacteria, whereas their spontaneous, acapsular mutants are not affected. All host strains are heavily encapsulated test strains for E. coli K antigens of the thermostable A type and they readily segregate acapsular mutants. In four of the phage-host systems, all secondary growth obtained was found to be acapsular. When tested for host-range mutants on 38 strains of E. coli and Klebsiella, less than one mutant per 10(5) plaque-forming units was found. No cross-reacting neutralizing antibodies were obtained when rabbits were immunized with the K phages. The latent periods (between 16 and 30 min) and average burst sizes (between 145 and 580) were determined by one-step growth experiments.     

Growth of bacteriophage Mu in Escherichia coli dnaA mutants.

In one-step growth experiments we found that bacteriophage Mu grew less efficiently in nonreplicating dnaA mutants than in dnaA+ strains of Escherichia coli. Phage development in dnaA hosts was characterized by latent periods that were 15 to 30 min longer and an average burst size that was reduced by 1.5- to 4-fold. The differences in phage Mu development in dnaA and dnaA+ strains were most pronounced in cells infected at a low multiplicity and became less pronounced in cells infected at a high multiplicity. Many of these differences could be eliminated by allowing the arrested dnaA cells to restart chromosome replication just before infection. In continuous labeling experiments we found that infected dnaA strains incorporated 5 to 40 times more [methyl-3H]thymidine than did uninfected cells, depending on the multiplicity of infection. DNA-DNA hybridization assays showed that greater than 90% of this label was contained in phage Mu DNA sequences and that only small amounts of the label appeared in E. coli sequences. In contrast, substantial amounts of label were incorporated into both host and viral DNA sequences in infected dnaA+ cells. Although our results indicated that phage Mu development is not absolutely dependent on concurrent host chromosomal DNA replication, they did strongly suggest that host replication is necessary for optimal growth of this phage.     

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.     

23Na NMR study of the interaction between hyaluronan and the bications Ca(++), Mg(++) and Cu(++)

The relaxation rate R = pi Delta nu(1/2) of the quadrupolar (23)Na nucleus was measured at pH approximately 7 using a 200 MHz NMR spectrometer with a view to observe the interaction between hyaluronan and its natural counterion Na(+) and the bications Ca(++), Mg(++) and Cu(++). An interpretation of our results, by means of the "entropy of fluctuations" concept of Na(+), is presented. We show that Cu(++) ions are more effective than Ca(++) and Mg(++). A possible model of complexation of Cu(++) in a cage formed by the 1-4 glycosidic bond, the carboxylate side-chain and the acetoamide side-chain is proposed, according to electrostatic potential computations using the ZINDO1 quantum semi empirical method.     

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.     

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.     

Susceptibility of biofilms to Bdellovibrio bacteriovorus attack

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that an initial titer of Bdellovibrio as low as 10(2) PFU/well or an exposure to the predator as short as 30 min is sufficient to reduce a preformed biofilm. The ability of B. bacteriovorus to reduce an existing biofilm was confirmed by scanning electron microscopy. The reduction in biofilm biomass obtained after the first 24 h of exposure to the predator remained unchanged even after longer exposure periods and reinoculation of the samples with fresh Bdellovibrio; however, no genetically stable resistant population of the host bacteria could be detected. Our data suggest that growth in a biofilm does not prevent predation by Bdellovibrio but allows a level of survival from attack greater than that observed for planktonic cells. In flow cell experiments B. bacteriovorus was able to decrease the biomass of both E. coli and Pseudomonas fluorescens biofilms as determined by phase-contrast and epifluorescence microscopy.     

An electron microscope study of the influence of divalent ions on myosin filament formation in chicken gizzard extracts and homogenates

Myosin as well as actin filaments could be seen in negatively stained preparations of fresh chicken gizzard homogenized in buffered KCI (I=0-12, pH 6.85), in a 1 : 1 ratio. Myosin filaments were also present when homogenates were diluted (1 : 9) with solutions containing additional Mg(++) and ATP provided naturally occurring traces of Ca(++) had not been chelated with EGTA. There were no myosin filaments when Mg(++), ATP, Ca(++) or Ca(++) + ATP were added respectively to the diluent. It is suggested that in vivo in relaxed muscles myosin is present in dimers, and only aggregates into filaments at the onset of contraction when Ca(++) are released.