Apparent contradiction: psychrotolerant bacteria from hydrocarbon-contaminated arctic tundra soils that degrade diterpenoids synthesized by trees.

Resin acids are tricyclic terpenoids occurring naturally in trees. We investigated the occurrence of resin acid-degrading bacteria on the Arctic tundra near the northern coast of Ellesmere Island (82 degrees N, 62 degrees W). According to most-probable-number assays, resin acid degraders were abundant (10(3) to 10(4) propagules/g of soil) in hydrocarbon-contaminated soils, but they were undetectable (<3 propagules/g of soil) in pristine soils from the nearby tundra. Plate counts indicated that the contaminated and the pristine soils had similar populations of heterotrophs (10(6) to 10(7) propagules/g of soil). Eleven resin acid-degrading bacteria belonging to four phylogenetically distinct groups were enriched and isolated from the contaminated soils, and representative isolates of each group were further characterized. Strains DhA-91, IpA-92, and IpA-93 are members of the genus Pseudomonas. Strain DhA-95 is a member of the genus Sphingomonas. All four strains are psychrotolerant, with growth temperature ranges of 4 degrees C to 30 degrees C (DhA-91 and DhA-95) or 4 degrees C to 22 degrees C (IpA-92 and IpA-93) and with optimum temperatures of 15 to 22 degrees C. Strains DhA-91 and DhA-95 grew on the abietanes, dehydroabietic and abietic acids, but not on the pimaranes, isopimaric and pimaric acids. Strains IpA-92 and IpA-93 grew on the pimaranes but not the abietanes. All four strains grew on either aliphatic or aromatic hydrocarbons, which is unusual for described resin acid degraders. Eleven mesophilic resin acid degraders did not use hydrocarbons, with the exception of two Mycobacterium sp. strains that used aliphatic hydrocarbons. We conclude that hydrocarbon contamination in Arctic tundra soil indirectly selected for resin acid degraders, selecting for hydrocarbon degraders that coincidentally use resin acids. Psychrotolerant resin acid degraders are likely important in the global carbon cycle and may have applications in biotreatment of pulp and paper mill effluents.     

Bacteria obtained from a sequencing batch reactor that are capable of growth on dehydroabietic acid.

Eleven isolates capable of growth on the resin acid dehydroabietic acid (DhA) were obtained from a sequencing batch reactor designed to treat a high-strength process stream from a paper mill. The isolates belonged to two groups, represented by strains DhA-33 and DhA-35, which were characterized. In the bioreactor, bacteria like DhA-35 were more abundant than those like DhA-33. The population in the bioreactor of organisms capable of growth on DhA was estimated to be 1.1 x 10(6) propagules per ml, based on a most-probable-number determination. Analysis of small-subunit rRNA partial sequences indicated that DhA-33 was most closely related to Sphingomonas yanoikuyae (Sab = 0.875) and that DhA-35 was most closely related to Zoogloea ramigera (Sab = 0.849). Both isolates additionally grew on other abietanes, i.e., abietic and palustric acids, but not on the pimaranes, pimaric and isopimaric acids. For DhA-33 and DhA-35 with DhA as the sole organic substrate, doubling times were 2.7 and 2.2 h, respectively, and growth yields were 0.30 and 0.25 g of protein per g of DhA, respectively. Glucose as a cosubstrate stimulated growth of DhA-33 on DhA and stimulated DhA degradation by the culture. Pyruvate as a cosubstrate did not stimulate growth of DhA-35 on DhA and reduced the specific rate of DhA degradation of the culture. DhA induced DhA and abietic acid degradation activities in both strains, and these activities were heat labile. Cell suspensions of both strains consumed DhA at a rate of 6 mumol mg of protein-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lessons learned from Sphingomonas species that degrade abietane triterpenoids

Abietane terpenoid-degrading organisms include Sphingomonas spp which inhabit natural environments and biological treatment systems. An isolate from the high Arctic indicates that these organisms occur far from trees which synthesize abietanes and suggests that some of these organisms can occupy a niche in hydrocarbon-degrading soil communities. Abietane-degrading Sphingomonas spp provide additional evidence that the phylogeny of this genus is independent of the catabolic capabilities of its members. Studies of Sphingomonas sp DhA-33 demonstrate that biological treatment systems for pulp mill effluents have the potential to mineralize abietane resin acids. On the other hand, these studies indicate that some chlorinated dehydroabietic acids are quite recalcitrant. Strain DhA-33 grows relatively well on some chlorinated dehydroabietic acids but transforms others to stable metabolites. Using strain DhA-33, a novel method was developed to measure the metabolic activity of an individual population within a complex microbial community. Oligonucleotide hybridization probes were used to assay the 16S rRNA:rDNA ratio of DhA-33 as it grew in an activated sludge community. However, this method proved not to be sufficiently sensitive to measure naturally occurring resin acid-degrading populations. We propose that the same approach can be modified to use more sensitive assays. Received 01 May 1999/ Accepted in revised form 19 July 1999     

Environmental Limits of Tall Shrubs in Alaska’s Arctic National Parks

We sampled shrub canopy volume (height times area) and environmental factors (soil wetness, soil depth of thaw, soil pH, mean July air temperature, and typical date of spring snow loss) on 471 plots across five National Park Service units in northern Alaska. Our goal was to determine the environments where tall shrubs thrive and use this information to predict the location of future shrub expansion. The study area covers over 80,000 km² and has mostly tundra vegetation. Large canopy volumes were uncommon, with volumes over 0.5 m³/m² present on just 8% of plots. Shrub canopy volumes were highest where mean July temperatures were above 10.5°C and on weakly acid to neutral soils (pH of 6 to 7) with deep summer thaw (>80 cm) and good drainage. On many sites, flooding helped maintain favorable soil conditions for shrub growth. Canopy volumes were highest where the typical snow loss date was near 20 May; these represent sites that are neither strongly wind-scoured in the winter nor late to melt from deep snowdrifts. Individual species varied widely in the canopy volumes they attained and their response to the environmental factors. Betula sp. shrubs were the most common and quite tolerant of soil acidity, cold July temperatures, and shallow thaw depths, but they did not form high-volume canopies under these conditions. Alnus viridis formed the largest canopies and was tolerant of soil acidity down to about pH 5, but required more summer warmth (over 12°C) than the other species. The Salix species varied widely from S. pulchra, tolerant of wet and moderately acid soils, to S. alaxensis, requiring well-drained soils with near neutral pH. Nearly half of the land area in ARCN has mean July temperatures of 10.5 to 12.5°C, where 2°C of warming would bring temperatures into the range needed for all of the potential tall shrub species to form large canopies. However, limitations in the other environmental factors would probably prevent the formation of large shrub canopies on at least half of the land area with newly favorable temperatures after 2°C of warming.     

Bacterial metabolism of chlorinated dehydroabietic acids occurring in pulp and paper mill effluents

Chlorinated dehydroabietic acids are formed during the chlorine bleaching of wood pulp and are very toxic to fish. Thus, destruction of these compounds is an important function of biological treatment systems for pulp and paper mill effluents. In this study, 12 strains of diverse, aerobic resin acid-degrading bacteria were screened for the ability to grow on chlorinated dehydroabietic acids as sole organic substrates. All seven strains of the class Proteobacteria able to use dehydroabietic acid were also able to use a mixture of 12- and 14-chlorodehydroabietic acid (Cl-DhA). None of the strains used 12,14-dichlorodehydroabietic acid. Sphingomonas sp. strain DhA-33 grew best on Cl-DhA and simultaneously removed both Cl-DhA isomers. Ralstonia sp. strain BKME-6 was typical of most of the strains tested, growing more slowly on Cl-DhA and leaving higher residual concentrations of Cl-DhA than DhA-33 did. Strains DhA-33 and BKME-6 mineralized (converted to CO(inf2) plus biomass) 32 and 43%, respectively, of carbon in Cl-DhA consumed. Strain DhA-33 produced a metabolite from Cl-DhA, tentatively identified as 3-oxo-14-chlorodehydroabietin, and both strains produced dissolved organic carbon which may include unidentified metabolites. Cl-DhA removal was inducible in both DhA-33 and BKME-6, and induced DhA-33 cells also removed 12,14-dichlorodehydroabietic acid. Based on activities of strains DhA-33 and BKME-6, chlorinated DhAs, and potentially toxic metabolite(s) of these compounds, are relatively persistent in biological treatment systems and in the environment.     

Extreme home range sizes among Eurasian lynx at the northern edge of their biogeographic range

Eurasian lynx (Lynx lynx) have a wide distribution across Eurasia. The northern edge of this distribution is in Norway, where they reach up to 72 degrees north. We conducted a study of lynx space use in this region from 2007 to 2013 using GPS telemetry. The home range sizes averaged 2,606 (± 438 SE) km² for males (n = 9 ranges) and 1,456 (± 179 SE) km² for females (n = 24 ranges). These are the largest home ranges reported for any large felid, and indeed are only matched by polar bears, arctic living wolves, and grizzly bears among all the Carnivora. The habitat occupied was almost entirely treeless alpine tundra, with home ranges only containing from 20% to 25% of forest. These data have clear implications for the spatial planning of lynx management in the far north as the current management zones are located in unsuitable habitats and are not large enough to encompass individual lynx movements.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.     

Accumulation of Pharmaceuticals,Enterococcus, and Resistance Genes in Soils Irrigated with Wastewater for Zero to 100 Years in Central Mexico

Irrigation with wastewater releases pharmaceuticals, pathogenic bacteria, and resistance genes, but little is known about the accumulation of these contaminants in the environment when wastewater is applied for decades. We sampled a chronosequence of soils that were variously irrigated with wastewater from zero up to 100 years in the Mezquital Valley, Mexico, and investigated the accumulation of ciprofloxacin, enrofloxacin, sulfamethoxazole, trimethoprim, clarithromycin, carbamazepine, bezafibrate, naproxen, diclofenac, as well as the occurrence of Enterococcus spp., and sul and qnr resistance genes. Total concentrations of ciprofloxacin, sulfamethoxazole, and carbamazepine increased with irrigation duration reaching 95% of their upper limit of 1.4 µg/kg (ciprofloxacin), 4.3 µg/kg (sulfamethoxazole), and 5.4 µg/kg (carbamazepine) in soils irrigated for 19–28 years. Accumulation was soil-type-specific, with largest accumulation rates in Leptosols and no time-trend in Vertisols. Acidic pharmaceuticals (diclofenac, naproxen, bezafibrate) were not retained and thus did not accumulate in soils. We did not detect qnrA genes, but qnrS and qnrB genes were found in two of the irrigated soils. Relative concentrations of sul1 genes in irrigated soils were two orders of magnitude larger (3.15×10⁻³±0.22×10⁻³ copies/16S rDNA) than in non-irrigated soils (4.35×10⁻⁵±1.00×10⁻⁵ copies/16S rDNA), while those of sul2 exceeded the ones in non-irrigated soils still by a factor of 22 (6.61×10^–4±0.59×10⁻⁴ versus 2.99×10⁻⁵±0.26×10⁻⁵ copies/16S rDNA). Absolute numbers of sul genes continued to increase with prolonging irrigation together with Enterococcus spp. 23S rDNA and total 16S rDNA contents. Increasing total concentrations of antibiotics in soil are not accompanied by increasing relative abundances of resistance genes. Nevertheless, wastewater irrigation enlarges the absolute concentration of resistance genes in soils due to a long-term increase in total microbial biomass.     

Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters

Deuterated styrene ([²H₈]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [²H₈]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [²H₈]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.     

Lichens buffer tundra microclimate more than the expanding shrub Betula nana

Background and AimsIn tundra systems, soil-borne lichens are often the dominant groundcover organisms, and act to buffer microclimate extremes within or at the surface of the soil. However, shrubs are currently expanding across tundra systems, potentially causing major shifts in the microclimate landscape.MethodsHere, we compared soil temperature and moisture underneath the dwarf birch Betula nana and seven abundant lichen species in sub-alpine Norway. We also examined mixtures of lichens and dwarf birch – an intermediate phase of shrubification – and measured several functional traits relating to microclimate.Key ResultsWe found that all lichen species strongly buffered the daily temperature range, on average reducing maximum temperatures by 6.9 °C (± 0.7 s.d.) and increasing minimum temperatures by 1.0 °C (± 0.2 s.d.) during summer. The dwarf birch had a much weaker effect (maximum reduced by 2.4 ± 5.0 °C and minimum raised by 0.2 ± 0.9 °C). In species mixtures, the lichen effect predominated, affecting temperature extremes by more than would be expected from their abundance. Lichens also tended to reduce soil moisture, which could be explained by their ability to intercept rainfall. Our trait measurements under laboratory conditions suggest that, on average, lichens can completely absorb a 4.09 mm (± 1.81 s.d.) rainfall event, which might be an underappreciated part of lichen–vascular plant competition in areas where summer rainfall events are small.ConclusionsIn the context of shrubification across tundra systems, our findings suggest that lichens will continue to have a large effect on microclimate until they are fully excluded, at which point microclimate extremes will increase greatly.     

Iron Superoxide Dismutase Protects against Chilling Damage in the Cyanobacterium Synechococcus species PCC7942

A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB⁻, was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB⁻ and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB⁻ strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB⁻ strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB⁻ strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.     

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid.

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55 degrees C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30 degrees C to 60 degrees C with maximum growth at 50 degrees C; while, DhA-73 grew on DhA from 37 degrees C to 60 degrees C with maximum growth at 55 degrees C. At 55 degrees C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in the Rubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H2-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H₂-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 ± 0.19 mol%), a phospholipid pattern of type II, MK-9(H₄) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t_d = 8 h), H₂ plus CO₂ (t_d = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H₂, nickel was not necessary. The optimum growth temperature was 65°C; no growth was observed below 40°C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70°C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K_m was 88 μl of CO liter⁻¹ and V_max was 20.2 μl of CO h⁻¹ mg of protein⁻¹. The threshold value of S. thermoautotrophicus of 0.2 μl of CO liter⁻¹ was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 μmol min⁻¹ mg of protein⁻¹. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Chlorobenzoate-Degrading Bacteria in Similar Pristine Soils Exhibit Different Community Structures and Population Dynamics in Response to Anthropogenic 2-, 3-, and 4-Chlorobenzoate Levels

A study was conducted to determine the diversity of 2-, 3-, and 4-chlorobenzoate (CB) degraders in two pristine soils with similar physical and chemical characteristics. Surface soils were collected from forested sites and amended with 500 g of 2-, 3-, or 4-CB g^–1 soil. The CB levels and degrader numbers were monitored throughout the study. Degraders were isolated, grouped by DNA fingerprints, identified via 16S rDNA sequences, and screened for plasmids. The CB genes in selected degraders were isolated and/or sequenced. In the Madera soil, 2-CB and 4-CB degraded within 11 and 42 d, respectively, but 3-CB did not degrade. In contrast, 3-CB and 4-CB degraded in the Oversite soil within 14 and 28 d, respectively, while 2-CB did not degrade. Approximately 10⁷ CFU g^–1 of degraders were detected in the Madera soil with 2-CB, and the Oversite soil with 3- and 4-CB. No degraders were detected in the Madera soil with 4-CB even though the 4-CB degraded. Nearly all of the 2-CB degraders isolated from the Madera soil were identified as a Burkholderia sp. containing chromosomally encoded degradative genes. In contrast, several different 3- and 4-CB degraders were isolated from the Oversite soil, and their populations changed as CB degradation progressed. Most of these 3-CB degraders were identified as Burkholderia spp. while the majority of 4-CB degraders were identified as Bradyrhizobium spp. Several of the 3-CB degraders contained the degradative genes on large plasmids, and there was variation between the plasmids in different isolates. When a fresh sample of Madera soil was amended with 50, 100, or 200 g 3-CB g^–1, 3-CB degradation occurred, suggesting that 500 g 3-CB g^–1 was toxic to the degraders. Also, different 3-CB degraders were isolated from the Madera soil at each of the three lower levels of 3-CB. No 2-CB degradation was detected in the Oversite soil even at lower 2-CB levels. These results indicate that the development of 2-, 3-, and 4-CB degrader populations is site-specific and that 2-, 3-, and 4-CB are degraded by different bacterial populations in pristine soils. These results also imply that the microbial ecology of two soils that develop under similar biotic and abiotic environments can be quite different.     

Effects of pH and temperature on the wild-type and a mutant form of Neurospora glutamate dehydrogenase

1. We confirm the observation of Bürk (1965) that Neurospora crassa NADP-linked glutamate dehydrogenase normally exists in an inactive form below pH7·0 and in a fully active form above pH8·0 in either tris or orthophosphate buffer. At pH7·4 the enzyme is about half activated at 25°. 2. The variety of the enzyme produced by the mutant am^2l shows a similar behaviour except that the transition is shifted about one pH unit in the alkaline direction. 3. The am^2l enzyme has previously been reported to be activated by brief warming to 30° in phosphate buffer at pH8·0. The wild-type enzyme shows a similar effect at pH7·0. In tris buffer this effect is much less pronounced. 4. The am^2l enzyme is extremely unstable at 47° at pH7·0; its stability is somewhat greater at lower pH, and is markedly increased by increasing the pH in the range 7·0–8·7. The wild-type enzyme also shows an indication of a stability minimum at pH7·0, but a temperature of 60° is needed for a measurable rate of inactivation. 5. The inactive form of the enzyme is much more subject to thermal irreversible denaturation than is the active form.     

Erythrocruorin of Marphysa sanguinea. Isolation of some physical, physicochemical and other properties

1. The polychaete worm Marphysa sanguinea has a circulating erythrocruorin of mol.wt. about 2·4×10⁶ (S⁰_20,w 58·2s, D_20,w 2·06×10⁻⁷ cm.²/sec). This is the predominant form existing at pH 6–8 and (non-protein) I 0·10–0·21, and also at approx. pH 6·7 and I 0·15–3·00. 2. The pigment contains 2·24% of protohaem. 3. The 58s protein has an electrophoretic mobility of 8·08×10⁻⁵ cm.²/v/sec. at pH 8·12, I 0·21 and 0°. The isoelectric point of suspended particles is 4·63 at I 0·16 and 21·5°. 4. At very low ionic strength and pH 6·7 (unbuffered) the 58s pigment associates reversibly to 97s and 150s forms, which are probably dimer and tetramer species. 5. At pH 10·0 and I 0·025, it dissociates irreversibly to give a small amount of 2–4s non-haem-containing protein and much 9s haem-enriched protein. These and the 58s pigment may correspond to structures found in Levin's (1963) electron-microscope studies of other erythrocruorins. 6. Absorption spectra of the 58s oxygenated erythrocruorin and the deoxygenated and carbon monoxide derivatives have been obtained.     

Flavobacterium plurextorum sp. nov. Isolated from Farmed Rainbow Trout (Oncorhynchus mykiss)

Five strains (1126-1H-08^T, 51B-09, 986-08, 1084B-08 and 424-08) were isolated from diseased rainbow trout. Cells were Gram-negative rods, 0.7 µm wide and 3 µm long, non-endospore-forming, catalase and oxidase positive. Colonies were circular, yellow-pigmented, smooth and entire on TGE agar after 72 hours incubation at 25°C. They grew in a temperature range between 15°C to 30°C, but they did not grow at 37°Cor 42°C. Based on 16S rRNA gene sequence analysis, the isolates belonged to the genus Flavobacterium. Strain 1126-1H-08^T exhibited the highest levels of similarity with Flavobacterium oncorhynchi CECT 7678^T and Flavobacterium pectinovorum DSM 6368^T (98.5% and 97.9% sequence similarity, respectively). DNA–DNA hybridization values were 87 to 99% among the five isolates and ranged from 21 to 48% between strain 1126-1H-08^T, selected as a representative isolate, and the type strains of Flavobacterium oncorhynchi CECT 7678^T and other phylogenetic related Flavobacterium species. The DNA G+C content of strain 1126-1H-08^T was 33.2 mol%. The predominant respiratory quinone was MK-6 and the major fatty acids were iso-C_15∶0 and C_15∶0. These data were similar to those reported for Flavobacterium species. Several physiological and biochemical tests differentiated the novel bacterial strains from related Flavobacterium species. Phylogenetic, genetic and phenotypic data indicate that these strains represent a new species of the genus Flavobacterium, for which the name Flavobacterium plurextorum sp. nov. was proposed. The type strain is 1126-1H-08^T ( = CECT 7844^T = CCUG 60112^T).     

Selective Medium for Isolating Phanerochaete chrysosporium from Soil

A selective medium was developed that is capable of isolating Phanerochaete chrysosporium from soil. This medium contains 15 ppm of benomyl (15 μg g⁻¹) and 550 ppm of streptomycin sulfate in 2% malt agar and is held at 39°C after inoculation. P. chrysosporium was isolated from three nonsterile forest soils to which the fungus had been added. These soils contained large microbial populations.     

Determination of base and backbone contributions to the thermodynamics of premelting and melting transitions in B DNA

In previous papers of this series the temperature-dependent Raman spectra of poly(dA)·poly(dT) and poly(dA–dT)·poly(dA–dT) were used to characterize structurally the melting and premelting transitions in DNAs containing consecutive A·T and alternating A·T/T·A base pairs. Here, we describe procedures for obtaining thermodynamic parameters from the Raman data. The method exploits base-specific and backbone-specific Raman markers to determine separate thermodynamic contributions of A, T and deoxyribosyl-phosphate moieties to premelting and melting transitions. Key findings include the following: (i) Both poly(dA)·poly(dT) and poly(dA–dT)· poly(dA–dT) exhibit robust premelting transitions, due predominantly to backbone conformational changes. (ii) The significant van’t Hoff premelting enthalpies of poly(dA)·poly(dT) [ΔH_vH^pm = 18.0 ± 1.6 kcal·mol^–1 (kilocalories per mole cooperative unit)] and poly(dA–dT)·poly(dA–dT) (ΔH_vH^pm = 13.4 ± 2.5 kcal·mol^–1) differ by an amount (~4.6 kcal·mol^–1) estimated as the contribution from three-centered inter-base hydrogen bonding in (dA)_n·(dT)_n tracts. (iii) The overall stacking free energy of poly(dA)· poly(dT) [–6.88 kcal·mol_bp^–1 (kilocalories per mole base pair)] is greater than that of poly(dA–dT)· poly(dA–dT) (–6.31 kcal·mol_bp^–1). (iv) The difference between stacking free energies of A and T is significant in poly(dA)·poly(dT) (ΔΔG_st = 0.8 ± 0.3 kcal· mol_bp^–1), but marginal in poly(dA–dT)·poly(dA–dT) (ΔΔG_st = 0.3 ± 0.3 kcal·mol_bp^–1). (v) In poly(dA)· poly(dT), the van’t Hoff parameters for melting of A (ΔH_vH^A = 407 ± 23 kcal·mol^–1, ΔS_vH^A = 1166 ± 67 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 60.0 ± 3.2 kcal·mol^–1) are clearly distinguished from those of T (ΔH_vH^T = 185 ± 38 kcal·mol^–1, ΔS_vH^T = 516 ± 109 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 27.1 ± 5.5 kcal·mol^–1). (vi) Similar relative differences are observed in poly(dA–dT)· poly(dA–dT) (ΔH_vH^A = 333 ± 54 kcal·mol^–1, ΔS_vH^A = 961 ± 157 cal·°K^–1·mol^–1, ΔG_vH(25°C)^A = 45.0 ± 7.6 kcal· mol^–1; ΔH_vH^T = 213 ± 30 kcal·mol^–1, ΔS_vH^T = 617 ± 86 cal·°K^–1·mol^–1, ΔG_vH(25°C)^T = 29.3 ± 4.9 kcal·mol^–1). The methodology employed here distinguishes thermodynamic contributions of base stacking, base pairing and backbone conformational ordering in the molecular mechanism of double-helical B DNA formation.