Spirillum volutans
1. Classification
a. Higher order taxa
Domain: Bacteria
Phylum: Pseudomonadota
Class: Betaproteobacteria
Order: Nitrosomonadales
Family: Spirillaceae
Species: Spirillum volutans
2. Description and significance
Spirillum volutans is a Gram-negative bacterium from the genus of Spirillum, a species of bacteria in the family Spirillaceae (Krieg, 1976). It is widespread in freshwater and marine environments (Hylemon et al., 1973) and is also the first species designated as the type of species of Spirillum. S. volutans stands out as one of the largest bacterial species (Hylemon et al., 1973). Members of the genus Spirillum were first described by van Leeuwenhoek in the 1670s and later by Otto Friedrich Muller (Krieg, 1976). S. volutans plays a crucial role in aquatic microbial communities, participating in nutrient cycling within aquatic ecosystems (Bowdre, 1976). It is suggested that S. volutans has adaptations related to aerotolerance (Bowdre et al., 1976). This could indicate a role in oxygen dynamics within aquatic environments. Studies initially found that S. volutans was one of the most difficult of all spirilla to preserve. However it can now be easily preserved by the use of dimethyl sulfoxide and liquid nitrogen. It commands attention not only for its unique morphology but also by how it is vastly outnumbered by other bacteria and fails to form colonies on solid media (Padgett et al., 1981).
3. Genome structure
The entire genome of S. volutans has not yet been sequenced, but scientists have sequenced 459-1,492 base pair snippets of its linear 16S ribosomal DNA. The 16S ribosomal DNA of S. volutans has a guanine plus cytosine content (G+C) of 36% (by thermal denaturation methods) or 38% (by buoyant density methods) that distinguishes the genera Spirillum from Aquaspirillum and Oceanospirillum (42-48% G+C) (Cole, 1972; Hylemon et al., 1973a; Hylemon et al., 1973b).
4. Cell structure
S. volutans cells are non-spore forming and helical in shape, with a length of 14 to 60 μm and a number of turns ranging from less than one to a maximum of five, in a counterclockwise direction (Hylemon et al., 1973; Swan, 1985). The bipolar flagella of this Gram-negative bacterium occur in a trailing configuration or leading configuration with flagellar filaments surrounded by a sheathlike structure (Swan, 1985). Cylindrical flagellar bases extend into the cytoplasm below the plasma membrane, and in some areas extend into the peptidoglycan layer of the cell wall. Cells additionally contain hook regions associated with the flagellar filaments, that may in part control for flagellar orientation (Swan, 1985). S. volutans cells contain numerous refractile intracellular granules of poly-𝛽-hydroxybutyrate, a biological polyester used for energy storage (Krieg, 2006; Elustondo et al., 2012).
S. volutans maintains a fairly rigid helical shape, but straighten out during rapid movement, and the cytoplasm separates into three or more pieces. This flexibility may be due in part to the mucopeptide layer of the envelope (Krieg, 2006).
S. volutans primarily use their flagella bundles for motility. The retention of polar flagella fascicles in non-helical mutant cells of S. volutans allows them to swim just as fast as helical cells (Padgett et al., 1983). The flagellar bundles beat in a helical fashion, and the aft flagella provides the propulsive force necessary to push through fluid (Winet & Keller, 1976). The hooks at the base of the flagella assist in controlling the direction of movement. Flagella occur in leading or trailing configurations, which do not alter velocity, but do alter the frequency of flagellar rotation and wave amplitude. In the leading configuration, higher flagellar wave amplitude increases cell propulsion, but simultaneously increases viscous drag, which offsets a lower frequency of flagellar rotation. (Swan, 1982).
5. Metabolic processes
S. volutans is microaerophilic, requiring an atmosphere of 1-9% oxygen (Cole & Rittenberg, 1971). It is also chemotrophic. It contains cytochrome b and cytochrome c, but does not contain cytochrome a (Krieg, 1976). However, its cytochrome levels are lower than normal (Bowdre, Hoffman, & Smibert, 1976), meaning that S. volutans has a low respiratory rate and that an excess of oxygen could be harmful to the cell. S. volutans has other enzymatic deficiencies in its metabolic pathway: S. volutans possesses superoxide dismutase, but lacks catalase (Padgett, Cover & Krieg, 1981). Because S. volutans lacks catalase, it is unable to degrade excess peroxide in the cell that is built up from aerobic metabolic processes contributing to its microaerophilic state (Padgett, Cover & Krieg, 1981).
S. volutans cannot catabolize sugars, but uses tricarboxylic acid intermediates, organic acids, and amino acids as primary carbon sources. The nitrogen sources for S.volutans are extremely limited, as most only get nitrogen from ammonium salts (Krieg, 1976). Existence of enzymes necessary for the Krebs Cycle as well as the Electron Transport Chain suggest that these are the primary pathways S. volutans uses to catabolize substrates (Cole & Rittenberg, 1971).
6. Ecology
S. volutans was discovered and refined from a sugar beet refinery in England and a polluted pond (Krieg, 1976). In general, S. volutans can be found in stagnant fresh-water bodies. S. volutans can be found near the top of the water, underneath biofilms due to its microaerophilic qualities. Additionally, it prefers fresh waters due to its intolerance of high sodium, phosphate, and pH levels above 8 (Cole & Rittenberg, 1971). Its ideal pH range is between 6.5 and 8, since it prefers neutral or slightly alkaline conditions and its ideal temperature range is from 30-32 °C (Krieg, 1976). Although there have been no studies that have focused on geographical locations for S. Volutans, it has been found in fresh water, stream water, and pond mud in Aransas, Texas, Long Island Sound, New York, and across England (Krieg, 1976).
7. Pathology
There haven’t been any recorded diseases caused by S. volutans, but the closely related Spirillum minus has been found to be pathogenic in mammals (Krieg, 1976). S. minus primarily infects rats, but can cause rat bite fever in humans if humans come into contact with an infected rodent.
8. Isolation and Growth Methods
S. volutans is isolated from freshwater samples using the capillary tube method (Padgett, 1981). It is most commonly grown in peptide-succinate-salts (PSS) broth under 1-9% atmospheric oxygen exposure (Padgett, 1981). To grow S. volutans under aerobic conditions of at least 12% atmospheric oxygen, a modified peptone-succinate-salts (MPSS) broth is required. This is composed of 5 g/L peptone, 1.0 g/L succinic acid, 1.0 g/L (NH4)2SO4, 1.0 g/L MgSO4⋅7H2O, 0.002 g/L FeCl3⋅6H2O, and 0.002 MnSO4⋅H2O (Padgett, 1981). Vitamin-free, acid-hydrolyzed casein broth with low Na+, or casein hydrolysate-succinate-salts (CHSS) agar media supplemented with catalase and SOD and incubated under minimal light exposure also supports aerobic growth (Padgett, 1981). These enzymes may also be added to the MPSS and casein broth to further support growth.
9. Current Research
There are mutant strains of S. volutans that have been found to be more resistant to H2O2. From isolated DNA and protein sequences, it was found that a portion of the sequence codes for a protein with a similar sequence to the proteins rubrerythrin and nigerythrin (Alban et al., 1998). Although this rubrerythrin/nigerythrin-like sequence was found in the H2O2-resistant S. volutans mutant, researchers were unable to determine how this protein was related to the organism’s ability to survive in a more oxygenated environment. However, since this was the only difference between S. volutans and the mutant strain, they hypothesized that this protein must play a role in the mutant strain’s ability to survive in a more oxygenated environment. Because rubrerythrin in the archaeon Pyrococcus furiosus, when combined with iron, works as a peroxidase (Weinberg et al., 2004), the rubrerythrin-like protein sequence found in S. volutans could play a role in breaking down hydrogen peroxide created by a highly oxygenated environment.
10. References
Alban, Popham, D. L., Rippere, K. E., & Krieg, N. R. (1998). Identification of a gene for a rubrerythrin/nigerythrin‐like protein in Spirillum volutans by using amino acid sequence data from mass spectrometry and NH2‐terminal sequencing. Journal of Applied Microbiology, 85(5), 875–882.
Bowdre, Krieg, N. R., Hoffman, P. S., & Smibert, R. M. (1976). Stimulatory effect of dihydroxyphenyl compounds on the aerotolerance of Spirillum volutans and Campylobacter fetus subspecies jejuni. Applied and Environmental Microbiology, 31(1), 127–133.
Carney, J.G., Trachtenberg, A.M., Rheaume, B.A., Linnana, J.D., Pitts, N.L., Mykles, D.L., & MacLea, K.S. (2017). Genome Sequence of a Marine Spirillum, Oceanospirillum multiglobuliferum ATCC 3336T, Isolated from Japan. Genome Announc 5(21): e00396-17
Cole, J.A. (1972). Base Composition of Deoxyribonucleic Acid from Spirillum volutans, S. serpens and S. itersonii. Journal of General Microbiology 72: 411-413
Cole, J.A. & Rittenberg S.C. (1971). A Comparison of Respiratory Processes in Spirillum volutans, Spirillum itersonii and Spirillum serpens. Microbiology Society. 69(3): 375-383
E. H. Pauley, N. R. Krieg (1974). “Long-Term Preservation of Spirillum volutans Free.” International Journal of Systematic Bacteriology, p. 292-293
Elustondo, P., Zakharian, E., & Pavlov, E. (2012). Identification of the Polyhydroxybutyrate Granules in Mammalian Cultured Cells. Chemistry & Biodiversity 9(11): 2597-2604
Hylemon, P.B., Wells Jr., J.S., Bowdre, J.H., Macadoo, T.O., & Krieg, N.R. (1973). Designation of Spirillum volutans Ehrenberg 1832 as Type Species of the Genus SPirillum Ehrenberg 1832 and Designation of the Neotype Strain of S. volutans. International Journal of Systematic Bacteriology 23(1): 20-27
Hylemon, P.B., Wells Jr., J.S., Krieg, N.R., & Jannasch, H.W. (1973b). The Genus Spirillum: a Taxonomic Study. International Journal of Systematic Bacteriology 23(4): 340-380
Krieg, N R. (1976). Biology of the chemoheterotrophic spirilla. Bacteriological reviews (40)1: 55-115.
Krieg, N.R. (2006). The Genus Spirillum. In: Dworkin, M., Flakow, S., Rosenberg, E., Schleifer, KH., Stackebrandt, E. (eds) The Prokaryotes. Springer, New York, NY
Padgett, P.J., et al. (1982). The Microaerophile Spirillum volutans: Cultivation on Complex Liquid and Solid Media. Applied and environmental microbiology. (43)2: 469-77.
Padgett, P.J., & Krieg, N. R. (1986). Factors Relating to Aerotolerance of Spirillum Volutans. Can. J. Microbiol. p. 548-552
Padgett, P.J., Cover, W.H., & Krieg, N.R. (1981). The Microaerophile Spirillum volutans: Cultivation on Complex Liquid and Solid Media. Applied and Environmental Microbiology p. 469-477
Padgett, P.J., Friedman, M.W., & Krieg, N.R. (1983). Straight Mutants of Spirillum volutans Can Swim. J. of Bacteriology p.1543-1544
Swan, M.A. (1982). Trailing Flagella Rotate Faster than Leading Flagella in Unipolar Cells of Spirillum volutans. J. of Bacteriology p. 3770380
Swan, M.A. (1985). Electron Microscopic Observations of Structures Associated with the Flagella of Spirillum volutans. J. of Bacteriology p. 1137-1145
Weinberg, Jenney, F., Cui, X., & Adams, M. (2004). Rubrerythrin from the Hyperthermophilic Archaeon Pyrococcus furiosus Is a Rubredoxin-Dependent, Iron-Containing Peroxidase. Journal of Bacteriology, 186(23), 7888–7895.
Winet, H., & Keller, S.R. (1976) Spirillum Swimming: Theory and Observations of Propulsion by the Flagellar Bundle. Journal of Experimental Biology 65: 577-602
