INTRODUCTION

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"Life is extremely conservative. On whatever levelthe individual organism, the species, the biota as a wholelife expends energy such that it preserves its past, even if, paradoxically, various threats force it to innovate."

Lynn Margulis and Dorion Sagan (279a)

The recent sequencing of the entire genomes of Mycoplasma genitalium (139) and M. pneumoniae (181) has attracted considerable attention among life scientists to the molecular biology of mycoplasmas, the smallest self-replicating organisms. It appears that we are now much closer to the goal of defining, in molecular terms, the entire machinery of a self-replicating cell. Considerable advances were also made toward a better understanding of mycoplasma pathogenesis. Most impressive are the findings concerning the interaction of mycoplasmas with the immune system, macrophage activation, cytokine induction, mycoplasma cell components acting as superantigens, and autoimmune manifestations. Evasion of the host immune system by antigenic variation of mycoplasmal surface components, as well as molecular definition of mycoplasmal adhesins, has also gained much attention recently. The demonstration of the ability of mycoplasmas to enter host cells and the possibility that several human mycoplasmas act as accessory factors in the activation of AIDS played a role in intensifying research on mycoplasma pathogenesis, bringing more researchers into the circle of those interested in this group of organisms. We were thus prompted to try and summarize within the framework of a single comprehensive review the cell biology and pathogenicity of the mycoplasmas, emphasizing when possible the lessons that can be learned from the mycoplasmal genome projects on the minimal complement of genes enabling life.

Mycoplasmas are distinguished phenotypically from other bacteria by their minute size and total lack of a cell wall. Taxonomically, the lack of cell walls is used to separate mycoplasmas from other bacteria in a class named Mollicutes (mollis, soft; cutis, skin, in Latin). The current classification of Mollicutes and the properties distinguishing the currently established taxa are presented in Table 1. While the trivial terms "mycoplasmas" or "mollicutes" have been used interchangeably to denote any species included in Mollicutes, the trivial names ureaplasmas, entomoplasmas, mesoplasmas, spiroplasmas, acholeplasmas, asteroleplasmas, and anaeroplasmas are routinely used for members of the corresponding genera. The preliminary molecular characterization of the uncultured plant and insect mycoplasma-like organisms (MLOs) has provided strong experimental support for their inclusion in the class Mollicutes. Consequently, the trivial term "phytoplasmas" has been proposed to replace the awkward name "mycoplasma-like organisms."

Because of the wide scope of the review and space limitations, emphasis will be put on the more recent findings, published since 1990. The interested reader is referred to a number of books on various aspects of mycoplasmology published during the last decade or so (210, 277, 365, 370, 395, 459, 473). A wealth of information can be found in the proceedings of the biannual meetings of the International Organization for Mycoplasmology (the last issues were named IOM Letters, volumes 1 through 4), and in a special issue of Journal of Clinical infectious Diseases (volume 17, supplement 1, 1993). Reviews covering different aspects of the molecular biology and genetics of mycoplasmas, as well as their general properties and taxonomy, are also available (30, 56, 111, 114, 356, 357, 359-361).

ECOLOGY AND HABITATS

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Distribution of Mollicutes

Mycoplasmas are widespread in nature as parasites of humans, mammals, reptiles, fish, arthropods, and plants (361). The list of hosts known to harbor mycoplasmas is continuously increasing, as is the number of established mollicute species, close to 180 at the time this review was written (Table 1). It is widely agreed that the mollicutes that have already been characterized and taxonomically defined constitute only a part, apparently a minor one, of the mollicutes living in nature. It appears as though the main factor for adding an animal or plant to the list of hosts is the willingness of a mycoplasmologist to invest the effort and funds required to isolate and taxonomically characterize the mycoplasmas from the tested host. The larger the number of established mollicute species, the harder it becomes to fulfill the minimal requirements for establishing new species (198). Particularly difficult is the requirement for serological differentiation of the new isolate from the other established species. A large battery of species-specific antisera and seed is needed for this task. The introduction of molecular tools to taxonomy, including the comparison of 16S rRNA and other conserved gene sequences, genomic restriction patterns, etc. (361, 363), has already been found effective in species and strain identification. The fast developments in genome-sequencing methodology are expected to facilitate the trend of basing bacterial taxonomy and species definition on molecular data and phylogeny, decreasing the weight of serology in species and strain identification and classification (see "Taxonomy and phylogeny" below).

The wide occurrence of mycoplasmas has frequently led researchers with little or no expertise in mycoplasmology to suggest that structures resembling mycoplasmas in tissues of oysters, bryozoans, and Giardia are mycoplasmas (reference 361 and references therein). While some of the structures may indeed represent mycoplasmas, as long as no culture was available, one could not be certain of their true nature. This statement has to be changed now. The successful application of genomic analysis methodology has led to the identification of the uncultured plant and insect MLOs as bona fide mycoplasmas, showing the way for confirmation of the identity of other MLOs as mollicutes. Thus, the grey lung "virus" disease of mice, caused by an agent that resembles mycoplasmas in thin sections of lung material but resists cultivation, has been identified as a mycoplasma based on its genome size and 16S rRNA sequence analysis. It has been proposed to classify this organism as "Candidatus Mycoplasma ravipulmonis", the "Candidatus" taxon being reserved to classification of uncultured procaryotes (312). Rather exciting is the recent finding, based on 16S rRNA sequence analysis, that Haemobartonella and Eperythrozoon, well known uncultured, wall-less pathogenic bacteria, infecting erythrocytes of a wide range of vertebrate hosts, are phylogenetically closely related to Mollicutes rather than to rickettsia and should therefore be reclassified as Mycoplasma species (313, 375). The inclusion of these hemotropic agents in Mollicutes represents an entirely new group of pathogens among the mycoplasmas, widening considerably the scope of mycoplasmology. These findings may encourage those promoting the idea that other mycoplasma-like structures observed in tissues of a variety of animals including humans, are in fact mollicutes. However, the claims by Wirostko's group that uncultured MLOs observed in leukocytes are mollicutes that cause uveitis and possibly other diseases in humans (208) have not been substantiated as yet by any of the molecular tools available for genomic DNA analysis (361). Nevertheless, the fact that in the human host, so extensively searched for mycoplasmas for decades, a new mycoplasma, M. penetrans, has recently been discovered (260) encourages the continued search for additional mycoplasma species in humans.

Mycoplasmas usually exhibit a rather strict host and tissue specificity, probably reflecting their nutritionally exacting nature and obligate parasitic mode of life. However, there are numerous examples of the presence of mycoplasmas in hosts and tissues different from their normal habitats (361). Of special interest is the host and tissue specificity of the human respiratory pathogen M. pneumoniae. While experimental respiratory infections by this mycoplasma can be induced in hamsters, the development of lung disease is different from in humans, and intratracheal inoculation of the hamsters is essential for successful infection. Only chimpanzees can be infected by droplet infection like humans, and this infection produces a respiratory disease remarkably similar to the naturally occurring pneumonia in humans (21, 138).

The primary habitats of human and animal mycoplasmas are the mucous surfaces of the respiratory and urogenital tracts, the eyes, alimentary canal, mammary glands, and joints. The obligatory anaerobic anaeroplasmas have so far been found in the bovine and ovine rumen only (Table 1). Spiroplasmas and phytoplasmas are widespread in the gut, hemocele, and salivary glands of arthropods. The spiroplasmas and phytoplasmas may be introduced via sap-sucking insects to the phloem tissues of plants, causing disease (473).

Mycoplasmas usually exhibit organ and tissue specificity. Thus, M. pneumoniae is found preferentially in the respiratory tract and M. genitalium is found primarily in the urogenital tract, but exceptions are possible, since M. genitalium has been isolated from the respiratory tract and M. pneumoniae has been isolated from the genital tract (163). These two mycoplasmas are genetically closely related, and their entire genomes have been sequenced (see "Genome-sequencing projects" below). Hence, it would be of interest to try and genetically define the factors responsible for tissue specificity of these mycoplasmas.

Accompanying the increasing numbers of patients suffering from various types of immunodeficiencies associated with hypogammaglobulinemia, AIDS, and treatment with immunosuppressive medication in patients undergoing organ transplantation, more and more reports appear on the isolation of mycoplasmas from organs different from their usual habitats. Thus, mycoplasmas and ureaplasmas belonging to the normal urogenital flora have been isolated from the blood of patients suffering from AIDS or treated with immunosuppressive drugs. Hypogammaglobulinemic and immunocompromised patients become susceptible to infections by the urogenital mycoplasmas M. hominis and U. urealyticum spreading into organs such as the respiratory tract and joints (152, 290) and causing disease in these organs. In some cases, the infecting mycoplasmas could not be cultivated, so that their identification was based on PCR amplification of their 16S rRNA genes (249, 363).

Cell cultures infected by mycoplasmas constitute an artificial unnatural habitat. The serious problems created by the persistent and elusive infections of cell cultures are reflected in the voluminous literature on this subject (22, 286). Reports from various countries show that 10 to 87% of cell cultures are infected by mycoplasmas (22, 53, 286). The percentage of infected cell cultures depends to a large extent on the population of cell cultures assayed, on the control practices used, and on the efficiency of the assay procedures applied. The mycoplasma species infecting cell cultures have remained essentially the same over the years, with M. hyorhinis, M. orale, M. arginini, and A. laidlawii being the dominant contaminants. The increasing percentage of cell cultures infected by M. fermentans (53), a mycoplasma incriminated as a cofactor in AIDS (see "Virulence factors" below), is of particular interest in terms of the possible origin of contamination. Another problem concerns the natural habitat of M. pirum, a mycoplasma initially detected only in cell cultures. The recent isolation of this mycoplasma from the blood of AIDS patients has been taken to indicate that humans are the natural hosts (164).

The intracellular location of mollicutes in insect tissues is well established. While human and animal mycoplasmas were shown to be taken up by polymorphonuclear leukocytes and macrophages (281), the question whether mycoplasmas can enter epithelial cells has not been easy to resolve, and for a long time this question was answered in the negative. The stimulus to reexamine this issue came from the studies by Lo (259) showing the intracellular location of M. fermentans incognitus in a variety of nonphagocytic cells in AIDS patients. This finding was strengthened by the discovery of a new human mycoplasma capable of entry into a variety of human cells in vivo and in vitro, named accordingly M. penetrans (261).

The mechanism of cell entry by mycoplasmas is still unclear. While mycoplasmas such as M. penetrans and M. genitalium appear to enter the cells through their specialized tip structure (206, 261), other mycoplasmas shown to internalize, such as M. fermentans and M. hominis, have no tip structures (447). Following contact of M. genitalium with human lung fibroblasts, the plasma membrane of the cells appeared to be forced inward to form a cup or a depression. The membrane pockets resembled clathrin-coated pits, suggesting that the mycoplasma might adhere to and enter the cells by a site-directed, receptor-mediated event resembling cell entry by chlamydias (289).

M. penetrans presents, perhaps, the strongest case for the ability of a mycoplasma to actively penetrate into a variety of different types of mammalian cells, many with minimal phagocytic ability (261). Internalization has a definite anatomical orientation, led by the unique structural component, the tip structure. The entry of the pathogen into a host cell is initiated by the binding of the pathogen onto the host cell surface followed by a dramatic rearrangement of microtubule and microfilament proteins (391). Ultrastructural studies of human larynx carcinoma cells infected with M. penetrans revealed, as early as 2 h postinfection, that the organisms were invaginated in the cell membrane or internalized in the cytoplasm, free or inside vesicles. Adherence triggered a signal that promoted cytoskeletal changes, namely, aggregation of tubulin and -actinin and condensation of phosphorylated proteins. Other cytoskeletal components, such as talin, tropomyosin, and vinculin did not appear to accumulate at the site of mycoplasma clustering, suggesting that M. penetrans selectively uses signals to induce specific cytoskeletal rearrangements (156). Along these lines, Andreev et al. (11) reported that the actin polymerization inhibitor cytochalasin D markedly inhibited the internalization of M. penetrans in HeLa cells whereas the tyrosine phosphorylase inhibitors staurosporin and genistein had only a slight effect. Invasion of enteropathogenic Escherichia coli depends on tyrosine phosphorylation of a 90-kDa HeLa cell protein. This was not found to be the case for M. penetrans, although tyrosine phosphorylation of another HeLa cell protein of 140 kDa could probably be associated with the internalization of M. penetrans (11).

The percentage of the mycoplasma cell population internalized is difficult to determine. Under electron microscopy, M. genitalium was demonstrated intracellularly in about 10% of Vero cells infected in vitro (206), indicating significant variability in the capacity of the cells to internalize mycoplasmas. It should be pointed out that electron microscopic observations may lead to conflicting interpretations. It can be argued that the mycoplasmas that appear to be located intracellularly in vacuoles are actually at the bottom of crypts formed by invagination of the cell membrane. To overcome this difficulty, during the preparation of thin sections Taylor-Robinson et al. (447) applied ruthenium red to stain the mucopolysaccharide surface components of both the HeLa cells and the mycoplasmas infecting them. The intracellular location of some mycoplasmas could be confirmed by exclusion of ruthenium red from their membrane surface. Another approach, based on confocal microscopy and flow cytometry of fluorochrome-labeled mycoplasmas, revealed that M. penetrans, M. pneumoniae, and M. genitalium entered the intracellular spaces and were located throughout the cytoplasm and perinuclear regions of cultured human cells (28). The mycoplasmas could be cultivated from the cytoplasmic and nuclear fractions 96 h after infection and persisted intracellularly for at least 7 days. Whether the mycoplasmas replicate intracellularly remains to be resolved. The finding by Baseman et al. (28) of M. pneumoniae mutants capable of cytadherence but defective in their capacity to invade cells is of particular interest, since it suggests that mycoplasma cytadherence and invasion are active but separable processes.

As might be expected, invasion of mycoplasmas into the host cell cytoplasm may affect cell function and integrity. Thus, Mernaugh et al. (289) showed by electron microscopy the complete lysis of human lung fibroblasts 96 h after infection by M. genitalium, accompanied by large numbers of mycoplasmas in the milieu. Lo et al. (261) also stated that extensive invasion of cells by M. penetrans eventually results in cell disruption and necrosis. The presence in the host cell cytoplasm of mycoplasmas, some of which may not even be enclosed within a vacuole, may expose the cytoplasm and the nucleus to mycoplasmal hydrolytic enzymes, such as proteases, nucleases, and phospholipases. The potent endonuclease of M. penetrans was suggested to cause chromosomal damage (36).

It should be emphasized that intracellular location, if even for a short period, may protect the mycoplasmas against the effects of the host immune system and antibiotics and may account to some extent for the difficulty of eradicating mycoplasmas from infected cell cultures. Thus, intracellular residence, which sequesters mycoplasmas, promotes the establishment of latent or chronic infection states and circumvents mycoplasmacidal immune mechanisms and selective drug therapies (30).

MORPHOLOGY AND ULTRASTRUCTURE

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Motility and Cytoskeletal Elements

Since mycoplasma cells are bounded by a plastic cell membrane only, their dominating shape is a sphere. However, many mollicutes exhibit a variety of morphological entities, including pear-shaped cells, flask-shaped cells with terminal tip structures, filaments of various lengths, and helical filaments. The ability to maintain such shapes in the absence of a rigid cell wall has long indicated the presence of a cytoskeleton in mycoplasmas (356). Some of the flask-shaped Mycoplasma species are capable of gliding on solid surfaces. The mechanism of this peculiar gliding motility is still unknown (224). Thus, although M. pneumoniae is known to be motile and to exhibit chemotactic behaviour, gliding motility genes could not be identified, since it is not yet known which genes have to be looked for. Furthermore, none of the components of the chemotactic signal pathway, the Che proteins, which are well conserved among bacteria, or any other "two-component signal transduction systems" could be identified in the sequenced M. pneumoniae genome (181).

Spiroplasma species are unique in having helical morphology, rotary motility, and chemotaxis. Upon cell lysis by deoxcholate, spiroplasmas release fibrils that are 4 nm in diameter. These fibrils, thought to function as a cytoskeletal element involved in the helical shape and motility of the organisms, consist of a major protein of 59 kDa. The gene for this protein (fib) has been cloned, facilitating the characterization of the structural features of the fibril protein (476). It appears that the protein possesses four -helices; axial projection of the three most prominent ones shows that the hydrophobic and hydrophilic amino acids are located on the opposite sides of the helices. It is therefore possible that the fibril protein attaches to the plasma membrane with the hydrophobic side while the hydrophilic side faces the cytoplasm (476). In light of the finding that reversible phosphorylation controls the assembly of intermediate filaments in eucaryotes, Platt et al. (339) investigated the interrelationship of protein phosphorylation and Spiroplasma melliferum fibrils. Their results showed the phosphorylation of a protein resembling the fibril protein in molecular mass, but it was not definitely identified as such. Some information on a gene involved in S. citri motility has recently been obtained through transposon mutagenesis (57, 203) (see "Gene transfer" below). An S. citri nonmotile mutant was generated by Tn4001 transposon mutagenesis. The transposon was shown to be inserted into a gene, scm1, encoding a putative polypeptide with no significant homology to any known protein. The scm1 gene was recovered from the wild motile strain and inserted into an S. citri cloning vector. Transfection of the nonmotile mutant with this recombinant plasmid restored motility, indicating that the scm1 gene product is indeed involved in S. citri motility.

Williamson et al. (476) have suggested that an actin-like protein found in spiroplasmas may be linked to the fibrils and associated with motility. However, they admit that there is really no evidence for such an association. It should be pointed out that the presence of actin-related proteins in procaryotes in general, and in mollicutes in particular, has long been suspected but was never proven (356). While some workers have claimed to identify actin or actin-related proteins in mollicutes (161), others have failed (77). The complete genomic analyses of M. genitalum and M. pneumoniae (139, 181) also failed to identify actin-related gene(s), leaving this issue unresolved. In fact, it now appears that the interest in this subject, strong in the 1970s (356), has dwindled. In general, bacterial interconnecting protein networks appear to be important in preserving the integrity of wall-less cells, also keeping an asymmetric distribution of membrane proteins. However, thus far, significant sequence homologies between eucaryotic cytoskeletal proteins and any bacterial protein have not been found (345), although Wasinger et al. (469), applying the proteome technology (see "Genome sequencing and the minimal cell concept" below), have suggested the presence of a tubulin-like protein in M. genitalium.

An important group of pathogenic mycoplasmas, including M. pneumoniae and M. genitalium, have a flask- or clublike cell shape with a protruding tip or bleb structure. These mycoplasmas attach to eucaryotic cells via the tip structure, serving as an attachment organelle (see "Adhesion to host cells" below). Scanning and transmission electron microscopy of M. pneumoniae cells grown on grids treated with 1% Triton X-100 revealed a rodlike tip structure and a network of filamentous strands (288). Proteins of this so-called Triton shell are apparently cytoskeleton-forming or cytoskeleton-associated proteins. The cytoskeleton-like structure is thought to function in modulating cell shape and to participate in cell division, gliding motility, and the proper localization of adhesins.

The genomic analysis of M. pneumoniae has enabled the identification and molecular characterization of major protein building blocks of the cytoskeleton of this mycoplasma. Some of these proteins function as surface-exposed adhesins, including proteins P1 and P30, while others, named accessory proteins (designated HMW1, HMW2, and HMW3 and A, B, and C) collectively maintain the proper distribution and/or disposition of the adhesins in the mycoplasma membrane (see "Adhesion to host cells" below). Additional proteins, named P65 and P200 (345, 346) share characteristic structural features with HMW1 and HMW3, suggesting their function as elements of the M. pneumoniae cytoskeleton, consistent with their presumed scaffolding role (236). HMW2 (about 215 kDa) is predicted to assume a coiled-coil conformation, similar to that of the filamentous portion of the myosin heavy chain, reflecting a likely structural role for this cytoskeletal mycoplasmal protein (237).

An interesting property of these cytoskeletal proteins (such as HMW1, HMW3, and P65) is their abnormal (slower than expected from their molecular weight) migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which is apparently due to proline-rich regions in their molecular structure (penta- and hexapeptide repeats forming a proline-rich acidic domain [345]). Many eucaryotic membrane-associated cytoskeletal proteins (such as spectrin) are characterized by similar internal repeats that function in the binding of accessory proteins of importance in the construction and maintenance of a cytoskeletal network. It should also be mentioned that membrane lipoproteins, common in mycoplasmas, exhibit abnormal electrophoretic migration due to the presence of the lipid moiety (478).

Another interesting feature of the M. pneumoniae cytoskeletal proteins concerns their phosphorylation. Protein phosphorylation is a widespread mechanism for regulating intracellular signaling. Through the action of kinases, phosphotransferases, and phosphatases, this posttranslational protein modification results in various reversible phosphorylation states, modulating cellular events by interconverting between active and inactive protein forms (218). Dirksen et al. (106) showed the HMW1 and HMW2 cytadherence-associated proteins of M. pneumoniae to be phosphorylated at threonine and serine residues in an ATP-dependent manner, implying that a classical protein kinase phosphatase system is functioning. Whether the phosphorylation state of these cytoskeletal proteins plays a role in regulating the dynamics of cytoskeletal interactions is a moot point, since the phosphorylation of HMW1 and HMW2 was generally poor. Rather, phosphorylation was associated at very high level with three unidentified proteins of 54, 57, and 60 kDa. Only when the Triton-soluble fraction was removed could phosphorylation be detected in the Triton-insoluble fraction (238).

The mode of reproduction of mycoplasmas is essentially not different from that of other procaryotes dividing by binary fission. For typical binary fission to occur, cytoplasmic division must be fully synchronized with genome replication, and in mycoplasmas the cytoplasmic division may lag behind genome replication, resulting in the formation of multinucleate filaments. The transformation of mycoplasma filaments into chains of cocci followed by microcinematography revealed the appearance of constrictions in the cell membrane at about equal distances along the entire length of the filament, a process that takes a few minutes. At the time these observations were discussed (356), one had no idea about the mechanism of this process. Recent genetic data throw some light on the genes and proteins involved in maintaining the peculiar cell shapes and the cell division process in mycoplasmas.

Since the mechanics of cell septation in conventional eubacterial species are believed to be mediated by cell wall constituents, there is no clear understanding of what coordinates that process in wall-less bacteria. Our knowledge of the genes and their products associated with cell division of walled bacteria, particularly of E. coli, is quite extensive. The recent sequencing of several mycoplasma genomes (55, 139, 181) has provided some information on mycoplasmal genes homologous to cell division genes of walled-covered bacteria. The comparative genomics data reveal the lack in mycoplasmas of a significant number of genes belonging to this category, findings which may be relevant in the consideration of the relative importance of the different genes in the procaryotic cell division process.

The most important finding, perhaps, is that of the ftsZ gene in mycoplasmas (55, 139, 181, 468). In eubacteria, the FtsZ protein is a polymer-forming, GTP-hydrolyzing protein with tubulin-like elements; it is localized to the site of septation and forms a constricting ring (the Z ring) between the dividing cells. The finding of ftsZ in the mollicutes indicates that it is a highly conserved and ubiquitous gene (found also in archeons and chloroplasts) fulfilling a key role in procaryote cell division. Of the additional genes associated with cell division in eubacteria (ftsA, ftsH, ftsI, ftsQ, ftsW, and ftsY) ftsH was identified in M. pneumoniae (181) and M. capricolum (55), and ftsY was identified in M. genitalium (139) and M. pneumoniae (181). More recently, the ftsY gene of M. mycoides subsp. mycoides was cloned and characterized and its role in protein targeting by complexing with the signal-recognition particle was investigated (269). FtsW has been suggested to initiate Z-ring formation, probably serving as a membrane target recognized by FtsZ. The fact that it is absent from the mycoplasmas argues against its being absolutely essential for the localization of FtsZ to the division site. FtsA, FtsI, and FtsQ are believed to act in the division process after the formation of the Z ring; their absence from the mycoplasmas also suggests that they are not absolutely essential for cell division (468). Applying the proteome approach (see "Genome sequencing and the minimal cell concept" below), Wasinger et al. (469) proposed the presence of a tubulin-like protein in M. genitalium. It has been suggested that the FtsZ protein of procaryotes is a tubulin homolog; however, the tubular structures formed by FtsZ in vitro are substantially different from microtubules, and sequence similarity between FtsZ and tubulin is barely significant (169).

An integral part of cell division concerns chromosome replication and partition into daughter cells. The mechanism of chromosome partitioning has not yet been examined in any mollicute species. The identification of parC and parE homologs in M. genitalium suggests that topoisomerase IV (a topoisomerase capable of relaxing superhelical DNA and converting knotted DNA to a simple ring in vitro) has been evolutionarily conserved because of its essential role in cell division (20). In conclusion, it appears that on the whole we are still rather far from clearly understanding the factors coordinating the cell division process in mollicutes, since genetic information has only just started to accumulate and has not been entirely analyzed and evaluated.

IN VITRO CULTURE

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Definition of Nutritional Requirements

A major impediment to mycoplasma research and laboratory diagnosis of mycoplasma infections has been the difficulty of their in vitro cultivation. There appears to be a consensus that only a minority of the mycoplasmas existing in nature have been cultivated so far. For example, despite many efforts for over 30 years, none of the phytoplasmas infecting insects and plants has been cultivated in vitro in an axenic culture (229). Some of the cultivable mycoplasmas also grow very poorly and slowly on the best mycoplasma media available (363). The recent mycoplasma genome projects have provided definitive genetic explanations of the above-mentioned difficulties by demonstrating the remarkable scarcity in mycoplasmas of genes involved in biosynthetic pathways. For example, both M. genitalium and M. pneumoniae lack all the genes involved in amino acid synthesis (139, 181), making them totally dependent on the exogenous supply of the complete spectrum of amino acids.

To overcome the assimilative deficiencies of the mycoplasmas, complex media are used for their cultivation. The media are usually based on beef heart infusion, peptone, yeast extract, and serum with various supplements (359). The use of these complex undefined growth media has interfered with the molecular definition of mycoplasmal metabolic pathways, genetic analysis, preparation of mycoplasmal antigens free of serum components, etc. Continual efforts to replace the serum component, with the aim of reaching a defined growth medium, have been made. Serum has been shown to provide, among other nutrients, fatty acids and cholesterol (required for membrane synthesis) in an assimilable nontoxic form. Efforts to replace the serum component with albumin, fatty acids, and cholesterol solubilized in Tween 80 or with liposomes made of phospholipids and cholesterol supplemented with serum albumin to neutralize free fatty acid toxicity (356) have been partially successful. In an attempt to replace the albumin component, cyclodextrins have been proposed as carriers of cholesterol and fatty acids (166). However, cyclodextrin derivatives may have too strong a lipid binding capacity, rendering the essential lipid unavailable to the mycoplasma and consequently inhibiting growth. It is difficult, therefore, to find a cyclodextrin derivative that combines a proper lipid binding capacity with nontoxicity.

The definition of lipid requirements, particularly for cholesterol, has served as an important taxonomic criterion distinguishing the sterol-nonrequiring mollicutes, particularly the Acholeplasma species, from the sterol-requiring ones. Testing for sterol requirement has been based on the use of a serum-free medium containing serum albumin, fatty acids, and various concentrations of cholesterol solubilized in Tween 80, yielding a final Tween concentration of 0.01% (356). The finding (385) that Tween 80 at a final concentration of 0.04% is essential for the growth of some of the sterol-nonrequiring mycoplasmas (later named Mesoplasma species [Table 1]) is still an enigma. It seems unlikely that requirement for this relatively high Tween 80 concentration can be explained by the provision of a required fatty acid component, since experiments to test this issue by adding a variety of fatty acids have failed.

One thing is certain: although the numerous nutritional requirements of mollicutes dictate the need for complex growth media, the notion that the richer the medium the better may be wrong. It appears, at least in some cases, that the lack of growth of a mycoplasma in a rich medium is not the result of the lack of a specific nutrient but, rather, is due to the presence of a component(s) toxic to the mycoplasma. Some M. hyorhinis strains, common contaminants of cell cultures, have been known to resist cultivation on conventional mycoplasma media, leading us to consider them particularly fastidious strains. This concept proved to be wrong, since these strains could grow well in a minimal serum-free medium, leading Gardella and Del Guidice (150) to suggest that these "noncultivable" strains are not particularly fastidious but are more sensitive to inhibitors found in the complex media, mostly as components of peptone and yeast extract. Hence, the terms "noncultivable" and "fastidious" should be considered relative terms, which take their meaning only in the context of a specific culture system where growth promoters and possibly inhibitors are present.

The possibility that the presence of inhibitory substances in complex mycoplasma media, rather than the lack of essential nutrients, is the reason for the continuous failure to cultivate phytoplasmas is unresolved. The notion that the failure to cultivate phytoplasmas may be associated with a need for anaerobic conditions has not been substantiated. Experiments with phytoplasma-infected Oenothera leaf tip cultures, growing under conditions of different levels of O₂ and CO₂ mixed in air, showed no consistent correlation between atmospheric conditions and phytoplasma abundance. However, the phytoplasmas grew better in photosynthesis-defective mutants of Oenothera, indicating that the pathogen is more successful in nonphotosynthetic tissue, with no generated O₂ (410). Generally, mollicutes differ markedly in their atmospheric requirements; while most mollicutes are facultative anaerobes and usually favor an anaerobic or low-redox, enhanced CO₂ atmosphere on primary isolation, the ruminal Anaeroplasma and Asteroleplasma species are strict anaerobes, very sensitive to oxygen (Table 1). Some mycoplasmas, such as M. hyorhinis, require an aerobic atmosphere (150).

A novel approach to improve the chances of in vitro cultivation of fastidious mollicutes is based on coculture with eucaryotic cell lines (cell-assisted growth). In this way, uncultivable spiroplasmas, such as the Colorado potato beetle spiroplasma, were first successfully cocultivated in insect cell lines. Subsequently (232), primary cultures of this spiroplasma could be obtained on cell-free media under a low-redox, enhanced CO₂ atmosphere and at a pH lower than 7.0 (for most mollicutes, the initial pH is adjusted to a slightly alkaline value), conditions which imitate those obtained in the insect cell cocultures (232).

A similar cell-assisted cocultivation, using Vero cell cultures, enabled several strains of the highly fastidious human pathogen M. genitalium to be cultured from clinical specimens (207). Also, in this case, the mycoplasmas grown in the cell culture (as was indicated by PCR monitoring) could be subsequently subcultured in a cell-free medium (207). The above method, as complex as it is, requiring "heroic" efforts, is certainly inadequate for routine cultivation of fastidious mycoplasmas, leaving the door open for the application of molecular techniques, such as PCR, for detection and identification of fastidious or so far uncultivable mycoplasmas (363) (see also "Taxonomy and phylogeny" below).

GENOME SIZE AND BASE COMPOSITION

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Genome Size

The application of pulsed-field gel electrophoresis to mollicute genome size determinations (314, 348) has provided a much more accurate and labor-saving procedure than the previously used renaturation kinetics method and has resulted in a wealth of genome size data (57, 71, 314). The data show a continuum of genome sizes among mollicutes, ranging from less than 600 to over 2,200 kb, with overlapping values between mollicute genera. Thus, the genome sizes of Mycoplasma species range from 580 kb for M. genitalium to 1,380 kb for M. mycoides subsp. mycoides LC, while for the helical Spiroplasma species the genome size ranges from 780 kb for S. platyhelix to 2,220 kb for S. ixodetis (57, 71) (Table 1). Clearly, genome size can no longer be taken as the definitive taxonomic criterion to distinguish higher taxa in Mollicutes (359). Yet, as a general rule, Acholeplasma and Spiroplasma species, considered phylogenetically to be "early" mollicutes, have larger genome sizes than Mycoplasma and Ureaplasma species, considered to be phylogenetically more "recent" mollicutes (see "Taxonomy and phylogeny" below). This is in agreement with the notion that Mollicutes have evolved by degenerative or reductive evolution, accompanied by significant losses of genomic sequences (479).

Genome sizes are variable not only within the same genus but even among strains of the same species (71, 191, 241, 382). One of the reasons for this variability is the frequent occurrence in mollicute genomes of repetitive elements, consisting of segments of protein genes, differing in size and number, or insertion sequence (IS) elements (see "Chromosomal rearrangements" below). Intraspecies variability of genome size may also be caused by integration into the chromosome of viral sequences, as was found in S. citri, where these sequences may account for up to 150 kb, 1/12 of the entire genome (32, 56, 481, 482). On the whole, in light of the highly dynamic nature of the mycoplasmal genome (see "Chromosomal rearrangements" below), one should be surprised not by intraspecies genomic size variations but, rather, by the relative conservation of genome size observed in mollicute species.

Is there any correlation between genome size and the ability of a mycoplasma to grow in vitro? Although, a priori, a larger genome and consequently a larger number of genes would be expected to endow the organism with better adaptability to in vitro cultivation, the available data do not support this expectation. Thus, S. ixodetis, the spiroplasma with the largest genome, is much harder to cultivate than spiroplasmas with much smaller genomes (460). As to the uncultured phytoplasmas, the limited genome size data available suggest that though they may be included among the mollicutes with the smaller genomes (311) such as the western X-disease phytoplasma with a 670-kb genome (127), animal mycoplasmas with even smaller genomes have been easily cultivated in vitro. The conclusion, therefore, is that there is no simple relationship between genome size and cultivability of mollicutes (see "In vitro culture," above, for a possible explanation). Interestingly, the obligate intracellular bacteria Chlamydia trachomatis, Rickettsia prowazekii, and Chlamydia burnetii have genomes of 1,000, 1,100, and 1,600 kb, respectively (10, 273), larger than the genomes of many of the self-replicating mollicutes, again strengthening the conclusion that there is no simple correlation between genome size and growth in axenic culture.

The mycoplasma genome has a characteristically low G+C content. With very few exceptions, the G+C content of mycoplasma genomes is within the range of 24 to 33 mol% (appendix in reference 459). The G+C distribution along the genome is uneven. Thus, while the average G+C content of the M. genitalium genome is 32 mol%, the G+C content of its rRNA genes is 44 mol% and that of its tRNA genes is 52 mol% (139). The M. pneumoniae adhesin genes, P1 and ORF6, and their repetitive sequences, exhibit a G+C content as high as 56 mol%, while on the other extreme the origin of replication of this mycoplasma has a G+C content of only 26 mol%, compared to 40 mol% of the entire M. pneumoniae genome (181, 182). Consequently, many of the mycoplasmal intergenic regions have a higher A+T content than do the coding regions, reaching values as high as 80 to 90 mol% (114, 305, 485). The variable G+C content of coding regions within the mycoplasmal genome has phylogenetic relevance, indicating the highly conserved nature of the rRNA and tRNA genes and the possible exogenous origin of the adhesin genes (see "Adhesion to host cells" below).

As in other procaryotes, some of the adenine and cytosine residues in mycoplasmal genomes may be methylated (114, 275, 357). In many mycoplasmas the adenine residues at the GATC site are methylated, while in others the cytosine residues are methylated. Of special interest is the exclusive methylation of the genomic cytosine residues of S. monobiae (MQ-1) when they are located 5' to guanine (CpG), a methylation trait considered unique to eucaryotes (374). The type of base methylated, the extent of methylation, and methylation sequence specificity have been suggested as markers in mollicute taxonomy (361).

Among the mollicutes, the spiroplasmas and acholeplasmas are the most frequently infected by a variety of viruses (phages), whereas very few viruses are known to infect Mycoplasma species (499). The characteristics of mollicute viruses have been described and discussed in great detail in previous reviews (271, 357, 372). The mollicute phage DNA genomes range in size from 4 to 40 kb and may be either circular or linear and single or double stranded (114). Of the very few phages infecting Mycoplasma species, the most recent phage discovered is the lysogenic phage MAV1, infecting M. arthritidis (465). The finding that this phage is associated with highly arthritogenic strains and that experimental infection of low-virulence M. arthritidis strains with this phage significantly increases their arthritogenicity suggests that MAV1 carries a virulence enhancing factor (see "Virulence factors" below).

Plasmids were detected in S. citri and more recently in M. mycoides subsp. mycoides (221, 222) and in phytoplasmas (229). The potential of the mollicute phages and plasmids to serve as cloning and shuttle vectors has been the main focus of interest in these elements (see "Gene transfer" below).

One of the first and most intriguing issues supported by the National Aeronautics and Space Administration was the search for extraterrestrial forms of life. The assumption that these living forms, if they exist, may be extremely simple, led Harold Morowitz to look for the simplest self-replicating cells existing on our planet. A short search aided by Mark Tourtellotte pointed at the mycoplasmas as the smallest and simplest self-replicating organisms. To attract attention to these organisms, in 1962 Morowitz and Tourtellotte published an article in Scientific American on mycoplasmas as the smallest living cells (298) and organized, with the support of the National Aeronautics and Space Administration, the first meeting on the molecular biology of mycoplasmas. We were then enchanted by the "crazy" idea of assembling a living cell from its components. It should be recalled that these were the 1960s, a period of revolutionary ideas including the trip to the Moon. The mycoplasmas, being built of the minimum set of organelles (a plasma membrane, ribosomes, and a circular double-stranded DNA molecule, the typical prokaryotic genome), were naturally selected by us as the best candidates for cell reassembly. Starting with detergent solubilization and reassembly of the cell membrane appeared to be the most logical step to initiate this venture. To make a long story short, we succeeded in reconstituting vesicular membranous structures from the solubilized components (369), but the resulting membranes differed in molecular organization from the native membranes. Nevertheless, our study had an impact, giving a considerable push to the field of membrane reconstitution (355). However, the failure to reconstitute a functional plasma membrane led us to abandon the idea of reconstituting a living cell in the laboratory.

Harold Morowitz did not entirely give up this idea. In 1984, 20 years after our pioneering membrane reconstitution experiments, Morowitz was invited to deliver a keynote lecture at the International Congress of Mycoplasmology in Jerusalem. This lecture has had a major impact on mycoplasma research, culminating in the significant achievements to be discussed below. This time, Morowitz proposed another, more practical and down-to-earth approach (297). His major idea was to define in molecular terms the entire machinery of a mycoplasma cell, in order to prove the dogma of the completeness of molecular biology, that is, that the "logic of life" is finite, relatively simple, and subject to full exploration. He proposed the launching of an international effort to accomplish this ambitious goal. The project appeared to require an enormous amount of work and generous funding, but even in 1984, it did not appear to involve conceptual and methodological difficulties. The proposed plan consisted of physical and functional mapping of a Mycoplasma genome including its complete sequencing, determining the open reading frames (ORFs), reading out the encoded amino acids, defining the genes and their products, and in this way achieving a complete molecular description of the cell machinery (297).

Although the proposal by Morowitz was met with enthusiasm, a collaborative international effort was not established. Instead, several laboratories initiated independent projects focusing on sequencing and genetic mapping of different mycoplasma genomes. Since the late 1980s, there has been a continuous flow of papers on the construction of physical maps of mollicute genomes, followed by the sequencing of genomic fragments and trials to identify the genes they carry (references 56, 360, and 364 and references therein). The first large-scale studies directed at sequencing entire mycoplasma genomes were initiated at about 1990. The Harvard Genome Lab chose to sequence the genome of M. capricolum. The selection of this mycoplasma was not optimal, as it carries a relatively large genome, of over 1,000 kb (294). By 1995, when the project terminated, only 214 kb of the M. capricolum genome had been definitely sequenced and genetically analyzed in collaboration with the Heidelberg European Molecular Biology Laboratory (55). Although this study was apparently the first to provide extensive genetic data based on sequences of a significant part of a mycoplasma genome, generalizations based on these partial data could have led to erroneous conclusions.

Richard Herrmann's laboratory at the University of Heidelberg chose to sequence the 800-kb genome of M. pneumoniae. Despite being small and limited in resources, this laboratory succeeded, in about 3 years of hard work, in fully sequencing and genetically characterizing the entire M. pneumoniae genome (181). They first constructed a cosmid library and then, by applying the sequence strategies and methods described by Hilbert et al. (180), sequenced both DNA strands in a directed fashion by primer walking, limiting random (shotgun) sequencing to a minimum.

However, Herrmann's group was not the first to publish the complete sequence of a mycoplasma genome. The first report was published in October 1995 by a large team from The Institute for Genomic Research (TIGR), Gaithersburg, Md., collaborating with teams from Johns Hopkins University and from the University of North Carolina at Chapel Hill (139). They selected apparently the most adequate mycoplasma for whole-genome sequencing: M. genitalium. This mycoplasma carries the smallest genome known so far for a self-replicating organism, a genome of 580 kb only. The foundations of the project were laid down by the Chapel Hill team, employing the conventional chromosome-walking strategy on a set of ordered cosmids. This led to the partial sequencing and genetic characterization of the M. genitalium genome (264, 334). However, the breakthrough occurred when the M. genitalium genome sequencing was moved to Gaithersburg in January 1995. The TIGR team sequenced the entire mycoplasma genome in less than 6 months. This remarkable achievement was made possible by application of the whole-genome shotgun sequencing strategy, based on the random fragmentation of genomic DNA to small segments of about 2 kb followed by their cloning and sequencing. The reassembly of the thousands of the sequenced overlapping fragments in the right order has been made possible by the TIGR Assembler software program. The predicted coding regions of the genome were defined with the GeneMark software. Role assignments were made, where possible, for each of the predicted coding regions by database matches to all proteins in the public archives (130, 138a, 139). The remarkable power and cost-effectiveness of the new genome sequencing technology mean that complete genome sequences of over 40 of major bacterial pathogens as well as archeons could be available by the year 2000 (96, 192). Included in these are several mollicutes, such as U. urealyticum (158), M. gallisepticum (192), and M. mycoides subsp. mycoides (192).

The complete sequence of the M. genitalium genome was reported only a few months after publication by the TIGR and Johns Hopkins teams of the first complete sequence of a bacterial genome, that of Haemophilus influenzae (130). The voluminous amount of genetic data provided by the genome projects has opened the way for "comparative genomics," by which the total genomic complements of organisms could be compared. This provides an opportunity to explore the functional content of genomes and evolutionary relationships between them at a new qualitative level, signifying the onset of a new era in biology. We intend to discuss in this section the cell biology of M. genitalium and its close relative M. pneumoniae, comparing it to that of H. influenzae and other eubacteria. The number of coding regions, the ORFs for proteins, in the M. genitalium genome is only 479, compared to 677 in M. pneumoniae, 1,703 in H. influenzae, and 4,288 in E. coli K-12 (Table 2).

Assimilative processes. Examination of the mycoplasmal genomic data to find where economization in genes took place may help us define the genes which are really essential for a minimal cell. Most striking are the results concerning genes of biosynthetic pathways. During their evolution, the two mycoplasmas M. pneumoniae and M. genitalium have apparently lost all the genes involved in amino acid biosynthesis and thus require the full spectrum of the essential amino acids from the host or from the artificial culture medium (Fig. 1). The mycoplasmas have also lost most of the genes involved in cofactor biosynthesis, so that to cultivate them in vitro, the medium has to be supplemented with essentially all the vitamins (Fig. 1). Very significant savings in genetic information have resulted through the loss of the cell wall during mycoplasma evolution. Compared with the 105 genes involved in the synthesis of the cell envelope (comprising the outer membrane and the cytoplasmic membrane) in the gram-negative H. influenzae, only 54 genes were found to be directly associated with the synthesis of the M. pneumoniae cytoplasmic membrane proteins, mostly lipoproteins and only 30 were found in M. genitalium (Fig. 1). Nevertheless, it should be mentioned in this context that in M. pneumoniae 275 predicted gene products carry transmembrane segments, in line with SDS-PAGE analysis showing that the membrane fraction of this mycoplasma contains about 50% of the cell proteins (181). Yet, each gene saving has its price. Being limited by a cell membrane only, the mycoplasmas are osmotically much more sensitive than the walled bacteria (356). Adaptation to a parasitic mode of life has provided the osmotically sensitive mycoplasmas with a rather osmotically constant milieu. M. genitalium is a parasite of the human urogenital tract, and its transmission by sexual contact ensures its minimal exposure to the external, osmotically variable, environment.

View larger version (43K): [in this window] [in a new window]   FIG. 1.   Biosynthetic pathways genes in the genomes of H. influenzae, M. pneumoniae, and M. genitalium. Numbers above the bars indicate the percentages of the total putatively identified genes. Based on data from Fleischmann et al. (130), Fraser et al. (139), and Himmelreich et al. (181, 182).

Cellular processes. The number of genes involved in cellular processes, such as the fts genes associated with cell division, heat shock proteins, and genes for chaperones functioning in protein secretion, is definitely smaller in the mycoplasmas than in H. influenzae (Fig. 2). The protein secretion system in M. pneumoniae is much less complex than in E. coli. The channel-forming proteins SecG, SecF, SecE, and SecD and the cytosolic receptor protein SecB were not identified in the mycoplasma. Also missing is the signal peptidase Spase I (181). The simplified protein export system might reflect the fact that the mycoplasma cell is bounded by a cytoplasmic membrane only. The problem concerns the refolding of the proteins which are exported in an unfolded configuration. Refolding is catalyzed by chaperones which have to function on the cell surface. This might impose a problem in mollicutes that lack a periplasmic space, a space which prevents the proteins from diffusing away. A way to solve this problem is by anchoring the proteins to the membrane surface via long acyl chains, and, indeed, M. pneumoniae, M. genitalium, and mycoplasmas in general are rich in cell surface lipoproteins (181) (see "Cell membrane" and "Antigenic variation" below).

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Cellular processes, metabolic pathways, and regulatory functions genes in the genomes of H. influenzae, M. pneumoniae, and M. genitalium. Numbers above the bars indicate the percentages of total putatively identified genes. Based on data from Fleischmann et al. (130), Fraser et al. (139), and Himmelreich et al. (181, 182).

Energy metabolism and transport. The genomes of the two mycoplasmas are deficient in genes coding for components of intermediary and energy metabolism (Fig. 2). Thus, the two mycoplasmas depend mostly on glycolysis as a means of synthesizing ATP. Genes that encode the components of the pyruvate dehydrogenase complex, phosphotransacetylase, and acetate kinase were also detected, as well as a deficient pentose phosphate pathway (139, 181). Most striking is the lack of many energy-yielding systems from the mycoplasmas. No tricarboxylic acid cycle, and no quinones and cytochromes were found in any of the mycoplasmas (see "Metabolism and Transport" below). The electron transport system in mycoplasmas is flavin terminated. Thus, ATP is produced in mycoplasmas by substrate-level phosphorylation, a less efficient mechanism than oxidative phosphorylation. However, it appears that the parasitic mycoplasmas grow well in vivo despite the truncated and inefficient ATP-yielding systems at their disposal. A major reason probably is the relatively small investment of ATP needed for the limited biosynthetic pathways characterizing mycoplasmas.

Replication, transcription, and translation. Moving on to the comparative genomics of genes involved in DNA replication, transcription, and translation, the picture is somewhat different (Fig. 3). The essential role of these basic processes in cell biology, leads us to expect that saving of genes in these categories will be more restricted as compared to metabolic processes. Cells are capable of importing and utilizing exogenous metabolites but not functional proteins; therefore, they have to rely on their own gene products to provide housekeeping functions. Figure 3 shows that while the absolute number of genes involved in DNA replication in the mycoplasmas is still much smaller than in Haemophilus, the percentage of genes devoted to DNA replication and degradation in M. genitalium and M. pneumoniae is higher than in H. influenzae, pointing to the essential biological role of these genes, so that evolutionary deletion of genes in this category had to be limited.

View larger version (60K): [in this window] [in a new window]   FIG. 3.   DNA replication, transcription, and translation genes in the genomes of H. influenzae, M. pneumoniae, and M. genitalium. Numbers above the bars indicate the percentages of total putatively identified genes. Based on data from Fleischmann et al. (130), Fraser et al. (139), and Himmelreich et al. (181, 182).

Parasitism-associated genes. As emphasized above, the considerable economization in genes depends primarily on the obligate parasitic mode of life of mycoplasmas. However, there is no "free lunch," and some genomic price has to be paid for parasitism; that is, mycoplasma cells must possess surface components enabling their attachment to the host cells. In some cases, including M. genitalium and M. pneumoniae, the mycoplasmas developed special attachment organelles (see "Morphology and ultrastructure" above). Obviously, a significant number of genes is involved in the construction of such an organelle (342a). Some of these genes have already been identified and characterized, but some have not and are apparently included in the parts of the mycoplasmal coding regions or genes that are unidentified as yet. Of those characterized, the genes for the major adhesins of M. pneumoniae and M. genitalium, P1 and MgPa, are well known. The two adhesin proteins resemble each other in structure, with both being large integral membrane proteins having regions exposed on the mycoplasma cell surface tha