Survival strategy of <Emphasis Type="Italic">Bdellovibrio</Emphasis>

The effects of cadmium and diuron, typical environmental pollutants, on the survival of predatory bacteria of the genus Bdellovibrio were studied. The adhesion and cohesion of bdellovibrios were shown to enhance cell resistance to xenobiotics. The viability of Bdellovibrio cells was shown to be higher at the stage of bdelloplasts. The obtained results confirm the concept of the surface-associated existence of Bdellovibrio in the natural environment and serve as a basis for the employment of predatory bacteria to solve the problems of public health, biological protection of ecosystems, and bioterrorism protection.     

A Comparative Study of the Effect of Certain Pollutants on Free-living and Immobilized Bdellovibrio

The paper deals with a comparative study of the growth of free-living and immobilized predatory bacteria of the genus Bdellovibrio in the presence of toxic concentrations of urea and phenol. It was found that the cell wall of bdelloplasts plays a protective role in the adaptation of bdellovibrios to xenobiotics. The attachment of bdellovibrios to solid surfaces allows them to survive under unfavorable environmental conditions.     

Dependence of Marine Bdellovibrios on Potassium, Calcium, and Magnesium Ions

Marine bdellovibrios show a specific requirement for K⁺, Ca²⁺, and Mg²⁺. Potassium is essential for high velocity and seems to be necessary for attachment of the free bdellovibrios. Calcium and magnesium are necessary for attachment and penetration. Magnesium also plays a role in maintaining the integrity of the bdelloplast. The adaptation of these bdellovibrios to the marine environment is manifested by their stringent cation requirements.     

Differentiation after premature release of intraperiplasmically growing Bdellovibrio bacteriovorous.

Bdellovibrio bacteriovorous attacks and penetrates other gram-negative bacteria, creating a growth chamber termed a bdelloplast. We have found that exposing the bdelloplasts to EDTA, followed by treatment with a lytic enzyme concentrate derived from bdellovirio cultures, prematurely released the intraperiplasmically growing bdellovibrios at any time during their growth cycle. Upon release, the growth-form bdellovibrios terminated any initiated rounds of DNA synthesis and differentiated into motile attack-form cells. The ability of growth-form cells to synthesize DNA appears to depend upon an initiation signal that is not received until about 60 min after attack. Each subsequent round of DNA synthesis by the growing bdellovibrio filaments seems to require an additional initiation signal that is provided by their intraperiplasmic environment. Differentiation included fragmentation into multiple progeny cells to a degree proportional to the extent of intraperiplasmic growth. This differentiation could be performed totally at the expense of cellular reserves. The significance of these data to an understanding of the regulation of differentiation in bdellovibrios is 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 × 10² to 6 × 10³ and 2.8 × 10² to 2.3 × 10⁴ 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.     

Growth Cycle of Predacious Bdellovibrios in a Host-Free Extract System and Some Properties of the Host Extract

Host-free growth and reproduction of a host-dependent strain of Bdellovibrio bacteriovorus incubated with an extract from host cells were studied. The morphological changes occurring in the cells were correlated with deoxyribonucleic acid (DNA) synthesis as measured by labeled nucleotide or orthophosphate incorporation. The host-free developmental cycle of Bdellovibrio is similar to that of the two-membered system; the early loss of flagella, the elongation into filaments, and multiple fission into flagellated progeny are typical for both host-free and intraperiplasmic development of bdellovibrios. Filament length and time of division appear to depend on the concentration of the host extract. Host extract was found to be heat stable and DNase stable, and Pronase sensitive and RNase sensitive. Addition of ribonucleic acid to the extract medium at various times during the Bdellovibrio growth cycle demonstrated that host extract is required continuously during the cycle for growth. The observations reported give a unified picture of Bdellovibrio development and allow for the suggestion that wild-type bdellovibrios depend upon the presence of some host factor for induction of DNA synthesis, whereas depletion of host factor triggers division. The ecological implications of such host dependence are discussed.     

The First Bite— Profiling the Predatosome in the Bacterial Pathogen Bdellovibrio

Bdellovibrio bacteriovorus is a Gram-negative bacterium that is a pathogen of other Gram-negative bacteria, including many bacteria which are pathogens of humans, animals and plants. As such Bdellovibrio has potential as a biocontrol agent, or living antibiotic. B. bacteriovorus HD100 has a large genome and it is not yet known which of it encodes the molecular machinery and genetic control of predatory processes. We have tried to fill this knowledge-gap using mixtures of predator and prey mRNAs to monitor changes in Bdellovibrio gene expression at a timepoint of early-stage prey infection and prey killing in comparison to control cultures of predator and prey alone and also in comparison to Bdellovibrio growing axenically (in a prey-or host independent “HI” manner) on artificial media containing peptone and tryptone. From this we have highlighted genes of the early predatosome with predicted roles in prey killing and digestion and have gained insights into possible regulatory mechanisms as Bdellovibrio enter and establish within the prey bdelloplast. Approximately seven percent of all Bdellovibrio genes were significantly up-regulated at 30 minutes of infection- but not in HI growth- implicating the role of these genes in prey digestion. Five percent were down-regulated significantly, implicating their role in free-swimming, attack-phase physiology. This study gives the first post- genomic insight into the predatory process and reveals some of the important genes that Bdellovibrio expresses inside the prey bacterium during the initial attack.     

Bacterial Predator-Prey Interaction at Low Prey Density

A bacterial predator-prey interaction was studied using Bdellovibrio and bioluminescent prey bacteria. The attacking bdellovibrio causes decay of bioluminescence, which is correlated with bdellovibrio penetration into the prey. The behavior of the prey and predator populations over time was found to be well described by a Lotka-Volterra model. By using this model, the probability of bdellovibrio penetration after encountering a prey cell was found to be approximately 3.0%. The prey density required to give the bdellovibrios a 50% chance of survival was calculated to be at least 3.0 × 10⁶ cells per ml, and the density required for population equilibria was calculated to be about 7 × 10⁵ prey bacteria per ml. These values, not generally characteristic of natural habitats, suggest that the existence of Bdellovibrio in nature is limited to special ecological niches.     

Effects of calcium and magnesium ions and host viability on growth of bdellovibrios

Bdellovibrio spp. strains 6-5-S, 100, 109 (Davis), and A3.12 multiply in the presence of viable but non-proliferating or heat-killed (70 or 100 C, 10 min; 121 C, 5 min) cells ofSpirillum serpens strain VHL suspended in buffers supplemented with Ca⁺⁺ and/or Mg⁺⁺. Ca⁺⁺ (optimal, 2 × 10⁻³ m) and Mg⁺⁺ (optimal, 2 × 10⁻⁵ m) independently stimulate the groth of bdellovibrios: additive effects are noted. Multiplication ofBdellovibrio in the presence of Ca⁺⁺ and Mg⁺⁺ is associated with the release into the culture supernatant solution of UV-absorbing materials and of amino sugars (presumably by activating or stabilizing lytic enzymes). The growth rate ofBdellovibrio strain 6-5-S in suspensions of heat-killed host cells is lower than in living but non-proliferating host cells. Bdellovibrio spp. strains 100, 109 (Davis), 109 (Jerusalem), A3.12, and 6-5-S all require added Ca⁺⁺ for growth in cell suspensions of homologous or heterologous host bacteria which have been grown in minimal medium.Bdellovibrio sp. strain 109 (Jerusalem) is capable of growing in the presence of the low level of Ca⁺⁺ boundin situ to the cells of its host,E. coli B, when the host cells had been cultivated in a complex medium but not when the host cells had been grown in a Ca⁺⁺-depleted minimal medium (except when Ca⁺⁺ is added). Addition of ethylenediaminetetraacetic acid (0.01m) preventsBdellovibrio growth, which is restored by addition of Ca⁺⁺ and Mg⁺⁺. The nonparasitic growth ofBdellovibrio spp. strains 100, 109, A3.12, and 6-5-S in heat-killed cell suspensions only in the presence of added cations indicates that, in this system, the cations are essential for activity of bacteriolytic and other enzymes and that they might also directly affectBdellovibrio growth rather than — as may be the case in other systems of live host cells plusBdellovibrio — only indirectly by affecting attachment to the host cell, maintaining integrity of the host spheroplasts, and increasing the burst size.     

Periplasmic enzymes in Bdellovibrio bacteriovorus and Bdellovibrio stolpii.

When cells of either Bdellovibrio bacteriovorus 109J or Bdellovibrio stolpii UKi2 were subjected to osmotic shock by treatment with sucrose-EDTA and MgCl2 solutions, only trace amounts of proteins or enzyme activities were released into the shock fluid. In contrast, when nongrowing cells were converted to motile, osmotically stable, peptidoglycan-free spheroplasts by penicillin treatment, numerous proteins were released into the suspending fluid. For both species, this suspending fluid contained substantial levels of 5'-nucleotidase, purine phosphorylase, and deoxyribose-phosphate aldolase. Penicillin treatment also released aminoendopeptidase N from B. bacteriovorus, but not from B. stolpii. Penicillin treatment did not cause release of cytoplasmic enzymes such as malate dehydrogenase. The data indicated that bdellovibrios possess periplasmic enzymes or peripheral enzymes associated with the cell wall complex. During intraperiplasmic bdellovibrio growth, periplasmic and cytoplasmic enzymes of the Escherichia coli substrate cell were not released upon formation of the spherical bdelloplast during bdellovibrio penetration. Most of the E. coli enzymes were retained within the bdelloplast until later in the growth cycle, when they became inactivated or released into the suspending buffer or both.     

The outer cyst wall ofBdellovibrio bdellocysts is made de novo and not from preformed units from the prey wall

Bdellovibrio sp. strain W bdellocysts were produced inEscherichia coli using three sources of³H-diaminopimelic acid (DAP) for incorporation into the cyst wall peptidoglycan: (a) labeledE. coli peptidoglycan, (b) labeledBdellovibrio peptidoglycan, and (c) exogenous³H-DAP in the encystment medium. After cysts were produced, they were either sonicated to remove the prey cell wall, or germinated to solubilize the cyst wall. The results show that label was incorporated into the cyst wall preferentially from the exogenous DAP in the medium, and not from the bdellovibrio or bdelloplast peptidoglycan. The encysting bdellovibrio does not therefore incorporate existing peptidoglycan units from the bdelloplast for synthesis of the cyst wall.     

Alterations in the cell wall of Spirillum serpens VHL early in its association with Bdellovibrio bacteriovorus 109D

In both freeze-etched and critical-point dried preparations examined by transmission and scanning electron microscopy, respectively, the outer surfaces of the cells of Spirillum serpens VHL assume a wrinkled appearance 10–15 min after challenge by Bdellovibrion bacteriovorus 109D. This wrinkling effect is believed (on circumstantial evidence) to be caused by the bdellovibrio's disruption of the cell wall lipoprotein of the Spirillum. With the exception of those topological changes caused by wrinkling, the outer membrane of the Spirillum cell wall retains a normal appearance as viewed in freeze-etched preparations, even after the Spirillum cell has been converted into a bdelloplast. Although the peptidoglycan layer of the Spirillum cell presumably is weakened somewhat by the invading Bdellovibrio, evidence obtained from freeze-fractured preparations of Spirillum bdelloplasts suggests that the peptidoglycan remains as a discrete cell wall layer, even though the Spirillum cell wall apparently has lost much of its rigidity. That the peptidoglycan backbone remains essentially intact, even after the Spirillum cell has been entered by the Bdellovibrio, is supported by the observation that the soluble amino sugar content of the culture medium, as determined by chemical analysis, does not rise even 5.0 h after the association of the Bdellovibrio with the Spirillum has begun.     

Design and Performance of a 16S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization

A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment.     

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.     

Relationships among the bdellovibrios revealed by partial sequences of 16S ribosomal RNA

Members of the genusBdellovibrio possess the unifying phenotypic trait of attacking and preying upon other Gram-negative bacteria. It has been suggested that this common lifestyle arose by convergent evolution. Physiological and G + C studies have led to the notion that bdellovibrios are a heterogeneous group of loosely related bacteria. We have inferred the phylogenetic relatedness of 12 strains ofBdellovibrio through the analysis of partial 16S ribosomal RNA sequences. Similarity and degree of homology were assessed, and a phylogenetic tree was constructed by the distance matrix method. One branch of the two-branched tree consisted ofB. bacteriovorus and related strains (W, 6-5-S, 109, 109D, 109J, 114, HI Ox9-2, and HI Ox9-3). The other branch was itself branched, withB. starrii, B. stolpii, and marine strain BM4 in separate sub-branches. AllBdellovibrio strains in turn clustered with representatives of the delta division of theProteobacteria. The results indicate that there are at least two subdivisions of the genusBdellovibrio and that present-day bdellovibrios arose from a common ancestor. The placement of the genusBdellovibrio within the delta division of theProteobacteria was confirmed.     

A small predatory core genome in the divergent marine Bacteriovorax marinus SJ and the terrestrial Bdellovibrio bacteriovorus

Bacteriovorax marinus SJ is a predatory delta-proteobacterium isolated from a marine environment. The genome sequence of this strain provides an interesting contrast to that of the terrestrial predatory bacterium Bdellovibrio bacteriovorus HD100. Based on their predatory lifestyle, Bacteriovorax were originally designated as members of the genus Bdellovibrio but subsequently were re-assigned to a new genus and family based on genetic and phenotypic differences. B. marinus attaches to Gram-negative bacteria, penetrates through the cell wall to form a bdelloplast, in which it replicates, as shown using microscopy. Bacteriovorax is distinct, as it shares only 30% of its gene products with its closest sequenced relatives. Remarkably, 34% of predicted genes over 500 nt in length were completely unique with no significant matches in the databases. As expected, Bacteriovorax shares several characteristic loci with the other delta-proteobacteria. A geneset shared between Bacteriovorax and Bdellovibrio that is not conserved among other delta-proteobacteria such as Myxobacteria (which destroy prey bacteria externally via lysis), or the non-predatory Desulfo-bacteria and Geobacter species was identified. These 291 gene orthologues common to both Bacteriovorax and Bdellovibrio may be the key indicators of host-interaction predatory-specific processes required for prey entry. The locus from Bdellovibrio bacteriovorus is implicated in the switch from predatory to prey/host-independent growth. Although the locus is conserved in B. marinus, the sequence has only limited similarity. The results of this study advance understanding of both the similarities and differences between Bdellovibrio and Bacteriovorax and confirm the distant relationship between the two and their separation into different families.     

Factors affecting growth of Bdellovibrio on Rhizobium

The extent of decline in the population density of Rhizobium sp. exposed to Bdellovibrio was markedly reduced in the presence of montmorillonite, kaolinite or vermiculite but not by a soil clay fraction. Increasing levels of montmorillonite reduced the numbers of vibrios that appeared in a two-membered culture and allowed for greater survival of the rhizobia. Bdellovibrio and not Rhizobium sp. was retained when mixed with the three clay minerals, but no appreciable retention was evident with the soil clay fraction. Suspensions of colloidal soil organic matter protected the hosts from parasitism, although aqueous extracts of soil did not affect the relationship. Cells from old Rhizobium sp. cultures were attacked only after a lag phase, but rhizobia that had been stored were more rapidly lysed than cells tested immediately after removal from the growth medium. The possible significance of these findings to the survival of rhizobia in soils containing Bdellovibrio is discussed.     

Possible enzymatic bases of bacteriolysis by bdellovibrios

Summary Bdellovibrio sp. strain 6-5-S grows on and lyses autoclaved cells of Spirillum serpens strain VHL. The dissolution of the S. serpens cells is accompanied by a decrease in optical density and by a release of reducing substances, amino sugars, amino groups, and muramic acid into the culture supernatant. S. serpens cells are degraded by Bdellovibrio sp. strain 6-5-S into fragments of various sizes of which 9% is dialyzable. Fractions of the clear lysate precipitated by ammonium sulfate or cold acetone show lytic activity against autoclaved cells of Micrococcus lysodeikticus or S. serpens and are capable of releasing reducing sugars or ¹⁴C-labeled materials from isolated unlabeled or ¹⁴C-labeled S. serpens peptidoglycan, respectively. The lysozyme-like enzyme has been partially purified from the ammonium sulfate-precipitated fractions by DEAE cellulose chromatography. The molecular weight of the lysozyme-like enzyme is about 12500 as determined by Sephadex G-100.Like Bdellovibrio sp. strain 6-5-S, Bdellovibrio spp. strains 100, 109 (Davis), and A 3.12 also produce proteolytic enzymes not only in living cells but also in autoclaved cells and in cell-free extracts of S. serpens. The multiplicity of infection affects the rate of proteolytic enzyme production. In all cases, lysis of S. serpens cells occurs before production of proteolytic enzyme is evident. Mutants of Bdellovibrio sp. strain 6-5-S, which no longer produce certain proteolytic enzymes, were obtained by nitrosoguanidine treatment and selected by inability to clear casein agar; these mutants grow more slowly and form smaller plaques on S. serpens lawns than the wild type. Enzymatic analysis shows that some mutants lack the capacity to hydrolyze Azocoll brand of collagen (Azocollase-negative) and casein (exopeptidase-negative) but, like the wild type, they possess carboxypeptidase (endopeptidase-positive). A sixty-two-fold purification of the Azocollase was achieved by passage of the acetone-precipitated fraction of a lysate through a DEAE cellulose column. The Azocollase liberated amino groups also from hemoglobin, bovine serum albumin, and gelatin. The Michaelis constant (K _m) for the Azocollase acting on N,N-dimethylcasein is 5.1×10^-5 M and the molecular weight of the enzyme is about 11000.A lipase, which hydrolyzes tributyrin incorporated into an agar medium, has been detected in the acetone-precipitated fraction and in a double-layer lawn containing non-lipolytic S. serpens and Bdellovibrio sp. strain 6-5-S.The lysozyme-like enzyme, Azocollase, peptidases, and lipase probably are all involved in the bacteriolysis caused by the bdellovibrios.     

Inhibition of the predatory activity ofBdellovibrio by various environmental pollutants

The predatory activity of bdellovibrios is affected by various environmental pollutants such as detergents, heavy metals, and pesticides. This was shown in a two-membered system ofBdellovibrio andPhotobacterium, in which the effect of the predator on the bioluminescence of the prey indicated the activity of the former. The high sensitivity of the bdellovibrios toward certain chemicals (e.g., CdCl₂) indicates the possibility of using the system for biological monitoring of those chemicals.     

Survival strategy of Bdellovibrio

The effects of cadmium and diuron, typical environmental pollutants, on the survival of predatory bacteria of the genus Bdellovibrio were studied. The adhesion and cohesion of bdellovibrios were shown to enhance cell resistance to xenobiotics. The viability of Bdellovibrio cells was shown to be higher at the stage of bdelloplasts. The obtained results confirm the concept of the surface-associated existence of Bdellovibrio in the natural environment and serve as a basis for the employment of predatory bacteria to solve the problems of human population health, biological protection of ecosystems, and bioterrorism protection.