Short Communication Isolation and Characterization of Phototrophic

Microbes Environ. Vol. 22, No. 4, 405–411, 2007 http://wwwsoc.nii.ac.jp/jsme2/ Short Communication

Isolation and Characterization of Phototrophic Purple Nonsulfur Bacteria from Chloroflexus and Cyanobacterial Mats in Hot Springs

TAKAYOSHI HISADA1,2, KEIKO OKAMURA1, and AKIRA HIRAISHI1*

1 Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi 441–8580, Japan 2 TechnoSuruga Co, Ltd., 330 Nagasaki, Shimizu-ku, Shizuoka 424–0065, Japan

(Received April 1, 2007—Accepted July 1, 2007)

Chloroflexus and cyanobacterial mats developing at 50–65°C in Nakanoyu and Nakabusa hot springs, Japan, were collected and cultivated phototrophically at 50 and 37°C. Chloroflexus organisms grew when incubated at 50°C, whereas the incubation at 37°C resulted in the enrichment of pink- to brown-colored cultures, from which different species of phototrophic purple nonsulfur (PPNS) bacteria were isolated. The majority of the isolates grew at temperatures of up to 45°C and optimally at around 40°C. The high frequencies of the occurrence of PPNS bacteria in hot spring microbial mats suggest that these environments offer a favorable ecological niche for these bacteria.

Key words: hot springs, purple nonsulfur bacteria, phototrophic bacteria, microbial mats

Among anoxygenic phototrophic bacteria, thermophily is which were isolated from soil and water samples collected found in species of limited genera, such as Chloroflexus, at the edge of the source pool at Thermopolis Hot Springs Heliobacterium, Roseiflexus, and Thermochromatium, the (Wyoming, USA), respectively. Also, thermotolerant strains main habitatts of which are sulfide-containing geothermal of aerobic anoxygenic phototrophic bacteria assigned to hot springs2,6,18). No thermophilic species have been Porphyrobacter species have been isolated from hot spring reported among phototrophic purple nonsulfur (PPNS) environments8,24). Moreover, both thermotolerant and meso- bacteria16). However, thermotolerant or mildly thermophilic philic strains of PPNS bacteria and aerobic phototrophic PPNS bacteria, which are defined to have an optimum bacteria have been found in hot springs in Russia5,21,34). Dur- growth temperature of around 40°C18), have been isolated ing the course of our research on the biodiversity of pho- from hot spring environments. A bacteriochlorophyll totrophic bacteria in hot springs in Japan, several strains of (BChl) b-containing bacterium, Blastochloris sp. strain GI, PPNS bacteria were successfully isolated from Chloroflexus was isolated from a hot spring microbial mat in the USA25). and cyanobacterial mats. In this study, we report the phylo- This bacterium grows at temperatures of up to 47°C and genetic diversity and growth temperature characteristics of optimally at around 42°C. Other thermotolerant strains of these novel isolates. Ecophysiological aspects of these PPNS bacteria described to date are “Rhodospirillum cente- PPNS bacteria are also discussed. num” 3,28), now classified as Rhodocista centenaria, and Chloroflexus and cyanobacterial mats were collected “Rhodopseudomonas cryptolactis” strain DSM 998729), from two hot springs, Nakanoyu (36°12'N, 137°36'E) and Nakabusa (36°24'N, 137°45'E), Nagano Prefecture, Japan in * Corresponding author. E-mail address: [email protected]; August 1999 and August 2000, respectively. The Nakanoyu Tel.: +81–532–44–6913; Fax: +81–532–44–6929. and Nakbusa hot springs as habitats of phototrophic bacteria 406 HISADA et al.

Table 1. List of the isolates of PPNS bacteria from hot-spring microbial mats and their growth temperature characteristics

° a Phylogenetic Location and characteristics of microbial mats Growth temperature ( C) affiliation and isolate Site Temp (°C) pH Type of mats Color Optimum Maximum Blastochloris sp. TUT3225 Nakanoyu 60 8.1 Chloroflexus Orange 40 45 Phaeospirillum sp. TUT3101 Nakabusa 60 8.1 Chloroflexus Brown 38 42 Rhodomicrobium sp. TUT3402 Nakanoyu 50 7.5 Cyanobacteria Dark-green 37 41 TUT3403 Nakanoyu 50 7.5 Cyanobacteria Dark-green 37 41 Rhodoplanes sp. TUT3521 Nakanoyu 65 8.3 Chloroflexus Orange-brown 40 45 TUT3522 Nakanoyu 65 8.3 Chloroflexus Orange-brown 40 45 TUT3523 Nakanoyu 65 8.3 Chloroflexus Orange-brown 40 45 TUT3524 Nakanoyu 65 8.3 Chloroflexus Orange-brown 40 45 TUT3525 Nakanoyu 65 8.3 Chloroflexus Orange-brown 40 45 TUT3526 Nakanoyu 65 8.3 Chloroflexus Orange-brown 40 45 TUT3527 Nakanoyu 65 8.3 Chloroflexus Orange-brown 40 45 Rhodopseudomonas spp. TUT3620 Nakanoyu 61 7.6 Chloroflexus Orange 37 42 TUT3621 Nakanoyu 61 7.6 Chloroflexus Orange 37 42 TUT3622 Nakanoyu 61 7.6 Chloroflexus Orange 40 42 TUT3623 Nakanoyu 58 7.9 Chloroflexus Orange 40 42 TUT3624 Nakabusa 58 7.9 Chloroflexus Orange 37 42 TUT3625 Nakabusa 58 7.9 Chloroflexus Orange 37 42 TUT3626 Nakabusa 58 7.9 Chloroflexus Orange 40 42 TUT3627 Nakanoyu 54 7.5 Cyanobacteria Dark-green 40 42 TUT3628 Nakanoyu 54 7.5 Cyanobacteria Dark-green 40 42 Rubrivivax sp. TUT3903 Nakabusa 58 8.1 Chloroflexus Orange 40 45 TUT3904 Nakabusa 58 8.1 Chloroflexus Orange 40 45 TUT3905 Nakabusa 58 8.1 Chloroflexus Orange 40 45 TUT3906 Nakabusa 55 8.0 Cyanobacteria Dark-green 40 45 TUT3907 Nakabusa 55 8.0 Cyanobacteria Dark-green 40 45 a The culture media used for growth tests were MGYS for the Blastochloris strain, MYS for the Pheospirillum, Rhodomicrobium, and Rubrivivax isolates, MSPYS for the Rhodopseudomonas isolates, and PYSV for the Rhodoplanes isolates.

as well as of (hyper-)thermophilic chemotrophic bacteria medium22). Then, the tubes were completely filled with the have been well studied14,19,20,30,33). The microbial mats we same medium and incubated in a water bath at 37 and 50°C sampled were developing in hot spring streams at 50–65°C. under incandescent illumination (ca. 8 W m−2). After 1 to 4 Detailed information about the characteristics of the micro- weeks of incubation, most of the cultures incubated at 37°C bial mats collected is shown in Table 1. Microbial mat and turned pink to brown. Microscopic observations confirmed hot water samples from Nakanoyu (6 samples) and Naka- that PPNS bacteria-like cells (i.e., non-filamentous rod-type busa (4 samples) were taken in polypropylene tubes, kept and oval cells) predominated in these cultures. On the other at ambient temperature during transportation, and tested hand, incubation of the test tubes at 50°C resulted in the immediately upon return to the laboratory. Small portions of enrichment of Chloroflexus organisms exclusively, which microbial mat samples were inoculated into 20-ml screw- were characterized by orange to green-colored massive capped tubes containing 10 ml of PE medium7) or SAYS growth and flexible, filamentous cell morphology. How- Phototrophs from Hot Springs 407 ever, when these Chloroflexus enrichment cultures were ATCC 17011T, and Rhodopseudomonas palustris strain transferred into fresh medium and incubated at 37°C after ATCC 17001T, respectively. On the other hand, all isolates subculturing for 1 year at 50°C, pink-colored cultures of Blastochloris, Phaeospirillum, and Rhodoplanes and the appeared unexpectedly. The overgrowth of PPNS bacteria other 7 isolates of Rhodopseudomonas showed lower levels in these pink cultures was also demonstrated under a phase- of similarity (<99.0%) to any previously established species contrast microscope. There were no marked differences in of these genera. All of the Rhodoplanes strains, isolated the efficiency for enrichment of PPNS bacteria between PE from the Chloroflexus enrichment culture, had identical and SAYS media. Consequently, all of the 10 mat samples sequences with “Rps. cryptolactis” strain DSM 9987. It is tested yielded PPNS bacteria. clear that “Rhodopseudomonas cryptolactis” is a misclassi- The pink and brown cultures enriched from the mat sam- fied species and should be reclassified as a distinct species ples were subjected to standard purification by the agar- of the genus Rhodoplanes. Also, the Blastochloris, shake dilution method and by streaking of agar plates using Phaeospirillum, and Rhodopseudomonas isolates noted the AnaeroPack system (Mitsubishi Gas Chemicals, Niigata, above may represent new species of respective genera, and Japan). As a result, 19 strains of PPNS bacteria were iso- require further study for official taxonomic proposals. lated directly from the microbial mat samples and 6 from Phototrophic growth of the PPNS isolates at different the Chloroflexus enrichment cultures maintained at 50°C. temperatures was studied using MYS10,12) and PYS13) media The new PPNS isolates were phylogenetically analyzed (pH 6.8), which contained 20 mM malate and pyruvate (fil- by 16S rRNA gene sequencing. 16S rRNA genes from the ter sterilized) as the carbon source, respectively. For this cell lysates prepared from the isolates were amplified by testing, authentic PPNS strains of our own collection and PCR with bacterial consensus universal primers32) as from the American Type Culture Collection (ATCC), described previously9). PCR products were purified by the Manassas, Virginia, USA, and the Deutsche Sammlung von polyethylene-glycol-precipitation method11), sequenced Mikroorganismen und Zellkulturen GmbH (DSM), Braun- directly with a Dye Terminator Cycle Sequencing kit, and schweig, Germany, were used for comparison. For growth analyzed using an ABI PRISM 3100 DNA sequencer of “Rps. cryptolactis” strain DSM 9987 and the new (Applied Biosystems, Foster City, USA). Sequence data Rhodoplanes isolates, PYS medium supplemented with 20 (1,302–1,485 bp) were compiled with the GENETYX-MAC µg of vitamin B12, 10 µg of nicotinic acid, 3 µg of p-ami- program ver. 13 (GENETYX Corporation, Tokyo, Japan) nobenzoic acid, and 0.5 g of Na2S2O3·5H2O (per liter) (des- and compared to those available from the DDBJ/EMBL/ ignated PYSV medium) was used. For the new GenBank nucleotide sequence database using the BLAST Rhodopseudomonas strains, MYS medium supplemented search system1). The multiple alignment of sequences, cal- with succinate and pyruvate (0.1% each) as the carbon culation of the corrected evolutionary distance17), and con- sources (MSPYS medium) was employed. MYS medium struction of a neighbor-joining phylogenetic tree26) were supplemented with 10 mM glucose and 0.5 mM sulfide performed using the CLUSTAL W program ver. 1.8331). (MGYS medium) was used for growing a Blastochloris The topology of the tree was evaluated by bootstrapping strain. The cultivation was performed anaerobically at 25– with 1,000 resamplings4). The 16S rRNA gene sequences 50°C under incandescent illumination (8 W m−2). Growth determined in this study have been deposited under DDBJ was monitored by measuring the optical density at 660 nm accession numbers AB087718 for “Rhodopseudomonas (OD660) with an Amersham-Pharmacia Novaspec Plus spec- cryptolactis” strain DSM 9987 and AB250613 to AB250625 trophotometer. In this study, thermotolerant or mildly ther- for other strains. mophilic strains were defined as being able to grow at 45°C As the result of sequence comparisons and a phylogenetic and above and optimally at around 40°C18). analysis, the PPNS isolates were affiliated with the genera The growth-temperature characteristics of the PPNS iso- of Alphaproteobacteria (i.e., Blastochloris, Phaeospirillum, lates as well as their sources are summarized in Table 1. All Rhodomicrobium, Rhodoplanes, and Rhodopseudomonas) tested isolates grew optimally at 37–40°C. The isolates of and Betaproteobacteria (i.e., Rubrivivax) (Fig. 1). All of the Phaeospirillum, Rhodomicrobium, and Rhodopseudomonas Rhodomicrobium and Rubrivivax isolates and one of the showed a growth-temperature limit of less than 43°C, Rhodopseudomonas isolates, strain TUT3620, had nearly whereas those of Blastochloris, Rhodoplanes, and Rubri- identical sequences (≥99.6%) with the type strains of the vivax were able to grow at higher temperature up to 45°C. type species of respective genera; i.e., Rhodomicrobium Therefore, at least, the 13 isolates of Blastochloris, vannielli strain DSM 162T, Rubrivivax gelatinosus strain Rhodoplanes, and Rubrivivax could be regarded as being 408 HISADA et al.

Fig. 1. Neighbor-joining phylogenetic tree of the PPNS isolates (in bold face) and related phototrophic bacteria based on 16S rRNA gene sequences. Escherichia coli was used as the outgroup to root the tree. The database accession numbers are given in parentheses following the strain names. Bootstrap confidence values (1000 bootstrap trials) are given at branched points. Bar=2% sequence divergence (Knuc). thermotolerant. for growth and about a 2-fold higher growth rate at the opti- Figure 2 shows the effects of temperature on pho- mal temperature than did the latter (Fig. 2c). On the other totrophic growth of the Rhodoplanes, Rhodopseudomonas, hand, the Rhodopseudomonas isolates, none of which were and Rubrivivax isolates compared to the type strains of rep- thermotolerant, showed a similar temperature range for resentative species of the respective genera. It was obvious growth to Rps. palustris strain ATCC 17001T (Fig. 2b). that the optimum and maximum temperatures for growth of These results suggest that the thermotolerant Rhodoplanes the thermotolerant Rhodoplanes isolates were 4–10°C and Rubrivivax strains have adapted to high-temperature higher than those for Rpl. roseus strain DSM 5909T and Rpl. environments as their natural habitats. elegans strain AS130T (Fig. 2a). Also, although the Rubri- Curiously, Rps. palustris strain DSM 123T had a lower vivax isolates were almost identical with Rvi. gelatinosus range of growth temperature than Rps. palustris ATCC strain ATCC 17011T in 16S rRNA gene sequences, the 17001T. In view of this observation, together with the fact former group of strains exhibited a 6°C higher temperature that the two strains had different 16S rRNA gene sequences Phototrophs from Hot Springs 409

nomic problem. As reported herein, we have successfully isolated PPNS bacteria from all of the microbial mat samples collected. The majority of the isolates were thermotolerant strains that grew at 45°C and optimally at 40°C. In view of our results and previous data obtained from the geographically different areas5,21,34), it is evident that hot spring environments are common sources of both mesophilic and thermotolerant PPNS bacteria. A puzzling question is why PPNS bacteria are able to inhabit microbial mats with high temperatures outside their growth temperature ranges. Except in the case of Rhodocista centenaria, which forms cysts able to tolerate high temperatures3,28), it seems difficult, at this stage, to find a plausible reason for this. Resnick and Madigan25) sug- gested that mesophilic PPNS bacteria derived from hot springs are not truly indigenous to these environments. Nev- ertheless, the frequent isolation of PPNS bacteria from geographically different hot springs implies that they are more than chance contaminants or transients. In fact, even mesophilic PPNS bacteria have been constantly isolated from the hot springs we studied (unpublished observations). In situ studies on hot-spring microbial communities have shown that the optimum temperature for their growth and decomposition activity is much lower than the temperature of the habitats21,27). Therefore, one can speculate that the temperature which microorganisms, including PPNS bacteria, in hot spring mats can tolerate varies depending upon a combina- tion of environmental factors, such as pH, Eh, nutrient con- centrations, and structure of biomats as the microhabitats23). The high frequencies of the occurrence of PPNS bacteria in the photosynthetic microbial mats in hot springs might suggest that these mats offer a favorable ecological niche for these bacteria. All of the PPNS bacteria isolated in this study have a tendency to photoorganotrophy as the pre- ferred mode of growth. In hot spring microbial mats, the Fig. 2. Temperature-dependent phototrophic growth of the isolates of Rhodoplanes (a), Rhodopseudomonas (b), and Rubrivivax (c) PPNS bacteria as well as Chloroflexus may grow photoor- compared to that of representative species of the respective gen- ganotrophically using organic substrates and growth factors era. Symbols in (a): closed circles, “Rps. cryptolactis” strain possibly excreted by co-existing cyanobacteria. Since the DSM 9987 and the 7 Rhodoplaens isolates (averages and stan- PPNS bacteria have BChl a or b as the major photopig- dard variations); open circles, Rpl. elegans strain AS130T; open triangles, Rpl. roseus strain DSM 5909T. Symbols in (b): closed ments, no competition takes place between PPNS bacteria circles, the 9 Rhodopseudomonas isolates (averages and standard and Chloroflexus in the utilization of light. The versatility of T variations); open circles, Rps. palustris strain DSM 123 ; open the PPNS bacteria with respect to energy metabolism may T triangles, Rps. palustris strain ATCC 17001 . Symbols in (c): enable them to compete with co-existing chemoorgano- closed circles, the 5 Rubrivivax isolates (averages and standard variations); open circles, Rvi. gelatinosus strain ATCC 17011T. trophic bacteria in such oligotrophic environments as geothermal hot springs. A stable ecosystem might be enhanced by the co-existence of diverse microorganisms, including (97.4% similarity to each other), it seems evident that Rps. PPNS bacteria, predatory bacteria, and other physiological palustris strains DSM 123T and ATCC 17001T were not groups of chemotrophic bacteria, in the photosynthetic identical. Further study is necessary to clarify this taxo- microbial mats possibly as shelters from outer stress15). 410 HISADA et al.

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