Proteome of the Bacterium Mycoplasma penetrans - American

A proteome map of Mycoplasma penetrans has been constructed using two-dimensional gel ... Mycoplasmas are the smallest and simplest self-replicating...
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Proteome of the Bacterium Mycoplasma penetrans Mario Ferrer-Navarro, Antonio Go´ mez, Oscar Yanes, Raquel Planell, Francesc Xavier Avile´ s, Jaume Pin ˜ ol, Josep Antoni Pe´ rez Pons, and Enrique Querol* Institut de Biotecnologı´a i de Biomedicina and Departament de Bioquı´mica i Biologı´a Molecular, Universitat Auto`noma de Barcelona. 08193, Bellaterra, Barcelona, Spain Received October 6, 2005

A proteome map of Mycoplasma penetrans has been constructed using two-dimensional gel electrophoresis in combination with mass spectrometry (MS). Mycoplasma penetrans infects the urogenital and respiratory tracts of humans. A total of 207 spots were characterized with MS and, in comparing the experimental data with the DNA sequence-derived predictions, it was possible to assign these 207 spots to 153 genes. The Pro-Q Diamond phosphoprotein dye technology was used for the fluorescent detection of 26 phosphoproteins in the 4-7 pH range. Keywords: proteomics • mass spectrometry • two-dimensional gel electrophoresis • phosphoproteome • genome annotation

Introduction Mycoplasmas are the smallest and simplest self-replicating bacteria. Mycoplasmas are bacteria with no cell wall and have the minimum range of genome sizes necessary for selfreplication.1 All known species of Mycoplasmataceae are obligate parasites of mammals and birds, and host specificity is quite strict. Mycoplasmal infections are most frequently associated with disease in the urogenital or repiratory tracts and, in most cases, mycoplasmas infect the host persistently. These parasites display antigenic diversity as a mechanism for evasion of host immune response, and in this way there are a number of studies that have demonstrated that Mycoplasma species can modify their surface antigenic molecules with high frequency.2-5 Mycoplasma penetrans was first isolated from urine samples from human immunodeficiency virus (HIV)-infected patients,6 and also from a patient with a case of primary antiphospholipid syndrome without HIV infection, suggesting that M. penetrans may be pathogenic without HIV.7 The hemolytic and hemoxidative activities were demonstrated in M. penetrans strains isolated from AIDS patients.8 It has been also demonstrated that some lipoproteins from Mycoplasma penetrans enhance HIV replication.9 This Mycoplasma has a morphology characterized by an elongated flask shape allowing the organism to penetrate eukaryotic cells, hence its name. The genome of Mycoplasma penetrans HF-2 has been sequenced by Sasaki et al.2 This genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs). This is the greatest genome of all the Mycoplasma species currently sequenced. Among the 1038 CDSs, 264 predicted proteins are common to four Mycoplasmataceae sequenced thus far and 463 are M. penetrans specific.2 The annotation of the sequenced Mycoplasma genomes assigns a function in 50-70% of the proposed open reading * To whom correspondence should be addressed. Tel: +34 935811429. Fax: +34 935812011. E-mail: [email protected].

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frames (ORFs). The assignment is based on sequence similarity to proteins. The functionally unassigned ORFs are the most interesting ones, since they are potential candidates for new functions. Proteomics can help to gain insight into the identification of proteins expressed in different cellular conditions thus helping in the functional annotation of hypothetical proteins. In addition, it can also help to identify proteins not disclosed by current ORF prediction algorithms. Moreover differential proteomics can help to identify proteins related to pathogenicity and virulence factors. For the identification of all expressed proteins of a cell, a combination of twodimensional gel electrophoresis (2-DE) with mass spectrometry is a widely used method. The combination of these methods allows a complex protein mixture, as a total cellular lysate, to be separated into individual protein spots and to identify proteins by peptide mass fingerprinting. As recently suggested by Roberts and by the American Society for Microbiology, a collaborative research to decipher the role of the “hypothetical proteins” and misannotated genes encoded in the microbial genomes10,11 identifying protein function should be started. The action should start with the best possible bioinformatics analysis to be followed by experimental corroboration of function. It is obvious that proteome analysis should contribute to this effort.

Material and Methods Materials and Equipment. The equipment for IEF and vertical electrophoresis (IPGphor, Ettan IPGphor Cup loading manifold, Ettan Dalt Twelve), ready-made IPG DryStrips, IPG Buffers, Ettan Dalt Gel 12.5, Ettan Dalt buffer kit, 2-D evaluation software (Labscan, Imagemaster 2D 5.0) and Protease Inhibitor Cocktail were from Amersham Biosciences (Uppsala, Sweden). An Umax Astra 4000U scanning device was used to acquire images from silver-stained gels, while a Typhoon scanner from Amersham Biosciences was used to acquire images from specific phosphoprotein-stained gels. SDS, thiourea, CHAPS, 10.1021/pr050340p CCC: $33.50

 2006 American Chemical Society

Mycoplasma penetrans Proteome

iodoacetamide, urea, water HPLC gradient, acetonitrile HPLC gradient and all silver-staining reagents were form SigmaAldrich (St Louis, MO), Pro-Q Diamond was from Invitrogen, Molecular Probes. TBP was from Fluka, ASB-14 from Pierce (Rockford, IL). Modified trypsin (sequencing grade) was from Promega (Madison, WI). ZipPlate C18 (Millipore, Bedford, MA) was used for tryptic digestions. A Bruker-Daltonics Ultraflex MALDI-TOF mass spectrometer (Bremen, Germany) with a 337 nm nitrogen laser was used. Strain and Growth Conditions. M. penetrans GTU-54, kindly provided by J. Baseman (University of Texas Health Science Center at San Antonio, USA), was grown in SP4 medium at 37 °C, 130 rpm for 36 h. The cells were cultured in 250 mL flasks containing 40 mL medium. Frozen cultures were inoculated into the medium. The color range of the phenol red was used to monitor the growth of the culture. Cultures were used when the medium turned orange (pH ≈ 6.5), corresponding to late exponential growth. One flask with 40 mL medium contained approximately 15 mg of protein. Extract Preparation. M. penetrans cells from 40 mL of culture were washed twice in PBS, and immediately suspended in 100 µL of 1% hot SDS and harvested with a little grinder. The sample was then boiled for 3 min. When the sample was at room temperature, 550 µL of lysis solution were added. Lysis solution composition was: 8.0 M urea, 2.0 M thiourea, 2.5% CHAPS, 2% ASB-14, 3 mM TBP, 40 mM Tris-HCl, 2% IPG buffer, and protease inhibitor cocktail (manufacturer’s recommendations were followed). Samples were disrupted by sonication for 5 min at 4 °C in ice water and centrifuged (10 min at 15 800 × g) to discard insoluble particles. The protein sample was immediately used or stored in aliquots at -20 °C until use. Protein concentration was measured with RC DC Protein Assay kit from Bio-Rad. 2-D Gel Electrophoresis. 2-D electrophoresis with immobilized pH gradients was carried out according to Go¨rg et al.12 with some minor modifications. The length of the IPG strips was 24 cm and the pH range was from 4 to 7, 6-9, and 7-11 NL. Equilibration of the focused IPG strips was carried out in a single step using TBP and iodoacetamide for 25 min. The second dimension was performed in a precast polyacrylamide gel (Ettan Dalt Gel 12.5) with a size of 25 × 20 cm. These gels were run according to the manufacturer’s instructions. Sample amounts loaded for silver stained and for specific phosphoprotein staining were the same, approximately 200 µg. Gel Staining and Detection of Proteins. 2-D gels were stained with silver according to manufacturer’s recommendations. Briefly, the gels were fixed in two steps in 40% ethanol and 10% acetic acid for 60 min each time. Sensitizing was carried out for 60 min in 30% ethanol, 5% (w/v) sodium thiosulfate and 6.8% (w/v) sodium acetate. After the gels were washed 5 times, for 15 min each time with water, they were incubated in 2.5% (w/v) silver nitrate for 1h. The gels were then washed in two steps for a minute each with water and then developed with 3% (w/v) Na2CO3 and 0.025% (v/v) formaldehyde in water until the desired contrast was reached. The reaction was stopped with for 45 min in 1.5% (w/v) EDTA-Na2. After stopping, the reaction gels were washed twice with water. For specific phosphoprotein staining, the gels were fixed in two steps in 500 mL fixation solution (50% methanol and 10% acetic acid). The first fixation step was carried out for during 60 min, with second step lasting overnight. The gels were washed three times with 500 mL of distilled H2O, for 15 min every wash, in gentle agitation (50 rpm). Once the gels were

research articles washed, they were incubated with 500 mL of Pro-Q Diamond phosphoprotein stain in the dark for 2 h and destained with 500 mL of destain solution (20% acetonitrile, 50 mM sodium acetate, pH 4), followed by three changes, 30 min per wash, in the dark and with gentle agitation. An image was acquired on a Typhoon scanner (Amersham Biosciences) with 532 nm laser excitation and a 555 nm band-pass emission filter. Image Analysis. The stained gels were immediately scanned using a Umax Astra 4000 U scanning device, and digitalized images were evaluated using ImageMaster 2D 5.0 from Amersham Biosciences. Molecular mass was automatically determined with ImageMaster 5.0 using a standard protein marker (Amersham) covering the range of 6.5-205 kDa during the second dimension. Isoelectric point was also automatically determined by bilinear interpolation between landmark features on each image by ImageMaster 5.0 In-Gel Tryptic Digestion. The silver-stained protein spots were excised from the acrylamide gel, using a cut tip, and immediately destained and digested. Montage In-Gel Digestzp Kit manufacturer’s recommendations were followed to destain and perform the digestion. Peptide elution was performed by centrifugation and the eluted peptides were stored at -20 °C until they were analyzed by mass spectrometry. MALDI-MS Analysis. A microliter of sample was mixed with the same volume of a solution of a-cyano-4-hydroxy-transcinnamic acid matrix (0.3 mgr/ml in ethanol: acetone 6:3), and spotted onto a 600 µm AnchorChip MALDI target plate (Bruker) and allowed to air-dry at room temperature. For phosphopeptides analysis 2,5-dihydroxybenzoic acid (2,5-DHB) matrix was used. Saturated 2,5-DHB matrix solution was prepared by dissolving 2,5-DHB in 50% acetonitrile in water with 1% phosphoric acid as additive. MALDI-mass spectra were recorded in the positive ion mode on an Ultraflex time-of-flight instrument. Ion acceleration was set to 25 kV. All mass spectra were externally calibrated using a standard peptide mixture containing angiotensin II (1046.54180), angiotensin I (1296.68480), substance P (1347.73540), bombesin (1619.82230), renin substrate (1758.93261), adrenocorticotropic hormone 1-17 (2093.08620), adrenocorticotropic hormone 18-39 (2465.19830), and somatostatin 28 (3147.47100). Spectra were also calibrated internally using the autolysis products of trypsin at m/z 842.50 and m/z 2211.10. For PMF analysis, the MASCOT search engine (Matrix Science. London, UK) was used with the following parameters: one missed cleavage permission, 50 ppm measurement tolerance, and at least five matching peptide masses. Positive identifications were accepted with P values higher than 0.05. In the searches, methionine residues modified to methionine sulfoxide were allowed and cysteine residues were allowed to be reduced and alkylated by iodoacetamide to carboxyamidomethyl cysteine wherever necessary. Treatment with Alkaline Phosphatase. Each selected spot was excised from the gel and digested as explained previously. Peptides from tryptic digestions were treated with alkaline phosphatase to eliminate phosphates. A five-hundred nanoliter portion of tryptic digestions was treated with 1 µL of alkaline phosphatase (0.06 u/µL in 100 mM ammonium carbonate, pH ) 8) and incubated for 1 h at 37 °C. Bioinformatics. Several spots in the 2-D gel correspond to proteins previously annotated as “hypothetical” (indicated with an asterisk in Supporting Information Table 1) from the original analysis of the genome sequence of M. penetrans.2 These sequences have been analyzed carefully and their functions Journal of Proteome Research • Vol. 5, No. 3, 2006 689

research articles predicted by combining several programs13-17 using the following web servers: PSI-BLAST (http://www.ncbi.nlm.nih.gov/BLAST); BYPASS (http://bioinf.uab.es/cgi-bin/bypass/bypass.pl?home)1); MODFUN (http://salilab.org/∼marcius/ModFun/); InterPro (http:// www.ebi.ac.uk/interpro); PROTLOC (http://bioinf.uab.es/ cgi-bin/trsdb/protloc.cgi); TRANSMEM (http://bioinf.uab.es/ cgi-bin/trsdb/transmem.cgi); TRANSCOUT (http://bioinf.uab.es/ TranScout/).

Results and Discussion The term “proteome” is defined as the total protein complement encoded by a genome.18 Nowadays there are different approaches in proteomics to study a proteome, but 2-DE is the tool of choice, because it delivers a map of intact proteins, reflecting changes in protein expression level, isoforms or PTM. The main objective of this study is to catalog the expressed gene products of M. penetrans because annotation of a gene within a DNA sequence itself is insufficient to make sure that the gene is expressed. The proteome analyzed belongs to M. penetrans GTU-54, a strain isolated from an acquired immunodefiency syndrome patient,6 while the genome sequence was obtained from M. penetrans HF-2, a strain isolated from tracheal aspirate of a non-HIV-infected patient.7 In a previous work, the protein electrophoresis patterns were compared to identify molecular differences between M. penetrans strains, and a 46 kDa protein that was only present in the HF-2 strain was found.3 All the spots analyzed came from 2-D gels with IPG gradients of pH 4-7, 6-9, and 7-11 NL stained with silver nitrate. To make sure of a great success rate on MALDI-MS identification, silver-stained gels were processed immediately after staining to impede protein modification. The success rate with silverstained spots was between 65% and 80%. About 470 protein spots could be resolved within a pI range of 4-7 and detected by silver staining. Using a pH gradient of 6-9, 108 additional spots were able to be visualized, and with a pH gradient 7-11 NL 84 additional spots can be visualized, most of them being ribosomal proteins. Altogether, proteins from 207 individually separated spots have been successfully determined by MS and assigned to 153 genes from M. penetrans (Supporting Information Table 1). Of the proteins annotated in the map reported here in, 27 proteins were classified in the Swiss-Prot database as hypothetical. We thus demonstrate, in this work, the expression of these proteins whose existence has so far not been demonstrated and we can predict a function for them. The 153 ORFs have been classified into 22 different functional categories (Table 1). For the categories signal transduction, cell motility, intracellular traffiking and coenzyme transport, we cannot detect any protein from those listed by authors of the genome sequence.2 In other Mycoplasma proteomes analyzed there are some proteins identified belonging to these categories.19-21 The expression of the proteins belonging to these categories in M. penetrans is probably very low or it depends on specific growth conditions. Among the most prominent spots are: the most abundant protein in the M. penetrans membrane; the two major antigens, P35 (Spot 4) and P38 (Spot 5), an ABC transporter (Spot 128), heat shock proteins GroEL (Spot 75), DnaK (Spot 78), elongation factor Tu (Spot 86) and two subunits of the pyruvate dehydrogenase (R, Spot 45 and dihydrolipoamide, Spot 46, subunits). The consistently high abundance of these proteins is indicative of a constitutive expression in the absence of any 690

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Ferrer-Navarro et al. Table 1. Functional Classification of Identified Proteinsa,b translation transcription replication, recombination and modification cell cycle control, mitosis and meiosis defense mechanisms signal transduction mechanisms cell wall/membrane biogenesis cell motility extracellular structures intracellular trafficking and secretion posttranslational modification, protein turnover, chaperones energy production and conversion carbohydrate transport and metabolism amino acid transport and metabolism nucleotide transport and metabolism coenzyme transport and metabolism lipid transport and metabolism inorganic ion transport and metabolism secondary metabolites biosynthesis, transport and catabolism general function prediction only function unknown not in COGs

25/110 7/42 11/114 3/30 2/40 0/21 1/21 0/14 1/10 0/48 16/37 12/36 18/76 8/52 10/39 0/16 2/20 1/49 1/8 15/123 2/33 18/264

a Functional classes adapted from COG.32 b The numbers indicate identified in this work/genome annotated proteins from each category.

recognized specific induction. The utility of their continued presence intracellularly has been commented upon elsewhere22 and is in agreement with that reported for the proteome of other microorganisms.19-21,23,24 All of these strongly expressed proteins were found as a “train of spots” (row of single spots, all independently identifiable as the same protein). In addition, P35 was found in three spots with decreasing Mr, probably representing stable, truncated P35 fragments. p35 forms the largest paralog family in this Mollicute, a total of 44 genes for p35 and p35 homologues have been identified. In our proteome analysis, only three members of this family have been detected. Other strongly expressed proteins were the predicted cytoskeleton components. There are four predicted cytoskeletal proteins. We found three of these four cytoskeletal proteins (Spots 13-15) in our proteome map. Of these, the MYPE1570 product (Spot 13), appears in two forms. This protein is similar to cytoskeletal protein HMW2 from Mycoplasma genitalium and seems to be important in the biogenesis of a functional attachment organelle25 that allows the attachment to host cells. One form of this protein appears with the predicted molecular weight, and three isoforms appear with approximately half of this molecular weight (Spots 13b, 13c, and 13d). No peptides were found in these three isoforms from the N-term half of the protein, indicating that this protein undergoes limited proteolysis. Mr and pI of protein spots were experimentally determined and compared with gene-deduced Mr/pI coordinates obtained from MASCOT (Supporting Information Table 1). The majority of gel-estimated and theoretical Mr/pI fit quite well. Lower Mr values could be due to post-translational processing or proteolysis, while higher Mr values could be the result of covalent binding of chemical groups. But there are some cases where a great difference is observed. An unexpected mass increase is observed in cell division protein FtsZ (Spot 19), whose predicted mass is 52 kDa, and it is detected with 87 kDa. ATP synthase subunit β (Spot 69) undergoes a 20 kDa mass increase. Other proteins also undergo an important mass modification. Subtilisin-like serine protease (Spot 81) has a theoretical mass of 127 kDa; 31 kDa being the observed mass. Autotransporter/

Mycoplasma penetrans Proteome

adhesion protein (Spot 8) has a theoretical mass of 126 kDa, while that observed is 36 kDa. The same modification is observed in the chromosomal replication initiator (Spot 36) which loses 35 kDa in its molecular weight. These fragments of these proteins may be stable fragments due to limited proteolysis but we are not able to detect the form with the predicted molecular weight. Twenty-six proteins were found to be present as multiple electrophoretic species. Most of this heterogeneity was mainly due to pI variability, but also variability is observed in Mr or a combination of pI and Mr. In this group of proteins, we do not include the proteins that appear as a “train of spots”, like GroEL or DnaK. In the case of GroEL protein, besides appearing as a “train of spots” we find a 36 kDa form. The presence of small GroEL homologues has been reported in other proteomes.21 Lipoprotein P35, elongation factor Tu, carbamate kinase (Spot 49), RNA polymerase sigma factor (Spot 120) and carboxymuconolactone decarboxylase (Spot 71) appear with more than four isoforms. These different forms probably represent stable fragments or they are the result of different posttranslational modifications, but we cannot explain the existence of these spots from the mass spectrometry analysis. Dihydroxyacetone kinase (Spot 66) is an enzyme involved in glycerol utilization. This enzyme acts as a homodimer and a form with twice the molecular weight is detected, which strongly suggests it is the homodimer. Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how and when protein phosphorylation takes place. The new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly on 2-D gels was used. Using this technology, we have detected 26 different proteins to be phosphorylated (Supporting Information Table 1, proteins indicated with + symbol), which represents 17% of the total proteins identified in our analysis. All of the spots analyzed came from 2-D gels with IPG gradients of pH 4-7. The appearance of a train of spots of the same molecular weight can be indicative of post-translational modifications. The proteins DnaK and EF-Tu were reported to be phosphorylated in E. coli,26 and we also found these two proteins to be phosphorylated and as a train of spots in the proteome of M. penetrans. Recently, it was reported that the binding of lipids to proteins might change the pI, as, for example, is the case of E. coli heat shock protein GroEL.27 In our analysis of phosphorylated proteins we find that GroEL appears as a train of spots, but only two spots of the train appear phosphorylated. Therefore, additional spots could be due to lipid binding. A clear proteolysis event is observed on the product of cytoskeletal protein HMW2 homolog. Only the three proteolyzed isoforms of cytoskeletal protein HMW2 homologue are phosphorylated, while the intact protein remains unphosphorylated. This organism, like mollicutes in general, has a very small number of regulatory proteins. We identified the key regulatory protein of carbon metabolism, the HPr protein (Spot 116). This protein is a component of the phosphoenolpyruvatedependent sugar phosphotransferase system (PTS). Activity of HPr is regulated by phosphorylation. In the absence of glucose, HPr is present, either nonphosphorylated or phosphorylated, at His-15. If glucose becomes available, HPr is phosphorylated

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Figure 1. 2-DE maps of proteins from Mycoplasma penetrans GTU-54. (A) Total protein extract separated on a 24 IPG strip (47) followed by vertical 12.5% polyacrylamide SDS gel (25 × 20 cm) and silver-stained. (B) Total protein extract separated on a 24 IPG strip (7-11 NL) followed by vertical 12.5% polyacrylamide SDS gel (25 × 20 cm) and silver-stained.

in Ser-46, and even a doubly phosphorylated HPr (His-15 and Ser-46).28 We found this protein to be phosphorylated which is in agreement with carbon metabolism regulation in other mycoplasmas. Some proteins detected as phosphorylated belong to energy metabolism, including the three detected subunits of pyruvate dehydrogenase. In an organism with a very low number of regulatory proteins, this suggests that the metabolic pathways of this organism are highly regulated by phosphorylation. To characterize the specific phosphorylation site in the phosphoprotein identified with Pro-Q Diamond phosphoprotein dye technology, de-phosphorylation with alkaline phosphatase combined with differential MALDI peptide mass mapping were carried out with an excised spot. 2,5-DHB with phosphoric acid as additive was used as a MALDI matrix, as it has been shown to enhance the relative abundance of phosphorylated peptides in the presence of nonphosphorylated peptides in MALDI-MS.29,30 Figure 2 shows the obtained spectra for an excised spot identified as a mixture of ABC transporter (MYPE5900) and chaperone DnaK (Spots 128 and 78) before and after treatment with alkaline phosphatase. The predicted heptaphosphorylated peptide from ABC transJournal of Proteome Research • Vol. 5, No. 3, 2006 691

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Figure 2. Detail of mass spectrum of a Pro-Q Diamond stained spot. This spot was identified as a mixture of a putative lipoprotein (MYPE5900) and chaperone dnaK. (A) 2,5-DHB spectrum of the excised spot before alkaline phosphatase treatment. (B) MALDI peptide mass map of the same peptide mixture after alkaline phosphatase treatment. The signal corresponding to the phosphorylated peptide from a putative lipoprotein (MYPE5900) in (A) (3032.913 Da) decrease 559.72 Da after de-phosphorylation and the nonphosphorylated peptide appears (2473.235) (B).

porter, 487(K)NGTTTSGTAGSSGTNGTTTGDLNTTAK(G)513 (m/z 3032.9), disappears from the mass spectrum after treatment with alkaline phosphatase. The MALDI peptide mass map obtained after de-phosphorylation shows a new peak not present in the control spectrum, corresponding to the loss of seven (m/z 2473.2) phosphate groups. Some new peptides corresponding to predicted nonphosphorylated peptides from the ABC transporter appear in the mass spectrum after alkaline phosphatase treatment, suggesting the presence of more phosphopeptides in the sample. The same procedure was carried out to characterize the specific phosphorylation site for Elongation Factor Tu. The phosphorylation of threonine 382 in Elongation Factor Tu was 692

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detected in E. coli and Thermus thermophilus by phosphoamino acid analysis.26 This post-translational modification abolishes its ability to bind aa-tRNA.31 Before de-phosphorylation treatment the predicted phosphorylated peptide from Elongation Factor Tu, 378(R)EGGRTVGAGTVTK(V)392 (m/z 1311.61) was detected. After de-phoshorylation treatment, 1311.61 Da peptide disappear from the spectrum and a 1231.65 Da appear, corresponding to the loss of one phosphate group.

Conclusions In this study, we report the first proteomic analysis of Mycoplasma penetrans, which is expected to provide the basis for more extensive proteomic studies addressing mycoplasmal

Mycoplasma penetrans Proteome

biology. As the number of sequenced genomes increases, the need of functional analysis becomes a priority aim. Of particular interest will be to gain insight into the identification of proteins expressed in different cellular conditions thus helping in the functional annotation of hypothetical proteins, identifying proteins not disclosed by current ORF prediction algorithms, identify proteins related to pathogenicity and virulence factors, etc. Some important findings are as follows: (i) The evidence of expression of many proteins previously annotated as hypothetical. (ii) The high number of energy-metabolizing proteins, as well as proteins involved in translation and transcription, and (iii) The relatively high presence of phosphoproteins suggests that the regulation of all cellular processes is higher than expected in such simple cells.

Acknowledgment. The authors thank J. B. Baseman from The University of Texas Health Science Center at San Antonio, Francesc Canals, from the Unitat de Proteo`mica of the Hospital Vall d′Hebron, for his assistance in phosphoprotein image analysis and finally Silvia Bronsoms is thanked for her valuable advice on MALDI-MS. This research was supported by Grant Nos. BFU2004-06377-C02-01 from the MCYT (Ministerio de Ciencia y Tecnologı´a, Spain) and by the Centre de Refere`ncia de R+D de Biotecnologia de la Generalitat de Catalunya. Supporting Information Available: Supporting Table 1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Pollack, J. D.; Williams, M. V.; McElhaney, R. N. The comparative metabolism of the Mollicutes (Mycoplasmas): The utility for taxonomic classification and the relationship of putative gene annotation and phylogeny to enzymatic function in the smallest free-living cells. Crit. Rev Microbiol. 1997, 23, 269-354. (2) Sasaki, Y.; Ishikawa, J.; Yamashita, A.; Oshima, K.; Kenri, T.; Furuya, K.; Yoshino, C.; Horino, A.; Shiba, T.; Sasaki, T.; Hattori, M. The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans. Nucleic Acids Res. 2002, 30, 5293-5300. (3) Horino, A.; Sasaki, Y.; Sasaki, T.; Kenri, T. Multiple Promoter Inversions Generate Surface Antigenic Variation inMycoplasma penetrans. J. Bacteriol. 2003, 185, 231-242. (4) Rosengarten, R.; Citti, C.; Glew, M.; Lischewsky, A.; Droesse, M.; Much, P.; Winner, F.; Brank, M.; Spergser, J. Host-pathogen interactions in mycoplasma pathogenesis: virulence and survival strategies of minimalist prokaryotes. Int. J. Med. Microbiol. 2000, 290, 15-25. (5) Rosengarten, R.; Wise, K. S. Phenotypic switching in mycoplasmas: phase variation of diverse surface lipoproteins. Science 1990, 247, 315-318. (6) Lo, S. C.; Hayes, M. M.; Tully, J. G.; Wang, R. Y.; Kotani, H.; Pierce, P. F.; Rose, D. L.; Shih, J. W. Mycoplasma penetrans sp. nov., from the urogenital tract of patients with AIDS. Int. J. Syst. Bacteriol. 1992, 42, 357-264. (7) Ya´n ˜ ez, A.; Cedillo, L.; Neyroller, O.; Alonso, E.; Prevost, M. C.; Rojas, J.; Watson, H. L.; Blanchard, A.; Cassell, G. H. Mycoplasma penetrans bacteremia and primary antiphospholipid syndrome. Emerg. Infect. Dis. 1999, 5, 164-167. (8) Kannan, T. R.; Baseman, J. B. Hemolytic and hemoxidative activities in Mycoplasma penetrans. Infect. Immun. 2000, 68, 6419-6422 (9) Shimizu, T.; Kida, Y.; Kuwano, K.; Lipid-associated membrane proteins of Mycoplasma fermentas and M. penetrans activate human immunodeficiency virus long-terminal repeats through Toll-like receptors. Immunology 2004, 113, 121-129. (10) Roberts, R. J. Identifying protein function-a call for community action. PloS Biol. 2004, 2, 293-294. (11) Roberts, R. J.; Karp, P.; Kasif, S.; Linn, S.; Buckley, M. R. An experimental approach to genome annotation. Am. Soc. Microbiol. 2004, Academy Colloquia Reports.

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