For the first time a single experimental approach, 16 S ribosomal RNA sequence characterization, has been used to develop an overview of phylogenetic relationships in the bacterial world. The technique permits the tracing of relationships back to the common ancestor of all extant life. This first glimpse of bacterial phylogeny reveals a world whose roots appear to span more than 3 billion years. A deep phylogenetic split exists among the bacteria, which necessitates their division into two major lines of descent, the archaebacteria and the true bacteria (or eubacteria). It is a general finding that the most ancient bacterial phenotypes are anaerobic, and that aerobic phenotypes have arisen a number of times. Photosynthetic phenotypes are also extremely ancient. Many nonphotosynthetic groups appear to have arisen from photosynthetic ancestry, which is reason to question the generally held belief that the first bacteria were anaerobic heterotrophs. The two ultimate lines of bacterial descent are no more closely related to one another than either is to the cytoplasmic aspect of the eukaryotic cell. However, in that the eukaryotic cell is a phylogenetic chimera, it itself cannot be seen as a line of descent comparable to the two bacterial lines—although some of its individual parts can be so viewed. In this way, the chloroplast and perhaps the mitochondrion are each eubacterial, and at least one ribosomal protein is archaebacterial. A third line of descent that is neither eubacterial nor archaebacterial is represented in the 18 S ribosomal RNA.

The 16S rRNA sequences were determined for species of Spirochaeta, Treponema, Borrelia, Leptospira, Leptonema, and Serpula, using a modified Sanger method of direct RNA sequencing. Analysis of aligned 16S rRNA sequences indicated that the spirochetes form a coherent taxon composed of six major clusters or groups. The first group, termed the treponemes, was divided into two subgroups. The first treponeme subgroup consisted of Treponema pallidum, Treponema phagedenis, Treponema denticola, a thermophilic spirochete strain, and two species of Spirochaeta, Spirochaeta zuelzerae and Spirochaeta stenostrepta, with an average interspecies similarity of 89.9%. The second treponeme subgroup contained Treponema bryantii, Treponema pectinovorum, Treponema saccharophilum, Treponema succinifaciens, and rumen strain CA, with an average interspecies similarity of 86.2%. The average interspecies similarity between the two treponeme subgroups was 84.2%. The division of the treponemes into two subgroups was verified by single-base signature analysis. The second spirochete group contained Spirochaeta aurantia, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirochaeta litoralis, and Spirochaeta isovalerica, with an average similarity of 87.4%. The Spirochaeta group was related to the treponeme group, with an average similarity of 81.9%. The third spirochete group contained borrelias, including Borrelia burgdorferi, Borrelia anserina, Borrelia hermsii, and a rabbit tick strain. The borrelias formed a tight phylogenetic cluster, with average similarity of 97%. The borrelia group shared a common branch with the Spirochaeta group and was closer to this group than to the treponemes. A single spirochete strain isolated from the shrew constituted the fourth group. The fifth group was composed of strains of Serpula (Treponema) hyodysenteriae and Serpula (Treponema) innocens. The two species of this group were closely related, with a similarity of greater than 99%. Leptonema ilini, Leptospira biflexa, and Leptospira interrogans formed the sixth and most deeply branching group. The average similarity within this group was 83.2%. This study represents the first demonstration that pathogenic and saprophytic Leptospira species are phylogenetically related. The division of the spirochetes into six major phylogenetic clusters was defined also by sequence signature elements. These signature analyses supported the conclusion that the spirochetes represent a monophylectic bacterial phylum.

An anaerobic, nonphototrophic bacterium that β-oxidizes saturated fatty acids (butyrate through octanoate) to acetate or acetate and propionate using protons as the electron acceptor (H 2 as electron sink product) was isolated in coculture with either a non-fatty acid-degrading, H 2 -utilizing Desulfovibrio sp. or methanogens. Three strains of the bacterium were characterized and are described as a new genus and species, Syntrophomonas wolfei. S. wolfei is a gram-negative, slightly helical rod with round ends that possesses between two to eight flagella laterally inserted along the concave side of the cell. It has a multilayered cell wall of the gram-negative type. The presence of muramic acid, inhibition of growth by penicillin, and increased sensitivity of the cells to lysis after treatment with lysozyme indicate that peptidoglycan is present in the cell wall. Cells of S. wolfei contain poly-β-hydroxybutyrate. Isoheptanoate was degraded to acetate, isovalerate, and H 2 . Carbohydrates, proteinaceous materials, alcohols, or other tested organic compounds do not support growth. Common electron acceptors are not utilized with butyrate as the electron donor. Growth and degradation of fatty acids occur only in syntrophic association with H 2 -using bacteria. The most rapid generation time obtained by cocultures of S. wolfei with Desulfovibrio and Methanospirillum hungatei is 54 and 84 h, respectively. The addition of Casamino Acids but neither Trypticase nor yeast extract stimulated growth and resulted in a slight decrease in the generation time of S. wolfei cocultured with M. hungatei. The addition of H 2 to the medium stopped growth and butyrate degradation by S. wolfei.

Anaerobic fungi were isolated from rumen digesta of sheep and cattle and were purified using a plate culture technique. The isolates were successfully cultured on a semi-defined medium which lacked rumen fluid, and on a defined medium.

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Differential carbohydrate media and anaerobic replica plating techniques were used to assess the degrees of diurnal variations in the direct and viable cell counts as well as the carbohydrate-specific subgroups within the mixed rumen bacterial populations in cattle fed maintenance (metabolizable energy) levels of either a high-forage or a high-concentrate diet once daily. The rumen was sampled at 1 h before feeding and 2, 4, 8, 12, and 16 h after feeding, and selected microbiological parameters of the isolated bacterial populations were assessed. Corresponding samples of ruminal fluid were assayed for fermentation acids, carbohydrate, ammonia, and pH changes. The data showed that regardless of diet, total bacterial numbers remained fairly constant throughout the day. The number of viable bacteria declined 40 to 60% after feeding and then increased to a maximum at 16 h postfeeding. Changes occurred in the carbohydrate-specific subgroups within the bacterial populations, and some of the changes were consistent with a predicted scheme of ruminal feedstuff carbohydrate fermentation. Regardless of diet, however, soluble-carbohydrate-utilizing bacteria predominated at all times. Xylan-xylose and pectin subgroups respectively comprised about one-half and one-third of the population when the high-forage diet was given. These subgroups, along with the cellulolytics, constituted lesser proportions of the population when the high-concentrate diet was given. The cellulolytic subgroup was the least numerous of all subgroups regardless of diet but followed a diurnal pattern similar to that predicted for cellulose fermentation. There were few diurnal variations or differences in bacterial cell compositions and ruminal fluid parameters between diets. The observed similarities and dissimilarities of the rumen bacterial populations obtained when the two diets were given are discussed. The data are consistent with the versatility and constancy of the rumen as a stable, mature microbial system under the specific low-level feeding regimens used.

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A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates, and a variety of differential (DC) media containing one or two carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (sl mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts. The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress. The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media. With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria. Bacteria. Pure cultures of anaerobic rumen bacteria were obtained from the culture collection of the Department of Dairy Science at the University of Illinois. The medium used for their maintenance was the RGCA slant medium of Bryant and Burkey (6) as modified by Bryant and Robinson (9). Bacterial populations in rumen contents were obtained from a fistulated, nonlactating Holstein cow fed a diet consisting of 10 kg of alfalfa hay and 3.6 kg of concentrate (75% forage/25% grain as dry matter). The diet was fed in equal portions twice daily at 6 a.m.