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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
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