Mycoplasma

Mycoplasma is a genus of bacteria that lack a cell wall. Because they lack a cell wall, they are unaffected by some antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. They can be parasitic or saprophytic. Several species are pathogenic in humans, including M. pneumoniae, which is an important cause of atypical pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved in pelvic inflammatory diseases. They may cause or contribute to some cancers.

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The genus Mycoplasma is one of several genera within the class Mollicutes. Mollicutes are bacteria which have small genomes, lack a cell wall and have low a GC-content (18-40 mol%). There are over 100 recognized species of the genus Mycoplasma. Their genome size ranges from 0.58 - 1.38 megabase-pairs. Mollicutes are parasites or commensals of humans, animals (including insects), and plants; the genus Mycoplasma is by definition restricted to vertebrate hosts. Cholesterol is required for the growth of species of the genus Mycoplasma as well as certain other genera of mollicutes. Their optimum growth temperature is often the temperature of their host if warmbodied (e.g. 37 degrees Celsius in humans) or ambient temperature if the host is unable to regulate its own internal temperature. Analysis of 16S ribosomal RNA sequences as well as gene content strongly suggest that the mollicutes, including the mycoplasmas, are closely related to either the Lactobacillus or the Clostridium branch of the phylogenetic tree (Firmicutes sensu stricto).

Mycoplasmas are often found in research laboratories as contaminants in cell culture. Mycoplasmal cell culture contamination occurs due to careless working or contaminated cell culture medium ingredients. The Mycoplasma cell is usually smaller than 1 µm and they are therefore difficult to detect with a conventional microscope. Their presence in cell cultures or media can skew experimental results.

History and general characteristics

The bacteria of the genus Mycoplasma (trivial name: mycoplasmas) and their close relatives are largely characterized by lack of a cell wall. Despite this, the shapes of these cells often conform to one of several possibilities with varying degrees of intricacy. For example, the members of the genus Spiroplasma assume an elongated helical shape without the aid of a rigid structural cell envelope. These cell shapes presumably contribute to the ability of mycoplasmas to thrive in their respective environments. M. pneumoniae cells possess an extension, the so-called 'tip-structure', protruding from the coccoid cell body. This structure is involved in adhesion to host cells, in movement along solid surfaces (gliding motility), and in cell division. M. pneumoniae cells are of small size and pleomorphic, but with a rough shape in longitudinal cross-section resembling that of a round-bottomed flask.

Mycoplasmas are unusual among bacteria in that most require sterols for the stability of their cytoplasmic membrane. Sterols are acquired from the environment, usually as cholesterol from the animal host. Mycoplasmas also generally possess a relatively small genome of 0.58-1.38 megabases, which results in drastically reduced biosynthetic capabilities and explains their dependance on a host. Additionally they use an alternate genetic code where the codon UGA is encoding for the amino acid tryptophan instead of the usual opal stop codon.

In 1898 Nocard and Roux reported the cultivation of the causative agent of contagious bovine pleuropneumonia (CBPP), which was at that time a grave and widespread disease in cattle herds. Today the disease is still endemic in Africa and Southern Europe. The disease is caused by M. mycoides subsp. mycoides SC (small-colony type), and the work of Nocard and Roux represented the first isolation of a mycoplasma species. Cultiviation was, and still is difficult because of the complex growth requirements. These researchers succeeded by inoculating a semi-permeable pouch of sterile medium with pulmonary fluid from an infected animal and depositing this pouch intraperitoneally into a live rabbit. After fifteen to twenty days, the fluid inside of the recovered pouch was opaque, indicating the growth of an microorganism. Opacitiy of the fluid was not seen in the control. This turbid broth could then be used to inoculate a second and third round and subsequently introduced into a healthy animal, causing disease. However, this did not work if the material was heated, indicating a biological agent at work. Uninoculated media in the pouch, after removal from the rabbit, could be used to grow the organism in vitro, demonstrating the possibility of cell-free cultivation and ruling out viral causes, although this was not fully appreciated at the time (Nocard and Roux, 1890). The name Mycoplasma, from the Greek mykes (fungus) and plasma (formed), was proposed in the 1950’s, replacing the term pleuropneumonia-like organisms (PPLO) referring to organisms similar to the causative agent of CBPP (Edward and Freundt, 1956). It was later found that the fungus-like growth pattern of M. mycoides is unique to that species.

This confusion about mycoplasmas and virus would surface again 50 years later when Eaton and colleagues cultured the causative agent of human primary atypical pneumonia (PAP) or 'walking pneumonia.' This agent could be grown in chicken embryos and passed through a filter that excluded normal bacteria. However, it could not be observed by high magnification light microscopy, and it caused a pneumonia that could not be treated with the antimicrobials sulphonamides and penicillin (Eaton, et al., 1945a). Eaton did consider the possibility that the disease was caused by a mycoplasma, but the agent did not grow on the standard PPLO media of the time. These observations led to the conclusion that the causative agent of PAP ia a virus. Researchers at that time showed that the cultured agent could induce disease in experimentally infected cotton rats and hamsters. In spite of controversy whether the researchers had truly isolated the causative agent of PAP (based largely on the unusual immunological response of patients with PAP), in retrospect their evidence along with that of colleagues and competitors appears to have been quite conclusive (Marmion, 1990). In the early 1960's, there were reports linking Eaton's Agent to the PPLOs or mycoplasmas, well known then as parasites of cattle and rodents, due to sensitivity to antimicrobial compounds (i.e. organic gold salt) (Marmion and Goodburn, 1961). The ability to grow Eaton's Agent, now known as Mycoplasma pneumoniae, in cell free media allowed an explosion of research into what had overnight become the most medically important mycoplasma and what was to become the most studied mycoplasma.

Recent advances in molecular biology and genomics have brought the genetically simple mycoplasmas, particularly M. pneumoniae and its close relative M. genitalium, to a larger audience. The second published complete bacterial genome sequence was that of M. genitalium, which has one of the smallest genomes of free-living organisms (Fraser, et al., 1995). The M. pneumoniae genome sequence was published soon afterwards and was the first genome sequence determined by primer walking of a cosmid library instead of the whole-genome shotgun method (Himmelerich, et al., 1996). Mycoplasma genomics and proteomics continue in efforts to understand the so-called minimal cell (Hutchison and Montague, 2002), catalog the entire protein content of a cell (Regula, et al., 2000), and generally continue to take advantage of the small genome of these organisms to understand broad biological concepts.

Scientists have also been exploring an association between mycoplasma and cancer. Despite a number of interesting studies, this cancer bacteria association hasn't been clearly established, and has yet to be fully elucidated (Ning and Shou, 2004), (Tsai, et al., 1995).

Taxonomy and phylogeny

The medical and agricultural importance of members of the genus Mycoplasma and related genera has led to the extensive cataloging of many of these organisms by culture, serology, and small subunit rRNA gene and whole genome sequencing. A recent focus in the sub-discipline of molecular phylogenetics has both clarified and confused certain aspects of the organization of the class Mollicutes, and while a truce of sorts has been reached, the area is still somewhat of a moving target (Johansson and Pettersson, 2002).

The name mollicutes is derived from the Latin mollis (soft) and cutes (skin), and all of these bacteria do lack a cell wall and the genetic capability to synthesize peptidoglycan. While the trivial name 'mycoplasmas' has commonly denoted all members of this class, this usage is somewhat imprecise and will not be used as such here. Despite the lack of a cell wall, Mycoplasma and relatives have been classified in the phylum Firmicutes consisting of low G+C Gram-positive bacteria such as Clostridium, Lactobacillus, and Streptococcus based on 16S rRNA gene analysis. The cultured members of Mollicutes are currently arranged into four orders: Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, and Mycoplasmatales. The order Mycoplasmatales contains a single family, Mycoplasmataceae, which contains two genera: Mycoplasma and Ureaplasma. Historically, the description of a bacterium lacking a cell wall was sufficient to classify it to the genus Mycoplasma and as such it is the oldest and largest genus of the class with about half of the class' species (107 validly described) each usually limited to a specific host and with many hosts harboring more than one species, some pathogenic and some commensal. In later studies, many of these species were found to be phylogenetically distributed among at least three separate orders. A limiting criterion for inclusion within the genus Mycoplasma is that the organism have a vertebrate host. In fact, the type species, M. mycoides , along with other significant mycoplasma species like M. capricolum, is evolutionarily more closely related to the genus Spiroplasma in the order Entomoplasmatales than to the other members of the Mycoplasma genus. This and other discrepancies will likely remain unresolved because of the extreme confusion that change could engender among the medical and agricultural communities. The remaining species in the genus Mycoplasma are divided into two non-taxonomic groups, hominis and pneumoniae, based on 16S rRNA gene sequences. The hominis group contains the phylogenetic clusters of M. bovis, M. pulmonis, and M. hominis, among others. The pneumoniae group contains the clusters of M. muris, M. fastidiosum, U. urealyticum, the currently unculturable haemotrophic mollicutes, informally referred to as haemoplasmas (recently transferred from the genera Haemobartonella and Eperythrozoon), and the M. pneumoniae cluster. This cluster contains the species (and the usual or likely host) M. alvi (bovine), M. amphoriforme (human), M. gallisepticum (avian), M. genitalium (human), M. imitans (avian), M. pirum (uncertain/human), M. testudinis (tortoises), and M. pneumoniae (human). Most if not all of these species share some otherwise unique characteristics including an attachment organelle, homologs of the M. pneumoniae cytadherence-accessory proteins, and specialized modifications of the cell-division apparatus.

A detailed analysis of the 16S rRNA genes from the order Mollicutes by Maniloff has given rise to a view of the evolution of these bacteria that includes an estimate of the time-scale for the emergence of some groups or features (Maniloff, 2002). This analysis suggests that about 600 million years ago (MYA), late in the Proterozoic era, Mollicutes branched away from the low G+C Gram-positive ancestor of the streptococci, losing their cell wall. At this time on Earth, molecular oxygen was present in the atmosphere at 1%, and the fossil record shows that multicellular marine animals had recently spread in the Cambrian explosion. One hundred million years later the requirement for sterols in the cytoplasmic membrane evolved along with the change to the alternate genetic code. Also, the ancestor of the genera Spiroplasma and Entomoplasma (primarily plant and insect pathogens) and Mycoplasma emerged at this time and would itself diverge into the Spiroplasma-Entomoplasma and Mycoplasma lineages approximately 100 million years after that. This diversity coincided with the origin of land plants 500 MYA. It appears that the calculated rate of evolution for the Mycoplasma group increased several fold about 190 MYA, soon after the appearance of vertebrates, while the Spiroplasma-Entomoplasma ancestor continued to evolve at the previously shared slower rate until about 100 MYA, when angiosperms and their associated pollinating insects appeared. Then the evolution rate of these bacteria appears to have also increased significantly. This is an attractive hypothesis, but while it tracks the emergence of several of the unusual characteristics of Mycoplasma and related organisms, it does not address the selective pressures driving their evolution, except perhaps the widespread close association of a parasite with a specific host. The advantages of a reduced genome, cell wall-less structure, and alternate genetic code remain murky.

References

• Eaton, M. D., G. Meiklejohn, W. van Herick, and M. Corey. 1945. Studies on the etiology of primary atypical pneumoniae. II. Properties of the virus isolated and propagated in chick embryos. J Exp Med 82:329-342.
• Edward, D. G., and E. A. Freundt. 1956. The classification and nomenclature of organisms of the pleuropneumonia group. J Gen Microbiol 14:197-207.
• Fraser, C. M., J. D. Gocayne, O. White, M. D. Adams, R. A. Clayton, R. D. Fleischmann, C. J. Bult, A. R. Kerlavage, G. Sutton, J. M. Kelley, and a. et. 1995. The minimal gene complement of Mycoplasma genitalium. Science 270:397-403.
• Himmelreich, R., H. Hilbert, H. Plagens, E. Pirkl, B. C. Li, and R. Herrmann. 1996. Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res 24:4420-4449.
• Hutchison, C. A. III, and M. G. Montague. 2002. Mycoplasmas and the minimal genome concept, p. 221-254. In Razin, S., and R. Herrmann (eds.), Molecular Biology and Pathogenicity of Mycoplasmas, Kluwer Academic/Plenum, New York.
• Johansson, K.-E., and B. Pettersson. 2002. Taxonomy of Mollicutes, p. 1-30. In Razin, S., and R. Herrmann (eds.), Molecular Biology and Pathogenicity of Mycoplasmas, Kluwer Academic/Plenum, New York.
• Maniloff, J. 2002. Phylogeny and Evolution, p. 31-44. In Razin, S., and R. Herrmann (eds.), Molecular Biology and Pathogenicity of Mycoplasmas, Kluwer Academic/Plenum, New York.
• Marmion, B. P. 1990. Eaton agent--science and scientific acceptance: a historical commentary. Rev Infect Dis 12:338-353.
• Marmion, B. P., and G. M. Goodburn. 1961. Effect of an organic gold salt on Eaton's primary atypical pneumonia agent and other observations. Nature 189:247-248.
• Nocard, Roux. 1990. The microbe of pleuropneumonia. 1896. Rev Infect Dis 12:354-358. English translation of original 1896 French article.
• Regula, J. T., B. Ueberle, G. Boguth, A. Gorg, M. Schnolzer, R. Herrmann, and R. Frank. 2000. Towards a two-dimensional proteome map of Mycoplasma pneumoniae. Electrophoresis 21:3765-3780.
• Ning, J.Y., Shou, C.C. 2004. Mycoplasma infection and cancer. Ai Zheng May;23(5):602-4, Department of Biochemistry and Molecular Biology, Beijing Institute for Cancer Research, School of Oncology, Peking University, Beijing, China.
• Tsai S, Wear D.J., Shih J.W., Lo, S.C. 1995. Mycoplasmas and oncogenesis: persistent infection and multistage malignant transformation. Proc Natl Acad Sci U S A; 92(22):10197-201