More than 40 isolates of a novel, ubiquitous, proteolytic, moderately alkaliphilic, thermophilic obligate anaerobe were obtained from geothermally and anthropogenically heated environments and mesobiotic environments located on three continents. Whole-cell protein sodium dodecyl sulfate gel electrophoresis revealed that most of these organisms are very similar. Eight of the isolates were characterized in detail; this analysis included 16s ribosomal DNA sequence analysis. The cells of those organisms are (depending on the isolate) 0.5 to 0.8 pm in diameter and 1.5 to 13 pm long, exhibit tumbling motility, and have a positive Gram stain reaction. The temperature range for growth is 43" to 75°C (optimum temperature, 66"C), and the pH range for growth is 5.4 to 9.5 (optimum pH, 8.2); the shortest doubling time is around 10 min. Yeast extract is required for growth, and (depending on the strain) glucose, sucrose, fructose, galactose, and ribose are utilized. The fermentation products from glucose in the presence of yeast extract are CO,, H2, acetate, formate, and ethanol. The G+C content is 30 to 31 mol%. On the basis of these properties, which differentiate these strains from all alkalitolerant thermophiles described previously, and the results of a comparison of the 16s ribosomal DNA sequences of these organisms with previously described sequences, we propose that our isolates be placed in a single species of the new genus Therrnobrachiurn; strain JWm-NZ35 is the type strain of the the type species, Them0 brachiurn celere.There are relatively few alkaliphilic environments that occur naturally (13, 34). The most common and best-studied alkaliphilic environments are the soda lakes and deserts that have pH values of 10 to 11.5 (10). These environments usually result from the presence of large amounts of sodium carbonate and other salts (17). However, alkaline conditions can also be found in niches of other environments in which the pH of a microenvironment can be different from the pH of the environment as a whole (17).Only a few thermophiles are able to grow at pH values of more than 9.0 (31). Aside from characterizations of recently described anaerobic alkalithermophiles (5,14, 15,34), our knowledge of alkaliphilic bacteria is based mainly on the characteristics of Bacillus species (13, 20, 21). In this paper, we describe a novel, ubiquitous, moderately alkaliphilic, anaerobic thermophile, Thermobrachium celere, which was isolated from samples obtained from various mesobiotic and thermobiotic environments on three continents; the pH values of these samples ranged from 4.8 to 9.0. MATERIALS AND METHODSSample collection. Separated and mixed sediment and water samples were obtained from various locations by completely filling 100-to 500-ml sterile glass jars which had been flushed with nitrogen and were sealed with butyl rubber stoppers. Although the samples were kept at temperatures below 10°C as much as possible during transport to the laboratory in Athens, Ga., they were temporarily exposed to ambient temperatures f...

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The industrial application of thermophilic (eu)bacteria is hampered by the lack of genetic systems for these bacteria. We report here the first unequivocal transformation of a Gram‐positive, thermophilic, anaerobic microorganism, Thermoanaerobacterium, with the kanamycin resistance‐mediating plasmid pIKM1. The construct pIKM1 is based on the Escherichia coli–Clostridium acetobutylicum shuttle vector pIMP1 and contains the thermostable kanamycin cassette from S. faecalis plasmid pKD102. Using electrotransformation, plasmid pIKM1 mediated kanamycin resistance in Thermoanaerobacterium sp. strain JW/SL‐YS485 up to 400 μg ml−1 at 48°C and 200 μg ml−1 at 60°C.

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Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.