The stability of the alkylating derivatives of oligodeoxyribonucleotides containing a cholesterol or phenazine radical added to the 3'-termination during their interaction with Acholeplasma laidlawii PG-8]

Stability of alkylating derivatives of decathymidylates protected on the 3'-terminal by cholesterol and phenazine residues has been studied in the process of their interaction with cells of Acholeplasma laidlawii PG-8. It is shown that the studied reagents are not split by nucleases of A. laidlawii PG-8 for the time necessary for alkylation of mycoplasma biopolymers.     

Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase

Maize polyamine oxidase (MPAO), the only member of the polyamine oxidase (PAO) family whose three-dimensional structure is known, is characterized by a 30 A long U-shaped catalytic tunnel located between the substrate binding domain and the FAD. To shed light on the MPAO ligand binding mode, we studied the inhibition properties of linear diamines, agmatine, prenylagmatine (G3), G3 analogues, and guazatine, and analyzed the structural determinants of their biological activity. Linear diamines competitively inhibited MPAO, with the inhibitory activity increasing as a function of the number of methylene groups. With regard to the guanidino competitive inhibitors, including agmatine, G3, and G3 analogues, the presence of a hydrophobic substituent constitutes the principal factor influencing MPAO inhibition, as the addition of a hydrophobic substituent to the guanidino group of both G3 and G3 analogues greatly increases the inhibitory activity. Moreover, results obtained by a molecular modeling procedure indicated that in their preferred orientation, G3 analogues point the ammonium group toward the narrow entrance of the tunnel, while the terminal hydrophobic group is located within the large entrance. The high binding affinity for MPAO exhibited by G3 and G3 analogues bearing a prenyl group as a substituent on the guanidino moiety is in agreement with the observation that the prenyl group binds in a well-defined hydrophobic pocket, mainly formed by aromatic residues. Finally, docking simulations performed with the charged and uncharged forms of MPAO inhibitors indicate that the stereoelectronic properties of the MPAO active site are consistent with the binding of inhibitors in the protonated form.     

Cellular re-distribution of flavin-containing polyamine oxidase in differentiating root and mesocotyl of <Emphasis Type="Italic">Zea mays</Emphasis> L. seedlings

Plant polyamine oxidases (PAOs; EC 1.5.3.11) are hydrogen peroxide-producing enzymes supposedly involved in cell-wall differentiation processes and defence responses. Maize (Zea mays L.) PAO (MPAO) is a 53 kDa secretory glycoprotein, abundant in primary and secondary cell walls of several tissues. Using biochemical, histochemical, ultrastructural and immunocytochemical techniques, the distribution and sub-cellular compartmentalisation of MPAO in the primary root and mesocotyl of seedlings at different maturation stages or after growth under varying light conditions were analysed. In apical root tissues, MPAO immunoreactivity was mainly detected in the cytoplasmic compartment, while a lower immunoreactivity was observed in the cell walls. In the more mature, basal part of the root, intense immunogold labelling was found in the primary and secondary walls of protoxylem precursors and vessels, while endodermal cells and living metaxylem precursors were immunopositive both in their walls and in their thin cytoplasmic compartments. A re-distribution of MPAO protein from the cytoplasm toward the primary and secondary walls was also recognised when immunoreactivity of basal root tissues from 3-day-old seedlings was compared with that detected in 11-day-old tissues. Accordingly, biochemical analyses revealed MPAO entrapment in the extracellular matrix of mature tissues. In the mesocotyl, an enrichment of MPAO immunolabelling in the cell wall of protoxylem, metaxylem and epidermal tissues, as a function of light exposure, was observed. Taken together, these data support the hypothesised role of PAOs in cell-wall maturation. Moreover, the relevant intraprotoplasmic MPAO localisation observed mainly in differentiating root tissues suggests an additional role in intracellular production of hydrogen peroxide.Alessandra Cona and Sandra Moreno have contributed equally to this paper.     

Involvement of Polyamine Oxidase in Abscisic Acid induced Cytosolic Antioxidant Defense in Leaves of Maize

Using pharmacological and biochemical approaches, the role of maize polyamine oxidase (MPAO) in abscisic acid (ABA)induced antioxidant defense in leaves of maize (Zea mays L.) plants was investigated. Exogenous ABA treatment enhanced the expression of the MPAO gene and the activities of apoplastic MPAO. Pretreatment with two different inhibitors for apoplastic MPAO partly reduced hydrogen peroxide (H2O2) accumulation induced by ABA and blocked the ABA-induced expression of the antioxidant genes superoxide dismutase 4 and cytosolic ascorbate peroxidase and the activities of the cytosolic antioxidant enzymes. Treatment with spermidine, the optimum substrate of MPAO, also induced the expression and the activities of the antioxidant enzymes, and the upregulation of the antioxidant enzymes was prevented by two inhibitors of MPAO and two scavengers of H2O2. These results suggest that MPAO contributes to ABA-induced cytosolic antioxidant defense through H2O2, a Spd catabolic product.     

Role of the membranes in lymphocyte interaction with mycoplasmas

The interaction of Acheleplasma laidlawii cells, derived membranes and liposomes with mouse spleen lymphocytes does not require the energy and participation of electrostatic coupling. The absence of pinocytosis of mycoplasma is demonstrated. Specific membrane receptors don't participate in mycoplasma and lymphocyte binding. The exchange by membrane lipids occurs during the interaction of mycoplasma, membranes and liposomes with lymphocytes; mycoplasma, membranes and liposomes lose saturated fat acids and enrich with cholesterol. On the contrary, lymphocytes lose cholesterol, but absorb fat acids. The intensity of lipid exchange depends on the degree of unsaturation of fat acids in mycoplasma. The carbohydrate transport into lymphocytes is stimulated considerably as a result of interaction of both cells.     

Inducible expression of maize polyamine oxidase in the nucleus of MCF-7 human breast cancer cells confers sensitivity to etoposide

Summary. In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H₂O₂, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine. Authors’ address: Dr. Paraskevi Tavladoraki, Department of Biology, University ‘Roma Tre’, Viale G. Marconi 446, 00146 Rome, Italy     

Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.     

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.     

Flavin-containing polyamine oxidase is a hydrogen peroxide source in the oxidative response to the protein phosphatase inhibitor cantharidin in Zea mays L

In this study, the specific contribution of polyamine oxidase (PAO), a hydrogen peroxide (H2O2)-producing enzyme, to the oxidative burst induced in maize mesocotyl by the phosphatase inhibitor cantharidin was examined. For this purpose, a pharmacological approach was applied using, either in vitro or in vivo, two strong inhibitors of maize PAO (MPAO), N-prenylagmatine (G3) and its structural analogue Ro5, as well as diphenyleneiodonium (DPI), an inhibitor of the phagocyte NAD(P)H oxidase. DPI was shown to be a good MPAO inhibitor in vitro. G3, Ro5, and DPI were very effective in inhibiting in vivo the extracellular accumulation of H2O2 that is released by mesocotyl segments upon spermidine supply. G3 and Ro5 did not show any inhibition in vitro of either horseradish peroxidase or barley oxalate oxidase. Moreover, G3 and Ro5 did not inhibit the extracellular accumulation of superoxide radical that is released in vivo upon NADH supply. G3, Ro5, and DPI strongly affected H2O2 production induced in maize mesocotyl by cantharidin. Histochemical localization of H2O2 in cantharidin-treated mesocotyl cross-sections revealed an increase of H2O2-specific staining in the epidermal and subepidermal tissues. The effect was also inhibited by G3 and DPI. Moreover, an increase in MPAO activity was observed in the same tissues upon cantharidin treatment. All these data suggest that G3 and Ro5 behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo and that MPAO activity contributes to a major part of the cantharidin-induced H2O2 synthesis in the apoplastic milieu of maize mesocotyl.     

Mycoplasma Suppression of THP-1 Cell TLR Responses Is Corrected with Antibiotics

Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 β was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1β and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.     

Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases

It was previously shown (Paddenberg et al (1996) Eur J Cell Biol 69, 105 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47 - 54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.     

Utilization of Neuraminic Acid Receptors by Mycoplasmas

Erythrocytes and H-HeLa cells were treated with neuraminidase and then compared with untreated cells for their ability to adsorb to mycoplasma colonies or be agglutinated by suspensions of the mycoplasmas. Of the 17 mycoplasma serotypes examined, only 4 were found to use neuraminic acid receptors; these were Mycoplasma pneumoniae, M. gallisepticum, M. synoviae, and mycoplasma WR1. Not all strains of a serotype behaved alike. Thus, removal of receptors on erythrocytes for one strain of M. gallisepticum required at least 100 times the concentration of neuraminidase needed to remove them for another strain. The mechanism of attachment of erythrocytes to mycoplasma colonies does not appear to be the same as that for attachment to mycoplasmas in suspension.     

Construction and analysis of a modified Tn4001 conferring chloramphenicol resistance in Mycoplasma pneumoniae

The Staphylococcus aureus transposon Tn4001 and derivatives thereof have been transformed successfully in several mycoplasma species. In order to expand the versatility of Tn4001 for other genetic manipulations and for use in mycoplasma species resistant to gentamicin (Gm), chloramphenicol acetyltransferase (Cat) from S. aureus was evaluated as a selectable marker. The cat gene was cloned in both orientations into a modified Tn4001 and transformed into Mycoplasma pneumoniae, conferring resistance to Cm and Gm. Replacement of the gene for GmR in Tn4001 with cat likewise conferred CmR when transformed into M. pneumoniae. The minimum inhibitory concentration to Cm in transformants with cat derivatives of Tn4001 was 300-500 microg/ml, and Cat enzyme activity was demonstrated by using a fluorescent substrate.     

A novel C-terminal sequence from barley polyamine oxidase is a vacuolar sorting signal

Barley contains two different isoforms of flavin-containing polyamine oxidase (BPAO1 and BPAO2). We have previously demonstrated that BPAO2 is a symplastic protein in barley leaves. On the contrary, maize polyamine oxidase (MPAO), the best characterized member of this enzyme class, is apoplastic. Comparison of the derived amino-acid sequences of BPAO2 and MPAO has revealed that both precursor proteins include a cleavable N-terminal signal peptide of 25 amino acid residues, but the barley enzyme shows an extra C-terminal extension of eight amino acids. By means of MPAO engineering with BPAO2 C-terminal tail (MPAO-T) and exploiting transient expression in Nicotiana tabacum protoplasts, we demonstrate that this oligopeptide is a signal for protein sorting to the plant vacuole. The vacuolar sorting of MPAO-T was saturable. Specific mutations of the C-terminal tail were constructed to determine which amino acid residues of this novel propeptide affect proper protein sorting. No consensus sequence or common structural determinant is required for the intracellular retention of the MPAO-T protein, but a gradual lowering of the efficiency was observed as a result of progressive deletion of the C-terminus.     

A barley polyamine oxidase isoform with distinct structural features and subcellular localization.

Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and the corresponding gene copies were isolated from barley cultivar Aura. Gene organization is not conserved between these two nonallelic coding sequences. Both precursor proteins include a cleavable N-terminal leader of 25 amino acids. N-terminal sequencing of PAO purified from barley seedlings reveals a unique amino-acid sequence corresponding to the BPAO2 N-terminus as predicted from the corresponding cDNA. BPAO2 has been purified, characterized and compared to maize PAO (MPAO), the best characterized member of this enzyme class. The two proteins show different pH optima for catalytic activity, Km and Vmax values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, substitution of the active site residue Phe403 by a tyrosine, provides a rationale for the different catalytic properties of the two enzymes. In barley leaves PAO-specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C-terminus of BPAO2 shows homology with the endoplasmic reticulum retention signal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Production of BPAO2 mRNA and the corresponding protein is induced by light, and has a different pattern of accumulation in leaves and coleoptiles.     

Rapid removal of mycoplasma from cell lines mediated by a direct effect of complement

Mycoplasma can be removed from the surface of contaminated human and murine cell lines by incubation for 4 h with human, rabbit, guinea pig, or mouse sera. Several lines of evidence suggest the involvement of complement in this process: (1) The activity can be abrogated by heat treatment (56 degrees C for 45 min). (2) Using monoclonal antibodies directed against C3a and C3b, the deposition of C3b fragments on the surface of mycoplasma-positive cells can be demonstrated after 1 h incubation with human serum. (3) Ca2+ depletion ablates the ability of serum to remove the activity. (4) C2def' sera are inactive while addition of purified C2 reconstitutes the activity. The latter two findings implicate that activation of the classical pathway of complement is responsible for the effect. Antibody, however, is not required as demonstrated by the uncompromised activity of Ig-deficient sera from bursectomized chicken. Treatment with human serum or rabbit serum was used successfully to permanently cleanse 10/10 tumor cell lines of human and of murine origin. The complete removal of mycoplasma was monitored over at least 8 weeks by direct DNA staining and confirmed by agar culture and transfer of supernatants to mycoplasma-free Vero cells followed by DNA staining. Thus the direct interaction of mycoplasma and complement appears to be an effective and rapid means of curing cell lines from mycoplasma.     

Recent news related to substrates and inhibitors of plant amine oxidases

Reactions of pea diamine oxidase (PSAO) and maize polyamine oxidase (MPAO) with 1,4-bis(3-aminopropyl)-piperazine (BAPP), diethylenetriamine (DETA), dipropylenetriamine (DPTA), dehydrospermine (DHSP) and 3-oxapentane-1,5-diamine (OPD) were studied and compared. These reactions were characterised by kinetic measurements (kinetic constants, stoichiometry) and by measurements of absorption spectra (reaction mechanisms). In the case of oxidised polyamine compounds, the corresponding reaction products were determined using analytical methods (coloured trapping reactions, mass spectrometry, IR spectroscopy, thin layer chromatography). Some of the compounds were found to be substrates of PSAO and relatively potent inhibitors of MPAO (and vice versa) all at once. The others showed the same effect on both enzymes. This may have an importance for designing of experiments in physiological studies in plants.     

Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas.

Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.     

Depletion of CD8+ T cells exacerbates CD4+ Th cell-associated inflammatory lesions during murine mycoplasma respiratory disease

Mycoplasma infection is a leading cause of pneumonia worldwide and can lead to other respiratory complications. A component of mycoplasma respiratory diseases is immunopathologic, suggesting that lymphocyte activation is a key event in the progression of these chronic inflammatory diseases. The present study delineates the changes in T cell populations and their activation after mycoplasma infection and determines their association with the pathogenesis of murine Mycoplasma respiratory disease, due to Mycoplasma pulmonis infection. Increases in T cell population numbers in lungs and lower respiratory lymph nodes were associated with the development of mycoplasma respiratory disease. Although both pulmonary Th and CD8(+) T cells increased after mycoplasma infection, there was a preferential expansion of Th cells. Mycoplasma-specific Th2 responses were dominant in lower respiratory lymph nodes, while Th1 responses predominated in spleen. However, both mycoplasma-specific Th1 and Th2 cytokine (IL-4 and IFN-gamma) responses were present in the lungs, with Th1 cell activation as a major component of the pulmonary Th cell response. Although a smaller component of the T cell response, mycoplasma-specific CD8(+) T cells were also a significant component of pulmonary lymphoid responses. In vivo depletion of CD8(+) T cells resulted in dramatically more severe pulmonary disease, while depletion of CD4(+) T cells reduced its severity, but there was no change in mycoplasma numbers in lungs after cell depletion. Thus, mycoplasma-specific Th1 and CD8(+) T cell activation in the lung plays a critical regulatory role in development of immunopathologic reactions in Mycoplasma respiratory disease.     

Unadapted and adapted to starvation Acholeplasma laidlawii cells induce different responses of Oryza sativa, as determined by proteome analysis

For the first time, we studied the phytopathogenicity toward Oryza sativa L. of unadapted and adapted to unfavorable environment (starvation) cells of Acholeplasma laidlawii PG8--ubiquitous mycoplasma found in the soil, waste waters, tissues of the highest eukaryotes and being the basic contaminant of cell cultures and a causative agent of phytomycoplasmoses. The features of morphology, ultrastructural organization and proteomes of unadapted and adapted cells of the mycoplasma and infected plants were presented. Using 2D-DIGE and MS, 43 proteins of O. sativa L. that were differentially expressed in the leaves of plants cultivated in media with A. laidlawii PG8 were identified. The qualitative and quantitative responses of the plant proteome toward adapted and unadapted mycoplasma cells differed. That may be explained by differences in the virulence of the corresponding bacterial cells. Using 2D-DIGE and MS, 82 proteins that were differentially expressed in adapted and unadapted mycoplasma cells were detected. In adapted cells of the mycoplasma, in comparison with unadapted ones, a significant increase in the expression of PNPase--a global regulator of virulence in phytopathogenic bacteria occurred; there was also decreased expression of 40 proteins including 14 involved in bacterial virulence and the expression of 31 proteins including 5 involved in virulence was not detected. We propose that differences in the phytopathogenicity of adapted and unadapted A. laidlawii PG8 cells may be related to features of their proteomes and membrane vesicles.