Structural components of alginic acid. I. The crystalline structure of poly-beta-D-mannuronic acid. Results of x-ray diffraction and polarized infrared studies

A Structure determination of the naturally occuring marine algal polysaccharide poly-β-D -mannuronic acid is described. The structure consists of 1e → 4e linked D -mannuronic acid chains with the monosaccharide units in the C1 chair conformation. The X-ray fiber diffraction photograph obtained from bundles of fibers prepared from Fucus vesiculosus has been indexed to an orthorhombic unit cell in which a =7.6 Å, b (fiber axis) = 10.4 Å, c = 8.6 Å, the unit cell containing two disaccharide chain segments with space group P2₁2₁2₁. A sheet-like structure involving one intra-chain, one intra-sheet, and one inter-sheet hydrogen bond per monosaccharide is proposed. Features of the chain-packing arrangement are compared with mannan.     

Efficiency of bacterial protein synthesis during anaerobic degradation of cattle waste

The rate of [15N]ammonia (15NH3) uptake or incorporation into bacterial cells was studied, using stirred, 3-liter benchtop digestors fed on a semicontinuous basis with cattle waste. The fermentations were carried out at 40 and 60 degrees C and at four different loading rates (3, 6, 9, and 12 g of volatile solids per liter of reactor volume per day). The rate of NH3-N incorporation for the period 1 to 5 h after feeding at the four different loading rates was 0.49, 0.83, 1.05, and 1.08 mg/liter per h in the mesophilic digestor and 0.68, 1.07, 1.17, and 1.21 mg/liter per h in the thermophilic digestor. Values were lower 7 to 21 h after feeding in both digestors and were related to the rate of fermentation or CH4 production. In the mesophilic digestors, the rate of bacterial cell production ranged from 3.97 to 8.72 mg of dry cells per liter per h, 1 to 5 h after feeding at the different loading rates. Corresponding values for the thermophilic digestors ranged from 5.46 to 9.77 mg of dry cells per liter per h. Cell yield values ranged from 2.3 to 3.1 mg of dry cells per mol of CH4 produced in the mesophilic and thermophilic digestors at the two lower loading rates. The values were higher (2.8 to 3.4) in the mesophilic digestors at the two higher loading rates because of the accumulation of propionate and a consequent reduction in CH4 production. Low cell yields such as those measured in this study are characteristic of low-specific-growth rates under energy-limited conditions.     

Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation.

The principal DNA restriction-modification system of the cellulolytic ruminal bacterium Ruminococcus flavefaciens FD-1 is described. The restriction endonuclease RflFI could be separated from cell extracts by phosphocellulose and heparin-sepharose chromatography. Restriction enzyme digests utilizing RflFI alone or in combination with SalI, a restriction enzyme isolated from Streptomyces albus G, showed that the DNA sequence recognized by RflFI either overlapped or was the same as that recognized by SalI. DNA sequence analysis confirmed that RflFI was identical in activity to SalI, with the recognition sequence being 5'-GTCGAC-3' and cleavage occurring between G and T. Adenine methylation within this sequence can be catalyzed in vitro by TaqI methylase, and this inhibited the cleavage of plasmid DNA molecules by RflFI and SalI. Chromosomal DNA from R. flavefaciens FD-1 is also methylated within this DNA sequence because neither restriction endonuclease could degrade this DNA substrate. These findings provide a means to protect plasmid molecules from degradation prior to gene transfer experiments with R. flavefaciens FD-1.     

Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Influence of translational efficiency on the stability of the mRNA for ribosomal protein S20 in Escherichia coli.

A set of plasmids was constructed so as to contain point mutations which limit the efficiency and/or extent of translation of the gene for ribosomal protein S20. These plasmids were transformed into strains carrying mutations in the genes for polynucleotide phosphorylase (pnp-7), RNase E (rne-1), or both. Subsequently, the effect of translational efficiency on mRNA abundance and chemical half-life was determined. The data indicated that mutations altering translational efficiency also affected mRNA levels over a 10-fold range. This variation in mRNA abundance occurred independently of mutations in either RNase E or polynucleotide phosphorylase, both of which determine the stability of the S20 mRNAs. Moreover, a mutation at codon 15 which caused premature termination of translation of the S20 mRNA did not significantly reduce its stability in different genetic backgrounds. We propose a model in which initiation of translation competes for early steps in mRNA turnover, which could be the binding of RNase E itself or as a complex to one or more sites near the 5' terminus of the S20 mRNA.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

3-Phenylpropanoic Acid Improves the Affinity of Ruminococcus albus for Cellulose in Continuous Culture

A continuous-culture device, adapted for use with solid substrates, was used to evaluate the effects of 3-phenylpropanoic acid (PPA) upon the ability of the South African strain Ruminococcus albus Ce63 to ferment cellulose. Steady states of fermentation were established with a dilution rate of 0.17 h⁻¹, and the extent and volumetric rates of cellulose fermentation were determined over four consecutive days. When the growth medium contained no additions (control), 25 μM phenylacetate alone, 25 μM PPA alone, or 25 μM each of phenylacetate and PPA, the extent of cellulose hydrolysis was determined to be 41.1, 35.7, 90.2, and 86.9%, respectively, and the volumetric rate of cellulose hydrolysis was 103.0, 97.9, 215.5, and 230.4 mg liter⁻¹ h⁻¹, respectively. To evaluate the effect of PPA availability on affinity for cellulose, the values for dilution rate and extent of cellulose hydrolysis were used in combination with values for maximum specific growth rate determined from previous studies of growth rates and kinetics of cellulose hydrolysis. The findings support the contention that PPA maintains a competitive advantage for R. albus when grown in a dynamic, fiber-rich environment.