Mass spectrometric mapping of the enzymes involved in the phenol degradation of an indigenous soil pseudomonad

Tsirogianni, Irene; Aivaliotis, Michalis; Karas, Michael; Tsiotis, Georgios

doi:10.1016/j.bbapap.2004.04.003

Cited by 22 publications

(30 citation statements)

References 27 publications

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“…On the contrary, Pseudomonas sp. strain phDV1 was shown to degrade phenol through the meta-cleavage pathway (Tsirogianni et al, 2004(Tsirogianni et al, , 2006. In a research work carried out by Kim et al (2004), both of the ortho-and meta-cleavage pathways were identified in the degradation of aniline by Pseudomonas sp.…”

Section: Identification Of Catabolic Enzymesmentioning

confidence: 99%

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

Cao

Loh

2008

Biotech & Bioengineering

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The catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate were studied through proteomics approach. Two-dimensional gel electrophoresis (2-DE) gel profiles of P. putida cells grown on 100 and 800 mg/L benzoate were quantitatively compared using threshold criteria and statistical tools. Protein spots of interest were identified through database searching based on peptide mass fingerprints (PMFs) obtained using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight catabolic enzymes involved in both the ortho-cleavage (CatB, PcaI, and PcaF) and the meta-cleavage (DmpC, DmpD, DmpE, DmpF, and DmpG) pathways for benzoate biodegradation were identified in P. putida grown on 800 mg/L of benzoate while no meta-cleavage pathway enzymes were observed in the 2-DE gel profiles of P. putida grown on 100 mg/L of benzoate. The activation of both the ortho- and the meta-cleavage pathways in P. putida P8 grown on high benzoate concentration was confirmed directly at the protein level. In addition, another 28 differentially expressed proteins were also identified, including proteins involved in (i) detoxification and stress response (AhpC, ATPase-like ATP-binding region, putative DNA-binding stress protein, SodB and catalase/peroxidase HPI); (ii) carbohydrate, amino acid/protein and energy metabolism (isocitrate dehydrogenase, SucC, SucD, AcnB, GabD, ArcA, ArgI, Efp and periplasmic binding proteins of several ABC-transporters); and (iii) cell envelope and cell division (bacterial surface antigen family protein and MinD). Based on the data obtained, physiological changes of P. putida in response to growth on benzoate at different concentrations were discussed.

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Section: Identification Of Catabolic Enzymesmentioning

confidence: 99%

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

Cao

Loh

2008

Biotech & Bioengineering

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“…Individual bands or proteins spots were excised from the Coomassie-stained gels, digested with trypsin (Roche, sequence grade) and PMFs were generated by a Voyager-DE STR MALDI-TOF instrument (Applied Biosystems) as earlier described (Tsirogianni et al 2004). MALDI-TOF mass spectra were externally calibrated with a peptide mixture containing der-Arg1-Bradykinin (904.47 Da), angiotensin I (1296.69 Da), glu1-fibrinopeptide B (1570.68 Da), ACTH (2093.0, 2465, and analyzed using the software Data Explorer 3.0 (Applied Biosystem).…”

Section: Mass Spectrometry For Protein Identificationmentioning

confidence: 99%

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

2006

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Chl. tepidum is a Gram-negative green-sulfur bacterium, which is strict by anaerobic and grows by utilizing sulfide or thiosulfate as an electron source. Blue native-polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes. In particular, the Chl. tepidum-soluble proteome was monitored under native condition by using BN-PAGE. The BN-PAGE protein complexes map was analyzed by MALDI-TOF MS after trypsin treatment and from 42 BN proteins bands, 62 different proteins were identified. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. One-hundred and seventy gel bands were spotted, out of which 187 different proteins were identified. The identified proteins belong to various functional categories like energy metabolism, protein synthesis, amino acid biosynthesis, central intermediate metabolism, and biosynthesis of cofactors indicating the potential of the method for elucidation of functional proteomes.

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“…Protein spots separated by 2DE were manually excised from the stained 2DE gels and subjected to in-gel tryptic digestion according to the procedure described by Tsirogianni et al (2004). Peptide mass fingerprinting (PMF) of tryptic peptides was detected by MALDI-TOF MS. Because the draft genome of P. stutzeri is poorly annotated and very few internal cellular proteins were present in the database, the protein identifications were assigned by comparing peak lists generated from PMF with a protein database of MASCOT using bacterial subset (www.matrixscience.com).…”

Section: Proteomic Analysismentioning

confidence: 99%

“…The 2-D electrophoresis (2DE) was performed on whole cell lysate according to a procedure described by Tsirogianni et al (2004) with minor modifications. About 350 lg proteins (350 ll) were pipetted into the strip holders (Bio-Rad) and 17 cm dry IPG strips with a linear immobilized pH 3-10 gradients (Bio-Rad) were loaded and rehydrated overnight.…”

Section: Proteomic Analysismentioning

confidence: 99%

“…To clarify the lactate-oxidation mechanism in P. stutzeri SDM, the major enzymes involved in pyruvate production were investigated based on the enzyme activity assay and proteomic analysis combined use of two-dimensional gel electrophoresis (2DE) with matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) (Tsirogianni et al 2004). 2,2¢-azinobis (3-ethylbenzothioazoline-6-sulfonic acid) were from Fluka.…”

Section: Introductionmentioning

confidence: 99%

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Pseudomonas stutzeri as a novel biocatalyst for pyruvate production from DL-lactate

Hao

Gao

et al. 2006

Biotechnol Lett

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Mass spectrometric mapping of the enzymes involved in the phenol degradation of an indigenous soil pseudomonad

Cited by 22 publications

References 27 publications

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of cytoplasmatic protein complexes from Chlorobium tepidum

Pseudomonas stutzeri as a novel biocatalyst for pyruvate production from DL-lactate

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