Reaction intermediate rotation during the decarboxylation of coproheme to heme b in C. diphtheriae

Monoderm bacteria utilize coproheme decarboxylases (ChdCs) to generate heme b by a stepwise decarboxylation of two propionate groups of iron coproporphyrin III (coproheme), forming two vinyl groups. This work focuses on actinobacterial ChdC from Corynebacterium diphtheriae (CdChdC) to elucidate the hydrogen peroxide-mediated decarboxylation of coproheme via monovinyl monopropionyl deuteroheme (MMD) to heme b, with the principal aim being to understand the reorientation mechanism of MMD during turnover. Wild-type CdChdC and variants, namely H118A, H118F, and A207E, were studied by resonance Raman and ultraviolet-visible spectroscopy, mass spectrometry, and molecular dynamics simulations. As actinobacterial ChdCs use a histidine (H118) as a distal base, we studied the H118A and H118F variants to elucidate the effect of 1) the elimination of the proton acceptor and 2) steric constraints within the active site. The A207E variant mimics the proximal H-bonding network found in chlorite dismutases. This mutation potentially increases the rigidity of the proximal site and might impair the rotation of the reaction intermediate MMD. We found that both wild-type CdChdC and the variant H118A convert coproheme mainly to heme b upon titration with H₂O₂. Interestingly, the variant A207E mostly accumulates MMD along with small amounts of heme b, whereas H118F is unable to produce heme b and accumulates only MMD. Together with molecular dynamics simulations, the spectroscopic data provide insight into the reaction mechanism and the mode of reorientation of MMD, i.e., a rotation in the active site versus a release and rebinding.     

Acetate Degradation at 70°C in Upflow Anaerobic Sludge Blanket Reactors and Temperature Response of Granules Grown at 70°C

Anaerobic acetate degradation at 70°C and at 55°C (as a reference) was studied by running laboratory upflow anaerobic sludge blanket (UASB) reactors inoculated with mesophilic granular sludge. In UASB reactors fed with acetate-containing media (3 g of chemical oxygen demand [COD] per liter, corresponding to 47 mM acetate) approximately 50 days was needed at 70°C and less than 15 days was needed at 55°C to achieve an effluent COD of 500 to 700 mg/liter. In the UASB reactors at both 70 and 55°C up to 90% of the COD was removed. Batch assays showed that sludges from two 70°C UASB reactors, one run at a low effluent acetate concentration and the other run at a high effluent acetate concentration, exhibited slightly different responses to temperatures in the range from 37 to 70°C. Both 70°C sludges, as well as the 55°C sludge, produced methane at temperatures of 37 to 73°C. The 55°C sludge exhibited shorter lag phases than the 70°C sludges and higher specific methane production rates between 37 and 65°C.     

Bacterial Activity at −2 to −20°C in Arctic Wintertime Sea Ice

Arctic wintertime sea-ice cores, characterized by a temperature gradient of −2 to −20°C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4′,6′-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O₂-based respiration), the abundances of total, particle-associated (>3-μm), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (−2 to −20°C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (−20°C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to −20°C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.     

Structural Changes in Mammalian Cells Associated with Cooling to -79°C

Suspensions of HeLa and S37 cells, with and without added glycerol, were cooled in stages to -79°C. and held at that temperature for 30 minutes. After warming to room temperature the cells were fixed, sectioned, and compared by phase contrast and electron microscopy with similar specimens kept at room temperature. Correlated viability tests were made. Abnormal cytological characteristics, visible with the phase contrast microscope, were clearly related to the sequence of freezing and thawing, and the proportion of altered cells was highest in specimens cooled without glycerol. Electron microscopy showed that even in the presence of glycerol all cells were markedly altered, with distinctive vesiculation and disruption of the various intracellular membranes. There is evidence that much cytoplasmic damage is compatible with survival, but it seems likely that separation of the two layers of the nuclear envelope and rearrangement of the nuclear contents are signs of irreversible damage. The findings lend some support to the belief that cell death on cooling is due largely to denaturation of semipermeable membranes, caused by the increasing concentration of electrolytes.     

Changes in Bacteria Recoverable from Subsurface Volcanic Rock Samples during Storage at 4°C

The abundance of viable microorganisms recovered from deep subsurface volcanic rock samples increased after rock perturbation and storage for 1 week at 4°C, while the diversity and evenness of recoverable heterotrophic bacterial communities generally decreased. One sample of each morphologically distinct colony type, recovered both before and after storage of U12n rock samples, was purified and characterized by fatty acid methyl ester (MIDI) and API rapid NFT strips. As determined by MIDI cluster analysis, the composition of the recoverable microbial communities changed with storage of rock samples; some groups of organisms were recovered only before, only after, or at both sample times. In general, the isolates recovered only after storage of rock samples had a greater ability to utilize the carbohydrates included in API test strips and had faster generation times than isolates recovered only on initial plating. The nutritional versatility and faster growth rates of organisms recovered in higher proportions after sample storage provide evidence that some microbial community changes may be due to the proliferation of a few bacterial types. However, because some new genera are recovered only after storage, the possibility also exists that dormant bacterial types are resuscitated during sample perturbation and storage.     

Initial Heat Production in Isometric Frog Muscle at 15°C

An infrared radiation-detecting system was used to measure initial heat production in bull frog sartorius muscle at 15°C. Numerous tests with the system showed that thermal artifacts were not noticeable. Many previous measurements with myothermic thermopiles were corroborated with this method. In addition, a cooling phase as large as 0.39 of peak exothermicity was found during and after relaxation. Cooling diminished with both increasing sarcomere length and increasing duration of mechanical activity. No large rapid increase in heat rate accompanied a 0.6 reactivation at the peak of twitch tension. Above rest length, initial heat rate and the heat produced up to the peak of tension decreased nearly proportionally with overlap of myofilaments, while the total twitch initial heat decreased slightly.     

Solid Medium for Culturing Black Smoker Bacteria at Temperatures to 120°C

A solid, highly thermostable medium, based on the new gelling agent GELRITE, was devised to facilitate the culturing of extremely thermophilic microorganisms from submarine hydrothermal vents. The medium remained solid at temperatures to 120°C at vapor pressures and hydrostatic pressures to 265 atm. It proved useful to its maximum tested limits in isolating colonies of black smoker bacteria from hydrothermal fluids recently collected at the Juan de Fuca Ridge in the Pacific Ocean.     

Growth of Rhizobium japonicum Strains at Temperatures Above 27°C

A study was conducted to examine the growth responses of different Rhizobium japonicum strains to increasing temperatures, determine the degree of variability among strains in those responses, and identify temperature-related growth characteristics that could be used to select temperature-tolerant strains. Each of 42 strains was grown in liquid culture for 96 h at 19 incubation temperatures ranging from 27.4 to 54.1°C in a temperature gradient apparatus. Growth was estimated by measuring the change in optical density over time. Strains differed in their responses to increasing temperatures. Three characteristic temperatures were determined for each strain: the temperature giving the maximum optical density at 96 h (optimum temperature), the maximum temperature allowing a continuous increase in optical density during the 96-h period (maximum permissive temperature), and the maximum temperature allowing growth of the cultures after they were transferred to a uniform incubation temperature of 28°C (maximum survival temperature). The three characteristic temperatures varied among strains and had the following ranges: optimum temperature, from 27.4 to 35.2°C; maximum permissive temperature, from 29.8 to 38.0°C; and maximum survival temperature, from 33.7 to 48.7°C. Significant positive correlations were found between maximum permissive temperature and optimum temperature and between maximum permissive temperature and maximum survival temperature. Eight strains which had the highest maximum permissive temperature, optimum temperature, and maximum survival temperature were considered tolerant of high temperatures and were able to grow at temperatures higher than those previously reported for the most tolerant R. japonicum strains. The strains were of diverse geographical origin, but the response to high temperatures was not related to their origin. Evaluation of the temperature responses in pure culture may be useful in the search for R. japonicum strains better suited to environments in which high soil temperature is a limiting factor.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Finite element modeling of α-helices and tropocollagen molecules referring to spike of SARS-CoV-2

The newly developed finite element (FE) modeling at the atomic scale was used to predict the static and dynamic response of the α-helix (AH) and tropocollagen (TC) protein fragments, the main building blocks of the spike of the SARS-CoV-2. The geometry and morphology of the spike’s stalk and its connection to the viral envelope were determined from the combination of most recent molecular dynamics (MD) simulation and images of cryoelectron microscopy. The stiffness parameters of the covalent bonds in the main chain of the helix were taken from the literature. The AH and TC were modeled using both beam elements (wire model) and shell elements (ribbon model) in FE analysis to predict their mechanical properties under tension. The asymptotic stiffening features of AH and TC under tensile loading were revealed and compared with a new analytical solution. The mechanical stiffnesses under other loading conditions, including compression, torsion, and bending, were also predicted numerically and correlated with the results of the existing MD simulations and tests. The mode shapes and natural frequencies of the spike were predicted using the built FE model. The frequencies were shown to be within the safe range of 1–20 MHz routinely used for medical imaging and diagnosis by means of ultrasound. These results provide a solid theoretical basis for using ultrasound to study damaging coronavirus through transient and resonant vibration at large deformations.     

Autumnal Biomass and Potential Productivity of Salt Marsh Fungi from 29° to 43° North Latitude along the United States Atlantic Coast

It has been established that substantial amounts of fungal mass accumulate in standing decaying smooth cordgrass (Spartina alterniflora) marshes in the southeastern United States (e.g., in standing decaying leaf blades with a total fungal organic mass that accounts for about 20% of the decay system organic mass), but it has been hypothesized that in marshes farther north this is not true. We obtained samples of autumnal standing decaying smooth cordgrass from sites in Florida to Maine over a 3-year period. The variation in latitude could not explain any of the variation in the living fungal standing crop (as determined by ergosterol content) or in the instantaneous rates of fungal growth (as determined by acetate incorporation into ergosterol at a standard temperature, 20°C), which led to the conclusion that the potential levels of fungal production per unit of naturally decaying grass are not different in northern and southern marshes. Twenty-one percent of the variation in the size of the living fungal standing crop could be explained by variation in the C/N ratio (the higher the C/N ratio the smaller the fungal crop), but the C/P ratio was not related to the size of the fungal crop. Instantaneous rates of fungal growth were negatively related to the size of the living fungal crop (r = −0.35), but these rates were not correlated with C/nutrient ratios. The same two predominant species of ascomycetes (one Phaeosphaeria species and one Mycosphaerella species) were found ejecting ascospores from standing decaying smooth cordgrass blades at all of the sites examined from Florida to Maine.     

Involvement of Two Specific Causes of Cell Mortality in Freeze-Thaw Cycles with Freezing to −196°C

The purpose of this study was to examine cell viability after freezing. Two distinct ranges of temperature were identified as corresponding to stages at which yeast cell mortality occurred during freezing to −196°C. The upper temperature range was related to the temperature of crystallization of the medium, which was dependent on the solute concentration; in this range mortality was prevented by high solute concentrations, and the proportion of the medium in the vitreous state was greater than the proportion in the crystallized state. The lower temperature range was related to recrystallization that occurred during thawing. Mortality in this temperature range was increased by a high cooling rate and/or high solute concentration in the freezing medium and a low temperature (less than −70°C). However, a high rate of thawing prevented yeast mortality in this lower temperature range. Overall, it was found that cell viability could be conserved better under freezing conditions by increasing the osmotic pressure of the medium and by using an increased warming rate.