Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperone protein, has broad oncogenic properties.

We isolated a strain of human mycoplasma that promotes lymphomagenesis in SCID mice, pointing to a p53-dependent mechanism similar to lymphomagenesis in uninfected p53-/- SCID mice. Additionally, mycoplasma infection in vitro reduces p53 activity. Immunoprecipitation of p53 in mycoplasma-infected cells identified several mycoplasma proteins, including DnaK, a member of the Hsp70 chaperon family. We focused on DnaK because of its ability to interact with proteins. We demonstrate that mycoplasma DnaK interacts with and reduces the activities of human proteins involved in critical cellular pathways, including DNA-PK and PARP1, which are required for efficient DNA repair, and binds to USP10 (a key p53 regulator), impairing p53-dependent anticancer functions. This also reduced the efficacy of anticancer drugs that depend on p53 to exert their effect. mycoplasma was detected early in the infected mice, but only low copy numbers of mycoplasma DnaK DNA sequences were found in some primary and secondary tumors, pointing toward a hit-and-run/hide mechanism of transformation. Uninfected bystander cells took up exogenous DnaK, suggesting a possible paracrine function in promoting malignant transformation, over and above cells infected with the mycoplasma. Phylogenetic amino acid analysis shows that other bacteria associated with human cancers have similar DnaKs, consistent with a common mechanism of cellular transformation mediated through disruption of DNA-repair mechanisms, as well as p53 dysregulation, that also results in cancer-drug resistance. This suggests that the oncogenic properties of certain bacteria are DnaK-mediated.

Mycoplasma fermentans deacetylase promotes mammalian cell stress tolerance.

Mycoplasma fermentans is a pathogenic bacterium that infects humans and has potential pathogenic roles in respiratory, genital and rheumatoid diseases. NAD+-dependent deacetylase is involved in a wide range of pathophysiological processes and our studies have demonstrated that expression of mycoplasmal deacetylase in mammalian cells inhibits proliferation but promotes anti-starvation stress tolerance. Furthermore, mycoplasmal deacetylase is involved in cellular anti-oxidation, which correlates with changes in the proapoptotic proteins BIK, p21 and BIM. Mycoplasmal deacetylase binds to and deacetylates the FOXO3 protein, similar with mammalian SIRT2, and affects expression of the FOXO3 target gene BIM, resulting in inhibition of cell proliferation. Mycoplasmal deacetylase also alters the performance of cells under drug stress. This study expands our understanding of the potential molecular and cellular mechanisms of interaction between mycoplasmas and mammalian cells.

Genomic features and insights into the biology of Mycoplasma fermentans.

We present the complete genomic sequence of Mycoplasma fermentans, an organism suggested to be associated with the pathogenesis of rheumatoid arthritis in humans. The genome is composed of 977,524 bp and has a mean G+C content of 26.95 mol%. There are 835 predicted protein-coding sequences and a mean coding density of 87.6 %. Functions have been assigned to 58.8 % of the predicted protein-coding sequences, while 18.4 % of the proteins are conserved hypothetical proteins and 22.8 % are hypothetical proteins. In addition, there are two complete rRNA operons and 36 tRNA coding sequences. The largest gene families are the ABC transporter family (42 members), and the functionally heterogeneous group of lipoproteins (28 members), which encode the characteristic prokaryotic cysteine 'lipobox'. Protein secretion occurs through a pathway consisting of SecA, SecD, SecE, SecG, SecY and YidC. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The genes encoding DnaK-DnaJ-GrpE and Tig, forming the putative complex of chaperones, are intact, providing the only known control over protein folding. Eighteen nucleases and 17 proteases and peptidases were detected as well as three genes for the thioredoxin-thioreductase system. Overall, this study presents insights into the physiology of M. fermentans, and provides several examples of the genetic basis of systems that might function as virulence factors in this organism.

The reducing antioxidant capacity of Mycoplasma fermentans.

Mycoplasma fermentans is an extracellular microorganism capable of adhering to the surface of host cells. It has been recently shown that plasminogen binding to M. fermentans in the presence of the urokinase-type plasminogen activator promotes the invasion of host cells by this organism. In this report, we show that viable mycoplasmas persist within the infected HeLa cells for prolonged periods of time despite the expectation that within host cells the organism may be exposed to oxidative stress. Using cyclic voltammetry and luminol-enhanced chemiluminescence assays, we detected a potent reducing antioxidant activity in M. fermentans. The reducing antioxidant activity was heat stable, not affected by proteolysis and was almost totally lost upon dialysis suggesting that the activity is due to a nonproteinaceus low molecular weight antioxidant. This antioxidant was partially purified by Bio-Gel column chromatography followed by high-pressure liquid chromatographic analysis. We suggest that the high reducing antioxidant capacity in M. fermentans is a principal defense mechanism playing a major role in the battle of the organism against oxidative stress within the host cells.

Mycoplasma fermentans binds to and invades HeLa cells: involvement of plasminogen and urokinase.

Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37 degrees C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with beta-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.

Lipid-associated membrane proteins of Mycoplasma fermentans and M. penetrans activate human immunodeficiency virus long-terminal repeats through Toll-like receptors.

Mycoplasmas are known to enhance human immunodeficiency virus (HIV) replication, and mycoplasma-derived lipid extracts have been reported to activate nuclear factor-kappaB (NF-kappaB) through Toll-like receptors (TLRs). In this study, we examined the involvement of TLRs in the activation of HIV long-terminal repeats (LTR) by mycoplasma and their active components responsible for the TLR activation. Lipid-associated membrane proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans and M. penetrans) that are associated with acquired immune-deficiency syndrome (AIDS), were found to activate HIV LTRs in a human monocytic cell line, THP-1. NF-kappaB deletion from the LTR resulted in inhibition of the activation. The LTR activation by M. fermentans LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and TLR6, whereas HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but not by DN TLR6. These results indicate that the activation of HIV LTRs by M. fermentans and M. penetrans LAMPs is dependent on NF-kappaB, and that the activation of HIV LTR by M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In contrast, the LTR activation by M. penetrans LAMPs is carried out through TLR1 and TLR2, but not TLR6. Subsequently, the active component of M. penetrans and M. fermentans LAMPs was purified by reverse-phase high-performance liquid chromatography (HPLC). Interestingly, the purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate NF-kappaB through TLR1 and TLR2. On the other hand, the activation of NF-kappaB by purified lipoprotein of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but not TLR1.

Presence of human mycoplasma DNA in gastric tissue samples from Korean chronic gastritis patients.

We aimed to determine whether mycoplasmas are present in Korean chronic gastritis, and to understand their roles in gastric cancer tumorigenesis, because mycoplasmas resemble Helicobacter pylori in terms of ammonia production and induction of inflammatory cytokines in immune and non-immune cells. The presence and identity of mycoplasmas were assessed by semi-nested PCR and sequencing, and the results were compared with pathologic data. Fifty-six samples collected from Korean chronic gastritis patients were used for this study. Twenty-three (41.1%) were positive for mycoplasmas. Eighteen sequenced samples contained a single human mycoplasma or two mycoplasmas, which were identified as Mycoplasma faucium (13/18), M. fermentans (3/18), M. orale (1/18), M. salivarium (2/18), and M. spermatophilum (1/18). Mycoplasma-infected chronic gastritis samples showed significantly more severe neutrophil infiltration than non-infected samples (P = 0.0135). Mycoplasma profiles in the oral cavity (M. salivarium is major) and stomach were different, and the presence of significant proinflammatory responses in mycoplasma-positive patients suggests that the mycoplasmas are not simply contaminants. Further studies are required to understand whether mycoplasmas play a role in gastric tumorigenesis.

Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CGDPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.

Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins of Mycoplasma fermentans induces apoptotic cell death after NF-kappa B activation.

Mycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Toll-like receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF-kappaB, but also initiate apoptotic responses. The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP-2, showed cytocidal activity towards HEK293 cells transfected with a TLR2-encoding plasmid. The activity was synergically augmented by co-expression of TLR6, but not by co-expression of other TLRs. Under the condition of co-expression of TLR2 and TLR6, the lipoproteins could induce maximum NF-kappa B activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively. Dominant-negative forms of MyD88 and FADD, but not IRAK-4, reduced the cytocidal activity of the lipoproteins. In addition, both dominant-negative forms also downregulated the activation of both NF-kappa B and caspase-8 in the cells. Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK. These findings suggest that mycoplasmal lipoproteins can trigger TLR2- and TLR6-mediated sequential bifurcate responses: NF-kappa B activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.

Chronic fatigue syndrome: a risk factor for osteopenia?

No data documenting a possible depletion of bone mineral density in patients with chronic fatigue syndrome (CFS) are currently available. However, recent pathophysiological observations in CFS patients may have deleterious consequences on bone density. Firstly, the deregulation of the 2,5A synthetase RNase L antiviral pathway and its associated channelopathy, implicates increased demands for calcium and consequent increased calcium-re-absorption from the skeletal system. Secondly, Mycoplasma fermentans which has been frequently associated with CFS, produces a lipopeptide, named 2-kDa macrophage-activating lipopeptide (MALP-2), which stimulates macrophages. MALP-2 has been shown to enhance bone resorption in a dose-dependent manner, at least in part by stimulating the formation of prostaglandins. Thirdly, decreased levels of insulin-like growth factor I (IGF-I) have been reported in CFS-patients. IGF-I is critical to the proliferation of osteoblasts. Consequently, depleted levels of IGF-I may shift the balance between osteoclastic and osteoblastic activity towards bone resorption.

Identification and functional mapping of the Mycoplasma fermentans P29 adhesin.

Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).

Choline-containing lipids in mycoplasmas.

Choline-containing lipids were identified and characterized in the cell membrane of Mycoplasma fermentans and were shown to participate in the adhesion to the surface of eukaryotic cells, to stimulate mycoplasma fusion with eukaryotic cells, and to induce cytokine secretion by cells of the immune system. These findings suggest that choline-containing lipids are important mediators of tissue pathology in the infectious process caused by M. fermentans.

Effect of Mycoplasma fermentans on brain PGE(2): role of glucocorticoids and their receptors.

OBJECTIVES: Mycoplasmas are a group of eubacteria, which cause various diseases in animals and in humans, and can contribute to diseases produced by other infectious agents, particularly HIV. We have recently reported that intracerebral administration of Mycoplasma fermentans (MF) produces both neuroendocrine and behavioral alterations. Some of these responses were mediated by MF-induced production of prostaglandin E(2 )(PGE(2)). The aim of this study was to examine the role of glucocorticoids (GC) in regulating MF-induced brain prostaglandin production. METHODS: Male rats were injected intracerebroventricularly with various doses of heat-inactivated MF, LPS or IL-1 beta and the following parameters were measured: (1) ex vivo production of hippocampal PGE(2), (2) serum levels of ACTH and corticosterone, and (3) binding capacity of [(3)H]-dexamethasone (DEX) to hippocampal cytosol. RESULTS: MF caused a small increase in hippocampal PGE(2) production, but higher doses failed to produce a further increase. In contrast, the effects of LPS or IL-1 beta on PGE(2) were dose-dependent. Removal of circulating GC by bilateral adrenalectomy significantly enhanced MF-induced brain PGE(2) production. The three immune stimulators increased serum levels of ACTH and corticosterone to the same extent. Finally, MF, but not IL-1 beta increased the specific binding of [(3)H]-DEX to hippocampal cytosol. CONCLUSIONS: Brain PGE(2) induced by MF is regulated by endogenous GC. These hormones have an attenuating effect on PGE(2 )production, probably through an MF-induced increase in GC binding by brain tissue. This mechanism may be important in the pathological effect of MF within the brain of AIDS patients.

The physico-chemical characteristics of the phosphocholine-containing glycoglycerolipid MfGL-II govern the permeability properties of Mycoplasma fermentans.

Mycoplasma fermentans seems to be involved in several pathogenic conditions in humans, and is among other things capable of fusing with T-cells and lymphocytes. The choline-containing phosphoglycolipid 6'-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-alpha-D-glucopyranosyl-(1'-->3)-1,2-diacylglycerol (MfGL-II) in the membrane of M. fermentans has been suggested to enhance the fusion process, and the characteristics of MfGL-II were therefore investigated. When a cell culture ages the fraction of MfGL-II increases, and the fraction of the other major membrane lipid, phosphatidylglycerol (PtdGro), decreases concomitantly. Swelling experiments showed that the permeability and osmotic fragility are markedly reduced in aged cells. MfGL-II is selectively released into the surrounding medium when aged M. fermentans cells are incubated in buffer containing EDTA. The physico-chemical properties of MfGL-II were studied by NMR spectroscopy and differential scanning calorimetry, and they can explain the biochemical results. The temperature for the transition between gel and lamellar liquid crystalline (Lalpha) phases is 35-45 degrees C higher for MfGL-II than for PtdGro, which most probably gives rise to the reduced permeability in aged cells. At high water contents MfGL-II forms an Lalpha phase and isotropic aggregates which were interpreted to be vesicles with a radius of approximately 450 A. It is proposed that MfGL-II forms vesicles in the surrounding medium when it is released from the cell membrane. Neither EDTA nor Ca2+ ions have a significant influence on the aggregate structures formed by MfGL-II. Our results indicate that MfGL-II has no fusogenic properties. It is more probable that a recently identified lysolipid in the M. fermentans membrane acts as a fusogen.

MALP-2, a Mycoplasma lipopeptide with classical endotoxic properties: end of an era of LPS monopoly?

Although some activities of LPS are shared by other bacterial components, for half a century LPS has been regarded as unique in displaying many pathophysiological activities. Here we report on a synthetic lipopeptide, MALP-2 from Mycoplasma fermentans, which expresses potent endotoxin-like activity and whose lethal toxicity is comparable to that of LPS. With the exception of the Limulus lysate gelation test, in which MALP-2 was approximately 1000-fold less active than LPS, the synthetic lipopeptide induced all activities tested for, and in most cases to an extent comparable to that of LPS. Unlike LPS, the biological activities of MALP-2 were expressed both in LPS-responder and in LPS-non-responder mice (BALB/c/l, C57BL10/ScCr), indicating that MALP-2 signaling, unlike that of LPS, is not transduced via the Toll-like receptor (Tlr) 4 protein.MALP-2 expressed no toxicity in normal or sensitized Tlr2 knockout (Tlr2(-/-)) mice indicating that its toxic activity is induced via Tlr2 signaling. The phenomenology of the lethal shock induced by MALP-2 in normal or sensitized mice, i.e. the kinetics of its development and symptoms of illness exhibited by the treated animals, was very reminiscent of the lethal shock induced by LPS.

Lack of serological evidence for Mycoplasma fermentans infection in army Gulf War veterans: a large scale case-control study.

Mycoplasma firmentans is suspected in the development of 'Gulf War illness' in veterans of Operation Desert Storm. We conducted a matched case-control study for the prevalence of M. firmentans-specific antibodies before and after the operation, as well as seroconversion rates in veterans with and without complaints of 'Gulf War illness'. Cases consisted of Gulf War veterans, who complained of various illnesses and were enrolled in the second phase of the health evaluation by the Army Comprehensive Clinical Examination Program (CCEP). Controls were selected from Gulf War veterans who did not participate in the registry and did not request a health evaluation by the CCEP. Before operation deployment, 34 out of 718 of the cases (48%) and 116 out of 2233 of the controls (5.2%) tested positive for M. fermentans-specific antibodies. There was no difference in rates of seroconversion between cases and controls (1.1 vs. 1.2%) to M. fermentans during Operation Desert Storm. Thus, there is no serological evidence that suggests infectionby M. fermentans is associated with development of 'Gulf War illness'.

Factores de riesgo para la colonización por micoplasmas genitales / Risk factors for colonization with genital mycoplasmas

Con el propósito de determinar los factores de riesgo para colonización por micoplasmas genitales, se procesaron 93 muestras de origen urogenital, provenientes de pacientes que acudieron a la Consulta de Microbiología de la Policlínica San Luis de Maracaibo entre marzo y diciembre de 1997. El cultivo, aislamiento y recuento de estos microorganismos, se realizó de acuerdo con la metodología recomendada por Biomerieux, a través de su equipo comercial, Mycoplasma-Lyo. Las frecuencias relativas de micoplasmas genitales fueron comparadas a través de la prueba de "z" para determinar diferencias entre proporciones de la población, con un porcentaje de significacia del 5 por ciento (alfa=0,05). Se obtuvo una proporción de 39,92 por ciento para M.hominis en mujeres y del 4.30 por ciento y 21.54 por ciento para U.urealyticum en el sexo masculino y femenino, respectivamente. No hubo aislamiento de M.fermentans. La edad, el sexo, la actividad sexual (sobre todo si es de inicio precoz), la promiscuidad sexual, la raza, el nivel socioeconómico y abortos previos, constituyen factores de riesgo para la colonización genital por micoplasmas; a diferencia de la anticoncepción, embarazos previos, instrumentación, alteraciones bioquímicas, fisiológicas y/o funcionales, así como los tratamientos previos, que al parecer, no son factores predisponentes para la colonización por estos microorganismos

Phosphocholine-containing glycoglycerolipids (GGPL-I and GGPL-III) are species-specific major immunodeterminants of Mycoplasma fermentans.

Mycoplasma fermentans has unique glycoglycerophospholipids (GGPLs: GGPL-I and GGPL-III). Previously, the structure of these lipids was determined as phosphocholine-6'-alpha-glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-I) and 1"-phosphocholine-2"-aminodihydroxypropane-3"-phospho-6'-alph++ + a- glucopyranosyl-(1'-3)-1, 2-diacyl-glycerol (GGPL-III). Thin-layer chromatography (TLC) immunostaining showed that the GGPLs were main lipid-antigens of the M. fermentans species. Anti-M. fermentans serum stained mainly the GGPLs, but the other anti-mycoplasma sera (anti-M. arginini, anti-M. hyorhinis, anti-M. pneumonia, anti-M. primatum, and anti-Acholeplasma laidlawii, anti-M. hominis, anti-M. orale, and M. salivarium) stained neither GGPL-I nor GGPL-III. The TLC analysis of glycolipids and phospholipids of various human related mycoplasmas showed clearly that GGPLs are specifically expressed in M. fermentans species. GGPL-I and GGPL-III ranged from 1.6 to 28% and from an undetectable level to 35% of total phospholipids, respectively. Although there was heterogeneity among the amounts of GGPL-I or GGPL-III of M. fermentans strains, all of the M. fermentans strains had GGPL-I and/or GGPL-III. These observations showed that GGPL structures are species-specific immunodeterminants of M. fermentans. The fact that the GGPLs are main phospholipid components of the M. fermentans species means the M. fermentans has a unique choline metabolic pathway. This observation may raise phylogenetic interest.