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Phylogenetic Diversity of Bacteria in an Earth-Cave in Guizhou Province, Southwest of China


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The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.
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The Journal of Microbiology, April 2007, p. 105-112
Copyright 2007, The Microbiological Society of Korea
Vol. 45 , No . 2
Phylogenetic Diversity of Bacteria in an Earth-Cave in Guizhou Province,
Southwest of China
JunPei Zhou, YingQi Gu, ChangSong Zou, and MingHe Mo*
Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming 650091, P. R. China
(Received December 27, 2006 / Accepted March 20, 2007)
The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil
of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were ampli-
fied directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and
cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and repre-
sentative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190
analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high
diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%),
Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes
(2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria,
6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were
affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of
the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed
similarities in the bacterial community components, especially in the high abundance of Proteobacteria and
Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes,
Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.
Keywords:16S rRNA gene, karst, cave, bacterial community, microbial ecology
The term of “cave” is defined as any natural space below
the Earth’s surface that extends beyond the twilight zone,
and that is accessible to humans (Gillieson, 1996). Most of
common types of caves in karst regions are those formed in
limestone and other calcareous rocks, and as lava tubes in
basaltic rock. Remaining types, including those formed in
gypsum, granite, talus, quartzite, ice, and sandstone are usually
limited in extent. Caves, with relatively limited nutrient in
organic matter, stable and low temperatures, high humidity
and mineral concentrations, can be considered extreme en-
vironments for life and provide ecological niches for highly
specialized microorganisms (Schabereiter-Gurtner et al., 2003).
Dripping water, visitors and animals can provide organic
input that facilitates life of heterotrophic microorganisms in
some caves (Groth and Saiz-Jimenez, 1999; Groth et al.,
1999, 2001).
Microorganism in caves is the main biological habitant
and remarkably contributes to cave ecology (Northup and
Lavoie, 2001). Presences of microorganisms in terrestrial
and aquatic cave environments around the world have been
reported using the cultivation method (Cunningham et al.,
1995; Gonzalez et al., 1999; Laiz et al., 1999, 2000; Groth
et al., 1999, 2001; Canˇaveras et al., 2001). Many microbes
have been identified to be related to dissolution and pre-
cipitation reactions that involves carbonates, moonmilk, sili-
cates, clays, iron, manganese, sulfur, and saltpeter (Northup
and Lavoie, 2001). Enrichment-based and cultural inves-
tigations on typical heterotrophic microbes have shown that
microbes grow in proportion to less than 1% in an environ-
ment (Amann et al., 1995).
Culture-independent 16S rRNA gene sequence analysis
has been employed to study bacterial communities in envi-
ronmental samples without prior cultivation. It has signifi-
cantly revealed a broader diversity of 16S rRNA gene
sequence types than culture-based studies (Amann et al.,
1995; Head et al., 1998; Hugenholtz et al., 1998). The com-
bination of phylogenetic sequence analysis with restriction
fragment length polymorphisms (RFLPs) of PCR-amplified
bacterial 16S rRNA genes has become a powerful tool to
investigate natural bacterial communities. However, the 16S
rRNA gene-based analysis of bacterial colonization in caves
has been restricted to the samples of rocks, paintings, drip-
ping waters, springs, and underwater passages, but not on
soil (Northup et al., 2000; Vlasceanu et al., 2000; Engel et
al., 2001, 2004; Holmes et al., 2001; Schabereiter-Gurtner et
al., 2002a, 2002b, 2003).
Guizhou, a province in southwest of China, is the center
of East-Asia developing karst area. As one of the three
developing karst areas, this area has a karst area of over
5.5×105 km2 and is the largest and the most complex devel-
oping karst area in the world (Smart et al., 1986; Zhang et
al., 1992). Niu Cave, an unusual cave in this karst area, is
composed of soil other than usual limestone or alcareous
rocks. So far we have limited knowledge to understand the
microbial community in an earth-cave. The aim of the pre-
sent study was to investigate the bacterial diversity in Niu
106 Zhou et al. J. Microbiol.
Table 1. Distribution of clones and phylotypes from the bacterial 16S rRNA gene library
Putative phylogenetic affiliationbNo. of clones % of clones No. of
phylotypesa% of
% of sequence
similarity to its
closest relativesb
No. of phylotypes
that exhibit <90%
similarities to its
closest relativesb
1. Proteobacteria 80 42.6 43 37.4 89-100 3
1.1 Alphaproteobacteria 21 11.2 12 10.4 91-98 -
1.2 Betaproteobacteria 14 7.5 7 6.1 94-100 -
1.3 Deltaproteobacteria 20 10.6 11 9.6 89-98 3
1.4 Gammaproteobacteria 25 13.3 13 11.3 92-99 -
2. Acidobacteria 35 18.6 21 18.3 91-98 -
3. Planctomycetes 17 9.0 14 12.2 87-95 1
4. Chloroflexi 14 7.5 8 7.0 88-94 2
5. Bacteroidetes 42.1 43.592-96 -
6. Gemmatimonadetes 52.7 54.492-99 -
7. Nitrospirae 15 8.0 6 5.2 89-98 1
8. Actinobacteria 12 6.4 10 8.7 90-99 -
9. Candidate division 6 3.2 4 3.5 89-95 2
a Sequences of RFLP types differing only slightly (3%) were considered as a phylotype (Huang et al., 2004).
b Closest relatives as determined by the BLAST method (Altschul et al., 1990; Engel et al., 2004).
Cave by culture-independent method and present the first
knowledge to understand microbial composition in such
Materials and Methods
Site description and environmental sample collection
Niu Cave (25° 57 N, 107° 48 E) is located in Dushan county,
south of Guizhou province, southwest of China. As a “karst
province”, Guizhou has a karst area of 1.3×105 km2,
comprising 73.8% of its total area. Niu Cave is formed by
soil, and with 5-10 m height, 4-50 m width. After a distance
of about 2 km from the entrance, the cave is divided into
three layers. Each layer has a long distance more than 5
km. Thirty soil samples, 10 from each layer, were sampled
and mixed thoroughly for bacterial community analysis. Soil
samples were stored at -20°C and analyzed within a month.
Soil DNA extraction, PCR amplification, and cloning
Soil DNA was extracted with a soil DNA isolation kit
(Catalog #12800-50, MO BIO Laboratories, USA) following
the manufacturer’s instructions. Bacterial 16S rRNA genes
were amplified by PCR using the combination of universal
primer 1492r and bacterial primer 27f (Lane, 1991). The
PCR reaction was performed with a thermal program,
which comprised preheating at 95°C for 2 min, 25 cycles at
98°C for 1 min, 50°C for 40 s, 72°C for 2 min and a final
extension of at 72°C for 10 min. The amplified products
were purified using an agarose gel DNA purification kit
(No. DV805A, Takara, Japan). Bacterial 16S rRNA gene
amplicon (ca. 1500 bases) was then excised from a 1%
agarose gel and eluted with the same kit. Finally, the puri-
fied product was ligated into the pMD 18 T-vector (Takara)
and the ligation product was transformed into Escherichia
coli DH5α competent cells with ampicillin and blue/white
screening following manufacturer’s instructions.
Screening of rRNA gene clones
Inserts of rRNA genes from recombinant clones were
reamplified with vector primers M13-M3 and M13-RV. The
amplifications were subjected to restriction fragment length
polymorphism (RFLP) by separate enzymatic digestions
with HhaI (Takara) and MspI (Takara) endonucleases fol-
lowing the manufacturer’s instructions, and the digested
DNA fragments were electrophoresed in 3% agarose gels.
After staining with ethidium bromide, the gels were photo-
graphed using an image-capture system UVITEC DBT-08,
and scanning image analyses were performed manually.
DNA sequencing and phylogenetic analysis
One to three representative clones from each unique RFLP
type were selected for sequencing. The 16S rRNA gene
inserts were sequenced using plasmid DNA as template
and M13-20 or M13-RV-P as sequencing primer. Sequencing
was done on an automated ABI 3730 sequencer by Beijng
Genomics Institute. The resulting sequences (next to the
primer 1492r and at least 600 bp) were compared with
those available in GenBank by use of the BLAST method
to determine their approximate phylogenetic affiliation and
16S rRNA gene sequence similarities (Altschul et al., 1990;
Engel et al., 2004). Chimeric sequences were identified by
use of the CHECK-CHIMERA program of the Ribosomal
Database Project (Maidak et al., 1997), and by independently
comparing the alignments at the beginning and the end of
each sequence and the alignments of the entire sequence
with sequences from public databases. Sequences differing
only slightly (3%) were considered as a phylotype, and
each phylotype was represented by a sequence (Huang et
al., 2004). Nucleotide sequences were initially aligned using
CLUSTAL X (Thompson et al., 1997) and then manually
Vol . 4 5 , N o. 2 Bacterial diversity in Niu Cave 107
Fig. 1. 16S rRNA gene-based dendrogram showing phylogenetic
relationships of bacterial phylotypes from Niu Cave (shown in
bold) to members of the Proteobacteria from public database.
Bootstrap values (n=1000 replicates) of 50% are reported as
percentages. The scale bar represents the number of changes per
nucleotide position. Thermosipho sp. MV1063 (AJ419874) was used
as outgroup. Sequences of RFLP types differing only slightly (3%)
are shown in parentheses. Accession numbers are given at the end
of each sequence.
adjusted. Distance matrices and phylogenetic trees were
calculated according to the Kimura two-parameter model
(Kimura, 1980) and neighbor-joining (Saitou et al., 1987)
algorithms using the MEGA (version 3.1) software packages
(Kumar et al., 2004). One thousand bootstraps were per-
formed to assign confidence levels to the nodes in the trees.
The 16S rRNA gene sequences have been deposited in the
GenBank nucleotide sequence database under accession num-
bers EF141837-EF141978.
A total of 190 recombinant clones were randomly selected,
and their rRNA gene inserts were subjected to restriction
endonuclease analysis (RFLP), resulting in 142 different
RFLP types. One to three representative clones of each
unique RFLP type were partially sequenced. The clones
showing sequence dissimilarity less than 3% among cloned
library were considered as a phylotype. As a result, 117
phylotypes were generated. Two chimeric sequences were
identified and excluded from subsequent analyses. Both of
them belonged to unique RFLP types represented by a single
clone. It was determined that most relatives of phylotypes
(87 sequences representing 133 clones) were related to envi-
ronmental clones and 9 phylotypes had relatively low levels
of similarity (<90%) with their closest counterparts in the
GenBank databases (Table 1). None of phylotypes were
closely related to bacterial sequences detected in other caves
or karst areas in the public databases except the clone
CV76 (DQ499315), relative of NC123. These indicated that
the bacterial community associated with Niu Cave was
novel and complex.
Phylogenetic analyses placed the 115 phylotypes in the
following 9 groups of the domain Bacteria: Proteobacteria,
Acidobacteria, Planctomycetes, Chloroflexi (Green nonsulfur
bacteria), Bacteroidetes, Gemmatimonadetes, Nitrospirae,
Actinobacteria (High G+C Gram-positive bacteria), and
candidate divisions (including the OP3, GN08, and SBR
1093). Among them, the Proteobacteria was the largest
group including 80 clones, followed by Acidobacteria (35
clones) and Planctomycetes (17 clones).
A total of 80 clones, represented by 43 phylotypes and ac-
counting for 42.6% of the clone library, were phylogenetically
associated with 4 classes of Proteobacteria with similarities
between 89%-100%: Alphaproteobacteria (number of phylotypes,
np=12, number of clones, nc=21), Betaproteobacteria (np=7,
nc=14), Deltaproteobacteria (np=11, nc=20), and Gamma-
proteobacteria (np=13, nc=25) (Table 1). Thirty-eight clones
were represented by 10 phylotypes each including at least 3
clones, while 24 clones were represented by phylotypes of
which each included a single clone.
Eight phylotypes of Deltaproteobacteria revealed less than
94% similarity. Three phylotypes (NC002, NC039, and NC
141), sharing less than 90% similarity to known sequences,
seemed to be representatives of novel taxa within Delta-
proteobacteria subdivisions, respectively (Table 1, Fig. 1).
Thirty-six clones, represented by 19 phylotypes, were
related to cultured members and belonged to putatively
108 Zhou et al. J. Microbiol.
Fig. 2. 16S rRNA gene-based dendrogram showing phylogenetic
relationships of bacterial phylotypes from Niu Cave (shown in
bold) to members of the Acidobacteria and Planctomycetes from
public database. Bootstrap values (n=1000 replicates) of 50%
are reported as percentages. The scale bar represents the number
of changes per nucleotide position. Thermosipho sp. MV1063
(AJ419874) was used as outgroup. Sequences of RFLP types
differing only slightly (3%) are shown in parentheses. Accession
numbers are given at the end of each sequence.
Sphingomonadales (NC006 and NC032), Rhizobiales (NC091
and NC076), Rhodospirillales (NC011), Nitrosomonadales
(NC017 and NC051), Burkholderiales (NC029, NC031, and
NC078), Desulfuromonales (NC002), Xanthomonadales (NC004,
NC018, NC027, NC030, NC060, NC083, and NC094) and
Oceanospirillales (NC127). The closest relatives of NC006
and NC032 were strains of Sphingomonas (Fig. 1). NC015,
NC141 affiliated to the members of Entotheonella (simi-
larity 95) which is a new genus belonging to unclassified
Deltaproteobacteria (Schirmer et al., 2005).
Thirty-five clones, represented by 21 phylotypes and account-
ing for 18.6% of the clone library, were clustered with the
uncultivated bacterial sequences of Acidobacteria with sim-
ilarities between 91%-98% (Table 1, Fig. 2). Fig. 2 showed
a phylogenetic tree of Acidobacteria, which was grouped
into at least 4 acidobacterial clusters. Acidobacteria form a
newly devised division of Bacteria, probably as diverse as
Proteobacteria or Gram-positive bacteria. The definition of
this phylum was based on the analysis of 16S rRNA gene
sequences retrieved from cloned rRNA genes and phyloge-
netically related to several cultivated species such as the
Fe(III)-reducing Geothrix fermentans (Ludwig et al., 1997;
Quaiser et al., 2003). The clones Y72 (AB116490) and Y190
(AB116442), relatives of NC065 and NC099 respectively,
were detected in coastal marine sediment beneath areas of
intensive shellfish aquaculture where sulfur cycle was accel-
erated (Asami et al., 2005).
Fourteen phylotypes, representing 17 clones and accounting
for 9.0% of the clone library, were grouped into at least 3
clusters with Planctomycetes. These clones were related with
relatively low similarities (in the range of 87%-95%) to cul-
tured and uncultured bacterial sequences listed in the
GenBank database (Table 1, Fig. 2). Molecular microbial ecol-
ogy has provided new evidence showing that Planctomycetes
bacteria are ubiquitous and constitute a representative part
of the natural bacteria population (Hugenholtz et al., 1998;
Neef et al., 1998). The clone 018 (AB252879), relative of NC057,
was a member detected in an iron-oxidation biofilm at
Shibayama lagoon. The retrieved three sequences NC010,
NC038, and NC052 were clustered with Pirellula staleyi
strain ATCC 35122 (AF399914) which can outlast periods of
nutrient depletion with the expression of genes for carbon
starvation (Glöckner et al., 2003).
Chloroflexi (Green nonsulfur bacteria)
Fourteen clones, represented by 8 phylotypes and accounting
for 7.5% of the clone library were related to the members
of the Chloroflexi phylum (88%similarity94%) (Table 1,
Fig. 3). The low similarities to closest members indicated
that the corresponding bacteria detected in Niu Cave be-
longed to putatively new taxonomic groups. The clone H5
(AF234688), close to NC130, was found in a nitrifying-
denitrifying activated sludge (Juretschko et al., 2002).
Bacteroidetes, Gemmatimonadetes, and Nitrospirae
Four phylotypes, each representing a single clone, were clus-
tered with Bacteroidetes. Five clones, each represented by a
phylotype and accounting for 2.7% of the clone library,
were grouped within Gemmatimonadetes phylum. These
clones were related to only uncultured bacterial sequences
listed in the GenBank database (Table 1, Fig. 3). Fifteen
clones, represented by 6 phylotypes and accounting for
8.0% of the clone library, were grouped with Nitrospirae.
Among them, 10 of the 15 clones were represented by 2
phylotypes (NC022 and NC132).
Vol . 4 5 , N o. 2 Bacterial diversity in Niu Cave 109
Fig. 3. 16S rRNA gene-based dendrogram showing phylogenetic
relationships of bacterial phylotypes from Niu Cave (shown in bold)
to members of the Chloroflexi, Nitrospirae, Gemmatimonadetes,
Actinobacteria, Bacteroidetes and Candidate divisions from public
database. Bootstrap values (n=1000 replicates) of 50% are reported
as percentages. The scale bar represents the number of changes
per nucleotide position. Thermosipho sp. MV1063 (AJ419874)
used as outgroup. Sequences of RFLP types differing only slightly
(3%) are shown in parentheses. Accession numbers are given at
the end of each sequence.
Actinobacteria (High G+C Gram-positive bacteria)
Ten phylotypes, representing 12 clones and accounting for
6.4% of the clone library, were associated with Actinobacteria
with 90%-99% similarities (Table 1, Fig. 3). Five phylotypes
were related to members of Actinomycetales (NC008, NC
079, and NC114) and Rubrobacterales (NC081 and NC085).
Terrestrial subsurface environments are often inaccessible
for study, limiting our understanding of ecosystem structure
and dynamics, elemental cycling, and the impacts to earth
and atmospheric biogeochemical processes. Geomicrobiological
activities in caves are no longer underestimated, since studies
showed that bacterial metabolism remarkably contributes to
cave ecology (Northup and Lavoie, 2001). However, no study
has been initiated to investigate the phylogenetic diversity
of bacteria in caves formed in soil, such as Niu Cave. We
firstly presented the knowledge on bacterial community and
revealed a considerable number of novel and unknown bac-
terial sequences and a high diversity of putative bacterial
communities in this environment.
Using culture-independent methods, previous studies (Holmes
et al., 2001; Schabereiter-Gurtner et al., 2002a, 2002b, 2003;
Northup et al., 2003; Engel et al., 2004) have revealed that
bacteria belonging to Proteobacteria, Bacteroidetes, and Actino-
bacteria were usually found in different substances of caves
(Table 2). Among them, Proteobacteria was reported to be
the dominant bacteria and could act as a key role in the
processes of biogeochemical cycle in caves. In Tito Bustillo
Cave, Altamira Cave and La Garma Cave, Acidobacteria
was the second dominating phylogenetic group after Proteo-
bacteria. Our result supported this conclusion. Higher than
7.0% of Chloroflexi was found in NC and Tito Bustillo Cave.
And Firmicutes had been found in Llonín Cave, La Garma
Cave and Lechuguilla and Spider Caves. In our study, there
were no phylotypes belonging to Firmicutes. Orders Rhizobiales,
Sphingomonadales, Rhodospirillales, Burkholderiales, Rhodocyclales,
Desulfuromonales, Pseudomonadales and Actinobacterales were
reported to be present in caves, including Niu Cave. However,
Myxococcales was found only in Altamira Cave, and Xantho-
monadales only in Llonín Cave.
The phylum Gemmatimonadetes, and the orders Nitroso-
monadales (Betaproteobacteria), Oceanospirillales (Gamma-
proteobacteria), and Rubrobacterales (Actinobacteria) found
in Niu Cave were never reported in the previous studies.
Six clones, NC091, NC031, NC051, NC017, NC029, and
NC022, were related to Mesorhizobium, Ralstonia, Nitrosomonas,
Nitrosospira, Alcaligenes, and Nitrospira with similarities higher
than 97%, respectively (Fig. 1, 3). These genera were reported
to involve in putatively nitrogen cycle. Mesorhizobium belongs
to members of nitrogen-fixing bacteria known as rhizobia
(Bottomley, 1992). Ralstonia taiwanensis was reported being
capable of nitrogen fixation (Chen et al., 2001), and Ralstonia
eutropha containing two nitric oxide reductases (Cramm et
al., 1997). Nitrosomonas and Nitrosospira are members of the
ammonia-oxidizing proteobacteria which can convert ammonia
to nitrite (Suzuki et al., 1974; Hiorns et al., 1995). Alcaligenes
sp. STC1 (AB046605) was a C1-using aerobic denitrifier
(Ozeki et al., 2001). Nitrospira was the dominant nitrite oxi-
dizers in most environmental samples tested so far (Burrell
et al., 1998). The bacteria involved in nitrogen cycle were
reported in all studies on bacterial diversity in caves.
NC076 and NC078 were related to Ped omicrob ium and
Leptothrix respectively (Fig. 1). Ped omicr obium plays an im-
portant role in iron- and manganese-oxidization. Ghiorse
and Hirsch (1979) observed that two Pedom icr obium-like
110 Zhou et al. J. Microbiol.
Table 2. Distribution of bacteria in caves investigated by culture-independent molecular method
% of clones
Nullarbor Caves
et al
Altamira Cave
et al
Tito Bustillo Cave
et al
Llonín Cave
et al
La Garma Cave
et al
Lechuguilla and
Spider Caves
et al
Lower Kane Cave
et al
Niu Cave
(this study)
Proteobacteria 40.0 52.3 48.8 59.3 32.8 35.0 92.7 42.6
Acidobacteria 23.8 29.2 24.1 5.6 18.6
Planctomycetes 5.7 4.8 2.4 9.0
Bacteroidetes 8.6 9.5 2.4 11.1 3.5 1.7 2.1
Chloroflexi 4.8 7.3 1.7 7.5
Nitrospirae 5.7 3.7 3.5 15.5 8.0
Actinobacteria 5.7 4.8 9.8 22.2 19.0 11.7 6.4
Firmicute s 3.7 13.8 37.9
Gemmatimonadetes 2.7
budding bacteria deposit Fe and Mn ions on their cell
walls. Peck (1986) reported iron-impregnated sheaths of
Leptothrix sp., which was inoculated with mud from Level
Crevice Cave. Moore (1981) found manganese-oxidizing
bacteria Leptothrix sp. in a stream in Matts Black Cave,
West Virginia, and attributed the formation of birnessite in
this cave to the precipitation of manganese around sheaths
of the bacteria. The concentrations of iron, manganese and
other elements have been found in Tito Bustillo and other
Spanish and Italian caves (Schabereiter-Gurtner et al., 2002a,
2002b, 2003).
The order Desulfuromonales included Desulfuromonas
(Liesack and Finster, 1994) related with sulfate/sulfur reduc-
tion and Geobacter with Fe(III)/Mn(IV) reduction (Nealson
and Saffarini, 1994). A pioneering study of Movile Cave,
Romania, by Sarbu et al. (1996) revealed sulfide/ sulfur oxida-
tion bacteria including species of Thiobacillus and Beggiatoa,
and sulfate-reducers including species of Desulfovibrio. Up
to now, sulfur and sulfide oxidizers, sulfate reducers, appear
abundant in caves (Schabereiter-Gurtner et al., 2003).
Acidobacteria and Planctomycetes were the second and
third dominating phylogenetic groups in Niu Cave respec-
tively. It confirmed members of the divisions are ecologi-
cally significant constituents of Niu Cave. Representatives
of the poorly studied phylogenetic divisions have been de-
tected in many clonal analyses and are thought to be of
great ecological significance to many ecosystems (Kuske et
al., 1997; Ludwig et al., 1997; Holmes et al., 2001). As limited
cultivated species, Acidobacteria and Planctomycetes’ ecologi-
cal functions, and possible impacts on caves remain unclear
at present. Two novel genera of Planctomycetes, Candidatus
Kuenenia stuttgartiensis, and Candidatus Brocadia annamoxidans
are capable of catalyzing the anaerobic oxidation of ammo-
nium (Schmid et al., 2000; Jetten et al., 2001).
All above-mentioned results in our study were ascribed
to culture-independent techniques including PCR amplifica-
tion of bacterial 16S rRNA genes and restriction fragment
length polymorphism (RFLP). The applied approach allows
the analysis of poorly studied environments where nothing
or little is known about bacterial diversity and natural
growing conditions. Our present study gave insight into the
great bacterial taxonomic diversity in Niu Cave, overcoming
some of the limiting factors of cultivation methods and allow-
ing the detection of putatively uncultivable and unexpected
However, studies based on culture-independent methods
make more difficult valid statements about the ecological
role that clones might play in the environment. To deter-
mine microbial contribution to cave ecology, we need to
draw on additional wealth of rigorous research done on
this system. Stable isotope techniques can provide informa-
tion on microbial contribution to mineral formation (Hose
et al., 2000) and ecosystem bioenergetics (Sarbu et al., 1996).
The PCR- and RFLP-based investigation of bacterial diversity
is not free from bias. Limitations such as inefficient cell
lysis, and the preferential and selective amplification of 16S
rRNA gene fragments may lead to an underestimation of
bacterial diversity (Liesack et al., 1997). Potentially, with
the development of molecular sequence-based techniques
and accumulation of information on the diversity and struc-
ture of bacterial communities in caves, more explanations
concerning contribution of microorganisms to cave ecosys-
tem will be presented.
This work was supported jointly by projects from NSFC
(30460078) and Department of Science and Technology of
Yunnan Province (2006XY41, 2006C0005M, 2004C0001Q,
2003RC03, and 2005NG05).
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... Caves, which are extreme environments in terms of microbial diversity, are ideal habitats for searching for novel microorganisms and, consequently, new compounds and proteins. Caves represent unique ecosystems with extreme conditions such as darkness, nutrient limitation, low oxygen level, high humidity, low temperature and high-level concentrations of minerals (Grothet et al., 1999;Schabereiter-Gurtner et al., 2003;Zhou et al., 2007). Because of these harsh conditions, caves contain rich and largely undiscovered microbial diversity and cave-dwelling microorganisms have unique properties from which to explore novel enzymes and different bioactive substances (Oliveira et al., 2017;Riquelme et al., 2017;Wiseschart et al., 2018). ...
... Metagenomic DNA from the cave soil samples was extracted according to a modified SDS-based method by Zhou et al. (2007). A 5 g soil sample was mixed with 13.5 ml of DNA extraction buffer (100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCl, 1% CTAB) and 100 ml of proteinase K (10 mg/ml) in sterile 50ml Falcon tubes by horizontal shaking at 225 rpm for 30 min at 37°C. ...
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Abstract: Turkey has a great number of karstic caves which are unexplored and have unknown microbial diversity. The biodiversity characterisation of these caves has not yet been systematically studied from the molecular point of view. Çal Cave in Trabzon, Turkey is one of the important karstic caves. In the present study, a metagenomic approach was used to explore the microbial diversity of Çal Cave for the first time to assess the potential of gene sources. Detailed taxonomic profiling was defined by sequencing all environmental genomes instead of a specific marker gene such as 16S rRNA which only targets prokaryotes. Taxonomic analysis revealed that the Çal Cave soil sample was represented as 98% Bacteria, 2% Eukaryota, 0.3% Archaea, and 0.01% Virus. Results showed that, the 31 distinct bacterial phyla represented in the Çal Cave soil sample were dominated by Actinobacteria (65%) and Proteobacteria (31%). The most dominant bacterial genus was Streptomyces. Among the 2% Eukaryotic population, the largest phylum was Ascomycota and it was mostly represented as Sordariomycetes. It was determined that 77% of Archaea was Halobacteria. The most abundant class of viruses dwelling in Çal Cave was Caudovirales. 91.61% of total readings could not be classified into any specific kingdom. Overall, classified and unclassified data verify that there exists vast microbial biodiversity in Çal Cave which could not be identified with classical microbiology techniques, and this microbial diversity provides a promising gene source for novel enzyme and bioactive compounds to be used in biotechnological applications
... The mouthwash with chamomile was considered satisfactory in reducing gingival inflammation, showing great performance in reducing bacterial plaque index [31]. [32][33][34]. The most characteristic constituents of chamomile are unstable oil, sesquiterpene lactones, ascorbic acid, and phenol compounds, primarily the flavonoids, apigenin, quercetin, patulin, luteolin, and glycosides. ...
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Twelve samples were collected from Baghdad city. From 12 samples, 17 colonies were obtained. Out of the 17 Actinomycetes colonies. sub-cultured on ISP2 for growth, and incubation of plates for 7 days, only three isolates demonstrated cultural characteristics similar to that of Streptomyces sp. three isolates were selected and purified by pure culture techniques of Streptomyces sp. All isolates were given a number as B1, B2, and B3. All Streptomyces sp isolates were screened for their antibacterial activity on Yeast extract-malt extract agar medium (ISP2) using scross-streak technique against two pathogenic bacteria include Gram-negative (Two pathogenic bacteria, including Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus). Among three Streptomyces sp isolates that obtained from Baghdad city (Al-Jadriya), one isolates (B2) didn’t show any antibacterial activity against any type of pathogenic bacteria (Gram-negative and Gram-positive bacteria), while two Streptomyces sp isolates (B1 and B3) showed antibacterial activity against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus. Screening was performed by Agar-Well Diffusion method and growth inhibition zones were measured in millimeters for each of the Streptomyces isolates (B1 and B3). Tested isolates have shown potent in vitro antibacterial activities against all tested pathogens. The highest activities were shown by isolate B1 against S. aureus 19.5 mm, Pseudomonas aeruginosa 14 mm. It is also evident that B3 isolate have shown activities against all pathogenic bacteria with inhibition zone diameters ranging between 17 and 13 mm against S. aureus and Pseudomonas aeruginosa respectively. Effect of Pomegranate peel and Matricaria chamomilla extracts (200μl, 300μl and 400μl) on the growth of Streptomyces sp were initially determined by the agar well-diffusion method, showed there is no diameters of inhibition zones exerted by the extract towards Streptomyces in different concentrations of Pomegranate peel and Matricaria chamomilla extracts.
... Gemmatimonadetes clone sequences are well adapted to not only arid but also oligotrophic conditions. They can survive the saline-alkali stress and starvation, possibly forming a resting stage (Zhou et al. 2007;Pasic et al. 2010). Research on grassland soil by Nacke et al. (2011) showed that relative abundances of Bacteroidetes and Actinobacteria in the analyzed soils significantly increased with higher pH values. ...
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The salinization of grassland in arid and semi-arid areas is a serious environmental issue in China. Halophytes show extreme salt tolerance and are grown in saline-alkaline environments. Their rhizosphere microorganisms contribute significantly to plant stress tolerance. To study bacterial and fungal community structure changes in Chinese ryegrass (Leymus chinensis) rhizosphere soil under salt and alkali stress, pot experiments were conducted with different salt and alkali stress intensities. High-throughput sequencing was conducted, and the microbial diversity, community structure, and driving factors were analyzed in rhizosphere soil. The salinization of grassland in arid and semi-arid areas is a serious environmental issue in China. Halophytes show extreme salt tolerance and grow in saline-alkaline environments. A total of 549 species of bacteria from 28 phyla and 250 species from 11 phyla of fungi were detected in the rhizosphere soil of Leymus chinensis with different saline-alkali gradients. Alpha diversity analysis along saline-alkali gradients showed that bacterial community richness and diversity were the highest in the moderate saline-alkali group (pH = 8.28, EC = 160.4 μS·cm⁻¹), while fungi had high richness and diversity in the control group (pH = 7.35, EC = 134.5 μS·cm⁻¹). The bacteriophyta Proteobacteria, Acidobacteria, Plantomycetes, and the eumycota Ascomycota, Basidiomycota, and Glomeromycota were found with relative abundances of more than 10%. Saline-alkali gradients had significant effects on the abundance of the bacterial and fungal groups in the rhizosphere. The distribution of bacterial colony structure was not significant at the species level (P > 0.05). However, there were significant differences in the distribution of fungal structure and considerable differences in the composition of fungal species among the moderate saline-alkali group, severe saline-alkali group, and control group (P < 0.05). Correlation analysis showed that the bacterial phylum Gemmatimonadetes had a highly significant positive correlation with pH and EC (P < 0. 01). Saline-alkali stress significantly inhibited the abundance of the bacteria Latescibacteria, Cyanobacteria, and Bacteroides, and the fungi Zoopagomycota, Mortierllomycota, and Cryptomycota (P < 0. 05). Compared with fungi, bacterial community composition was most closely correlated with soil salinization. This report provided new insights into the responses of relationships between rhizosphere soil microorganisms and salt and alkali tolerance of plants.
... Some taxa were exclusive of one substratum (e.g., Microbacterium sp., Pseudomonas sp. and Staphylococcus sp.); others were present in two different substrata (e.g., Bacillus megaterum and Bacillus mycoides). The bacterial taxa observed have been also documented in other caves [41,43] as well as on mural paintings [44,45] and considered by some authors as the first colonizers of these environments [46,47]. The algal component of the biofilm mainly constituted green algae, and occurred in sites nearby the entrance of the cave lit by direct or indirect sunlight (Table 3). ...
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Caves present unique habitats for the development of microbial communities due to their peculiar environmental conditions. In caves decorated with frescoes, the characterization of microbial biofilm is important to better preserve and safeguard such artworks. This study aims to investigate the microbial communities present in the Fornelle Cave (Calvi Risorta, Caserta, Italy) and their correlation with environmental parameters. The cave walls and the wall paintings have been altered by environmental conditions and microbial activity. We first used light microscopy and scanning electron microscopy (SEM) and X-ray diffraction to characterise the biofilm structure and the mineral composition of substrata, respectively. Then, using both culture-dependent (Sanger sequencing) and culture-independent (automated ribosomal intergenic spacer analysis, ARISA) molecular methods, we demonstrated that the taxonomic composition of biofilms was different across the three substrata analysed and, in some cases, positively correlated with some environmental parameters. We identified 47 taxa in the biofilm samples, specifically 8 bacterial, 18 cyanobacterial, 14 algal and 7 fungal taxa. Fungi showed the highest number of ARISA types on the tuff rock, while autotrophic organisms (cyanobacteria and algae) on the frescoes exposed to light. This study confirms that caves constitute a biodiversity-rich environment for microbial taxa and that, in the presence of wall paintings, taxonomic characterization is particularly important for conservation and restoration purposes.
... The most extensively studied caves for their microbial diversity are karstic or limestone caves. High humidity (90-100%), relatively stable, and low temperature inside with limited temperature fuctuation during the seasons and low to no light conditions are the main characteristics of caves studied so far (Schabereiter-Gurtner et al. 2003, Zhou et al. 2007. From the geomicrobiological aspect, caves are nutrient-limited habitats and have low biotic potential (Barton and Jurado 2007). ...
... Bacterial genera present in moonmilk are usually found within the epilithic and endolithic microbial communities of the macco and sabbione (Zhou et al., 2007;Antony et al., 2012;Chan et al., 2012;Mogul et al., 2017). The pores and fissures of calcite rocks facilitate the ingress of water which, in turn, supports the proliferation of microorganisms (Meslier and DiRuggiero, 2019). ...
Earth's microbial biosphere extends down through the crust and much of the subsurface, including those microbial ecosystems located within cave systems. Here, we elucidate the microbial ecosystems within anthropogenic 'caves'; the Iron-Age, subterranean tombs of central Italy. The interior walls of the rock (calcium-rich macco) were painted 2500 years ago and are covered with CaCO 3 needles (known as moonmilk). The aims of the current study were to: identify biological/geochemical/biophysical determinants of and characterize bacterial communities involved in CaCO 3 precipitation; challenge the maxim that biogenic activity necessarily degrades surfaces; locate the bacterial cells that are the source of the CaCO 3 precipitate; and gain insight into the kinetics of moonmilk formation. We reveal that this environment hosts communities that consist primarily of bacteria that are mesophilic for temperature and xerotolerance (including Actinobacteria, Bacte-roidetes and Proteobacteria); is populated by photo-synthetic Cyanobacteria exhibiting heterotrophic nutrition (Calothrix and Chroococcidiopsis); and has CaCO 3 precipitating on the rock surfaces (confirma-tion that this process is biogenic) that acts to preserve rather than damage the painted surface. We also identified that some community members are psychrotolerant (Polaromonas), acidotolerant or aci-dophilic (members of the Acidobacteria), or resistant to ionizing radiation (Brevundimonas and Truepera); elucidate the ways in which microbiology impacts mineralogy and vice versa; and reveal that biogenic formation of moonmilk can occur rapidly, that is, over a period of 10 to 56 years. We discuss the paradox that these ecosystems, that are for the most part in the dark and lack primary production, are apparently highly active, biodiverse and biomass-rich.
This study aimed to identify the bacteria which take part in the CaCO3 precipitation on the speleothem surfaces of Dupnisa Cave. In addition, this study highlighted the CaCO3 precipitation ability of the bacteria with negative urease activity. 150 isolates with microbial induced calcium carbonate precipitation features were selected and identified. They were belonging to Proteobacteria (53.3%), Firmicutes (32.7%) and, Actinobacteria (4.7%) phyla. The dominant bacterial species on all surface samples were Bacillus mycoides (9.3%), Bacillus zhangzhouensis (5.3%), and Serratia quinivorans (4%). Our results showed that most of the bacteria which can precipitate calcium carbonate on the B4 medium at the first 3 days, have urease negative activity. Within this study, it has been emphasized that other mechanisms enabling the precipitation of CaCO3 besides the urease mechanism should also be investigated. EDS analyses confirmed that the crystals were predominantly composed of calcium, carbon, and oxygen. In addition, the EDS highlighted that the two strains of Bacillus mycoides, isolated from two different surfaces, produced crystals of different morphology. Our study results to the identification of the bacteria which contribute to the Dupnisa Cave walls formation. Besides, our results showed that the Dupnisa Cave is housing bacteria with biotechnological and engineering applications potentials.
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In this paper we describe a new species of cave fungus belonging to Sporocadaceae (Amphisphaeriales), collected from Gem Cave, Fumin County, Yunnan Province, China. Initial morphological observations confirmed that our fungal collection is a pestalotioid species. Phylogenetic analyses of combined internal transcribed spacer (ITS), β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1α) gene sequence dataset confirmed that our fungus forms an independent branch within Neopestalotiopsis. Thus, we describe our fungus as a new species of Neopestalotiopsis based on both morphology and multigene phylogeny. This is the first-ever report of Neopestalotiopsis from a cave habitat. A full description, micrographs and a phylogenetic tree showing the placement of the new species are provided.
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Moonmilk are cave carbonate deposits that host a rich microbiome including antibiotic-producing Actinobacteria making these speleothems appealing for bioprospecting. Here we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave “Grotte des Collemboles” via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9 to 23% of the total bacterial population. Next to actinobacterial OTUs attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with Rhodoccocus (4 OTUs, 17%) and Pseudonocardia (9 OTUs, ~16%) as the most abundant in terms of absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of OTUs unlinked to the culturable Streptomyces strains previously isolated from the same deposits. 43% of OTUs were shared between the three studied collection points while 34% were exclusive to one deposit indicating that distinct speleothems host their own population despite their nearby localization. This important spatial diversity suggests that prospecting within different moonmilk deposits should result in the isolation of unique and novel Actinobacteria. These speleothems also host a wide range of non-streptomycetes antibiotic-producing genera, and should therefore be subjected to methodologies for isolating rare Actinobacteria.
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Cone karst of all types occurs, and is dissected by deeply incised fluvial canyons. River capture, above and below ground, is a recurring feature in the karst, and is spectacularly developed along the incised Sancha River. Morphometric analysis of the karst cones reveals a remarkable uniformity and only limited geological control. The critical factor in the evolution of the karst is the rate of tectonic uplift. -from Authors
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Caves in the Guadalupe Mountains offer intriguing examples of possible past or present geomicrobiological interactions within features such as corrosion residues, pool fingers, webulites, u-loops, and moonmilk. Scanning electron microscopy, transmission electron microscopy, molecular biology techniques, enrichment cultures, bulk chemistry, and X-ray diffraction techniques have revealed the presence of iron- and manganese-oxidizing bacteria in corrosion residues, which supports the hypothesis that these organisms utilize reduced iron and manganese from the limestone, leaving behind oxidized iron and manganese. Metabolically active populations of bacteria are also found in "punk rock" beneath the corrosion residues. Microscopic examination of pool fingers demonstrates that microorganisms can be inadvertently caught and buried in pool fingers, or can be more active participants in their formation. Enrichment cultures of moonmilk demonstrate the presence of a variety of microorganisms. Humans can have a deleterious impact on microbial communities in Guadalupe caves.
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With its theoretical basis firmly established in molecular evolutionary and population genetics, the comparative DNA and protein sequence analysis plays a central role in reconstructing the evolutionary histories of species and multigene families, estimating rates of molecular evolution, and inferring the nature and extent of selective forces shaping the evolution of genes and genomes. The scope of these investigations has now expanded greatly owing to the development of high-throughput sequencing techniques and novel statistical and computational methods. These methods require easy-to-use computer programs. One such effort has been to produce Molecular Evolutionary Genetics Analysis (MEGA) software, with its focus on facilitating the exploration and analysis of the DNA and protein sequence variation from an evolutionary perspective. Currently in its third major release, MEGA3 contains facilities for automatic and manual sequence alignment, web-based mining of databases, inference of the phylogenetic trees, estimation of evolutionary distances and testing evolutionary hypotheses. This paper provides an overview of the statistical methods, computational tools, and visual exploration modules for data input and the results obtainable in MEGA.
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
Deposits of hydrated iron and manganese oxides are common as stalactites, wall crusts, and as thick layers in sediment in caves at Dubuque, Iowa. The metal oxide deposits are concluded to be at least partly the result of organic (bacterial) rather than inorganic precipitation reactions. -from Author
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