A review of the salt sensitivity of the Australian freshwater biota

In Victoria, Australia, both dryland salinity and salinity in irrigation regions are serious agricultural problems. One option to control the latter is to pump groundwater to maintain it below the surface. However, this leaves a saline wastewater for disposal, probably into local streams or wetlands. This review of the salt sensitivity of the biota of Australian streams and wetlands gives information of interest to those responsible for developing controls on these discharges. The review addresses the lethal and sub-lethal effects of salinity on microbes (mainly bacteria), macrophytes and micro-algae, riparian vegetation, invertebrates, fish, amphibians, reptiles, mammals, and birds. Data suggest that direct adverse biological effects are likely to occur in Australian river, stream and wetland ecosystems if salinity is increased to around 1 000 mg L⁻¹. The review highlights a general lack of data on the sensitivity of freshwater plants and animals to salinity increases.     

A Pseudomonas strain accumulating polyesters of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids

Summary A citronellol-utilizing bacterium was isolated that accumulated a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from various carbon sources up to approximately 70% of the cellular dry matter if the cells were cultivated in ammineral salts medium under nitrogen limitation. In octanoate-grown cells, for instance, the polyester consisted of 87.5 mol% 3HB and 12.5 mol% 3-hydroxyoctanoic acid (3HO), whereas it consisted of 10.3 mol% 3HB, 16.7 mol% 3HO and 73.0 mol% 3-hydroxydecanoic acid (3HD) in gluconate-grown cells. However, the results of various experiments indicated that a blend rather than a copolyester was synthesized in the cell. It was the only strain among 45 different recently isolated citronellol-utilizing bacteria that accumulated such a polyester. All other citronellol-utilizing bacteria behaved like Pseudomonas aeruginosa with respect to their polyhydroxyalkanoic acid (PHA) biosynthetic capabilities and accumulated PHA consisting of 3HA_MCL with 3HO and 3HD as the main constituents from octanoate or gluconate, respectively, whereas 3HB was never present. None of 232 different heavy-metal-resistant bacteria was able to accumulate PHA composed of 3HB plus, for example, 3HO. Only 20.3% did not accumulate any PHA at all, 44.8% accumulated PHB from gluconate, and 34.9% behaved like P. aeruginosa. Many bacteria belonging to the latter group were distinguished from the other by rapid growth in nutrient broth and in gluconate mineral salts medium and by their ability to grow in the presence of a high concentration (up to 1.5%, w/v) of octanoate. Correspondence to: A. Steinbüchel     

Isolation and characterization of a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-manno-octulosonate-8-phosphate synthesis.

A new mutant of Salmonella typhimurium was isolated which possesses a temperature-sensitive defect in the synthesis of 3-deoxy-D-manno-octulosonic acid. The defect in 3-deoxy-D-manno-octulosonic acid synthesis is due to a temperature-sensitive 3-deoxy-D-manno-octulosonate-8-phosphate synthetase, and the mutant accumulates an incomplete lipid A under nonpermissive conditions. Evidence is presented which indicates that the incomplete lipid A molecule is most likely identical in structure to the lipid A precursor synthesized by previously characterized mutants conditionally defective in 3-deoxy-D-manno-octulosonic acid synthesis. However, unlike related mutants which undergo growth stasis under nonpermissive conditions, the accumulation of lipid A precursor in the new mutant results in cell death at elevated temperatures.     

A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum

The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum.     

An Analysis of γ-Aminobutyrate Receptors and Uptake by Isolated Purkinje Cells

Abstract: An isolated fraction of Purkinje perikarya was prepared. This fraction had [³H]GABA receptor binding and [³H]muscimol binding analogous to that reported for heterogeneous cerebellar membranes. The finding of two binding components with each ligand suggests that these components do not represent two binding sites, one presynaptic and the other postsynaptic, since both are clearly seen on purified Purkinje cell bodies. Subcellular fractionation indicates that synaptic endings and plasma membranes are enriched in GABA receptors compared with intracellular organelles. Purkinje cell bodies were also found to possess a high-affinity transport system for GABA, which was sensitive to inhibition by diaminobutyric acid (DABA) but not by β-alanine. They showed no evidence of homoexchange (the movement of label without net transport). This supports our suggestion that homoexchange is an artifact of synaptic particle formation.     

Enhancement of cellulose accessibility and enzymatic hydrolysis by simultaneous wet milling

It has been shown that the rate of enzymatic saccharification of cellulosic materials including “pure” cellulose (Whatman CF?11 cellulose), newsprint, lignocellulose (prehydrolyzed to remove hemicelluloses), and wood can be substantially increased by simultaneous wet milling. An enhanced hydrolysis rate was sustained above that observed for ball milling: providing a more extensive saccharification. The cellulosic substrates were wet milled with a variety of grinding elements, such as sand, glass beads, and stainless-steel beads, agitated in a shaker bath. Simultaneous hydrolysis was achieved with a 2% substrate slurry in a 0.1M acetate buffer at 45°C and pH 5. The effectiveness of this process was dependent upon the lignified matrix of the cellulose microfibrils, the grinding elements, and the oscillation frequency of the shaker bath. Wet milling “pure” cellulose for 48 hr, with 3.5 mm glass beads and 200 oscillations/min (opm), yielded 1031 mg reducing sugar/g substrates (93% saccharification) as compared to 483 mg (44%) for the ball-milled sample and 253 mg (23%) for the unmilled material. With the lignified substrates stainless-steel beads (3.5 mm) were more effective than glass. For lignocellulose 529 mg sugar/g substrate (93% saccharification) could be obtained by wet milling with cellulase for 24 hr. This was about three times greater than that of the ball milled (169 mg, 30%) and 10 times greater than that of the unmilled (52 mg, 9%) substrates. The method was also effective for wood particles (60 mesh) giving 143 mg sugar/g wood (approximately 38% saccharification) in 48 hr, whereas the ball-milled sample gave only 79 mg (21%) and the unmlilled substrate 38 mg (10%). These observations can be explained on the basis of the current crystalline theory for the morphology of the cellulosic microfibrils. The advantage of wet milling and simultaneous hydrolysis apparently depends on a continuous generation of accessible sites and sustained rapid hydrolysis rate as the saccharification proceeds, where in the pretreated substrates the hydrolysis rate slow down as the active sites are reduced.     

Population studies in Artemisia tridentata ssp. vaseyana: Chromosome numbers and sesquiterpene lactone races

The sesquiterpene lactones and chromosome numbers for three chemical races of Artemisia tridentata ssp. vaseyana have been examined from four populations in western Montana. TLC analysis of the sesquiterpene lactones in the seeds and seed producing parents demonstrated that genetic exchange does occur between sympatric sesquiterpene lactone chemical races. However, other evidence suggests that introgression between these races is restricted to zones of sympatry. There appears to be no correlation between chromosome numbers and sesquiterpene lactone races.     

Characterization of functionally distinct mitochondrial subpopulations

Mitochondrial stress results in changes in mitochondrial function, morphology and homeostasis (biogenesis, fission/fusion, mitophagy) and may lead to changes in mitochondrial subpopulations. While flow cytometric techniques have been developed to quantify features of individual mitochondria related to volume, Ca²⁺ concentration, mtDNA content, respiratory capacity and oxidative damage, less information is available concerning the identification and characterization of mitochondrial subpopulations, particularly in epithelial cells. Mitochondria from rabbit kidneys were stained with molecular probes for cardiolipin content (nonyl acridine orange, NAO) and membrane potential (tetramethylrhodamine, TMRM) and analyzed using flow cytometry. We validated that side scatter was a better indicator of volume and that as side scatter (SSC) decreased mitochondrial volume increased. Furthermore, those mitochondria with the highest NAO content had greater side scattering and were most highly charged. Mitochondria with average NAO content were of average side scattering and maintained an intermediate charge. Those mitochondria with low NAO content had minimal side scattering and exhibited minimal charge. Upon titration with the uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), it was found that the high NAO content subpopulations were more resistant to uncoupling than lower NAO content populations. Ca²⁺-induced swelling of mitochondria was evaluated using probability binning (PB) analyses of SSC. Interestingly, only 30 % of the mitochondria showed changes in response to Ca²⁺, which was blocked by cyclosporine A. In addition, the small, high NAO content mitochondria swelled differentially in response to Ca²⁺ over time. Our results demonstrate that flow cytometry can be used to identify mitochondrial subpopulations based on high, mid and low NAO content and/or volume/complexity. These subpopulations showed differences in membrane potential, volume, and responses to uncoupling and Ca²⁺-induced swelling.