Complete genome sequence of Methanoculleus marisnigri Romesser et al. 1981 type strain JR1

Methanoculleus marisnigri Romesser et al. 1981 is a methanogen belonging to the order Methanomicrobiales within the archaeal phylum Euryarchaeota. The type strain, JR1, was isolated from anoxic sediments of the Black Sea. M. marisnigri is of phylogenetic interest because at the time the sequencing project began only one genome had previously been sequenced from the order Methanomicrobiales. We report here the complete genome sequence of M. marisnigri type strain JR1 and its annotation. This is part of a Joint Genome Institute 2006 Community Sequencing Program to sequence genomes of diverse Archaea.     

Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility

A protein spin label and lipid spin probes were used to study the temperature-dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20°C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12°C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14°C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes.     

A mutational analysis of active site residues in trans-3-chloroacrylic acid dehalogenase

trans-3-Chloroacrylic acid dehalogenase (CaaD) catalyzes the hydrolytic dehalogenation of trans-3-haloacrylates to yield malonate semialdehyde by a mechanism utilizing βPro-1, αArg-8, αArg-11, and αGlu-52. These residues are implicated in a promiscuous hydratase activity where 2-oxo-3-pentynoate is processed to acetopyruvate. The roles of three nearby residues (βAsn-39, αPhe-39, and αPhe-50) are unexplored. Mutants were constructed at these positions (βN39A, αF39A, αF39T, αF50A and αF50Y) and kinetic parameters determined along with those of the αR8K and αR11K mutants. Analysis indicates that αArg-8, αArg-11, and βAsn-39 are critical for dehalogenase activity whereas αArg-11 and αPhe-50 are critical for hydratase activity. Docking studies suggest structural bases for these observations.     

Naphthalene biosorption in soil/water systems of low or high sorptive capacity

 A model system was developed for evaluating naphthalene biosorption based on the use of a mutant (strain TG-5 Nah^-) derived from a naphthalene-degrading Pseudomonas fluorescens isolate. Cells of strain TG-5 had a sorptive capacity for naphthalene (partition coefficient of 380 cm³/g) significantly higher than a soil with a 5.1% organic carbon content (partition coefficient of 41 cm³/g). However, experimental results and a mass balance model demonstrated that, in soil systems of high organic carbon content, the mass of naphthalene associated with biological solids is insignificant. In contrast, in a soil system of nonsorptive Ottawa sand, up to 10% of the initial naphthalene was demonstrated experimentally, and by modeling, to be associated with cells of strain TG-5. Received: 6 June 1994/Received revision: 12 September 1994/Accepted: 28 September 1994     

Why rods and cones?

In continuation of a previous paper, the auxiliary signal focussing properties of more complicated spinal neuronal networks are considered here. Special emphasis is put on the distributive function of the recurrent feedback system of -motoeurones, but also the inhomogeneous distribution of excitatory and inhibitor input to motoneurones is taken into account as an essential prerequisite for signal focussing. Simple hypothetical calculations for steady-state conditions yield a more vivid insight into the interaction of the two types of neuronal circuitry contributing to signal focussing.     

Presence of coenzyme M derivatives in the prosthetic group (coenzyme MF430) of methylcoenzyme M reductase from Methanobacterium thermoautotrophicum

2-Mercaptoethanesulfonic acid (coenzyme M), or a derivative of it, and a yellow chromophore, known as the nickel-containing tetrapyrrole factor F₄₃₀, occur in the prosthetic group of methylcoenzyme M reductase in an equimolar amount, and bound to each other; this enzyme catalyzes the final step of methane production. The prosthetic group, which is called coenzyme MF₄₃₀, was isolated from the purified enzyme and was extracted from cells. The presence of coenzyme M was confirmed by a bioassay using Methanobrevibacter ruminantium and by the use of chemical and physicochemical analyses.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.     

Effect of four antimicrobials against an Encephalitozoon sp. (Microsporidia) in a grasshopper host

Encephalitozoon spp. are the primary microsporidial pathogens of humans and domesticated animals. In this experiment, we test the efficacy of 4 commercial antimicrobials against an Encephalitozoon sp. infecting a grasshopper (Romalea microptera) host. Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts (93% and 88% respectively), whereas spore counts of grasshoppers fed quinine produced a non-significant 53% reduction in spores, and those fed streptomycin a non-significant 29% increase in spores, compared to the control. We observed a moderate dose-response effect for thiabendazole, whereby spore count decreased as drug consumption increased. No thiabendazole-treated animals died, whereas 27% of streptomycin-treated animals died, suggesting that thiabendazole was not toxic at the doses administered. The deaths among streptomycin-treated animals may have been caused by drug toxicity, parasite burden, or both. Although fumagillin and thiabendazole significantly reduced spore counts, in no individual was the pathogen totally eliminated. Our data confirm that microsporidia are difficult to control and that fumagillin and thiabendazole are partially effective antimicrobials against this group. Our study suggests that quinine and related alkaloids should be further examined for antimicrosporidial activity, and streptomycin should be examined as a possible enhancer of microsporidiosis.