Multiple independent losses of sporulation and peptidoglycan in the Mycoplasmatales and related orders of the class Bacilli.

Many peptidoglycan-deficient bacteria such as the Mycoplasmatales are known host-associated lineages, lacking the environmental resistance mechanisms and metabolic capabilities necessary for a free-living lifestyle. Several peptidoglycan-deficient and non-sporulating orders of interest are thought to be descended from Gram-positive sporulating Bacilli through reductive evolution. Here we annotate 2650 genomes belonging to the class Bacilli, according to the Genome Taxonomy Database, to predict the peptidoglycan and sporulation phenotypes of three novel orders, RFN20, RF39 and ML615J-28, known only through environmental sequence surveys. These lineages are interspersed between peptidoglycan-deficient non-sporulating orders including the Mycoplasmatales and Acholeplasmatales, and more typical Gram-positive orders such as the Erysipelotrichales and Staphylococcales. We use the extant genotypes to perform ancestral state reconstructions. The novel orders are predicted to have small genomes with minimal metabolic capabilities and to comprise a mix of peptidoglycan-deficient and/or non-sporulating species. In contrast to expectations based on cultured representatives, the order Erysipelotrichales lacks many of the genes involved in peptidoglycan and endospore formation. The reconstructed evolutionary history of these traits suggests multiple independent whole-genome reductions and loss of phenotype via intermediate transition states that continue into the present. We suggest that the evolutionary history of the reduced-genome lineages within the class Bacilli is one driven by multiple independent transitions to host-associated lifestyles, with the degree of reduction in environmental resistance and metabolic capabilities correlated with degree of host association.

A phylogenomic and molecular markers based taxonomic framework for members of the order Entomoplasmatales: proposal for an emended order Mycoplasmatales containing the family Spiroplasmataceae and emended family Mycoplasmataceae comprised of six genera.

The "Spiroplasma cluster" is a taxonomically heterogeneous assemblage within the phylum Tenericutes encompassing different Entomoplasmatales species as well as the genus Mycoplasma, type genus of the order Mycoplasmatales. Within this cluster, the family Entomoplasmataceae contains two non-cohesive genera Entomoplasma and Mesoplasma with their members exhibiting extensive polyphyletic branching; additionally, the genus Mycoplasma is also embedded within this family. Genome sequences are now available for all 19 Entomoplasmataceae species with validly published names, as well as 6 of the 7 species from the genus Mycoplasma. With the aim of developing a reliable phylogenetic and taxonomic framework for the family Entomoplasmataceae, exhaustive phylogenetic and comparative genomic studies were carried out on these genome sequences. Phylogenetic trees were constructed based on concatenated sequences of 121 core proteins for this cluster, 67 conserved proteins shared with the phylum Firmicutes, 40 ribosomal proteins, three major subunits of RNA polymerase (RpoA, B and C) by different means and also for the 16S rRNA gene sequences. The interspecies relationships as well as different species groups observed in these trees were identical and robustly resolved. In all of these trees, members of the genera Mesoplasma and Entomoplasma formed three and two distinct clades, respectively, which were interspersed among the members of the other genus. The observed species groupings in the phylogenetic trees are independently strongly supported by our identification of 103 novel molecular markers or synapomorphies in the forms of conserved signature indels and conserved signature proteins, which are uniquely shared by the members of different observed species clades. To account for the different observed species clades, we are proposing a division of the genus Mesoplasma into an emended genus Mesoplasma and two new genera Tullyiplasma gen. nov. and Edwardiiplasma gen. nov. Likewise, to recognize the distinct species groupings of Entomoplasma, we are proposing its division into an emended genus Entomoplasma and a new genus Williamsoniiplasma gen. nov. Lastly, to rectify the long-existing taxonomic anomaly caused by the presence of genus Mycoplasma (order Mycoplasmatales) within the Entomoplasmatales, we are proposing an emendation of the family Mycoplasmataceae to include both Entomoplasmataceae plus Mycoplasma species and an emendation of the order Mycoplasmatales, which now comprises of the emended family Mycoplasmataceae and the family Spiroplasmataceae. The taxonomic reclassifications proposed here accurately reflect the species relationships within this group of Tenericutes and they should lead to a better understanding of their biological and pathogenic characteristics.

On molecular taxonomy: what is in a name?

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.

Role of genital mycoplasmata and other bacteria in urolithiasis.

Urease-producing bacteria have been shown to affect the formation of infection stones by splitting urea into ammonia, bicarbonate and carbonate. An increase in alkaline pH results in urinary supersaturation of the ions. The increase in ammonia also causes injury to the urothelial glycosaminoglycan layer. Non-urease-producing bacteria have been speculated to form urinary stones. Midstream voided bladder urine and fractured stone nidus samples from 72 patients undergoing surgery for urolithiasis were cultured on specific media for genital mycoplasmata and on conventional media. Urine samples were obtained from a control group of 40 healthy subjects. Genital mycoplasmata and other bacteria were evaluated with regard to the composition of urinary stones. Compared with other origins of stones, the relation between isolation of Ureaplasma urealyticum and infection stone disease was statistically proven. Isolation of genital mycoplasmata was significantly higher in women than in men in the study group. The urinary stones comprised 84.7% calcium stones, 8.3% uric acid stones and 6.9% infection (magnesium ammonium phosphate) stones. Coagulase-negative Staphylococci, Escherichia coli, Corynebacterium spp., Enterobacterium spp. and U. urealyticum were cultured from stone samples. The results suggests that non-urease-producing bacteria, as well as urease-producing bacteria, may influence the formation of urinary stones.

Phylogenetic depth of the bacterial genera Aquifex and Thermotoga inferred from analysis of ribosomal protein, elongation factor, and RNA polymerase subunit sequences.

The phylogenetic placement of the Aquifex and Thermotoga lineages has been inferred from (i) the concatenated ribosomal proteins S10, L3, L4, L23, L2, S19, L22, and S3 encoded in the S10 operon (833 aa positions); (ii) the joint sequences of the elongation factors Tu(1alpha) and G(2) coded by the str operon tuf and fus genes (733 aa positions); and (iii) the joint RNA polymerase beta- and beta'-type subunits encoded in the rpoBC operon (1130 aa positions). Phylogenies of r-protein and EF sequences support with moderate (r-proteins) to high statistical confidence (EFs) the placement of the two hyperthermophiles at the base of the bacterial clade in agreement with phylogenies of rRNA sequences. In the more robust EF-based phylogenies, the branching of Aquifex and Thermotoga below the successive bacterial lineages is given at bootstrap proportions of 82% (maximum likelihood; ML) and 85% (maximum parsimony; MP), in contrast to the trees inferred from the separate EF-Tu(1alpha) and EF-G(2) data sets, which lack both resolution and statistical robustness. In the EF analysis MP outperforms ML in discriminating (at the 0.05 level) trees having A. pyrophilus and T. maritima as the most basal lineages from competing alternatives that have (i) mesophiles, or the Thermus genus, as the deepest bacterial radiation and (ii) a monophyletic A. pyrophilus-T. maritima cluster situated at the base of the bacterial clade. RNAP-based phylogenies are equivocal with respect to the Aquifex and Thermotoga placements. The two hyperthermophiles fall basal to all other bacterial phyla when potential artifacts contributed by the compositionally biased and fast-evolving Mycoplasma genitalium and Mycoplasma pneumoniae sequences are eschewed. However, the branching order of the phyla is tenuously supported in ML trees inferred by the exhaustive search method and is unresolved in ML trees inferred by the quartet puzzling algorithm. A rooting of the RNA polymerase-subunit tree at the mycoplasma level seen in both the MP trees and the ML trees reconstructed with suboptimal amino acid substitution models is not supported by the EF-based phylogenies which robustly affiliate mycoplasmas with low-G+C gram-positives and, most probably, reflects a "long branch attraction" artifact.

'Candidatus phytoplasma japonicum', a new phytoplasma taxon associated with Japanese Hydrangea phyllody.

A phytoplasma was discovered in diseased specimens of field-grown hortensia (Hydrangea spp.) exhibiting typical phyllody symptoms. PCR amplification of DNA using phytoplasma specific primers detected phytoplasma DNA in all of the diseased plants examined. No phytoplasma DNA was found in healthy hortensia seedlings. RFLP patterns of amplified 16S rDNA differed from the patterns previously described for other phytoplasmas including six isolates of foreign hortensia phytoplasmas. Based on the RFLP, the Japanese Hydrangea phyllody (JHP) phytoplasma was classified as a representative of a new subgroup in the phytoplasma 16S rRNA group I (aster yellows, onion yellows, all of the previously reported hortensia phytoplasmas, and related phytoplasmas). A phylogenetic analysis of 16S rRNA gene sequences from this and other group I phytoplasmas identified the JHP phytoplasma as a member of a distinct sub-group (sub-group Id) in the phytoplasma clade of the class Mollicutes. The phylogenetic tree constructed from 16S rRNA gene sequences was consistent with the hypothesis that the JHP phytoplasma and its closest known relatives, the Australian grapevine yellows (AUSGY), Phormium yellow leaf (PYL), Stolbur of Capsicum annuum (STOL) and Vergilbungskrankheit of grapevine (VK) share a common ancestor. The unique properties of the DNA from the JHP phytoplasma clearly establish that it represents a new taxon, 'Candidatus Phytoplasma japonicum'.

Entomoplasma freundtii sp. nov., a new species from a green tiger beetle (Coleoptera: Cicindelidae).

A mollicute (strain BARC 318T) isolated from gut tissue of a green tiger beetle (Coleoptera: Cicindelidae) was found by dark-field microscopy to consist of non-helical, non-motile, pleomorphic coccoid forms of various sizes. In ultrastructural studies, individual cells varied in diameter from 300 to 1200 nm, were surrounded by a cytoplasmic membrane and showed no evidence of cell wall. The organisms were readily filterable through membrane filters with mean pore diameters of 450 and 300 nm, with unusually large numbers of organisms filterable through 200 nm pore membrane filters. Growth occurred over a temperature range of 15-32 degrees C with optimum growth at 30 degrees C. The organism fermented glucose and hydrolysed arginine but did not hydrolyse urea. Strain BARC 318T was insensitive to 500 U penicillin ml-1 and required serum or cholesterol for growth. It was serologically distinct from all currently described sterol-requiring, fermentative Mycoplasma species and from 12 non-sterol-requiring Mesoplasma species, 13 non-sterol-requiring Acholeplasma species and 5 previously described sterol-requiring Entomoplasma species. Strain BARC 318T was shown to have a G + C content of 34 mol% and a genome size of 870 kbp. The 16S rDNA sequence of strain BARC 318T was compared to 16S rDNA sequences of several other Entomoplasma species and to other representative species of the genera Spiroplasma and Mycoplasma, and to other members of the class Mollicutes. These comparisons indicated that strain BARC 318T had close phylogenetic relationships to other Entomoplasma species. On the basis of these findings and other similarities in morphology, growth and temperature requirements and genomic features, the organism was assigned to the genus Entomoplasma. Strain BARC 318T (ATCC 51999T) is designated the type strain of Entomoplasma freundtii sp. nov.

Effect of natural amphipathic peptides on viability, membrane potential, cell shape and motility of mollicutes.

The antibiotic activity of ten amphipathic peptides was investigated in six species of mollicutes belonging to the genera Acholeplasma, Mycoplasma and Spiroplasma. A. laidlawii was the most sensitive and M. mycoides subsp. mycoides SC the most resistant. Animal defence peptides (cecropins A and P1, and magainin 2) proved to be less potent than bee-venom mellitin and most of the peptides produced by bacteria (globomycin, gramicidin S, surfactin and valinomycin) or fungi (alamethicin). Gramicidin S was by far the most active peptide, with minimal inhibitory concentrations ranging from 2 to 50 nM. Alamethicin, gramicidin S, mellitin and surfactin had a cidal effect, whilst cecropins, globomycin, magainin 2, polymyxin B and valinomycin proved to be static. The peptides altered the membrane potential of spiroplasma cells with a potency independent of their linear or cyclic structure. However, globomycin depolarized the plasma membrane only weakly, whilst polymyxin B, in order to be active, required prior hyperpolarization of the membrane. The peptides also induced the loss of cell motility and helicity in spiroplasmas, suggesting that motility and cell shape in these bacteria are coupled to the transmembrane electrochemical gradient. Globomycin, an inhibitor of signal-peptidase II, prevented the growth of spiroplasmas, M. gallisepticum, and M. genitalium, but not that of A. laidlawii and M. mycoides subsp. mycoides SC, although the latter also synthesized membrane lipoproteins. Inhibition of spiralin processing by globomycin was demonstrated in S. citri and S. melliferum, with a more pronounced effect in the second species.

Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA.

The phylogenetic relationships of 17 phytopathogenic mycoplasmalike organisms (MLOs) representing seven major taxonomic groups established on the basis of MLO 16S ribosomal DNA (rDNA) restriction patterns were examined by performing a sequence analysis of the 16S rDNA gene. The sequence data showed that the MLOs which we examined are members of a relatively homogeneous group that evolved monophyletically from a common ancestor. In agreement with results obtained previously with other MLOs, our results also revealed that the organisms are more closely related to Acholeplasma laidlawii and other members of the anaeroplasma clade than to any other mollicutes. A phylogenetic tree based on 16S rDNAs showed that the MLOs which we examined can be divided into the following five primary clusters: (i) the aster yellows strain cluster; (ii) the apple proliferation strain cluster; (iii) the western-X disease strain cluster; (iv) the sugarcane white leaf strain cluster; and (v) the elm yellows strain cluster. The aster yellows, western-X disease, and elm yellows strain clusters were divided into two subgroups each. MLOs whose 16S rDNA sequences have been determined previously by other workers can be placed in one of the five groups. In addition to the overall division based on 16S rDNA sequence homology data, the primary clusters and subgroups could be further defined by a number of positions in the 16S rDNAs that exhibited characteristic compositions, especially in the variable regions of the gene.

The nasal mycoplasmal flora of healthy calves and cows.

The nasal mycoplasmal flora of 270 healthy cows from 27 herds in the Netherlands and 35 healthy calves from 7 of these herds was examined. Various methods for isolating mycoplasmas were compared. The prevalence of the various species was as follows: Ureaplasma diversum in 3 (9%) calves; Mycoplasma dispar in 14 (40%) calves; M. bovis in 1 (3%) calf; M. bovirhinis in 23 (66%) calves and 16 (6%) cows; M. bovoculi in 8 (23%) calves and 53 (20%) cows; M. canis in 1 (3%) calf; M. equirhinis in 2 (1%) cows; M. conjunctivae in 2 (1%) cows; Acholeplasma laidlawii in 1 (3%) calf and 3 (1%) cows; and A. axanthum in 7 (3%) cows. The noses of healthy calves were less frequently colonized by the pathogenic species U. diversum and contained fewer U. diversum and M. dispar organisms than the noses of pneumonic calves. We concluded that the mycoplasmal flora of calves and healthy cows was quite different and also that cows play only a minor role in the epidemiology of pathogenic mycoplasma species of calves in the Netherlands.

Evidence for superoxide dismutase and catalase in mollicutes and release of reactive oxygen species.

The presence of superoxide dismutase was demonstrated in 21 strains of mollicutes, including achuloplasmas, mycoplasmas and ureaplasmas. Additionally, catalase activities were demonstrated in nearly 50% of the cell lysates, whereas no peroxide activities were detectable. The production of O2- and H2O2 with glucose as substrate was demonstrated for 8 strains of 10 strains tested. Anaerobic mycoplasmas showed the highest amount of radical production, whereas superoxide dismutase and catalase activities were in the range of activities estimated for aerobic mollicutes. Some pathogenic strains additionally released compounds into the culture medium, which stimulated O2- production by PMNs.

Mollicutes DNA polymerases: characterization of a single enzyme from Mycoplasma mycoides and Ureaplasma urealyticum and of three enzymes from Acholeplasma laidlawii.

The DNA polymerase activity of different members of Mollicutes was studied. A single DNA polymerase was found in Mycoplasma mycoides and Ureaplasma urealyticum, type species of the genera Mycoplasma and Ureaplasma, and was compared with the previously described Mycoplasma orale enzyme. Most of their properties were comparable; an immunological relationship was demonstrated between M. orale and M. mycoides enzymes by immunoblotting. In contrast to these results, three different DNA polymerases were purified in Acholeplasma laidlawii, type species of the genus Acholeplasma which, in this aspect, resembles the genus Spiroplasma. A 3'-5' exonuclease activity was found in the different purified preparations. In M. mycoides, M. orale and one of the three A. laidlawii preparations, the 3'-5' exonuclease could be separated from the DNA polymerase by non-denaturing PAGE. The presence of a single DNA polymerase seems to be a typical feature of the Mycoplasmataceae, which include the genera Mycoplasma and Ureaplasma, in contrast to the occurrence of three enzymes within the Acholeplasmataceae and Spiroplasmataceae. These results are in agreement with the phylogenetic tree of Mollicutes proposed from their 5 S and 16 S rRNA sequence comparisons, in which the evolution of Acholeplasma and Spiroplasma branches led, by genome reductions, to Mycoplasma and Ureaplasma species.

Crohn's disease uveitis. Parasitization of vitreous leukocytes by mollicute-like organisms.

Crohn's disease (CD) is an idiopathic chronic inflammatory gut disease with frequent extragut inflammatory manifestations in the eyes, orbit, lungs, joints, and skin. A bacterial cause of CD is suspected, but cultivation of a specific pathogen has not been forthcoming. Mollicute-like organisms (MLOs) were recently reported to cause human chronic ocular inflammatory disease. Inoculation of this MLO into mouse eyelids produced chronic progressive granulomatous ocular and orbital inflammatory disease. In addition, MLOs disseminated to produce similar disease in the gut, heart, and lungs. MLOs are noncultivable cell wall-deficient bacterial pathogens. Because they also pass bacteria-retaining 0.450-micron filters, they can be overlooked or confused with viruses. Because MLOs have a characteristic ultrastructural appearance, they can be identified in diseased cells with the use of a transmission electron microscope. MLOs parasitize and destroy leukocytes. They alter the nucleus, replace the cytoplasm, and destroy organelles. MLO-caused disease is treatable by certain antibiotics. This report describes MLO-parasitized vitreous lymphocytes, monocytes, and polymorphonuclear leukocytes from three patients with CD who had chronic uveitis. The results indicate that MLOs probably caused the uveitis of these patients with CD. The gut as the possible source of the MLO is suggested. Rifampin therapy of Crohn's and MLO-caused disease is discussed.

Mouse interstitial lung disease and pleuritis induction by human Mollicute-like organisms.

Mollicute-Like Organisms (MLO) are cell-wall deficient intracellular bacterial pathogens. As MLO are non-cultivable, detection is based on finding typical Mollicute bodies within the host cell using a transmission electron microscope. Extracellular Mollicutes cause disease by a variety of mechanisms. MLO cause disease by similar mechanisms, and in addition directly alter the host cell nucleus, replace the cytoplasm, and destroy the organelles. MLO parasitization of plant cells causes a well studied chronic vascular disease reversible by tetracycline antibiotics. Recently similar MLO were reported to cause human chronic ocular vasculitis. As it parasitizes, lyses, and destroys leucocytes, it has been termed Leucocytoclastic MLO. Inoculation of this MLO into mouse eyelids produced delayed onset chronic ocular and lethal cardiac vasculitis. All lesions demonstrated tissue lysis with leucocytic infiltrates and MLO parasitized leucocytes. MLO-caused human and mouse disease responds to Rifampin. This report describes the 40 interstitial lung disease lesions in 21 of 100 of those MLO inoculated mice vs 0 in 200 controls (P less than 0.05) and 27 pleuritis lesions in 17 mice vs 0 control mice (P less than 0.05). The lung and pleural disease were associated in 13 lesions and unassociated in 41 lesions. MLO parasitized leucocytes were found in both the lung and pleural lesions from six of six MLO inoculated mice versus none of six controls. As most human interstitial lung and pleural diseases are idiopathic and closely resemble this mouse disease, they may be induced by MLO and treatable by Rifampin.

Isolation of anaerobic mollicutes from the intestine of swine.

Anaerobic mollicutes were isolated from the intestinal content and the epithelium of the caecum and colon of 4 of 8 swine. All of the isolates belonged to the same species. This species was resistant to digitonin and polyanethol sulfonate and was able to ferment glucose, hemolyse erythrocytes and lyse horse serum. For testing the cholesterol requirement the strain 4 A 11 was selected which was independent of cholesterol. The agent was different in the immunofluorescence test from the mollicutes reported previously in swine and quite different in its nutritional requirements from cholesterol-independent mollicutes isolated from the rumen of cattle and sheep. Thus the isolated mollicutes seem to represent a new species.