1 Institute for Veterinary Bacteriology, University of Berne, Längass-Strasse 122, CH-3012 Berne, Switzerland
2 Wildvet Projects, CH-7605 Stampa, Switzerland
Correspondence:
Joachim Frey
joachim.frey{at}vbi.unibe.ch
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: Fab, antigen-binding fragment; IKC, infectious keratoconjunctivitis
The EMBL/GenBank DNA sequence accession number of the sequenced lppSlppT operon is AJ318939.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mycoplasma cells are only delimited by a single trilaminar membrane consisting of lipids and proteins with a small amount of polysaccharide. As mycoplasmas lack a cell wall and outer membrane, their single membrane must contain the necessary factors for adherence to, and colonization of, host tissues, as well as structures capable of preventing damage by the humoral immune response. Several recent reports show the prevalence of genes capable of encoding antigenically variable surface proteins in several mycoplasma species (Rosengarten & Wise, 1990; Yogev et al., 1991
; Bhugra et al., 1995
; Simmons et al., 1996
; Lysnyansky et al., 1996
; Citti & Rosengarten, 1997
). Mycoplasma surface proteins are often amphiphilic integral membrane proteins, many of which are covalently modified by lipid (Jan et al., 1996
). Among them, adhesin proteins play an important role in pathogenicity (Hu et al., 1982
; Krause, 1998
; Boguslavsky et al., 2000
; Seto et al., 2001
).
Recent studies detected several specific antigens of M. conjunctivae, including proteins of 175, 73, 68, 60 and 33 kDa, which have been exploited to develop specific serological tests to detect M. conjunctivae infection (Degiorgis et al., 2000; Belloy et al., 2001
). To study the antigenic proteins in more detail, we screened a bacteriophage
-based expression gene library of the M. conjunctivae type strain HRC/581T using rabbit serum raised against a whole cell preparation of M. conjunctivae. The clones were then analysed further by DNA sequence analysis and a clone containing a gene for a potential adhesin was selected and characterized in detail.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For gene cloning, Escherichia coli strains XL-1 Blue MRF' and XLOLR (Stratagene) were used. These strains were grown on LuriaBertani broth at 37 °C in an orbital shaker-incubator (Sambrook et al., 1989). Antibiotics (ampicillin, 100 µg ml-1; kanamycin, 50 µg ml-1) were added when needed to enable selection for E. coli carrying recombinant plasmids.
DNA extraction and DNA manipulation.
DNA from M. conjunctivae type strain HRC/581T was extracted by the guanidium thiocyanate method (Pitcher et al., 1989). Ligation, subcloning, plasmid extraction of the DNA fragments, agarose gel electrophoresis (0·7 %) and photography were performed as described previously (Ausubel et al., 1999
). Plasmid extraction from recombinant E. coli clones was done by alkaline lysis using Miniprep kits (Qiagen).
Construction of genomic library, cloning and DNA sequence analysis.
Genomic DNA of M. conjunctivae HRC/581T was partially digested with Sau3AI and fragments from 2 to 10 kb were selected to construct a genomic library, using BamHI-digested -ZAP-express vector arms, which was packaged with the Gigapack-11 packaging system (Stratagene). The library was plated using standard protocols on the E. coli strain XL-1 Blue MRF'. Immunoscreening was carried out by blotting phage plaques onto nitrocellulose membranes and reacting them with rabbit hyperimmune serum directed against whole cell antigens of M. conjunctivae HRC/581T (Belloy et al., 2001
). Positive clones were purified and subjected to in vivo excision using the f1 helper phage in E. coli strain XLOLR. Both ends of the fragments inserted into the excised phagemid vector pBK-CMV were sequenced using an ampli-Taq FS dye terminator kit (Applied Biosystems) and the universal primers complementary to the T3 and T7 promoters flanking the multiple cloning site of pBK-CMV. The complete sequence of the insert of the selected plasmid was obtained by deriving a double-stranded nested-deletion series using exonuclease III (Pharmacia Biotech), from approximately 500 ng plasmid DNA following the manufacturer's protocol. DNA analysis of segments adjacent to the cloned fragment was performed using the Vectorette II System (Genosys Biotechnologies). This system is based on a unidirectional approach and the previously determined DNA sequences from M. conjunctivae allowed the design of specific primers. Briefly, 100 ng genomic DNA from M. conjunctivae was digested with different enzymes (BclI, HaeIII, HindIII, PvuII or RsaI) and ligated to the corresponding Vectorette units as described in the protocol. Thereafter, unidirectional PCR amplification using one primer specific for the Vectorette unit and one primer complementary to the known mycoplasmal sequence (Table 1
) produced a fragment which was then entirely sequenced by primer walking. Sequences were determined with an ABI Prism model 3100 genetic analyser (Applied Biosystems). DNA sequences were assembled and edited with the program Sequencher 3.0 (GeneCode).
|
Colony-blot analysis.
Nitrocellulose membranes (0·45 µm; Millipore) were applied to plates with phage-infected E. coli MRF' strains for 15 min and blocked with 1 % milk buffer (100 mM Tris/HCl, pH 7·5, 150 mM NaCl, 0·5 % Tween 20, 1 %, w/v, skimmed milk powder) for 1 h at room temperature. Then, membranes were incubated in rabbit hyperimmune serum against total antigens from M. conjunctivae HRC/581T diluted 1 : 2000 in 1 % milk buffer, for 90 min at room temperature. The membranes were then washed with 1 % milk buffer and incubated with monoclonal goat anti-rabbit phosphatase-labelled antibodies (Kirkegaard & Perry Laboratories) at a dilution of 1 : 2000 in 1 % milk buffer for 90 min at room temperature with shaking. The bound conjugate was detected by incubation in nitro blue tetrazolium and bromochloroindolyl phosphate (NBT/BCIP) in alkaline substrate buffer (7 mM Na2CO3, 3 mM NaHCO3, pH 9·6, 1 mM MgCl2).
Bioinformatic analysis.
Comparisons of nucleotide sequences and deduced amino acid sequences with the nonredundant GenBank, EMBL, DDBJ, SWISS-PROT and PDB databases were done using the NCBI programs BLASTN, BLASTX and BLASTP (Altschul et al., 1990). Promoter regions were predicted using the promoter predictor program available on the web site http://www.fruitfly.org/seq_tools/promoter.html, selecting prokaryote as the type of organism. For the antigenicity-immunogenicity analysis of amino acid sequences, we used standard methods to locate the most antigenic region based on the hydrophilicity scores and the charged amino acid content (http://www.expasy.ch/cgi-bin/protscale.pl) (Bairoch et al., 1995
). Further investigations of secondary and tertiary protein structures were performed, including coiled-coil analysis (Lupas et al., 1991
) (http://www.ch.embnet.org/software/COILS_form.html), a method for prediction of transmembrane domains (Hofmann & Stoffel, 1993
; Reithmeier, 1995
) to reveal potential exposed domains of peptides, and a program for prediction of signal sequences (Nielsen et al., 1997
) (http://www.ch.embnet.org/software/TMPRED_form.html).
Expression and purification of His-tailed LppS.
Primers 2EN-terml and 2EC-terml (Table 1) containing HindIII and NotI restriction endonuclease cleavage sites, respectively, and genomic DNA of strain HRC/581T were used to amplify the 5'-terminal part of the lppS gene, corresponding to aa 475 to 1400. This region is orthologous to the Mycoplasma hyopneumoniae adhesin-like protein P146 and was predicted to be strongly antigenic. It does not contain TGATrp codons. Plasmid pJFFLppS-His, encoding the polyhistidine-tailed N-terminal part of LppS, was constructed by ligating the amplicon between the HindIII and NotI sites of the T7 promoter-based expression vector pETHIS-1 (Schaller et al., 1999
). The plasmid was purified using the QIAprep Spin Plasmid kit (Qiagen), sequenced using primers complementary to the T7 promoter and to the 3'-terminal region flanking the multi-cloning site of the vector to confirm that it contained the expected insert, and used to transform E. coli BL21(DE3) cells (Novagen) for expression. A positive clone was inoculated into 50 ml LB broth containing 50 µg ampicillin ml-1 and incubated at 37 °C to an OD600 of 0·45. Expression was induced by addition of 1 mM IPTG and incubation continued for another 3 h. The cells were sedimented by centrifugation at 3000 g for 10 min, resuspended in 5 ml PN buffer (50 mM NaH2PO4, pH 8·0, 300 mM NaCl), sonicated with a microtip for 4 min with the power output control at 7 and a duty cycle of 50 % (1 s pulse) in a Branson Sonifier 250 (Branson Ultrasonics), and then centrifuged at 15 000 g for 20 min. The supernatant containing the cytosolic fraction was kept and the pelleted cell debris was resuspended in 5 ml PN buffer. Analysis of the sonicated fraction on SDS/10 % acrylamide gels (Laemmli, 1970
) showed that the induced protein was in the pellet. Guanidine hydrochloride was added to the pelleted cell debris to a final concentration of 6 M and the mixture was loaded onto a prewashed 2·5 ml bed volume Ni/nitrilotriacetic acid/agarose column (Qiagen) and the column washed once with 30 ml PNG buffer (50 mM NaH2PO4, pH 8·0, 300 mM NaCl, 6 M guanidine hydrochloride). Step elution of the protein was performed with 10 ml PNG buffer at pH 7·0, 6·0, 5·5, 5·0 and 4·5, collecting 1 ml fractions. The fractions were dialysed and analysed on SDS/10 % acrylamide gels. The purified fusion protein eluted at pH 5·0 was dialysed overnight against PN buffer and was designated LppS'-His.
Production of monospecific rabbit anti-LppS antibodies, IgG purification and Fab (antigen-binding fragment) preparation.
Monospecific, polyclonal antibodies directed against LppS were obtained by immunizing a rabbit subcutaneously with 50 µg LppS'-His protein in 500 µl PN buffer mixed with 500 µl adjuvant 10 (GERBU Biotechnik) followed by two booster immunizations with the same amount of protein and the same adjuvant 3 and 6 weeks later. The animal was bled 8 days after the second booster immunization. Serum was prepared from the blood sample and stored at -20 °C.
Immunoglobulin (IgG) fractions from serum obtained from the rabbit prior to immunization and from monospecific anti-LppS'-His serum were purified using the HiTrap Protein G HP kit (Amersham Pharmacia Biotech) as indicated by the manufacturer. Briefly, HiTrap Protein G HP was swollen and washed extensively with binding buffer (20 mM sodium phosphate, pH 7·0). After the addition of 4 ml serum and extensive washing of the column with binding buffer, the bound IgG was eluted with 20 ml elution buffer (0·1 M glycine/HCl, pH 2·7). The protein in the eluate was precipitated with 6·4 g ammonium sulfate. After 10 min centrifugation at 12 000 g, the pellet was resuspended in 1 ml dialysis buffer (50 mM NaHCO3, pH 8·0, 125 mM NaCl) and then dialysed at 4 °C overnight against 1000 vols dialysis buffer.
Fab fragments were prepared using the ImmunoPure Fab Preparation kit (Pierce) following the manufacturer's instructions. Briefly, Fab and Fc (crystallizable fragment) fragments were generated from anti-LppS'-His IgG by incubating 1·7 mg purified IgG with immobilized papain at 37 °C for 5 h. The crude digest was then applied to a column of immobilized protein A. Separation of 1 mg Fab fragments from the Fc fragments bound to protein A was achieved by washing the column. The Fab fragments were then dialysed overnight against PBS buffer (140 mM NaCl, 2·7 mM KCl, 15 mM KH2PO4, 8 mM Na2HPO4, pH 7·4). Protein concentrations were determined by the method of Bradford (1976).
Hyperimmune rabbit serum directed against M. conjunctivae HRC/581T and sera from chamois free of IKC or with IKC have been described earlier (Belloy et al., 2001; Degiorgis et al., 2000
).
Extraction of Tween 20 soluble proteins.
Tween 20 solubilization of cells was done as described previously (Belloy et al., 2001). Briefly, M. conjunctivae HRC/581T cells were harvested by centrifugation and then resuspended in TES buffer at 1 mg wet cells ml-1. Subsequently, Tween 20 was added to a final concentration of 1 % (v/v). The suspension was then incubated at 37 °C for 90 min with gentle mixing and cleared by ultracentrifugation at 48 000 g at 4 °C for 60 min. The supernatant was then filtered through a low protein-binding membrane (0·2 µm, PALL Gelman Laboratory). This procedure resulted in a fraction of dissolved mycoplasma membrane proteins and was referred to as the Tween 20 membrane protein fraction.
Culture of lamb joint synovial cells.
Cell cultures (LSM 192) from lamb carpal joint synovial tissue were prepared in six-well tissue culture plates. The tissue was mechanically minced and subsequently incubated at 37 °C in tissue culture medium [MEM (Biochrom) supplemented with 10 % foetal calf serum, 2·5 mM L-glutamine, 100 U penicillin ml-1 and 100 µg streptomycin ml-1] in a CO2 incubator. The medium was changed every 3 days. After reaching confluence, the cells were trypsinized and transferred into 75 cm2 tissue culture flasks. The cells were passaged at weekly intervals and transferred into 24-well plates. Synovial cells were used in adherence assays when they had reached confluence, with 105 cells per 2 cm2 well. Non-specific binding was blocked by incubation of the cells with a solution of 0·1 % BSA for 15 min at 37 °C prior to addition of the mycoplasma suspensions for adherence assays.
Adherence and inhibition assays.
For adherence assays, M. conjunctivae type strain HRC/581T was grown in 200 ml standard mycoplasma culture medium containing 20 µCi (740 kBq)[U-14C] palmitic acid for 3 days at 37 °C with 5 % CO2. The mycoplasmas were washed three times in buffer A (50 mM Tris/HCl, pH 7·2, 100 mM NaCl and 1 mM CaCl2) to remove unincorporated [14C]palmitic acid. Cultures were frozen at -80 °C in small aliquots until used. M. conjunctivae were used immediately after thawing and were not frozen again. In each case, three parallel trials were carried out.
To determine the optimal concentration of mycoplasmas for the adhesion assay, dilutions (1 : 2 to 1 : 32) of 1010 c.f.u. ml-1 culture were prepared in buffer A (Sachse, 1998). Two hundred microlitres of each dilution was transferred onto each LSM 192 monolayer and incubated for 2 h at 37 °C. After removing excess liquid, the LSM 192 cells were washed three times with 500 µl buffer A to remove non-adherent mycoplasmas and then solubilized by incubation with 100 µl 1 % (w/v) SDS and 500 µl buffer A for 2 h at 37 °C with shaking. The lysed suspension of LSM 192 cells from each well was then transferred into a vial containing 3 ml Emulsifier Scintillator Plus (Packard Instrument Company) and decays per minute were counted using a scintillation spectrometer (Wallac 1410 Liquid Scintillation Counter, Perkin Elmer). Relative adherence (percentage of mycoplasmas attached to LSM 192 cells) was expressed as the ratio of decays min-1 in the lysed samples to the decays min-1 measured in 200 µl of the original mycoplasma suspension at the corresponding dilution. A pilot experiment with freshly grown and labelled mycoplasma cells showed that adherence was not affected by freezing and thawing.
For inhibition experiments a 1 : 5000 dilution of purified IgG from rabbit monospecific anti-LppS serum at a concentration of 3·4 mg ml-1, a 1 : 500 dilution of serum from an uninoculated rabbit at a concentration of 0·38 mg ml-1, as well as dilutions of 1 : 10, 1 : 40, 1 : 100 and 1 : 200 of the Fab fraction from anti-LppS IgG at a concentration of approximatively 0·1 mg ml-1 and from the serum IgG from the rabbit prior to immunization were prepared in buffer A. Each dilution was incubated with the appropriate concentration of 14C-labelled M. conjunctivae HRC/581T in a final volume of 200 µl for 2 h at room temperature with shaking. The mycoplasma/antibody mix was then transferred into wells containing LSM 192 cells and incubated for an additional 2 h at 37 °C. Cells were then treated as described above.
To measure the adhesion capacity of purified recombinant LppS protein, approximately 25 µg of the LppS'-His protein was incubated with LSM 192 cells for 2 h. After three washes, the cells were lysed using a solution of 60 µl 1 % (w/v) SDS and 240 µl buffer A. The resulting lysate containing bound LppS'-His and disrupted LSM 192 cells was concentrated to 100 µl and mixed with 2x times; SDS-PAGE sample buffer, boiled for 5 min, proteins separated in 10 % polyacrylamide gels using SDS-PAGE and blotted onto nitrocellulose membrane (0·2 µm pore size, Bio-Rad). The membranes were then incubated with the rabbit monospecific anti-LppS'-His serum. The specificity of LppS'-His binding to LSM 192 cells was assessed by inhibition of binding of LppS'-His by different dilutions of purified IgG directed against LppS. Purified IgG from the serum of the rabbit prior to immunization and anti-His antibody (Amersham Pharmacia Biotech) were used as controls.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The entire 3·5 kb insert in plasmid pJFF2E was sequenced in both directions and the DNA primers p2ET71 and p2ET31 derived from its sequence (Table 1) were used in a PCR with genomic DNA of M. conjunctivae to confirm the integrity of the clone. The clone pJFF2E was found to contain ORFs encoding the carboxy-terminal part of a putative Ser-rich adhesin-like protein and the amino-terminal part of a putative lipoprotein. Both ORFs were in the same orientation (Fig. 1
). Based on this sequence, primers were designed to complete the DNA sequence of both ORFs. These primers were used with the Vectorette System. Five different, complementary Vectorette libraries were made. Only PCR products between approximately 400 and 3000 bp were amplified and sequenced. Based on each newly sequenced fragment, new primers were designed and novel Vectorette-based amplifications were performed until a DNA segment of 7693 bp, including the complete ORF for each of the two genes was sequenced. This segment contained two genes encoding predicted lipoproteins. The protein of 1405 aa (152 kDa) was named LppS and the protein of 947 aa (105 kDa) was named LppT. Upstream of the ORF encoding LppS, two putative -35 and two putative -10 boxes were found (Fig. 1
). In addition, putative ribosome-binding sites were found upstream of the ATG initiation codon of the two ORFs. The two ORFs with their promoter region were flanked by sequences predicted to form mRNA structures very similar to rho-independent transcriptional stop signals (Fig. 1
). The calculated free energy was -3·5 kcal mol-1 for the hairpin preceding the promoter region and -13·5 kcal mol-1 for the hairpin downstream of the two complete ORFs. Southern blot analysis of HindIII-digested genomic DNA of M. conjunctivae HRC/581T using the lppS gene probe detected a single copy of the lppS gene (Fig. 2
).
|
|
Immunoblots containing M. conjunctivae HRC/581T membrane protein antigens prepared by extraction with the neutral detergent Tween 20 were reacted with monospecific polyclonal antibodies directed against the recombinant LppS'-His peptide. The antibodies bound to two bands of 150 kDa in the detergent-associated fraction, confirming that LppS was a membrane protein (Fig. 3). The doublet band may be due to the presence of both unprocessed pre-LppS and mature LppS in the fraction. Rabbit anti-M. conjunctivae hyperimmune antiserum and serum from a chamois that had been infected with M. conjunctivae also detected the distinct doublet of 150 kDa, but at a weaker intensity (Fig. 3a
). Sera from sheep with IKC reacted strongly with purified LppS'-His in Western blots, while sera from IKC-free sheep did not react (Fig. 3b
).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adhesion of recombinant LppS'-His to lamb synovial membrane cells and inhibition of adhesion by monospecific, polyclonal antibodies directed against LppS showed that LppS could adhere specifically to ovine cells. This adhesion was not influenced by the poly-His tail on the recombinant protein. The lamb synovial membrane cells were chosen as sheep are the natural host of M. conjunctivae. Cells from passage 415 were used, as higher passages of these cells were known to show a reduced capacity to bind other mycoplasma species that are pathogenic in Caprinae. Adhesion experiments with radioactively labelled M. conjunctivae and inhibition experiments using Fab fragments from IgG directed against LppS revealed that LppS was involved in adhesion of M. conjunctivae to sheep cells. The inhibition of adhesion by Fab fragments of anti-LppS IgGs was up to 80 % at a dilution of 1 : 10, and diminished with increasing serum dilutions. Fab fragments derived from the serum of the rabbit prior to immunization were only able to inhibit 6·7 % of the adherence. Hence, we consider LppS to be an adhesion protein of M. conjunctivae.
Serological analysis revealed that chamois and sheep that had suffered from IKC had immunological reactions to LppS, while animals from areas without IKC did not have anti-LppS antibodies. The second gene, lppT, in the same operon, encoded a protein of 947 aa with a calculated molecular mass 105 kDa. This gene product was not investigated further in this study. However, sequence data indicate that the gene product encoded by lppT is also a membrane protein with a signal sequence of 34 aa at the amino-terminal end, followed by two transmembrane structures. It shows significant similarity to the membrane proteins P76 and P110 of M. hyopneumoniae (GenBank accession numbers AAF87782 and AAF87780). The lppT gene lacked a promoter and is likely to be co-transcribed with lppS, thus suggesting a functional relationship between LppS and LppT.
In summary, we developed a binding assay and showed that the protein encoded by the lppS gene was strongly implicated in the adhesion of M. conjunctivae to its host. Hence, LppS is likely to play an important role in mycoplasmahost cell interactions and can be considered a virulence factor of M. conjunctivae.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1999). Current Protocols in Molecular Biology, vol. 4. New York: Wiley
Bairoch, A., Bucher, P. & Hofmann, K. (1995). The PROSITE database, its status in 1995. Nucleic Acids Res 24, 189196.[CrossRef]
Belloy, L., Giacometti, M., Abdo, E.-M., Nicolet, J., Krawinkler, M., Janovsky, M., Bruderer, U. & Frey, J. (2001). Detection of specific Mycoplasma conjunctivae antibodies in the sera of sheep with infectious keratoconjunctivitis. Vet Res 32, 155164.[Medline]
Bhugra, B., Voelker, L. L., Zou, N., Yu, H. & Dybvig, K. (1995). Mechanism of antigenic variation in Mycoplasma pulmonis: interwoven, site-specific DNA inversions. Mol Microbiol 18, 703714.[Medline]
Boguslavsky, S., Menaker, D., Lysnyansky, I., Liu, T., Levisohn, S., Rosengarten, R., Garcia, M. & Yogev, D. (2000). Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein. Infect Immun 68, 39563964.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Citti, C. & Rosengarten, R. (1997). Mycoplasma genetic variation and its implication for pathogenesis. Wien Klin Wochenschr 109, 562568.[Medline]
Degiorgis, M. P., Abdo, E.-M., Nicolet, J., Frey, J., Mayer, D. & Giacometti, M. (2000). Immune responses to Mycoplasma conjunctivae in Alpine ibex, Alpine chamois, and domestic sheep in Switzerland. J Wildl Dis 36, 265271.
Giacometti, M., Nicolet, J., Frey, J., Krawinkler, M., Meier, W., Welle, M., Johansson, K. E. & Degiorgis, M. P. (1998). Susceptibility of Alpine ibex to conjunctivitis caused by inoculation of a sheep-strain of Mycoplasma conjunctivae. Vet Microbiol 61, 279288.[CrossRef][Medline]
Giacometti, M., Nicolet, J., Johansson, K. E., Naglic, T., Degiorgis, M. P. & Frey, J. (1999). Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene. J Vet Med B Infect Dis Vet Public Health 46, 173180.
Giacometti, M., Janovsky, M., Jenny, H., Nicolet, J., Belloy, L., Goldschmidt-Clermont, E. & Frey, J. (2002). Mycoplasma conjunctivae infection is not maintained in alpine chamois in eastern Switzerland. J Wildl Dis 38, 297304.
Hofmann, K. & Stoffel, W. (1993). TMbase - A database of membrane spanning proteins segments. Biol Chem Hoppe-Seyler 347, 166.
Hsu, T., Artiushin, S. & Minion, F. C. (1997). Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae. J Bacteriol 179, 13171323.[Abstract]
Hu, P. C., Cole, R. M., Huang, Y. S., Graham, J. A., Gardner, D. E., Collier, A. M., & Clyde, W. A., Jr (1982). Mycoplasma pneumoniae infection: role of a surface protein in the attachment organelle. Science 216, 313315.[Medline]
Jan, G., Fontenelle, C., Verrier, F., LeHenaff, M. & Wroblewski, H. (1996). Selective acylation of plasma membrane proteins of Mycoplasma mycoides subsp. mycoides SC, the contagious bovine pleuropneumonia agent. Curr Microbiol 32, 3842.[CrossRef][Medline]
Janovsky, M., Frey, J., Nicolet, J., Belloy, L., Goldschmidt-Clermont, E. & Giacometti, M. (2001). Mycoplasma conjunctivae infection is self-maintained in the Swiss domestic sheep population. Vet Microbiol 83, 1122.[CrossRef][Medline]
King, K. W., Faulds, D. H., Rosey, E. L., & Yancey, R. J., Jr (1997). Characterization of the gene encoding Mhp1 from Mycoplasma hyopneumoniae and examination of Mhp1's vaccine potential. Vaccine 15, 2535.[CrossRef][Medline]
Krause, D. C. (1998). Mycoplasma pneumoniae cytadherence: organization and assembly of the attachment organelle. Trends Microbiol 6, 1518.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lupas, A., Van, D. M. & Stock, J. (1991). Predicting coiled coils from protein sequences. Science 252, 11621164.[Medline]
Lysnyansky, I., Rosengarten, R. & Yogev, D. (1996). Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements. J Bacteriol 178, 53955401.[Abstract]
Mayer, D., Degiorgis, M. P., Meier, W., Nicolet, J. & Giacometti, M. (1997). Lesions associated with infectious keratoconjunctivitis in Alpine ibex. J Wildl Dis 33, 413419.[Abstract]
McDevitt, D., Francois, P., Vaudaux, P. & Foster, T. J. (1994). Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol Microbiol 11, 237248.[Medline]
Nielsen, H., Engelbrecht, J., Brunak, S. & von-Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 16.[Abstract]
Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8, 151156.
Reithmeier, R. A. (1995). Characterization and modeling of membrane proteins using sequence analysis. Curr Opin Struct Biol 5, 491500.[CrossRef][Medline]
Rosengarten, R. & Wise, K. S. (1990). Phenotypic switching in mycoplasmas: phase variation of diverse surface lipoproteins. Science 247, 315318.[Medline]
Sachse, K. (1998). Detection and analysis of mycoplasma adhesins. Methods Mol Biol 104, 299307.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schaller, A., Kuhn, R., Kuhnert, P., Nicolet, J., Anderson, T. J., MacInnes, J. I., Segers, R. P. A. M. & Frey, J. (1999). Characterization of apxIVA, a new RTX determinant of Actinobacillus pleuropneumoniae. Microbiology 145, 21052116.[Abstract]
Seto, S., Layh-Schmitt, G., Kenri, T. & Miyata, M. (2001). Visualization of the attachment organelle and cytadherence proteins of Mycoplasma pneumoniae by immunofluorescence microscopy. J Bacteriol 183, 16211630.
Simmons, W. L., Zuhua, C., Glass, J. I., Simecka, J. W., Cassell, G. H. & Watson, H. L. (1996). Sequence analysis of the chromosomal region around and within the V-1-encoding gene of Mycoplasma pulmonis: evidence for DNA inversion as a mechanism for V-1 variation. Infect Immun 64, 472479.[Abstract]
Wilton, J. L., Scarman, A. L., Walker, M. J. & Djordjevic, S. P. (1998). Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae. Microbiology 144, 19311943.[Abstract]
Yogev, D., Rosengarten, R., Watson-McKown, R. & Wise, K. S. (1991). Molecular basis of Mycoplasma surface antigenic variation: a novel set of divergent genes undergo spontaneous mutation of periodic coding regions and 5' regulatory sequences. EMBO J 10, 40694079.[Abstract]
Received 8 July 2002;
revised 15 August 2002;
accepted 13 September 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |