Hugh W. Morgan

16S rDNA Analysis of Spirochaeta thermophila: Its Phylogenetic Position and Implications for the Systematics of the Order Spirochaetales

Summary The 16S rRNA gene of Spirochaeta thermophila DSM 6578 was amplifed by the polymerase chain reaction and analysis of a 1333 nucleotide long stretch performed. The sequence was aligned to the homologous region of 13 representatives from six spirochete genera, and the phylogenetic position of S. thermophila determined. This species constitutes a deep-branching member of a cluster that is defined by representatives of Spirochaeta and Treponema and by Borellia burgdorferi . The genera Serpulina, Leptonema and Leptospira are more ancient representatives of the spirochete line of descent. The branching pattern confirms earlier results of phylogenetic studies which showed the genus Spirochaeta to be heterogeneous, with S. zuelzerae and S. stenostrepta displaying a higher degree of relatedness to treponemas than to the main (authentic) Spirochaeta group.

Very stable enzymes from extremely thermophilic archaebacteria and eubacteria

SummaryThirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria have been screened on solid media for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities. Extracellular enzymes were detected in 14 archaebacteria belonging to three different orders. Twelve of these were able to degrade starch and casein and the two Thermofilum strains were able to degrade starch, xylan and carboxymethylcellulose. Three of the eubacteria could degrade only starch. The other six (including four Thermotoga strains) all had activity against starch, xylan and carboxymethylcellulose, and one also had activity against casein. Some of the amylolytic archaebacteria released α-glucosidase, β-glucosidase, amylase and transglucosylase activities into liquid media containing starch or maltose. Thermotoga strain FjSS3B.1 released amylase, xylanase, cellulase and β-glucosidase activities into the medium when grown in the presence of substrates. When the partially purified enzymes from Thermotoga and some of the archaebacteria were compared with known thermostable enzymes the majority were found to be the most thermostable of their type. The β-glucosidase, xylanase and cellulase from Thermotoga and two α-glucosidases, a β-glucosidase, an amylase and a pullulanase from archaebacteria all have half-lives of at least 15 min at 105°C.

A Numerical Classification of Some Thermus Isolates

Summary: A numerical classification was performed on a collection of 45 Thermus isolates recovered from New Zealand hot pools and on six type strains including T. aquaticus and “T. thermophilus”. Unweighted average linking (UPGMA) and single linkage clustering methods were applied to similarity matrices derived from simple matching (SSM ) and Jaccard similarity (SJ ) coefficients. Differences were observed between phenograms derived from SSM and SJ coefficients, indicating that some of the clusters formed were derived from a significant component of negative matches. Test error was estimated at 2.9%. In the UPGMA/SSM phenogram, seven clusters were formed. A majority of the New Zealand isolates did not cluster with non-New Zealand isolates. Analysis of variance showed that there was a relationship between the composition of the clusters and the temperature and pH of the source of the isolate. Chi-squared testing showed that, within New Zealand, the geographical source of the isolate had no bearing on the clusters formed.

Clostridium fervidus sp. nov., a new chemoorganotrophic acetogenic thermophile

Clostridium fervidus sp. nov. was isolated from a hot spring in New Zealand. The cells were strictly anaerobic, gram negative, sporeforming, and sluggishly motile rods (0.65 to 0.75 μ wide and 2 to 3 μm long). The spherical spores were subterminal to terminal and did not distend the sporangium. Lysis of the culture occurred at the onset of stationary phase. The deoxyribonucleic acid guanine-plus-cytosine content was 39 mol%. The temperature optimum was 68°C (range, > 37 and 5.5 and < 9.0). Growth occurred on Trypticase peptone (BBL Microbiology Systems) or yeast extract. However, with the exception of serine, which could be catabolized as the sole carbon source, either peptone or yeast extract was essential for the fermentation of carbohydrates including glucose, maltose, mannose, xylan, starch, and pyruvate. Acetate was always the major fermentation end product. CO2, H2, and minor quantitites of valerate, butyrate, ethanol, and lactate were also produced. C. fervidus (type strain Rt4-B1T) has been deposited with the American Type Culture Collection (ATCC 43204).

Thermophilic proteases: Properties and potential applications

Abstract Proteases are arguably the most important group of industrial enzymes and certainly form a major portion of world-wide enzyme sales. Thermophilic proteases, with their high specific activities and their superior chemical and physical stability characteristics, would seem to be good candidates for current and future biotechnological applications.

Thermus filiformis sp. nov., a filamentous caldoactive bacterium

In a preliminary investigation the isolation of a caldoactive filamentous microorganism from a New Zealand hot spring was reported. This organism is described here as a new species belonging to the genus Thermus, namely, Thermus filiformis, based on ultrastructural, phenotypic, and anomalous Gram type characteristics. The cell wall of T. filiformis resembles that of Thermus aquaticus apart from the presence of an extra layer. The Thermus species tested, including T. filiformis, are negative for the aminopeptidase test, which is unusual for a gram-negative genus. T. filiformis is nonproteolytic, unlike most other Thermus strains, and also differs radically from other strains in morphology when it is observed by using phase-contrast microscopy. The single strain of the species has been deposited with the American Type Culture Collection as strain ATCC 43280T (T = type strain).

A highly stable pullulanase from Thermus aquaticus YT-1

Abstract A cell-associated pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) from Thermus aquaticus YT-1 has been detergent solubilized and purified by a method involving dispersion into ethanediol followed by gel filtration and ion-exchange chromatography. Molecular weight was determined as 83 000 by SDS-PAGE and 80 000 by steric exclusion chromatography. The enzyme had an optimum pH of 6.4 and exhibited debranching activity towards starch (Km = 3.4 × 10−3%) and amylopectin. (Km = 2.5 × 10−3%). Hydrolysis of pullulan was specific to α(1→6) glycosyl linkages (Km = 6.3 × 10−3%). Activity was highly thermostable with over 80% activity remaining after 10 h at 85°C and t 1 2 = 4.5 h at 95°C. Thermostability was Ca2+ dependent and influenced by pH ( t 1 2 at 100°C = 13.5 min at pH = 7.0, 6.5 min at pH 5.9 and 1.5 min at pH 4.7 in the presence of Ca2+).

The phylogenetic position of the Thermococcus isolate AN1 based on 16S rRNA gene sequence analysis : a proposal that AN1 represents a new species, Thermococcus zilligii sp. nov.

Abstract The 16S rRNA gene from the Thermococcus New Zealand isolate AN1 was cloned and sequenced. Analysis of the gene revealed the presence of signature sequences, indicating that strain AN1 represents a new species of the genus Thermococcus. Since the isolate AN1 differed from other thermococci in both its lower optimal NaCl concentration and generally lower optimal temperature for growth, in its unusual lipid membrane composition, and in its sensitivity to antibiotics, we propose that strain AN1 represents a new species of Thermococcus. The proposed name is Thermococcus zilligii, and the type strain is DSM 2770.

Characterization of a New Obligately Anaerobic Thermophile, Thermoanaerobacter wiegelii sp. nov.

An obligately anaerobic, extremely thermophilic Thermoanaerobacter species was isolated from a freshwater pool formed from a geothermally heated (56 to 69 degrees C) water outlet in Government Gardens, Rotorua, New Zealand. This organism was a spore-forming, gram-negative, rod-shaped bacterium. Strain Rt8.B1T (= DSM 10319T) (T = type strain) fermented a wide variety of mono-, di-, and polysaccharides and produced ethanol, acetate, lactate, propionate, and hydrogen. Sugar alcohols were also fermented, but organic acids and amino acids were not utilized. On the basis of its morphological characteristics, DNA G + C content, obligately anaerobic, thermophilic, polysaccharolytic nature, and levels of 16S rRNA sequence homology, we propose that strain Rt8.B1T should be classified in the genus Thermoanaerobacter as a new species, Thermoanaerobacter wiegelii.

Numerical Classification of Thermus Isolates from Globally Distributed Hot Springs

Summary A numerical classification was carried out on a collection of 131 Thermus strains isolated predominantly from geothermal sources in New Zealand (North and South Islands), Iceland and Yellowstone National Park (USA) with some strains from New Mexico (USA), Japan, Fiji, USSR and the UK. The reference strains comprised T. aquaticus YT-1 T , T. ruber T , T. filiformis Wai33 A1 T , “T. thermophilus” HB8, “T. flavus” AT-62, “T. lacteus” and “T. rubens” . Results from unweighted average linkage (UPGMA) clustering applied to a similarity matrix derived from the simple matching (S SM ) coefficient showed the formation of 14 clusters at the 73% S SM level. These clusters showed a clear relationship to the geographical sources of the isolates. Analysis of variance also showed a relationship between the cluster composition and the pH and temperature values of the isolate sources. The recognised species were separated at 65% S SM and if this value is taken to define the species level then there appear to be 8 species among the strains studied.