Analysis of aromatic catabolic pathways in <b><i>Pseudomonas putida</i></b> KT 2440 using a combined proteomic approach: 2‐DE/MS and cleavable isotope‐coded affinity tag analysis

Kim, Young Hwan; Cho, Kun; Yun, Sung-Ho; Kim, Jin Young; Kwon, Ki Young; Yoo, Jong Shin; Kim, Kyung Sik

doi:10.1002/pmic.200500329

Cited by 150 publications

(96 citation statements)

References 29 publications

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“…M1 and KT2440 when grown on or in the presence of phenol (Santos et al, 2004(Santos et al, , 2007. Multiple stress-related proteins, such as AphC, SodB and other antioxidants, are induced in P. putida KT2440 cells in the presence of a range of aromatic substrates (Kim et al, 2006). The presence of stress-related proteins in cells grown on styrene supports the claim that styrene has a toxic effect on bacteria cells (Ramos et al, 2001).…”

Section: Stress-related Proteinsmentioning

confidence: 50%

Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions

et al. 2009

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Pseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating mediumchain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogenlimited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth). INTRODUCTIONPseudomonas putida CA-3 has been reported to utilize styrene as a sole source of carbon and energy and to accumulate the biodegradable polymer medium-chainlength polyhydroxyalkanoate (mclPHA) when nitrogen (N) becomes limited in the growth medium (O'Connor et al., 1996;. These two abilities have been employed in the two-step chemo-biotechnological conversion of polystyrene, a non-biodegradable polymer, to mclPHA at laboratory scale (Ward et al., 2006). The process has been improved through the controlled feeding of N to the growth medium, which results in a twofold increase in biomass and a 1.4-fold increase in mclPHA accumulation (Goff et al., 2007).Molecular investigations of the styrene degradation pathway in this organism have been reported (Mooney et al., 2006b;O'Connor et al., 2001). Styrene metabolism in P. putida CA-3 proceeds via initial side chain oxidation and involves an upper pathway converting styrene to phenylacetic acid (PA) (O'Connor et al., 1995), and a lower pathway initiated via activation of PA to phenylace...

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Section: Stress-related Proteinsmentioning

confidence: 50%

Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions

et al. 2009

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“…The cell lysate was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 30 fractions were obtained based on molecular size. They were then in-gel digested using trypsin (Promega, WI) (17), and tryptic digests were dissolved in 0.5% trifluoroacetic acid solution to be analyzed by mass spectrometry (Thermo Finnigan LTQ, CA). The identities of peptides were determined by using the Sequest program (Thermo Finnigan, CA).…”

Section: Methodsmentioning

confidence: 99%

The Complete Genome Sequence of Thermococcus onnurineus NA1 Reveals a Mixed Heterotrophic and Carboxydotrophic Metabolism

et al. 2008

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Members of the genus Thermococcus, sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO 2 , thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus.Deep-sea hydrothermal vents comprise a plethora of potential habitats, with gradients of nutrient and extreme physicochemical conditions that vary from high to low with respect to temperature (350 to 2°C), oxygenation states, and fluid velocities (13). Many multidisciplinary studies have been carried out to understand the complexities of hydrothermal vent systems. Biological studies have also been accomplished using samples collected from hydrothermal vent areas and culture-dependent and culture-independent techniques, revealing the presence of physiologically, metabolically, and phylogenetically diverse microorganisms (15). These findings have been followed by characterization of many bacterial and archaeal thermophiles (and hyperthermophiles), including both chemolithoautotrophic and chemoorganoheterotrophic strains. Among representative species of the Archaea, sulfur-reducing heterotrophs belonging to the order Thermococcales (encompassing the genera Thermococcus, Pyrococcus, and Palaeococcus) have been reported to be one of the predominant groups (20, 25). Notably, members of the species of Thermococcus were found to be more abundant in the vent ecosystem, with such isolates more frequently reported than the Pyrococcus species (9,11,23,24). Such large populations indicate some significance for the presence of Thermococcus in the microbial consortia that make up the microbial ecology of hydrothermal vent systems.In addition to ecological significance, the hyperthermophilic feature of Thermococcales has fascinated microbiologists interested in fundamental and/or application-based research. To date, the complete genome sequences of three Pyrococcus species, i.e., Pyrococcus horikoshii (16), Pyrococcus furiosus (26), and Pyrococcus abyssi (5), and a Thermococcus strain, Thermococcus kodakaraensis KOD1 (7), have been determined. Analysis of the sequences and the physiolo...

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“…In-Gel Digestion and MALDI-TOF/MS Protein spots were excised from silver-stained 2-DE gels, and peptides digested with trypsin according to previously described methods [9,14]. Digested peptides were redissolved using 0.1% trifluoroacetic acid (TFA).…”

Section: Methodsmentioning

confidence: 99%

Antibacterial Effects of Green Tea Polyphenols on Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

Cho

Schiller

2008

Curr Microbiol

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The antibacterial effects of tea polyphenols (TPP) extracted from Korean green tea (Camellia sinensis) against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) were evaluated. Characterization of the minimal inhibitory concentration (MIC) of oxacillin for 30 S. aureus strains isolated from patients treated with oxacillin identified 13 strains with an oxacillin MIC C 4 lg/mL as methicillinresistant Staphylococcus aureus (MRSA) (range: 8 to 512 lg/ mL), while 17 strains were methicillin-susceptible Staphylococcus aureus (MSSA) (range: 0.25-0.5 lg/mL). The MICs of TPP ranged from 50 to 180 lg/mL for both the MSSA and the MRSA strains. The MICs of oxacillin for each of the 13 MRSA strains were reduced between 8-and 128-fold when these strains were coincubated with sub-MIC (B0.59 MIC) levels of TPP, demonstrating that the combination of TPP plus oxacillin was synergistic for all of the clinical MRSA isolates. Two-dimensional polyacrylamide gel electrophoresis identified 14 extracellular proteins of MRSA-13 down-regulated and 3 proteins up-regulated by exposure to TPP. These studies demonstrate that TPP can differentially stimulate the expression of various proteins in these bacteria and synergize the bactericidal activity of oxacillin for MRSA.

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Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2‐DE/MS and cleavable isotope‐coded affinity tag analysis

Cited by 150 publications

References 29 publications

Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions

Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions

The Complete Genome Sequence of Thermococcus onnurineus NA1 Reveals a Mixed Heterotrophic and Carboxydotrophic Metabolism

Antibacterial Effects of Green Tea Polyphenols on Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

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