333 research outputs found
Methanogenesis by Methanosarcina acetivorans involves two structurally and functionally distinct classes of heterodisulfide reductase
Biochemical studies have revealed two distinct classes of Coenzyme B-Coenzyme M heterodisulfide (CoB-S-S-CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methaneproducing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane-bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic methanogenesis, based on reduced growth and methanogenesis rates of an hdrA1C1B1 mutant on methylotrophic substrates and downregulation of the genes during growth on acetate. This conclusion is further supported by phylogenetic analysis showing that the presence of hdrA1 in an organism is specifically correlated with the presence of genes for methylotrophic methanogenesis. Examination of mRNA abundance in methanol-grown DhdrA1C1B1 strains relative to wild-type revealed upregulation of genes required for synthesis of (di)methylsulfide and for transport and biosynthesis of CoB-SH and CoM-SH, suggesting that the mutant has a defect in electron transfer from ferredoxin to CoB-S-S-CoM that causes cofactor limitation
Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase
The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F_(420)-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H_2-CO_2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO2 in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H_2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech Δfrh and Δech Δfrh Δvht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H_2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO_2 produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F_(420) dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F_(420). The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed
An Optimal Design for Universal Multiport Interferometers
Universal multiport interferometers, which can be programmed to implement any
linear transformation between multiple channels, are emerging as a powerful
tool for both classical and quantum photonics. These interferometers are
typically composed of a regular mesh of beam splitters and phase shifters,
allowing for straightforward fabrication using integrated photonic
architectures and ready scalability. The current, standard design for universal
multiport interferometers is based on work by Reck et al (Phys. Rev. Lett. 73,
58, 1994). We demonstrate a new design for universal multiport interferometers
based on an alternative arrangement of beam splitters and phase shifters, which
outperforms that by Reck et al. Our design occupies half the physical footprint
of the Reck design and is significantly more robust to optical losses.Comment: 8 pages, 4 figure
Virus in Water. II. Evaluation of Membrane Cartridge Filters for Recovering Low Multiplicities of Poliovirus from Water
The efficiency of a Millitube MF cartridge filter, a membrane filter, for recovery of poliovirus from 100-gal volumes of both fresh (tap) and estuarine water was determined. In the high multiplicity of virus input-output experiments, recovery of 97% or greater of input virus was achieved in both types of water when the final concentration of divalent cation as Mg2+ was 1,200 ÎĽg/ml and the pH was 4.5. Virus was effectively eluted from the membrane cartridge with 5Ă— nutrient broth in 0.05 M carbonate-bicarbonate buffer at pH 9.0. Four elutions of 250 ml each were used. In the low multiplicity of virus input-output experiments under the same cationic and pH conditions, up to 67% of the input virus was recovered when the virus was further concentrated from the eluates by the aqueous polymer two-phase separation technique. The volume reduction was 126,000-190,000 to 1. The use of the combined techniques, i.e., membrane adsorption followed by aqueous polymer two-phase separation, provided a highly sensitive, simple, and remarkably reliable sequential methodology for the quantitative recovery of poliovirus occurring at multiplicities as low as 1 to 2 plaque-forming units per 5 gal of water
Reply to letter from William W. Eustis, December 9, 1940.
https://digitalmaine.com/alien_corresp/1021/thumbnail.jp
New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species
A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage PhiC31. Host strains expressing the PhiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetRgene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion
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