Peculiar properties of mycoplasmas: The smallest self-replicating prokaryotes

Mycoplasmas are the smallest and simplest prokaryotes capable of self-replication, with information provided by a genome which may be as small as 600 kb, estimated to carry less than 500 genes. Keeping the number of structural elements, metabolic pathways and components of the protein synthesizing machinery to an essential minimum places mycoplasmas closest to the concept of 'minimum cells'. Mycoplasmas are, therefore, most adequate candidates for the complete deciphering of the machinery of a self-replicating organism, and studies towards this goal are already underway. Living as 'minimum cells' was made possible by adopting a parasitic mode of life, securing from the host the many nutrients which cannot be synthesized by the mycoplasmas themselves. When pathogenic, infections by mycoplasmas usually follow a chronic course, with host immune reactions playing an important role in symptom production. Recent studies on the possible association of mycoplasmas with rheumatoid arthritis and AIDS are reviewed.     

Tetrazolium reduction methods for assessment of substrate oxidation and strain differentiation among mycoplasmas, with particular reference to Mycoplasma bovigenitalium and some members of the Mycoplasma mycoides cluster

Aims:  To apply a rapid nitroblue tetrazolium (NBT) reduction assay of substrate metabolism by mycoplasmas that would help to differentiate Mycoplasmas .
Methods and Results:  Growth, substrate preferences and tetrazolium reduction were assessed for 18 strains of Mycoplasma bovigenitalium and Mycoplasma ovine serogroup 11. NBT reduction was detectable in 1 h with 10⁸ CFU ml⁻¹. Use of α-ketobutyrate, lactate and pyruvate to support growth and NBT reduction were correlated: pyruvate was preferred and lactate was used by only four of the 18 strains. Selected members of the Mycoplasma mycoides cluster were also assessed and monotetrazoles tested as alternatives to NBT. The NBT method was applied to a further 19 species.
Conclusions:  This simple and reproducible method requires only small amounts of cells, enabling routine assessment of substrate use within 1 h, and the rapid assignment of numerous mycoplasmas to one of six physiological groups. The four physiological groups of M. bovigenitalium and Mycoplasma serogroup 11 strains were indistinguishable from each other, which supports the view that these belong to the same species.
Significance and Impact of the Study:  Strain-specific substrate-utilization patterns by mycoplasmas can be obtained rapidly and reliably. The method has potential as a large-scale semi-automated procedure to monitor numerous strains and substrates simultaneously.     

Alternatives to arginine as energy sources for the non-fermentative Mycoplasma gallinarum

Abstract In contrast to previously studied non-fermentative arginine-hydrolysing (F-/A+) Mycoplasma species, M. gallinarum cells suspended in salts solution oxidised ethanol and L-lactic, pyruvic and 2-oxobutyric acids. The organic acids were additionally shown effectively to replace arginine as energy sources in growth media. However, their presence did not inhibit arginine hydrolysis, nor did arginine inhibit organic acid catabolism. The ability to oxidase organic acids is a potentially useful diagnostic character enabling sub-division of the F-/A+ Mycoplasma species. M. gallinarum also differed from previously studied F-/A+ mycoplasmas in possessing relatively high NADH oxidase activity and producing H₂O₂ as only a minor product of NADH oxidation.     

Metal-induced extracellular protein excretion in Arthrobacter aurescens

Abstract Mycoplasma contamination of cell cultures is a menace to diagnostic and research procedures. Rapid and reliable detection methods are, therefore, sorely needed. After comparing 16S rRNA sequences from those mycoplasmas that contaminate cell cultures, three different oligonucleotide probes were constructed. Two of these probes were designed to be group-specific and one to be species-specific. The three oligonucleotide probes were designed to cover all mycoplasmas commonly isolated from cell cultures. Contaminated cell lines could easily be detected by a direct filter hybridization assay in which the probes were incubated jointly. The assay proved to be rapid and sensitive with the possibility to perform and evaluate the mycoplasma testing within one working day.     

Chemotaxis away from thiocyanic and isothiocyanic esters in Pseudomonas aeruginosa

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.     

The phylogeny of Mycoplasma bovis as determined by sequence analysis of the 16S rRNA gene

Abstract The nucleotide sequence of the 16S rRNA gene of Mycoplasma bovis has been determined. Comparisons with other 16S rRNA sequences of mycoplasmas showed that Mycoplasma agalactiae is phylogenetically the closet relative. In total, only eight nucleotides differed between the M. bovis and M. agalactiae 16S rRNA sequences. The phylogenetic position of M. bovis with respect to other mycoplasmas was determined by sequence comparisons and from features in the secondary structure of 16S rRNA.     

What are mycoplasmas: The relationship of tempo and mode in bacterial evolution

Summary In phenotype the mycoplasmas are very different from ordinary bacteria. However, genotypically (i.e., phylogenetically) they are not. On the basis of ribosomal RNA homologies the mycoplasmas belong with the clostridia, and indeed havespecific clostridial relatives. Mycoplasmas are, however, unlike almost all other bacteria in the evolutionary characteristics of their ribosomal RNAs. These RNAs contain relatively few of the highly conserved oligonucleotide sequences characteristic of normal eubacterial ribosomal RNAs. This is interpreted to be a reflection of an elevated mutation rate in mycoplasma lines of descent. A general consequence of this would be that the variation associated with a mycoplasma population is augmented both in number and kind, which in turn would lead to an unusual evolutionary course, one unique in all respects. Mycoplasmas, then, are actually tachytelic bacteria. The unusual evolutionary characteristics of their ribosomal RNAs are the imprints of their rapid evolution.     

Contrasting effects of Mycoplasma fermentans and M. felis on the viability and chemiluminescence response of human polymorphonuclear leukocytes

Abstract Trypan blue exclusion was used to estimate the viability of human polymorphonuclear leukocytes (PMNL) in the presence of Mycoplasma felis and two strains of M. fermentans (PG18 and incognitus). The competence of PMNL to mount a respiratory burst when challenged with the mycoplasmas was also monitored by luminol-dependent chemiluminescence (CL). Both un-opsonised and non-immune human serum opsonised M. felis cells had little effect on PMNL viability. In contrast, PMNL viability was reduced markedly by un-opsonised cells of M. fermentans strain incognitus and, to a lesser extent, strain PG18, and opsonisation of these mycoplasmas further enhanced killing. Death of PMNL in the presence of M. fermentans was not associated with the autonomous production of active oxygen species during the respiratory burst as M. felis induced a high CL response from PMNL, whereas that induced by M. fermentans strain incognitus was significantly lower. M. fermentans may invade mammalian cells and it is suggested that the mechanism of PMNL death could be related to the ability of M. fermentans to penetrate host cell membranes.     

Interactions between mycoplasma lipoproteins and the host immune system

Mycoplasmas typically have a number of distinct lipoproteins anchored on the outer face of the plasma membrane. These surface antigens have a potent modulin activity and are preferential targets of the host immune response. However, the variation of some of these lipoproteins provides mycoplasmas with an effective means of evading the host immune defence system.     

Re-suspension of T(1)44 vaccine cultures of Mycoplasma mycoides subsp mycoides SC in 1 molar MgSO(4) causes a drop in pH and a rapid reduction in titre

We report Mycoplasma bovis induces apoptotic death of bovine lymphocytes. Using flow cytometry analyzed propidium iodide inclusion we observed a loss in viable lymphocytes upon incubation of freshly isolated bovine PBMCs with M. bovis. The use of annexin V staining as well as TUNEL assays corroborated these findings. In addition, these assays indicated that the M. bovis induced lymphocyte death is apoptotic in nature. Subsequent experiments demonstrated that the prokaryotic protein production inhibitor chloramphenicol inhibited lymphocyte death induced by M. bovis, showing that M. bovis protein production is necessary for the induction of lymphocyte death, and that the death is not dependent upon the addition of apoptotic inducers as shown with other mycoplasmas. We also show that M. bovis is different from other bovine mycoplasmas (both pathogenic and non-pathogenic) with regards to this characteristic.     

ISMmy1, a novel insertion sequence of Mycoplasma mycoides subsp. mycoides small colony type

A new insertion sequence, ISMmy1, has been identified in the bovine pathogen Mycoplasma mycoides subsp. mycoides biotype small colony (MmymySC). The occurrence of ISMmy1 in 15 MmymySC strains and 12 other mycoplasmas was examined by Southern blotting. All MmymySC strains showed identical hybridisation patterns except for the type strain PG1(T), the vaccine strain T1Sr49, and the strain Afadé, which all had unique patterns. ISMmy1-like sequences were also found in the bovine pathogen Mycoplasma bovis strain Donetta(T) while mycoplasmas that are phylogenetically closer to MmymySC lack ISMmy1. This observation suggests horizontal transfer between MmymySC and M. bovis.     

Internalization and intracellular survival of Mycoplasma pneumoniae by non-phagocytic cells

Current theory holds that mycoplasmas remain attached to the surface of epithelial cells although some mycoplasmas have evolved mechanisms for entering host cells that are not naturally phagocytic. The ability of Mycoplasma pneumoniae strain M129 to invade and survive within host cells was studied using a HeLa cell line and a human lung carcinoma cell line (A549). The invasion process into the eukaryotic cells was studied qualitatively by confocal laser scanning microscopy and quantitatively by the gentamicin resistance assay. Internalization was found with A549 cells but not with HeLa cells. Internalization was dependent on the duration of the infection and on temperature. The organism, detected in the cytoplasm and perinuclear regions, survived within the host cells for prolonged periods of time. The intracellular location of M. pneumoniae is obviously a privileged niche, well protected from the immune system and from the action of many antibiotics and may explain the pathogenic potential of this organism.     

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell’s interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.     

Characterization of membrane surface proteins of Mycoplasma agalactiae during natural infection

We have analyzed antigenic variation of seven M. agalactiae wild strains using different sera from naturally infected sheep. Only 30 day sera recognized all surface proteins and inhibited the growth of mycoplasmas. Furthermore, we have observed that two strongly immunogenic proteins: 55 and 35 kDa were digested using 500 μg/ml of trypsin. These two bands are immunoprecipitated together with four other proteins but only the 35 kDa protein is recognized by eluted antibodies.     

Development of two PCR assays for the identification of mycoplasmas causing contagious agalactia

Abstract A new detection test for the mycoplasmas causing contagious agalactia, Mycoplasma agalactiae , M. capricolum subsp. capricolum and M. mycoides subsp. mycoldes L.C., was developed. It was based on two polymerase chain reaction assays: the Ma-PCR for the detection of M. agalactiae and the MYC-PCR for the 'mycoides cluster' thus including M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L.C. An M. agalactiae strain was identified by a 933-bp Ma-PCR product and no amplification with the MYC-PCR. In contrast, a 460-bp MYC-PCR product and a negative or a 350-bp Ma-PCR product characterized a 'mycoides cluster' strain. M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L.C. were identified by their species-specific Asel pattern of the 460-bp MYC-PCR product.     

Mechanisms of genetic instability of mollicutes (mycoplasma)

Mollicutes are unique microorganisms characterized by a great extent for the reduction in genetic material, which retained the capability of independent division on acellular nutrient media. Phenotypically mycoplasmas differed from other bacteria by their small size and lack of a cell wall (mollis, soft; cutis, skin). High dependence on metabolism components utilized in the cultivation medium and high metabolic plasticity due to the absence of many genome regulatory elements make mycoplasmas perfect parasites for cells of the eukaryotic origin. The ability of these microorganisms to pass through host cells and their assumed participation in AIDS activation facilitate the study of mycoplasma pathogenesis. Another important feature of mycoplasmas, which is expressed during their interaction with a macroorganism, is their ability to escape from the immune response of a host due to surface antigen variation. These adaptation capacities of mycoplasmas ensuring their life in various biological niches, given a limited genome and the direct metabolic dependence on an environment, cannot be adequately explained at present. In this review, we attempted to collect and systematize data that contribute to our understanding of the important feature of mycoplasmas, genetic instability, which may underlie many of their adaptive responses.     

Mechanisms of Genetic Instability in Mollicutes (Mycoplasmas)

Mollicutes are unique microorganisms characterized by a great extent for the reduction in genetic material, which retained the capability of independent division on acellular nutrient media. Phenotypically mycoplasmas differed from other bacteria by their small size and lack of a cell wall (mollis, soft; cutis, skin). High dependence on metabolism components utilized in the cultivation medium and high metabolic plasticity due to the absence of many genome regulatory elements make mycoplasmas perfect parasites for cells of the eukaryotic origin. The ability of these microorganisms to pass through host cells and their assumed participation in AIDS activation facilitate the study of mycoplasma pathogenesis. Another important feature of mycoplasmas, which is expressed during their interaction with a macroorganism, is their ability to escape from the immune response of a host due to surface antigen variation. These adaptation capacities of mycoplasmas ensuring their life in various biological niches, given a limited genome and the direct metabolic dependence on an environment, cannot be adequately explained at present. In this review, we attempted to collect and systematize data that contribute to our understanding of the important feature of mycoplasmas, genetic instability, which may underlie many of their adaptive responses.