The Genus Selenomonas

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Species of Selenomonas are defined as anaerobic, Gram-negative, curved or crescent-shaped rods that are motile by means of a tuft of flagella originating from the inner curvature of the cell. Selenomonads have been isolated from the rumen and ceca of mammals, and the human oral cavity. Depending upon the health or diet of the host, these bacteria can constitute a significant proportion of the total microbial population. In general, selenomonads are obligately saccharolytic, although some strains ferment lactate or amino acids. It has been suggested that the role of ruminal and intestinal selenomonads involves the fermentation of soluble sugars and lactate in their natural environments. Oral selenomonads may play a role in the pathogenesis of periodontal disease in humans. The first classification of selenomonads was by Miller (1887), who designated strains from the human mouth as Spirillum sputigenum, but the present classification system places these organisms in the genus Selenomonas as S. sputigena (Bryant, 1984; Johnson et al., 1985). As a result of recent findings, however, at least six species of oral selenomonad have been recognized (Moore et al., 1987). Phylogenetic studies based on 16S ribosomal RNA sequence analysis have shown that species of Selenomonas are closely related to the bacteria Centipeda periodontii, Pectinatus cerevisiiphilus, and Sporomusa paucivorans. The selenomonads are more distantly related to anaerobic Gram-negative cocci of the genera Veillonella and Megasphaera. Selenomonads, Veillonella and related bacteria comprise a phylogenetic grouping which is more closely related to Gram-positive bacteria than to typical Gram-negative bacteria.

Habitats

Selenomonads have been observed in and isolated primarily from the rumen, the human mouth, and the cecum of mammals (Table 1). Gram-negative organisms having the cell morphology and flagellar arrangement typical for selenomonads have also been observed in river water (Leifson, 1960), and recently selenomonads have been isolated from anaerobic sewage sludge (Nanninga et al., 1987) and bog water (Harborth and Hanert, 1982). Selenomonads were probably first observed by Antonie van Leeuwenhoek in gingival scrapings from the human mouth (Dobell, 1960). Traditionally, speciation within Selenomonas has been based upon the habitat from which the strain was isolated (Buchanan and Gibbons, 1974; Lessel and Breed, 1954). Although helpful, this criterion has been largely supplanted by more adequate cytological, biochemical, and molecular criteria.

Table 1. Strains, sources, and DNA base composition of Selenomonas species.

Selenomonads appear to be part of the normal indigenous microflora of human gingival crevices and are often more abundant in those persons having clinically detectable gingivitis or peridontal disease. Oral selenomonads may play a role in peridontal disease, inasmuch as lipopolysaccharide purified from these bacterial species has been shown to possess several endotoxic properties in mice (Kurimoto et al., 1986). Some occurrences of S. sputigena and other selenomonads in human septicemia have been reported (MacCarthy and Carlson, 1981; Pomeroy et al., 1987).

Selenomonads isolated from the rumen are usually shown to be strains of S. ruminantium. These organisms are routinely observed and isolated from rumen contents of cows and sheep. (Bryant, 1956; Hobson and Mann, 1961; Prins, 1971). In general selenomonads are more numerous in animals fed rations such as grains, which contain rapidly fermentable carbohydrates, than they are in animals fed silage or straw (Caldwell and Bryant, 1966). In addition to the fermentation of soluble carbohydrates, rumen selenomonads have other important roles in the rumen. These organisms are among the most important members of the glycerol-fermenting species of ruminal bacteria in sheep (Hobson and Mann, 1961) and in cattle (Bryant, 1956). While lactate is fermented by only a few species of ruminal bacteria, many ruminal selenomonad strains ferment lactate. Often, these strains are designated as S. ruminantium subspecies lactilytica and are almost phylogenetically identical to S. ruminantium (Fig. 1). Lower ruminal pH and increased lactate formation resulting from bloat or high-grain feeding, can lead to a substantial increase of ruminal selenomonads. Urea present in saliva and feedstuffs is degraded in the rumen by urease to form ammonia, a major nitrogen source for growth of many rumen microoganisms. S. ruminantium probably contributes to the hydrolysis of urea because urease-producing strains are commonly isolated (Wozny et al., 1977).

Fig. 1.

Phylogenetic tree for the selenomonads and related bacteria. The scale indicates a 5% difference in nucleotide sequence, as determined by taking the sum of all branch lengths connecting two species. (Sequences for S. paucivorans and M. elsdenii courtesy of C. R. Woese.)

Selenomonad strains that have been phenotypically classified as S. ruminantium constitute a significant part of the intestinal microbial populations of swine. Approximately 21% of the total bacterial isolates from cecal contents of healthy swine were strains of S. ruminantium (Robinson et al., 1981). S. ruminantium strains were found to represent about 5% and 15% of the total bacteria associated with the epithelial tissue of the colon in healthy and dysentery-infected swine, respectively (Robinson et al., 1984). Although this last study did not find selenomonad strains in luminal contents of the colon, these organisms have been detected in swine feces (Salanitro et al., 1977). Presumably, a functional role for selenomonads in the swine intestine involves fermentation of soluble sugars as occurs with these organisms in the rumen. The dominance of S. ruminantium strains in the swine intestine may be related to the availability of lactate. The swine of the small intestine can contain high levels of lactate (15 to 20 mM) and lactate levels decrease almost 10-fold upon reaching the cecum (Imoto and Namioka, 1978). In this regard, all of the selenomonad strains isolated from cecal contents have been lactate-fermenting strains, namely S. ruminantium subsp. lactilytica (Robinson et al., 1981). Lactate may also be available to Selenomonas strains that adhere to the colon wall since other adherent bacteria include lactate-producing species such as Lactobacillus and Enterococcus (Robinson et al., 1984).

Selenomonas species have frequently been observed and isolated from the cecum of a number of small rodents. The study by Ogimoto (1972) indicated that about 5% of the total bacterial isolates from rat cecal contents were selenomonads. All their rat strains could ferment any one of a number of sugars, including cellobiose; propionate was the major fermentation acid. With freeze-fracture electron microscopic techniques, selenomonads could be seen in the rat cecum submucosa. Gram-negative organisms that have the cell morphology and flagellar arrangement typical for selenomonads have been observed in cecal contents of squirrels. These organisms were enumerated and isolated from cecal contents of the 13-lined ground squirrel (Citellus tridecemlineatus) and shown to be as high as 1 × 10⁹ to 1 × 10¹⁰ cells per gram of cecal contents (Barnes and Burton, 1970). In addition, it was shown that these selenomonads constituted 18% or more of the total viable cells of the cecal contents of both active and hibernating squirrels. All selenomonad strains from squirrels could ferment glucose and some strains could ferment starch, but further biochemical characteristics of these strains have not been determined. Many investigators have observed selenomonads in the cecal contents of guinea pigs (Kingsley and Hoeniger, 1973; Robinow, 1954). These organisms have been designated as S. palpitans (Simons, 1922), but since the organisms have not been isolated or grown in pure culture, this classification is questionable. However, electron micrographs of “S. palpitans” indicate it is a selenomonad which differs in some respects in cell morphology from S. ruminantium (Kingsley and Hoeniger, 1973).

The isolation of selenomonads from nonmammalian environments has been reported only on one occasion; using lactate and sulfate enrichment cultures, a selenomonad strain was isolated from ditch water from a bog habitat (Harborth and Hanert, 1982). This strain was phenotypically similar to S. ruminantium except it has a lower optimal growth temperature (25°C) and produces catalase. The authors suggested it be considered S. ruminantium subspecies psychrocataligenes. Selenomonad strain DSM 3853 (strain DKglu16) was isolated from glutamate- plus aspartate-limited chemostat cultures inoculated with anaerobic sewage sludge (Nanninga et al., 1987). Because this strain fermented only amino acids and differed in many other ways from other Selenomonas species, it was named S. acidaminophila. These studies suggest that selenomonads may exist in a number of natural, anaerobic habitats.

Isolation

Selective Enrichment and Isolation

Enrichment and selection procedures have been effectively developed only for the isolation of selenomonad strains from ruminal contents. Ruminal organisms can be selectively isolated using SS medium that has mannitol as the only added carbohydrate (Tiwari et al., 1969). The selective factors of SS medium are: 1) the use of mannitol as the main energy source, (since few species of ruminal bacteria can ferment this sugar); 2) the pH of the medium is 6.0, which does not affect selenomonad growth, but inhibits growth of many other species; and 3) the medium contains no branched-chain volatile fatty acids or heme, and either or both of these compounds are required for the growth of many other ruminal bacteria. Substitution of glycerol or lactate for mannitol in SS medium might allow for preferential isolation of selenomonad strains commonly designated as S. ruminantium subspecies lactilytica.

Selective Medium (SS) for Isolation of Selenomonas ruminantium (Modified from Tiwari et al., 1969)

Mannitol	0.2 g
Trypticase	0.5 g
Yeast extract	0.1 g
Sodium acetate	0.1 g
FeSO4−·−7H2O	0.1 g

Mineral S solution (see below)	4.1 ml
n-Valeric acid	0.05 ml
Distilled water	92.5 ml
L-Cysteine−·−HCl solution (2.5%) (see below)	1.0 ml
Sodium carbonate solution (8%) (see below)	2.5 ml

The L-cysteine solution is prepared with oxygen-free distilled water under a nitrogen-gas phase. Both the sodium carbonate solution and final SS medium are prepared and equilibrated under a carbon dioxide gas phase. The L-cysteine and sodium carbonate solutions are autoclaved separately and added to the cooled medium. The composition of the mineral S solution is: monobasic potassium phosphate, 12.0 g; ammonium sulfate, 6 g; sodium chloride, 12.0 g; magnesium sulfate heptahydrate, 2.5 g; calcium chloride dihydrate, 1.6 g; and distilled water to a final volume of one liter. This solution is stable when stored at 5°C.

In regard to other species of Selenomonas, some enrichments for these organisms appear possible from samples of anaerobic sludge, bog water, or highly eutrophic waters. With such samples, the use of glutamate or aspartate as the main carbon source may lead to enrichment of S. acidaminophila strains (reference is not an exact match Nanninga et al., 1982), whereas use of lactate may result in enrichment of S. ruminantium strains (Harborth and Hanert, 1982).

MacDonald and Madlener (1957) examined several methods for isolating oral selenomonads. These authors found that complex media containing sodium lauryl sulfate (0.01%) or sodium oleate (0.15%) in addition to sheep serum (10%) would inhibit the growth of many microorganisms present in gingival scrapings, but would not affect selenomonad growth. Nonselective media, such as commercially available blood agar, can sometimes be used for the isolation of oral selenomonads. In certain subgingival sites that possess clinical signs of periodontitis, oral selenomonads can represent 10–30% of the total bacterial population (S. S. Socransky, personal communication). Appropriate dilutions of clinical samples are plated onto agar media and incubated under an atmosphere of 80% N₂/10% CO₂/10% H₂. Identification of these motile bacteria is initially based upon colony morphology. Since cells are able to migrate through solid medium with lowered concentrations of agar, colonies with a spreading, fuzzy morphology form. Some species of Selenomonas or Centipeda form a hazy zone of growth on the surface of the agar medium (Lai et al., 1983).

Cultivation Media

The nutrient requirements of S. ruminantium strains are rather simple, and most, if not all, strains can be grown anaerobically in chemically defined media containing glucose, minerals, B vitamins, ammonia, sulfide, and a volatile fatty acid (usually n-valerate), under a carbon dioxide atmosphere (Bryant and Robinson, 1962; Kanegasaki and Takahashi, 1967; John et al., 1974; Tiwari et al., 1969). More complex media containing yeast extract and trypticase, such as those used for Succinivibrio dextrinosolvens (see The Family Succiaivibrionaceae in Volume 3), will support growth of S. ruminantium strains. These complex media probably can support growth of S. acidaminophila, but glutamate, aspartate, pyruvate, or lactate would be needed as the energy source (Nanninga et al., 1987). For S. ruminantium, sulfide or cysteine serve as sole sulfur sources. Substances that can serve as sole nitrogen sources include ammonia, urea, certain single amino acids (cysteine, serine, threonine, aspartate, histidine, glutamate, and valine), or the purines adenine and uric acid, Some strains utilize urea or ammonia as a sole nitrogen source, but many strains do not. When lactate is the energy source, biotin and p-aminobenzoic acid satisfy the vitamin requirements; aspartate, malate, or fumarate are required for growth and aromatic amino acids may be stimulatory to growth (Linehan et al., 1978).

The nutritional features of most human oral species of Selenomonas have not been studied in detail, as has been done with S. ruminantium. Most of these species have been isolated on very complex media, such as brain-heart infusion agar or on this medium supplemented with 5% rabbit blood or serum. Rich media such as peptone-yeast extract-glucose often support growth of many of these species (Moore et al., 1987).

Preservation of Cultures

Most species can be maintained for long periods by storing cultures in liquid nitrogen or ultracold freezers (Hespell and Canale-Parola, 1970). Depending upon the strain, preservation by lyophilization under anaerobic conditions may be possible. Alternatively, short-term storage (6 to 15 months) may be possible by placing glycerol-containing cultures in normal (−20°C) freezers (Teather, 1982).

Identification

Phenotypic Properties

Quite often, newly isolated bacterial strains can be identified as Selenomonas based on the characteristic cell shape and tumbling motility of these species. All species are obligately anaerobic, motile, nonsporeforming, Gram-negative rods. The cells are usually curved or cresent-shaped and have a tuft of flagella that originates from the concave side of the cell, as revealed by Leifson flagella staining or by electron microscopy. Strains of the related genus Pectinatus have a similar curved-cell morphology, but the flagella are arranged linearly along the entire length of the concave side of the cell (Lee et al., 1978). Centipeda strains are Gram-negative, curved or helical rods. However, the flagella Centibeda are inserted in a line or stop that spirals around the cell, resulting in bundles that arise from both cell sides, giving a centipede-like appearance to the cell (Lai et al., 1983). A major characteristic of all known Selenomonas species is that they produce both acetate and propionate as major fermentation acids. Many strains also form small amounts of lactate and/or succinate (Table 2).

Table 2. Characteristics that differentiate species of Selenomonas, Pectinatus, and Centipeda. ^a

Species of Selenomonas have varying types of colony morphologies and cell sizes. Most strains of S. ruminantium produce large colonies (3- to 6-mm diameter) that are smooth, entire, slightly convex, and light tan to white in color. Often, these colonies have a gray to black appearance (due to hydrogen sulfide production) starting in the center of the colony. The cells are usually 1 µm by 2.0 to 4.0 µm long, but S. ruminantium subspecies lactilytica strains can be 2.0–3.0 µm by 5.0–10.0 µm long. Many S. ruminantium strains have carbohydrate granules in the cytoplasm, and cells may be strongly iodophilic (Prins, 1971) but no capsular material is present.

With newly isolated strains, S. sputigena colonies on blood-agar media are generally small (0.5 to 1.2 mm in diameter), smooth, convex, and gray to gray-yellow in color. Larger colonies tend to have an irregular edge and translucent appearance. Most of the other oral selenomonad species form minute colonies (0.5–1.0 mm in diameter) that are shiny, smooth, and colorless to white. A spreading growth over the entire plate is not uncommon for several species. The cell sizes of most oral selenomonad species range from about 1.0–1.4 µm wide by 3.0–5.5 µm long.

Species of Selenomonas, Pectinatus, and Centipeda can be differentiated from one another on the basis of a number of phenotypic traits (see Table 2). C. periodontii is the only species listed that has a bilateral flagella arrangement and that does not form acetate as a major fermentation acid, although trace amounts of acetate can be made by some strains. P. cerivisiiphilus and S. ruminantium are the only listed species to produce hydrogen sulfide. P. cerivisiiphilus differs from S. ruminantium by its linear array of flagella and its inabilities to ferment sucrose and hydrolyze esculin (Lee et al., 1978). S. acidaminophila differs from all other listed species by its inability to ferment sugars, by using only lactate, pyruvate, glutamate, and aspartate, and by being able to hydrolyze gelatin. S. ruminantium is the only Selenomonas species capable of fermenting cellobiose. S. infelix and S. dianae are the only oral Selenomonas species capable of esculin hydrolysis, and these two species can be separated on the basis of acid production on trehalose. With respect to the esculin-negative oral Selenomonas species, S. flueggei and S. sputigena are both positive for acid from lactose and can be separated on the basis of acid from mannitol, whereas S. artemidis is negative for acid from lactose. S. noxia is negative for all of the previously mentioned traits of Selenomonas species, but growth is abundant in a peptone-yeast extract medium containing glucose, mannose, sorbitol, or sorbose.

Phylogeny

The GC content of the DNA from Selenomonas species ranges from 48 to 58 mol% (see Table 1). S. acidaminophila has the lowest value, whereas S. ruminantium strains vary from 49 to almost 54 mol%. The oral species of Selenomonas have only a narrow range of 53 to 58 mol%, but it is clear from the results of DNA-DNA hybridization (Table 3) that these strains are, in fact, separate species.

Table 3. Percentage of homology from DNA-DNA hybridizations of oral Selenomonas species.

The phylogeny of species of Selenomonas and related bacteria has been determined by using 16S rRNA sequence analysis (Dewhirst et al., 1989). Complete 16S rRNA sequences of these bacteria were compared with the rRNA sequences of over 250 other bacterial species (Paster and Dewhirst, unpublished observations). From these data, a phylogenetic tree was constructed (Fig. 1). The microorganisms tested fall into two major groups-Selenomonas and related bacteria occupy one branch, and Veillonella and related bacteria are on the other. The Selenomonas group is phylogenetically coherent with interspecies homology levels of 90 to 99%. Selenomonas dianae, S. infelix, S. flueggei, S. noxia, S. artemidis, and Centipeda periodontii form a very tight cluster with a homology range of 96 to 99%. Selenomonas sputigena and S. ruminantium have an average sequence homology of 94% with members of this cluster. Aside from its unusual flagellation, C. periodontii is phenotypically similar to other members of the genus Selenomonas.

Pectinatus cerevisiiphilus, a Gram-negative, anaerobic, motile rod originally isolated from spoiled beer, is related to the Selenomonas group, but with an average homology value of only 91% (for further information see Chapter 91). Sporomusa paucivorans is an anaerobic, Gram-negative, sporeforming rod. This organism is related to the selenomonads at an average homology level of 90%.

In the other major branch of this tree, Veillonella dispar and V. parvula-åerobic, Gram-negative cocci isolated from humans-are very closely related to each other with 99% sequence homology. Megasphaera elsdenii (see Pectinatus, Megashaera and Zymophilus in this Volume), an anaerobic, large, Gram-negative coccus isolated from humans and the ovine rumen, is related to Veillonella at a level of 92% homology. Species within these two bacterial genera are related to members of the Selenomonas branch with an average homology of 88%. The close relationship between motile, curved rods and nonmotile cocci may seem unusual, but there are phenotypic traits that unify this diverse group. One of the more convincing characteristics is that species of Selenomonas, Sporomusa, Veillonella, and Megasphaera all possess the one of the diamines, either cadaverine or putrescine, which are covalently bound to their peptidoglycan (Stackebrandt et al., 1985).

It has been previously shown that the genera Selenomonas and Veillonella share a branch with the Gram-positive bacteria (Stackebrandt et al., 1985). Members of these two groups both have an average 16S rRNA sequence homology of 85% with the Gram-positive bacteria, as represented by species of Clostridium, Bacillus, and Enterococcus, but only 79% sequence homology with Gram-negative bacteria such as E. coli and related bacteria (Fig. 2). Other bacterial groups, such as those containing the spirochetes, bacteroides, and radiation-resistant bacteria, branch deeper in the tree (unpublished observations) with even lower sequence homologies (e.g., <75%) with the selenomanads). From these data, it is evident that the selenomonads should be removed from the Bacteroidaceae, their current taxonomic placement in Bergey’s Manual of Systematic Bacteriology. In general, a sequence homology of less than 80% indicates that the bacteria compared are in different major taxonomic divisions.

Fig. 2.

Phylogenetic tree for the selenomonads, related bacteria, and distantly related bacteria. (Sequences for C. perfringens and E. faecalis courtesy of C. R. Woese.)

Now that 16S rRNA sequences are known (see below), it will be possible to develop short DNA probes targeted for signature regions of 16S rRNA. These probes can be used to identify new strains or species isolated from environmental samples. Ultimately, these probes will allow for the rapid identification of selenomonads directly from samples without in vitro cultivation. Family-specific and species-specific DNA probes have been already used to identify RNA isolated from selenomonads (Dewhirst et al., 1989).

Physiological Traits

Many biochemical and physiological studies have been done with S. ruminantium, but not with the oral species of Selenomonas. Early studies on isolation of ruminal bacteria did not indicate that S. ruminantium produced hydrogen gas as a fermentation product. However, when this organism was grown in the presence of a hydrogen-utilizing bacterium, namely a methanogen, (Scheifinger et al., 1975b). As a consequence, considerably less lactate and propionate were produced from glucose fermentation. S. ruminantium also changes the array of fermentation acids made as function of growth rate, with acetate and propionate dominating at low growth rates and lactate at high growth rates (Hobson, 1965). These shifts in fermentation products are correlated with less ATP formation and can be largely explained by the effects of pyruvate on the activity of the intracellular lactic acid dehydrogenase (Scheifinger et al., 1975a; Wallace, 1978). At higher growth rates, intracellular pyruvate pools probably increase and this compound causes a homotropic activation of lactate acid dehydrogenase, resulting in higher rates of lactate formation. The other major fermentation acid, propionate, has been shown to be formed via the succinate pathway (Paytner and Elsden, 1970). Propionate formation most likely involves generation of ATP via formation of a proton-motive force, since S. ruminantium was one of the first anaerobes to be shown to possess cytochromes, mainly of the cytochrome-b type (DeVries et al., 1974).

Ammonia and urea play central roles in ruminal nitrogen metabolism. S. ruminantium is one of the major ureolytic bacteria in the rumen (Wozny et al., 1977). Studies on ammonia assimilation and glutamate formation in S. ruminantium indicate this organism possesses both the glutamate dehydrogenase and glutamine synthetase pathways for ammonia assimilation (Smith et al., 1980). In fact, this study was the first to show presence of a nonadenylylation control mechanism for glutamine synthetase in a Gram-negative organism. However, it was shown later that this enzyme and urease were coordinately controlled (Smith et al., 1981), as in other bacteria.

One of the first detailed studies on survival of anaerobic bacteria under nongrowing conditions was with S. ruminantium. Washed cell suspensions obtained from glucose-limited chemostats were found to lose viability very rapidly, with about a 50% loss in 2.5 hours (Mink and Hespell, 1981). The viability losses could not be attributed to cell lysis but correlated with rapid declines in cellular DNA, RNA, and protein. Similar effects were found with cells grown under nitrogen-limited conditions but better survival times were observed that correlated with growth rates (Mink et al., 1982). During starvation, the cells produced trace amounts of acetate as the only fermentation product. In addition, it was shown that the cellular levels of urease, glutamine synthetase, and glutamate dehydrogenase remained relatively stable despite loss of cell viability.

Applications

At present, no biotechnological applications have been made with Selenomonas species. However, a recent paper on the kinetics of glucose fermentation by S. ruminantium suggests that this organism might have commercial application for the production of lactic acid (Shimizu et al., 1989). A new species of the genus Selenomonas has been described by Schleifer et al. 1990. Selenomonas lacticifex was isolated from pitching yeast and can ferment glucose to lactic acid as major product.