Adaptive Response to Cold Temperatures in Vibrio vulnificus

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35°C to 6°C conditions, it underwent transition to a non-culturable state. Cells adapted to 15°C prior to change to 6°C condition, however, remain viable and culturable. In addition, cultures adapted to 15°C were able to survive better upon freezing at −78°C compared with cultures frozen directly from 35°C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive “protective” proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2′-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with ³⁵S-methionine during exposure at 15°C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15°C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6°C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters. Received: 30 July 1998 / Accepted: 1 October 1998     

Cultivation of low-temperature (15°C), anaerobic, wastewater treatment granules

Aims: Anaerobic sludge granules underpin high‐rate waste‐to‐energy bioreactors. Granulation is a microbiological phenomenon involving the self‐immobilization of several trophic groups. Low‐temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low‐temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15°C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. Methods and Results: One mesophilic (R1; 37°C) and two low‐temperature (R2 and R3, 15°C) laboratory‐scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0·4 mm) granules and were fed a glucose‐based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low‐temperature (LT) bioreactors. Conclusions: Granulation was observed at 15°C in anaerobic bioreactors and was associated with H₂/CO₂‐mediated methanogenesis and distinct community structure development. Significance and Impact of the Study: Granulation underpins high‐rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20°C was not previously documented.     

Methanol as the Primary Methanogenic and Acetogenic Precursor in the Cold Zoige Wetland at Tibetan Plateau

Previous studies suggested that methanol and acetate were the likely methanogenic precursors in the cold Zoige wetland. In this study, the contribution of the two substances to methanogenesis and the conversion in Zoige wetland were analyzed. It was determined that methanol supported the highest CH₄ formation rate in the enrichments of the soil grown with Eleocharis valleculosa, and even higher at 15°C than at 30°C; while hydrogenotrophic methanogenesis was higher at 30°C. Both methanol- and acetate-using methanogens were counted at the highest (10⁷ g⁻¹) in the soil, whereas methanol-using acetogens (10⁸ g⁻¹) were ten times more abundant than either methanol- or acetate-using methanogens. Both methanol and acetate were detected in the methanogenesis-inhibited soil samples, so that both could be the primary methanogenic precursors in E. valleculosa soil. However, the levels of methanol and acetate accumulated in 2-bromoethane-sulfonate (BES)- and CHCl₃-treated soils were in reverse, i.e., higher methanol in CHCl₃- and higher acetate in BES-treated soil, so that methanol-derived methanogenesis could be underestimated due to the consumption by acetogens. Analysis of the soil 16S rRNA genes revealed Acetobacterum bakii and Trichococcus pasteurii to be the dominant methanol-using acetogens in the soil, and a strain of T. pasteurii was isolated, which showed the high conversion of methanol to acetate at 15°C.     

Processing of the 5′-UTR and existence of protein factors that regulate translation of tobacco chloroplast psbN mRNA

The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b ₆ /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5′-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5′-UTRs, the psbN 5′-UTR has two processing sites, at ?39 and ?24 upstream from the initiation site. Processing at ?39 enhanced the translation rate fivefold. In contrast, processing at ?24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5′-UTR has no Shine–Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5′-UTR or with deleted psbN 5′-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5′-UTR. Mobility shift assays using 10 other chloroplast 5′-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.     

Aeromycoflora in the Central Milk Dairy of Calcutta, India

Intramural aeromycological survey was performed at the Central Milk Dairy, Calcutta, covering eight locations within the Dairyusing Burkard personal volumetric air sampler. The locations were butter cold storage (−2 °C), cold store (8 °C), packaging section (23 °C), milk processing section (24 °C), reconstituent of skimmed milk (24 °C), quality control lab (25 °C), raw milk reception (28 °C) and loading dock (26 °C). A number of fungal spores, conidia and mycelia were recorded in different rooms: the highest spore quantity was recorded in the packaging section (23 °C) and the minimum at the butter cold store (−2 °C). The dominant spores consisted of Aspergillus niger, A flavus,Cladosporium sp., Fusarium sp., Curvularia sp.,Alternaria sp., Torula sp., Myrotheciumsp., Helminthosporium sp., Periconia sp.,Nigrospora sp. and Pithomyces sp. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of the molecular conformation of beta-D-O2,2'-cyclouridine in aqueous solution by proton magnetic resonance spectroscopy

The solution conformation of β-D -O²,2′-cyclouridine has been determined at 27 and 88°C in D₂O by proton magnetic resonance spectroscopy. The conformation is described in terms of a fixed syn-like sugar-base torsional angle, a type S furanose ring conformation (similar to 2′-endo), and a temperature-dependent exocyclic C(4)′–C(5′) rotamer population containing approximately 50% of the gauche-gauche form at 27°C. β-D -O²,2′-Cyclouridine 5′-phosphate likewise possesses a type S furanose ring conformation.     

Conformational analysis of the 2'-deoxyribofuranose ring from proton-proton coupling constants: analysis of a nucleoside-carcinogen adduct formed from 2-acetylaminofluorene utilizing a three-state model

The conformation of the sugar moiety of 8-(N-fluoren-2-ylamino)-2′-deoxyguanosine in solution has been examined as a function of temperature by ¹H-nmr spectroscopy. Analysis of coupling constants shows that lowering the temperature to ?50°C in methanol shifts the conformational equilibrium of the sugar ring resulting in a C2′-endo conformation at a mole fraction of 0.97. The computed phase angle of pseudorotation and amplitude of pucker are 154° and 36°, respectively, with very little discrepancy between the five calculated coupling constants and coupling constants extrapolated from the temperature profiles. A computer program has been written enabling a three-state best-fit analysis. The three-state analysis indicates an equilibrium between C2′-endo, C3′-endo, and 04′-endo conformations. In aqueous solution, the computed mole fraction of the 04′-endo form is 0.18 at 30°C. The conformation associated with the sugar ring and the C4′? C5′ bond is compared to that of 2′-deoxyguanosine.     

Functional and structural response of the methanogenic microbial community in rice field soil to temperature change

The microbial community in anoxic rice field soil produces CH₄ over a wide temperature range up to 55°C. However, at temperatures higher than about 40°C, the methanogenic path changes from CH₄ production by hydrogenotrophic plus acetoclastic methanogenesis to exclusively hydrogenotrophic methanogenesis and simultaneously, the methanogenic community consisting of Methanosarcinaceae, Methanoseataceae, Methanomicrobiales, Methanobacteriales and Rice Cluster I (RC‐1) changes to almost complete dominance of RC‐1. We studied changes in structure and function of the methanogenic community with temperature to see whether microbial members of the community were lost or their function impaired by exposure to high temperature. We characterized the function of the community by the path of CH₄ production measuring δ¹³C in CH₄ and CO₂ and calculating the apparent fractionation factor (α_app) and the structure of the community by analysis of the terminal restriction fragment length polymorphism (T‐RFLP) of the microbial 16S rRNA genes. Shift of the temperature from 45°C to 35°C resulted in a corresponding shift of function and structure, especially when some 35°C soil was added to the 45°C soil. The bacterial community (T‐RFLP patterns), which was much more diverse than the archaeal community, changed in a similar manner upon temperature shift. Incubation of a mixture of 35°C and 50°C pre‐incubated methanogenic rice field soil at different temperatures resulted in functionally and structurally well‐defined communities. Although function changed from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a rather narrow temperature range of 42–46°C, each of these temperatures also resulted in only one characteristic function and structure. Our study showed that temperature conditions defined structure and function of the methanogenic microbial community.     

Untenability of the Heteronomous DNA Model for Poly(dA) · Poly(dT) in Solution. This DNA Adopts a Right-Handed B-DNA Duplex in Which the Two Strands are Conformationally Equivalent. A 500 MHz NMR Study Using One Dimensional NOE

Abstract

ID NOE ¹H NMR spectroscopy at 500 MHz was employed to examine the structure of poly(dA)·poly(dT) in solution. NOE experiments were conducted as a function of presaturation pulse length (50, 30, 20 and 10 msec) and.power (19 and 20 db) to distinguish the primary NOEs from spin diffusion. The 10 msec NOE experiments took 49 hrs and over 55,000 scans for each case and the difference spectra were almost free from diffusion.

The spin diffused NOE difference spectra as well as difference NOE spectra in 90% H₂O + 10% D₂O in which TNH3 was presaturated enabled to make a complete assignment of the base and sugar protons. It is shown that poly(dA) ·poly(dT) melts in a fashion in which single stranded bubbles are formed with increasing temperature.

Extremely strong primary NOEs were observed at H2′/H2″ when AH8 and TH6 were presaturated. The observed NOEs at AH2′ and that AH2″ were very similar as were the NOEs at TH2′ and TH2″. The observed NOEs at AH2′ and AH2″when AH8 was presaturated were very similar to those observed at TH2′ and TH2″ when TH6 was presaturated. In addition, presaturation of H1′ of A and T residues resulted in similar NOEs at AH2′/H2″ and TH2′/H2″ region and these NOEs at H2′ and H2″ were distinctly asymmetric as expected in a C2′-endo sugar pucker. There was not a trace of NOE at AH8 and TH6 when AH3′ and TH3′ were presaturated indicating that C3′-endo, × = 30–40° conformation is not valid for this DNA. From these NOE data, chemical shift shielding calculations and stereochemistry based computer modellings, we conclude that poly(dA)·poly(dT) in solution adopts a right- handed B-DNA duplex in which both dA and dT strands are conformationally equivalent with C2′-endo sugar pucker and a glycosyl torsion, ×, of ?73°, the remaining backbone torsion angles being φ′ = 221°, ω′ = 212°, ω = 310°, φ = 149°, ψ = 42°, ψ′ = 139°. The experimental data are in total disagreement with the heteronomous DNA model of Arnott et. al. proposed for the fibrous state. (Arnott, S., Chandrasekaran, R., Hall, I.H., and Puigjaner, L.C., Nucl. Acid Res. 11, 4141, 1983).     

Activity of Stress-related Antioxidative Enzymes in the Invasive Plant Crofton Weed （Eupatorium adenophorum）

Crofton weed is an invasive weed in southwestern China. The activities of several antioxidative enzymes involved in plant protection against oxidative stress were assayed to determine physiological aspects of the crofton weed that might render the plant vulnerable to environmental stress. Stresses imposed on crofton weed were heat （progressively increasing temperatures： 25 ℃, 30 ℃, 35 ℃, 38℃ and 42 ℃ at 24 h intervals）, cold （progressively decreasing temperatures： 25 ℃, 20 ℃, 15℃, 10 ℃ and 5℃ at 24h intervals）, and drought （without watering up to 4days）. The three stresses induced oxidative damage as evidenced by an increase in lipid peroxidation. The effect varied with the stress imposed and the length of exposure. The activity of superoxide dismutase （SOD） increased in response to all stresses but was not significantly different from the controls （P 〈 0.05） when exposed to cold stress. Catalase （CAT） activity decreased in response to heat and drought stress but increased when exposed to cold conditions. Guaiacol peroxidase （POD） and glutathione reductase （GR） activities increased in response to cold and drought but decreased in response to heat stress. The activity of ascorbata peroxidase （APX） responded differently to all three stresses. Monodehydroascorbate reductase （MDHAR） activity decreased in response to heat and drought, and slightly increased in response to the cold stress but was not significantly different from the controls （P 〈 0.05）. The activity of dehydroascorbata reductase （DHAR） increased in response to all three stresses. Taken together, the co-ordinate increase of the oxygen-detoxifying enzymes might be more effective to protect crofton weed from the accumulation of oxygen radicals at low temperatures rather than at high temperatures.     

Adult cold tolerance and potential North American distribution of Myllocerus undecimpustulatus undatus (Coleoptera: Curculionidae)

Cold tolerance and potential distribution of Myllocerus undecimpustulatus undatus Marshall, a polyphagous pest in the United States, were investigated. Adult survivorship after 2 days at 0 °C and ??5 °C averaged 60% and 18%, respectively. Four days of exposure resulted in survivorship of 11% at 0 °C and 4% at ??5 °C, respectively. Summer-collected weevils at ??5 °C through repeated cold exposure of 2 h survived 3 times longer than those subjected to sustained cold period of 10 h. Leaf consumption did not differ among summer-collected weevils at constant 20 °C and repeated cold exposure treatments; weevils under sustained cold exposure consumed less than weevils in repeated cold exposure treatments. Leaf area consumed after cold exposure was 2–4 times greater in winter-collected weevils compared to summer-collected weevils. Leaf consumption by winter-collected weevils decreased as the number of repeated cold exposure periods increased. Locality data from collections in Florida during 2000–2012 were used to produce a correlative model complemented by a mechanistic model from the cold tolerance data to project the potential distribution of M. undecimpustulatus undatus in North America. The models support the hypothesis that M. undecimpustulatus undatus could spread to areas of the southeastern and western United States. The predicted northern distribution followed an isothermal line about 33° North. The niche model defined an area along the western Gulf Coast as unsuitable for the weevil, possibly because the area receives greater annual rainfall than other areas of the southeastern United States and has aquic or udic soil unlike the well-drained sandy soil of peninsular Florida.

     

Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases

Seventeen psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activity were isolated from deep sea sediment of Prydz Bay, Antarctic. They were affiliated with γ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequencing. The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus, and Nocardiopsis. Two Pseudomonas strains, 7193 and 7197, which showed highest amylolytic activity were chosen for further study. The optimal temperatures for their growth and amylase-producing were between 15 and 20°C. Both of the purified amylases showed highest activity at 40°C and pH 9.0, and retained 50% activity at 5°C. The SDS-PAGE and zymogram activity staining showed that the molecular mass of strain 7193 and 7197 amylases were about 60 and 50 kDa respectively. The Pseudomonas sp. 7193 amylase hydrolyzed soluble starch into glucose, maltose, maltotriose, and maltotetraose, indicating that it had both activities of α-amylase and glucoamylase. The product hydrolyzed by Pseudomonas sp. 7197 amylase was meltotetraose.     

Stoichiometry and temperature sensitivity of methanogenesis and CO2 production from saturated polygonal tundra in Barrow,Alaska

Arctic permafrost ecosystems store ~50% of global belowground carbon (C) that is vulnerable to increased microbial degradation with warmer active layer temperatures and thawing of the near surface permafrost. We used anoxic laboratory incubations to estimate anaerobic CO₂ production and methanogenesis in active layer (organic and mineral soil horizons) and permafrost samples from center, ridge and trough positions of water‐saturated low‐centered polygon in Barrow Environmental Observatory, Barrow AK, USA. Methane (CH₄) and CO₂ production rates and concentrations were determined at ?2, +4, or +8 °C for 60 day incubation period. Temporal dynamics of CO₂ production and methanogenesis at ?2 °C showed evidence of fundamentally different mechanisms of substrate limitation and inhibited microbial growth at soil water freezing points compared to warmer temperatures. Nonlinear regression better modeled the initial rates and estimates of Q₁₀ values for CO₂ that showed higher sensitivity in the organic‐rich soils of polygon center and trough than the relatively drier ridge soils. Methanogenesis generally exhibited a lag phase in the mineral soils that was significantly longer at ?2 °C in all horizons. Such discontinuity in CH₄ production between ?2 °C and the elevated temperatures (+4 and +8 °C) indicated the insufficient representation of methanogenesis on the basis of Q₁₀ values estimated from both linear and nonlinear models. Production rates for both CH₄ and CO₂ were substantially higher in organic horizons (20% to 40% wt. C) at all temperatures relative to mineral horizons (<20% wt. C). Permafrost horizon (~12% wt. C) produced ~5‐fold less CO₂ than the active layer and negligible CH₄. High concentrations of initial exchangeable Fe(II) and increasing accumulation rates signified the role of iron as terminal electron acceptors for anaerobic C degradation in the mineral horizons.     

Crystal structure of guanylyl-2′,5′-cytidine dihydrate: An analogue of msDNA-RNA junction in stigmatella aurantiaca

Sequence analysis of msDNA from bacterium such as Stigmatella aurantiaca, Myxococcus xanthus and Escherichia coli B revealed that the guanine residue of the single-stranded RNA is linked to the cytosine residue of the msDNA through a 2′–5′ instead of a conventional 3′–5′ phosphodiester bond. We have now obtained the crystal structure of the self-complementary dimer guanylyl-2′,5′-cytidine (G2′p5′C) that occurs at the msDNA-RNA junction. G2′p5′C crystallizes in the orthorhombic space group P2₁2₁2₁ with a = 8.376(2), b = 16.231(5), c = 18.671(4). CuK ∝ intensity data were collected on a diffractometer in the ω ?2θ scan mode. The amount of 1699 out of 2354 reflections having I ≥ 3σ (F) were considered observed. The structure was solved by direct methods and refined by full-matrix least squares to a R factor of 0.054. The conformation of the guanine base about the glycosyl bond is syn (χ₁ = ?54°) and that of cytosine is anti (χ₂ = 156°). The 5′ and 2′ and ribose moieties show C2′-endo and C3-endo mixed puckering just like in A2′p5′A, A2′p5′C, A2′p5U, and dC3′p5′G. Charge neutralization in G2′p5′C is accomplished through protonation of the cytosine base. An important feature of G2′p5′C is the stacking of guanine on ribose 04′ of cytosine similar to that seen in other 2′–5′ dimers. G2′p5′C, unlike its 3′–5′ isomer, does not form a miniature double helix with the Watson-Crick base-pairing pattern. Comparison of G2′p5′C with A2′p5′C reveals that they are isostructural. A branched trinucleotide model for the msDNA-RNA junction has been postulated. © 1994 John Wiley & Sons, Inc.     

Methanogenesis from methanol at low temperatures by a novel psychrophilic methanogen, "Methanolobus psychrophilus" sp. nov., prevalent in Zoige wetland of the Tibetan plateau

The Zoige wetland of the Tibetan plateau is at permanent low temperatures and is a methane emission heartland of the plateau; however, cold-adaptive methanogens in the soil are poorly understood. In this study, a variety of methanogenic enrichments at 15 degrees C and 30 degrees C were obtained from the wetland soil. It was demonstrated that hydrogenotrophic methanogenesis was the most efficient type at 30 degrees C, while methanol supported the highest methanogenesis rate at 15 degrees C. Moreover, methanol was the only substrate to produce methane more efficiently at 15 degrees C than at 30 degrees C. A novel psychrophilic methanogen, strain R15, was isolated from the methanol enrichment at 15 degrees C. Phylogenetic analysis placed strain R15 within the genus Methanolobus, loosely clustered with Methanolobus taylorii (96.7% 16S rRNA similarity). R15 produced methane from methanol, trimethylamine, and methyl sulfide and differed from other Methanolobus species by growing and producing methane optimally at 18 degrees C (specific growth rate of 0.063 +/- 0.001 h(-1)) and even at 0 degrees C. Based on these characteristics, R15 was proposed to be a new species and named "Methanolobus psychrophilus" sp. nov. The K(m) and V(max) of R15 for methanol conversion were determined to be 87.5 +/- 0.4 microM and 0.39 +/- 0.04 mM h(-1) at 18 degrees C, respectively, indicating a high affinity and conversion efficiency for methanol. The proportion of R15 in the soil was determined by quantitative PCR, and it accounted for 17.2% +/- 2.1% of the total archaea, enumerated as 10(7) per gram of soil; the proportion was increased to 42.4% +/- 2.3% in the methanol enrichment at 15 degrees C. This study suggests that the psychrophilic methanogens in the Zoige wetland are likely to be methylotrophic and to play a role in methane emission of the wetland.