Treponema saccharophilum sp. nov., a large pectinolytic spirochete from the bovine rumen

A large, obligately anaerobic spirochete (strain PB) was isolated from bovine rumen fluid by a procedure involving rifampin as a selective agent. The helical cells measured 0.6 to 0.7 micron by 12 to 20 micron and possessed approximately 16 periplasmic flagella inserted near each end of the protoplasmic cylinder. The periplasmic flagella were arranged in a bundle wound around the cell body. Strain PB utilized as fermentable substrates various plant polysaccharides (e.g., pectin, arabinogalactan, starch, and inulin) as well as pentoses, hexoses, disaccharides, and uronic acids. Glucose was fermented to acetate, formate, and ethanol, whereas the fermentation of pectin or glucuronic acid yielded only acetate and formate as major end products. Determinations of radioactivity in end products and assays of enzymatic activities indicated that strain PB catabolized glucose via the Embden-Meyerhof pathway. Extracts of cells grown in pectin-containing media possessed relatively high levels of phospho-2-keto-3-deoxygluconate aldolase activity, an enzymatic activity typical of the Entner-Doudoroff pathway. The guanine-plus-cytosine content of the DNA of strain PB (54 mol%) was considerably higher than that of known host-associated anaerobic spirochetes. This study indicates that strain PB represents a new species of Treponema, for which we propose the name Treponema saccharophilum.     

Treponema saccharophilum sp. nov., a large pectinolytic spirochete from the bovine rumen.

A large, obligately anaerobic spirochete (strain PB) was isolated from bovine rumen fluid by a procedure involving rifampin as a selective agent. The helical cells measured 0.6 to 0.7 micron by 12 to 20 micron and possessed approximately 16 periplasmic flagella inserted near each end of the protoplasmic cylinder. The periplasmic flagella were arranged in a bundle wound around the cell body. Strain PB utilized as fermentable substrates various plant polysaccharides (e.g., pectin, arabinogalactan, starch, and inulin) as well as pentoses, hexoses, disaccharides, and uronic acids. Glucose was fermented to acetate, formate, and ethanol, whereas the fermentation of pectin or glucuronic acid yielded only acetate and formate as major end products. Determinations of radioactivity in end products and assays of enzymatic activities indicated that strain PB catabolized glucose via the Embden-Meyerhof pathway. Extracts of cells grown in pectin-containing media possessed relatively high levels of phospho-2-keto-3-deoxygluconate aldolase activity, an enzymatic activity typical of the Entner-Doudoroff pathway. The guanine-plus-cytosine content of the DNA of strain PB (54 mol%) was considerably higher than that of known host-associated anaerobic spirochetes. This study indicates that strain PB represents a new species of Treponema, for which we propose the name Treponema saccharophilum.     

Enumeration and selective isolation of rumen spirochetes.

Enumeration by means of light microscopy showed that from 0.4 x 10(8) to 2.0 x 10(8) spirochetes were present per ml of bovine rumen fluid. Viable cell counts yielded slightly lower values, ranging from 0.1 x 10(8) to 1.2 x 10(8) spirochetes per ml of rumen fluid. The antibiotic rifampin, which served as a selective agent for rumen spirochetes, was added to agar media used in the estimation of viable spirochete numbers in rumen fluid. Morphologically diverse spirochetes were isolated from rumen fluid by means of a procedure involving the use of rifampin as a selective agent in agar media. The strains isolated represented seven morphological types of spirochetes differing in cell size, cell coiling pattern, and number of periplasmic fibrils per cell. Electron microscopy showed that the number of periplasmic fibrils present in the different morphological types of rumen spirochetes ranged from 2 to more than 20 per cell. The results of this study indicate that the bovine rumen is a highly favorable environment for a number of morphologically diverse spirochetes.     

Treponema succinifaciens sp. nov., an anaerobic spirochete from the swine intestine

The morphology, the general physiological characteristics, and the energy-yielding metabolism of an obligately anaerobic spirochete isolated from the colon of a swine were studied. Electron microscopy showed that the helical spirochetal cells possessed an outer sheath, a protoplasmic cylinder, and 4 periplasmic fibrils in a 2-4-2 arrangement. The spirochete grew in an atmosphere of N₂ in prereduced media containing a carbohydrate, NaHCO₃, rumen fluid, yeast extract, peptone, l-cysteine, and inorganic salts. The spirochete fermented carbohydrates and required substrate amounts of CO₂ (HCO ₃ ^- ) for growth. Amino acids were not fermented. Major fermentation products of cells growing with glucose as the substrate and in the presence of CO₂ were acetate, formate, succinate, and lactate. Small amounts of 2,3-butanediol, pyruvate, and acetoin were also formed. Determinations of enzymatic activities in cell extracts, and of radioactivity in products formed by growing cells from [1-¹⁴C]glucose, indicated that this sugar was dissimilated to pyruvate via the Embden-Meyerhof pathway. The spirochetes used a coliform-type clastic reaction to metabolize pyruvate. Determinations of radioactivity in products formed from [¹⁴C]NaHCO₃ indicated that CO₂ was assimilated and used in succinate production. The guanine+cytosine content of the DNA was 36 mol%. This study indicates that this intestinal spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema succinifaciens.Abbreviations cpm counts per minute - DTT dithiothreitol - EM Embden-Meyerhof - GC guanine plus cytosine - IgG immunoglobulin G - PC protoplasmic cylinder - PF periplasmic fibrils (axial fibrils) - OS outer sheath     

Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon.

Ten Bacteroides species found in the human colon were surveyed for their ability to ferment mucins and plant polysaccharides ("dietary fiber"). A number of strains fermented mucopolysaccharides (heparin, hyaluronate, and chondroitin sulfate) and ovomucoid. Only 3 of the 188 strains tested fermented beef submaxillary mucin, and none fermented porcine gastric mucin. Many of the Bacteroides strains tested were also able to ferment a variety of plant polysaccharides, including amylose, dextran, pectin, gum tragacanth, gum guar, larch arabinogalactan, alginate, and laminarin. Some plant polysaccharides such as gum arabic, gum karaya, gum ghatti and fucoidan, were not utilized by any of the strains tested. The ability to utilize mucins and plant polysaccharides varied considerably among the Bacteroides species tested.     

Isolation and characterization of an h(2)-oxidizing thermophilic methanogen

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55 degrees C). This isolate exhibited a temperature optimum of 55 to 60 degrees C and a maximum near 70 degrees C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth near pH 7.2. Although 4% salt was present in the isolation medium, salt was not required for optimal growth. The thermophile utilized formate or H(2)-CO(2) but not acetate, methanol, or methylamines for growth and methanogenesis. Growth in complex medium was very rapid, and a minimum doubling time of 1.8 h was recorded in media supplemented with rumen fluid. Growth in defined media required the addition of acetate and an unknown factor(s) from digester supernatant, rumen fluid, or Trypticase. Cells in liquid culture were oval to coccoid, 0.7 to 1.8 mum in diameter, often occurring in pairs. The cells were easily lysed upon exposure to oxygen or 0.08 mg of sodium dodecyl sulfate per ml. The isolate was sensitive to tetracycline and chloramphenicol but not penicillin G or cycloserine. The DNA base composition was 59.69 mol% guanine plus cytosine.     

Strain selection for the purpose of alcohol production from starch substrates

Summary Yeast strains capable of fermenting starch were selected. Among 90 strains able to assimilate starch, only two could appreciably ferment soluble starch. The study of these strains showed that it was possible to ferment starch solution up to 150 g soluble starch/l.     

Isolation and Characterization of an H2-Oxidizing Thermophilic Methanogen

A thermophilic methanogen was isolated from enrichment cultures originally inoculated with sludge from an anaerobic kelp digester (55°C). This isolate exhibited a temperature optimum of 55 to 60°C and a maximum near 70°C. Growth occurred throughout the pH range of 5.5 to 9.0, with optimal growth near pH 7.2. Although 4% salt was present in the isolation medium, salt was not required for optimal growth. The thermophile utilized formate or H₂-CO₂ but not acetate, methanol, or methylamines for growth and methanogenesis. Growth in complex medium was very rapid, and a minimum doubling time of 1.8 h was recorded in media supplemented with rumen fluid. Growth in defined media required the addition of acetate and an unknown factor(s) from digester supernatant, rumen fluid, or Trypticase. Cells in liquid culture were oval to coccoid, 0.7 to 1.8 μm in diameter, often occurring in pairs. The cells were easily lysed upon exposure to oxygen or 0.08 mg of sodium dodecyl sulfate per ml. The isolate was sensitive to tetracycline and chloramphenicol but not penicillin G or cycloserine. The DNA base composition was 59.69 mol% guanine plus cytosine.     

Taxonomy of the Lyme disease spirochetes.

Morphology, physiology, and DNA nucleotide composition of Lyme disease spirochetes, Borrelia, Treponema, and Leptospira were compared. Morphologically, Lyme disease spirochetes resemble Borrelia. They lack cytoplasmic tubules present in Treponema, and have more than one periplasmic flagellum per cell end and lack the tight coiling which are characteristic of Leptospira. Lyme disease spirochetes are also similar to Borrelia in being microaerophilic, catalase-negative bacteria. They utilize carbohydrates such as glucose as their major carbon and energy sources and produce lactic acid. Long-chain fatty acids are not degraded but are incorporated unaltered into cellular lipids. The diamino amino acid present in the peptidoglycan is ornithine. The mole % guanine plus cytosine values for Lyme disease spirochete DNA were 27.3-30.5 percent. These values are similar to the 28.0-30.5 percent for the Borrelia but differed from the values of 35.3-53 percent for Treponema and Leptospira. DNA reannealing studies demonstrated that Lyme disease spirochetes represent a new species of Borrelia, exhibiting a 31-59 percent DNA homology with the three species of North American borreliae. In addition, these studies showed that the three Lyme disease spirochetes comprise a single species with DNA homologies ranging from 76-100 percent. The three North American borreliae also constitute a single species, displaying DNA homologies of 75-95 percent. Lyme disease spirochetes and Borrelia exhibited little or no DNA homology (0-2 percent) with the Treponema or Leptospira. Plasmids were present in the three Lyme disease spirochetes and the three North American borreliae.     

Characterization of rat cecum cellulolytic bacteria.

Cellulose-degrading bacteria previously isolated from the ceca of rats have been characterized and identified. The most commonly isolated type was rods identified as Bacteroides succinogenes. These bacteria fermented only cellulose (e.g., pebble-milled Whatman no. 1 filter paper), cellobiose, and in 43 of 47 strains, glucose, with succinic and acetic acids as the major products. The only organic growth factors found to be required by selected strains were p-aminobenzoic acid, cyanocobalamine, thiamine, and a straight-chain and a branched-chain volatile fatty acid. These vitamin requirements differ from those of rumen strains of B. succinogenes, indicating the rat strains may form a distinct subgroup within the species. The mole percent guanine plus cytosine was 45%, a value lower than those (48 to 51%) found for three rumen strains of B. succinogenes included in this study. Cellulolytic cocci were isolated less frequently than the rods and were identified as Rumminococcus flavefaciens. Most strains fermented only cellulose and cellobiose, and their major fermentation products were also succinic and acetic acids. Their required growth factors were not identified but were supplied by rumen fluid.     

Pectin-fermenting Bacteria Isolated from the Bovine Rumen

Thirty-two strains of pectin-fermenting rumen bacteria were isolated from bovine rumen contents in a rumen fluid medium which contained pectin as the only added energy source. Based on differences in morphology and the Gram stain, 10 of these strains were selected for characterization. Two strains were identified as Lachnospira multiparus, four strains were identified as Butyrivbrio fibrisolvens, and three strains were identified as Bacteroides ruminicola. Characteristics of the remaining strain did not correspond with any previously described species. It was a gram-positive anaerobic coccus, 1.0 to 1.2 mum in diameter, and occurred primarily as single cells or diplococci. The strain fermented pectin rapidly but showed little or no growth on any other energy sources tested. The only detectable end products were acetic acid and gas, a portion of which was identified as hydrogen. Although the physiological characteristics of this organism differ markedly from other described species, it has been placed in the genus Peptostreptococcus on the basis of morphology, Gram stain, relations to oxygen, and the occurrence of cell division in only one plane. End products of fermentation are somewhat similar to those of the cellulolytic ruminococci. Eight previously characterized strains of cellulolytic bacteria isolated in nonselective media were unable to ferment pectin, whereas ten strains of hemicellulolytic rumen bacteria, eight of which were isolated with a xylan medium, showed considerable variation in this characteristic.     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Pectinolytic enzymes of oral spirochetes from humans

Five strains of obligately anaerobic, pectin-fermenting spirochetes were isolated from the subgingival plaque of humans. The strains produced two extracellular enzymatic activities that functioned in pectin degradation. One of these enzymatic activities was pectin methylesterase (EC 3.1.1.11), and the other was pectate lyase (EC 4.2.2.2) of the endo type. The data indicate that the cumulative action of these two enzymatic activities brought about depolymerization of pectin in spirochete cultures. Pectin- or polygalacturonate-degrading hydrolases were not detected. A cell-associated lyase activity that catalyzed polygalacturonate breakdown was present in one of the spirochete strains. In addition to pectin, the isolates utilized polygalacturonic, glucuronic, or galacturonic acid as fermentable substrate but did not neutral sugars, amino acids, or other substrates tested. Although the oral spirochetes did not ferment hyaluronic acid, one of the strains grew in coculture with a hyaluronidase-producing Peptostreptococcus strain in a medium containing hyaluronic acid as fermentable substrate. Two of the isolates were identified as Treponema pectinovorum strains on the basis of their substrate utilization pattern, end products of fermentation, other phenotypic characteristics, and the guanine-plus-cytosine content of their DNA. Even though the pectinolytic isolates were specialized with respect to the fermentable substrates they utilized, they appeared to compete successfully with other microorganisms in their habitat.     

Fermentation of 2-methoxyethanol by Acetobacterium malicum sp. nov. and Pelobacter venetianus

Anaerobic bacteria degrading 2-methoxyethanol were enriched from freshwater sediments, and three strains were isolated in pure culture. Two of them were Grampositive non-spore-forming rods and grew strictly anaerobically by acetogenic fermentation. Optimal growth occurred at 30°C, initial pH 7.5–8.0. 2-Methoxyethanol and 2-ethoxyethanol were fermented to acetate and corresponding alcohols. Hydrogen plus carbon dioxide, formate, acetoin, l-malate, lactate, pyruvate, fructose, and methoxyl groups of 3,4,5-trimethoxybenzoate and 3,4,5-trimethoxycinnamate were fermented to acetate. 1,2-Propanediol was fermented to acetate, propionate, and propanol. Strain MuME1 was described as a new species, Actetobacterium malicum. It had a DNA base composition of 44.1 mol% guanine plus cytosine. The third strain, which was identified as Pelobacter venetianus, fermented 2-methoxyethanol to methanol, ethanol, and acetate.     

Fermentation of S-citramalate,citrate, mesaconate,and pyruvate by a gram-negative strictly anaerobic non-spore-former,Formivibrio citricus gen. nov., sp. nov.

Two strains of S-citramalate-fermenting strictly anaerobic non-spore-formers were isolated in pure culture from anoxic mud samples of a creek and from a pond. One of them (strain CreCit 1) was studied in detail. It stained gram-negative, and contained β-hydroxymyristic acid. Nitrate, sulfate and other sulfur compounds were not utilized as electron acceptors. S-citramalate, citrate, mesaconate, and pyruvate were utilized as substrates; but R-citramalate, citraconate, l-glutamate, and carbohydrates not. S-citramalate was fermented to acetate, formate, and hydrogen. Citrate, mesaconate, and pyruvate were fermented to acetate and formate. The DNA base ratio was 59 mol% guanine plus cytosine. Strain CreCit 1 is described as a member of a new genus and a new species in the family Bacteroidaceae, Formivibrio citricus gen. nov., sp. nov.     

Deoxyribonucleic Acid Hybridization Studies on Flavobacterium meningosepticum

Seventeen flavobacteria, including 12 strains of Flavobacterium meningosepticum, were investigated to determine their genetic relationships by deoxyribonucleic acid (DNA)-DNA hybridization and guanine plus cytosine content. The percentage of binding among the strains tested ranged from 6 to 87%. There was no correlation between the serotype and DNA-duplex formation. The guanine plux cytosine content of the representative strains of the five serotypes ranged from 31 to 50%.     

Isolation and characterization of an obligately anaerobic,pectinolytic, member of the genus Eubacterium from mullet gut

A gram positive, motile rod-shaped strictly anaerobic non sporulating bacterium was isolated from an enrichment initiated with mullet gut contents. The organism grew optimally at 30°C at pH 6.5 and at a salinity of 10/10³. Out of a variety of mono-, di-, and polysaccharides tested only pectin, cellobiose and starch actively supported growth in either semi defined medium or peptone-yeast extract (PY) medium. Galacturonic acid and maltose were less effective as substrates. Mol product per 100 mol of pectin monomer degraded were: acetate, 163; ethanol, 30; methanol, 88 and formate, 48. Per 100 mol of hexose in cellobiose or starch degraded, the amounts were acetate, 39; ethanol, 128 and formate, 41. Hydrogen was not detectable in the incubations (detection limit, <10^-5 atm) and propionate, butyrate, lactate or succinate were not produced as fermentation end-products (<2 mol per 100 mol monomer). The guanine plus cytosine content of DNA from the bacterium was 31 mol%, and the cell walls contained meso-diaminopimelic acid. A phylogenetic analysis of the organism by 16S rDNA sequencing and DNA-DNA homology indicated that the organism grouped more closely with several species of Clostridium than with Eubacterium. The phenotypic characteristics of the organism indicated that it did not fit within the genus Clostridium and more closely resembled Eubacterium. The organism is therefore designated as a species of Eubacterium; the type strain is P-1 (DSM 6788).     

Isolation and Characterization of a Methylotrophic Marine Methanogen, Methanococcoides methylutens gen. nov., sp. nov

A new genus of marine methanogenic bacteria is described that utilizes trimethylamine, diethylamine, monomethylamine, and methanol as substrates for growth and methanogenesis. Methane was not produced from H₂-CO₂, sodium formate, or sodium acetate. Growth on trimethylamine was stimulated by yeast extract, Trypticase (BBL Microbiology Systems, Cockeysville, Md.), rumen fluid, or B vitamins. The optimal growth temperature was 30 to 35°C. The maximum growth rate was between pH 7.0 and 7.5. Na⁺ (0.4 M) and MgSO₄ (0.05 M) were required for maximum growth. Colonies of the type strain, TMA-10, were yellow, circular, and convex with entire edges. Cells were nonmotile, nonsporeforming, irregular cocci 1 μm in diameter which stained gram negative and occurred singly or in pairs. Micrographs of thin sections revealed a monolayered cell wall approximately 10-nm thick which consisted of protein. Cells were lysed in 0.01% sodium dodecyl sulfate or 0.001% Triton X-100. The DNA base composition was 42 mol% guanine plus cytosine. Methanococcoides is the proposed genus and Methanococcoides methylutens is the type species. TMA-10 is the type strain (ATCC 33938).     

Formate as an Intermediate in the Bovine Rumen Fermentation

An average of 11 (range, 2 to 47) mumoles of formate per g per hr was produced and used in whole bovine rumen contents incubated in vitro, as calculated from the product of the specific turnover rate constant, k, times the concentration of intercellular formate. The latter varied between 5 and 26 (average, 12) nmoles/g. The concentration of formate in the total rumen contents was as much as 1,000 times greater, presumably owing to formate within the microbial cells. The concentration of formate in rumen contents minus most of the plant solids was varied, and from the rates of methanogenesis the Michaelis constant, K(m), for formate conversion to CH(4) was estimated at 30 nmoles/g. Also, the dissolved H(2) was measured in relation to methane production, and a K(m) of 1 nmole/g was obtained. A pure culture of Methanobacterium ruminantium showed a K(m) of 1 nmole of H(2)/g, but the K(m) for formate was much higher than the 30 nmoles for the rumen contents. It is concluded that nonmethanogenic microbes metabolize intercellular formate in the rumen. CO(2) and H(2) are the principal substrates for rumen methanogenesis. Eighteen per cent of the rumen methane is derived from formate, as calculated from the intercellular concentration of hydrogen and formate in the rumen, the Michaelis constants for conversion of these substrates by rumen liquid, and the relative capacities of whole rumen contents to ferment these substrates.     

Mesophilic cellulolytic clostridia from freshwater environments

Eight strains of obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from mud of freshwater environments. The isolates (C strains) were rod-shaped, gram negative, and formed terminal spherical to oval spores that swelled the sporangium. The guanine plus cytosine content of the DNA of the C strains ranged from 30.7 to 33.2 mol% (midpoint of thermal denaturation). The C strains fermented cellulose with formation primarily of acetate, ethanol, CO(2), and H(2). Reducing sugars accumulated in the supernatant fluid of cultures which initially contained >/=0.4% (wt/vol) cellulose. The C strains resembled Clostridium cellobioparum in some phenotypic characteristics and Clostridium papyrosolvens in others, but they were not identical to either of these species. The C strains differed from thermophilic cellulolytic clostridia (e.g., Clostridium thermocellum) not only in growth temperature range but also because they fermented xylan and five-carbon products of plant polysaccharide hydrolysis such as d-xylose and l-arabinose. At 40 degrees C, cellulose was degraded by cellulolytic mesophilic cells (strain C7) at a rate comparable to that at which C. thermocellum degrades cellulose at 60 degrees C. Substrate utilization and growth temperature data indicated that the C strains contribute to the anaerobic breakdown of plant polymers in the environments they inhabit.