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Applied and Environmental Microbiology, June 2000, p. 2365-2371, Vol. 66, No. 6
Department of Plant Pathology and
Microbiology, Faculty of Agricultural, Food and Environmental Quality
Sciences, The Hebrew University of Jerusalem, Rehovot
76100,1 and Soil, Water and
Environmental Sciences Institute, Agricultural Research Organization,
Volcani Research Center, Bet-Dagan 50250,2
Israel
Received 19 October 1999/Accepted 22 March 2000
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 Bdellovibrio
spp. are small, very motile gram-negative bacteria that exhibit a
unique and obligate requirement for other gram-negative cells, which
they invade and use as substrates (22). These organisms were
first isolated from soil (25), where they are commonly encountered. They can also be found in freshwater, brackish water, seawater, sewage, water pipes, and water reservoirs (13,
22). It has been shown that marine
Bdellovibrio species preferentially associate
with surfaces, where they are components of biofilms (13,
31).
The biphasic growth cycle of Bdellovibrio
species includes a free-swimming attack phase and an intraperiplasmic
growth phase; this growth cycle distinguishes this group of bacteria
from all other bacterial parasites of bacteria (29). Three
Bdellovibrio species,
Bdellovibrio bacteriovorus,
Bdellovibrio stolpii, and Bdellovibrio starrii, were described first on
the basis of their G+C contents and DNA relative association data
(23, 26) and then on the basis of 16S rRNA analysis data
(6). Because of the large distances between the species,
Baer et al. (2) recently proposed that the genus should be
split and the new genus Bacteriovorax should be created and
should include two species, Bacteriovorax starrii and
Bacteriovorax stolpii.
Although bdellovibrios survive in nature, the activity of
these organisms and their influence on bacterial communities are still
controversial. The intrinsic ability of bdellovibrios to parasitize and lyse prey cells makes them attractive potential biocontrol agents which could be used against gram-negative root phytopathogens (9, 28). However, until now, there has been no study in which researchers have compared soil and rhizosphere bdellovibrios and examined the activities of these
organisms against agriculturally important microbes.
It has been shown that the structure of the bacterial communities found
in soil and the structure of the bacterial communities found in the
rhizosphere are different (5, 14, 17).
Bdellovibrios use members of these communities as
substrates and have specific host ranges, and whether
bdellovibrio populations in the rhizosphere environment and
in soil are different is not known.
In this study, using collection and newly isolated soil and rhizosphere
bdellovibrios in conjunction with molecular and
culture-based techniques, we found that coexisting
Bdellovibrio soil subpopulations and rhizosphere
bdellovibrio populations are different.
Bacterial strains, media, isolation procedure, and
maintenance.
The Bdellovibrio and prey
strains used are shown in Tables 1 and
2, respectively; the origins of the
strains are also shown. Strains whose designations begin with SRP, SRA,
and SRE, strains whose designations begin with BRP, and strains whose
designations begin with BEP were isolated from soil, from the
rhizosphere, and from the total root extract of common bean in four,
two, and two independent isolation events, respectively. Strain TRA2
was isolated from the total root extract of a tomato plant.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Prey Range Characterization, Ribotyping, and
Diversity of Soil and Rhizosphere Bdellovibrio
spp. Isolated on Phytopathogenic Bacteria
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bdellovibrio strains used in
this study
TABLE 2.
Bacterial strains used as potential prey
80°C.
Plant and soil material and rhizosphere extraction.
The soil
used was Rehovot sand, which was obtained from the experimental farm of
The Hebrew University of Jerusalem and contained 95% sand, 5% silt,
and no clay. The organic matter content was 0.9%, the pH was 7.3, and
the electrical conductance was 112 µS · cm1.
Common bean (Phaseolus vulgaris cv. Bulgarian) (Gedera
Seeds, Gedera, Israel) and tomato (Lycopersicon esculentum
cv. M-82) (Gedera Seeds) were grown in 1-kg pots in a greenhouse for 3 and 4 weeks, respectively. The soil used to grow the tomato plants was
amended with 30% sphagnum peat. Rhizosphere samples were obtained by
gently rinsing the roots with sterile water, leaving the adhering particles and small clumps, and blotting them to remove excess liquid.
The roots (5 g) were shaken for 30 min at 200 rpm in 40 ml of 0.1 M
phosphate buffer (pH 7.2) at room temperature, and the resulting
suspension was treated as described above. To include the
endorhizosphere, washed roots were ground with a mortar and pestle in
0.2% polyvinylpolypyrrolidone in sterile distilled water, which
yielded the total root bacteria. Bdellovibrios were
isolated from this slurry as described above.
Electron microscopy. One drop of fresh lysate was placed on a microscope grid, and excess liquid was removed by blotting. The sample was then counterstained with a 1% (wt/vol) solution of uranyl acetate for 30 min and examined with a JEOL model 100-CX transmission electron microscope.
Determination of prey range and kinetics of lysis.
Cultures
of the prey bacteria tested were grown in nutrient broth until the
stationary phase and then were pelleted by centrifugation at
4,400 × g for 10 min at 4°C. Each pellet was washed
once with 25 mM HEPES buffer containing 2 mM CaCl2 · 2H2O (pH 7.8) and resuspended in the same buffer. Then the
optical density at 570 nm of the suspension was adjusted to 0.55 to
0.6, which corresponded to a concentration of 108 to 5 × 108 cells · ml1 for most strains,
by using a cuvette with a 1-cm light path and a Genesis 5 spectrophotometer (Spectronic, Rochester, N.Y.). Fresh lysates of the
bdellovibrios which were going to be tested were passed
through a 0.45-µm-pore-size filter in order to remove residual prey
and cell debris, centrifuged at 10,000 × g for 15 min
at 4°C, and resuspended in HEPES to a final concentration of
106 to 5 × 106 cells · ml1. The supernatant was filtered through a
0.22-µm-pore-size membrane filter and used as a control. Prey
suspensions (150 µl) and predator suspensions (60 µl) were added to
96-well microtiter plates. The controls included
Bdellovibrio-free prey suspensions amended with buffer and prey suspensions amended with filtered lysates. The plates
were incubated overnight at 30°C on a rotary shaker at 200 rpm.
Turbidity (cell density) was determined at 570 nm by using a model
MR5000 enzyme-linked immunosorbent assay plate reader (Dynatech,
Denkerdorf, Germany). Six replicates of each predator-prey combination
were prepared, and each experiment was carried out at least twice.
Primers and PCR conditions. A Bdellovibrio-specific oligonucleotide sequence designed to represent almost all bdellovibrios, including representative of all species of this bacterial parasitic genus, was constructed by aligning the 12 Bdellovibrio sequences (two complete sequences and 10 partial sequences) present in databases. Seven sequences exhibited no mismatches, three exhibited mismatches caused by undefined bases (indicated by N), and two exhibited one defined base substitution (11). This conserved sequence was analyzed to determine whether it was specific by using CHECKPROBE software (11, 16), and then it was used as a Bdellovibrio-specific primer (primer 842R; 5'-CGWCACTGAAGGGGTCAA-3') in conjunction with Bacteria-specific primer 63F (18) in PCR. Primer 799F (5'-GGTAGTCCACGCCGTAAACGATG-3'), which was designed on the basis of partial Bdellovibrio sequences, is not specific for this group and was used in PCR along with primer 1492R (1).
Primers 176F (5'-GTGGCTTCAAACGGAGTGGA-3') and 887R (5'-ACGACTGTGAACGGCAACG-3'), which are specific for the hit locus, were designed based on a previously published nucleotide sequence (4). hit is a genetic locus in B. bacteriovorus 109J which appears to be involved in the ability of this strain to form axenic mutants (4). The templates used for PCR were either 1 or 10 ng of DNA, purified as described by Tsai and Olson (27), or individual plaques. In the latter case, plaques were resuspended in 100 µl of sterile double-distilled water and vortexed at a high speed. The liquid phase was transferred to a new tube and subjected to three cycles of freezing in liquid nitrogen followed by 3 min of heating in boiling water. The tubes were then cooled on ice, and 10% dimethyl sulfoxide was added. PCR (50-µl mixtures) were performed by using a programmable cyclic reactor (Ericomp Inc., San Diego, Calif.) or a Mastercycler gradient (Eppendorff, Hamburg, Germany). Each reaction mixture contained 3 mM MgCl2, each deoxyribonucleoside triphosphate at a concentration of 20 µM, 1× reaction buffer, 1.25 U of Taq polymerase, and each primer at a concentration of 1 µM. When the Ericomp cycler was used, the samples were covered with 2 drops of mineral oil (Sigma Chemical Co., St. Louis, Mo.). Amplification was started by using a denaturation cycle consisting of 4 min at 94°C, 50°C for 1 min, and 72°C for 1 min, and this was followed by 34 cycles consisting of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min and a final elongation step consisting of 72°C for 5 min. Most of the steps were only 45 s long when the Mastercycler gradient was used; the only exceptions were the first denaturation step and the last elongation step.Restriction analysis. Products obtained from PCR performed with primers 63F and 842R were purified by using a High Pure PCR purification kit (Roche Molecular Biochemicals, Mannheim, Germany) and then were enzymatically digested by using BamHI, EcoRI, SacII, and XbaI as recommended by the manufacturers. Digests were electrophoresed in 1% agarose gels and stained with ethidium bromide. A computer-assisted restriction analysis of sequences obtained from databases and in this study was performed by using the GCG package (Genetics Computer Group, Madison, Wis.).
DNA sequencing and sequence analysis. Products obtained from PCR performed with primers 63F and 842R or with primers 799F and 1492R (1) were purified as described above. A Big Dye terminator kit (Perkin-Elmer Inc., Branchburg, N.J.) was used in conjunction with these primer pairs to sequence the 16S ribosomal DNA (rDNA) fragments. The sequences were determined by using a model Prism 377 DNA sequencer (Applied Biosystem Inc., Foster City, Calif.).
Phylogenetic analysis. 16S rDNA sequence analyses were performed by using the program package ARB (http://www.biol.chemie.tu-muenchen.de/pub/ARB/). Sequences were aligned by using the implemented ARB automated alignment tool, and the alignment was refined manually by visual inspection and by secondary-structure analysis. Phylogenetic analyses were performed by using ARB parsimony, distance matrix, and maximum-likelihood methods. To determine the robustness of phylogenetic trees, analyses were performed both with the original data set and with a data set from which highly variable positions were removed by using a 50% conservation filter for members of the genus Bdellovibrio in order to reduce potential tree artifacts that may have resulted from multiple base changes. The consistency of the phylogenic tree was also verified by bootstrapping (n = 100).
Southern blot analysis. A Southern blot analysis was performed by using total DNA from strains SRP1, BEP2, BRP4, TRA2, and 109J after enzymatic digestion with SauIIIA and with a hit probe obtained by PCR digoxigenin labelling (Roche Molecular Biochemicals, Mannheim, Germany). Prehybridization was performed at 68°C, and hybridization was performed at 60°C. Detection was performed by using CSPD as recommended by the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany) after low-stringency washing (0.5 × SSC, 60°C) and high-stringency washing (0.1 × SSC, 68°C) (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate).
Nucleotide sequence accession numbers. Sequences obtained in this study have been deposited in the GenBank database under accession no. AF148938, AF148939, AF148940, and AF148941.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolation and quantification of soil and root-associated
bdellovibrios.
Thirty strains were isolated from soil
and from the root systems of common bean and tomato plants. The
isolation schemes used resulted in efficient separation of
bdellovibrios and other plaque-forming organisms, such as
bacteriophages and bacterium-consuming protozoans. Microscopic
observations and the presence of very active, swimming, small, usually
vibrioid cells established that lysates obtained from all of the
plaques tested resulted from bdellovibrio activity. Nine soil
strains isolated by using P. corrugata PC as prey
(strains SRP8E to SRP21 [Table 2]), three soil strains isolated by
using Erwinia carotovora subsp. carotovora 24 as
prey (strains SRE8, SRE9, and SRE12), and two soil strains isolated by
using Agrobacterium tumefaciens C58 as prey (strains SRA9
and SRA10), all of which originated from the same isolation event, were
tested to determine whether they exhibited activity with each of
these three substrate organisms in liquid culture. While the SRP and
SRE isolates induced lysis of both P. corrugata PC and
E. carotovora subsp. carotovora 24, they did not
utilize A. tumefaciens C58 as prey. The SRA isolates were
inactive when strain PC or strain 24 was used as a substrate. Moreover,
strains SPR1 to SPR4 did not prey on E. carotovora subsp. carotovora. Table 3 shows the
numbers of Bdellovibrio plaques retrieved from
soil, from the rhizosphere, and from a total extract of washed roots of
common bean.
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Determination of prey range and kinetics of prey lysis.
Prey
cell lysis data were validated by performing experiments in which the
changes in the optical densities of mixed suspensions containing
P. corrugata PC or Escherichia coli ML35 and
strain SRP1 were determined both in microtiter plates and in Erlenmeyer flasks. The cultures behaved similarly; P. corrugata PC
was cleared within 30 h, while E. coli ML35 was
resistant to the predator (data not shown). The
bdellovibrios examined (strains BEP2, BRP4, and SRP1,
which were isolated from a common bean rhizosphere, a total root
extract, and soil, respectively, by using P. corrugata; TRA2, which originated from tomato roots when A. tumefaciens
was used as prey; and collection strain 109J) lysed a wide range of selected gram-negative bacteria, including both bacteria that enhance
plant growth and phytopathogens (Table
4). In some instances, increases in
optical density were observed in control wells that were not inoculated
with bdellovibrios or in prey suspensions that were not
attacked by the predator; these increases may have been due to
utilization of capsule or storage materials. Controls, which were
prepared by using cell-free lysate of each of the
Bdellovibrio strains tested and each type of
prey, were always negative; i.e., lysis did not occur. Strains BEP2 and
BRP4 exhibited the same prey range and grew on 9 of the 22 potential
prey species tested, while SRP1 and TRA2 grew on six different prey
species. The most versatile predator was strain 109J, which grew at the
expense of 10 of the 22 prey cell suspensions tested. None of the
substrate cell suspensions tested supported growth of all five
Bdellovibrio strains, while Azospirillum
brasilense, Vibrio fluvialis, E. carotovora subsp. carotovora 2, and the gram-positive organism
Bacillus megaterium were resistant to attack by each of the
predators (Table 4). Large differences were observed in this lysis
fingerprinting analysis, even when closely related substrate
strains were used; while E. carotovora subsp.
carotovora 24 was preyed upon by strains BEP2, BRP4, and
109J, E. carotovora subsp. carotovora 2 was not.
Similarly, the Bdellovibrio strains exhibited
particular abilities to grow at the expense of various rhizobia and
fluorescent pseudomonads. A. tumefaciens C58 was
resistant to all of the bdellovibrios except TRA2, but
A. tumefaciens IDI was lysed by
bdellovibrio strains BEP2 and BRP4. Although strain
TRA2 produced relatively large plaques on A. tumefaciens
C58, it was not able to lyse cell suspensions of this bacterium in
HEPES and only partially lysed such suspensions in DNB. However,
efficient lysis was observed when Sinorhizobium meliloti
(which is phylogenetically closely related to A. tumefaciens) was used as prey in HEPES preparations. This
bacterium, as well as Rhizobium cicer and Xanthomonas
campestris pv. vesicatoria, produces copious amounts of
extracellular material; however, as previously reported, this material
did not protect the cells against Bdellovibrio
attack (15).
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Electron microscopy.
Transmission electron microscopy of
negatively stained preparations of attack cells was used to
measure the sizes of isolate BEP2, BRP4, SRP1, and TRA2 cells. As shown
in Fig. 2, BEP2 and BRP4 cells were
the smallest and the same size (0.85 by 0.2 µm), and SRP1 and
TRA2 cells were larger (1 by 0.25 and 1.15 by 0.4 µm, respectively).
A Bdellovibrio-containing bdelloplast is evident in Fig. 2d.
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Restriction enzyme profiles of 16S rDNA sequences amplified with a
Bdellovibrio-specific primer pair.
A PCR
product was obtained with all of the isolates, as well as with the
collection strains, when we used primers 63F and 842R and
Bdellovibrio DNA or individual plaques as
templates. No products were obtained when plugs of prey cell lawns or
prey cell DNA was used as the template (data not shown). The PCR
products of soil isolates yielded two restriction patterns, one
identical to the restriction pattern of B. stolpii UKi2
(strains SRP 1 to SRP4 [Fig. 3a]) and
one that was represented by 18 other SRP, SRE, and SRA strains (five of
which are shown in Fig. 3b) and was identical to the restriction
pattern of collection strain B. bacteriovorus 109J. Most of
the rhizosphere strains produced a third pattern, which was identical
to the restriction pattern of the cyst-forming organism B. bacteriovorus W (Fig. 3c and d); the only exception was
strain TRA2, whose pattern was like that of B. bacteriovorus 109J.
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Sequence analysis and phylogenetic tree.
The almost complete
16S rRNA sequences of four Bdellovibrio strains
are available from databases; these sequences include two very recently
completed collection strain sequences (2). We also
determined that sequences of two clones obtained from marine snow, one
clone that originated from cold sediments, and an unidentified
soil bacterium exhibited homology to the 16S rRNA gene of
Bdellovibrio spp. Only short fragments, mostly
of poor quality, were available for the other strains. Therefore, we
decided to use only the four long previously described
Bdellovibrio sequences and the data obtained in
this study to construct a reliable phylogenetic tree based on the
almost complete 16S rRNA gene sequence. Our phylogenetic analysis
confirmed that the bdellovibrios are members of the 
subclass of the class Proteobacteria (32) and
that these organisms are related to Myxococcus xanthus
(6) and Nitrospina gracilis. Strains BEP2
and BRP4 were found to be identical. Along with soil strains SAR9 and
SRE7 and the unidentified soil bacterial sequence whose accession
number is AF0128654, they clustered with B. bacteriovorus 109J (Fig. 4).
Although isolate TRA2 belonged to the B. bacteriovorus
group, it was more distantly related, exhibiting 92.4% identity with
strain 109J. This bacterium and type strain 100 are almost identical;
they differ only in one substitution and in a small number of undefined
bases. Isolate SRP1 was found to be very similar to B. starrii; the sequences of these organisms exhibited 99.2%
identity. Along with two sequences of uncultured bacteria that
originated from marine snow in the Adriatic Sea and one uncultured
clone obtained from marine sediment, the B. stolpii-B.
starrii cluster formed a rather heterologous group, which was
distantly related to B. bacteriovorus. Our sequence analysis
confirmed that B. stolpii, B. starrii, and SRP1
produce the same restriction profile. Restriction analysis of the
sequenced isolates clustering with B. bacteriovorus was also
confirmed.
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PCR and Southern analysis of the hit locus.
hit is a genetic locus in B. bacteriovorus 109J
which appears to be involved in the ability of this organism to form
axenic mutants (4). No PCR product was obtained from strain
BEP2, BRP4, SRP1, or TRA2 under the various amplification conditions tested (different temperatures and Mg concentrations) when we used
hit-specific primers, while an 810-bp fragment was amplified from B. bacteriovorus 109J and was sequenced in order to
confirm its identity. The hit locus was then subjected to
Southern analysis by using strains SRP1, BEP2, BRP4, and TRA2. No
signal was obtained with any of these strains after hybridization with
a hit-derived probe when the blot was washed under
high-stringency conditions. Under lower-stringency conditions, signals
that were clearly different from the 109J signal were obtained with
strains BEP2 and BRP4 (Fig. 5).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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New Bdellovibrio strains isolated from an agricultural soil, from plant roots grown in the same soil, and from various sources were examined. Based on restriction, phylogenetic, and prey range analyses, we found that the root bdellovibrios and the rhizosphere bdellovibrios were different. Moreover, we found that various populations coexisted in the soil at different levels.
The bdellovibrios accounted for only a small fraction of the total bacterial population in an agricultural soil and in the rhizosphere of common bean, and the numbers of Bdellovibrio plaques per gram of sample were similar to values reported previously (29). The bdellovibrios are a genetically heterologous group of bacteria that must invade bacterial hosts to survive (22). Therefore, they can be isolated and grown only in two-member cultures with selected prey organisms. This characteristic makes estimating Bdellovibrio population sizes and isolating large numbers of isolates difficult. We obtained a PCR fragment from all of the strains isolated in this study and from collection strains by using a Bdellovibrio-specific oligonucleotide (11) and primer 63F, which targets the domain Bacteria (18). Restriction analysis of the products revealed that the soil isolates belonged to two different ribotypes, one represented by B. stolpii UKi2 and the other represented by B. bacteriovorus 109J; SRP strains (isolated by using P. corrugata cells) belong to both ribotypes. Almost all of the rhizosphere bdellovibrios belonged to a different ribotype identical to the ribotype of B. bacteriovorus W.
Members of a group of strains that were isolated on and preyed on both P. corrugata and E. carotovora subsp. carotovora 24 and members of another group of strains that were isolated on A. tumefaciens C58 produced the same restriction pattern but could not utilize the prey used to isolate members of the other group. A phylogenetic analysis of isolates SRE7 and SRA9, which belonged to the former group and the latter group, respectively, revealed that these organisms clustered with B. bacteriovorus but were different from each other. Also, the number of bdellovibrios that were active on A. tumefaciens C58 and were retrieved from the soil was significantly lower than the number of bdellovibrios that were able to grow at the expense of P. corrugata PC or E. carotovora subsp. carotovora 24. These findings (prey range, ribotype, phylogenetic affiliation, and level in soil samples) together suggest that the soil strains which we isolated belonged to at least three distinct populations.
Strains BEP2 and BRP4, which were analyzed further, were indistinguishable; their 16S rDNA sequences, their low levels of homology to the hit locus, their growth kinetics in liquid and solid media, and their prey ranges were identical. Since they originated from the same root system, they probably were clones of the same population.
The heterogeneity of the genus Bdellovibrio is reflected by the distance between the B. stolpii-B. starrii group and the B. bacteriovorus groups (Fig. 5), as well as the distances within these groups (6; this study). On the basis of the great phylogenetic distances between Bdellovibrio species, Baer et al. (2) recently proposed that the new genus Bdellovorax should be formed and should include B. stolpii and B. starrii. The results presented in this study support this proposal.
Both the B. bacteriovorus branch and the B. stolpii-B. starrii branch contain distantly related organisms, based on sequences, such as isolate TRA2 (which is 92.4% identical to the strain 109J sequence). We also detected one uncultured soil bacterium and three uncultured marine clones which clustered with B. bacteriovorus and B. stolpii, respectively, and increased the heterogeneity in these groups. However, whether these uncultured bacteria are true bacterial parasites is not known.
The 959-bp hit locus, which is associated with the host-independent phenotype in strain 109J (4), is the only genetic locus that has been defined in members of the genus Bdellovibrio. No PCR product was obtained with strains BEP2, BRP4, SRP1, and TRA2 under any of the amplification conditions tested when hit-specific primers were used. The results of Southern analysis suggest that a heterologous, cross-hybridizing sequence is present in strains BEP2 and BRP4, which are closely related to strain 109J, but not in TRA2, a more distantly related strain in the B. bacteriovorus cluster, or in SRP1, which is related to B. stolpii UKi2. Since axenic mutants can be obtained readily from isolated bdellovibrios (24), there may be divergent but functionally equivalent loci in other strains. Therefore, this tool may become a useful tool for characterization of bdellovibrios. Phylogenetic affiliations cannot be inferred on the basis of prey range, which also depends on the experimental conditions used (26). However, such information still provides a useful tool for characterizing and distinguishing between bdellovibrios obtained from a particular environment and for assessing the impact of these bacterial predators on microbial communities. In this study, common bean rhizosphere isolates BEP2 and BRP4 and the two groups of soil SRP strains exhibited different prey spectra, in spite of the fact that they were originally isolated with the same substrate cells. Also, strain TRA2, which was isolated by using A. tumefaciens as prey, utilized five of the seven members of the family Rhizobiaceae tested and differed substantially from all of the other isolates. The biological basis for prey specificity is not known, although it has been suggested that the R antigen is involved (29). However, Rhizobium etli and S. meliloti differ markedly in this respect (12), although both of these organisms are lysed by strain TRA2. Moreover, the rough mutant AK 631 and the smooth wild-type strain S. meliloti Rm41 were also attacked by strain TRA2, in spite of the fact that this kind of phenotype may result from a change in the molecular structure of the R antigen (3).
Soil strain SRP1 and rhizosphere strains BEP2 and BRP4 behaved differently; while the former grew faster, forming larger plaques in double-agar plates, and swam freely in wet mounts, the latter grew more slowly and adhered to the glass. It has been shown that bdellovibrios are components of biofilms (13, 31) and exhibit different tendencies to adhere to glass (21), which may reflect adaptation to specific biotas. In a recent study, Markelova and Kerzhentsev showed that a rhizosphere Bdellovibrio isolate adhered to a plastic surface and completed its growth cycle when it was presented with adsorbed prey cells (19).
Bacterial rhizosphere and soil populations differ in relative composition or in structure, as recently shown by a denaturing gradient gel electrophoresis analysis of samples obtained from both microenvironments (7, 14). Rice et al. (20) showed that marine bdellovibrios obtained from various biotas are able to attack up to 85% of isolated autochthonous gram-negative bacteria. Therefore, a shift in bacterial composition from one biota to another, such as the rhizosphere to bulk soil, may provide an advantage to a subpopulation of bdellovibrios best adapted to use the potential prey found in the root microhabitat. This may reflect adaptations to different niches. Moreover, when cabbage was inoculated with fluorescent pseudomonads, an increase in the number of recoverable rhizosphere bdellovibrios able to use this group of bacteria was detected (8). The possibility that the root-associated bdellovibrios examined in this study originated from the seeds and developed along with the root bacterial populations cannot be ruled out as members of the genus Bdellovibrio have been shown to survive in dry soil for extended periods (10), probably in association with soil particles. However, in view of the lengthy storage the seeds used and no previous reports of seed colonization by bdellovibrios, we assumed that the root-associated bdellovibrios originated in the soil and that the bacterial substrates present were a determining factor for selecting rhizosphere-competent bdellovibrios.
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ACKNOWLEDGMENTS |
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We thank R. Guvrin for technical assistance. We also thank Y. Okon and the participants in our weekly discussion group for their helpful remarks and suggestions.
This work was supported by grants from the Israeli Ministry of Agriculture (grant 823-0138-98) and the Israel Science Foundation (grant 132/99).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O.B. 12, Rehovot 76100, Israel. Phone: 972 8 9489167. Fax: 972 8 9466794. E-mail: jurkevi{at}agri.huji.ac.il.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, June 2000, p. 2365-2371, Vol. 66, No. 6
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