Proposal to transfer some members of the genera Haemobartonella and Eperythrozoon to the genus Mycoplasma with descriptions of 'Candidatus Mycoplasma haemofelis', 'Candidatus Mycoplasma haemomuris', 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'.

Cell-wall-less uncultivated parasitic bacteria that attach to the surface of host erythrocytes currently are classified in the order Rickettsiales, family Anaplasmataceae, in the genera Haemobartonella and Eperythrozoon. Recently 16S rRNA gene sequences have been determined for four of these species: Haemobartonella felis and Haemobartonella muris and Eperythrozoon suis and Eperythrozoon wenyonii. Phylogenetic analysis of these sequence data shows that these haemotrophic bacteria are closely related to species in the genus Mycoplasma (class Mollicutes). These haemotrophic bacteria form a new phylogenetic cluster within the so-called pneumoniae group of Mycoplasma and share properties with one another as well as with other members of the pneumoniae group. These studies clearly indicate that the classification of these taxa should be changed to reflect their phylogenetic affiliation and the following is proposed: (i) that Haemobartonella felis and Haemobartonella muris should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemofelis' and 'Candidatus Mycoplasma haemomuris' and (ii) that Eperythrozoon suis and Eperythrozoon wenyonii should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'. The former Haemobartonella and Eperythrozoon species described here represent a new group of parasitic mycoplasmas that possess a pathogenic capacity previously unrecognized among the mollicutes. These haemotrophic mycoplasmas have been given the trivial name haemoplasmas. These results call into question the affiliation of the remaining officially named species of Haemobartonella and Eperythrozoon which should be considered species of uncertain affiliation pending the resolution of their phylogenetic status.

Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups.

ABSTRACT Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.

An approach to characterizing uncultivated prokaryotes: the Grey Lung agent and proposal of a Candidatus taxon for the organism, 'Candidatus Mycoplasma ravipulmonis'.

An approach to characterizing uncultivated bacteria which combines a PFGE procedure for obtaining purified full-length chromosomes with PCR amplification is described. Isolated chromosomes from uncultivated organisms provide a specifically identifiable source material for hybridization, amplification and cloning. The availability of purified chromosomes for DNA amplification should aid in examining the diversity of microbial populations and should also reduce the possibility of forming hybrid DNA artifact molecules. The approach is illustrated by isolating the chromosome of the uncultivated agent of rodent Grey Lung disease and using the purified chromosomes to amplify and directly sequence the evolutionarily conserved 16S rRNA gene. The Grey Lung agent (GLA) contains a 650 kb chromosome and is shown to be a Mycoplasma sp. located phylogenetically in the hominis group of mycoplasmas. If a simple genomic lesion(s) is responsible for the unculturability of GLA, it is conceivable that complementation with DNA from a close relative could permit growth on artificial media.

The cell wall-less rickettsia Eperythrozoon wenyonii is a Mycoplasma.

The 16S rRNA gene of Eperythrozoon (Haemobartonella) wenyonii, a wall-less hemotrophic prokaryote currently classified as a rickettsia, was sequenced to determine the relationship of this organism to other wall-less prokaryotes. Comparison to the GenBank data base showed that this hemotrophic organism is a Mycoplasma (family Mollicutes). Phylogenetic analysis of 16S rRNA genes indicated that this and other recently sequenced 16S rRNA genes of hemotrophic bacteria form a new, separate branch which shares a node in common with the pneumoniae group of mycoplasmas. This result will require that Eperythrozoon wenyonii be reclassified as a Mycoplasma. A main point of this study is that this and related hemotrophic bacteria represent an entirely new group of pathogens among the mycoplasmas.

Direct identification and typing of Mycobacterium tuberculosis by PCR.

We have developed a rapid PCR assay that types strains of Mycobacterium tuberculosis by generating distinct DNA fingerprints directly from primary cultures. This assay allows strain identification analogous to that achieved by the standard restriction fragment length polymorphism method, and fingerprints are obtained in less than 8 h. This assay does not require subculturing, DNA purification, restriction digestion, Southern blotting, or nucleic acid hybridization. Rapid and precise identification of M. tuberculosis strains permits immediate molecular epidemiologic studies. The assay can be converted to a computer-automated system by employing fluorescently labeled PCR primers and the Perkin-Elmer DNA sequencer so that unknown-specimen fingerprints are identified by computer comparison to a database of M. tuberculosis strain fingerprints.

Effect of methylation on the electrophoretic mobility of chromosomal DNA in pulsed-field agarose gels.

Factors other than molecular weight are known to affect DNA electrophoretic mobility. DNA methylation has been found to affect the curvature of DNA, causing anomalous mobility in polyacrylamide gels; the effect of methylation on the mobility of large DNA molecules in agarose gels was unknown. Chromosomal DNA from Mycoplasma capricolum, a wall-less prokaryote which has a low intrinsic methylation rate, was methylated in agarose blocks by SssI methylase, a de novo methylase with a CpG recognition sequence. (A surprising finding was that SssI methylase altered the structure of InCert, but not SeaKem Gold, agarose.) Restriction enzyme analysis was used to estimate the extent of CpG methylation. DNA methylation was found to have no effect on the electrophoretic mobility of full-length chromosomal DNA (1,120 kbp) in agarose gels. Therefore, methylation is not a source of error in PFGE-based size estimation for chromosomal DNA molecules less than 1.12 Mbp in agarose gels.

Isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic Mycoplasma-like organisms.

We describe the isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic, mycoplasma-like organisms (MLOs). MLO chromosomes are circular and their sizes (640 to 1185 kbp) are heterogeneous. Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents. MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non-culturability. Hybridization with cloned MLO-specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO 'type' strains. Large DNA molecules that appear to be MLO megaplasmids were also demonstrated. The ability to characterize full-length chromosomes from virtually any non-culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.

Invasion of the central nervous system by Borrelia burgdorferi in acute disseminated infection.

OBJECTIVE: To determine central nervous system (CNS) involvement in acutely disseminated Borrelia burgdorferi infection by measurement of borrelia-specific DNA using the polymerase chain-reaction (PCR) assay and to compare the results of this with standard serological tests. DESIGN: Prospective study with laboratory investigators blinded to clinical data. SETTING: Multicenter office practice with a central reference laboratory. PATIENTS: Cerebrospinal fluid (CSF) was collected from 12 patients with acute disseminated Lyme borreliosis with less than 2 weeks of active disease. The normal control specimens came from 16 patients whose CSF samples had been sent to the clinical laboratory for tests unrelated to the present study. MAIN OUTCOME MEASURES: Clinical evidence of disease and laboratory abnormalities. RESULTS: Eight of the 12 patients (four of six with multiple areas of erythema migrans and four of six with cranial neuritis without erythema migrans) had B burgdorferi-specific DNA in their CSF. Among the 12 patients studied, nine had acute cranial neuritis and six had multiple erythema migrans lesions. Just four of the eight who were found to have spirochetal DNA in their CSF had complaints suggestive of CNS infection. In three of the PCR-positive CSF samples, no other abnormalities were noted. None of 16 samples from controls were positive in the PCR assay. CONCLUSION: B burgdorferi can invade the CNS early in the course of infection. Careful consideration should be given to choosing antibiotics that achieve adequate CSF levels in patients with disseminated infection.

Pulse-field electrophoresis indicates full-length Mycoplasma chromosomes range widely in size.

Full-size linear chromosomes were prepared from mycoplasmas by using gamma-irradiation to introduce one (on average) double-strand break in their circular chromosomes. Chromosome sizes were estimated by pulsed-field gel electrophoresis (PFGE) from the mobilities of these full-length molecules relative to DNA size references. Sizes estimated for Ureaplasma urealyticum T960 and 16 Mycoplasma species ranged from 684 kbp (M. hominis) to 1315 kbp (M. iowae). Using this sample, we found no correlation between the mobility of the full-size linear chromosomes and their G + C content. Sizes for A. laidlawii and A. hippikon were within the range expected from renaturation kinetics. PFGE size estimates are in good agreement with sizes determined by other methods, including electron microscopy, an ordered clone library, and summation of restriction fragments. Our estimates also agree with those from renaturation kinetics for both the largest and some of the smallest chromosomes, but in the intermediate size range, renaturation kinetics consistently provides lower values than PFGE or electron microscopy. Our PFGE estimates show that mycoplasma chromosomes span a continual range of sizes, with several intermediate values falling between the previously recognized large and small chromosome size clusters.

Broad range DNA probes for detecting and amplifying eubacterial nucleic acids.

In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.

Deletions, duplications and rearrangements in mycoplasma ribosomal RNA gene sequences.

Mycoplasma genomes lack several entire ribosomal RNA gene sets, and the remaining RNA genes lack some nucleotides. Analysis of mycoplasma 5S RNAs utilizing generalized models for prokaryotic 5S RNA secondary structure allows us to specify the molecular locations and identify many of the nucleotides missing from mycoplasma 5S RNAs and to detect the presence of unusual structural features. The most extensive alterations occur in the 5S RNAs from M. mycoides subsp. capri and M. capricolum, which have a similar pattern. Each of these 5S RNAs, as well as the Spiroplasma BC3 5S RNA, has a concentration of deletions in helix V, and they also share several unusual structures. We also noted the occurrence in mycoplasma 5S RNAs of numerous repeated sequences. The genetic process of unequal crossing-over is suggested as the explanation for the repeated sequences observed in mycoplasma RNAs.

Unusual structural features of the 5S ribosomal RNA from Streptococcus cremoris.

The nucleotide sequence of the 5S ribosomal RNA of Streptococcus cremoris has been determined. The sequence is 5' (sequence in text) 3'. Comparison of the S. cremoris 5S RNA sequence to an updated prokaryotic generalized 5S RNA structural model shows that this 5S RNA contains some unusual structural features. These features result largely from uncommon base substitutions in helices I, II and IV. Some of these unusual structural features are shared by several of the known 5S RNA sequences from mycoplasmas. However, the characteristic bloc of deletions found in helix V of these mycoplasma 5S RNAs is not present in the 5S RNA of S. cremoris.

Mycoplasma and bacterial proteins resembling contractile proteins: a review.

The basis of gliding motility in prokaryotes including certain mycoplasmas and the ability of mycoplasmas to retain their characteristic cell shapes in the absence of a supporting cell wall is unexplained. This review examines the available studies describing proteins resembling contractile proteins and cytoskeletal proteins in prokaryotes. Proteins with a significant degree of amino acid sequence homology to the myofibrillar proteins actin and myosin Al light chain and to tropomyosin have been described in prokaryotes. In addition, protein preparations from Mycoplasma pneumoniae have been shown to bind heavy meromyosin fragments, anti-actin antibody, and phalloidin; however, it remains to be proved that proteins in these preparations sharing properties with actin are synthesized by the mycoplasma.

Evolution of mycoplasmas and genome losses.

Streptococci and acholeplasmas have a close evolutionary relationship. We examined their genomes to determine what chromosomal losses occurred to produce the smaller acholeplasma genomes and found by RNA-DNA hybridization that Streptococcus cremoris and S. lactis possess at least five or at least six ribosomal RNA gene sets, respectively, while acholeplasmas have only two rRNA gene sets. Other important deficiencies identified in mycoplasmas are associated with envelope or RNA genes, and analysis of these chromosomal deletions may lead to an understanding of how mycoplasmas evolved from walled bacteria.

Origins of the mycoplasmas: sterol-nonrequiring mycoplasmas evolved from streptococci.

We report the establishment of a phylogenetic relationship between the sterol-nonrequiring mycoplasmas (Acholeplasma species) and streptococci. Three specific antisera prepared against purified Streptococcus faecalis fructose diphosphate aldolase and glyceraldehyde-3-phosphate dehydrogenase and Pediococcus cerevisiae glyceraldehyde-3-phosphate dehydrogenase were used for comparative enzyme immunological studies; the Ouchterlony double-diffusion technique and the quantitative microcomplement fixation procedure were employed. The reactions obtained provide evidence showing that all seven ACholeplasma species studied (A. laidlawii, A. granularum, A. modicum, A. oculi, A. axanthum. A. hippikon, and A. equifetale) are phylogenetically related to streptococci and that they evolved from streptococci. The data strongly suggest that the acholeplasmas comprise a distinct evolutionary group that has diverged from streptococci belonging to Lancefield group D or N. No reactions were observed between these enzyme antisera and cell extracts from six fermentative Mycoplasma species. These results support the view that mycoplasmas are derived from various bacteria.

Extraction of an actin-like protein from the prokaryote Mycoplasma pneumoniae.

An actin-like protein has been identified in cell extracts from the prokaryote Mycoplasma pneumoniae. This protein bears a striking resemblance to actin from vertebrates: (i) the solubility of the protein during isolation is analogous to that of actin bound to myosin (soluble in high ionic strength salt solution and insoluble at low ionic strength), (ii) sodium dodecyl sulfate treatment of the partially purified M. pneumoniae extract produces a protein with an electrophoretic mobility very close to that of vertebrate actin in sodium dodecyl sulfate/polyacrylamide gels, (iii) treatment of preparations with ATP-Mg2+ allows separation of long curvilinear filaments, 5-6 nm wide, that closely resemble eukaryotic filamentous actin, and (iv) the prokaryotic filamentous actin binds vertebrate heavy meromyosin fragments to form hybrid compleexes with the characteristic shape of periodic repeating arrowheads, and no heavy meromyosin is bound in the presence of ATP.

Properties of a fructose-1,6-diphosphate-activated lactate dehydrogenase from Acholeplasma laidlawii type A.

Acholeplasma laidlawii A possesses a nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactate dehydrogenase (LDH) which is activated specifically by low concentrations of fructose-1, 6-diphosphate (FDP). Studies with partially purified enzyme show that the kinetic response to FDP is hyperbolic. The enzyme is inhibited by inorganic phosphate, adenosine triphosphate, and high concentrations of reduced NAD (NADH). Low activity is demonstrable in the absence of FDP at pH 6.0 to 7.2, but FDP is absolutely required in the region of pH 8. FDP causes an upward shift in the optimum pH of the enzyme, which is near 7.2 in tris (hydroxymethyl)aminomethane buffer. Activation of the enzyme by FDP is markedly affected by substrate concentration; FDP lowers the apparent K(m) for pyruvate and NADH. The affinity of the enzyme for pyruvate is also influenced by H(+) concentration. The pyruvate analogue alpha-ketobutyrate serves as an effective substrate for the enzyme; when it is utilized, the enzyme is still activated by FDP. Reversal of the pyruvate reduction reaction catalyzed by the enzyme can be demonstrated with the 3-acetylpyridine analogue of NAD. The catalytic properties of the A. laidlawii enzyme and the known FDP-activated LDHs which occur among lactic acid bacteria are discussed.

Occurrence and properties of lactic dehydrogenases of fermentative mycoplasmas.

Eight fermentative mycoplasmas differing in genome size, deoxyribonucleic acid (DNA) base composition, or sterol dependence were examined for lactic dehydrogenase composition by spectrophotometric assay and polyacrylamide gel electrophoresis. Three completely different patterns of lactic dehydrogenase composition were found. (i) A nicotinamide adenine dinucleotide (NAD)-dependent l(+)-lactic dehydrogenase was found in Mycoplasma pneumoniae, M. gallisepticum, M. mycoides var. mycoides, mycoplasma UM 30847, M. neurolyticum, and Acholeplasma axanthum. Electrophoresis of cell-free extracts of each of these mycoplasmas produced, with the exception of M. mycoides var. mycoides and UM 30847, single, different enzyme bands. M. mycoides var. mycoides and UM 30847 were similar and formed multiple bands of enzyme activity. We were unable to establish whether these multiple bands were due to lactic dehydrogenase isoenzymes or artifacts. (ii) An NAD-dependent d(-)-lactic dehydrogenase which could not be reversed to oxidize lactate was found in M. fermentans. (iii) A. laidlawii A possessed an NAD-independent d(-)-lactic dehydrogenase capable of reducing dichlorophenol-indophenol, and an NAD-dependent l(+)-lactic dehydrogenase which is specifically activated by fructose-1,6-diphosphate. Heretofore, this enzyme regulatory mechanism was known to occur only among the Lactobacillaceae. No yeast-type lactic dehydrogenase activity was found in any of the mycoplasmas examined. The stereoisomer of lactic acid accumulated during growth correlated perfectly with the type of NAD-dependent lactic dehydrogenase found in each mycoplasma. The types of lactic dehydrogenase activity found in these mycoplasmas were not related to genome size, DNA base composition, or sterol dependence.