Bacterial
diversity
Australian
in a soil sample
environment
from
a subtropical
as determined
by 16S rDNA
analysis
E. STACK1BRANIYr
W. LIESACX,1
Department
of Mkrobiolog
Lucia,
Qld. 4072, Australia
Centre
AND
B. M. GOEBEL
for Bacterial
Diversity
ABSTRACT
In order to investigate
the genetic
diversity of streptomycetes
in an acid forested
soil sample from
Mt. Coot-tha,
Brisbane,
Australia,
cells were mechanically lysed within
the soil matrix and genomic
DNA was
isolated
and purified.
16S ribosomal
(r)DNA
was amplified
by the polymerase
chain reaction
(PCR)
method
using one primer
conserved
for members
of the domain
Bacteria
and a second designed
specifically
for streptomycetes and related
taxa. PCR amplification
products
were
cloned
into phage vector M13 mpl9 and the diversity
of
16S rDNA
genes was determined
by sequence
analysis
and oligonucleotide
probing
of the resultant
clone library.
Comparison
of partial
16S rDNA
sequences
with published
sequences
revealed
that few sequences
originated
from streptomycetes.
The majority
of sequences
belonged
to members
of the alpha subclass of Proteobacteria.
Other
clones were related
to planctomycetes,
actinomycetes,
or
represented
novel lines of descent.
Bacteria
that are customarily
isolated
from soil of pH 4-7 such as thiobacilli,
bacilli,
spore- and nonsporeforming
actinomycetes,
and
pseudomonads
are represented
in the clone library
in
small numbers
or were not detected
at all. Parameters
influencing
the recovery,
amplification,
quantification,
and interpretation
of genetic
information
from natural
sites
are discussed.Stackebrandt,
E., Liesack,
W.,
Goebel,
B. M. Bacterial
diversity
in a soil sample from a
subtropical
Australian
environment
as determined
by
16S rDNA analysis.
FASEBJ
7: 232-236;
1993.
Key Words:
phylogenetic
analysis
genetic
and Identification,
St.
MATERIAL
AND
METHODS
diversity
STUDIES
THAT MEASURE
THE physiological
activities of microorganisms
in situ are typical
examples
of a
“black
box”
approach
to understanding
the role
and
significance
organisms
play in ecosystem
functioning.
Measurements
of actual microbial
and genetic diversity,
population size, spatial distribution,
and the fraction
of metabolically active members
within a complex microbial
community
have proved
to be difficult
using conventional
enrichment
and isolation
techniques.
A combination
of modern
molecular ecological
approaches
designed
to reveal a more accurate
reflection
of the microbial
and genetic diversity,
with classical and novel isolation
techniques,
will allow a more directed
approach
to environmental
studies.
Ultimately,
the fluctuations occurring
within a natural
microbial
community
due to
changes
in physicochemical
conditions
could be monitored,
quantified,
and even manipulated.
The failure of traditional
enrichment
techniques
to cultivate the majority
of prokaryotes
that were shown to exist in
natural
samples-estimates
of unculturable
and
as yet
232
of Queensland,
unknown
prokaryotic
species
are as high as 99% of the
known diversity
(1, 2)-calls
for novel approaches.
The first
step was the application
of molecular
techniques
to the
characterization
of microbial
diversity
in the environment.
This was a logical consequence
of strategies
developed
for
the determination
of relationships
between
pure cultures,
i.e., the elucidation
of the primary
structure
of ubiquitously
distributed
genes that were also evolutionary
and functionally conserved
such as ribosomal
RNA and rRNA genes (3).
The first of such strategies
was introduced
by Pace and coworkers
in the middle
1980s (4, 5). Variations
thereof were
later applied
to a variety of samples
such as the marine
environment
(6-10),
hot springs
(11, 12), and soil (13, 14).
Results of these studies confirmed
earlier suggestions
that the
number
of described
prokaryotic
species (about
3000) is a
gross underestimation
of the diversity
that actually
exists.
Molecular
evidence
indicates
that of the rDNA
sequences
recovered
from biomass,
not only was none identical
to those
of described
species, but most of the sequences
were not even
related to culturable
species at the genus level. In this communication
we summarize
(13, 14) and provide
new data on
the microbial
diversity
of a random
soil sample from a subtropical
Australian
region
that indicates
the presence
of
several novel types of prokaryotic taxa within the domain Bacteria.
DNA isolation,
sequencing
ECOLOGICAL
The University
16S rDNA
amplification,
cloning,
and
A subsurface
sample,
5 through
10 cm in depth,
was collected from the Mount
Coot-tha
region,
Brisbane,
Queensland, Australia.
The methods
applied for the molecular
analysis
of microbial
diversity,
including
extraction
and
purification
of bulk DNA,
PCR-mediated
amplification
of
16S rDNA, cloning of the amplified
fragments,
and sequencing analysis,
have been described
(14). Here only the important steps of the strategy
will be summarized:
genomic
bulk
DNA released
from strains
after mechanical
disruption
of
the cells within their natural habitat with glass beads was isolated and purified
according
to Steffan
et al. (15). As a
modification,
the hydroxyapatite
column
chromatography
‘To whom correspondence
should be addressed, at: Max-PlanckInstitut f#{252}r
Terrestrische
Mikrobiologie,
D-W 3550 Marburg,
Federal Republic of Germany.
2Abbreviation:
PCR,
polymerase
chain reaction.
0892-6638/93/0007-0232/$01
.50. © FASEB
step was replaced
by a preparative
agarose
gel (0.7%) electrophoresis
to remove
low-molecular-weight
DNA.
The 5 and 3’ primers
used for polymerase
chain reaction
(PCR)2-mediated
amplification
of a large region
of 16S
rDNA were 5-’ CCCCCATCC/GAGTVII3ATCCTGGCThAG3 (Escherichia coli positions
9 through
27, IUB nomenclature)
and 5- CCCCCTCCAC/GCCATTGTAGCACGTGTGCA3
(positions
1224 through
1243).
The
underlined
regions
represent
overhangs
with a BamHI
and a Sail restriction
site, respectively. During the course of this investigation,
a
survey
of about
400 16S rDNA
sequences
for internal
BamHI/SalI
restriction
sites indicated
that a higher proportion of rDNA operons
was susceptible
to these enzymes
than
was originally
expected.
Between 1000 and 1130 nucleotides
were determined
for
clones MCs 4, 9, 13, 19, 22, 24, 26, 27, 58, 74, 77, and 106.
After an initial phylogenetic
survey demonstrated
that additional clone sequences
were related to the long sequence
versions, the analysis
of the new clones was restricted
to about
450 to 500 nucleotides
(MC’S 42, 47, 64, 65, 66, 87, 101, 103,
and 114). The sequences
for the 20 sequenced
clones have
been deposited
in EMBL
(Heidelberg)
under the accession
numbers
X68454
to X68474.
Phylogenetic
analyses
The
16S rDNA
clone sequences
were entered
into the
Ribosomal
RNA Database
Project (16), supplemented
with
previously
published
sequences
from the same clone library
(13, 14). Evolutionary
distance
values were calculated
by the
algorithm
of Jukes
and Cantor
(17) using only those sequence positions
for which all strains or clones had data and
could be aligned unambiguously,
omitting
undetermined
positions
and alignment
gaps. Phylogenetic
trees were constructed
from dissimilarity
matrices
by the Neighbor-joining
method
of de Soete (18). The reproducibility
of the branching nodes was examined
by changing
the number
and taxa
of reference
organisms
as well as by the neighbor-joining
bootstrapping
program
PDFIND
and NJBOOT
(kindly
provided
by T S. Whittam,
Institute
of Molecular
Evolution
and Genetics,
Pennsylvania
State University).
One thousand
bootstrap
trees were generated
and examined.
Analyses
were
done on a SUN Sparc ICP Workstation.
Cultivation
land,
Australia
(G. Kervin,
Department
of Agriculture,
University
of Queensland,
St. Lucia,
QId. Australia,
personal communication).
Slightly elevated
aluminum
(128 sM)
and iron (14.8 tM) levels were present
in solution,
which can
be due to the low pH ofthis
soil. Nitrate (1233 tiM) and ammonium
(151 ,sM) levels were also quite high, which could
indicate
an active nitrifying
population
uninhibited
by the
low pH. The dissolved
organic
carbon
concentration
was
178.4 mg/I, of which only a small percentage was measured
as aromatic
or aliphatic
acids. Although
not determined,
humic acids were probably present in high levels as indicated
by the dark brown color of the extracted
soil solution
(G.
Kervin, personal communication).
Enrichment
cultures
More than 50 strains of the genus Streptomyces were isolated
and the 16S rDNA
of most of them were subsequently
sequenced.
None of these sequences
were identical
to the two
streptomycetes
clones
recovered
from
the clone
library
(Naomi
Ward, personal
communication).
Although
bacterial
cells were observed
in the primary
enrichment
cultures,
set up for the TH3-type
organisms,
chemical
data indicated
that the supplied
inorganic
substrates
were not oxidized
by bacterial
action. The pH of
sulfur-containing
enrichments
did not fall below that of the
negative controls, nor was the ferrous iron oxidation
rate of
the enrichments
ever greater than chemical oxidation
rate in
the uninoculated
control.
No growth
or iron oxidation
was
observed on agarose-gelled
media.
Phylogenetic
sequences
analysis
of the novel
clone
16S rDNA
As demonstrated
previously,
the majority
of 113 analyzed
clones could be allocated
to three major clusters:
ct-2 Proteobacteria (cluster I; [13]), planctomycetes,
and a novel group
that was found to be remotely related to planctomycetes
and
chlamydiae
(clusters
II and
III,
respectively;
[14]).
Phylogenetic
analysis
of 20 additional
clones revealed the
presence of three novel clusters (clusters IV-VI).
The position of each cluster within
the radiation
of its phylum
remained unaltered
whether the analyses were based on about
procedures
Streptomycetes
were
isolated
using
standard
medium
(growth
medium
No. 65, Catalog
of Strains
of the German
Collection
for Microorganisms)
and isolation
protocols.
Several of the clones shared a distinct but remote relationship to the bacterial strain TH3, a moderately
thermophiic,
iron-oxidizing
organism
originally
isolated
from a copperleaching
dump (19). Protocols
used for the enrichment
of
bacteria with physiologies
similar to that of this strain were
comparable
to the original
isolation
methods (19, 20).
AcidiphiliuR
AcidiphiliuR
cryptuL
rubruR
RESULTS
Chemical
analysis
of the soil
A SOO-g sample of freshly collected
soil, which represents
a
5- to 10-cm surface horizon,
was chemically
analyzed using
a soil solution technique
(21). This method
of analysis
was
chosen as it was assumed that the soluble fraction
of the soil
would have the greatest effect on the bacterial
population.
The acid soil sample,
pH 4.2, was not unusual in composition when compared with other acid soils found in Queens-
GENETIC DIVERSITY OF A NATURAL MICROBIAL
COMMUNITY
5%
1. Dendrogram
of 16S rDNA relatedness between a-i Proteobacteria
(cluster IV) and their cultured relatives. Bootstrap
values (in percent) areindicated at branching points. Bar represents
5% nucleotide differences.
Figure
233
1000 or 450 nucleotides.
Cluster
IV represents
clones
originating
from members
of the cr-i subgroup
of the Proteobacteria (Fig. 1). Clones MC74 and MCIO6 show between 5.0
and
5.8%
sequence
difference
to Rhodopila
globfonnis,
whereas
slightly
lower values
are found
with Thiobacillus
acidophilus and strains ofAcidiphilium
(5.8-8.2%).
The finding
that certain
signature
nucleotides
published
for members
of
the a subclass of Proteobacteria
(22) are missing
in the clone
sequences
and in Rpl. globformis
strengthens
the relationship
between these sequences.
Clone MC77 stand isolated within
this subclass.
Sequence
dissimilarity
values between
8.9 and
10.7%, and the low bootstrap
value, however,
indicate
that
the degree
of relatedness
between
clone
MC77
and its
phylogenetic
neighbors
is remote
and ill defined.
The remaining
16 clones exhibited
the 165 rDNA nucleotide signatures
of Gram-positive
bacteria
(23) but did not
cluster with any of the described
taxa of the subphyla
of actinomycetes or Bacillus/Clostridium.
As shown by their branching points (Fig. 2), two clusters
(V and VI) emerged
from
the phylogenetic
analysis.
Individual
clone groups occur within each cluster. The bootstrap value of287/1000
(Fig. 2) indicates an extremely
low degree of possibility
that members
of the two clusters are actually
descendents
of a common
ancestor.
The eight clones of cluster
V, ranging
from MC65
to
MC87 in Fig. 2, appear
to be remotely
related
to the iron
oxidizing
Gram-indeterminate
strain TH3 (19, 24). Based
on the distribution
of signature
nucleotides,
this organism
has recently been shown to represent a deep branch of the
actinomyces
subphylum
(24). The range of genetic diversity
between
strain TH3 and the relevant clone sequences is approximately
as low as those found between members of two
distantly
related ta.xa of the actinomycetes
subphylum,
i.e.,
Art hrobacter and B1dobaathum
(13-17% dissimilarity).
On the
other hand, as judged from the presence of signature nucleotides and bootstrap values, the phylogenetic
coherency
of the
enlarged TH3 cluster is apparent,
and even more so for each
of the three clone groups, i.e., MC66 and MC47 (cluster Va)
and MC19 (cluster Vb). Clone sequences of subcluster
Vb
appear slightly more closely related to strain TH3 (11.5% sequence
divergence)
than
the other
two clone
groups
(13-16.7% sequence divergence).
Members
of cluster Va and
Vb can be defined by oligonucleotides
5’ T1EGGC[C,T]’TCC
3’ (positions
204 through
219, B. subtilis nomenclature
(25)
and 5’ TGGATTCC
3’ (positions
202 through
209), respectively.
The eight clone sequences
of cluster VI (MCIO1 to MC 103
in Fig. 2) form an individual
line of descent,
the coherency
of which
is reflected
by a number
of common
signature
nucleotides
and the presence
of a diagnostic
oligonucleotide
5’ AGAAAG[T,G]GGAGCAAICC
[A,C]TGAGTAC
3’ (positions 70 through
100 B. subtilis nomenclature).
However,
neither its common
branch with Lactobacillus
minulus nor its
membership
to the actinomycetes
subphylum
is reproducible
by bootstrap
analysis.
The clone numbers
indicated
above are not a reflection
of
the actual numerical
distribution
of taxa within the sample.
As outlined
below,
several
selective
parameters
prevent
quantifiable
data from being obtained.
DISCUSSION
The phylogenetic
analysis
acid forested
soil sample
microbial
diversity. Most
234
Vol. 7
January 1993
of genomic
DNA isolated from an
revealed
a significant
degree of
of 113 16S rDNA clone sequences
Bifidob.
bifidum
globi
Lactobacillus
-MCi 01
subtilis
Clostridium
Clostridium
formis
minutus
aminova1erjcun
perfringens
10%
Figure 2. Dendrogram
of 165 rDNA relatedness between clones of
clusters V, VI, and members
of the phylum of Gram-positive
bacteria. Bootstrap
values (in percent) are indicated
at branching
points. Bar represents
10% nucleotide differences.
analyzed
belong to novel types of organisms,
with no close
relatedness
to sequenced,
culturable
representatives.
In this
respect
our results
are in accord
with the information
obtained from studies about the genetic diversity
of unculturable organisms
in marine and hot spring environments
(6-12).
The highest
degree
of relationship
we could detect was
that between
a small fraction of clone sequences
and the a-2
Proteobacteria,
e.g., Rhodopseudomonas
palustris, members
of
Rhizobium
and “Photorhizobium” (13) and related taxa, such as
Nitrobacter
(for which no complete
16S rDNA
sequence
exists). The rather high content of ammonium
and nitrate
in
the soil sample (nitrite
has not been analyzed)
could suggest
the presence
of nitrifying
bacteria.
Sequences
of the beta
subclass of Proteobacteria, to which ammonium
oxidizing
bacteria belong, have not been detected
in the clone library, and
Nitrobacter
species-specific
oligonucleotides
(26) were absent
in the short stretches
of about 200 nucleotides
available
for
the eight a-2 clone sequences.
Certain
clone sequences
branch
adjacent
to members
of
genera which themselves
stand phylogenetically
isolated,
i.e.,
Gemrnata, Planctomyces,
and
Isosphaera
(14), as well as
Rhodopila
globformis
and the iron-oxidizing
actinomycete
strain TH3.
Whether
or not these sequences
represent
organisms
that resemble
the culturable
strains phenotypically
and physiologically
remains
an open question
until the relevant organisms
are cultured.
Attempts
to enrich and cultivate relatives
of planctomycetes
(14), Acidiplzilium,
and strain
TH3 under conditions
optimal for these bacteria have so far
been unsuccessful.
This may not be surprising
considering
The FASEB journal
STACKEBRANDT
ET AL.
that the metabolic
variation
within taxa, separated
by these
sequence
differences
of more
than 5%, embraces
a wide
range of physiological
types.
Compared
with the diversity
of prokaryotes
that have been
cultivated
from acidic soils, e.g., thiobacilli
and streptomycetes, the diversity
of bacteria
from the Mt. Coot-tha
acidic
forested
soil appears
to be significantly
different.
From information
available
from
similar
soil types,
not a single
representative
sequence
of the culturable
strains
shows a
close relatedness
to the clone sequences
that would point
toward membership
of the same species (extensive
attempts
to determine
the diversity
of culturable
prokaryotes
within
this soil are currently
under investigation),
although
the 16S
rDNA of acidophilic
streptomycetes
is yet to be analyzed.
A
simple explanation
would characterize
the Australian
habitat
as so unique
that the microbial
diversity
is markedly
different from those populations
isolated
from acidic environments in other parts of the world. However,
it seems to be
more appropriate
to think of alternate
solutions.
First, the
typical acidiphilic
microflora
is actually
present,
but too low
in numbers,
to be detected
in only a limited
clone library
of
about
110 clones.
Second,
the analysis
represents
a single
time point sampling
taken after a long period of no rain, thus
favoring
organisms
that survive as resting forms. Third,
the
presence
of a high concentration
of ammonium
in the Mt.
Coot-tha
soil sample
would
indicate
that microsites
are
present
in the soil matrix with higher pH values than that of
the pooled soil sample.
The question
is raised as to whether
the strategy
applied
in this study is actually
apt to determine
the whole range of
genomic
diversity,
or, alternatively,
do certain
factors
influence
a selective
recovery
of specific bacterial
taxa? A
number
of problems
associated
with molecular
methods
in
community
analysis have been recognized
and need to be addressed;
in addition
to unknown
factors such as differential
amplification
rates due to the base composition
of DNA, the
following
technical
problems
arise.
1) Isolated
bulk DNA should reflect the actually
existing
genetic diversity.
Two different
strategies
for isolation
of bulk
DNA from soil have been applied:
the separation
of cells
from the soil matrix
(cell extraction
technique
[27, 28]) and
the direct lysis of bacteria
within
their natural
habitat
by
mechanical
disruption
(direct lysis technique
[15, 29]). Because previous
research
suggests
(15, 30) that direct lysis
technique
recovers
a more representative
fraction
of the
genetic diversity
than the cell extraction
technique,
this approach was applied in this study. One drawback
of mechanical lysis is the substantial
shearing
of extracted
DNA (14). A
final electrophoretic
purification
step therefore
had the additional
advantage
of removing
low-molecular-weight
DNA
that would otherwise
increase
the possibility
of chimeric
(shuffle gene) products
(6, 31).
2) Oligonucleotide
primers
used for PCR-mediated
amplification
of 16S rDNA should cover the phylogenetic
diversity present
in a sample.
The primer
pair used in this study
represents
a compromise
between
detection
of a broad range
of bacteria
combined
with the aim to favor the amplification
of 16S rRNA genes from streptomycetes.
The 5’ oligonucleotide primer targets a highly conserved
stretch within the 16S
rDNA of members
of the domain
Bacteria,
but does not amplify 16S rRNA genes from archaeae
(it is worth noting that
archaeal
rDNA was not detected
in the genomic
DNA recovered from the sample
as determined
by oligonucleotide
probing
with an archaeal
consensus
probe
[Fred Rainey,
personal
communication]).
The 3’ oligonucleotide
primer
was designed
to be completely
complementary
to the 16S
rDNA
target
sequence
of the streptomycetes.
As a consequence,
mismatches
exist
between
this
oligonucleotide
primer and target stretches
from almost all representatives
of
other phyla of the domain
Bacteria
(Fig. 3). Studies
of the
influence
of primer-template
mismatches
on the polymerase
chain reaction
have since reyealed
that 1) a single base mismatch between
the PCR primer
and template
that is either
one, two, or three bases from the 3’ nucleotide
of the primer
will be extended
without
a significant
effect on the overall
PCR yield, and 2) the presence
of two mismatches
within the
last four bases of the 3’ nucleotide
of a primer
is mostly
detrimental
to the PCR reaction
unless the terminal
base is
a Thymidin
(32). Consequently,
as judged from the comparison of primer
and target sites, the 3’ primer
used will allow
amplification
of 16S rDNA spanning
a broad range of taxa
of the domain
Bacteria.
3’ primer
(target
region
1224-1243)
#{149}
ACGTGTGCACGATGTTACCG
Streptomyces
Uycobacterlum
Bacillus
5,
ambofaclens
bovis
subtllls
ATGTCTTGGGC
TGCACACGTGCTACAATGOC
CGGTACAA3
ATGTCCA000C
-T
CGGTACA&
ATGACTTGGGC
-A
nidulans
Agrobacterium
tumefaciens
ACATCCT000C
-A
AC000CTOGOC
-A
Camonaa
ATAGOTOGGOC
-A
ACGACCA000C
-A
Anacy.tls
testosteroni
Eacherichia
coil
Desulfovlbrjo
desulfuricans
ACGCCTAGGGC
-A
Campylobactar
jejuni
ATGCCCA000C
GA
-A
Chiamydia
paittaci
ATGCCCA000C
Pirellula
ataleyi
ATGAT?A000C
Bacteroidea
fragilla
C.blorobium
vlbriofoxiie
Leptospira
.illlni
Delnococcua
radiodurans
Thermcalcrobium
roseum
Thermotoga
maritime
A
A CAGAACAA
CT CCGGACAG
A
t
A
GGTGACAA
TGGTACAA
GCATACAA
A
OCGCACAA
ATATACAA
CAGTACAG
t
C
GCACACAA
ACGTCC0000C
-A
t
0 000TACAG
ACO?CC0000C
-A
A
T
AACTACAO
ATOTCC0000C
-A
T
CGGTACA0
ACGTCCT000C
-A
A TAGGACAA
ACGCCC?000C
GA
ATGCCCT000C
GA
AC
C
C000ACAO
g--A-
0
COGTACAA
Figure 3. Comparison
of the nucleotide sequences between the 3’ PCR primer used in the amplification
of 16S rDNA and its target sites
in 16S rDNA from representatives
of bacterial phyla. The primer was designed to amplify streptomycetes
rDNA but was shown to amplify
DNA from other firmicutes, Proteobacteria, planctomycetes, and chlamydiae and relatives as well (see text).
GENETIC DIVERSITY OF A NATURAL MICROBIAL
COMMUNITY
235
3) The applied cloning
strategy
should be a reliable
tool
with
a low risk of loosing
detectable
genetic
diversity.
Problems
encountered
with “sticky end” cloning
technique
can be avoided by “blunt end” cloning (33). However,
the low
yield of recombinants
is a major technical
disadvantage.
The detection
of genetic diversity
within
a natural
environment
can be considered
the very first step toward
the
understanding
ofthe
role that bacteria
play in an ecosystem.
The power of this approach
hopefully
allows the progression
from a collection
of sequence
data to the isolation
of novel
taxa. If these sequences
are very similar to those of culturable strains,
the chance
for providing
adequate
cultivation
conditions
is increased.
The use of sequence
information
in
the development
of diagnostic
probes may also be the starting point for an almost unexplored
avenue,
namely,
the detection of organisms
within the sample matrix,
the determination of growth dynamics,
and the response toward changes.
This work
from
was supported
the Australian
Toalster
by grants
research
for her assistance
Council
AD
891593
and A 19031196
to E. S. We thank
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