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Vol. 18, No. 2, 51–61, 2003
Minireview
Filamentous Anoxygenic Phototrophs in Hot Springs
S ATOSHI H ANADA1*
1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1–1–1 Higashi, Tsukuba, Ibaraki 305–8566, Japan
(Received April 30, 2003—Accepted May 12, 2003)
Microbial mats often seen in neutral and alkaline hot springs are generally formed by phototrophic bacteria. These mat-forming thermophiles comprise oxygenic phototrophs, namely cyanobacteria, and the other group of phototrophic bacteria, so-called filamentous anoxygenic phototrophs (FAPs). FAPs contain bacteriochloro-phyll(s) as photosynthetic pigments and conduct photosynthesis without producing oxygen. They are all affiliat-ed to the order Chloroflexales in the phylum Chloroflexi, and are classified into three genera, Chloroflexus, Heliothrix and Roseiflexus. Chloroflexus species have greenish brown to brown filaments and a typical light-harvesting apparatus called the chlorosome, while the others are red to reddish orange and completely lack chlorosomes.FAPs form colorful and variously shaped microbial mats with cyanobacteria.Distinctive microbial mats composed of these bacteria, e.g. complexly layered or ruffled fur-like mats, can be seen in some Japanese hot springs. While FAPs phylogenetically differ from all other phototrophs, the group is known to have a “chimeric” photosynthetic system: The photochemical core complex in the FAPs, essential to photosynthesis, resembles that in the purple bacteria; their light-harvesting apparatus is similar to that in green sulfur bacteria. The photosynthetic peculiarity found in the FAPs is important to solving the evolution of photosynthesis.
Key words: filamentous anoxygenic phototroph, Chloroflexi, hot spring, microbial mat, photosynthsis
In neutral and alkaline hot springs at 40 to 70°C, one can often see “microbial mats” or “biomats” that structurally resemble a sponge in tidal pools or a rug on the living room floor.These microbial mats are generally green,brown, orange and red in color. Such colored microbial mats are formed by several kinds of photosynthetic bacteria in general. Important members of the microbial mat-forming phototrophs are thermophilic species categorized as cyanobacteria6). They contain chlorophyll (Chl) a and phycobilin as photosynthetic (and light-harvesting) pig-ments,a nd l ook d ark g reen.C yanobacteria are principally o xy-genic phototrophic organisms that perform photosynthesis with oxygen evolution, like higher plants and algae. These phototrophic bacteria can grow autotrophically with water and carbon dioxide as the sole electron donor and carbon source, respectively. Cyanobacteria often play a leading role in neutral and alkaline hot springs as primary producers and supply organic substrates and vitamins to neighboring heterotrophic organisms.
In addition to thermophilic cyanobacteria, there is anoth-er type of phototrophic bacteria in hot springs. Organisms of this type grow by photosynthesis without producing oxygen, have a multicellular filamentous morphology and contain bacteriochlorophyll(s) (BChls) as photosynthetic pigments. Thus, they are called filamentous anoxygenic phototrophs (FAPs). The microbial mats formed mainly by FAPs vary in morphology and color, depending on tempera-ture, pH and sulfide concentration8,11,33).
FAP mats appear in hot spring effluents as orange-brown or reddish colored layers within dark green cyanobacterial mats. FAPs occa-
*Corresponding author; E-mail: jp, Tel: +81–29–861–6591, Fax: +81–29–861–6587
Abbreviations: FAP, filamentous anoxygenic phototroph; BChl, bacteriochlorophyll; Chl, chlorophyll.
H ANADA 52
sionally form yellow-orange streamers on the surface of cyanobacterial mats.
General characteristics
FAPs found in hot springs are taxonomically classified into three genera, i.e., the genera Chloroflexus42), Heliothrix45) and Roseiflexus28). The differential phenotypic characteristics of these organisms are summarized in Table 1. All strains belonging to these genera have morphological-ly unbranched multicellular filaments with a diameter of 0.7 to 1.5 m m. A distinctive motility, called gliding, is found in all strains. This motility is a smooth movement on a solid or semisolid surface without flagella-like propulsive organs7). All strains are thermophilic and grow best at 50 to 55°C. Good growth occurs under photoheterotrophic conditions. Although FAPs of the three genera share a numbe
r of com-mon phenotypic features, pigmentary traits clearly differen-tiate the genus Chloroflexus from the others. While all these organisms contain BChl a as an essential photosynthetic pigment, another type of chlorophyll, BChl c, is also found in the genus Chloroflexus. A large amount of BChl c is densely packed into an intracellular organelle, named the chlorosome, which functions as a light-harvesting apparatus in Chloroflexus cells51). The other genera of FAPs, i.e., Heli-othrix and Roseiflexus, however, have neither chlorosome nor BChl c. These chlorosome (and BChl c)-less FAPs also differ from Chloroflexus species in carotenoid composition. While Chloroflexus species contain b-carotene as a main component of carotenoids, this type of carotene is not found in strains of the genera Heliothrix and Roseiflexus23,60,61). The chlorosome-less FAPs have g-carotene derivatives (keto-3’,4’-didehydro-OH-g-carotene;myxocoxanthin deriv-atives) as the major carotenoids60). The difference in pigmentary profiles affects their coloration in natural habitats: microbial mats mainly formed by Chloroflexus species are greenish brown to yellowish brown; those com-posed of the chlorosome-less FAPs are reddish orange to red. Further respective characteristics and habitats of these three genera are described below.
Table1.Differential phenotypic characteristics of the thermophilic filamentous anoxygenic phototrophs (FAPs). Genus Chloroflexus Heliothrix Roseiflexus Species    C. aurantiacus    C. aggregans H. oregonensis R. castenholzii
Type strain (accession no.)J-10-fl
(ATCC 29366, DSM 635)MD-66 (DSM 9485)IS/F-1 [co-culuture with
Isophaera pallida]
HLO8 (DSM 13941, JCM
11240, NBRC 100045)
Morphology Multicelular filaments Multicelular filaments Multicelular filaments Multicelular filaments Cell diameter (m m)0.7–1.2  1.0–1.5  1.50.8–1.0 Sheath± (occ. thinly sheathed)---Optimal growth tempera-
ture (°C) [temperature
range for the growth]
5555[40–55]50
Metabolism:
Photoheterotroph++++ Photoautotroph± (in some strains)-ND-
O2 respiration++ND+ Chlorosomes++--Bacteriochlorophyll(s)a and c a and c a a Remarkable peaks (nm) in
in vivo absorption spectra
of IR region
740, 808, 868740, 808, 868795, 865801, 878
Major carotenoids g-carotene, b-carotene,
OH-g-caroteneglucoside
ester g-carotene, b-carotene,
OH-g-caroteneglucoside
ester
keto-OH-g-carotene,
keto-myxocoxanthin,
myxobactene
methoxy-keto-
myxocoxanthin, keto-
myxocoxanthin glycoside
ester
Major cellular fatty acids C18:0, C16:0, C18:1ND ND C16:0, C14:0, C15:0 Major quinone MK-10MK-10 (and MK-4)ND MK-11
DNA G+C content (mol %)53.1–54.9 (Bd),
56.9–57.1 (HPLC)
56.7–570 (HPLC)ND62.0 (HPLC) Symbols: +, positive; -, negative; ND, not determined. Data were taken from the following reference numbers: 23, 24, 28, 42, 44, 45, 60, and 61.
Filamentous Anoxygenic Phototrophs53
Genus Chloroflexus
The genus Chloroflexus was proposed by Pearson and Castenholtz in 197442) with Chloroflexus aurantiacus as the type species. This is the first described genus of FAPs. C. aurantiacus is a thermophilic filamentous bacterium with a diameter of approximately 1 m m. The organism contains BChls a and c as photosynthetic pigments. The latter pig-ment is present in intracellular vesicles, chlorosomes, and shows a conspicuous in vivo absorption maximum at 740 nm in the near infrared (IR) region. The organism also has absorption peaks at 808 and 868 nm in the near IR region due to the presence of BChl a. It contains b- and g-carotenes and OH-g-carotene glucoside esters as major carotenoid pigments. C. aurantiacus can grow photoheterotrophically in light and chemoheterotrophically in darkness. Some strains, e.g., strain OK-70-fl, are able to grow photo-autotrophically with sulfide as a electron donor and carbon dioxide as a sole carbon source30). The main respiratory and photosynthetic quinone is menaquinone with ten isoprene units (MK-10), and the major cellular fatty acids are palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1)22,25,31,35).
buchi
Another species belonging to the genus, C. aggregans, was proposed in 199524). This organism was isolated from Japanese hot springs (Okukinu Meotobuchi hot spring, Na-gano Pref. and Yufuin hot spring, Oita Pref.). C. aggregans is phenotypically similar to the type species, C. aurantiacus. However, the former differs from the latter in exhibiting a rapid formation of bacterial mat-like dense cell aggregates in an axenic culture27). Marked differences are also found between the two species in cell morphology and carotenoid and quinone compositions. In addition to these phenotypic differences, the sequence similarity of the 16S rRNA gene between the two is low enough (approx. 93%) to warrant classification into different species.
Almost all strains of the genus Chloroflexus are thermo-philic which grow best at around 55°C and inhabit hot springs all over the world. In neutral and alkaline hot springs, the organisms generally form yellow-orange-green-ish microbial mats with cyanobacteria. In North America, Chloroflexus populations are found at a temperature of up to 70 to 72°C; the lower temperature limits are 30 to 40°C. Al-so, they occur in a pH range of 6 to 1043). The temperature and pH ranges in which Chloroflexus species can be detect-ed are the same in other geographic regions, such as Japan, Italy and Iceland. In Japanese hot springs, Chloroflexus strains were isolated at temperatures of 50 to 70°C and a pH of 6.4 to 8.225). In an extensive survey with quinones as markers, menaquinone-10 (typical
of Chloroflexus species) was dominant at between 61 and 65°C in Japanese sulfide-containing neutral-pH hot springs, and was even found in a microbial mat at 78°C (Yufuin hot spring, Oita)29). Pentecost41) reported that, in Italian hot springs, Chloro-flexus strains were mainly observed at 40 to 60°C and were successfully isolated from 39.5 to 63.4°C. In Icelandic sulfide-rich hot springs at 55 to 66°C and pH 7.5 to 8.5, Chloroflexus microbial mats were well developed33).
In natural hot springs, Chloroflexus species generally grow photoheterotrophically or chemoheterotrophically us-ing organic substrates excreted by co-existing cyanobacte-ria. However, there are reports of autotrophic Chloroflexus mats growing independent of the presence of cyanobacteria.
A dark green microbial mat of Chloroflexus without cyano-bacteria was found in Yellowstone National Park9). The hot water contains a large amount of sulfide (up to 1000 m M), and this Chloroflexus mat would grow photoautotrophically using dissolved sulfide as an electron donor. The Chloro-flexus mat showed sulfide-dependent photoautotrophic growth but not respiratory growth in the presence of oxygen17). A similar autotrophic Chloroflexus mat devoid of cyanobacteria was found in Nakabusa hot spring, Nagano Prefecture, Japan59). Indeed, some authentic strains of C. aurantiac us showed significant photoautotrophic growth30,32).
To date, no Chloroflexus strains have been detected in acidic or brackish-saline hot springs. It has also been report-ed that the organism are absent in Japanese hot springs with a high iron content25). In North America, however, a Chloroflexus strain was found in an iron-depositing hot spring containing ferrous iron at a concentration of more than 100 m M (Chocolate Pots, Yellowstone National Park). The Chloroflexus strain formed a microbial mat with a cyanobacterium, Synechococcus sp., in a hot water stream at 49 to 54°C47). Pentecost41) also detected Chloroflexus strains in Italian hot springs with travertine deposits. Genus Heliothrix
The first chlorosome-less FAP was discovered and named Heliothrix oregonensis by Pierson et al.44,45). Since H. oregonensis strain F-1 was isolated as a co-culture with a non-phototrophic aerobic bacterium, Isosphaera pallida, which belongs to the phylum Planctmycetes18), no axenic (pure) culture of this phototrophic organism has been ob-tained. However, the co-culture was stable enough to inves-tigate the physiological and phylogenetic features of H. oregonensis. It grow best at 40 to 55°C, suggesting that H.
H ANADA 54
oregonensis is thermophilic. Microautoradiography using a labeled carbon source demonstrated that p
hotoheterotrophy was the preferred mode of growth. The phylogenetic analy-ses based on 5S rRNA45) and 16S rRNA gene63) sequences revealed that the organism is related to Chloroflexus species and should be classified into the phylum Chloroflexi. H. oregonensis has morphologically multicellular filaments with a diameter of 1.5 m m. A septum (a cross-wall between individual cells) is clearly visible under a phase-contrast microscope. Cells often contain a large amount of poly-hydroxybutyrate inclusions. The photosynthetic apparatus has conspicuous in vivo absorption maxima at 795 and 865 nm in the IR region due to the presence of BChl a. In natural hot springs, H. oregonensis forms bright orange microbial mats on dark green cyanobacterial mats. Where the mat is exposed to saturated oxygen, its surface has a puffy and tufty structure9). The organism would be unable to form its own microbial mat completely independent of cyanobacteria, since organic compounds excreted by cyanobacteria appear to be necessary for its growth. H. oregonensis is remarkably predominant in thermal alkaline pools at Warm Springs Indian Reservation (Oregon, USA). Microbial mats formed by similar organisms are commonly observed in several hot springs in Yellowstone National Park (Western North America) at 55°C and below and pH 8.5, without hydrogen sulfide44,45).
Genus Roseiflexus
Recently, a novel chlorosome-less FAP was described with the name Roseiflexus c astenholzii28). Although this organism is quite similar to H. oregonensis in lacking chlorosomes and having BChl a as the sole photosynthetic pigment, phylogenetic and phenotypic comparisons have revealed that it is not closely related to H. oregonensis. R. castenholzii has filamentous cells with a diameter of 0.8 to 1.0 m m and grows at 45 to 55°C and pH 7 to 9. The organ-ism is able to grow photoheterotrophically in light without oxygen and also by respiration in darkness at a full atmo-spheric oxygen tension. Photoautotrophic growth is not ob-served under any conditions. These metabolic profiles sug-gest that, in its natural habitats, the organism grows photo-and/or chemo-heterotrophically using organic substrates supplied by neighboring autotrophs such as cyanobacteria. The cell suspension shows absorption maxima at 801 and 878 nm in the IR region, because of the presence of BChl a. The photosynthetic pigment is produced under both aero-bic-dark and anaerobic-light conditions, while it has been reported that oxygen suppresses BChl synthesis in Chloro-flexus species52–54). The cellular fatty acids consist of C16:0, C14:0, and C15:0. No unsaturated fatty acids are detected, un-like the case of Chloroflexus species, which contain a sig-nificant amount of unsaturated fatty acid, C18:1. The respira-tory and photosynthetic quinone is MK-11, which is one isoprene unit longer than that found in Chloroflexus species. R. castenholzii strain HLO8T was isolated from Nakabusa hot spring, Nagano Pref., Japan28). In this hot spring, the or-ganism forms a dense red mat underneath the micro
bial mats of cyanobacteria and Chloroflexus species. The Rosei-flexus mat is found at 45.5 to 68.5°C and pH 7.8 to 8.259). Similar red microbial mats probably formed by R. casten-holzii have been observed in other Japanese hot springs, such as Meotobuchi, Tochigi Prefecture (57°C, pH 6.8) and Atagawa, Shizuoka Prefecture (44°C, pH 8.0). Roseiflexus-like microbial mats have also been found in alkaline hot springs of Yellowstone National Park; the temperature and pH ranges of the hot springs were 30 to 50°C and 7.5 to 8.7, respectively2,3). A culture-independent molecular technique based on 16S rRNA gene sequences revealed that the Yel-lowstone filaments were closely related to, but different in part from, R. castenholzii. There were also clear differences between the Yellowstone filaments and R. c astenholzii in morphological and spectroscopic characteristics. These dif-ferences suggest that the Yellowstone filaments reported by Boomer et al. should be classified into a distinctive species related to R. castenholzii. Using a similar culture-indepen-dent molecular technique, Nübel et al.39) retrieved a number of R. castenholzii-related 16S rDNA clones from the micro-bial mats in Mushroom spring, Yellowstone National Park (at 60 and 70°C, pH 8.3). Almost all uncultured clones obtained from Mushroom spring were closely related to R. c astenholzii and Boomer’s filaments. However, the sequence similarities between these uncultured clones and R. castenholzii were not so high (less than 94%), suggesting the filaments in Mushroom spring to be a new species of the genus Roseiflexus.
FAP mats in natural hot springs
In general, the thermophilic FAPs form a microbial mat with photoautotrophic organisms, cyanobacteria, occasion-ally developing without cyanobacteria. The microbial mats vary in morphology and color, depending on the tempera-ture, pH and sulfide concentration of the hot water. Several on-site studies have been made on the pattern of mat-formation and behavior of FAPs in hot springs worldwide3,8,11,33,46).
Since there are a wide variety of microbial mats in Japan,
Filamentous Anoxygenic Phototrophs 55
I show here typical but appealing microbial mats found in two Japanese hot springs where Chloroflexus aggregans  or Roseiflexus castenholzii  were isolated.
1) Okukinu Meitobuchi hot spring, Tochigi Prefecture, Japan
In Okukinu Meotobuchi hot spring, a distinctive dark green mat covered with yellowish streamers has been ob-served. The microbial mat has developed in hot spring water running along a drain made of bamboo (Fig. 1A). The microbial mat collected from the drain (57.1 to 57.4°C,pH 7.0) is dark green and
dense, the top surface covered with yellow-orange filaments (Fig. 1B).
Microscopic observations (Fig. 1C) revealed that the dark green basal part was predominantly composed of filamen-tous and oval-shaped unicellular cyanobacteria with a cell diameter of 3 to 4 m m. The filamentous and oval-shaped organisms would be classified into the Form-genera Geitlerinema  and Synechococcus , respectively, based solely on their morphology and growth temperature. The basal part also contained finer filaments of Chloroflexus  species.The yellowish streamers are dominantly occupied by Chloroflexus  species. Two kinds of filaments of different thickness (less than 1 m m or 1.5 m m) were present in the
streamers, suggesting that at least two species of the genus Chloroflexus  reside there. C. aggregans  MD-66T  was isolated from the same microbial mat sample 24). The thicker filament  would  be  C. aggregans , and  the  thinner  one  appears to be C. aurantiacus , the type species of the genus. These Chloroflexus  species were closely associated with the cyanobacteria in this hot spring, and appeared to grow by photoheterotrophy being dependent on cyanobacterial products as carbon substrates.
2) Nakabusa hot spring, Nagano Prefecture, Japan
In Nakabusa hot spring, thick and dense microbial mats are formed mainly by three kinds of phototrophic , cyanobacteria, Chloroflexus  species and Roseiflexus castenholzii , with non-phototrophic thermophilic bacteria (such as organisms that belong to the genus Thermus , and some  sulfur-oxidizing  bacteria)37,59). In fact, the chlorosome-less filamentous anoxygenic phototroph, Roseiflexus castenholzii  strain HLO8T  was first isolated from a micro-bial mat in this hot spring 28).
Figure 2A is a shot of Nakabusa hot spring showing a sand-trap dam filled with sand and gravel. There is a con-crete wall with a height of about 4 meters standing on a riv-er. A hot spring is beyond this wall, and hot water falls on the concrete. The temperature and pH of the hot water were 40 to 58°C and 7.8 to 8.0, respectively. Microbial mats were well developed on the wall, the surface of which was cov-ered with several groups of bacteria (Fig. 2B). The dark green area was formed mainly by species of cyanobacteria,the brown part was due to a predominance of Chloroflexus species, and the white filamentous streamers appeared to be a non-photosynthetic sulfide-oxidizing bacterium. To the eye, there is no red microbial mat typical of R. castenholzii in this hot spring. The fact was, however, that a distinct red mat existed underneath the dark green and brown microbial mats.
A cross section and structural model of the Nakabusa microbial mat is shown in Fig. 3. Three phototrophs were present in layers within the microbial mat. The red layer predominantly comprised of R. castenholzii  adhered to the concrete wall under the brown and dark green layers of Chloroflexus  species and cyanobacteria.
A question arising here is whether the phototrophic growth of R. castenholzii  can be supported even under such thick layers of other phototrophic bacteria. Spectroscopic analysis of the three layers in the microbial mats implied a peaceful co-existence of these phototrophs for the following reasons.
The in vivo  absorption spectrum of each layer is shown in
Fig.1.Microbial mats in Okukinu Meotobuchi hot spring, Tochigi
Pref., Japan. (A) microbial mats grown in hot water flowing along a drain made of bamboo (at 57.1 to 57.4°C, pH 7.0). (B) The col-lected microbial mats formed mainly by thermophilic cyanobac-teria and Chloroflexus  species. (C) A micrograph of the mat sam-ple showing several kinds of filaments: Green thick filaments are cyanobacteria; thin filaments would be Chloroflexus aurantiacus (less than 1 m m) and C. aggregans  (approx. 1.5 m
m).
H ANADA
56Fig. 4. The cyanobacterial layer had a distinctive absorption peak at 670 nm derived from Chl a . The brown layer of Chloroflexus  species showed a large peak of BChl c (chlorosomes) at 740 nm and
small peaks of BChl a  at 800and 868 nm. The R. c astenholzii  population possessed no absorption peaks due to Chl a  and BChl c . But it had distinctive peaks at 801 and 878 nm due to the presence of BChl a . The absorption ranges of the three phototrophs are,therefore, completely different from each other. Since near-infrared light passes through cyanobacterial mats (even with a thickness of 5 mm, the transmitted radiance is more than 1% of the incident light)33), R. c astenholzii  could grow phototrophically underneath the thick cyanobacterial layer.These spectroscopic findings strongly suggest that light can be equally shared out among the three kinds of phototrophs in the microbial mats without competition in terms of the wavelengths to be used for photosynthesis. It can be said
that they do not compete for the light, and live together in harmony.
In Nakabusa hot spring, another type of microbial mat, a sulfur-turf microbial mat, is also frequently observed. Sul-fur-turf microbial mats (covered with massive filamentous streamers) are mainly composed of a large sausage-shaped bacterium that would be a sulfur- and/or hydrogen-oxidiz-ing autotrophic bacterium 65). Sugiura et al.59) found a Chloroflexus  strain in these mats as well. This strain was growing in a high temperature zone (71 to 77°C) beyond the tolerance of any cyanobacterium. The strain might grow by sulfide- and/or hydrogen-dependent photoautotrophy,
Fig.2.Microbial mats in Nakabusa hot spring, Nagano Pref., Japan. (A) A shot of the sand-trap dam covered with microbial mats taken on May
14, 1999. The temperature and pH of the area where microbial mats developed were 40 to 58°C and 7.8 to 8.0, respectively. (B) Thick and dense microbial mats on the concrete wall mainly composed of several phototrophic bacteria. The arrow indicates a part where the surface is ripped. Reddish mats of Roseiflex us castenholzii  were visible from under green and brown mats formed mainly by cyanobacteria and Chloroflexus
species.
Fig.3.  A cross section (inset) and structural model of Nakabusa
microbial mats. Three phototrophic bacteria formed layered
and complex microbial mats with a maximal thickness of 10 mm.
Fig.4.In vivo  absorption spectra of three phototrophic layers,
cyanobacteria (green line), Chloroflexus  species (orange), and Roseiflexus castenholzii  (red) in Naka
busa microbial mats. Each layer of the mats was ultrasonically disrupted in buffer (100 mM KCl, 3 mM KH 2PO 4, and 2 mM K 2HPO 4; pH 7.0), and then
measured with a spectrophotometer.