Thermorudis pharmacophila sp. nov., a novel member of the class Thermomicrobia isolated from geothermal soil, and emended descriptions of Thermomicrobium roseum, Thermomicrobium carboxidum, Thermorudis peleae and Sphaerobacter thermophilus.

An aerobic, thermophilic and cellulolytic bacterium, designated strain WKT50.2T, was isolated from geothermal soil at Waikite, New Zealand. Strain WKT50.2T grew at 53-76 °C and at pH 5.9-8.2. The DNA G+C content was 58.4 mol%. The major fatty acids were 12-methyl C18 : 0 and C18 : 0. Polar lipids were all linked to long-chain 1,2-diols, and comprised 2-acylalkyldiol-1-O-phosphoinositol (diolPI), 2-acylalkyldiol-1-O-phosphoacylmannoside (diolP-acylMan), 2-acylalkyldiol-1-O-phosphoinositol acylmannoside (diolPI-acylMan) and 2-acylalkyldiol-1-O-phosphoinositol mannoside (diolPI-Man). Strain WKT50.2T utilized a range of cellulosic substrates, alcohols and organic acids for growth, but was unable to utilize monosaccharides. Robust growth of WKT50.2T was observed on protein derivatives. WKT50.2T was sensitive to ampicillin, chloramphenicol, kanamycin, neomycin, polymyxin B, streptomycin and vancomycin. Metronidazole, lasalocid A and trimethoprim stimulated growth. Phylogenetic analysis of 16S rRNA gene sequences showed that WKT50.2T belonged to the class Thermomicrobia within the phylum Chloroflexi, and was most closely related to Thermorudis peleae KI4T (99.6% similarity). DNA-DNA hybridization between WKT50.2T and Thermorudis peleae DSM 27169T was 18.0%. Physiological and biochemical tests confirmed the phenotypic and genotypic differentiation of strain WKT50.2T from Thermorudis peleae KI4T and other members of the Thermomicrobia. On the basis of its phylogenetic position and phenotypic characteristics, we propose that strain WKT50.2T represents a novel species, for which the name Thermorudis pharmacophila sp. nov. is proposed, with the type strain WKT50.2T ( = DSM 26011T = ICMP 20042T). Emended descriptions of Thermomicrobium roseum, Thermomicrobium carboxidum, Thermorudis peleae and Sphaerobacter thermophilus are also proposed, and include the description of a novel respiratory quinone, MK-8 2,3-epoxide (23%), in Thermomicrobium roseum.

Effect of long-term starvation on the survival, recovery, and carbon utilization profiles of a bovine Escherichia coli O157:H7 isolate from New Zealand.

The ability to maintain a dual lifestyle of colonizing the ruminant gut and surviving in nonhost environments once shed is key to the success of Escherichia coli O157:H7 as a zoonotic pathogen. Both physical and biological conditions encountered by the bacteria are likely to change during the transition between host and nonhost environments. In this study, carbon starvation at suboptimal temperatures in nonhost environments was simulated by starving a New Zealand bovine E. coli O157:H7 isolate in phosphate-buffered saline at 4 and 15°C for 84 days. Recovery of starved cells on media with different nutrient availabilities was monitored under aerobic and anaerobic conditions. We found that the New Zealand bovine E. coli O157:H7 isolate was able to maintain membrane integrity and viability over 84 days and that the level of recovery depended on the nutrient level of the recovery medium as well as the starvation temperature. In addition, a significant difference in carbon utilization was observed between starved and nonstarved cells.

Draft genome sequence of the thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1.

The genes and molecular machines that allow for a thermoalkaliphilic lifestyle have not been defined. To address this goal, we report on the improved high-quality draft genome sequence of Caldalkalibacillus thermarum strain TA2.A1, an obligately aerobic bacterium that grows optimally at pH 9.5 and 65 to 70°C on a wide variety of carbon and energy sources.

The major lipid cores of the archaeon Ignisphaera aggregans: implications for the phylogeny and biosynthesis of glycerol monoalkyl glycerol tetraether isoprenoid lipids.

The lipid cores from Ignisphaera aggregans, a hyperthermophilic Crenarchaeon recently isolated from New Zealand hot springs, have been profiled by liquid chromatography-tandem mass spectrometry. The distribution revealed includes relatively high proportions of monoalkyl (also known as H-shaped) tetraether cores which have previously been implicated as kingdom-specific biomarkers for the Euryarchaeota. Such high expression of monoalkyl tetraether lipids is unusual in the archaeal domain and may indicate that formation of these components is an adaptive mechanism that allows I. aggregans to regulate membrane behaviour at high temperatures. The observed dialkyl tetraether and monoalkyl tetraether lipid distributions are similar but not fully concordant, showing differences in the average number of incorporated rings. The similarity supports a biosynthetic route to the ring-containing dialkyl and monoalkyl tetraether lipids via a dialkyl tetraether core containing zero rings, or a closely related structural relative, as an intermediate. Currently, however, the precise nature of the biosynthetic route to these lipids cannot be deduced.

Venenivibrio stagnispumantis gen. nov., sp. nov., a thermophilic hydrogen-oxidizing bacterium isolated from Champagne Pool, Waiotapu, New Zealand.

A novel thermophilic, hydrogen-oxidizing bacterium, designated strain CP.B2(T), was isolated from a terrestrial hot spring in Waiotapu, New Zealand. Cells were motile, slightly rod-shaped, non-spore-forming and Gram-negative. Isolate CP.B2(T) was an obligate chemolithotroph, growing by utilizing H(2) as electron donor and O(2) as corresponding electron acceptor. Elemental sulfur (S(0)) or thiosulfate ( ) was essential for growth. Microbial growth occurred under microaerophilic conditions in 1.0-10.0 % (v/v) O(2) [optimum 4-8 % (v/v) O(2)], between 45 and 75 degrees C (optimum 70 degrees C) and at pH values of 4.8-5.8 (optimum pH 5.4). The DNA G+C content was 29.3 mol%. 16S rRNA gene sequence analysis demonstrated that strain CP.B2(T) belonged to the order Aquificales, with a close phylogenetic relationship to Sulfurihydrogenibium azorense (94 % sequence similarity to the type strain). However, genotypic and metabolic characteristics differentiated the novel isolate from previously described genera of the Aquificales. Therefore, CP.B2(T) represents a novel species in a new genus, for which the name Venenivibrio stagnispumantis gen. nov., sp. nov. is proposed. The type strain of Venenivibrio stagnispumantis is CP.B2(T) (=JCM 14244(T) =DSM 18763(T)).

The microbial ecology of a high-temperature near-neutral spring situated in Rotorua, New Zealand.

A combination of both culture and culture-independent techniques were used to investigate the microbial ecology of a near-neutral, high-temperature hot spring (designated AQ1) in Rotorua, New Zealand. The active microbial members of the community were targeted by analyzing biofilms that developed on surfaces incubated in situ in AQ1. Colonization of surfaces was rapid as indicated by ATP assay and microscopic observation. DNA-based analysis of both colonized surfaces and pool water from AQ1 revealed an exclusively archaeal community. Different colonization patterns were observed on glass slides incubated near the pool surface or at depth. Slides incubated at the surface were colonized exclusively by Pyrobaculum species, while at greater depth a novel coccus was also observed and detected by DGGE. Sequence analysis revealed the coccus was related to Aeropyrum pernix. Two microorganisms were isolated from AQ1 pool water, namely Ignisphaera aggregans AQ1.S1T and a species of Pyrobaculum, isolate AQ1.S2.

Isolation and characterization of aerobic thermophilic bacteria from the savusavu hot springs in fiji.

The relative isolation and unique physical properties of the Savusavu Hot Springs in Fiji may yield unique thermophiles. This study was conducted to determine the presence of aerobic thermophilic bacteria in these hot springs. A total of 104 thermophilic bacterial isolates were characterized and using Thermus and Bacillus strains as controls, 58% of the isolates were identified as Anoxybacillus flavithermus, 19% as Geobacillus stearothermophilus/Bacillus licheniformis, 10% as Thermus sp. TG153 and 10% as Thermus sp. TG206. Four isolates were unique in their molecular patterns suggesting there may be novel bacteria in the Savusavu hot springs.

Microbial life in Champagne Pool, a geothermal spring in Waiotapu, New Zealand.

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

Removal of contaminating DNA from polymerase chain reaction using ethidium monoazide.

The presence of exogenous DNA in PCR reagents and DNA polymerase is a common occurrence. In particular, the amplification of 16S rRNA genes with universal primers for non-culture-based study is often hampered by the formation of false positives. Here, we describe the use of ethidium monoazide (EMA) to eliminate contaminating DNA in a polymerase chain reaction. The advantage of the proposed methodology is the retention of the highly sensitive nature of PCR with the ability to amplify template DNA at concentrations lower than those of contaminating DNA. The treatment of PCR master mix with EMA concentrations that exceeded those required to remove contaminating DNA can interfere with the amplification of low-template concentrations. The methodology presented is straightforward and can be accomplished within 10 min.

Development of a real-time PCR assay targeting the sporulation gene, spo0A, for the enumeration of thermophilic bacilli in milk powder.

Thermophilic bacilli, such as Anoxybacillus, Geobacillus and Bacillus, are common contaminants growing within the processing lines of milk powder producing factories. These contaminants are used as indicator organisms for plant hygiene and specification limits based on their numbers have been implemented to ensure milk powder quality. In this study, we present a SYBR Green-based real-time PCR assay for the rapid detection and enumeration of these thermophilic bacilli in milk powder using the spo0A sporulation gene as quantification target. With this method the detection of thermophilic bacilli in milk powder can be accomplished within 1 h. The detection limit for reconstituted and inoculated milk was 80 vegetative cfu ml(-1) and 640 spores ml(-1), respectively.

Cadmium ion biosorption by the thermophilic bacteria Geobacillus stearothermophilus and G. thermocatenulatus.

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl2 (as high as 400 to 3,200 microM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd2+ adsorption experiments to characterize Cd2+ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd2+ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.

Ignisphaera aggregans gen. nov., sp. nov., a novel hyperthermophilic crenarchaeote isolated from hot springs in Rotorua and Tokaanu, New Zealand.

Consortia containing a novel coccus-shaped, anaerobic heterotroph together with Pyrobaculum rods were cultivated from geothermal environments in New Zealand. Pure cultures of the cocci were only obtained from one such consortium, despite extensive attempts. Cells of this strain (AQ1.S1T) were regular to irregular cocci in morphology and occasionally formed large aggregates, especially when utilizing polysaccharides such as konjac glucomannan as a carbon source. Strain AQ1.S1T is a hyperthermophile, with an optimal temperature for growth between 92 and 95 degrees C (range 85-98 degrees C), and a moderate acidophile, with optimal growth occurring at pH 6.4 (range 5.4-7.0). Growth was inhibited by the addition of sulphur and NaCl (optimal growth occurred without addition of NaCl) and an electron acceptor was not required. Strain AQ1.S1T utilized starch, trypticase peptone, lactose, glucose, konjac glucomannan, mannose, galactose, maltose, glycogen and beta-cyclodextrin as carbon sources. The G+C content was 52.9 mol%. Based on 16S rRNA gene sequence analysis and physiological features it is proposed that isolate AQ1.S1T (=DSM 17230T=JCM 13409T) represents the type strain of a novel species of a new genus within the Crenarchaeota, Ignisphaera aggregans gen. nov., sp. nov.

Survival of thermophilic spore-forming bacteria in a 90+ year old milk powder from Ernest Shackelton's Cape Royds Hut in Antarctica.

Milk powder taken to Antarctica on Shackelton's British Antarctic Expedition in 1907 was produced in New Zealand by a roller drying process in the first factory in the world dedicated to this process. Thermophilic bacilli are the dominant contaminants of modern spray-dried milk powders and the 1907 milk powder allows a comparison to be made of contaminating strains in roller-dried and spray-dried powders. Samples of milk powder obtained from Shackelton's Hut at Cape Royds had low levels of thermophilic contamination (< 500 cfu ml-1) but the two dominant strains (Bacillus licheniformis strain F and Bacillus subtilis) were typical of those found in spray-dried powders. Soil samples from the floor of the hut also contained these strains, whereas soils distant from the hut did not. Differences in the RAPD profiles of isolates from the milk powder and the soils suggest that contamination of the milk from the soil was unlikely. It is significant that the most commonly encountered contaminant strain in modern spray-dried milk (Anoxybacillus flavithermus strain C) was not detected in the 1907 sample.

Development of a rapid detection and enumeration method for thermophilic bacilli in milk powders.

Thermophilic strains of Geobacillus, Anoxybacillus and Bacillus that are able to grow at 55 degrees C and above are recognized as commonly occurring contaminants during the production of milk powders. In particular, Anoxybacillus flavithermus strain C and Bacillus licheniformis strain F are often the most prevalent. We describe here the development of a TaqMan-based real-time-PCR assay using a small amplicon of the ribosomal 16S rRNA gene for the selective and quantitative detection of thermophilic bacilli in milk powders. We further present an effective, rapid and inexpensive method for the isolation of total bacterial DNA from milk powder for quantitative PCR analysis within 20 min. With this method, the detection of thermophilic bacilli in milk powder can be accomplished within 1 h. The detection limit for reconstituted and inoculated milk was 8 vegetative cfu ml(-1) and 64 spores ml(-1), respectively.

A RAPD-based survey of thermophilic bacilli in milk powders from different countries.

Twenty-eight milk powders from 18 different countries were examined for the number and type of contaminating thermophilic bacilli. Of 742 isolates examined, 96.8% were assigned to the same strains of bacilli as previously found in New Zealand powders. The dominant isolate was Anoxybacillus flavithermus strain C followed by Bacillus licheniformis strain F. The former was also prevalent in New Zealand powders and the results demonstrate that A. flavithermus represents a widespread contaminant, seemingly ubiquitous in factories producing milk powder. The presence of thermophilic strains of Geobacillus stearothermophilus and to a lesser extent of Bacillus subtilis in milk powders was reconfirmed.

Cloning and biochemical characterization of a novel mouse ADP-dependent glucokinase.

Glycolysis, the catabolism of glucose to pyruvate, is an iconic central metabolic pathway and often used as a paradigm for explaining the general principles of the regulation/control of cellular metabolism. The ubiquitous mammalian ATP-dependent hexokinases I-III and hexokinase IV, also termed glucokinase, initiate the process by phosphorylating glucose to glucose-6-phosphate. Despite glycolysis having been studied extensively for over 70 years and the last new mammalian ATP-dependent hexokinase isotype having been described in the 1960s, we report here the biochemical characterization of a recombinant ADP-dependent glucokinase cloned from a full-length Mus musculus cDNA, identified by sequence analysis. The recombinant enzyme is quite specific for glucose, is monomeric, has an apparent Km for glucose and ADP of 96 and 280 microM, respectively, and is inhibited by both high concentrations of glucose and AMP. The metabolic role of this enzyme in cells would be dependent on the relative level of its activity to those of the ATP-dependent hexokinases. The greatest advantage of an ADP-GK would clearly be during ischemia/hypoxia, clinically relevant conditions in multiple major disease states, by decreasing the priming cost for the phosphorylation of glucose, saving ATP.

Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.

We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

A RAPD-based comparison of thermophilic bacilli from milk powders.

The similarity of strains of thermophilic Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Anoxybacillus flavithermus (formerly Bacillus flavothermus), Bacillus licheniformis and Bacillus subtilis isolated from separate milk powder production runs from multiple factories was examined using a random amplified polymorphic DNA (RAPD) protocol. As a result of the analysis of the RAPD fingerprints and data relating to general growth and biochemical tests, over 98% of the 1470 isolates examined (grown at 55 degrees C) were assigned to the species G. stearothermophilus, A. flavithermus, B. licheniformis and B. subtilis. The G. stearothermophilus isolates were identified as being nearly identical to G. stearothermophilus (DSMZ 22; equivalent to ATCC 12980), or G. stearothermophilus var. calidolactis (DSMZ 1550). Three groups of isolates were found to be related to A. flavithermus (DSMZ 2641) by partial small ribosomal subunit (16S) sequence comparisons and shown to be interrelated by RAPD analyses with multiple primer sets. The thermophilic isolates of B. licheniformis were positively identified by comparison with type strains of B. licheniformis DSMZ 13 and DSMZ 8785. All of the B. subtilis strains shared bands in their RAPD profiles and were similar to a common B. subtilis type strain (DSMZ 10 and DSMZ 347). Overall, the most common and prevalent group of strains (group A) was demonstrated to be closely related to G. stearothermophilus (DSMZ 22).

Bioenergetic properties of the thermoalkaliphilic Bacillus sp. strain TA2.A1.

The thermoalkaliphilic Bacillus sp. strain TA2.A1 was able to grow in pH-controlled batch culture containing a nonfermentable growth substrate from pH 7.5 to 10.0 with no significant change in its specific growth rate, demonstrating that this bacterium is a facultative alkaliphile. Growth at pH 10.0 was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that a proton motive force (Deltap) generated via aerobic respiration was an obligate requirement for growth of strain TA2.A1. Strain TA2.A1 exhibited intracellular pH homeostasis as the external pH increased from 7.5 to 10.0; however, the maximum DeltapH generated over this pH range was only 1.1 units at an external pH of 9.5. The membrane potential (Deltapsi) was maintained between -114 mV and -150 mV, and little significant change was observed over the pH range for growth. In contrast, the Deltap declined from -164 mV at pH 7.5 to approximately -78 mV at pH 10.0. An inwardly directed sodium motive force (DeltapNa(+)) of -100 mV at pH 10.0 indicated that cellular processes (i.e., solute transport) dependent on a sodium gradient would not be affected by the adverse Deltap. The phosphorylation potential of strain TA2.A1 was maintained between -300 mV and -418 mV, and the calculated H(+)/ATP stoichiometry of the ATP synthase increased from 2.0 at pH 7.5 to 5.7 at pH 10.0. Based on these data, vigorous growth of strain TA2.A1 correlated well with the DeltapNa(+), phosphorylation potential, and the ATP/ADP ratio, but not with Deltap. This communication represents the first report on the bioenergetics of an extremely thermoalkaliphilic aerobic bacterium.