Article Figures & Data
Figures
- FIG 1
Coomassie-stained two-dimensional gels of L. biflexa proteins from exponentially growing cells. Soluble proteins (A) and membrane-associated proteins (B) were separated by two-dimensional PAGE and stained with Coomassie dye. The pH range for all gels was 3 (left) to 10 (right). Proteins analyzed by LC-MS/MS are indicated with numbered arrows, and identifications are provided in Tables 2 and 3. Ovals encompass the gene products selected for transcript analysis and normalized to FlaB1 (spot 113); results are shown in Fig. 2. Molecular weight standards are indicated to the left of each gel, in thousands.
- FIG 2
Relative transcript levels of selected genes. RNA was isolated from triplicate cultures of L. biflexa grown to mid-exponential phase (∼5 × 108 cells/ml). Transcript levels of 5 different genes were determined by qRT-PCR analysis and standardized to flaB1. Transcript levels for proteins present in the soluble fraction are shaded and on the left, to differentiate them from those enriched in the membrane-associated fraction (right). Numbers below the gene names refer to the protein spot numbers from Fig. 1 and Tables 2 and 3.
- FIG 3
Differential in-gel electrophoresis comparison of proteins isolated from exponential growth and stationary phase. Differentially labeled proteins from exponential and stationary phases were separated by 2-dimensional PAGE, and the merged image is shown. Proteins synthesized in equivalent amounts from each growth phase appear yellow, those with higher levels in exponential phase appear green, and those with higher levels in stationary phase appear red. Soluble proteins (A) and membrane-associated proteins (B) display posttranslational modifications. Identifications of proteins from panel B are provided in Table 4. Molecular weight standards are indicated to the left of each gel, in thousands.
- FIG 4
Detection of posttranslational modifications of L. biflexa proteins. Immunoblots of protein lysates detected trimethylated lysine residues (A) and acetylated lysine residues (B), mainly in the membrane-associated fractions, are shown. Protein concentrations were determined by Lowry assays, and equivalent quantities (20 μg) were loaded per lane. (C) Phosphorylated proteins were primarily detected among the soluble proteins using the Pro-Q Diamond phosphorylation stain (left). The same gel was subsequently stained for total proteins using the Sypro Ruby stain (middle), and a merged image is shown on the far right. Molecular weight standards are indicated to the side of each gel, in thousands. SP, soluble protein; MP, membrane-associated protein; Exp, exponential phase; Stat, stationary phase.
- FIG 5
The theoretical proteomes of saprophytic and pathogenic leptospires are similar. All the annotated proteins for each organism are plotted by isoelectric point (pI) on the x axis and by molecular weight on the y axis. The yellow-filled circles in the L. biflexa theoretical proteome indicate proteins identified in this study. The dashed vertical line indicates the median isoelectric point. The genomes of the three Leptospira species were reanalyzed by Bulach et al. to apply consistency to the annotations (32).
Tables
- TABLE 1
Oligonucleotide primers and probes used in this study
a Oligonucleotide probes were labeled at the 5′-end with FAM (6-carboxyfluorescein) and at the 3′-end with TAMRA (6-carboxytetramethylrhodamine).
b Originally described by Stewart et al. (30).
- TABLE 2
Soluble proteins identified from L. biflexa during exponential phasea
Spot Description (Uniprot accession no.) % coverage No. of unique peptides Mass (kDa)b pIb 1* Acyl carrier protein (B0SDK7) 65 14 8.50 3.99 2 50S ribosomal protein L7/L12 (B0SAG2) 91 118 12.65 5.20 3* Thioredoxin (B0SBX2) 83 74 11.41 5.04 5* Nucleoside diphosphate kinase (B0S9J3) 84 57 15.28 6.90 6* Acyl dehydratase MaoC family (B0SGL8) 94 113 14.95 6.83 7* Uncharacterized protein (B0SGL7) 94 70 16.95 5.75 8 Peptidyl-prolyl cis-trans isomerase (B0SGI0) 68 60 18.13 4.91 9 ATP-dependent Clp protease proteolytic subunit (B0S8T0) 61 32 22.42 4.92 10* Adenylate kinase (B0SA26) 88 107 20.49 5.52 12 Superoxide dismutase (B0S932) 58 10 21.29 6.22 13 Elongation factor Ts (B0SDN6) 77 83 21.83 6.23 14 Hydroxyethylthiazole kinase (B0SE82) 73 126 27.61 6.83 15 Short-chain dehydrogenase (B0SDL1) 82 128 26.26 7.69 16 Short-chain dehydrogenase (B0S953) 93 122 27.01 8.25 17* Electron transfer flavoprotein alpha subunit (B0SF29) 88 208 32.72 8.63 19* Spermidine synthase (B0SI93) 86 94 31.82 5.87 20* Enoyl-CoA hydratase/isomerase family protein (B0SDX5) 80 111 27.35 5.53 21 Electron transfer flavoprotein beta subunit (B0SF30) 82 211 27.32 5.34 22* Elongation factor P (B0SII2) 78 69 21.30 4.93 23 Uncharacterized protein (B0SE41) 88 271 39.19 4.95 24 DNA-directed RNA polymerase subunit alpha, RpoA (B0SA20) 88 163 36.60 4.86 25 Acyl-CoA dehydrogenase (B0S960) 77 161 42.32 5.75 26* Ketol-acid reductoisomerase (B0SCQ5) 77 107 36.16 6.03 27 Citrate lyase beta subunit (B0SCW2) 45 16 37.07 6.10 28 Malate dehydrogenase (B0SF41) 51 15 34.89 6.25 29* Succinyl-CoA ligase [ADP-forming] subunit alpha (B0SAP1) 26 6 30.54 6.66 Uncharacterized protein (B0SHJ5) 58 15 31.50 8.26 30 Aldolase (B0SGS2) 61 18 38.40 7.71 31 Aspartate-semialdehyde dehydrogenase (B0SIQ0) 44 15 37.80 7.09 Glyceraldehyde 3-phosphate dehydrogenase (B0SB20) 52 14 36.50 6.55 32 Glyceraldehyde 3-phosphate dehydrogenase (B0SB20) 90 37 36.50 6.55 34 Acyl dehydratase (B0SD87) 84 30 44.00 6.77 35* Acyl-CoA dehydrogenase (B0SF91) 41 11 42.52 6.43 Glyceraldehyde 3-phosphate dehydrogenase (B0SB20) 47 9 36.44 6.55 36 S-Malonyltransferase [acyl carrier protein] (B0SA97) 74 23 43.11 5.35 37 ATP synthase subunit beta (B0SDA5) 63 22 51.07 5.28 38 Acetyl-CoA acetyltransferase (B0SCQ4) 74 25 47.26 5.66 39 ATP synthase subunit alpha (B0SDA3) 67 34 55.00 6.05 40 Uncharacterized protein (B0SCE1) 57 51 63.20 7.97 41 Aconitate hydratase (B0SIM5) 55 35 81.10 6.25 42 Polyribonucleotide nucleotidyltransferase (B0SH22) 59 41 75.90 5.85 43 Phosphoglycerate mutase (B0SBJ6) 49 20 61.40 5.66 44 60-kDa chaperonin GroEL (B0SCC0) 77 48 58.10 5.35 45 Trigger factor (B0SME8)c 80 36 50.70 5.19 46 Chaperone protein DnaK (B0SHT1) 52 34 68.99 5.04 47 Elongation factor G (B0SF49) 63 36 78.80 5.39 48* Methionine synthase (B0SIG4) 31 33 137.58 5.40 49 Uncharacterized protein (B0SH48) 46 8 19.41 8.95 51 Phosphoribosylformylglycinamidine cyclo-ligase (B0SDW2) 64 18 36.71 5.38 52* Thioredoxin reductase (B0SCD5) 38 8 33.66 6.19 53 S-Adenosylmethionine synthase (B0SFH3) 55 23 42.20 6.13 54 3-Hydroxyacyl-CoA dehydrogenase (B0SC49) 61 25 47.63 8.47 55* Acetyl-CoA carboxylase, carboxyltransferase component subunits alpha and beta (B0SHH5) 35 17 59.86 6.14 ↵a Proteins were identified by LC-MS/MS on two independently isolated samples except for those marked with an asterisk, which indicates only a single LC-MS/MS identification. Numbers provided are from a single, representative run. CoA, coenzyme A.
↵b Calculated.
↵c Identified only in the L. biflexa Paris strain; no hit was obtained against the Ames strain.
- TABLE 3
Membrane-associated proteins identified during exponential phasea
Spot Description (Uniprot accession no.) % coverage No. of unique peptides Mass (kDa)b pIb 100 50S ribosomal protein L7/L12 (B0SAG2) 96 92 12.65 5.20 101* 30S ribosomal protein S6 (B0SB40) 91 27 10.43 6.29 103 OmpA-family lipoprotein (B0SGK2) 85 114 20.61 6.62 104* LipL21 lipoprotein (B0S915) 51 42 20.41 7.98 OmpA-family lipoprotein (B0SGK2) 71 22 20.61 6.62 105 ATP synthase subunit delta (B0SDA2) 89 58 20.89 6.18 106 Electron transfer flavoprotein beta subunit (B0SF30) 91 136 27.32 5.34 107 Lipoprotein LipL45 (B0SGV4) 61 116 42.92 5.79 Electron transfer flavoprotein beta subunit (B0SF30) 80 29 27.32 5.34 108 Electron transfer flavoprotein beta subunit (B0SF30) 90 98 27.32 5.34 109 Peroxiredoxin (B0SID1) 85 67 24.57 5.97 110 Uncharacterized protein (B0SG33) 73 49 27.05 7.82 111 OmpL1 (B0SDB5) 73 92 31.64 9.02 112 Hypothetical lipoprotein (B0SGX1) 80 97 33.99 8.83 113 Endoflagellar filament core protein, FlaB1 (B0SHI7) 83 112 31.38 7.03 114 Flagellar filament 35-kDa core protein, FlaB2 (B0SSZ5) 86 64 31.23 6.55 115 Endoflagellar filament core protein, FlaB-2 (B0SB70) 68 69 31.39 9.22 116 Uncharacterized protein (B0STJ8) (Paris)c 74 134 35.82 7.71 117 Uncharacterized protein (B0SHJ5) 68 54 31.52 8.26 118* Uncharacterized protein (B0SHJ5) 67 55 31.52 8.26 119* DNA-directed RNA polymerase subunit alpha, RpoA (B0SA20) 66 54 36.60 4.86 120 Elongation factor Tu (B0SAF6) 50 35 43.93 5.73 121 Elongation factor Tu (B0SAF6) 83 146 43.93 5.73 122 Periplasmic protein of an ABC transporter complex (B0SE25) 74 85 43.09 6.35 123 Bifunctional dihydrolipoyllysine-residue acetyltransferase/dihydrolipoyllysine-residue succinyltransferase (B0SEK8) 56 50 43.73 6.29 124 Conserved hypothetical lipoprotein (B0SB57) 63 40 44.62 8.52 Endoflagellar motor protein (B0S9G1) 61 56 41.94 6.67 GDP-mannose 4,6-dehydratase (B0S9S0) 66 43 38.63 6.42 125* Conserved hypothetical lipoprotein (B0SB57) 73 57 44.62 8.52 Heavy metal efflux pump (B0SHU0) 63 57 42.28 7.11 NAD(P)+ transhydrogenase (AB-specific) alpha subunit (B0SH36) 71 40 40.45 6.78 126 Uncharacterized protein (B0SC05) 69 68 43.00 8.79 127 ATP synthase subunit beta (B0SDA5) 89 142 51.12 5.28 128 Acetyl-CoA acetyltransferase (B0SCQ4) 75 75 47.31 5.66 129 LipL71 lipoprotein (B0SCA6) 69 75 51.11 5.42 130 60-kDa chaperonin GroEL (B0SCC0) 58 76 58.11 5.35 131 Hypothetical lipoprotein (B0S8T9) 66 69 57.39 6.29 132 ATP synthase subunit alpha (B0SDA3) 64 56 55.08 6.05 133 Chaperone protein DnaK (B0SHT1) 61 91 69.05 5.04 134 Fe-S-cluster-containing hydrogenase (B0SGB2) 42 78 113.43 8.48 135* Lipoprotein LipL45 (B0SGV4) 27 39 42.92 5.79 136* Conserved hypothetical lipoprotein (B0SCF9) 65 61 22.43 7.79 OmpA-family lipoprotein (B0SGK2) 66 13 20.61 6.62 137 Hypothetical lipoprotein (B0SGB7) 75 37 19.32 8.98 138* 50S ribosomal protein L9 (B0SB37) 89 56 16.15 9.75 139* Type II secretory pathway component protein G (B0S8Q5) 65 45 17.29 5.39 140 Acetyl-CoA acetyltransferase (B0SC48) 59 36 46.06 6.69 141 Acetyl-CoA carboxylase carboxyltransferase component subunits alpha and beta (B0SHH5) 69 89 59.92 6.14 142 Actin-like ATPase involved in cell morphogenesis (B0SBE9) 72 84 36.77 5.30 143* TolC-like protein (B0SAP2) 65 43 58.43 8.24 144* OMA87 related protein (B0SC13) 80 45 56.87 9.07 145* Hypothetical lipoprotein (B0SCN0) 82 49 13.91 9.05 146* Uncharacterized protein (B0SGS8) 89 18 14.16 6.34 147* Succinate dehydrogenase/fumarate reductase subunit B (B0S8S3) 91 30 26.89 6.31 ↵a Proteins were identified by LC-MS/MS runs on two or three independently isolated samples except for those marked with an asterisk, which indicates only a single LC-MS/MS identification. Numbers provided are from a single representative run.
↵b Calculated.
↵c Protein with the highest match was identified in the L. biflexa Paris strain.
- TABLE 4
Identification of membrane-associated proteins of L. biflexa with different isoforms as determined by DIGE analysis (Fig. 3B)
Spot Description (Uniprot accession no.) % coverage No. of unique peptides Mass (kDa)c pIc Corresponding spot in Fig. 1a 1a Chaperone protein DnaK (B0SHT1) 75 74 69.05 5.20 MP 133 1b Chaperone protein DnaK (B0SHT1) 76 83 69.05 5.20 MP 133 2 Glutamine-fructose-6-phosphate aminotransferase (B0SBL5) 15 8 67.23 5.75 3 Glutamine-fructose-6-phosphate aminotransferase (B0SBL5) 38 16 67.23 5.75 4 Glutamine-fructose-6-phosphate aminotransferase (B0SBL5) 23 10 67.23 5.75 5a 60-kDa chaperonin GroEL (B0SCC0) 83 72 58.11 5.35 MP 130 5b 60-kDa chaperonin GroEL (B0SCC0) 79 66 58.11 5.35 MP 130 5c 60-kDa chaperonin GroEL (B0SCC0) 81 78 58.11 5.35 MP 130 6a LipL71 lipoprotein (B0SCA6) 67 50 51.11 5.42 MP 129 6b LipL71 lipoprotein (B0SCA6) 67 68 51.11 5.42 MP 129 7a Hypothetical lipoprotein (B0S8T9) 80 66 57.39 6.29 MP 116 7b Hypothetical lipoprotein (B0S8T9) 81 62 57.39 6.29 MP 116 7c Hypothetical lipoprotein (B0S8T9) 89 105 57.39 6.29 MP 116 8a ATP synthase subunit alpha (B0SDA3) 66 40 55.08 6.05 MP 132 8b ATP synthase subunit alpha (B0SDA3) 69 51 55.08 6.05 MP 132 8c ATP synthase subunit alpha (B0SDA3) 76 68 55.08 6.05 MP 132 10a ATP synthase subunit beta (B0SDA5) 66 34 51.12 5.28 MP 127 10b ATP synthase subunit beta (B0SDA5) 89 54 51.12 5.28 MP 127 10c ATP synthase subunit beta (B0SDA5) 94 74 51.12 5.28 MP 127 11a Acetyl-CoA acetyltransferase (B0SCQ4) 83 57 47.31 5.66 MP 128 11b Acetyl-CoA acetyltransferase (B0SCQ4) 83 77 47.31 5.66 MP 128 11c Acetyl-CoA acetyltransferase (B0SCQ4) 90 95 47.31 5.66 MP 128 12a Conserved hypothetical lipoprotein (B0SI06) 71 41 49.76 8.75 12b Conserved hypothetical lipoprotein (B0SI06) 66 38 49.76 8.75 12c Conserved hypothetical lipoprotein (B0SI06) 67 45 49.76 8.75 12d Conserved hypothetical lipoprotein (B0SI06) 80 51 49.76 8.75 14a Elongation factor Tu (B0SAF6) 87 74 43.93 5.73 MP 120 14b Elongation factor Tu (B0SAF6) 90 97 43.93 5.73 MP 120 14c Elongation factor Tu (B0SAF6) 88 134 43.93 5.73 MP 120 15a Acyl-CoA dehydrogenase (B0SF91) 45 14 42.56 6.43 SP 35 15b Acyl-CoA dehydrogenase (B0SF91) 69 35 42.56 6.43 SP 35 16a Uncharacterized protein (B0SC05) 65 37 43.00 8.79 MP 126 16b Uncharacterized protein (B0SC05) 77 45 43.00 8.79 MP 126 16Y Uncharacterized protein (B0SC05) 76 52 43.00 8.79 MP 126 18a Periplasmic protein of an ABC transporter complex (B0SE25) 77 25 43.09 6.35 MP 122 18b Periplasmic protein of an ABC transporter complex (B0SE25) 87 40 43.09 6.35 MP 122 18c Periplasmic protein of an ABC transporter complex (B0SE25) 89 59 43.09 6.35 MP 122 18d Periplasmic protein of an ABC transporter complex (B0SE25) 88 79 43.09 6.35 MP 122 19a Aldolase (B0SGS2) 48 14 38.45 7.71 SP 30 UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (B0SDI2) 57 20 37.06 6.76 N-Acetyl-gamma-glutamyl-phosphate reductase (B0SGX7) 52 15 38.12 6.71 19b Aldolase (B0SGS2) 81 37 38.45 7.71 SP 30 ATP-binding protein of an ABC transporter complex (B0SE24) 79 29 37.98 7.81 19c Aldolase (B0SGS2) 87 38 38.45 7.71 SP 30 ATP-binding protein of an ABC transporter complex (B0SE24) 65 20 37.98 7.81 20a Uncharacterized protein (B0STJ8)–Parisb 68 34 35.82 7.71 MP 116 20b Uncharacterized protein (B0STJ8), Parisb 69 41 35.82 7.71 MP 116 Citrate lyase beta subunit (B0SCW2) 67 26 37.10 6.10 SP 27 20c Uncharacterized protein (B0STJ8), Parisb 76 72 35.82 7.71 MP 116 21a Uncharacterized protein (B0SHJ5) 89 39 31.52 8.26 MP 117 21b Uncharacterized protein (B0SHJ5) 91 67 31.52 8.26 MP 117 22a Endoflagellar filament core protein, flaB1 (B0SHI7) 74 50 31.38 7.03 MP 113 22b Endoflagellar filament core protein, FlaB1 (B0SHI7) 92 114 31.38 7.03 MP 113 24a OmpA-family lipoprotein (B0SGK2) 76 28 20.61 6.62 MP 103 24b OmpA-family lipoprotein (B0SGK2) 88 100 20.61 6.62 MP 103 26a 50S ribosomal protein L7/L12 (B0SAG2) 93 12 12.65 5.20 MP 100 26b 50S ribosomal protein L7/L12 (B0SAG2) 94 23 12.65 5.20 MP 100 27a Hypothetical lipoprotein (B0SCM9) 56 27 16.65 7.67 Cyclic nucleotide-binding protein (B0SC72) 65 11 16.19 5.71 27b Uncharacterized protein (B0S8Z7) 54 8 12.68 5.70 Hypothetical lipoprotein (B0SCM9) 50 6 16.65 7.67 Lipoprotein LipL45 (B0SGV4) 35 44 42.92 5.79 MP 107 28a Hypothetical lipoprotein (B0SGB7) 63 19 19.32 8.98 MP 137 28b Hypothetical lipoprotein (B0SGB7) 79 43 19.32 8.98 MP 137
Supplemental material
- Supplemental file 1
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Spreadsheet of raw peptide/protein data from all LC-MS/MS analyses performed in this study (Data Set S1).
XLSX, 8.0M
- Supplemental file 1
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