A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

Small-subunit rRNA sequences were determined for almost 50 species of mycoplasmas and their walled relatives, providing the basis for a phylogenetic systematic analysis of these organisms. Five groups of mycoplasmas per se were recognized (provisional names are given): the hominis group (which included species such as Mycoplasma hominis, Mycoplasma lipophilum, Mycoplasma pulmonis, and Mycoplasma neurolyticum), the pneumoniae group (which included species such as Mycoplasma pneumoniae and Mycoplasma muris), the spiroplasma group (which included species such as Mycoplasma mycoides, Spiroplasma citri, and Spiroplasma apis), the anaeroplasma group (which encompassed the anaeroplasmas and acholeplasmas), and a group known to contain only the isolated species Asteroleplasma anaerobium. In addition to these five mycoplasma groups, a sixth group of variously named gram-positive, walled organisms (which included lactobacilli, clostridia, and other organisms) was also included in the overall phylogenetic unit. In each of these six primary groups, subgroups were readily recognized and defined. Although the phylogenetic units identified by rRNA comparisons are difficult to recognize on the basis of mutually exclusive phenotypic characters alone, phenotypic justification can be given a posteriori for a number of them.

Twenty-five phenotypically and genotypically similar strains of a fastidious, xylem-limited bacterium were isolated from 10 plant disease sources including Pierce's disease of grapevines, phony disease of peach, periwinkle wilt, and leaf scorches of almond, plum, elm, sycamore, oak, and mulberry. The cells were single (occasionally filamentous), nonmotile, aflagellate straight rods (0.25 to 0.35 by 0.9 to 3.5 pm). They were gram negative, catalase positive, and oxidase negative, utilized hippurate, and produced gelatinase and often beta-lactamase but not beta-galactosidase, coagulase, lipase, amylase, phosphatase, indole, or HZS. The bacteria were strict aerobes with optimum growth at 26 to 28°C and pH 6.5 to 6.9 and had doubling times of 0.45 to 1.98 days in periwinkle wilt broth. Monoclonal antibodies prepared against the Pierce's disease bacterium reacted with all strains. DNA composition was 51 to 53 mol% guanine plus cytosine, and strains were at least 85% related in DNA hybridization. Sequencing of 16s ribosomal ribonucleic acid related them to the xanthomonads. These bacteria form a distinct group, and the name XyZeZZa fastidiosa is proposed, establishing a new genus with one species in the gamma subgroup of the eubacteria. Strain PCE-RR (ATCC 35879) is designated as the type strain. (13,16,26,27,33,36,37,41,44,45; J. Sherald, unpublished data).

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.

Small subunit rRNA sequences have been determined for representative strains of six species of the family Rickettsiaceae: Rickettsia rickettsii, Rickettsia prowazekii, Rickettsia typhi, Coxiella burnetii, Ehrlichia risticii, and Wolbachia persica. The relationships among these sequences and those of other eubacteria show that all members of the family Rickettsiaceae belong to the so-called purple bacterial phylum. The three representatives of the genus Rickettsia form a tight monophyletic cluster within the a subdivision of the purple bacteria. E. risticii also belongs to the a subdivision and shows a distant yet specific relationship to the genus Rickettsia. However, the family as a whole is not monophyletic, in that C. burnetii and W. persica are members of the -y subdivision. The former appears to show a specific, but rather distant, relationship to the genus Legionella.The order Rickettsiales (32) comprises a collection of procaryotes that share the property of intimate association with eucaryotic cells. The relationship in most cases is obligate intracellular parasitism, although a few of these bacteria have been grown in complex host-cell-free culture media. Although this order contains notorious pathogens of humans and animals, some of these bacteria cause no obvious damage to their host, and the relationship can be regarded as commensal. The Rickettsiales are clearly separate from the Chlamydiales (17), a narrowly defined group of energy-parasitizing, obligately intracellular bacteria. Certain fastidious parasites of plant vascular tissues and arthropods, sometimes referred to as rickettsialike (5), are not included in either category. Also, the highly heterogeneous group of bacteria that have established an endosymbiotic relationship with their hosts and for the most part have not been cultivated, including the hydrothermal-vent-associated symbionts (3,4,21), are viewed as distinct from the Rickettsiales (32) in the latest edition of Bergey's Manlual of Systematic Bacteriology.The order Rickettsiales contains three families: Rickettsiaceae, Bartonellaceae, and Anaplasmataceae. Our present study is confined to the family Rickettsiaceae, which is divided into three tribes: Rickettsieae, Ehrlichieae, and Wolbachieae (32). In the family Rickettsiaceae, rigorous criteria of classical taxonomy have been applied to the definition of most species and, in some but not in all cases, the genera. For example, good evidence was obtained by phenotypic analysis, DNA base ratio determinations, and DNA-DNA hybridization studies that in the genus Rickettsia, members of the typhus and spotted fever groups are related (32). However, the degree of relatedness of these microorganisms to the scrub typhus rickettsia, Rickettsia tsutsugamushi, remains unknown. Similarly, phenotypic analysis links the monocytic erhlichiae (Ehrlichia canis, Ehrlichia sennetsu, and Ehrlichia risticii) to each other (10,22,23), but their relationships to the granulocytic ehrlicheae * Corresponding author. t Present address: Gene-Trak Systems,

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