Spirillum Volutans Research Articles

ATP‐TOX system—a new, rapid, sensitive bacterial toxicity screening system based on the determination of ATP

AbstractA new toxicity screening test, based on the inhibition of bacterial growth and luciferase activity by toxicants was developed. In the ATP‐TOX System, chemical toxicity was found to be time‐dependent and increased with increasing exposure time up to 5 hours. Three organisms were evaluated in this study: E. coli K‐12 PQ37, Pseudomonas fluorescens and Salmonella typhimurium. E. coli K‐12 PQ37 was found to be the most sensitive organism. It was also shown that P. fluorescens was more sensitive to toxicants when grown in minimal medium than in nutrient broth, suggesting that nutrients may have a protective effect on the bacterium. In comparative studies using selected toxic chemicals the ATP‐TOX System was found to be more sensitive than the Spirillum volutans test and comparable to the Microtox test. Toxicant activity in sediment samples was found to be time dependent and increased with increasing exposure time in both ATP‐TOX (E. coli) System and Microtox. The ATP‐TOX System is complementary to the Microtox test as it also provides indications of low grade toxicant activity which is only manifested in actively growing cells over several life cycles. Thus, the ATP‐TOX System appears to be an ideal screening test for sediment toxicity. The data indicate that the ATP‐TOX System is sensitive, rapid, reproducible, economical and has great potential in applied studies.

Method for determining acute toxicant activity in water, effluents and leachates using spirillum volutans

Method for determining acute toxicant activity in water, effluents and leachates using <i>spirillum volutans</i>

Factors relating to the aerotolerance of Spirillum volutans

A variant of the microaerophile Spirillum volutans capable of growth under an air atmosphere (21% oxygen) was obtained by sequential selection and stabilization of colonies able to grow on a casein hydrolysate – succinate – potassium metabisulfite medium under an atmosphere of 14, 16, 18, 20, and finally 21% O2. A small amount of bisulfite (0.0025%) was still required, however, for aerobic growth. Both the variant and the wild type lacked catalase activity, and they had the same levels of superoxide dismutase activity. However, the mutant possessed 13 times more donor: hydrogen-peroxide oxidoreductase (peroxidase) activity (0.008 IU) than the wild type (0.0006 IU). An aerotolerant mutant was also isolated in one step by treatment with the mutagenic agent ethyl methane sulfonate, followed by incubation under 21% O2; it was found to possess approximately three times the peroxidase activity of the wild type. This mutant lacked catalase activity and the level of superoxide dismutase activity it contained was comparable with the wild type.

Microtox and Spirillum volutans tests for assessing toxicity of environmental samples.

Two short-term bacterial toxicity tests that have received a great deal of attention during the last few years are the Microtox and Spirillum volutans assays. The former involves monitoring changes in the light output from luminescent bacteria (Photobacterium phosphoreum) in a temperature controlled photometer when exposed to various concentrations of toxic substances. In the S. volutans assay, motility patterns of this large aquatic bacterium are used as the test endpoints. As part of the authors' program in assembling and establishing a battery of short-term bacterial toxicity tests, they examined the use of Microtox and S. volutans for determining the toxicity of a wide variety of environmental samples. Samples examined included potable and surface waters, industrial effluents, soil/sludge extracts and landfill leachates. Some of the results are presented in this paper.

Electron microscopic observations of structures associated with the flagella of Spirillum volutans.

Electron microscopy of thin-sectioned Spirillum volutans (ATCC 19554) showed that at the insertion site of the flagellum there was a cylindrical structure with a diameter of ca. 36 nm which extended ca. 19 nm into the cytoplasm. This structure, termed a cytoplasmic flagellar base, enclosed a central rod which was continuous with the hook. There was a continuation of the flagellar base into the peptidoglycan layer, enclosing ringlike structures and the central rod. The flagellar hook and proximal part of the flagellar filament contained a central channel which was large enough to accommodate the flagellin subunit. The flagella of fixed cells may project perpendicularly from the outer membrane in a position corresponding to a trailing, swimming orientation or may bend almost parallel to the membrane in a leading orientation. Maximum bending occurred in the hook region, which may be the structure responsible for executing changes in swimming direction.

Straight mutants of Spirillum volutans can swim.

Nonhelical mutant cells of Spirillum volutans ATCC 19554 can swim as fast as the helical cells. Consequently, a helical cell shape is not required for motility of this species, and the function of the polar flagellar fascicles is not merely to cause rotation, and therefore translocation, of the corkscrew-shaped cell.

Comparison of several microbiological toxicity screening tests

Four short-term microbiological toxicity screening tests were compared using the following test chemicals: 3,5 dichlorophenol, cetyl trimethyl ammonium chloride, sodium lauryl sulfate, phenol, copper(II) sulfate, mercury(II) chloride and zinc(II) sulfate. These seven chemicals represent a wide range of toxicity. The methods examined were Beckman's Microtox system, the Spirillum volutans motility test, inhibition of respiratory activity of activated sludge, and inhibition of activated sludge TTC-dehydrogenase activity. The results obtained indicate that each method has its own toxicity sensitivity pattern, and among the substances tested, only mercury(II) chloride and phenol were ranked equally by the four methods as the most and the least toxic chemical, respectively. In a tentative ranking of the methods, according to sensitivity, the Microtox test came out as the most sensitive test, followed by the Spirillum test, which in turn appeared more sensitive than the two sludge tests. This ranking has meaning only in a statistical sense, however, and the variable nature of the results support the philosophy that for assessing toxicity more thoroughly, a battery of several tests is required.

Application of four bacterial screening procedures to assess changes in the toxicity of chemical in mixtures

Four microbiological acute toxicity screening test methods with Microtox, Spirillum volutans, Pseudomonas fluorescens and Aeromonas hydrophila were compared using solutions of mixed toxic chemicals. Each toxicity screening test appeared to have its own sensitivity pattern and it would appear unwise to try to assess the presence of toxicants in waters or effluents by a single species test. The battery approach, encompassing two or three genera and involving two to four species, is recommended to assess the potential presence of toxicants more thoroughly.

Trailing flagella rotate faster than leading flagella in unipolar cells of Spirillum volutans.

In unipolar cells of Spirillum volutans, the flagellar rotation frequency is halved, approximately, when the flagellar bundle reorientates to rotate about the cell body and reverse the swimming direction. The viscous drag resulting from a concomitant increase in flagellar wave amplitude is probably responsible for the reduced frequency of flagellar rotation.

Distribution of Mutagens and Toxicants in Lake Ontario Waters as Assessed by Microbiological Procedures

Water samples collected from inshore and offshore waters in Lake Ontario, Toronto waterfront and Hamilton Bay were tested for mutagenic activity (Ames’ Test) and presence of acute toxicants ( Spirillum volutans test). Data indicate many inshore waters contain mutagenic compounds which cause reversion of Salmonella tester strain TA 1535. The majority of samples tested did not contain acute toxicants based on the Spirillum volutans test.

Stimulatory effect of dihydroxyphenyl compounds on the aerotolerance of Spirillum volutans and Campylobacter fetus subspecies jejuni.

The aerotolerance of the microaerophilic bacterium Spirillum volutans was greatly stimulated in a defined medium by the presence of dihydroxyphenyl ferric iron-binding compounds such as nor-epinephrine at 10(-5) to 10(-6) M. Dihydroxyphenyl compounds at 2 X 10(-4) M, or iron salts (ferrous or ferric) at high concentration, greatly increased the aerotolerance of a strain of Campylobacter fetus subsp. jejuni when grown on streak plates of Brucella agar. The results suggest that the microaerophilism of these organisms might in part be caused by a failure to synthesize microbial ferric iron-binding compounds at sufficient levels to support aerobic growth.

Aerotaxis in Spirillum volutans

Spirillum volutans responded aerotactically to self-created oxygen gradients in capillary tubes. Band formation required an oxidizable substrate, inorganic ions, and a chelating agent. The higher the respiratory rate, the faster the band migrated. Random motility occurred in the anaerobic region behind the band. Inhibitor studies indicated the existence of two alternative energy pathways for motility: one involved the oxidation of exogenous substrates while the other allowed energy production in the absence of oxygen uptake and was apparently linked to intracellular poly-β-hydroxybutyrate breakdown. Respiratory inhibitors such as KCN caused decreases in ATP and prevented band formation but allowed motility. 2,4-Dinitrophenol stimulated oxygen uptake but caused decreases in ATP and prevented band formation. Oxidation of substrates without aeration caused decreases in ATP while aeration increased ATP. Aerotactic band formation therefore appeared to result from an avoiding reaction in which cells moved away from the anaerobic environment created by the band, thereby resulting in an increase of cellular ATP. Results with 2,4-dinitrophenol suggested that not only is oxygen uptake required for band formation but also oxidative phosphorylation. Intracellular ATP levels may possibly serve to regulate the aerotactic response.

Long-Term Preservation of Spirillum volutans

Strains of Spirillum volutans, the type species of the genus Spirillum, have not been satisfactorily preserved in the past except by serial transfer. Long-term preservation of such strains can be achieved in liquid nitrogen by the use of dimethyl sulfoxide and a low-salt suspending medium. Glycerol is not satisfactory as a cryoprotective agent.

Abnormally low activities of fumarate hydratase and malate dehydrogenase in oxygen-sensitive cultures of Spirillum volutans.

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The Genus Spirillum: a Taxonomic Study

The morphological, physiological, and nutritional characteristics and the deoxyribonucleic acid (DNA) base compositions of 39 strains of Spirillum (including the available type strains) were determined. A uniform methodology provided a basis for comparison of the strains. The results, together with previous serological data and with previous results for two strains of Spirillum volutans, indicate that the present genus Spirillum should be divided into three genera, with the original name being restricted to obligately microaerophilic freshwater forms having a large cell diameter and a DNA base composition of 36 to 38 mol % guanine + cytosine (G + C). This genus would contain at present only one species, the type species, S. volutans. The generic name Aquaspirillum is proposed for the aerobic, freshwater forms having a DNA base composition of 49 to 65 mol % G + C. Thirteen species are included in this genus: the type species A. serpens (Müller) comb. nov.; A. metamorphum (Terasaki) comb. nov.; A. putridiconchylium (Terasaki) comb. nov.; A. sinuosum (Williams and Rittenberg) comb. nov.; A. gracile (Canale-Parola et al.) comb. nov.; A. itersonii (Giesberger) comb. nov.; A. dispar sp. nov.; A. polymorphum (Williams and Rittenberg) comb. nov.; A. aquaticum sp. nov.; A. delicatum (Leifson) comb. nov.; A. peregrinum (Pretorius) comb. nov.; A. anulus (Williams and Rittenberg) comb. nov.; and A. giesbergeri (Williams and Rittenberg) comb. nov. Strains of the latter two species, plus an additional strain (ATCC 12289), appear to be related to one another but should not be included together in a single species without additional supporting evidence (e.g., DNA homology studies). The following are proposed as type or neotype strains: A. serpens ATCC 12638 (neotype); A. metamorphum ATCC 15280 (type); A. putridiconchylium ATCC 15279 (type); A. gracile ATCC 19624 (type); A. itersonii ATCC 12639 (neotype); A. dispar ATCC 27510 (type); and A. aquaticum ATCC 11330 (type). The generic name Oceanospirillum is proposed for marine forms which do not attack carbohydrates and which possess a DNA base composition of 42 to 48 mol % G + C; five species are included. These are as follows: the type species O. linum (Williams and Rittenberg) comb. nov.; O. minutulum (Watanabe) comb. nov.; O. beijerinckii (Williams and Rittenberg) comb. nov.; O. japonicum (Watanabe) comb. nov.; and O. maris sp. nov. The following are proposed as type or neotype strains: O. minutulum ATCC 19192 (neotype); O. japonicum ATCC 19191 (neotype); and O. maris ATCC 27509 (type).

Designation of Spirillum volutans Ehrenberg 1832 as Type Species of the Genus Spirillum Ehrenberg 1832 and Designation of the Neotype Strain of S. volutans: Request for an Opinion

In 1832 Ehrenberg transferred Vibrio spirillum Müller and V. undula Muller to a new genus, Spirillum. Müller’s two species were renamed Spirillum volutans (Muller) Ehrenberg and S. undula (Müller) Ehrenberg, respectively. A comparison of Müller’s description of V. spirillum with Ehrenberg’s description of S. volutans, however, strongly indicates that the organisms described were not the same; therefore the name S. volutans should be related to the organism described by Ehrenberg. It is therefore requested that the Judicial Commission delete Müller’s name from the author citation for S. volutans (i.e., S. volutans Ehrenberg, not S. volutans [Müller] Ehrenberg). Although Ehrenberg included S. undula and S. volutans in his new genus, he did not designate a type species. Stiles (1905) was apparently the first to designate the type species (S. volutans), but the later designation of S. undula as the type by Vuillemin (1913) is the designation that has been generally recognized. The acceptance of S. undula as the type species is not only contrary to the rules of nomenclature [Rule 9c(3)] but also unfortunate because the name is a source of confusion and error. It is therefore requested that the Judicial Commission issue an opinion establishing S. volutans Ehrenberg as the type species of Spirillum Ehrenberg and placing S. undula on the list of rejected names. Wells’ strain (ATCC 19554) is designated as the neotype strain of S. volutans.

Uncoordination and recoordination in Spirillum volutans.

The quantitative effects of several new uncoordinating agents for Spirillum volutans were studied. A new type of uncoordination, "dual-straight-tail," was described for polyethylene glycol or HgCl2. Certain agents caused a mixture of more than one type of uncoordination. Exposure of normal cells to pH 4.4 caused dual-tail uncoordination, while pH 9.9 caused dual-head. Recoordination of cells was accomplished by washing cells free of agent, application of metal-complexing agents, or, in the case of uncoordination by pH, readjustment of pH. Protein synthesis was not required for recoordination. All agents tested caused substantial decreases in oxygen consumption at their uncoordinating concentrations, but this was not the cause of uncoordination since normal coordinated states could be maintained with every cell for prolonged periods in the absence of detectable respiration, even in the presence of cyanide. Stroboscopic measurements indicated decreased flagellar rotational speeds in uncoordinated cells. In an electric field, the act of reversing polarity or of breaking or closing the circuit caused motile coordinated cells to instantly reverse whatever direction in which they had been previously swimming. Electric fields did not cause alignment of the motile spirilla.

Mode of extension of cell surface during growth of Spirillum volutans.

Mode of extension of cell surface during growth of Spirillum volutans.

The growth of Spirillum volutans Ehrenberg in mixed and pure cultures.

The growth of Spirillum volutans in mixed culture was studied and liquid media were developed in which it grew rapidly and attained populations of over a million cells per ml. By taking advantage of their rapid and unidirectional motility, spirilla were separated from the mixed population by migration in capillary tubes. Pure cultures were achieved by growing the separated spirilla inside of dialysis sacks in contact with mixed cultures on the outside, and also by growth in an asparagine-mineral salts medium supplemented with an extract of Escherichia coli.

Purification of Water by Infusoria

Infusoria have the same bactericidal power as flagellates. Emulsions containing Bacillus typhosus, Vibrio cholerae, V. Dunbar, V. El Tor, B. megatherium and Spirillum volutans, to which Colpoda cucullus is added, are soon cleared. Before the clearage the Colpodae multiply actively.This bactericidal effect does not depend upon the production of toxic substances by the Infusoria. The fluid obtained from filtered cultures of the Infusoria exerts no bactericidal effect. Only living Colpodae are able to clear the emulsions.Direct sunlight does not prevent the clearing of the emulsions by the Infusoria, but temperatures below 10°C. and above 30°C. and absence of oxygen (anaerobic culture) are unfavourable to their exerting a bactericidal effect.The polluted water of the canals of Amsterdam slightly delayed the clearage of the emulsions; the sewage of starch factories and gas factories completely prevented this clearage.Consequently under natural conditions Infusoria will only play a part in purifying river water (1) if the temperature is above 10° and under 30°C., (2) if the aquatic vegetation is rich enough to supply the necessary quantity of oxygen, (3) if the water is not highly polluted by adjoining factories.