Download 16S rDNA sequence analysis of environmental Bdellovibrio-and

International Journal of Systematic and Evolutionary Microbiology (2002), 52, 2089–2094
NOTE
1,2
Marine Estuarine
Environmental Sciences
Program1 and
Department of
Craniofacial Biological
Sciences2 , University of
Maryland, Baltimore, 666
West Baltimore Street,
Baltimore, MD 21201,
USA
3
Biology Department,
Shippensburg University,
Shippensburg, PA 17257,
USA
4
Department of Medical
and Research Technology,
University of Maryland
School of Medicine,
Allied Health Building,
100 Penn Street,
Baltimore, MD 21201,
USA
5
Department of
Epidemiology and
Preventative Medicine,
University of Maryland
School of Medicine,
10 South Pine Street,
Baltimore, MD 21201,
USA
DOI : 10.1099/ijs.0.02261-0
16S rDNA sequence analysis of environmental
Bdellovibrio-and-like organisms (BALO) reveals
extensive diversity
Andrew R. Snyder,1 Henry N. Williams,1,2 Marcie L. Baer,3
Kimberly E. Walker4 and O. Colin Stine5
Author for correspondence : Henry N. Williams. Tel : j1 410 706 7211. Fax : j1 410 706 0193.
e-mail : hnw001!dental.umaryland.edu
Bdellovibrio-and-like organisms (BALO) are Gram-negative, predatory bacteria
that inhabit terrestrial, freshwater and salt-water environments. Historically,
these organisms have been classified together despite documented genetic
differences between isolates. The genetic diversity of these microbes was
assessed by sequencing the 16S rRNA gene. Primers that selectively amplify
predator 16S rDNA, and not contaminating prey DNA, were utilized to study 17
freshwater and terrestrial and nine salt-water BALO isolates. When the 16S
rDNA sequences were compared with representatives of other bacterial
classes, 25 of the 26 BALO isolates clustered into two groups. One group,
supported 100 % by bootstrap analysis, included all of the Bdellovibrio
bacteriovorus isolates. Each member of this group was isolated from either a
freshwater or terrestrial source. The genetic distance between these isolates
was less than 12 %. The other group, supported 94 % by bootstrap analysis,
includes Bacteriovorax starrii, Bacteriovorax stolpii and the salt-water isolates.
The salt-water isolates form a subgroup (83 % by bootstrap) and differ within
the subgroup by less than 11 %. This observation implies that the salt-water
isolates arose from Bacteriovorax progenitors. The difference between isolates
in different clades is over 17 %, a quantity similar to differences between
bacterial species in different classes. However, both the Bdellovibrio and
Bacteriovorax clades were closest to other representatives of the δProteobacteria using maximum-likelihood. One freshwater isolate, James
Island, was distinct from all other BALO (S 19 %), but differed from
Pseudomonas putida, a member of the γ-Proteobacteria, by only 3 %. Thus, by
16S rDNA sequence analysis, the BALO appear to have multiple origins,
contrary to the unified taxonomic grouping based on morphology and natural
history. These observations are consistent with the need to review and revise
the taxonomy of these organisms.
Keywords : Bdellovibrio-and-like organisms, 16S rDNA, environmental diversity
Bdellovibrio-and-like organisms (BALO) are Gramnegative, predatory bacteria that have a biphasic
lifestyle that includes a free-living attack form and an
intraperiplasmic growth phase that occurs within the
cell wall of a prey bacterium. These organisms are
.................................................................................................................................................
Abbreviations : BALO, Bdellovibrio-and-like organisms ; JC, Jukes–Cantor ;
K2, Kimura’s two-parameter.
The GenBank accession numbers for the 16S rRNA sequences obtained in
this study are AY094106–AY094131.
02261 # 2002 IUMS Printed in Great Britain
small, vibrioid-shaped and exhibit rapid motility during their attack phase form. In this report, BALO
refers to the bacteria within the genera Bdellovibrio
and Bacteriovorax (Baer et al., 2000) and undefined
isolates of intraperiplasmic, predatory prokaryotes.
Isolated from soil, sewage, fresh and salt-waters,
BALO may have an ecological role as contributors to
bacterial mortality in nature.
BALO represent a poorly defined, diverse group of
bacteria that exhibit wide ranges in salt tolerances,
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A. R. Snyder and others
Table 1. Strains used in these experiments
.................................................................................................................................................................................................................................................................................................................
BALO were isolated from water samples from around the world using standard culturing techniques for BALO. In all, 17
freshwater\terrestrial and nine marine\estuarine isolates were used in the study.
Isolate
Bdellovibrio bacteriovorus
109J
Ox9-2 (l ATCC 25630)
E (l ATCC 25634)
2484Se2 (l ATCC 25635)
109D*
Bacteriovorax stolpii ATCC 27052T
ARL-12*
P*
6-5-S*
Vietnam*
Tu-113*
Mil-1
ARL-1*
Lanham
NASA
Hefner
James Island
SJ
JS5
OC2
OC4
Cancun1B
Cancun2
Cancun6
Cancun7
Annapolis
Escherichia coli ML-35
Vibrio parahaemolyticus P-5
Fresh/salt-water
Source/origin
Reverse primer used
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Salt
Salt
Salt
Salt
Salt
Salt
Salt
Salt
Salt
Fresh
Salt
Dr M. Thomashow
ATCC
ATCC
ATCC
Dr M. P. Starr
ATCC
Sewage-treatment plant, Russia
Pond, Russia
Dr S. Straley
River, Vietnam
Dr S. Straley
Milwaukee, WI, USA
Russia
River, Prince George’s Co., MD, USA
River, Prince George’s Co., MD, USA
Lake Hefner, OK, USA
South Carolina, USA
St John, US Virgin Islands
Crab gill, Patuxent River, MD, USA
St Martin’s River, MD, USA
Ocean City, MD, USA
Biofilm, Cancun, Mexico
Cancun, Mexico
Cancun, Mexico
Cancun, Mexico
Severn River, MD, USA
Dr R. K. Nauman, UM, Baltimore, USA
Dr D. Johnson, VAMHCC
Bact-rev
Bact-rev
Bact-rev
Bact-rev
Bact-rev
Stolpii-rev
Bact-rev
Bact-rev
Bact-rev
Bact-rev
Bact-rev
Stolpii-rev
Bact-rev
Bact-rev
Bact-rev
Bact-rev
Stolpii-rev
Saltwater-rev
Saltwater-rev
Saltwater-rev
Saltwater-rev
Saltwater-rev
Saltwater-rev
Saltwater-rev
Saltwater-rev
Saltwater-rev
1492-R(2)
1492-R(2)
* Isolates kindly donated by Dr A. Afinogenova.
GjC ratios, fatty-acid profiles, membrane protein
compositions, antigenic variations and prey ranges
(Guether et al., 1993 ; Kramer & Westergaard, 1977 ;
Marbach et al., 1976 ; Park & Mahadevan, 1988 ;
Ruby, 1991 ; Seidler et al., 1969, 1972 ; Severin et al.,
1981 ; Sutton & Besant, 1994 ; Taylor et al., 1974). The
taxonomic status of the organisms remains ill defined.
Analysis of the diversity of BALO by molecular
techniques is just beginning (Baer, 1998 ; Baer et al.,
2000 ; Donze et al., 1991 ; Hespell et al., 1984 ; Snyder,
2000) and has already demonstrated its value. 16S
rDNA sequencing and DNA–DNA hybridization
studies revealed sufficient diversity among the genus
Bdellovibrio to place the species Bdellovibrio starrii and
Bdellovibrio stolpii into a newly established genus,
Bacteriovorax (Baer et al., 2000). Additionally, several
soil isolates of BALO were reported to be divergent
based on ribotyping, partial 16S rDNA sequencing
and prey range (Jurkevitch et al., 2000 ; Jurkevitch &
Ramati, 2000).
In this study, we amplified and sequenced 16S rRNA
2090
genes from 17 freshwater and nine marine\estuarine
isolates of BALO recovered from various locations
around the world (Table 1).
To prepare isolates for PCR amplification, stock
cultures were subcultured into fresh broth medium.
Freshwater and terrestrial isolates were cultured with
Escherichia coli ML-35 as prey growing in dilute
nutrient broth (DNB) (Ruby, 1991). Salt-tolerant
isolates were cultured with Vibrio parahaemolyticus P5 as prey in Pp20 medium (Ruby, 1991). Cultures were
incubated for 2–4 days at either 30 mC for terrestrial
and freshwater isolates or 25 mC for salt-tolerant
isolates. When the prey had been nearly exhausted, as
indicated by clearing of the initially turbid broth
suspension, 10-fold serial dilutions were made and
plated using the double-agar-overlay method to yield
well-isolated plaques. Plates were incubated at the
appropriate temperature until well-defined plaques
were observed in the top agar.
A single plaque excised from a culture plate was
International Journal of Systematic and Evolutionary Microbiology 52
16S rDNA sequence diversity among BALO
.................................................................................................................................................................................................................................................................................................................
Fig. 1. Neighbour-joining tree of BALO isolates. A neighbour-joining tree was constructed for the 17 salt-water and nine
freshwater isolates by aligning these sequences with other selected members from the prokaryotic domain. Listed beside
each organism or strain name is the GenBank accession number (in parentheses). Numbers at branch-points represent
confidence values obtained after bootstrap analysis of the neighbour-joining tree using 1000 replicates.
transferred into 5n0 ml of appropriate medium (DNB
or 70 % artificial sea water) with a minimal amount of
fresh prey cell suspension. After incubation (48–72 h),
http://ijs.sgmjournals.org
cell suspensions were centrifuged for 20 min at 12 000 g
and cell pellets were resuspended in 1n0 ml sterile PBS.
The cell suspension was then filtered through a 0n45 µm
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A. R. Snyder and others
syringe filter to eliminate prey cells. Samples were
centrifuged at 18 000 g for 15 min and the supernatant
was decanted. The tubes were then vortexed and the
cells were used as template for PCR.
16S rDNA PCR and sequencing
PCR primers were designed to amplify target predator
genes selectively while avoiding prey cell DNA amplification. A panel of four species-specific reverse
primers was synthesized to be used individually with a
universal Bdellovibrio forward primer. The primers
were as follows : bdello-forward (5h-GCGTGCCTAATACATGCAAG-3h, Tm l 59n1 mC, 3 mismatches in
comparison with the sequences of the prey, E. coli and
V. parahaemolyticus), annealing to bases 15 to 34 (all
E. coli numbering) ; bact-rev (5h-AGATAGCTTTTAAGCGATTTGCTCTAC-3h, Tm l 60n9 mC, 15 mismatches), annealing to bases 1258 to 1284 ; saltwaterrev (5h-TGCTAACTGTCACCAGATCGCTT-3h, Tm
l 63n3 mC, 9 mismatches), annealing to bases 1243 to
1265 ; stolpii-rev (5h-CGGTTTTTTGAGATTGGCTC-3h, Tm l 59n5 mC, 9 mismatches), annealing to bases
1261 to 1280 ; and starrii-rev (5h-CCGAACTGAGGCGCGC-3h, Tm l 65n4 mC, 9 mismatches), annealing
to bases 1277 to 1292. The placement of the primers
along the 16S rRNA gene allowed for amplification of
approximately 1250 bases in all cases. PCRs were
performed in a total volume of 25 µl containing Ready
to Go PCR beads (Amersham Pharmacia Biotech),
10 pmol of each primer, 2 µl cell suspension and 19 µl
sterile distilled water. The PCR cycle consisted of a
95 mC denaturation step (5 min), 30 cycles of 95 mC
(30 s), a 45 mC annealing step (30 s) and a 72 mC
extension step (30 s) and a final extension step for
7 min at 72 mC. PCR products were electrophoresed on
a 0n7 % agarose gel to check for successful amplification.
Amplified PCR products were purified and separated
from PCR primers using the Qiaquick PCR purification kit (Qiagen). Purified products were electrophoresed on a 2 % agarose gel to check for purity and
band intensity. When there was insufficient amplification, a larger amount of the purified PCR product
was used for sequencing. Up to 7 µl of each product
was used in 12 µl sequencing reactions that included
1 µl of 3n3 µM primer and 4 µl Big Dye Terminator
cycle sequencing mix (Perkin-Elmer). Sequencing PCR
conditions were 94 mC for 1 min, 25 cycles of 96 mC
(30 s), 50 mC (5 s) and 60 mC (4 min) and a final
extension step of 72 mC (2 min). All PCR products
were sequenced in both directions.
Sequence analysis and dendrogram construction
Raw sequence data were compiled using the
\\ package (Ewing & Green,
1998 ; Ewing et al., 1998 ; Gordon et al., 1998). The
low-quality bases from the ends were removed and the
resulting sequences were aligned with representative
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16S rDNA sequences from representative bacterial
and archaeal isolates selected from GenBank (see Fig.
1). All of the sequences were aligned using  
version 1.64 (Jeanmougin et al., 1998). Analysis of the
aligned sequences was performed using distance
matrices, UPGMA, neighbour-joining and bootstrapping algorithms as implemented in  (version
3.1 ; Sinauer Associates). Trees were constructed using
three methods, maximum-parsimony, distance and
maximum-likelihood methods, and three substitution
models, uncorrected ‘ p ’, Jukes–Cantor (JC) and
Kimura’s two-parameter (K2). The sequence data have
been submitted to GenBank as accession numbers
AY094106–AY094131.
Phylogenetic interpretation
The 16S rRNA gene was selectively amplified by PCR
using BALO-specific primers that did not amplify prey
sequences even when the prey DNA was the only
template for PCR. Phylogenetic relatedness of the 16S
rRNA gene from the 26 BALO isolates was examined
in relation to 63 sequences from selected representative
bacterial and archaeal isolates from GenBank (Fig. 1).
The phylogenetic tree based on the GenBank
sequences is similar to published trees (Dang & Lovell,
2000). There are two main clusters of BALO isolates,
containing 25 of the 26 isolates. A branch of one of
these clusters contains BALO that clustered away
from all other bacteria 100 % of the time in bootstrap
analysis regardless of whether the substitution model
was uncorrected ‘ p ’, JC or K2. This group included all
five Bdellovibrio bacteriovorus isolates and seven additional isolates that all diverge from each other by less
than 5n2 %. These isolates included ones from Russia,
Vietnam and the United States, indicating a widespread distribution of the species. Two additional
isolates, Nasa and Lanham collected in Maryland,
differ from each other by 0n3 % and from the
Bdellovibrio bacteriovorus isolates by 6n8–11n1 %.
These two isolates cluster together at least 99 % of the
time by bootstrap analysis regardless of the substitution model. All members of this clade were isolated
from freshwater or terrestrial environments.
The second clade of BALO contains the freshwater
isolates Bacteriovorax starrii and Bacteriovorax stolpii
and Mil1 and the nine estuarine and marine isolates,
referred to as salt-water isolates in Fig. 1. The second
clade was distinct in 94 % of 1000 replicates by
bootstrap analysis using the uncorrected ‘ p ’ substitution model, in 98 % of 100 replicates using a JC
model and in 100 % using a K2 model. Within this
clade, the nine salt-water isolates form a monophyletic
subgroup (83 % by bootstrap using ‘ p ’, 95 % by JC
and 98 % by K2). They differ from each other by less
than 15 %. Some BALO isolates from both the Atlantic
Ocean and the Caribbean Sea had similar sequences,
indicative of being members of the same species, while
the Cancun isolates from a single sample show enough
diversity potentially to represent different species. The
International Journal of Systematic and Evolutionary Microbiology 52
16S rDNA sequence diversity among BALO
salt-water isolates differ from the freshwater\
terrestrial isolates within this clade by 12n2–25 %. Our
observations imply that the salt-water isolates arose
from Bacteriovorax progenitors.
The BALO isolate outside the major clades for BALO
is the freshwater James Island isolate. Its 16S rDNA
sequence differs by 3n2 % from that of Pseudomonas
putida, a representative of the γ-Proteobacteria. It is
separated from all of the other BALO by a minimum
of 19 % sequence divergence. James Island and P.
putida cluster together in 96 % of 1000 bootstrap
replicates using uncorrected ‘ p ’ and 100 % of 100
replicates using either the JC or K2 substitution model.
The closeness of this sequence to Pseudomonas was
unexpected and requires explanation. The primers
were selected to be mismatched to E. coli and V.
parahaemolyticus and would not be expected to
amplify Pseudomonas-like sequences. However, there
is no indication of divergence near the ends of the
BALO sequence that might imply a chimera. One
implication of the closeness of the 16S sequences is that
systematic surveys using probes for 16S rDNA from
either Bdellovibrio or from Bacteriovorax will miss
these potentially important predators, and the best
method for screening for BALO is therefore the
traditional test for plaques on several distinct prey
species. One alternative explanation that cannot be
ruled out by the data is that this BALO lineage is the
result of a recombination of the 16S rDNA between a
BALO and a pseudomonad (Yap et al., 1999). BALO
reside in the periplasmic space, and the potential for
transformation of genes is likely to be higher than for
bacteria separated by cell walls ; P. putida is a known
prey of BALO. Whether or not this has occurred could
be tested by examination of housekeeping genes and
the genes involved in predation. Alternatively, BALO
with Pseudomonas-like sequences may be a case of
independent evolution of the predatory phenotype.
BALO are placed taxonomically in the δProteobacteria, along with Desulfovibrio desulfuricans,
Myxoccus xanthus, Syntrophobacter fumaroxidans and
Desulfobacter vibrioforme. These members of the δProteobacteria do not form a cluster under bootstrap
analysis, regardless of the model of substitution. The
differences between the sequences are too large. However, the δ-Proteobacteria are related more closely to
each other by maximum-likelihood analysis than they
are to members of other subdivisions like the εProteobacteria, which contains Campylobacter, Arcobacter, Wolinella and Helicobacter and clusters
together 100 % of the time by bootstrap analysis in all
trees. Based on these data from 16S rDNA sequences,
whether or not the two BALO clusters are monophyletic can not be resolved unambiguously.
The diversity of 16S rDNA sequences was greater than
expected based on the current taxonomic designation
of Bdellovibrio and Bacteriovorax as sibling genera.
Although Bdellovibrio and Bacteriovorax were part of
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separate monophyletic groups, the distance between
these clades was as large as those between species in
different bacterial subdivisions. The diversity among
prokaryotic divisions ranges from 13 to 26 % (Dang &
Lovell, 2000). The large ( 17 %) genetic distances
between the groups of BALO are consistent with either
a very ancient origin or two independent origins.
Although these two clades are distinct by bootstrapping, a single origin of BALO may be argued,
since they are both members of the δ-Proteobacteria. If
so, the original ancestor would have lived in a
freshwater or terrestrial environment, since the saltwater isolates are a monophyletic subgroup in the
cluster containing the freshwater Bacteriovorax isolates. Whether these clades represent a single origin or
two independent origins may be resolved by
sequencing either the whole genome or selected
sequences like those involved in DNA repair or
predation from isolates from each clade.
Our observation of substantial genetic diversity among
Bdellovibrio and Bacteriovorax is consistent with recent
work from PFGE and ribotyping (Baer, 1998).
Although PFGE and ribotyping clearly distinguish
between the two groups and separate the James Island
isolate, the distance between isolates and groups
cannot be assessed as accurately as with sequencing
results.
The taxonomy of Bdellovibrio and Bacteriovorax may
warrant revision based on our observations from 16S
rDNA sequences. At the very least, the taxonomy of
the salt-water isolates needs to be revised, since they
cluster with Bacteriovorax on the basis of the genetic
relatedness of 16S rDNA, contrary to the current
taxonomy, which assigns them to the genus
Bdellovibrio. If the criterion that a species consists of
organisms with 95 % or greater 16S rDNA similarity is
invoked (Amann et al., 1995) for BALO, at least two
species of Bdellovibrio and four species of
Bacteriovorax could be defined. In addition, by putting
the isolates into context with other members of the
eubacterial kingdom, it is evident that the extent of
nucleotide difference between Bacteriovorax isolates
may be equal to or greater than that measured between
different genera of bacteria. As a singlet, the James
Island isolate will remain a taxonomic curiosity.
However, if additional related isolates are found, a
new taxonomic designation will be required. Because
BALO cannot be cultured like most bacteria, the
traditional rules for taxonomic designation do not
apply for identifying novel species. The use of genomic
sequences or sequence data from other loci may
provide suitable data for revising the taxonomic
designations for BALO.
Acknowledgements
This work has been supported by NSF grant OCE-9731055.
Special thanks to Lily Zheng, Albina Afinogenova, Ruby
Singh and those who kindly donated water samples to this
study.
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A. R. Snyder and others
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