Effect of salinity on growth of four strains of Rhizobium and their infectivity and effectiveness on two species of Acacia

Two Rhizobium strains (WU1001 and WU1008) were isolated from nodules of Acacia redolens growing in saline areas of south-west Australia, and two strains selected from the University of Western Australia's culture collection (WU429 isolated from A. saligna and WU433 from A. cyclops). The growth of each in buffered, yeast extract mannitol broth culture was largely unaffected by salt up to 300 mM NaCl. A slight increase in lag time occurred at concentrations of 120 mM NaCl and above, but cell number at the static phase was not affected. Each of the four Rhizobium strains tested accumulated Na⁺ but showed decreasing levels of sugar with increasing salt in the external medium. Amino acid levels also increased, in some cases by more than tenfold. However, the relative proportion of each remained fairly constant in the bacteria, irrespective of salt treatment. Only trace quantities of proline were detected and there was no increase in this amino acid with salt. Acidic amino acids (glutamate and aspartate) remained as a constant proportion.Rhizobium strains WU429, WU1001 and WU1008 produced effective nodules on both A. cyclops and A. redolens grown in sand with up to 80 mM NaCl (added in nutrient solutions free of nitrogen). Strain WU433 was highly infective on both Acacia species tested at low salt concentrations (2–40 mM NaCl), but infection was sensitive to salt levels at 120 mM NaCl and above. Nodules formed with strain WU433 were, however, ineffective on both A. redolens and on A. cyclops and showed nil or negligible rates of acetylene reduction at all salt concentrations. Strains WU429, WU1001 and WU1008 in combination with a highly salt-tolerant provenance of A. redolens formed symbioses which did not vary significantly in nodule number and mass, specific nodule activity or total N content irrespective of salt level up to 160 mM NaCl. On a more salt sensitive provenance of A. redolens and on A. cyclops the infectivity and effectivity of the Rhizobium strains tested usually decreased as the external salt concentration increased. These data are interpreted to indicate that tolerance of the legume host was the most important factor determining the success of compatible Rhizobium strains in forming effective symbioses under conditions of high soil salinity.     

Adaptation of Nodulated Soybean (Glycine max L. Merr.) to Growth in Rhizospheres Containing Nonambient pO(2)

Nodulated soybean (Glycine max L. Merr. cv White Eye inoculated with Bradyrhizobium japonicum strain CB 1809) plants were cultured in the absence of combined N from 8 to 28 days with their root systems maintained continuously in 1, 2.5, 5, 10, 20, 40, 60, or 80% O₂ (volume/volume) in N₂. Plant dry matter yield was unaffected by partial pressure of oxygen (pO₂) and N₂ fixation showed a broad plateau of maximum activity from 2.5 to 40 or 60% O₂. Slight inhibition of nitrogenase activity occurred at 1% O₂ and as much as 50% inhibition occurred at 80% O₂. Low pO₂ (less than 10%) decreased nodule mass on plants, but this was compensated for by those nodules having higher specific nitrogenase activities. Synthesis and export of ureides in xylem was maintained at a high level (70-95% of total soluble N in exudate) over the range of pO₂ used. Measurements of nitrogenase (EC 1.7.99.2) activity by acetylene reduction indicated that adaptation of nodules to low pO₂ was largely due to changes in ventilation characteristics and involved increased permeability to gases in those grown in subambient pO₂ and decreased permeability in those from plants cultured with their roots in pO₂ greater than ambient. A range of structural alterations in nodules resulting from low pO₂ were identified. These included increased frequency of lenticels, decreased nodule size, increased volume of cortex relative to the infected central tissue of the nodule, as well as changes in the size and frequency of extracellular voids in all tissues. In nodules grown in air, the inner cortex differentiated a layer of four or five cells which formed a band, 40 to 50 micrometers thick, lacking extracellular voids. This was reduced in nodules grown in low pO₂ comprising one or two cell layers and being 10 to 20 micrometers thick in those from 1% O₂. Long-term adaptation to different external pO₂ involved changes which modify diffusive resistance and are additional to adjustments in the variable diffusion barrier.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.     

Neutron and cobalt-60 gamma irradiation produce similar changes in DNA supercoiling

The effects of 60Co gamma-ray and d(20 MeV)Be neutron irradiation on DNA supercoiling have been studied using a nucleoid rewinding technique. Irradiation of viable CHO AA8 cells on ice with from 4 to 25 Gy of either radiation produced a similar resistance to rewinding of nuclear supercoils after treatment with ethidium bromide. The restitution from the effects of 12 Gy of either radiation was also similar, leaving no detectable residual damage. The discrepancy between these data and the reduced ability of neutrons to produce DNA breaks, as defined by the alkaline elution assay, is explained by the discontinuous deposition of dose associated with neutron irradiation. It is suggested from a microdosimetric analysis that the neutron radiation interacts with DNA at sites on average 5-10 times further apart than the interactions with gamma rays. The long DNA sequences which results after neutron irradiation are consequently eluted inefficiently during alkaline elution, giving a reported RBE of approximately 0.3. Restrictions in the rewinding of individual supercoils are not dependent on the interionization distance and thus give rise to an RBE of approximately 1. Furthermore, the complete removal of DNA damage, as measured by this technique, supports the hypothesis that neutron toxicity is associated with incorrect, not incomplete, rejoining of the DNA molecule.     

Some chemical and physical properties of extracellular polysaccharides produced by Butyrivibrio fibrisolvens strains

Most strains of Butyrivibrio fibrisolvens are known to produce extracellular polysaccharides (EPs). However, the rheological and functional properties of these EPs have not been determined. Initially, 26 strains of Butyrivibrio were screened for EP yield and apparent viscosities of cell-free supernatants. Yields ranged from less than 1.0 to 16.3 mg per 100 mg of glucose added to the culture. Viscosities ranged from 0.71 to 5.44 mPa.s. Five strains (CF2d, CF3, CF3a, CE51, and H10b) were chosen for further screening. The apparent viscosity of the EP from each of these strains decreased by only 50 to 60% when the shear rate was increased from 20 to 1,000 s-1. Strain CE51 produced the EP having the highest solution viscosity. A detailed comparison of shear dependency of the EP from strain CF3 with xanthan gum showed that this EP was less shear sensitive than xanthan gum and, at a shear rate of 1,000 s-1, more viscous. EPs from strains CF3 and H10b were soluble over a wide range of pH (1 to 13) in 80% (vol/vol) ethanol-water or in 1% (wt/vol) salt solutions. The pH of 1% EP solutions was between 4.5 and 5.5. Addition of acid increased solution viscosities, whereas addition of base decreased viscosity. EPs from strains CF3, CE51, and H10b displayed qualitatively similar infrared spectra. Calcium and sodium were the most abundant minerals in the three EPs. The amounts of magnesium, calcium, and iron varied considerably among the EPs, but the potassium contents remained relatively constant.     

Time-resolved fluorescence studies of tryptophan mutants of Escherichia coli glutamine synthetase: conformational analysis of intermediates and transition-state complexes.

Single tryptophan-containing mutants of low adenylylation state Escherichia coli glutamine synthetase have been studied by frequency-domain fluorescence spectroscopy in the presence of various substrates and inhibitors. At pH 6.5, the Mn-bound wild-type enzyme (wild type has two tryptophans/subunit) and the mutant enzymes exhibit heterogeneous fluorescence decay kinetics; the individual tryptophans are adequately described by a triple exponential decay scheme. The recovered lifetime values are 5.9 ns, 2.6 ns, and 0.4 ns for Trp-57 and 5.8 ns, 2.3 ns, and 0.4 ns for Trp-158. These values are nearly identical to the previously reported results at pH 7.5 (Atkins, W.M., Stayton, P.S., & Villafranca, J.J., 1991, Biochemistry 30, 3406-3416). In addition, Trp-57 and Trp-158 both exhibit an ATP-induced increase in the relative fraction of the long lifetime component, whereas only Trp-57 is affected by this ligand at pH 7.5. The transition-state analogue L-methionine-(R,S)-sulfoximine (MSOX) causes a dramatic increase in the fractional intensity of the long lifetime component of Trp-158. This ligand has no effect on the W158S mutant protein and causes a small increase in the fractional intensity of the long lifetime component of the W158F mutant protein. Addition of glutamate to the ATP complex, which affords the gamma-glutamylphosphate-ADP complex, results in the presence of new lifetime components at 7, 3.2, and 0.5 ns for Trp-158, but has no effect on Trp-57. Similar results were obtained when ATP was added to the MSOX complex; Trp-57 exhibits heterogeneous fluorescence decay with lifetimes of 7, 3.5, and 0.8 ns. Decay kinetics of Trp-158 are best fit to a nearly homogeneous decay with a lifetime of 5.5 ns in the MSOX-ATP inactivated complex. These results provide a model for the sequence of structural and dynamic changes that take place at the Trp-57 loop and the central loop (Trp-158) during several intermediate stages of catalysis.     

Thermotropic lipid phase separation in the human immunodeficiency virus

The presence of thermodependent lipid domains in the envelope of the human immunodeficiency virus (HIV) was studied. HIV was propagated in Hut-78 cells and purified by differential-gradient centrifugation. Since the virus was highly infectious in cell culture and Western blots of detergent-inactivated HIV showed envelope proteins when exposed to sera containing anti-HIV antibodies, this viral preparation was not deficient in 'spike' or 'knob' particles. Electron spin resonance (ESR) studies of intact HIV labeled with 5-nitroxide stearate (5-NS) indicated that a temperature-dependent lipid phase separation occurs with a high onset at approx. 42 degrees C and a low onset at approx. 15 degrees C. Cooling below 42 degrees C induces 5-NS clustering. Similar phase separations with high onsets at approx. 37-38 degrees C were previously identified in 5-NS labeled human erythrocytes (cholesterol/phospholipid (C/P) molar ratio = 0.90) and cholesterol-loaded (C/P = 0.85-0.98) rat liver plasma membranes. These were attributed to a temperature-sensitive redistribution of endogenous lipid components such that 5-NS is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains at low temperatures. Since HIV has a lipid envelope with a similarly high C/P of 0.88 (Aloia et al. (1988) Proc. Natl. Acad. Sci. USA 85, 900-904), cholesterol-rich and cholesterol-poor domains also probably exist in HIV at physiologic temperatures. The reduced stability and infectivity of HIV noted on heating above 42 degrees C may be due, in part, to the abolition of these thermodependent domains.     

The structure of crystalline Escherichia coli-derived rat intestinal fatty acid-binding protein at 2.5-A resolution

Rat intestinal fatty acid-binding protein (I-FABP) is an abundant cytoplasmic protein which is synthesized in the small intestinal lining cell where it is thought to participate in the absorption and intracellular metabolism of fatty acids. Each mole of this 132-residue polypeptide binds 1 mol of long chain fatty acid in a noncovalent fashion. Because of its small size and single ligand-binding site, I-FABP represents an attractive model for defining the molecular details of long chain fatty acid-protein interactions. The structure of Escherichia coli-derived rat I-FABP has now been solved to 2.5 A resolution using three isomorphous heavy atom derivatives. The protein consists of 10 anti-parallel beta-strands present as two orthogonal beta-sheets. Together a "clam shell-like" structure is formed with an opening located between two beta-strands and an interior that is lined with the side chains of nonpolar amino acids. The bound fatty acid ligand is located in the interior of the protein and has a bent conformation, possibly reflecting the presence of several gauche bonds in the hydrocarbon tail. Our present interpretation of the electron density map suggests that the fatty acid is oriented with its carboxylate group facing the guanidinium group of Arg127, whereas the end of its hydrocarbon tail is in close proximity to Val106. The indole side chain of Trp83 forms the molecular framework around which the principal bend of the hydrocarbon chain occurs.