Dependence of plastoquinol diffusion on the shape, size, and density of integral thylakoid proteins.

The diffusion of plastoquinol in the chloroplast thylakoid membrane is modelled using Monte Carlo techniques. The integral proteins are seen as obstacles to diffusion, and features of percolation theory emerge. Thus, the diffusion coefficient diminishes with increasing distance and there is a critical threshold of protein concentration, above which the long-range diffusion coefficient is zero. The area occupied by proteins in vivo is assessed and appears to be around this threshold, as determined from calculations assuming randomly distributed noninteracting proteins. Slight changes in the protein arrangement lead to pronounced changes in diffusion behaviour under such conditions. Mobility of the proteins increases the protein occupancy threshold, while boundary lipids impermeable to PQ diffusion decrease it. Further, the obstruction of plastoquinone/plastoquinol binding sites in a random arrangement is evaluated.

Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone.

We have determined the stoichiometric composition of membrane components (lipids and proteins) in spinach thylakoids and have derived the molecular area occupied by these components. From this analysis, the lipid phase diffusion space, the fraction of lipids located in the first protein solvation shell (boundary lipids), and the plastoquinone (PQ) concentration are derived. On the basis of these stoichiometric data, we have analyzed the motion of PQ between photosystem (PS) II and cytochrome (cyt.) bf complexes in this highly protein obstructed membrane (protein area about 70%) using percolation theory. This analysis reveals an inefficient diffusion process. We propose that distinct structural features of the thylakoid membrane (grana formation, microdomains) could help to minimize these inefficiencies and ensure a non-rate limiting PQ diffusion process. A large amount of published evidence supports the idea that higher protein associations exist, especially in grana thylakoids. From the quantification of the boundary lipid fraction (about 60%), we conclude that protein complexes involved in these associations should be spaced by lipids. Lipid-spaced protein aggregations in thylakoids are qualitatively different to previously characterized associations (multisubunit complexes, supercomplexes). We derive a hierarchy of protein and lipid interactions in the thylakoid membrane.

Control of the photosynthetic electron transport by PQ diffusion microdomains in thylakoids of higher plants.

We investigate the role of plastoquinone (PQ) diffusion in the control of the photosynthetic electron transport. A control analysis reveals an unexpected flux control of the whole chain electron transport by photosystem (PS) II. The contribution of PSII to the flux control of whole chain electron transport was high in stacked thylakoids (control coefficient, CJ(PSII) =0.85), but decreased after destacking (CJ(PSII)=0.25). From an 'electron storage' experiment, we conclude that in stacked thylakoids only about 50 to 60% of photoreducable PQ is involved in the light-saturated linear electron transport. No redox equilibration throughout the membrane between fixed redox groups at PSII and cytochrome (cyt) bf complexes, and the diffusable carrier PQ is achieved. The data support the PQ diffusion microdomain concept by Lavergne et al. [J. Lavergne, J.-P. Bouchaud, P. Joliot, Biochim. Biophys. Acta 1101 (1992) 13-22], but we come to different conclusions about size, structure and size distribution of domains. From an analysis of cyt b6 reduction, as a function of PSII inhibition, we conclude that in stacked thylakoids about 70% of PSII is located in small domains, where only 1 to 2 PSII share a local pool of a few PQ molecules. Thirty percent of PSII is located in larger domains. No small domains were found in destacked thylakoids. We present a structural model assuming a hierarchy of specific, strong and weak interactions between PSII core, light harvesting complexes (LHC) II and cyt bf. Peripheral LHCII's may serve to connect PSII-LHCII supercomplexes to a flexible protein network, by which small closed lipid diffusion compartments are formed. Within each domain, PQ moves rapidly and shuttles electrons between PSII and cyt bf complexes in the close vicinity. At the same time, long range diffusion is slow. We conclude, that in high light, cyt bfcomplexes located in distant stromal lamellae (20 to 30%) are not involved in the linear electron transport.

[100 years of Mycoplasma--pathogenicity for domestic and farm animals]. / 100 Jahre Mykoplasmen--Pathogenität für Nutz- und Haustiere.

The paper presents a short description of the history of mycoplasmology, the systematic and characteristics of mycoplasmas and the diseases caused by mycoplasmas in cattle, sheep and goats, swine, poultry, and rats and mice. The pathogenicity of mycoplasmas for cats, dogs and horses is discussed.

Detection of Mycoplasma hyopneumoniae in bronchoalveolar lavage fluids of pigs by PCR.

In the present investigation we developed a method for the detection of Mycoplasma hyopneumoniae in bronchoalveolar lavage fluid (BALF) of pigs by PCR with a primer pair flanking a DNA fragment of 853 bp specific for M. hyopneumoniae. Several methods were tested to eliminate the amplification inhibitors present in BALFs. The best results were obtained by the extraction of the DNA from the BALFs. By the PCR performed with the extracted DNA, 10(2) CFU of M. hyopneumoniae could be detected in 1 ml of BALF from specific-pathogen-free swine experimentally inoculated with M. hyopneumoniae. DNA from 11 other mycoplasma species and 17 cell-walled bacterial species colonizing the respiratory tracts of pigs was not amplified. In a field study BALFs from 40 pigs from farms with a history of chronic pneumonia were tested for M. hyopneumoniae by cultivation and by PCR (i) with BALFs incubated in Friis medium and (ii) with DNA extracted from the BALFs. In addition, PCR was performed with postmortem lung washings from 19 of the 40 pigs, and immunofluorescence tests were carried out with sections of lungs from 18 of the 40 pigs. M. hyopneumoniae could not be detected in 18 of the 40 pigs by any of the five methods tested. The remaining 22 pigs showed a positive reaction by the PCR with DNA extracted from the BALFs and variable positive reactions by the other tests. A complete correspondence could be observed between the immunofluorescence test result and the result of PCR with DNA. The investigation shows that the PCR with DNA extracted from BALFs is a suitable technique for the sensitive and specific in vivo detection of M. hyopneumoniae.

Mycoplasma lagogenitalium sp. nov., from the preputial smegma of Afghan pikas (Ochotona rufescens rufescens).

Organisms with characteristics typical of mycoplasmas were isolated from the preputial smegma of Afghan picas (Ochotona rufescens rufescens). The results of growth inhibition tests, metabolic inhibition tests, and immunobinding assays showed that the isolated strains were identical and that they were distinct from previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. These organisms represent a new species, for which the name Mycoplasma lagogenitalium is proposed. M. lagogenitalium ferments glucose, does not hydrolyze arginine or urea, reduces tetrazolium chloride, possesses phosphatase activity, does not digest gelatin or casein, and does not produce films or spots. It lyses sheep erythrocytes and does not adsorb sheep, rabbit, or horse erythrocytes. Cholesterol or serum is required for growth. The growth temperature is 37 degrees C. The guanine-plus-cytosine content of the DNA is 23.0 +/- 1.0 mol%. The type strain is M. lagogenitalium 12MS (= ATCC 700289T).

Mycoplasma crocodyli sp. nov., a new species from crocodiles.

Organisms with the typical characteristics of mycoplasmas were isolated from joints and lungs of crocodiles. The results of growth inhibition tests and immunobinding assays showed that the 24 mycoplasma strains isolated were identical and distinct from previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. These organisms represent a new species, for which the name Mycoplasma crocodyli is proposed. M. crocodyli ferments glucose and maltose, does not produce films and spots, does not hydrolyze arginine, esculin, and urea, reduces tetrazolium chloride, and possesses phosphatase activity. It lyses and adsorbs bovine, ovine, and rabbit erythrocytes. Cholesterol or serum is required for growth. The optimum growth temperature is 37 degrees C. The G + C content of the DNA is 27.6 mol%. This organism causes exudative polyarthritis in crocodiles. The type strain of M. crocodyli is strain MP145 (= ATCC 51981).

Detection of Ureaplasma urealyticum in urine of patients with systemic lupus erythematosus and healthy individuals by culture and polymerase chain reaction.

A method was developed to detect Ureaplasma urealyticum in urine by the polymerase chain reaction (PCR). A 457-bp fragment of the urease gene of U. urealyticum was amplified by PCR. Before PCR, components disturbing the amplification had to be reduced. This was possible by diluting the urine 1 in 10 with distilled water and by the extraction of the U. urealyticum DNA. Urine specimens from 41 patients with systemic lupus erythematosus (SLE) and 21 healthy individuals were treated by the dilution method and investigated by PCR for U. urealyticum DNA. The results were compared with those obtained by culture and the detection rates of PCR and culture were found to be identical. Also there was no difference in the detection rates of U. urealyticum from urine of SLE patients and healthy individuals; 10 (24.4%) of the 41 urine specimens from SLE patients and five (23.8%) of the 21 urine specimens from healthy individuals gave positive results for U. urealyticum. The results of this study do not indicate a decisive role for U. urealyticum in SLE.

Immunoelectron microscopic localization of variable proteins on the surface of Mycoplasma bovis.

The ultrastructural distribution and immunological accessibility of the variable surface proteins VspA, VspB, VspC and VspD were determined by immunoelectron microscopy on the surface of negatively stained cells of Mycoplasma bovis PG45 and 18 subclones, expressing either one or two of the Vsps. The variable proteins VspA, VspB, VspC and VspD, recognized by two monoclonal antibodies (mAb 1E5 and mAb 87-2) and visualized by goat-anti-murine-lgM labelled with gold particles, showed identical distribution patterns on the surfaces of the cells of all M. bovis clones investigated. Gold particles were distributed over the whole cell surface, arranged in clusters. The cell form seemed not to have an influence on the decoration pattern. Gold particles were also observed in irregular distributions around the cells. All clones showed unlabelled cells as well as strongly and weakly labelled cells. There were in general, however, no significant differences in the percentages of unlabelled, weakly labelled and strongly labelled cells, either between clones expressing different Vsps or between individual clones. No correlations were found between the numbers of labelled cells in immunoelectron microscopy and the numbers of labelled colonies in immunobinding assay (IBA) originating from the same broth cultures. The percentage of positive colonies in IBA was generally much higher than the percentage of positive cells in immunoelectron microscopy. The results show that the cells of the M. bovis clones are not identical, but differ in their surface antigens, and reveal the high variable potential of this species.

Major membrane proteins and lipoproteins as highly variable immunogenic surface components and strain-specific antigenic markers of Mycoplasma arthritidis.

Surface antigenic variation was investigated in Mycoplasma arthritidis, an agent that produces chronic arthritis in rats which shares several features with many mycoplasma-induced diseases and thus defines a well-characterized model system. Hyperimmune rabbit antisera (anti-ISR1, anti-PG6, anti-H606 and anti-158p10) to whole M. arthritidis organisms were used as immunological probes in Western immunoblots of four M. arthritidis prototype strains (ISR1, PG6, H606 and D263) and five rat-passaged substrains (ISR1p1, ISR1p7, ISR1p8, 158p10 and D263p1). Several prominent antigens were identified that varied in expression. By Triton X-114 phase fractionation and treatment of whole cells with trypsin and carboxypeptidase Y, these strain-variant antigens were shown to be integral membrane proteins with C-termini and portions of the polypeptide chains oriented outside the membrane. Western blot immunoscreening of a large number of randomly selected clonal isolates and well-established clonal lineages from stock cultures of M. arthritidis ISR1p7, 158p10, PG6 and H606 revealed an expanded repertoire of variant membrane proteins whose expression was subject to independent, reversible phase variation. Colony immunoblots of these clonal populations with a hyperimmune rabbit antiserum to a gel-purified variant membrane protein (P36) showed that this phase switching occurred at a high frequency (10(-4) to 10(-2) per generation). Detailed immunological and biochemical characterization of the phase-variant membrane proteins demonstrated that they are: (i) antigenically related or distinct; (ii) apparently specific to particular strain populations; (iii) proteins or lipoproteins; (iv) major immunogens of M. arthritidis, recognized by serum antibodies from convalescent rat; and (v) able to undergo variation in expression during in vivo passage. Thus, M. arthritidis possesses a complex system capable of creating large repertoires of cell surface phenotypes which may affect the multiple interactions of this organism with its host and dictate its potential as a successful infectious agent and pathogen.

Investigation into the origin of the antigens of Mycoplasma arthritidis cross-reacting with host tissue antigens.

The electrophoretical separations of Mycoplasma arthritidis and the serum used in the cultivation medium show a high number of protein bands with identical molecular weights. Proteins with molecular weights of 84, 72 and 52 kDa also appeared to be identical with proteins of Mycoplasma arthritidis in their antigenic properties as demonstrated by Western blotting with rat-anti-Mycoplasma arthritidis serum. The autoradiography of electrophoretically separated Mycoplasma arthritidis cells metabolically labeled with 35S-methionine and 35S-cysteine revealed that the proteins of Mycoplasma arthiritidis identical in molecular weight and antigenic structure with serum proteins are synthesized by Mycoplasma arthritidis, and represent true translation products.

Piscine gill epithelial cell necrosis due to Mycoplasma mobile strain 163 K: comparison of in-vivo and in-vitro infection.

This paper describes experiments with Mycoplasma mobile 163 K in tench inoculated via the gills, skin, peritoneal cavity or whole body surface and kept at two different temperatures (20 and 25 degrees C). Gill tissues from experimentally infected tench and rainbow-trout gill tissue explants infected in vitro were compared by transmission electron microscopy, revealing that M. mobile was capable of producing gill epithelial cell necrosis in both, but that it was much more severe in the explants. M. mobile was found attached to chloride cells in the tench and between necrotic epithelial cells in the trout gill explants. M. mobile was recovered from the gills for up to 28 days after inoculation, from the skin and swim bladder for up to 14 days, and from the hind gut, kidneys and spleen for up to 8 days. There was no significant difference between the results at 20 and 25 degrees C.

Attachment of Mycoplasma mobile 163 K to piscine gill arches and rakers--light, scanning and transmission electron microscopic findings.

Explant cultures of gill arches and rakers were established to evaluate the attachment and colonization characteristics as well as the cytotoxic effects of the piscine bacterium, Mycoplasma mobile 163 K on piscine gill epithelium. Light, scanning and transmission electron microscopy were applied in this study and revealed heavy colonization of mycoplasmas on gill rakers, resulting in severe tissue damage of the gill epithelium. The complications for the function of the gills during breathing, the mechanisms of cytotoxicity, and the validity of this newly-established in vitro model are discussed in detail. In addition, anatomical specialities designated as spikes were identified on the inner surface of the gill rakers from trout; these could be used in the differentiation of fish.

A surface epitope undergoing high-frequency phase variation is shared by Mycoplasma gallisepticum and Mycoplasma bovis.

We have recently reported that three distinct size- and phase-variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis possess a common epitope recognized by monoclonal antibody 1E5. In the present study, we show that this epitope is also present on a size-variant protein (PvpA) of the avian pathogen Mycoplasma gallisepticum. Application of monoclonal antibody 1E5 in Western immunoblot analysis of Triton X-114 phase-fractionated proteins and in colony immunoblots, as well as in trypsin and carboxypeptidase digestion experiments, has demonstrated that (i) PvpA is an integral membrane protein with a free C terminus, (ii) the shared epitope is surface exposed, and (iii) PvpA is subjected to high-frequency phase variation in expression. By using serum antibodies from M. gallisepticum-infected chickens, we were able to demonstrate the immunogenic nature of PvpA and identify three additional highly immunogenic Triton X-114 phase proteins (p67, p72, and p75) also undergoing high-frequency phase variation spontaneously and independently. Metabolic labeling experiments with [14C]palmitate and [14C]oleate revealed that PvpA, in contrast to p67, p72, and p75, is not lipid modified. Southern blot hybridization with restriction fragments carrying the pvpA gene of M. gallisepticum or the vspA gene of M. bovis against digested genomic DNA of the two Mycoplasma species indicated the absence of genetic relatedness between the pvpA and vspA genes. The apparent complexity of the antigenic variation phenomenon in M. gallisepticum is discussed.

Antigen heterogeneity among isolates of Mycoplasma bovis is generated by high-frequency variation of diverse membrane surface proteins.

The protein and antigen profiles of 11 isolates of Mycoplasma bovis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of whole organisms. The isolates examined included the type strain PG45 and 10 other filter-cloned strains or purified isolates both from animals without clinical signs and from clinical cases of bovine mastitis, arthritis, or pneumonia. While the overall protein patterns visualized by silver staining were very similar, marked differences in the antigen banding profiles were detected by rabbit antiserum prepared against whole organisms from one of the strains analyzed. This antigenic heterogeneity was shown to be independent of the geographical origin, the type of clinical disease, and the site of isolation and was also observed among serial isolates from a single animal. Antigen profiles were further monitored throughout sequentially subcloned populations of the PG45 strain. This clonal analysis revealed a high-frequency variation in the expression levels of several prominent antigens. All of these variable antigens were defined by detergent-phase fractionation with Triton X-114 as amphiphilic integral membrane proteins. A subset of different-sized membrane proteins was identified by a monoclonal antibody raised against a PG45 subclone expressing a 63- and a 46-kDa variant antigen within that set. The selective susceptibility of these proteins to trypsin treatment of intact organisms and their ability to bind the monoclonal antibody in colony immunoblots demonstrated that they were exposed on the cell surface. In addition, their preferential recognition by serum antibodies from individual cattle with naturally induced M. bovis mastitis or arthritis confirmed that they were major immunogens of this organism. These studies establish that the apparent antigenic heterogeneity among M. bovis isolates reported here does not represent stable phenotypic strain differences generated from accumulated mutational events but reflects distinct expression patterns of diverse, highly variable membrane surface proteins.

A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis.

A set of strain- and size-variant highly immunogenic membrane surface protein antigens of Mycoplasma bovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family of antigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). By systematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this family have been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequency changes in size as well as noncoordinate phase variation between ON and OFF expression states. The monoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface in most, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragment of each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished from VspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibody against M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by their characteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structural fingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vsp product, they showed distinct differences among variants differing in size. This variability included gain or loss of individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns up- or downwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locations either within the C-terminal domain or within other regions of these proteins. This was similarly confirmed by comparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants. The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenic variation in mycoplasmas displaying features which closely resemble but are also different from the characteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineages established here provide key populations for subsequent analysis of corresponding genes to further elucidate Vsp structure and variation, which may have important relevance for a better understanding of the pathogenicity of this agent.

[Detection of mycoplasmas in horses with respiratory diseases and their biochemical and serologic characterization]. / Nachweis von Mykoplasmen bei Pferden mit respiratorischen Erkrankungen und deren biochemische und serologische Charakterisierung.

Tracheal swabs were taken from 25 horses with respiratory diseases and investigated for mycoplasmas using three different media. Mycoplasmas could be isolated from 5 horses. The isolates were characterized by serological and biochemical methods. Four isolates could be identified as Mycoplasma equirhinis. The fifth isolate could not be typed. It did not react with antisera against mycoplasmas found in the respiratory tract of horses and its biochemical characteristics were different from the mycoplasmas described so far. It may represent a new species.

The Mycoplasma arthritidis infection in congenitally athymic nude rats.

Fifteen young (47-81 days old) and 5 adult (360 days old) Rowett nude rats (rnu/rnu) and 12 euthymic littermates (rnu/+) (56 days old) were infected with Mycoplasma (M.) arthritidis ISR1. The polyarthritis developing after the infection was more severe in the athymic than in the euthymic rats. In the nude rats the disease was progressive and led to the death of 6 animals whereas in the euthymic littermates the disease was resolved within 70 days after infection. In contrast to euthymic rats athymic animals did not develop antibodies against M. arthritidis within 6 weeks (young nude rats) or 23 weeks (adult nude rats) after infection, indicating that M. arthritidis is a thymus dependent antigen. However, low antibody titers were found in the nude rats infected at an age between 47 and 81 days at 340 days after infection, probably due to the development of T-cells which occur in older nude rats. The euthymic as well as the young athymic rats showed an increase in antibody titers after a challenge infection 345 days after the first infection and both were resistant to challenge infection. On the contrary the adult nude rats infected with M. arthritidis did not develop antibodies against the mycoplasmas and were highly susceptible to a second infection after 198 days. This indicates that antibodies play an important role in the defence of the M. arthritidis infection in rats.

Analysis of membrane proteins of Mycoplasma capricolum strains by SDS-PAGE and immunoblotting.

Sixteen moroccan and five other Mycoplasma (M.) capricolum strains were characterized by SDS-PAGE as well as by the immunobinding assay and immunoblotting, using antisera against M. capricolum California Kid (CK) and M. mycoides YG. There was a strong reaction of all 21 strains with antiserum against M. capricolum CK and of 18 strains with antiserum against M. mycoides YG in the immunobinding assay, confirming the cross-reactivity between these two species observed earlier. A marked heterogeneity among the M. capricolum strains appeared in SDS-PAGE and in immunoblotting, characterized by different protein patterns and different strengths of identical protein bands. All M. capricolum strains investigated revealed, however, at least three strong protein bands with mol. weights of about 30, 42 and 52 KD reacting in immunoblotting with antiserum against M. capricolum CK but not with antiserum against M. mycoides YG. These proteins may represent antigens suitable for the identification and differentiation of M. capricolum strains.