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Applied and Environmental Microbiology, January 1999, p. 198-205, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Changes in Quinone Profiles of Hot Spring
Microbial Mats with a Thermal Gradient
Akira
Hiraishi,1,*
Taichi
Umezawa,1
Hiroyuki
Yamamoto,2
Kenji
Kato,3 and
Yonosuke
Maki4
Department of Ecological Engineering,
Toyohashi University of Technology, Toyohashi
441-8580,1
Department of Microbiology,
St. Marianna University School of Medicine, Kawasaki
216-8511,2
Laboratory of Biology,
School of Allied Medical Sciences, Shinshu University, Matsumoto
390-0802,3 and
Laboratory of
Biology, Faculty of Humanities & Social Sciences, Iwate
University, Morioka 020-8550,4 Japan
Received 6 July 1998/Accepted 20 October 1998
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ABSTRACT |
The respiratory and photosynthetic quinones of microbial mats which
occurred in Japanese sulfide-containing neutral-pH hot springs at
different temperatures were analyzed by spectrochromatography and mass
spectrometry. All of the microbial mats that developed at high
temperatures (temperatures above 68°C) were so-called sulfur-turf
bacterial mats and produced methionaquinones (MTKs) as the major
quinones. A 78°C hot spring sediment had a similar quinone profile.
Chloroflexus-mixed mats occurred at temperatures of 61 to
65°C and contained menaquinone 10 (MK-10) as the major component
together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to
56°C were all cyanobacterial mats, in which the photosynthetic
quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next
most abundant component in most cases. Ubiquinones (UQs) were not found
or were detected in only small amounts in the biomats growing at
temperatures of 50°C and above, whereas the majority of the quinones
of a purple photosynthetic mat growing at 34°C were UQs. A numerical
analysis of the quinone profiles was performed by using the following
three parameters: dissimilarity index (D), microbial
divergence index (MDq), and bioenergetic
divergence index (BDq). A D matrix
tree analysis showed that the hot spring mats consisting of the
sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and
purple phototrophic bacteria formed distinct clusters. Analyses of
MDq and BDq values indicated that the microbial diversity of hot spring mats decreased as
the temperature of the environment increased. The changes in quinone
profiles and physiological types of microbial mats in hot springs with
thermal gradients are discussed from evolutionary viewpoints.
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INTRODUCTION |
Geothermal hot spring streams
provide favorable conditions for the development of microbial mats,
which contain physiologically and phylogenetically different groups of
procaryotes, such as chemotrophic sulfur bacteria, cyanobacteria, and
anoxygenic phototrophic bacteria, depending on the temperature, pH,
sulfide concentration, and some other environmental conditions (for
reviews see references 4, 8-10, and
37). The different types of hot spring microbial mats have been studied extensively as simple and stable systems in
order to understand the ecology and evolution of microbial communities.
The major constituents of hot spring microbial mats are not cultured in
many cases. Therefore, non-culture-dependent approaches are essential
to study the microbial communities of hot spring environments.
One of the most promising culture-independent approaches in this
research area is an rRNA approach which involves selective cloning of
naturally occurring 16S rRNA as cRNA or PCR cloning of environmental
16S rRNA genes (2, 3, 24, 40, 41, 44). In a previous study,
we used the 16S ribosomal DNA (rDNA) clone library method to perform a
community analysis of the so-called sulfur-turf bacterial mats and
found that novel 16S rDNA phylotypes that represent a deeply branching
clade within the Aquifex-Hydrogenobacter complex
(Aquificales) predominated in the sulfur-turf mats
(46).
Another culture-independent approach useful for hot spring community
analysis is a chemical biomarker approach which involves direct
extraction and identification of cell envelope lipid constituents (36). Quinone profiling, which is a chemical method for
detecting various structural types of respiratory and photosynthetic
quinones in microbial cells, not only has been useful in microbial
chemotaxonomy (11, 12) but also has potential for estimating
microbial redox states (16) and community structures
(17-23, 27) in the environment. The fact that
representative species of thermophilic and hyperthermophilic bacteria
contain specific quinone homologs provides a basis for using the
quinone profile method for analyzing the communities of hot spring
bacterial mats. For example, Hydrogenobacter species, which
are the hot spring-inhabiting chemolithotrophs of the earliest branching eubacterial lineage, the Aquificales (5,
39), contain novel sulfur-containing naphthoquinones called
methionaquinones (MTKs) (26, 28, 43).
Chloroflexus species, which are widespread in thermophilic
photosynthetic bacterial mats (8-10, 38), contain menaquinones (MKs) with 10 isoprene units in the side chain as the main
components (13-15). Cyanobacteria are common photosynthetic procaryotes in hot spring microbial mats as well (6-10) and
contain plastoquinones (PQs) and phylloquinone (K1)
(11). Moreover, purple phototrophic bacteria, which
occasionally exhibit massive red growth in hot spring streams (4,
30, 31, 33), contain ubiquinones (UQs) with eight isoprene units
in most cases, and some of these organisms also contain MKs
(25).
In this study, the quinone profile method was used to characterize
biomat communities in sulfide-containing neutral-pH hot springs which
are growing at different temperatures in geographically different areas
of Japan. Relationships between microbial quinone profiles and thermal
gradients of hot springs are discussed below from evolutionary and
ecological viewpoints. To our knowledge, this report is the first
description of the quinone profiles of thermal microbial communities.
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MATERIALS AND METHODS |
Mat samples.
Microbial mats were collected from the
following four hot springs in Japan: Ganiba (39°47'N, 140°48'E) in
Akita Prefecture; Nakanoyu (36°12'N, 137°36'E) in Nagano
Prefecture; Yumata (36°24'N, 137°41'E) in Nagano Prefecture; and
Nikko-Yumoto (36°48'N, 139°25'E) in Tochigi Prefecture. A sediment
sample was also collected from a hot spring in Yufuin (33°15'N,
131°20'E) in Oita Prefecture. For comparison, a sewage mat sample was
collected from a sewage ditch in Toyohashi in Aichi Prefecture.
Detailed information concerning these samples is shown in Table
1. Temperature and pH were measured in
situ with a portable thermometer and pH meter. Surface material was
collected from microbial mats and sediments in sterile polyethylene bottles and was transported in an insulated cooler to the laboratory. Biomass and suspended solids were harvested by centrifugation, washed
once with 50 mM phosphate buffer (pH 7.0), and stored at 
20°C until
they were analyzed.
Microscopic and spectroscopic studies.
Portions of washed
mat samples were appropriately diluted with 50 mM phosphate buffer (pH
7.0) and observed with an Olympus model BX-50 phase-contrast
microscope. Pigments were extracted from washed biomass with an
acetone-methanol mixture (7:2, vol/vol), and absorption spectra were
measured with a Shimadzu model BioSpec-1600 spectrophotometer.
Quinone extraction and fractionation.
Washed samples (ca. 1 to 5 g [wet weight] for mats; 40 g [wet weight] for
sediments) were resuspended in 50 mM phosphate buffer (pH 7.0). Each
suspension was mixed with 2.5 volumes of a chloroform-methanol mixture
(2:1, vol/vol), sonicated for 1 min on ice (20 kHz; output power, 100 W), and centrifuged at 5,000 × g for 10 min. The
resulting upper aqueous layer was discarded, and the lipid layer was
collected with a pipette. The residue was extracted once with acetone
and then twice (30 min each) with the chloroform-methanol mixture. All
extracts were combined, evaporated under a vacuum, and reextracted three times with n-hexane-1% saline (1:1, vol/vol). The
hexane extract was concentrated and then applied to a chromatography column with Sep-Pak Vac cartridges (Waters Corp., Milford, Mass.) to
separate the MK and UQ fractions. Detailed information concerning the
procedures used has been given elsewhere (22, 27).
Analysis of quinones.
Quinone components were separated and
identified by spectrochromatography with a Beckman model 110B liquid
chromatograph or a Shimadzu model LC-10 liquid chromatograph equipped
with a diode array detector. Mass spectrometry was used to confirm the
chemical structures of quinones separated by high-performance liquid
chromatography (HPLC). Details of the analytical methods used have been
described previously (22). For quantification of quinones,
detector response factors were determined on the basis of HPLC data
obtained by using a known concentration of quinones which had been
determined by the reduced-minus-oxidized difference spectrum method
(29). Since the extinction coefficients of MTKs are not
known, MTKs were quantified by assuming that they have the same
extinction coefficients at 263 nm as their analogs, the MKs.
Abbreviations of quinones and standard quinones.
UQs, PQs,
MKs, and MTKs with n isoprene units in their side chains
were abbreviated Q-n, PQ-n, MK-n, and
MTK-n, respectively. Partially hydrogenated MKs and MTKs
were designated MK-n(Hx) and
MTK-n(Hx), where x
indicates the number of hydrogen atoms saturating the side chain.
Standard quinones were prepared from raw sewage and activated sludge as
described previously (22). In addition, MK-10 and
MTK-7(H4) were purified from Chloroflexus aurantiacus J-10-fl and Hydrogenobacter thermophilus
JCM 7687, respectively, and were used as the standard quinones.
Numerical analysis.
Differences in the community structures
of the microbial mats were estimated simply by visually interpreting
quinone profile data. To enhance the objectivity of the information and
to make quantitative estimates of population shifts over time and
space, however, it was necessary to process data by performing an
appropriate numerical cluster analysis (20, 27). Three
parameters, the microbial divergence index
(MDq), the bioenergetic divergence index
(BDq), and the dissimilarity index
(D) (27), were used in the numerical analysis of
quinone profiles. MDq is given by:
where xk 
0.001 and
xk indicates the molar ratio of the content of
the quinone homolog k to the total quinone content (defined as 1). Since MDq values indicate the divergence
of the quinone structural types detected, this parameter indicates the
extent of the diversity of microbial taxa (23, 27).
BDq is given by:
where UQ, (PQ + K1), MK 0.001, and UQ, PQ,
K1, and MK indicate the molar
fractions of UQs, PQs, K1, and MKs (plus their derivatives), respectively, compared to the total quinone content. Therefore, BDq is a reflection of the divergence
of bioenergetic modes of microbes (i.e., the balance of UQ-mediated
aerobic respiration, oxygenic photosynthesis, and anaerobic and/or
MK-mediated aerobic respiration) (27). D is given
by:
where xik, xjk 0.01, 
xjk = xjk = 100,
and xjk and xjk indicate
the levels (expressed as moles percent) of the quinone homolog
k in samples i and j, respectively.
The D values can be used as indicators of differences in
community structure among samples (20). The numerical
analysis was performed with a personal computer program, BioCLUST,
written by one of us (A.H.) for use with an IBM personal computer and
other compatible computers (27). The algorithm of the
neighbor-joining (NJ) method (42) was used to construct a
dendrogram based on D matrix data. A dendrogram was drawn by
using the TreeView program (35).
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RESULTS |
Microscopic and spectroscopic observations.
On the basis of in
situ observations and microscopic and spectroscopic studies, the hot
spring microbial mats from high-temperature environments (temperatures,
>45°C) were classified into the following three major types:
sulfur-turf mats, Chloroflexus-mixed mats, and
cyanobacterial mats. In addition to these types, a purple photosynthetic mat (NIK-3) was found in the Nikko-Yumoto spa
(temperature, 34°C (Table 1 and Fig.
1).
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FIG. 1.
Phase-contrast micrographs showing typical cell
morphologies of the predominant bacteria in hot spring microbial mats.
(A) Sulfur-turf mat (YUM-2). (B) Chloroflexus-mixed mat
(NAK-2). (C) Cyanobacterial mat (NAK-3). (D) Purple photosynthetic mat
(NIK-3). Bars = 10 µm.
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All of the sulfur-turf mats studied contained great numbers of
relatively large sausage-shaped bacteria (Fig.
1A); this morphotype
has
been described previously as the typical morphotype of the
sulfur-turf
bacteria (
32,
46). The cells of some of these
sausage-shaped
bacteria were accompanied by elemental sulfur granules.
In the
Chloroflexus-mixed mats, flexible filamentous bacteria,
possibly
Chloroflexus species, were abundant (Fig.
1B), and
the
sausage-shaped sulfur-turf bacteria were present in smaller
numbers.
The cyanobacterial mats contained large rod-shaped
cyanobacteria
that occurred singly and occasionally in chains (Fig.
1C). In
the purple photosynthetic mat, elemental sulfur-containing
large
cells, possibly
Chromatium cells or cells of related
purple sulfur
bacteria, occurred in large numbers together with much
smaller
rods and cocci (Fig.
1D).
Spectrophotometric measurements showed that all of the
Chloroflexus-mixed mats and cyanobacterial mats studied
contained a
photosynthetic pigment having an absorption maximum at 660 nm
in the acetone-methanol mixture, indicating that bacteriochlorophyll
c and/or chlorophyll
a was present (data not
shown).
HPLC elution patterns.
HPLC experiments revealed that most hot
spring samples contained quinones consisting of both UQ and MK
fractions; the only exception was sediment sample YUF, which contained
no UQs. In the UQ fractions, it was easy to identify each component by
spectrochromatography alone, because all of the samples produced a
simple elution pattern, such as that found in wastewater sludges, which
in general produce three major components, Q-8, Q-9, and Q-10
(17-22). On the other hand, the MK fractions from all of
the hot spring samples produced simple but characteristic HPLC patterns
which have never been found in wastewater environments.
The MK fractions of the microbial mat samples from the high-temperature
environments (temperatures, >45°C) produced three
major HPLC
patterns depending on the type of microbial mats and
the temperature at
which they occurred in situ. Examples of the
three patterns which were
obtained from the Yumata spa samples
(samples YUM-2, YUM-3, and YUM-4)
are shown in Fig.
2. Sample
YUM-2, which
was a typical sulfur-turf mat sample obtained from
a 68°C stream,
produced a major component having an HPLC retention
time of 13.0 min
(Fig.
2A) and absorption maxima at 240, 261 (maximum),
and 311 nm (Fig.
2A, inset). This spectral pattern was the same
as that of
MTK-7(H
4) purified from
H. thermophilus.
However, the
HPLC elution time of the main component of the YUM-2
sample differed
from the HPLC elution time of MTK-7(H
4),
which eluted at 17.9
min (data not shown). This suggested that the main
YUM-2 component
was an MTK homolog that differed from
MTK-7(H
4) in the structure
of the multiprenyl side chain.
MK-7 eluted at 12.2 min as the
second most abundant component in sample
YUM-2. Sample YUM-3 was
from a
Chloroflexus-mixed mat
developing at 65°C and produced
a major MK component that eluted at
27 min in addition to the
MTK component noted above (Fig.
2B). This MK
component was identified
as MK-10 by comparing its HPLC elution time
and UV absorption
spectrum with the HPLC elution time and UV absorption
spectrum
of the MK standards. In sample YUM-4, which was from a
cyanobacterial
mat growing at 50°C, PQ-9 was detected as the
predominant component
together with significant amounts of MK-10 and
K
1 (Fig.
2C).
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FIG. 2.
HPLC elution profiles of the MK fractions of three
microbial mats from the Yumata hot spring stream growing at different
temperatures. The UV absorption spectra of the predominant components
(indicated by arrows) are shown in insets. (A) Components of
sulfur-turf mat growing at 68°C (YUM-2). (B) Components of
Chloroflexus-mixed mat growing at 65°C (YUM-3). (C)
Components of cyanobacterial mat growing at 50°C (YUM-4).
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Mass spectrometry.
The chemical structures of all of the
quinone components detected by HPLC were confirmed by mass
spectroscopy. For example, a mass spectroscopy analysis of the MK
fraction of YUM-2 showed that the major MK component that eluted at
12.2 min had a molecular ion peak (M+1) at m/z 649.6, thereby confirming that it was MK-7. The predominant MTK component that
appeared at 13.0 min had a molecular ion peak (M+1) at m/z
681.6, a value which is 4 mass units lower than that of
MTK-7(H4) (26). Thus, on the basis of the
results of both spectrochromatography and mass spectrometry, the major
MTK component detected in this study was identified tentatively as
fully unsaturated MTK-7. A detailed structural determination analysis
of this MTK component will be described elsewhere. The minor component
that eluted at 17.9 min had a molecular ion peak (M+1) at
m/z 685.6 and was identified as MTK-7(H4).
Quinone compositions.
The quinone contents of all test
samples, as determined by HPLC and mass spectrometry, are summarized in
Table 2. There were marked differences in
quinone patterns, as well as in the total quinone contents, among
the samples examined. MTKs (mostly MTK-7) predominated (>68%) in the
sulfur-turf (GANI, YUM-1, YUM-2, and NAK-1) and sediment (YUF)
samples. In most of the sulfur-turf mats, considerable amounts of MK-7
also occurred, but other quinone homologs were not detected or were
present at only low levels. In the Chloroflexus-mixed mats
(YUM-3 and NAK-2), MK-10 was a major component (37 to 55%)
comparable to MTKs. All of the cyanobacterial mat samples (YUM-4,
NAK-3, NAK-4, NIK-1, and NIK-2) produced the photosynthetic quinones
(PQ-9 and K1) as the primary components (34 to 79%). In
these green mats at temperatures of 50°C, MK-10 also constituted a
significant proportion of the total content (17 to 27%), suggesting
that Chloroflexus spp. were present in the cyanobacterial
mats. In all samples from mats at temperatures of 50°C, UQs
constituted small fractions of the total content (<10%), whereas in
the 34°C purple photosynthetic mat (NIK-2), UQs (with Q-8
predominating) accounted for the majority of the total quinone content.
Numerical analysis.
Differences in quinone profiles among the
mat samples were quantitatively estimated by using D (Table
3). Also, the quinone profiles obtained
were evaluated by using MDq and
BDq, which were considered reflections of the
diversity of the microbial taxa and the diversity of their bioenergetic
modes, respectively. The MDq values for
microbial mats seemed to increase with decreasing in situ temperature,
as the MDq values were 1.57 to 3.59 for the sulfur-turf mats (temperatures of most mates, 68°C; GANI
temperature, 50°C), 3.44 to 6.44 for the
Chloroflexus-mixed mats (61 to 65°C), 4.75 to 8.59 for the
cyanobacterial mats (45 to 55°C), and 7.65 for the purple
photosynthetic mat (34°C). This was also the case for the
BDq values.
On the basis of the
D matrix data shown in Table
3, we
constructed an NJ dendrogram which grouped the hot spring microbial
mats from environments having different temperatures (Fig.
3).
The four physiologically different
types of microbial mats (i.e.,
the sulfur-turf bacterial,
Chloroflexus, cyanobacterial, and purple
phototrophic
bacterial mats) occurred in distinct clusters on
the dendrogram. The
branching order of the clusters was correlated
with temperatures from
which the samples were collected.
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FIG. 3.
NJ dendrogram showing relationships among community
structures of different microbial mats based on D value
matrix data. The ranges of temperatures from which the mat samples were
collected are shown. Sample SEW was used as an outgroup to root the
tree.
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Relationship between quinone profiles and temperature.
To
clarify the relationship between quinone profiles and temperature in
hot spring environments, the contents of the major quinone ring
structural types in the hot spring environments were plotted as a
function of in situ temperature (Fig. 4).
It was clear that MTKs predominated only in high-temperature
environments with temperatures above 65°C, although we found that one
50°C sample (GANI) contained a high proportion of MTKs. The maximum proportion of MKs was found at temperatures around 60°C. PQs and K1 were the most frequent structural types at temperatures
between 50 and 60°C. UQs were abundant only in the environments where the temperature was less than 40°C.
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FIG. 4.
Changes in the proportions of major quinone ring
structural types detected in hot spring mats as a function of
temperature. ×, data for the 50°C sulfur-turf mat sample (sample
GANI).
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DISCUSSION |
We found that quinone profiling of the hot spring bacterial mats
from environments having different temperatures resulted in some
interesting observations. One of our most striking findings is that
MTKs predominated in all of the sulfur-turf bacterial mats studied,
despite the fact that samples were collected from geographically
different areas in Japan. Previously, MTKs have been found to be the
sole quinones in the thermophilic chemolithotrophic bacteria
Hydrogenobacter spp., and MTK-7(H4) is the major
homolog in all of these bacteria (26, 28, 43). On the other
hand, this study showed that all of the major MTK types of the
sulfur-turf bacteria were MTK-7 types.
A previous molecular approach showed that a new 16S rDNA phylotype that
branches deeply from the Aquificales lineage predominates in
sulfur-turf mats (46). A concurrent in situ hybridization assay performed with a 16S rRNA-targeted oligonucleotide probe demonstrated that the sausage-shaped large bacteria that commonly predominate in sulfur-turf mats (32) correspond to this
phylotype (46). Our quinone profile findings strongly
suggest that the sulfur-turf bacteria having the phylotype and
morphotype mentioned above contain MTK-7 as their major quinone
component, and this chemotaxonomic feature is consistent with the
phylogenetic positions of these organisms as members of the
Aquificales. Although there is no information available
concerning the quinone systems of members of the Aquificales
other than Hydrogenobacter spp. and the sulfur-turf
bacteria, it has been suggested that MTKs are essential components of
the respiratory chains in this (hyper-)thermophilic phylogenetic
group. Recently, MTKs have also been found in an aerobic
hyperthermophilic archaeon (34).
Another interesting observation is that the quinone profiles and the
physiological types of microbial mats were dramatically different
depending on the temperature at which the mats developed in situ and
that these differences occurred independent of the geographical
locations of the mats sampled. The predominant structural types of
quinones detected changed with decreasing temperature of the hot
springs, as follows: >68°C, MTK-7; 61 to 65°C, MK-10; 45 to
56°C, PQ-9 plus K1; <40°C, Q-8. These differences in
the main structural types of quinones in response to temperature were a
reflection of the development of physiologically and phylogenetically different bacterial communities; namely, specific detection of MTK-7,
MK-10, PQ-9, and Q-8 as the major quinone types at the different
temperatures indicated that the sulfur-turf bacteria, Chloroflexus spp., thermophilic cyanobacteria, and purple
phototrophic bacteria, respectively, were the predominant organisms. An
exception was the 50°C GANI sample, which contained a high proportion
of MTKs. Since the GANI sampling site was in a thermal stream pipe not
exposed to sunlight, it did not provide favorable conditions for
photosynthetic growth. This explains why the MTK-producing bacteria
predominated at such a low temperature.
MK homologs other than MK-10, such as MK-7, were found to be widespread
in the hot spring mats independent of temperature. Detection of these
quinone homologs may have been due to the presence of phylogenetically
diverse thermophilic chemotrophs. In fact, microscopic studies revealed
that all of the microbial mats studied contained various morphological
types of unknown bacteria in addition to the typical sulfur-turf
bacteria (sausage-shaped bacteria), Chloroflexus spp.,
cyanobacteria, and purple phototrophic bacteria. Also, at this time, we
cannot exclude the possibility that archaeal species, which produce MKs
in general, coexist in hot spring microbial mats.
The numerical analysis based on the D value matrix data
revealed that the microbial mats containing the sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and purple phototrophic
bacteria form distinct clusters. These four physiologically different
types of mats could also be characterized by using
MDq and BDq values. Interestingly, the microbial diversity of hot spring biomats shown by
MDq and BDq values was
related to the temperatures of the environments from which the biomats
were taken. The extent of hot spring microbial diversity with respect
to both species composition and energy-yielding modes of the
inhabitants decreased as the in situ temperature increased.
In conclusion, quinone profiling of hot spring environments is useful
not only for characterization of microbial community structures but
also for evaluation of the energy-yielding modes of the microbes
present, both of which change sharply in response to the temperature of
the environment. The changes occur with decreasing temperature in the
following order: MTK-mediated respiration by members of the
Aquificales
MK-involved anoxygenic photosynthesis by
Chloroflexus spp.PQ-involved photosynthesis by
cyanobacteriaUQ-involved anoxygenic photosynthesis by purple
bacteria. The phylogenetic tree of modern organisms provides
circumstantial evidence that life on Earth arose from
hyperthermophilic ancestors (1, 5, 45) and that
the four phylogenetic groups described above branch more deeply in the
following order:
Aquificales
Chloroflexuscyanobacteriapurple bacteria. Thus, the succession of different quinone-containing bacterial populations found in extant hot spring environments with a
thermal gradient may have some evolutionary implications.
| |
ACKNOWLEDGMENTS |
We thank S. Higuchi, Nagano Research Institute for Health and
Pollution, for his help with sampling and S. Ishii, Department of
Biotechnology, The University of Tokyo, for making his unpublished data
on Hydrogenobacter quinones available to us. We especially thank K. Matsuura, Department of Biology, Tokyo Metropolitan
University, for his interest and stimulating discussions.
This study was supported in part by the Decoding the Earth Evolution
Program, Intensified Study Area Program of the Ministry of Culture,
Science, Sports and Education, Japan (grant 259, 1955-1997).
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Ecological Engineering, Toyohashi University of Technology,
Tenpaku-cho, Toyohashi 441-8580, Japan. Phone: 81-532-44-6913. Fax:
81-532-44-6929. E-mail: hiraishi{at}eco.tut.ac.jp.
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Applied and Environmental Microbiology, January 1999, p. 198-205, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
