Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting l-methionine, l-norleucine and l-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.     

Serological Cross-Reaction Between O-Antigens of Shigella dysenteriae Type 4 and an Environmental Escherichia albertii Isolate

An environmental freshwater bacterial isolate, DM104, appearing as Shigella-like colonies on selective agar plates was found to show strong and specific serological cross-reactivity with Shigella dysenteriae type 4. Biochemical identification according to the analytical profile index, molecular serotyping by restriction of the amplified O-antigen gene cluster (rfb-RFLP), together with phylogenetic analysis of the 16S rRNA gene and multi-locus sequence analysis, identified the isolate as Escherichia albertii. rfb-RFLP of DM104, revealed a profile different from that of S. dysenteriae type 4. However, western blot analysis of extracted lipopolysaccharides demonstrated strong cross-reactivity with S. dysenteriae type 4 using specific monovalent antisera and a lipopolysaccharide gel banding profile similar to that of S. dysenteriae type 4. The observed O-antigen cross-reaction between an E. albertii isolate and S. dysenteriae extends our knowledge of the extent of O-antigen cross-reaction within the Escherichia/Shigella group of organisms, and offers the possibility of using DM104 and similar cross-reacting strains as shigellosis vaccine candidates.     

Plankton community structure and size spectra in the Georgian Bay and North Channel ecosystems

Patterns in the size distribution and taxonomic composition of phytoplankton and zooplankton communities for 1974 in Georgian Bay and the North Channel are described. The Diatomeae predominate the phytoplankton in both areas. Copepods, particularly Calanoida, comprise the greatest fraction of the zooplankton biomass. Normalized plankton biomass spectra for both ecosystems are typical of those found in Lake Superior and offshore Lake Huron. The plankton communities of Georgian Bay and the North Channel are thus similar to the most oligotrophic of the Laurentian Great Lakes.     

Exploring aquatic ecosystem health: a multi-trophic and an ecosystemic approach

The field of aquatic ecosystem health is a new and developing discipline. The restoration and recovery of habitats is extremely complex and requires a clear understanding of a desirable and maximum/minimum set of conditions which is acceptable, achievable, and cost-effective for implementation. Since this field of research is still in its infancy, the technology for an integrative and innovative assessment will require a combination of physical, chemical, and biological methods and researchers will have to adopt and use some of the routine chemical, limnological, physiological, ecological, and toxicological procedures. A multi-disciplinary, multi-trophic and an ecosystemic approach has been initiated and applied in the North American Great Lakes during the past several years. This strategy, consisting of structural and functional indicators and endpoints, was implemented in the Great Lakes Areas of Concern adopting a field to laboratory approach for a holistic and integrated evaluation of the ecosystem. Some examples from our Great Lakes experience are presented. The ecosystem health technology should look beyond the traditional approach and must develop innovative, sensitive, automated, and cost-effective procedures including computer-assisted techniques to deal with the problems of stress, healing, recovery, and remediation.     

Assessing the impact of sewage effluent on the ecosystem health of the Toronto Waterfront (Ashbridges Bay), Lake Ontario

The ecosystem health of the Toronto Waterfront (Ashbridges Bay), Lake Ontario which receives treated sewage effluent was investigated during 1987 and 1988 by means of a functional and structural battery of tests. The functional tests included in situ size-fractionated primary productivity, Algal Fractionation Bioassays (AFBs), unfiltered and filtered bioassays, and sediment assays with Daphnia magna and Hyalella azteca. The structural evaluation involved the biomonitoring of the components of the microbial loop, such as bacteria, autotrophic picoplankton, heterotrophic nanoflagellates, and protozoa. The experimental results reveal a diversity of physiological responses to the complex nutrient and contaminant regimes by the indigenous phytoplankton. There was no evidence of the impact of chlorination on the primary productivity of the Bay. The overall productivity was higher during the post-chlorination period than the pre-chlorination phase. The high rates of microplankton + netplankton productivity near the outfall have been attributed to the bioavailability of nutrients which, quite possibly, exert ameliorating effects on metal toxicity. In contrast, the low ultraplankton rates have been interpreted to be due to their well-known sensitivity to contaminants. The Effluent Receiving Water Bioassays (ERWB) with filtered and unfiltered experiments provided interesting insight and appear to be a potentially useful assessment tool. Generally, the unfiltered water compared to the filtered was toxic to the offshore test phytoplankton. This demonstrates a unique ecological adaptation to the prevailing in situ conditions by the Bay community which might be important from the restoration point of view. However, the offshore population was found to be sensitive to the particulate-bound toxicity as indicated by the unfiltered bioassays. Consequently, it is essential to probe the complexity of nutrient-contaminant interactions which ultimately appear to determine the toxicity and the resulting health of the biota. Furthermore, our experiments have shown that the particulate-matter is an important carrier of both nutrients and contaminants in Ashbridges Bay. The sediment bioassays for Station 419 indicated that sediments were toxic during both the pre- and post-chlorination phases. Both solid and liquid phase testing indicated toxicity of sediment to the acute Daphnia test. The Hyalella chronic assay showed good survival during the 4-wk period of the experiment, in contrast to the toxicity observed for phytoplankton and Daphnia. This may be due to large mounts of organic matter available in the Bay. The invertebrate bioassays confirmed the lack of impact of chlorination. Finally, the microbial loop seems to be a sensitive, rapid, and an early warning bioindicator of anthropogenic stress. The multi-trophic battery of structural and functional strategy adopted in our laboratory appear to be holistic and effective. The strategy has a considerable potential for developing eco-technology for a badly needed assessment and restoration of ecosystem health of the Great Lakes as well as other perturbed environments in the world.     

A sensitive screening bioassay technique for the toxicological assessment of small quantities of contaminated bottom or suspended sediments

Biological impact assessment of sediment-bound contaminants is currently considered essential in addition to routine chemical characterization. Conventional methods of sediment bioassessment require relatively large quantities of the sample. When sample sizes are limited however, these methods become ineffective. To overcome this problem, the Limited Sample Bioassay (LSB) technique was developed. Bioassays conducted with bottom sediments and suspended particulates from western, central and Arctic regions of Canada indicated that the newly developed LSB technique successfully evaluated the contaminant/nutrient impact on laboratory-grown cultures of algae. The LSB method produced results which were comparable to those from standard elutriate bioassays conducted with natural phytoplankton size assemblages. The data suggest that the LSB can play an important role in providing a quick, simple, sensitive, and inexpensive screening technique for assessing the bioavailability of contaminants (or nutrients) from sediment of limited quantity.     

Enhancement in the germination, growth and yield of okra (Abelmoschus esculentus) using pre-sowing magnetic treatment of seeds

The effect of pre-sowing magnetic treatments was investigated on germination, growth and yield of okra (Abelmoschus esculentus cv. Sapz pari). The dry okra seeds were exposed to sinusoidal magnetic field induced by an electromagnet. The average magnetic field exposure was 99 mT for 3 and 11 min and seeds with no magnetic field treatment were considered as control. Both treated and non-treated seeds were sown in experimental plots (120 m2) under similar conditions. Samples were collected at regular intervals for statistical analysis. A significant increase (P < 0.05) was observed in germination percentage, number of flowers per plant, leaf area (cm2), plant height (cm) at maturity, number of fruits per plant, pod mass per plant and number of seeds per plant. The 99 mT for 11 min exposure showed better results as compared to control.     

A comparative study on the effects of selected pesticides on hemato-biochemistry and tissue histology of freshwater fish Cirrhinus mrigala (Hamilton, 1822)

The aim of the present study was to investigate the comparative effects of pesticides Chlorfenapyr, Dimethoate and Acetamiprid on the health of Cirrhinus mrigala under long term exposure. Eighty C. mrigala were divided in four equal groups; one control and three treated groups. The blood was collected from both control and treated groups at intervals of 10th, 20th and 30th days for hemato-biochemistry and histopathological alterations. The result indicates significant difference (P < 0.05) in RBCs, Hb, PCV and MCHC whereas elevation in WBCs and Platelets counts were recorded. In 10th day sampling, MCV value of Dimethoate and Acetamiprid treatment had no difference in comparison with the control group, however it is significantly increased (P < 0.05) in rest of sampling. The MCH value of exposed fish showed significant increased (P < 0.05) after 20th and 30th days for Chlorfenapyr and after 30th days for Acetamiprid exposure while insignificantly increased for rest of sampling. It was also found that these pesticides significantly decrease (p < 0.05) the T3 and T4 levels while increase in the TSH, cortical, ALP, AST, ALT and LDH levels in the serum of the treated fishes in contrast to control group. Similarly, histopathological analysis of gills and liver showed significant alterations in all the treated groups. Toxicity trends of these pesticides was ranked as Chlorfenapyr > Acetamiprid > Dimethoate. It is concluded that indiscriminate use of such pesticides poses a noxious threat to non-target organisms, harm the ecosystems and jeopardizes human health.     

A preliminary study of vertical movement of Eudorina elegans and trinema lineare during a bloom caused by them

Summary The paper describes a polluted pond from India, which exhibited a well marked thermal stratification in summer. The surface and bottom strata of water exhibited a temperature difference of 8° C. The paper also describes the diurnal movement of Eudorina elegans and Trinema lineare which formed a bloom in the pond. The movements of these organisms are interpreted on the basis of fluctuations in temperature and light conditions. The possibility of the competition for space is also discussed.

---

Sommaire On a décrit un cas de stratification thermale bien marquée dans un étang pollué en dépit de peu de profondeur de son bassin, dans un été en Inde. Les couches superficielles et de fond de l'eau ont montré une différence de temperature de 8° C. On décrit également le mouvement diurnal d'Eudorina elegans et Trinema lineare qui ont formé une floraison dans l'étang. Le déplacement de ces organismes est interprété en fonction du changement de temperature et de la luminosité. La possibilité d'une concurrence pour l'espace est discutée.

---

---

Hydrobiological Laboratory, Department of Botany, Osmania University, Hyderabad-7 (A.P.), India.