Pseudomonas putida - Wiley Online Library

Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 200583466478Original ArticleProteome of P. putida KT2440 with different carbon sourcesL. Kurbatov, D. Albrecht, H. Herrmann and L. Petruschka

Environmental Microbiology (2006) 8(3), 466–478

doi:10.1111/j.1462-2920.2005.00913.x

Analysis of the proteome of Pseudomonas putida KT2440 grown on different sources of carbon and energy Leonid Kurbatov,1,2 Dirk Albrecht,3 Heidrun Herrmann1 and Lothar Petruschka1* 1 Ernst-Moritz-Arndt-University, Institute for Microbiology, Department of Genetics and Biochemistry, Greifswald, Germany. 2 Institute of Biomedical Chemistry, Moscow, Russia. 3 Ernst-Moritz-Arndt-University, Institute for Microbiology, Department of Microbial Physiology, Greifswald, Germany. Summary Using 2D electrophoresis the protein expression pattern during growth on carbon sources with different impact on carbon catabolite repression of phenol degradation was analysed in a derivative of Pseudomonas putida KT2440. The cytosolic protein pattern of cells growing on phenol or the non-repressive substrate pyruvate was almost identical, but showed significant differences to that of cells growing with the repressive substrates succinate or glucose. Proteins, which were mainly expressed in the presence of phenol or pyruvate, could be assigned to the functional groups of transport, detoxification, stress response, amino acid, energy, carbohydrate and nucleotide metabolism. The addition of succinate to cells growing with phenol (‘shift-up’) resulted in the inhibition of the synthesis of these proteins. Proteins with enhanced expression at growth with succinate or glucose were proteins for de novo synthesis of nucleotides, amino acids and enzymes of the TCA cycle. The synthesis of proteins, necessary for phenol catabolism was regulated in different manners following the addition of succinate. Whereas the synthesis of Phl-proteins (subunits of the phenolhydroxylase) only decreased slowly, was the translation of the Catproteins (catechol 1,2-dioxygenase, cis,cis-muconate cycloisomerase and muconolactone isomerase) repressed immediately and the synthesis of the β-ketoadipate enolactone hydrolase, Pca-proteins (β β-ketoadipate succinyl-CoA transferase and βketoadipyl CoA thiolase) remained unaffected. Received 2 May, 2005; accepted 27 July, 2005. *For correspondence. E-mail [email protected]; Tel. (+49) 3834 864153; Fax (+49) 3834 864172.

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd

Introduction Bacteria of the species Pseudomonas putida belong to those microorganisms that can utilize a variety of organic compounds as sole source of carbon and energy. They are able to colonize different environments and have been isolated from water, soil and the plant rhizosphere. Many P. putida strains have acquired the capability to use exotic or toxic growth substrates, like aromatic compounds or hydrocarbons. Therefore, they play a key role in the elimination of organic wastes in their natural habitats, frequently, polluted soils. The utilization of a broad range of carbon and energy sources by pseudomonads requires the coordinated control of metabolic (catabolic) pathways. The expression of these pathways is (i) regulated by global control mechanisms, which monitor (sense) the physiological state of the bacterial cell, and (ii) by a pathway specific control response. A series of factors and sensing mechanisms like the Crc protein, the integration host factor, the alarmone (p)ppGpp and components of the electron transport system are probably involved in the global cellular response to environmental signals (for review see Shingler, 2003; Morales et al., 2004; Ruiz-Manzano et al., 2005). As aromatic compounds are not the preferred growth substrates for pseudomonads, the expression (induction) of genes coding for these degradative pathways is regulated (repressed) depending on the availability and/or concentration of other carbon sources (Duetz et al., 1994; 1996; Collier et al., 1996; Shingler, 2003). This regulatory mechanism, usually called carbon catabolite repression (CR), is well described for Escherichia coli and Bacillus subtilis, whereby CR is only a part of the global regulatory response of the bacteria to environmental signals. Although CR is the result of a complex global regulatory response, there are pronounced differences in these processes between E. coli and B. subtilis (Saier, 1998; Stulke and Hillen, 2000). In pseudomonads, the mechanisms responsible for global regulatory control of the metabolism are different from either E. coli or B. subtilis, and also CR proceeds in a completely different manner. The metabolic response of pseudomonades to the concomitant presence of aromatic compounds and other, preferred growth substrates in the environment, was intensively investigated for the catabolism of toluene (XylR/Pu), methyl-phenol (DmpR/Po), phenol (PhlR/PA) and alkanes

Proteome of P. putida KT2440 with different carbon sources 467

Results and discussion Growth characteristics of the strain PG150 The strain PG150 is able to grow on phenol as sole source of carbon and energy and phenol is converted to catechol by the action of the phenol hydroxylase. Catechol is then metabolized via the chromosomally encoded ortho cleavage pathway (cat and pca genes) to the TCA cycle intermediates acetyl- and succinyl-CoA. In order to analyse the impact of carbon sources on the protein pattern of the strain PG150, cells were cultivated in modified M9 minimal medium with phenol, the non-repressive substrate pyru-

10.0

OD600

(AlkS/PalkB) (Müller et al., 1996; Sze and Shingler, 1999; Cases et al., 2001; Dinamarca et al., 2002). In all these systems CR acts as the repression of transcription of the respective operons during growth on preferred carbon sources, even in the presence of the specific inductors. As shown in earlier studies, the regulation of the σ54dependent PhlR/PA system with respect to CR is the same in P. putida KT2440 as in the natural host bacterium of the phl-operon, P. putida H. Both, succinate and glucose, cause strong CR of the operon, whereas pyruvate has only a minor repressive effect. In this system the activator itself is the target of yet unknown factor(s), which generate the CR control of transcription (Müller et al., 1996). As the genome sequence of P. putida KT2440 is now known (Nelson et al., 2002), it should be possible to use a proteomic approach to search for proteins, which are affected by carbon CR and for proteins, which could be involved in regulatory mechanisms concerning CR. In this study we have analysed the cytosolic protein pattern of a P. putida KT2440 strain, harbouring the essential genes for phenol hydroxylation, using 2D electrophoresis and MALDI-TOF analysis. This strain, PG150, contains the genes encoding phenol hydroxylase (phlA-F), the specific activator gene (phlR) and their common promoter/operator region on a mini-Tn5 (Müller et al., 1996). Sequence analysis of the insertion site showed that the mini-Tn was integrated in the GPP-t-RNA-Val-5 gene. In a first approach, we have investigated a proteome of cells growing on phenol, the non-repressive substrate pyruvate and the repressive substrates succinate and glucose, to analyse the general differences in the protein expression pattern due to the carbon source used for cell growth. The synthesis of proteins, which might be involved in the global regulation of CR or in the regulation of the PhlR/PA system, should be either induced or repressed following the addition of a preferred carbon source to cells growing with phenol. To search for such proteins, changes in the protein synthesis pattern after the addition of a preferred carbon source (succinate) to cells growing with phenol were analysed.

1.0

0.1 0

2

4

6

8

10

Time (h) Pyruvate

Succinate

Glucose

Phenol

Fig. 1. Growth of strain PG150 in M9 minimal medium with 40 mM pyruvate, succinate and glucose, respectively, and with 2.5 mM phenol. Samples for 2D electrophoresis were taken at an OD600 of 0.4– 0.6 from the phenol culture and 0.5–0.8 from the pyruvate, succinate and glucose cultures respectively.

vate or with the repressive carbon sources succinate and glucose (see Experimental procedures). Although approximate growth rates are important for the comparability of protein expression profiles in bacterial cells (Blencke et al., 2003), it was not possible to reach similar growth rates with all carbon sources (Fig. 1). Cells growing in minimal medium containing succinate or glucose have a doubling time (td) of about 1 h whereas cells growing with pyruvate or with phenol have a td of about 1.8 h and 2.7 h respectively. Thus, in addition to the specific effects of the carbon sources themselves, the different growth rates and the concentration of the carbon source probably affect the protein expression pattern (Duetz et al., 1996; Blencke et al., 2003). As batch cultivation was used in these experiments, the limitation of nutrients and oxygen are factors, which influence the regulation of the metabolism, too. To minimize these complications, cells were harvested for 2D gel analysis during exponential growth, when cultures reached constant, substrate specific growth rates. Analysis of protein patterns and protein synthesis patterns About 1000 cytosolic proteins in the pI range 4–7, detectable by silver staining, were expressed in cells of the strain PG150, independently of the carbon source used in the growth medium. The analysis of 2D gels showed significant similarities in the expression pattern of cytosolic pro-

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 466–478

468 L. Kurbatov, D. Albrecht, H. Herrmann and L. Petruschka teins in P. putida PG150 at cultivation with phenol and with the non-repressing carbon source pyruvate. Next to the Phl-, Cat- and Pca-proteins, only 14 proteins were absent or diminished in their expression when grown with pyruvate, compared with cells grown with phenol (Fig. 2A and C, Table 1). In comparison to cells growing with succinate or glucose, there are about 90 proteins (31 identified), which were exclusively expressed in the phenol culture.

In addition to the phenol specific proteins, these proteins could be assigned to various functional groups (Table 1). The 2D pattern of cytosolic proteins from cells growing in minimal salts medium containing the preferred substrates succinate and glucose, respectively, were very similar to each other. No succinate specific protein spots were found, and only 10 specific protein spots were visible on 2D gels of cells growing with glucose. The protein spot

Fig. 2. Reference 2D gels of cytosolic protein extracts of P. putida PG150 grown in M9 minimal medium with 2,5 mM phenol (A), 40 mM succinate (B), 40 mM pyruvate (C) or 40 mM glucose (D). Proteins, involved in phenol degradation are indicated by black arrows. Further proteins, identified by MALDI_TOF analysis are indicated by white arrows and Arabic numbers are referring to Table 1 in gel A (phenol) and to Table 2 in gel B (succinate) respectively. Unidentified proteins, which are expressed predominantly at growth with a specific carbon source, are indicated with open circles (phenol), squares (succinate), diamonds (glucose) and triangles (pyruvate). © 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 466–478

Proteome of P. putida KT2440 with different carbon sources 469 pathway could be identified in phenol growing cells. Furthermore, the gene products of pcaB and pcaC, which are located in a gene cluster with pcaF and pcaD, were detected. These results were confirmed by the analysis of the protein synthesis pattern. During growth on phenol, strong incorporation of 35-S methionine was found in all proteins necessary for its utilization. The synthesis of the various proteins, however, responded in different manners to the addition of succinate to a culture growing with phenol. Syntheses of the components of the phenol hydroxylase decreased only slowly. In the first hour after addition of succinate their synthesis was reduced to about 25% of that of the phenol culture, and incorporation of 35S methionine was detectable even 1 h later (Fig. 3). The slow reduction of the synthesis rate of the phenol hydroxylase subunits might be a result of the repression of general transcriptional or translational processes. The phl-

patterns, expressed with these two carbon sources showed significant differences to those observed with phenol and pyruvate (Fig. 2B and D).

Proteins, upregulated at growth with phenol and pyruvate Proteins involved in phenol degradation. The proteins involved in the degradation of phenol, PhlA-F, CatA-C, Pca D, IJ and F, were solely expressed at growth on phenol and were not detectable on 2D gels of cells grown on pyruvate, succinate or glucose (Fig. 2). Due to their low molecular weight the subunits A (10.6 kDa) and E (13.2 kDa) were not detectable on our 2D gels. Also the subunits PhlB and PhlF were not distinguishable due to their almost identical pI and molecular weight. With the exception of PcaD and PcaJ, all proteins of the ortho-

Table 1. Cytosolic proteins of P. putida PG150, upregulated at growth with phenol and pyruvate.

Functional group/protein name Transport Amino acid ABC transporter, periplasmic amino acidbinding Glycine betaine/L-proline ABC transporter, periplasmic binding Polyamine ABC transporter, periplasmic polyaminebinding protein Branched-chain amino acid ABC transporter Outer membrane protein General amino acid ABC transporter, periplasmic binding protein Polyamine ABC transporter, periplasmic polyaminebinding protein Sugar ABC transporter Phosphate ABC transporter Branched-chain amino acid ABC transporterd Branched-chain amino acid ABC transporterd Putrescine ABC transporter, periplasmic putrescinebinding Nucleotide metabolism Adenylate kinase N-carbamoyl-beta-alanine amidohydrolase, putative

Decrease of synthesis after succinate addition (min)

Accession number (TIGR/SP)a

Phenol

Pyruvate

Succinate

Glucose

++

++

+

+

30

PP0282

+

+

−

(+)

15

PP0296

+++

++

(+)

(+)

30

PP0412

+++ ++ +++

+++ ++ ++

+ + (+)

++ + (+)

15 15 15

PP1141 PP1206 PP1297

++

+

(+)

+++

15

PP1486

+ + +++ +++ +++

+ (+) + +++ ++

− (+) − + +

− (+) − + +

ns nsb 15 15 30

PP2264 PP2656 PP4867 PP4867 PP5181

++ +++

++ +

+ (+)

+ −

30 15

PP1506 PP4034

b

Gene name

braC oprD aapJ

potF-2

adk

Amino acid metabolism N-acetyl-gamma-glutamyl-phosphate reductase, putative Arginine deiminase Betaine aldehyde dehydrogenase, putative Glutamate synthase, small subunit, putatived Glutamate synthase, small subunit, putatived Ornithine carbamoyltransferase, catabolic

++

+

−

−

120

PP0432

++ + + +++ ++

+ + + ++ ++

+ − − + +

+ − − + +

15 30 15 30 nsb

PP1001 PP1481 PP4037 PP4037 PP1000

arcA

Carbohydrate metabolism Phosphoenolpyruvate carboxylase Glucose-6-phosphate isomerase Acetyl-coA synthetased Acetyl-coA synthetased

+ ++ ++ ++

(+) ++ + +

(+) + + +

(+) + + +

30 30 15 15

PP1505 PP4701 PP4487 PP4487

ppc


argl

acsA

470 L. Kurbatov, D. Albrecht, H. Herrmann and L. Petruschka Table 1. cont. Decrease of synthesis after succinate addition (min)


Functional group/protein name

Phenol

Pyruvate

Succinate

Glucose

Antioxidants, detoxcification, chaperones Thiol–disulfide interchange protein, DsbA family Medium-chain-fatty-acid CoA ligase Antioxidant, AhpC/Tsa family Xenobiotic reductase, putative Quinoprotein ethanol dehydrogenase Thiol peroxidase Catalase/peroxidase HPI DnaK proteind DnaK proteind

++ ++ ++ ++ + ++ + + ++

+ ++ ++ ++ (+) + + + ++

− + − + − − − (+) +

+ + − + + (+) (+) (+) +

15 15 nsb 30 ndc 30 nsb nsb

PP0127 PP0763 PP1084 PP1478 PP2674 PP3587 PP3668 PP4727 PP4727

Others Aldehyde dehydrogenase family proteind Aldehyde dehydrogenase family proteind DNA-binding stress protein, putative D-hydantoinase, authentic frameshift Translation elongation factor G

++ + ++ +++ +

++ + +++ +++ +

+ (+) (+) − −

+ (+) + − −

60 30 15 15 30

PP0545 PP0545 PP1210 PP4036 PP4111

Conserved hypothetical proteins Homology to putative serine protein kinase PrkA (3) Homology to kinase autophosphorylation inhibitor KipI Homology to lactam utilization protein

+++ + +

+ ++ +++

(+) (+) +

(+) (+) +

15 30 30

PP0397 PP4576 PP4577

Phenol degradation Subunit of phenolhydroxylase (PhlB) Subunit of phenolhydroxylase (PhlC) Subunit of phenolhydroxylase (PhlD) Muconate cycloisomerase Muconolactone isomerase Catechol 1,2-dioxygenase Beta-ketoadipyl CoA thiolase 3-oxoadipate CoA-transferase, subunit A

+++ +++ + +++ ++ +++ ++ ++

− − − − (+) (+) − −

− − − − − − − −

− − − − − − − −

30 30 60 15 15 15

Q52162 Q52163 Q52164 PP3715 PP3714 PP3713 PP1377 PP3951

Gene name

tpx dnaK dnaK

fusA-2

phlB phlC phlD catB catC catA pcaF pcaI

a. Institute for Genomic Research (TIGR); Swiss-Prot/TrEMBL (SP). b. Not sure, conflicting results in single experiments. c. Synthesis not detectable. d. Double spots of a certain protein showing different molecular weight, expression level and/or synthesis rate are listed as single proteins.

genes, located in an operon, are cotranscribed and their transcription is activated by PhlR, a sigma-54-dependent activator protein (Müller et al., 1996; Burchhardt et al., 1997). Previous results have shown, that the transcription of the PA-promoter is not induced by phenol as long as cells grow exponentially in the presence of 40 mM succinate, glucose or lactate and the transcription of the promoter is silenced if a preferred carbon source is added to a phenol culture (Müller et al., 1996; Petruschka et al., 2001). The results obtained in the ‘shift-up’ experiments might indicate, that the translation of still present phlmRNA continued and was not or only slightly affected by the availability of succinate. In contrast to the synthesis of the phl-gene products, the translation of the cat-gene products were drastically reduced. Especially the synthesis of catechol 1,2-dioxygenase (CatA) ceased rapidly. The reduction of 35-S methionine incorporation was recognized already 15 min after the addition of succinate.

After 1 h, the incorporation of radiolabelled methionine into CatA was no longer detectable (Fig. 3). These results point to a regulation on post-transcriptional level, too. They could be explained by a cat-mRNA, highly unstable at least in the presence of a preferred substrate, or by an influence of the availability of a preferred carbon source on the translation of cat-mRNA. The regulation of Cat-enzymes in various P. putida strains was previously analysed mainly on the level of transcription. The cat-genes (catBCA) are activated by catR in response to the presence of cis-cis-muconate (Rothmel et al., 1990), and are subjected to global control mediated by succinate, glucose and by components of rich media (Tover et al., 2001). Furthermore, Crc (for catabolite repression control), an integral part of global control signal transduction pathways, is involved in the regulation of cat-genes (Morales et al., 2004), although its mode of action is yet unknown.


Proteome of P. putida KT2440 with different carbon sources 471

A

B

Fig. 3. Repression of protein synthesis in P. putida PG150 grown with phenol after the addition of 40 mm succinate. A. Synthesis of PhlB (subunit B of phenolhydroxylase) and CatA (catechol 1,2-dioxygenase). B. Synthesis of PcaF (beta-ketoadipyl CoA thiolase). C. Synthesis of various transport proteins (1 PP4867, 2 BraC, 3 PP1486, 4 PotF-2). Autoradiographs of representative gels are shown. The lower panels show the 35-S-methionine incorporation into proteins 15, 30 and 60 min after succinate addition. The upper panels show the 35-S methionine incorporation in cells of the control culture (phenol only) at the same timepoints.

C

In P. putida PG150 the pca-genes, which code for the proteins of the central β-ketoadipate pathway (pcaD, I, J and F), were only expressed in the presence of phenol (Fig. 2). They are located in two operons (pcaRKFTBDCP and pcaIJ), which are probably controlled by the IclR-type regulator PcaR (Jimenez et al., 2002). In contrast to the phl- and cat-genes, their translation remained derepressed upon succinate addition to cells growing with phenol (Fig. 3). As previously shown, the transcription of two other pca-genes (G and H), encoding the protocatechuate 3,4-dioxygenase, seemed to be modulated by the Crc protein (Morales et al., 2004). These genes form an operon and are regulated by their own regulatory protein, probably PcaQ (Jimenez et al., 2002). Apparently, the expression of the genes of the central β-ketoadipate pathway is not subjected to CR in P. putida, as was also shown for the induction of pcaF by 4-hydroxybenzoate in the presence of succinate in P. putida PRS2000 by Nichols and Harwood (1995). Thus, the degradation of aromatic compounds in P. putida is rather controlled by regulating the expression of the convergent pathways, which feed into the central pathway via β-ketoadipate enol-lactone (Jimenez et al., 2002; Morales et al., 2004). Transport proteins. KT2440 contains a large number of cytoplasmic membrane transport systems which facilitate the uptake of a multitude of organic compounds and reflect its metabolic flexibility in the natural environment (Nelson et al., 2002). We have identified one porin (OprD) and the periplasmic binding proteins of nine ABC transporters, whose expression were different in respect to the

carbon source used for cell growth. All ABC transporters were synthesized at growth on phenol and pyruvate and not or only slightly at growth with succinate and glucose. The ABC transporters expressed in the presence of phenol or pyruvate are involved in the transport of amino acids, peptides, polyamines, sugars and phosphate. As the growth rate of strain PG150 with phenol and pyruvate, doubling time (td) of about 2.7 h and 1.8 h, respectively, were about half of those achieved with succinate or glucose, about 1 h, respectively, cells probably activate their transport systems for the uptake of additional growth substrates. Also the lack of trace elements might be responsible for an increased synthesis of transport proteins. Heim and colleagues (2003), reported an upregulation of some transport and binding proteins, induced by iron starvation, both in P. putida and Pseudomonas aeruginosa. They could also show, that some of the transport proteins (braC, aapJ, potF), which were not observed here (or which were only slightly expressed) at growth with succinate or glucose (40 mM each), were expressed in KT2440 at growth in low-phosphate minimal medium with 20 mM succinate or 10 mM glucose. Sonawane and colleagues (2003) showed that the putrescine binding protein PotF (PP5181) was also induced at growth with glucose (22 mM). This peptide was found to be expressed also with glucose (40 mM) in our experiments, but it is present in a much higher concentration in cells grown with phenol or pyruvate. Toxic stress seems to be responsible for an increased expression of transporters of small molecules. Santos and colleagues (2004) found the upregulation of nine transporters adding higher doses (6.3 and 8.5 mM)


472 L. Kurbatov, D. Albrecht, H. Herrmann and L. Petruschka of phenol to P. putida KT2440 cells growing with 20 mM succinate. Three of these transporters (PP1141, 1297 and 4867) were induced already at growth with 2.5 mM phenol or 40 mM pyruvate in our experiments. The investigation of protein synthesis showed that the expression of transport proteins was obviously subjected to CR. In the ‘shiftup’ experiments the translation of all ABC-transporters and of OprD was reduced within 15 min after the addition of succinate (examples are shown in Fig. 3). This unspecific repression of transport systems with different specificity probably points to a global acting signal, which will be triggered by the media composition used for cell growth, especially by the concentration and the quality of the carbon source, but also by starvation conditions and toxic stress (Heim et al., 2003; Santos et al., 2004). Amino acid synthesis/energy metabolism. At poor growth conditions, the metabolism of cells is oriented towards an economical utilization of substrates by usage of the available metabolites for the synthesis of proteins and nucleic acids. As shown in Table 1, a set of proteins involved in the metabolism of amino acids, purines, pyrimidines and carbohydrates was preferentially synthesized at growth with phenol or pyruvate. Some of these proteins like a putative glutamate synthase (PP4037), the ornithine carbamoyltransferase (PP1000), the arginine deiminase (PP1001) and the N-acetyl-gamma-glutamyl-phosphate reductase (PP0432) are involved in the interconversion of glutamate and in the synthesis of aspartate and arginine respectively. Furthermore, the synthesis of a putative Ncarbamoyl-beta-alanine-amidohydrolase (PP4034) was enforced, which probably catalyses reactions connecting the degradation of pyrimidines to the metabolism of alanine and aspartate and valine, leucine and isoleucine respectively (West, 2001). The synthesis of all these proteins was reduced by the addition of succinate to phenol growing cells (not shown). This may indicate a switch of the metabolism to de novo synthesis reactions as suggested by the enhanced synthesis of key enzymes of anabolic pathways (see below). Detoxification/stress response. A number of proteins involved in detoxification, oxidative stress response and protein folding mechanisms were identified in cells grown both with phenol and pyruvate but not in cells grown with succinate or glucose. Among these were a protein of the AhpC/Tsa family (PP1084), a catalase protein (PP3668), a putative xenobiotic reductase (PP1478) and DnaK (PP4727), which was expressed both with pyruvate and phenol. Other proteins of this group, a quinoprotein ethanol dehydrogenase (PP2674), a thiolperoxidase (PP3587), and a thiol–disulfide interchange protein of the DsbA family (PP0127) were recognized on gels from phenol cultures. As revealed by the analysis of autoradio-

graphs from phenol cultured cells (‘shift-up’-experiments), the synthesis rate of these proteins was very low (not shown). The detected protein amounts in silver gels seemed to be an effect of gradual accumulation during cell growth. An influence of succinate addition on protein synthesis was not detectable. Some proteins involved in detoxification (PP1084, PP14778 and PP3668) or protein folding (DnaK) may be expressed independently of the growth substrate but always in the case of, poor carbon sources like phenol or pyruvate (Table 1), whereas others (PP3587, PP0127 and PP2674) will be expressed for protection against the abnormal formation of reactive oxygen species (ROS) induced by phenol (Winn et al., 2003; Santos et al., 2004). Proteins, upregulated at growth with succinate and glucose Overall, about 60 proteins (25 identified) were found with enhanced expression at growth with succinate or glucose. Particularly proteins for the de novo synthesis of nucleotides and amino acids and proteins of the TCA cycle, ribosomal proteins and t-RNA synthetases were detected. Proteins which realize early or key reactions of biosynthetic pathways, e.g. ribose-phosphate pyrophosphokinase, CTP-synthase, uridylate kinase (purines, pyrimidines, histidine, tryptophane), ketol-acid reductoisomerase (leucine, isoleucine, valine) and D-3-phosphoglycerate dehydrogenase (serine, glycine) were expressed in higher amounts (Table 2). Some enzymes of the TCA cycle like aconitate hydratase (AcnA) and isocitrate dehydrogenase showed identical expression with all growth substrates, while other proteins like succinate dehydrogenase (SdhA), succinylCoA synthetase (SucC,D), the dihydrolipoamide succinyltransferase (KdgB) and the lipoamide dehydrogenase (LpdG) component of the 2-oxoglutarate dehydrogenase were better expressed in the presence of succinate, glucose or pyruvate. Probably, at growth with phenol, acetylCoA was metabolized via glyoxylate rather than via the TCA cycle utilizing the end products of phenol degradation to provide metabolites (oxaloacetate, malate, pyruvate and phosphoenolpyruvate) for anabolic pathways. The synthesis of almost all proteins, exclusively or more expressed with preferred growth substrates, was induced by the addition of succinate in ‘shift-up’ experiments. Primarily, within 15 min, the synthesis of proteins which were required for the metabolization of succinate (SdhA) and for de novo synthesis pathways (SerA, PrsA, PyrH) was enhanced. The synthesis of other proteins involved in the TCA cycle (SucC, SucD, LpdG, kgdB) and protein synthesis (ribosomal proteins, t-RNA synthetases) increased gradually within 30–60 min after succinate addition, indicating the change of metabolic processes to


Proteome of P. putida KT2440 with different carbon sources 473 Table 2. Cytosolic proteins of P. putida PG150, upregulated at growth with succinate and glucose.

Functional group/protein name

Phenol

Pyruvate

Glucose

Succinate

Increase of synthesis after succinate addition (Min)

++

++

+++

+++

60

PP0338

aceF


Gene name

Amino acid/energy metabolism Pyruvate dehydrogenase, dihydrolipoamide acetyltransferase comp. Ketol-acid reductoisomerasec Ketol-acid reductoisomerasec Malate–quinone oxidoreductase Sulfat adenylyltransferase, subunit 1/ adenylylsulfate kinase Glutamate dehydrogenase D-3-phosphoglycerate dehydrogenasec D-3-phosphoglycerate dehydrogenasec Acetolactate synthase, catabolic, putative ATP synthase F1, delta subunit

++ + − +

+++ + − +

+++ + ++ ++

+++ + ++ ++

60 30 nsb nsb

PP4678 PP4678 PP1251 PP1304

ilvC ilvC mqo-2 cysNC

+ + ++ + +

+ + ++ + ++

+ ++ +++ ++ +++

+ ++ +++ ++ +++

30 15 15 15 nsb

PP0675 PP5155 PP5155 PP1157 PP5416

gdhA serA serA

Cofactor biosynthesis 5,10-methylenetetrahydrofolate reductase Riboflavin synthase, beta subunit

(+) +

+ ++

+ +++

+ +++

15 nsb

PP4977 PP0517

metF ribH

++

++

++

++

60

PP4012

+

++

++

++

15

PP4191

sdhA

+

++

+++

+++

60

PP4187

lpdG

atpH

TCA cycle Isocitrate dehydrogenase, NADPdependent, monomeric-type Succinate dehydrogenase, flavoprotein subunit 2-oxoglutarate dehydrogenase, lipoamide dehydrogenase comp. 2-oxoglutarate dehydrogenase, dihydrolipoamide succinyltransf. Succinyl CoA synthetase, a-chain Succinyl CoA synthetase,,-chain

+

+++

++

++

30

PP4188

kgdB

++ ++

+++ +++

+++ +++

+++ +++

60 60

PP4185 PP4186

sucD sucC

t-RNA synthesis Phenylalanyl-tRNA synthetase, beta subunit Threonyl-tRNA synthetase

+ +

++ +

++ ++

++ ++

30 30

PP2470 PP2465

pheT thrS

(+) (+) ++

+ + ++

+++ +++ +++

+++ +++ +++

15 15 60

PP0722 PP4016 PP4822

prsA purB purH

(+) +

+ ++

++ ++

++ ++

15 nsb

PP1593 PP1610

pyrH pyrG

+ ++ +++ ++

+ ++ +++ ++

+ +++ +++ +++

++ +++ +++ ++

60 60 15 60

PP4877 PP1772 PP4874 PP0721

rpsF rpsA rpll

+ +

+ +

+ +

+ +

15 30

PP1638 PP2928

fpr

Nucleotide metabolism Ribose-phosphate pyrophosphokinase Adenylosuccinate lyase Phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP Uridylate kinase CTP synthase Ribosomal proteins 6S ribosomal protein 30S ribosomal protein Ribosomal protein L9 Ribosomal 5S rRNA E-loop binding protein Ctc/L25/TL5 Others Ferredoxin-NADP reductase Conserved hypothetical protein

a. Institute for Genomic Research (TIGR); Swiss-Prot/TrEMBL (SP). b. Not sure, conflicting results in single experiments. c. Double spots of a certain protein showing different molecular weight, expression level and/or synthesis rate are listed as single proteins.

the utilization of succinate (Fig. 4). The intensity of the spots for the Fpr protein and the conserved hypothetical protein PP2928 (No. 30 and 31 in Table 2) was not increased in silver stained 2D gels after cultivation of cells with succinate or glucose. However, in the ‘shift-up’ exper-

iments the translation of these proteins was enhanced 15 and 30 min after succinate addition respectively. The physiological function of these two proteins is not known as yet. The ferredoxin-NADP reductase (Fpr) may play a role as electron donor in the oxidative stress response


474 L. Kurbatov, D. Albrecht, H. Herrmann and L. Petruschka Fig. 4. Induction of protein synthesis of selected proteins after the addition of 40 mM succinate to P. putida PG150 grown with phenol. Autoradiographs of representative gels are shown. The lower panels for each protein show the 35-S-methionine incorporation into proteins 15, 30 and 60 min after succinate addition. The upper panels show the 35-S methionine incorporation in cells of the control culture (phenol only) at the same timepoints.

(Krapp et al., 2002) and in different oxidoreductase reactions (Nam et al., 2002). Proteins with a putative regulatory role From the proteins which differed in their expression during growth on various carbon sources, three (PP1210, PP0397, PP4576) could be involved in regulatory mechanisms. They were absent (PP0397 and PP4576) or only slightly expressed (PP1210) in succinate growing cells. The synthesis of these proteins in phenol growing cells was repressed by the addition of succinate, with differences in the time course of downregulation. While the synthesis of the prkA homologue (PP0397) and of the

putative stress protein (PP1210) is completely repressed after 15 and 30 min, respectively, the synthesis of the kipI homologue (PP4576) was only slightly repressed (Fig. 5). The putative DNA-binding stress protein PP1210 contains a DPS-like domain (DNA-protecting protein under starved conditions) and belongs to the superfamily of ferritin-like diiron-carboxylate proteins. These proteins protect DNA from cleavage caused by ROS and probably have cellular functions associated with protein-DNA binding, metal chelation, ferroxidase activity, and regulation of gene expression (Nair and Finkel, 2004). PP1210 has 75% identity (amino acid sequence) to the DpsA protein, which mediates the resistance to organic oxidants in Burkholderia pseudomalle (Loprasert et al., 2004). Never-


Proteome of P. putida KT2440 with different carbon sources 475 Fig. 5. Protein synthesis of conserved hypothetical proteins after the addition of 40 mM succinate to P. putida PG150 grown with phenol. Autoradiographs of representative gels are shown. The lower panels for each protein show the 35-S-methionine incorporation into proteins 15, 30 and 60 min after succinate addition. The upper panels show the 35-S methionine incorporation in cells of the control culture (phenol only) at the same timepoints.

theless it was expressed also in the absence of phenol at cultivation with pyruvate (Table 1). In the ‘shift-up’ experiment the translation of PP1210 was repressed very fast, although the concentration of phenol did not change in the growth medium after succinate addition. The fast downregulation of translation of PP1210 and its expression at growth with pyruvate may indicate, that in P. putida this protein might have functions in addition to its involvement in DNA protection processes. PP0397 and PP4576 are conserved hypothetical proteins, the function of which is unknown in P. putida. Using a BLAST search against the NCBI database of microbial genomes (http://www.ncbi.nlm.nih.gov/sutils/genom_ table.cgi) homology to proteins which may be involved in regulatory processes were found. PP0397 had homology (33% identity) to a serine protein kinase (prkA) of B. subtilis, which is involved in the phosphorylation of an unknown protein (Fischer et al., 1996). The conserved hypothetical protein PP4576 showed homology (35% identity) to an autophosphorylation inhibitor (kipI) of kinase A (kinA), a sensor kinase of the signal cascade of sporulation in B. subtilis (Burbulys et al., 1991; Wang et al., 1997). These three proteins could thus be candidates for proteins involved in regulatory circuits. The respective genes will be cloned and introduced into reporter strains to further investigate the influence of their products on the regulation of the catabolic phenol operon.

Concluding remarks The proteome of the P. putida KT2440 derivative PG150 was investigated by means of 2D electrophoresis and MALDI-TOF analysis. Seventy-eight proteins, differentially expressed at growth in minimal medium with phenol, pyruvate, succinate or glucose were identified. These differences might be not only due to the carbon source itself, but also to the growth rate of the culture and to the energy state of the bacterial cells. The impact of the different carbon sources on the expression of particular functional protein groups, like stress-, detoxification- and transportproteins and on the amino acid, nucleotide and energetic metabolism was recorded. The analysis of protein synthesis following shift-up from a poor (phenol) to a preferred carbon source (succinate) showed a differential regulation of genes, involved in the catabolism of aromatics (phenol) via the ortho-pathway in P. putida KT2440, by the mechanism of carbon CR. Furthermore, a set of genes, involved in transport, stress response and nucleotide and amino acid metabolism, as shown to be most likely regulated by CR too. The data, obtained in these experiments, provide only a first insight into the metabolic changes of P. putida cells, which were exposed to drastic changes of their nutritional environment. To achieve more information on the metabolic processes and interactions, induced by these changes, investigations of the transcriptom and the metabolom are intended.


476 L. Kurbatov, D. Albrecht, H. Herrmann and L. Petruschka Experimental procedures Bacterial strains, media and growth conditions The construction of the strain P. putida PG150 was described by (Müller et al., 1996). Cells of PG150 were grown either in rich medium (preculture) or in modified M9 minimal medium (Miller, 1972), containing 2 mM MgSO4 and 10 µM FeSO4. Carbon sources were added to M9 medium to a final concentration of 40 mM, except phenol, which was used at 2.5 mM. Neomycin was used at concentrations of 50 µg ml−1 in M9 medium and 100 µg ml−1 in rich medium respectively. Cells were cultured at 30°C and growth was monitored by measuring the optical density (OD) at 600 nm.

Repression experiments (‘shift-up’) and 35-S methionine labelling of cellular proteins Bacteria were grown overnight in M9 medium containing phenol. Thereafter, fresh phenol (2.5 mM) was added and the culture was incubated for additional 3 h. Then the culture was diluted to an OD600 of 0.4–0.5 with fresh M9 medium and phenol was added to a concentration of 2.5 mM. The culture was grown for 30 min and was split in two parts and succinate (40 mM) was added to one part of the culture. Five millilitre aliquots of these cultures were incubated for 5 min with 35-S-methionine (15 mCi ml−1) at time zero and 15, 30, 60 and 120 min after the addition of succinate. The cultures were chased on ice with 0.06 mg ml−1 unlabelled methionine and 0.04 mg ml−1 chloramphenicol. The cells were harvested by centrifugation and treated as described below. Samples for 2D electrophoresis were prepared from three independent growth experiments.

Sample preparation Bacteria were harvested in the logarithmic growth phase (OD600 of 0.5–0.8) by centrifugation at 4°C, washed twice with cold Ws buffer (10 mM Tris, 1 mM EDTA, 1 mM PMSF pH 8) and then either used immediately or stored at −80°C. For extract preparation the pellet was resuspended in 500 µl icecold Ws buffer and disrupted by sonication on ice (Sonoplus, Bandelin; 5 × 20 s). The extracts were cleared by centrifugation for 30 min at 20 000 g and 4°C. The protein content of cell extracts was determined by the method of Lowry and colleagues (1951), with bovine serum albumin (BSA) as standard. The supernatants were kept on ice, supplemented with DNase K (Roche) for 1 h and then desalted using NAP-10 columns (Amersham). Aliquotes of the eluates were dried under vacuum at room temperature and stored at −80°C. Samples for the preparation of the reference gels for each carbon source were harvested from at least three independent cell cultures.

10 and bromphenol blue. IPG-Strips (18 cm, pI range 4–7; Amersham) were rehydrated at room temperature for 24 h in the appropriate protein containing solutions. Isoelectric focusing was performed in a MultiphorII system (Amersham) at 20°C using the following parameters: (1) gradient 0–500 V for 500 Vh, (2) a constant potential of 500 V for 2500 Vh, (3) gradient 500–3500 V for 10 kVh, (4) a constant potential of 3500 V for 35 kVh. After IEF, the individual strips were incubated at room temperature in equilibration solution A (50 mM Tris, 6 M Urea, 30% glycerin, 10% SDS containing 3.5 mg ml−1 DTT) for 15 min and in equilibration solution B (containing 45 mg ml−1 iodoacetamide instead of DTT) for additional 15 min. In the second dimension the proteins were separated on a 12.5% SDS-polyacrylamide gel with the Investigator(tm) 2D electrophoresis system (Perkin Elmer Life Sciences) in a standard Laemmli-buffer system. The electrophoresis was performed for 5 min at 8 W/gel, for 2 h 5 W/gel and the rest of the run was completed at 2 W/gel. Protein spots were visualized by silver staining and by Coomassie staining for MALDI-TOF analysis. The stained gels were scanned and analysed with Melanie 4 2D PAGE image analysis software (Free Viewer, Swiss Institute of Bioinformatics, Switzerland). Gels, containing radiolabelled proteins (samples from repression experiments) were silver stained, scanned, dried with a Vacuum-Gel Dryer at 75°C and autoradiographed on a Phosphoimager (Molecular Dynamics) with an exposure time between 2 and 7 days.

Protein identification and database search For protein identification, spots from preparative, Coomassie stained gels were cut manually, digested in gel with trypsin and MALDI-TOF measurements were carried out on a Proteome – Analyzer 4700 (Applied Biosystems, Foster City, CA, USA) as described by (Krah et al., 2003). The identification of spots was performed via batch mode using the Mascot protein identification system (Matrix Science, London, UK) applying the recent P. putida KT2240 protein database downloaded from The Institute for Genomic Research (TIGR, http:/ /www.tigr.org/), supplemented with peptide sequences of the phl-genes from P. putida H (Herrmann et al., 1995). Optimal search parameters were 30 p.p.m. peptide mass tolerance, fixed oxidation of methionine, and one missed trypsin cleavage. The criterion for reliable identification was a significant Mascot score >45 (P < 0.05) (Perkins et al., 1999). A peptide hold for identified if the same result was reached in at least two different experiments/analyses.

Acknowledgements We thank P. Stremlow and B. Rietow for excellent technical assistance, K. Buettner and U. Maeder for helpful discussions concerning 2D electrophoresis and MALDI-TOF analysis and H. Lehnherr for critical reading of the manuscript.

2D electrophoresis For isoelectric focusing (IEF), dried protein samples, 80 µg for silver-stained or 500 µg for Coomassie-stained gels, were solubilized in 450 µl rehydration buffer containing 8 M urea, 2 M thiourea, 1% CHAPS, 20 mM DTT, 0.5% Pharmalyte 3–

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