Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes.
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ABSTRACT: We studied the changes in structure and functioning of the microbial community in a degraded agricultural soil after the addition of two composts, obtained from cattle manure or pig slurry anaerobic digestate, and the use of rosemary plants for restoring soil quality. The compostswere added at lowor high doses to soil samples (30 or 60 t ha−1, respectively),whichwere kept in microcosms for 6 months. Some soil microcosms were treated with inorganic fertiliser and other non-treated soils were used as microbiological controls. Rosemary plants, used both for their ability to grow in semi-arid regions and for the capacity of their root system to protect soil from erosion, were planted in half of the entire microcosm set up. At different times (0–180 days) microbial abundance and dehydrogenase activity were measured in the various experimental treatments. Total and water-soluble soil organic carbon and nitrogen contentswere assessed at 0 and 180 days.With an increase in carbon and nitrogen soil content, a rise in microbial abundance was also observed in the presence of both composts. However, microbial activity was significantly influenced by the presence of rosemary, without considering the allochthonous carbon and nitrogen input. Microbial community structure and diversity were also assessed by Fluorescence In Situ Hybridization in the different treatments. The highest values for microbial community biodiversity were found in the co-presence of rosemary and at low concentrations of both composts. The overall results suggest that the use of composts togetherwith plant species suited toMediterranean areas seems to be an appropriate strategy for restoring soil quality and the ecosystem services provided by microorganisms.Geoderma 01/2015; 245-246(245-246):89-97. · 2.51 Impact Factor
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ABSTRACT: The response of soil microbes to elevated atmospheric CO 2 is fundamental to understanding how ecosystems will respond to global change. Here, we investigated soils that had been exposed to the ambient atmospheric CO 2 concentration or to 12 years of elevated atmospheric CO 2 in a Free Air Carbon Dioxide Enrichment (FACE) experiment in New Zealand. Through pyrosequencing, we obtained an average of 3,458 sequences per soil. We then assigned the sequencing data of 16S rRNA genes to RDP taxonomical hierarchy based on nomenclatural taxonomy and Bergey's Manual with a minimum support threshold of 80%. The result of classification at different levels (such as phylum-, class-, subclass-, order-, suborder-, family-and genus) showed that the most taxonomic groupings were present in both treatments but there were marked changes in relative abundance within the groupings. For example, under elevated CO 2 there was an increase in the abundance of Actinobacteria and Planctomycetes , no change in Proteobacteria and a decrease of Frimicutes. We expect these changes have functional consequences and this is an area for future research. Introduction The CO 2 concentration in the atmosphere has increased by >30% since the industrial revolution due to anthropogenic interference, and is predicted to be 450-600 ppm in 2050 (Houghton et al., 2001; Keeling & Whorf, 2004). Elevated CO 2 not only leads to climate change and global warming, but also has a direct impact on biological systems. While much is known about the stimulating effects of increasing CO 2 on aboveground plant growth and primary productivity (25th Annual FLRC Workshop; 02/2012
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ABSTRACT: To investigate vertical changes of bacterial communities from living plants to the associated sediments and bacterial biogeochemical roles in peatland ecosystem, samples of different part of individual Sphagnum palustre and the different layers of the underlying sediments were collected from Dajiuhu Peatland in central China. All samples were subject to 16S rRNA gene clone libraries and quantitive PCR analysis. Even though bacteria vary in abundance at the same order of magnitude in all samples, they show great profile difference in composition from the top part of S. palustre to the low layer of the sediments. Cyanobacteria and alpha-Proteobacteria dominate at the top part whereas Acidobacteria at the middle part of S. palustre. Alpha-Proteobacteria and Acidobacteria are the dominant phyla at the bottom part of S. palustre and in the surface peat sediment. In contrast, bacterial communities in the subsurface sediments are dominated by Acidobacteria. These profile distributions of different bacterial communities are closely related to their ecological functions in the peatland ecosystem. Specifically, most Cyanobacteria were observed at the top green part of S. palustre, a horizon where the active photosynthesis of the moss occurs, which infers their endosymbiosis. In contrast, Acidobacteria, dominant in the subsurface sediments, are able to decompose the specific compounds on the cell wall of Sphagnum moss and thus might play an important role in the formation of the peatland, including the acidic condition. Methane oxidizing process might have been underestimated in Sphagnum peatland due to the identification of Methylocystaceae in all parts of the moss investigated here. The vertical difference in bacterial composition and bacterial ecological functions presented here sheds light on the understanding of biogeochemical processes, in particular the CH4 flux, in peat ecosystems.Science China Earth Science 05/2013; 57(5):1013-1020. · 1.34 Impact Factor
2006, 72(3):1719. DOI:
Appl. Environ. Microbiol.
Peter H. Janssen
in Libraries of 16S rRNA and 16S rRNA
Identifying the Dominant Soil Bacterial Taxa
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2006, p. 1719–1728
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 3
Identifying the Dominant Soil Bacterial Taxa in Libraries
of 16S rRNA and 16S rRNA Genes
Peter H. Janssen*
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia
From near to far,
from here to there,
funny things are everywhere.
—Dr. Seuss, One Fish Two Fish Red Fish Blue Fish
In 1909, H. Joel Conn (25) expressed the hope that methods
would soon be at hand by which the significance of the different
bacteria present in any soil could be determined. However, by
1918 he was pointing out that the methods available to him,
which relied on cultivation of bacteria on artificial media, re-
sulted in the formation of colonies by only 1.5 to 10% of the
bacterial cells in soil (26). Fifty years later, Vagn Jensen con-
cluded a review of cultivation-based methodologies by stating
his suspicion that those cells that were forming colonies were
unrepresentative of the total bacterial community (59). This
was confirmed when cultivation-independent methods began
to be used to study soil bacteria (see below). Even so, in the
absence of better methods, the pure cultures derived from the
colonies that did form were extensively and successfully stud-
ied throughout the 20th century. Much of our basic knowledge
of soil bacteria, as well as the discovery of many important
antibiotics, came from investigations of pure cultures (2, 86,
104). Cultured isolates are still very important in developing
our understanding of bacterial physiology, genetics, and ecol-
ogy (85, 122).
Beginning in the 1990s, the application of molecular ecolog-
ical methods, especially those based on surveys of genes after
PCR amplification, has allowed cultivation-independent inves-
tigations of the microbial communities of soils to be made.
The power of these methods has largely rendered obsolete the
plate count approach to detecting and enumerating subsets
of soil bacteria, and a range of diagnostic and quantitative
methods that target functional genes, phylogenetically infor-
mative genes, or RNAs has been developed (49, 69). In par-
ticular, 16S rRNA and its gene have proven to be useful and
powerful markers for the presence of bacteria in samples (36,
56, 88). The utility of these markers is facilitated by the avail-
ability of primers that allow amplification of almost the com-
plete gene or its RNA product (66) and by the phylogenetic
inferences that can be made from the resultant nucleotide
sequences, permitting placement of the host organism within a
phylogenetic framework even if closely related cultured organ-
isms are lacking (36, 56, 74). Since the initial pioneering studies
to survey soil bacterial communities using molecular ecological
surveys (13, 68, 79, 91, 111), a number of libraries of 16S rRNA
and 16S rRNA genes derived from soils have become available
It is important to realize that libraries of PCR-amplified 16S
rRNA and 16S rRNA genes may not represent a complete or
accurate picture of the bacterial community. Firstly, the spe-
cies diversity is so great (28, 46, 109) that libraries of ?400
cloned sequences must represent only an incomplete sampling.
Even all of the currently published sequences combined would
seem to constitute an incomplete census of all of the 16S rRNA
genes on earth (98). In addition, there may be biases in the
contributions of the various bacterial groups to libraries. The
efficiencies of nucleic acid extraction may be different for dif-
ferent bacteria, the number of copies of 16S rRNA or 16S
rRNA genes per cell varies, and there may be preferential
amplification of some sequence types relative to others by PCR
(36, 43, 113). Some sequences may arise from contaminating
DNA and may not represent bacteria actually present in the
sample being studied (108). Assigning physiologies and func-
tions to the hosts of 16S rRNA gene sequences is complicated
in many cases by the lack of characterized close relatives (e.g.,
see references 31, 57, 81, and 88) and by the diversity of
phenotypes among close relatives in some groups (1, 95).
Some, but not all, of these biases may be overcome as meta-
genomic data sets accumulate (71, 87, 110). In the meantime,
the available libraries of 16S rRNA and 16S rRNA genes
permit an initial survey of the global soil bacterial community
SEQUENCE DATA SETS
Thirty-two libraries of 16S rRNA and 16S rRNA genes of
members of the domain Bacteria, prepared from a variety of
soils, were analyzed to gain an understanding of the general
composition of soil bacterial communities (Table 1). Libraries
or sequences from rhizosphere samples were not included in
this synthesis. Libraries consisting predominantly of sequences
of ?300 nucleotides were also excluded, as phylogenetic as-
signment from very short sequences can be unreliable (36, 74),
and sequences of ?300 nucleotides were removed from the
libraries that were included. Some published libraries were
* Mailing address: Department of Microbiology and Immunology,
University of Melbourne, Parkville, Victoria 3010, Australia. Phone: 61
(3) 8344 5706. Fax: 61 (3) 9347 1540. E-mail: firstname.lastname@example.org
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generated with primers that could not be expected to sample
most known bacteria or were screened in such a way that the
total number of clones belonging to each group could not be
deduced from the published data. These were not included in
this survey. Two potentially interesting libraries containing un-
expectedly large numbers of sequences assignable to the genus
Escherichia were excluded because these may not have origi-
nated from DNA from the soil being investigated (30). As a
consequence of the exclusions, the final data set is not as
geographically comprehensive as it might have been, but a
number of different vegetation types are included (Table 1).
Some libraries consist of data from multiple reports in which
the sample site or the sample itself appeared to be the same. In
a few cases, multiple libraries that originated from highly sim-
ilar replicate samples were pooled to increase the library size.
A total of 3,240 sequences from the 32 libraries were assigned
to genus level groupings by the “Classifier” program of Ribo-
somal Database Project II (24) and then weighted for multiple
clone assignments to one sequence type, and this pooled data
set of 3,398 clones was treated as one global set.
The contribution of phylum level groupings to soil bacterial
communities was calculated only from the 21 libraries with
?90 clones. Smaller libraries (?90 clones) contained repre-
sentatives of very few phyla. It was felt that the smaller libraries
might skew the outcomes, since the contributions were nor-
malized to compensate for library size and then analyzed fur-
ther. For some of the better-characterized dominant phyla
(Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Pro-
teobacteria, and Verrucomicrobia), the clones were also as-
signed to subphylum groups (class, subclass, or subdivision).
Some of these subphylum groups are organizational rather
than evolutionarily equivalent lineages, especially the classes of
the phylum Proteobacteria (74), but are useful for the purposes
of surveying the global data set. These assignments were based
on results obtained with Classifier (17), published phylogenetic
trees and tables in publications or their supporting material,
BLAST (3) in GenBank databases (8), Ribosomal Database
Project II databases (24), and phylogenetic analyses of se-
quences against references of known affiliation (e.g., see ref-
erences 60, 93, and 96).
HISTORICALLY IMPORTANT SOIL BACTERIA
In his landmark book, the second edition of Introduction to Soil
Microbiology, Martin Alexander (2) listed what were then consid-
TABLE 1. Libraries of 16S rRNA or 16S rRNA genes prepared from RNA and DNA extracted from soilsa
Canada, forest, DNA
Canada, forest, DNA
Canada, forest, DNA
Canada, forest, DNA
Canada, forest, DNA
Canada, forest, DNA
Brazil, forest, DNA
Brazil, pasture, DNA
United States, arid woodland, DNA
United States, arid woodland, DNA
United States, arid woodland, DNA
United States, arid woodland, DNA
United Kingdom, DNA*
United States, cropping rotation, DNA
Switzerland, RNA ? DNA*
Austria, forest, DNA
Austria, forest, DNA
Austria, forest, DNA
Australia, arid landscape, DNA
United States, DNA*
United States, DNA*
United States, alpine meadow, DNA
Switzerland, pasture, DNA
Switzerland, pasture, DNA
United Kingdom, pasture, DNA
United Kingdom, pasture, DNA
Germany, moorland, RNA ? DNA
Germany, forest, DNA
United States, wheat, DNA
United States, grassland, DNA
United States, grassland, DNA
Australia, pasture, DNA
32, 33, 65
32, 33, 65
32, 33, 65
32, 33, 65
aAll libraries were used to ascertain the genus and family affiliations of soil bacteria, but only libraries with ?90 clones were used to determine phylum and class
b?, details of vegetation not given.
cL. Schoenborn and P. H. Janssen, unpublished data (GenBank accession no. AY395320 to AY395454).
1720MINIREVIEWSAPPL. ENVIRON. MICROBIOL.
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ered to be important genera of soil bacteria, based on cultivation
studies. He suggested that members of nine genera were signifi-
cant in soils: Agrobacterium, Alcaligenes, Arthrobacter, Bacillus,
Flavobacterium, Micromonospora, Nocardia, Pseudomonas, and
Streptomyces (Table 2). Since 1977, two things that have changed
the validity of this list have occurred. First, many of the genera
have undergone taxonomic revision, and some of their species
have been reclassified into new or other genera. This is especially
true for the genera Flavobacterium and Pseudomonas (4, 9). Sec-
ond, surveying of 16S rRNA genes in soils has permitted a more
direct census of soil bacteria, without the limitations inherent in
cultivation-based studies. These surveys suggest that members of
Alexander’s nine genera, as they are currently defined, together
make up only 2.5 to 3.2% of soil bacteria (Table 2). Of the nine
genera, Pseudomonas spp. are the most abundant in soil bacterial
communities, contributing 1.6% of the cloned sequences from
soils (Table 2).
Recently, Floyd et al. (41) presented a breakdown of cul-
tures of prokaryotic organisms in the American Type Culture
Collection (ATCC). The 14 genera of soil bacteria with the
most deposited cultures, together encompassing over half of all
soil-derived isolates in the ATCC, are also not common among
the clones detected in libraries (Table 2). Together, members
of these genera make up only 2.7 to 3.7% of soil bacteria. This
shows that the cultured part of soil bacterial diversity is not
representative of the total diversity, as suggested by Jensen
(59) and many others since.
GENUS LEVEL DIVERSITY
The Classifier algorithm (24) returns a confidence value with
which a 16S rRNA gene sequence can be assigned to a taxon
(genus and higher) that is represented by a set of sequences,
based on the number of times, out of 100 trials, that random
subsets of the query sequence match sequences assigned to
that taxon. The algorithm also returns the name of the taxon to
which the sequence was most often assigned in those 100 trials.
Using Classifier, as many as 17% of the sequences could be
assigned to a known genus with 100% confidence; this in-
creased to 32% with a confidence level of 80% or greater.
However, these outcomes were greatly influenced by se-
quences falling into poorly defined groups with very few de-
scribed species. Sequences affiliated with these classes or phyla
tended to be identified as members of the one genus or few
genera in them, because there were no other genera to draw
the sequences during bootstrap analysis. Nearly all of these
assignments were spurious, as the sequence similarities to the
few named species were ?96%. Everett et al. suggest that 16S
rRNA gene sequence similarities of ?96% are indicative of the
hosts of the genes belonging to different genera (37). When the
genera Acidobacterium (phylum Acidobacteria), Alterococcus,
Verrucomicrobium, Xiphinematobacter (phylum Verrucomicrobia),
Gemmatimonas (phylum Gemmatimonadetes), and Conexibacter
and Rubrobacter (subclass Rubrobacteridae of the phylum
Actinobacteria) were removed, the number of sequences able
to be assigned to known genera decreased. In their absence,
only 11% of sequences could be assigned to known genera with
100% confidence and 21% at ?80% confidence. These figures
are still perhaps surprisingly high, given that soil bacterial
diversity is high and our ability to culture these bacteria is
generally considered to be poor (28, 46, 88, 98, 109, 122). It
seems that our ability to culture representatives of the phylo-
genetic diversity of soil bacteria, at least as judged at the genus
level, is better than the 1% often quoted, even if culturability
as a function of cell numbers is low.
The majority (79 to 89%) of 16S rRNA gene sequences are
from bacteria that are not affiliated with known genera. Some of
TABLE 3. Assignment of cloned 16S rRNA and 16S rRNA genes
affiliated with well-characterized subphylum groupings
(class or subclass) to described genera
Proportion of clones
assignable to genera
(%) at different
Total no. of
aConfidence levels calculated by the program Classifier of the Ribosomal
Database Project (24). The percentage of sequences affiliated with each class that
were able to be assigned to a genus is given in parentheses.
bData from Garrity et al. (48).
TABLE 2. Abundance of members of well-known genera of soil
bacteria in libraries of 16S rRNA and 16S rRNA genes compared
with their historical significance as colony-forming soil bacteria
and their contribution to soil isolates held in the ATCC
libraries (%) at
Range of abundance
soil isolates in
aConfidence levels calculated by the program Classifier of the Ribosomal
Database Project (24).
bCalculated from the data of Alexander (2), assuming a mean of one-third of
colonies being actinomycetes (filamentous members of the subclass Actinobac-
teridae, including Micromonospora, Nocardia, and Streptomycetes) and the re-
maining two-thirds being other non-actinomycete bacteria (2).
cData from Floyd et al. (41).
d—, no data.
VOL. 72, 2006MINIREVIEWS1721
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these are associated with well-studied lineages of bacteria, such as
Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria,
and Deltaproteobacteria. In some of these groups, the number of
bacteria affiliated with known genera is high. For example, up to
60% of sequences from members of the class Sphingobacteria and
up to 76% of sequences from members of Flavobacteria may
come from described and named genera (Table 3). Among se-
quences assigned to Actinobacteridae this is lower, with less than
half belonging to described genera, while in the Deltaproteobac-
lower (Table 3). Since members of the latter two groups have
been the source of many chemically novel bioactive compounds
(104), this indicates considerable scope for more discovery. In
some groups well represented by cultured isolates, such as in the
classes Alphaproteobacteria, Betaproteobacteria, and Gammapro-
teobacteria, less than half of all sequences could be assigned to
these described and named genera (Table 3). Overall, only 20 to
40% of sequences affiliated with well-characterized groups of
bacteria can be assigned to known genera. Of the genera in the
well-defined groups, the three most abundant at 100% assign-
ment confidence are Burkholderia (class Betaproteobacteria),
Pseudomonas (class Gammaproteobacteria), and Chitinophaga
(class Sphingobacteria), which constitute 2.7, 1.6, and 1.0% of all
the sequences, respectively.
Although recognized as the essential basis of bacterial system-
atics (115), the genus rank has not been well defined. In essence,
though, genera tend to consist of species that share major phe-
notypic characteristics that differentiate them from species of
related genera. The consequence of being able to assign only 10
to 21% of sequences to known genera is that the broad charac-
teristics of most of the soil bacteria are not known.
ABUNDANCE OF DIFFERENT PHYLA
16S rRNA genes from soil bacteria are affiliated with at
least 32 phylum-level groups. The contributions that mem-
bers of different phyla make to the different soil bacterial
communities vary (Fig. 1). The dominant phyla in the librar-
ies are Proteobacteria, Acidobacteria, Actinobacteria, Verruco-
microbia, Bacteroidetes, Chloroflexi, Planctomycetes, Gemmati-
monadetes, and Firmicutes (Table 4). Members of these nine
phyla make up an average of 92% of soil libraries (normalized
for the size of the individual libraries). Although there are at
least 52 bacterial phyla (88), and 24 are recognized by Bergey’s
FIG. 1. Contributions of 16S rRNA and 16S rRNA genes from
members of different phyla in libraries prepared from soil bacterial
communities (2,920 clones in 21 libraries). The horizontal line in the
middle of each block indicates the mean, the block represents 1 stan-
dard deviation on either side of the mean, and the vertical lines ex-
tending above and below each block indicate the minimum and max-
imum contributions of each phylum.
TABLE 4. Contribution of 16S rRNA and 16S rRNA genes from
members of different phyla and subphylum groups (class,
subdivision, or subclass) to soil bacterial communitiesa
aOnly the 21 libraries with ?90 clones were included in this survey (total,
1722MINIREVIEWSAPPL. ENVIRON. MICROBIOL.
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Manual (48), soils seem to be dominated by only a small num-
ber of these.
Members of the phyla Proteobacteria and Acidobacteria are the
most abundant soil bacteria, as judged by the occurrence of 16S
rRNA and 16S rRNA genes that are assignable to these groups
(Table 4). All of the libraries surveyed contained some sequences
assignable to these two phyla. The other dominant phyla are not
found in all libraries, but this is likely to be a consequence of
library size. Given that there is variation in the contribution of
different phyla and classes, abundances at the lower end of any
range may mean that no sequences are detected if the library is
too small. In addition to representatives of the nine major phyla,
members of a number of other phylum-level lineages, such as
Chlamydiae, Chlorobi, Cyanobacteria, Deinococcus-Thermus, Fi-
brobacteres, Nitrospirae, BRC1, NKB19, OP10, OP11, OS-K, SC3,
SC4, termite group I, TM6, TM7, WS2, and WS3, are present in
the global data set. Some members of a few of these phyla are
quite well studied, but in general very little is known about the
soil-inhabiting members of these groups. Most of these phyla are
virtually unstudied and have few or no known pure culture rep-
resentatives from soils (74).
A number of studies using methods other than analysis of
libraries have estimated the contribution of members of different
bacterial groups to the microbial population of soils (Table 5).
These studies support the general trends observed in clone librar-
ies (Fig. 1, Table 4) that Alphaproteobacteria, Acidobacteria, and
Actinobacteria are often abundant in soils and that members of
Bacteroidetes, Firmicutes, and Planctomycetes are generally less
abundant (Table 5). They also support the observation that the
estimated abundance of the major phyla varies between different
soils (or samples). It is not possible to state to what degree the
variations are method based. Fluorescence in situ hybridization
(FISH) and other hybridization methods may detect bacteria
other than the intended target group, or the phylogenetic cover-
age of oligonucleotide probes may not be comprehensive. The
same applies to oligonucleotides designed for quantitative PCR
approaches. Detection of extracted rRNA is affected by ribosome
levels in bacteria, while clone library compositions are influenced
by PCR steps and by rrn copy number. The results obtained with
all the methods are affected by the physical nature of bacterial
cells, which may vary between groups and under different condi-
tions, affecting oligonucleotide probe permeability and successful
nucleic acid extraction.
PHYLUM LEVEL DIVERSITY
Members of the phylum Proteobacteria make up an average of
39% (range, 10 to 77%) of libraries derived from soil bacterial
communities (Fig. 1). Most soil-dwelling members of the phylum
Proteobacteria can be classified within the classes Alpha-
proteobacteria, Betaproteobacteria, Gammaproteobacteria, and
Deltaproteobacteria (Table 4). The phylum Proteobacteria cur-
rently contains some 528 named and described genera (48), but
the number of proteobacterial sequences that can be confidently
assigned to known genera is relatively low. Depending on the
level of confidence used, only 19 to 36% of the proteobacterial
sequences can be assigned to a known genus (Table 3), indicating
that many proteobacterial groups still remain to be described and
named. Libraries from soils reveal the existence of lineages not
affiliated with known isolates (e.g., see references 5, 37, and 77).
Given the extent of physiological diversity within the phylum, no
guesses as to their general metabolism can be made. At present,
research interest seems to be directed toward the as-yet-uncul-
tured groups within phyla such as Acidobacteria and Verrucomi-
TABLE 5. Estimates of abundance of the members of different bacterial groups made by cultivation-independent methods
other than clone library analysis
Source (site designation)a
No. of members of indicated groupc
ACIACTALF BETGAM DELVERBAC FIR PLA
Organic soil, Norway
Mineral soil, Germany
Tilled cropland, United States (CT)
Tilled cropland, United States (NI)
No-till cropland, United States (AF)
No-till cropland, United States (NT)
Abandoned field, United States (HCS)
Abandoned field, United States (LS)
Tilled grassland, United States (HCST)
Meadow, United States (NCS)
Tree plantation, United States (PL)
Meadow, The Netherlands
Desert, United States
Forest, United States
Prairie, United States
aThe site designations are those used by authors to identify particular sources within studies with multiple soil samples.
bFISH, counting of cells in soil samples with group-specific oligonucleotide probes; rRNA, estimation of abundance of rRNA in total rRNA by hybridization with
group-specific oligonucleotide probes; qPCR, quantitative PCR estimate of 16S rRNA genes using group-specific assays, relative to estimates of total bacteria using
cACI, phylum Acidobacteria; ACT, phylum Actinobacteria; ALF, class Alphaproteobacteria; BET, class Betaproteobacteria; GAM, class Gammaproteobacteria; DEL,
class Deltaproteobacteria; VER, phylum Verrucomicrobia; BAC, phylum Bacteroidetes; FIR, phylum Firmicutes; PLA, phylum Planctomycetes.
VOL. 72, 2006MINIREVIEWS1723
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crobia, which constitute smaller parts of the soil bacterial com-
munity; the uncultured proteobacteria have been largely ignored.
Members of some cosmopolitan family or order level groups
of the Alphaproteobacteria and Gammaproteobacteria without
named and described genera have been isolated but are not yet
formally named (29, 60, 94). 16S rRNA genes affiliated with
these isolates have been found in most of the libraries listed in
Members of the phylum Acidobacteria make up an average
of 20% (range, 5 to 46%) of soil bacterial communities (Fig. 1).
The phylum Acidobacteria is divided into at least eight subdi-
visions that may have class level rank (56). Three of these are
particularly abundant in soils: subdivisions 1, 4, and 6 (Table
4). There are, however, only three formally described genera in
the phylum (48). Members of subdivision 1 have been shown to
be readily culturable. Kishimoto and Tano (62) originally cul-
tured eight isolates, one of which was named Acidobacterium
capsulatum (63). Since then, at least 99 further isolates that
span the phylogenetic breadth of the subdivision have been
reported (29, 52, 58, 60, 93, 94, 103). A few isolates of members
of subdivisions 2, 3, and 4 have been obtained in pure culture
(29, 60, 78, 92, 94). All isolates appear to be aerobic hetero-
trophs, but this may be a result of the cultivation strategies
employed to date. The phylogenetic breadth of subdivisions 3
and 4 in particular is much greater than that covered by the few
isolates (e.g., see references 5, 7, 50, 56, 71, 87, and 121). To
date, no isolates of subdivisions 6 and 7 have been reported,
even though members of these subdivisions are common in
soils (Table 4). Members of subdivision 6 may be aerobes,
since they were not detected in a permanently anoxic soil
system (116) and colonies have recently been found to form on
plates incubated under air (K. E. R. Davis and P. H. Janssen,
unpublished data). The phylogenetic depth of the phylum
Acidobacteria is nearly as great as in the phylum Proteobacteria,
and the different class rank subdivisions may contain bacteria
with very different physiologies (31, 56, 75). For example, the
only known members of subdivision 8 are obligate anaerobes,
with two different basic physiologies, which contrast with the
known aerobes of subdivisions 1 to 4 (23, 63, 70, 93).
Members of the phylum Actinobacteria make up an average of
13% (range, 0 to 34%) of soil bacterial communities (Fig. 1). The
phylum Actinobacteria contains three subclasses that are common
in soil: Actinobacteridae, Acidimicrobidae, and Rubrobacteridae
(Table 4). For the purposes of this synthesis, the subclasses are
used as subphylum groupings. The majority of described genera
of the phylum Actinobacteria are within the subclass Actinobacte-
ridae. This group consists of some 158 genera, many of which are
well known and well studied (48). Although members of the
subclass Actinobacteridae have been extensively investigated, only
26 to 47% of sequences could be assigned to described genera
and there remains considerable scope for the isolation of novel
members of this group, especially new rare genera that may yield
novel bioactive compounds (67).
In addition to members of the subclass Actinobacteridae,
soils contain many members of the less-studied subclasses
Rubrobacteridae and Acidimicrobidae (Table 4). To date, there
are no validly named and described members of the subclass
Acidimicrobidae from soil, and only five isolates from soil been
reported (29, 60). The only named and characterized members
of this subclass are Acidimicrobium ferrooxidans and Ferromi-
crobium acidophilus, which are ferrous-iron-oxidizing acido-
philes, and Microthrix parvicella, an as-yet-uncultured filamen-
tous bacterium found in activated sewage sludge (6, 11, 22).
Only Acidimicrobium ferrooxidans is currently recognized by
Bergey’s Manual (48).
Two genera of aerobic heterotrophs from soil in the sub-
class Rubrobacteridae have been described. These are So-
lirubrobacter and Conexibacter, each represented by one species
with one strain each (80, 100). Twenty further isolates, some
phylogenetically distant from these two genera, have been ob-
tained from soil (29, 58, 60, 94, 96). These are all aerobic
heterotrophs. The few other members of this subclass are
aquatic thermophiles of the genera Rubrobacter and Thermo-
leophilum (19, 21, 106, 117). Overall, there are many lineages
without cultured representatives in all three subclasses of
soil-inhabiting actinobacteria, especially in the subclasses Rubro-
bacteridae and Acidimicrobidae, but also some in the subclass
Actinobacteridae (e.g., see references 5, 50, 54, 73, 79, 89, and
90). Recently, members of some of the previously uncultured
lineages of the Actinobacteridae have been shown to be cultur-
able (60). The phylogenetic depth of the phylum Actinobacteria
appears to be lower than that of other major phyla, but the
degree of phenotypic diversity in this phylum is high (31, 47).
The as-yet-uncultured actinobacterids can be expected to be aer-
obic heterotrophs, but the subclasses Rubrobacteridae and Aci-
dimicrobidae may yet contain other metabolic types.
Members of the phylum Verrucomicrobia make up an aver-
age of 7% (range, 0 to 21%) of soil bacterial communities (Fig.
1). The phylum has been divided into five major class level
subdivisions (56). The major group of Verrucomicrobia found
in soil is the class Spartobacteria (Table 4), which is the name
proposed for subdivision 2 of Verrucomicrobia (97). Chthonio-
bacter flavus is the first named cultured isolate of the class
Spartobacteria (58, 97). C. flavus and a further nine isolates
belong to at least two genera within the family Chthoniobacter-
aceae in the class Spartobacteria (96). These are all aerobic
heterotrophs. The class Spartobacteria also contains bacterial
symbionts of nematodes in the family Xiphinematobacteraceae
(112), and the cloned sequences detected in soils may there-
fore have come from free-living or symbiotic bacteria. The 10
isolates of the class Spartobacteria do not cover the phyloge-
netic breadth of the class (96), and many unrepresented line-
ages (e.g., see references 5 and 56), indicative of novel genera
and families, remain to be cultured. There is no indication of
what the physiologies of members of those families could be.
Only six isolates of subdivision 3 of the phylum Verrucomicro-
bia have been obtained (60, 96). These are aerobic hetero-
trophs, but they also do not cover the full phylogenetic breadth
of the subdivision and so do not yet give a complete picture of
the phenotypes of members of this group (96).
Members of the phylum Bacteroidetes make up an average of
5% (range, 0 to 18%) of soil bacterial communities (Fig. 1).
There is some evidence suggesting that members of this phy-
lum may be underrepresented in libraries of PCR-amplified
16S rRNA genes (27, 34). Even so, members of the class
Sphingobacteria of the phylum Bacteroidetes are common in
soils (Table 4). Some members of this group are aerobes, while
others are anaerobes or facultative anaerobes, and so the spe-
cies composition of members of this class within a soil may
depend on oxygen levels or the amount of variation in oxygen
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availability. Members of the class Flavobacteria are less com-
mon (Table 4), and members of the third class, Bacteroidetes,
seem to be absent from soils. Of all the sequences affiliated
with the phylum Bacteroidetes, 34 to 62% could be assigned
to known genera, including Chitinophaga, Flavobacterium,
Hymenobacter, and Pedobacter, suggesting that this is one of
the few groups of dominant soil bacteria that is readily cultur-
able. However, given the high diversity of soil bacteria, we
should not be surprised if many novel genera, albeit in low
abundance, exist in soil. Lineages without cultured represen-
tatives have been detected (e.g., see references 72 and 77).
Members of the phylum Chloroflexi make up an average of
3% (range, 0 to 16%) of soil bacterial communities (Fig. 1).
The phylum Chloroflexi consists of perhaps eight candidate
classes, and the phylogenetic depth is comparable with the
phylum Proteobacteria (31, 88). Only eight described genera
are known, and these are not evenly distributed within the
classes (48). Rappe ´ and Giovannoni (88) and Hugenholtz et al.
(56) recognize the Thermomicrobia as a class of the phylum
Chloroflexi, with one described genus, but Garrity et al. (48)
accord the Thermomicrobia separate phylum status. The diver-
sity of phenotypes in this phylum is high, even among the
relatively few isolates that have been cultured to date (88). 16S
rRNA genes from uncultured soil bacteria belonging to the
phylum Chloroflexi are affiliated with a number of the candi-
date classes and so may display quite different physiologies
(88). Only one isolate from soil has been reported (29). It is a
filamentous aerobic heterotroph, but no conclusions can be
drawn yet about the general properties of soil chloroflexi.
There is evidence that other members of this group are cul-
turable (Davis and Janssen, unpublished data).
Members of the phylum Planctomycetes make up an average
of 2% (range, 0 to 8%) of soil bacterial communities (Fig. 1).
The planctomycetes are a group of budding bacteria that lack
peptidoglycan and possess membrane-bound intracellular com-
partments (44). The phylogenetic depth within the group is
sufficient to suggest that the phylum could be composed of at
least three classes (31, 88). One of these could consist of bacteria
such as those of the candidates Brocadia and Kuenenia, which
are involved in anaerobic nitrification (88). The other two
major groups are defined by the relatively well studied genera
classified in the class Planctomycetacia (48) and by sequences
affiliated with the WPS-1 lineage (82). Soil bacteria are affili-
ated with all three major groups, and there are many lineages
without any cultured representatives (e.g., see references 50,
68, and 88). Most isolates of this phylum are from aquatic
sources, and it is not clear whether these are physiologically
and genetically good models for soil planctomycetes. Isolates
from soil, including a few members of the WPS-1 lineage, have
been reported (29, 60, 114), but these do not represent the
full phylogenetic breadth suggested by the sequences de-
tected in soils.
Members of the phylum Gemmatimonadetes make up an
average of 2% (range, 0 to 4%) of soil bacterial communities
(Fig. 1). The phylum Gemmatimonadetes contains only one
named and described species, Gemmatimonas aurantiacus
(120). This bacterium is a gram-negative aerobic heterotroph
isolated from an anaerobic-aerobic sequential batch reactor
and belongs to subdivision 1, also known as the class Gemma-
timonadetes (48, 120). Four isolates from soil have been ob-
tained (29, 60). They too belong to subdivision 1 and display an
aerobic, heterotrophic phenotype. Soil-inhabiting representa-
tives of the phylum are found through most of the phylogenetic
breadth of the group, which may contain a number of discrete
class rank taxa (81, 88, 120). The diversity of general physiol-
ogies of this group remains to be ascertained.
Members of the genera Bacillus and Clostridium have long
been considered to be common members of the soil bacterial
community, but the classes Bacilli and Clostridia of the phylum
Firmicutes, together comprising of some 214 genera, including
Bacillus and Clostridium (48), contribute only a mean of 2%
(range, 0 to 8%) to the libraries (Fig. 1). It is possible that
members of this group are underrepresented in libraries be-
cause the cells or spores may be difficult to lyse and so are not
detected in PCR-based analyses that rely on DNA extraction
from soil. Until evidence for such a bias is available, members
of this group must be considered to be relatively minor com-
ponents of soil bacterial communities. They may, however, be
locally abundant, such as in a grassland soil in The Netherlands
(38, 39). Of the sequences affiliated with the phylum Firmicutes
in the 32 libraries analyzed, 17 to 52% could be assigned to
known genera, suggesting that a number of new genera remain
to be isolated, named, and described.
This review deals with members of the domain Bacteria, but
members of the domain Archaea have also been detected in soils
(10, 12, 17, 61, 111), although their abundance is generally low
(17, 84, 99). These studies also reveal the presence of high-rank
taxa of the domain Archaea with no cultured representatives.
Some of these soil archaea may prove to have unexpected phys-
iologies (42), and they appear to be culturable (99).
CHALLENGES AND GOALS
There is considerable variability in the abundance of mem-
bers of different phyla and classes in different soils, judged by
the abundance of 16S rRNA or 16S rRNA genes in libraries. It
is not yet clear to what extent the variations are systematic, in
response to conditions in the soil environment, and to what
degree method-induced artifacts impact the data. The number
of different biological, chemical, and physical factors that may
influence the abundance of different bacterial groups could be
very large. It has been suggested that the abundance of verru-
comicrobia is influenced by soil moisture (14), and the abun-
dance of members of subdivision 1 of the acidobacteria ap-
pears to be controlled by soil pH (93). It is not known whether
the abundance of members of other high-rank taxa is con-
trolled by single, readily identifiable factors. The degree of
phenotypic variation within some of the groups must mean that
the total abundance of a particular group may not change as
much as the representation of species within that group, and so
the abundance of such phenotypically diverse groups cannot be
expected to be controlled by single variables.
Regardless of whether one is interested in functional or
phylogenetic groupings, it is clear that the physiologies and
characteristics of the poorly studied groups of soil bacteria
must be of interest to soil microbiologists (49, 55, 85, 88, 122).
Those interested in functions will want to identify all the major
contributors to that function and will not want to disregard the
possibility that bacteria among the poorly characterized part of
the community are involved. The complexity of soil microbial
VOL. 72, 2006 MINIREVIEWS1725
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communities means that metagenomic approaches to studying
soil bacteria and assembling genomes of uncultured bacteria
to understand their physiologies seem impractical at present
(110). Although the assignment of functions and associated
genes to phylogenetic markers is possible in the absence of
cultures (49, 69), it can be achieved more easily with cultured
Culturing of the total diversity of species, estimated at 104to
106per 10-g soil sample (28, 46, 109), currently seems an
unreachable goal, given that such large numbers of isolates are
not routinely cultured and identified and that the rarer species
can be expected to be difficult to find among the colonies that
do form. An initial objective should be to obtain a range of
isolates from representatives of members of the different phyla
and classes and to determine to what extent there is functional
and genetic diversity within these groups. This will give us an
initial overview of the potential roles of different soil bacterial
groups and will also enable us to learn the tricks required to
culture their more recalcitrant relatives. This strategy has been
successfully applied to verrucomicrobia and acidobacteria (93,
96). At the same time, genome sequences of selected isolates
will help fill in the bacterial genome tree (55).
Some advances in culturing soil bacteria have been made in the
last few years (29, 58, 60, 93, 94, 96, 103, 107, 119). These recent
advances mean that it is probably incorrect to speak of the ma-
jority of bacterial species in soil as being unculturable. Instead, we
should be aware that isolating them will take patience and careful
selection of appropriate strategies. Many of the isolates of rarely
isolated groups are very slow growing and are difficult to maintain
in the laboratory (29). The formation of visible colonies requires
weeks or months rather than hours or days. Although the growth
rates are low, they are still much higher than their likely growth
rates in soil, where cells may divide only a few times per year (53).
It is likely that the few isolates of Acidobacteria, Verrucomicrobia,
Planctomycetes, Gemmatimonadetes, Chloroflexi, Acidimicrobidae,
and Rubrobacteridae, as well as many of the isolates of the better-
studied Proteobacteria, Bacteroidetes, and Actinobacteridae, are ac-
tually the more readily cultured representatives of these groups.
To isolate type strains and a representative collection of related
strains, and to deposit them in culture collections as required for
the valid description of new species (101), will require great ded-
ication and a high level of commitment and expertise from soil
microbiologists and from culture collections. Successful ap-
proaches to culturing these organisms require patience, but the
outcomes are immensely satisfying to microbiologists who enjoy
the challenge and savor the reward of observing the colony on the
plate, seeing the cells of the pure culture under the microscope,
elucidating the bacterium’s physiology, or releasing its genome
sequence into public databases.
The results obtained in the author’s laboratory have been due to
the intellectual and experimental efforts of Sally Cairnduff, Kathyrn
Davis, Bronwyn Grinton, Shayne Joseph, Suzana Kovac, Matthew
O’Neill, Catherine Osborne, Michelle Sait, Parveen Sangwan, Liesbeth
Schoenborn, and Penelope Yates.
Research has been supported by grants from the Australian Re-
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