During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

The distribution of inorganic nitrogen and phosphorus and bioassay experiments both show that nitrogen is the critical limiting factor to algal growth and eutrophication in coastal marine waters. About twice the amount of phosphate as can be used by the algae is normally present. This surplus results from the low nitrogen to phosphorus ratio in terrigenous contributions, including human waste, and from the fact that phosphorus regenerates more quickly than ammonia from decomposing organic matter. Removal of phosphate from detergents is therefore not likely to slow the eutrophication of coastal marine waters, and its replacement with nitrogen-containing nitrilotriacetic acid may worsen the situation.

https://science.sciencemag.org/content/sci/171/3975/1008.full.pdf

Nitrogen, Phosphorus, and Eutrophication in the Coastal Marine Environment

Bacterial plasmids encode resistance systems for toxic metal ions including Ag+, AsO2-, AsO4(3-), Cd2+, CO2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, Sb3+, TeO3(2-), Tl+, and Zn2+. In addition to understanding of the molecular genetics and environmental roles of these resistances, studies during the last few years have provided surprises and new biochemical mechanisms. Chromosomal determinants of toxic metal resistances are known, and the distinction between plasmid resistances and those from chromosomal genes has blurred, because for some metals (notably mercury and arsenic), the plasmid and chromosomal determinants are basically the same. Other systems, such as copper transport ATPases and metallothionein cation-binding proteins, are only known from chromosomal genes. The largest group of metal resistance systems function by energy-dependent efflux of toxic ions. Some of the efflux systems are ATPases and others are chemiosmotic cation/proton antiporters. The CadA cadmium resistance ATPase of gram-positive bacteria and the CopB copper efflux system of Enterococcus hirae are homologous to P-type ATPases of animals and plants. The CadA ATPase protein has been labeled with 32P from gamma-32P-ATP and drives ATP-dependent Cd2+ uptake by inside-out membrane vesicles. Recently isolated genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson's disease, encode P-type ATPases that are more similar to the bacterial CadA and CopB ATPases than to eukaryote ATPases that pump different cations. The arsenic resistance efflux system transports arsenite, using alternatively either a two-component (ArsA and ArsB) ATPase or a single polypeptide (ArsB) functioning as a chemiosmotic transporter. The third gene in the arsenic resistance system, arsC, encodes an enzyme that converts intracellular arsenate [As (V)] to arsenite [As (III)], the substrate of the efflux system. The three-component Czc (Cd2+, Zn2+, and CO2+) chemiosmotic efflux pump of soil microbes consists of inner membrane (CzcA), outer membrane (CzcC), and membrane-spanning (CzcB) proteins that together transport cations from the cytoplasm across the periplasmic space to the outside of the cell. Finally, the first bacterial metallothionein (which by definition is a small protein that binds metal cations by means of numerous cysteine thiolates) has been characterized in cyanobacteria.

BACTERIAL HEAVY METAL RESISTANCE: New Surprises

A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Role of assisted natural remediation in environmental cleanup

The fingerlike projections, which have been considered to be characteristic of the genus Zoogloea, appear to consist of generally globular packets of cells, each surrounded by capsular matrix. The individual packets which surround Z. ramigera 115 cells appear to adhere one to another by intermeshed fibrils that measure 2 to 5 nm in diameter. The fibril polymer appears to be polyglucose that is susceptible to cellulase. The polymer resembles cellulose in several respects, with the exception that it is soluble in 1 n NaOH. Although Z. ramigera I-16-M does not possess an observable zoogloeal matrix when viewed under a light microscope, chemical data indicate that it does produce cellulase-susceptible polymer. Fibrils can be observed under the electron microscope, which are resistant to 1 n NaOH and may be cellulose. Less polymer fibrils are observed around isolate I-16-M cells than around isolate 115 cells; the inability to observe the I-16-M material as a zoogloeal matrix under light microscopy may be due to lack of sufficient amount of polymer.

Fine Structure and Composition of the Zoogloeal Matrix Surrounding Zoogloea ramigera

Several gram-negative, polarly flagellated rods were isolated on the basis of their flocculent growth habit. Some of the isolates possessed a capsular matrix which is composed of exocellular fibrils. Other isolates did not appear to have a capsular matrix when examined with a bright-field microscope with or without the aid of stains. However, these latter type isolates did possess exocellular material which can be demonstrated by adsorption of a fluorescent dye under an ultraviolet microscope. Electron microscopic examination demonstrated that the exocellular material around all isolates examined is fibrillar. The fibrils were susceptible to cellulase although all fibrils did not appear to be identical. It is postulated that the exocellular polymers were responsible for the flocculent growth habit of the bacteria, and that the process of bacterial flocculation produced by synthetic polyelectrolytes was essentially the same as that caused by naturally produced exopolymers.

Structure of Exocellular Polymers and Their Relationship to Bacterial Flocculation

Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product

Biological nitrogen fixation, as determined by acetylene reduction, occurs in Lake Erie Fixation potential by blue-green algae in situ in water and by bacteria in collected sediments was demonstrated Nitrogen-fixing activity occurred from June through November suggesting that it is significant over the extremes of seasonal variation in light, temperature, and nutrients

http://science.sciencemag.org/content/sci/169/3940/61.full.pdf

Biological Nitrogen Fixation in Lake Erie

The effects of cadmium on the growth and respiration of two strains of Bacillus subtilis are compared to the accumulation of Cd by viable and cyanide-killed cells, protoplasts and cell fractions of the strains. Growth and respiration of strain 1A1 were significantly inhibited at 10μg Cd2+/ml while the growth and respiration of strain 1A1R, a selected mutant of 1A1, were only slightly affected. Similarly, 1A1R protoplasts were more resistant to Cd than were 1A1 protoplasts. The differential resistance of the strains correlates with the accumulation of Cd by the two strains, with 1A1 accumulating approximately 10 times the level of Cd after a 4 h exposure to 1 μg Cd2+/ml. The distributions of Cd throughout the cells, however, were similar between strains. Based on the accumulation of Cd by cyanide-killed protoplasts, uptake of Cd by 1A1 appears to be an active process, while for 1A1R, Cd accumulation is independent of protoplast viability.

Robert M. Pfister

Papers

Fine Structure and Composition of the Zoogloeal Matrix Surrounding Zoogloea ramigera

Structure of Exocellular Polymers and Their Relationship to Bacterial Flocculation

Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product

Biological Nitrogen Fixation in Lake Erie

Effects of cadmium accumulation on growth and respiration of a cadmium-sensitive strain of Bacillus subtilis and a selected cadmium resistant mutant