1 Philipps-University Marburg, Institute of Physiological Chemistry, D-35032 Marburg, Germany
2 Philipps-University Marburg, Department of Biology, Laboratory for Microbiology, D-35032, Marburg, Germany
3 Max-Planck-Institute for Terrestrial Microbiology, D-35043 Marburg, Germany
4 Ernst-Moritz-Arndt-University, Medical Faculty, Laboratory for Functional Genomics, D-17487 Greifswald, Germany
Correspondence
Klaus-Heinrich Röhm
roehm{at}staff.uni-marburg.de
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ABSTRACT |
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INTRODUCTION |
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The main organic components of root exudates are sugars, various organic acids and a number of amino acids (Fan et al., 1997). Although sugars account for most of the organic matter in exudates, there is no evidence indicating that they play a major role in plantbacterial interactions. Lugtenberg et al. (1999)
could not find a significant contribution of sugars to tomato root colonization by a well-studied Pseudomonas biocontrol strain. On the other hand, it was shown that root exudates can induce bacterial enzymes that are involved in the metabolism of amino acids such as proline (Vilchez et al., 2000a
, b
) or lysine (Espinosa-Urgel & Ramos, 2001
).
The predominant amino acids in root exudates are the acidic amino acids aspartate (Asp) and glutamate (Glu) and their amides asparagine (Asn) and glutamine (Gln) (Barber & Gunn, 1974; Jones & Darrah, 1993
). Glu and Gln are key intermediates in nitrogen metabolism. In E. coli and other enterobacteria all nitrogen-containing compounds derive their nitrogen from Glu or Gln (Reitzer, 1996a
, b
). Nevertheless, Glu and Gln are inferior to
in supporting the growth of enteric bacteria. In pseudomonads the situation is different. We have recently shown that several strains of Pseudomonas fluorescens and Pseudomonas putida rapidly grow on acidic amino acids and their amides, even when supplied as the sole source of carbon and nitrogen (Sonawane et al., 2003
). All of these amino acids strongly and specifically induce periplasmic glutaminase/asparaginase (PGA) (Hüser et al., 1999
). On the other hand, PGA is subject to carbon catabolite repression by glucose and dicarboxylic acids such as succinate, fumarate and 2-oxoglutarate. A PGA knockout mutant was unable to utilize Gln whereas growth on Glu, Asn and Asp was unimpaired.
In order to examine whether the acidic amino acids and their amides, in addition to regulating PGA expression, induce a more general response in pseudomonads, we used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to compare gene expression in pseudomonads in the presence and absence of these amino acids.
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METHODS |
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PGA assay.
Glutaminase/asparaginase (PGA) activities were measured with L-aspartic -hydroxamate as the substrate (Derst et al., 1992
). Briefly, 20 µl enzyme solution was added to 30 µl of 1 mM substrate in 50 mM MOPS, pH 7·0. After an incubation for 560 min at room temperature, the reaction was terminated and colour developed by adding 240 µl stop solution (1 M Na2CO3 containing 2 %, w/v, 8-hydroxyquinoline in dimethyl sulfoxide and 1 %, w/v, NaIO4). After 5 min, the A655 was measured in a Bio-Rad 3550-UV microplate reader. Protein concentrations were determined by the BCA method using bovine serum albumin as the standard. One unit of PGA activity is the amount of enzyme hydrolysing 1 mmol L-aspartic acid
-hydroxamate min-1 under these conditions.
Sample preparation for 2D gel electrophoresis.
Bacteria were usually harvested 46 h after transfer to fresh medium and collected by centrifugation. After washing with M9 salt solution, the pellet was resuspended in TE/PMFS buffer (10 mM Tris, 1 mM EDTA, 0·1 mM PMSF, pH 7·5) cooled on ice and disrupted by sonication (Sonoplus, Bandelin; 15x4 s). The homogenate was centrifuged for 10 min at 10 000 r.p.m. and then twice for 30 min at 14 000 r.p.m. Proteins were precipitated by the addition of 5 vols ice-cold acetone (analytical grade), incubated overnight at -20 °C, and then collected by centrifugation at 0 °C.
2D gel electrophoresis.
For isoelectric focussing (IEF), proteins were solubilized in a rehydration solution containing 8 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 28 mM DTT, 1·3 % (v/v) Pharmalytes, pH 310 and bromophenol blue. After rehydration for 24 h under low-viscosity paraffin oil, Immobiline DryStrips (IPG strips 18 cm NL; Amersham Biosciences) covering a pH range of 310 were subjected to isoelectric focussing with the following voltage/time profile: linear increase from 0 to 500 V for 1000 V h, 500 V for 2000 V h, linear increase from 500 to 3500 V for 10 000 V h and a final phase of 3500 V for 35 000 V h up to a total of 48 000 V h. After IEF, the individual strips were consecutively incubated in equilibration solutions A and B, each for 15 min [50 mM Tris/HCl, pH 6·8, 6 M urea, 30 % (v/v) glycerol, 4 % (w/v) SDS, with 3·5 mg DTT ml-1 (solution A); or 45 mg iodoacetamide ml-1 instead of DTT (solution B)]. In the second dimension, proteins were separated on 12·5 % SDS-polyacrylamide gels with the Investigator System (Perkin Elmer Life Sciences) at 2 W per gel. For routine use proteins were visualized by silver staining. Gels intended for MALDI-TOF analysis were stained with PhastGel Coomassie R350 according to the manufacturer's instructions (Amersham BioSciences). Scanned images were analysed with the Melanie3 software package (Bio-Rad) to facilitate identification of differentially expressed spots. For quantitative densitometry the BandLeader program (Magnitec) was used. Separate gels of each condition were analysed and only spots were labelled that displayed the same pattern (i.e. up- or down-regulation) in all replicates.
Protein identification by N-terminal sequencing.
Spots of interest were transferred to a PVDF membrane by electroblotting using the semi-dry method (Kyhse-Anderson, 1984). Sequencing was performed by Dr D. Linder, Giessen.
Protein identification by peptide mass fingerprinting.
Protein spots were excised from stained 2D gels. Pooled extracts from three to nine gels were destained and digested with trypsin (Promega). Peptides were extracted according to Otto et al. (1996). They were purified with C18 tips according to the manufacturer's instructions (Millipore) and eluted with 75 % acetonitrile/2 % trifluoroacetic acid (v/v). Peptide solutions were mixed with an equal volume of saturated
-cyano-3-hydroxycinnamic acid solution in 50 % acetonitrile/0·1 % trifluoroacetic acid (v/v) and applied to a sample template for a MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer. Peptide masses were determined in the positive ion reflector mode in a Voyager DE RP mass spectrometer (PerSeptive Biosystems) with internal calibration. Mass accuracy was usually in the range between 10 and 50 p.p.m. Peptide mass fingerprints were compared to databases using the program MS-Fit (http://prospector.ucsf.edu). Spots that could not be identified by the above method were further analysed by MALDI-Post Source Decay (PSD) sequencing (Protagen AG, Bochum, Germany).
Semi-quantitative RT-PCR.
RNA was isolated from mid-exponential-phase cells using the RNeasy minikit (Qiagen). Residual DNA was removed by digestion for 30 min at 37 °C with 1 U µl-1 RNase-free DNase (Promega). The reaction was stopped by adding 1 µl RQ1 DNase stop solution and incubated at 65 °C for 10 min. The reverse transcription was carried out using 1 µg RNA in a 20 µl reaction mixture containing 1x RT buffer, 0·1 mM dNTP, 50 pmol oligo(dT) primer, 5 mM MgCl2, 20 mM DTT, 2 U RNaseOUT Recombinant RNase inhibitor, 0·25 U SUPERSCRIPT II RT (Invitrogen). PCR was carried out in 50 µl reaction mixtures, containing 2 µl of the RT reaction as template for Pfu Turbo DNA polymerase (0·5 U, Stratagene), 100 pmol of each primer (Table 1), 0·2 mM dNTP, and amplified for 26 cycles. The PCR sequence used was: 94 °C, 60 s; 54 °C, 30 s; 72 °C, 4 min and 72 °C, 5 min. cDNA-specific primers (listed in Table 1
) were derived from the P. putida KT2440 genome (Nelson et al., 2002
). Ten microlitres of RT-PCR products was then subjected to electrophoresis in a 1·5 % agarose gel and visualized by staining with ethidium bromide.
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RESULTS |
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Regulation of PGA expression by amino acids and alternative carbon sources
As reported previously, the expression of periplasmic glutaminase/asparaginase (PGA) in pseudomonads is strongly and specifically enhanced by acidic amino acids and their amides, while good carbon sources such as glucose or tricarboxylic acid cycle intermediates repress PGA production (Hüser et al., 1999). Surprisingly, in P. fluorescens ATCC 13525, Asp and Glu rather than Asn and Gln were found to be the actual inducers, while in P. putida KT2440 the time-courses of PGA induction by Asn and Asp on the one hand, or Gln and Glu on the other, were almost the same (Sonawane et al., 2003
).
As in P. fluorescens ATCC 13525, the expression of PGA in P. putida KT2440 is subject to carbon catabolite repression by good carbon sources. Fig. 1 shows the effects of sugars (glucose, sucrose) and intermediates of the citric acid cycle (2-oxoglutarate, fumarate) on PGA expression. Glucose, fumarate and 2-oxoglutarate almost completely prevented PGA induction by Glu, while sucrose, which is not metabolized by P. putida KT2440, was much less effective as a repressor. As described elsewhere (Sonawane et al., 2003
), PGA induction by Asn and Asp is delayed as compared to that by Glu and Gln. Therefore, full PGA induction by Asp or Asn was only seen in samples taken after 24 h.
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Spots Pf1Pf6 responded in a similar fashion to different carbon and nitrogen sources. Fig. 3 compares their relative densities estimated by quantitative densitometry. Although the extent of induction and carbon catabolite repressions varied to some extent, the same general pattern was seen with all spots examined. As compared to growth on
/glucose, their expression was strongly upregulated by Asn and Asp, while the effect of Asn was markedly reduced by fumarate.
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DISCUSSION |
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The Rho protein (spot Pp1) is known to participate in the regulation of tryptophanase expression in E. coli: tryptophan enhances the transcription of tryptophanase by Rho-mediated antitermination (Konan & Yanofsky, 2000). It is unknown whether comparable mechnisms also operate in the regulation of Gln and Glu metabolism of other bacteria. The essential role of PGA (Pf2; Pp3/4) in the utilization of Gln by P. putida KT2440 has been established previously (Sonawane et al., 2003
). The origin of the minor spot Pp4 (cf. Fig. 4
) has not yet been investigated in detail. It may correspond to a PGA variant in which one or more Asn or Gln residues have been degraded to the respective dicarboxylates. However, the existence of a phosphorylated or otherwise covalently modified form of the enzyme cannot be excluded.
PGA hydrolyses Asn and Gln at similar rates and thus can generate both dicarboxylates (Glu and Asp) for uptake by transport systems in the inner membrane. The amino-acid-binding protein Pf2 (encoded by PP10171 in P. putida KT2440) and the ATP-binding protein Pp5 (endoded by PP1068) could both belong to such a transport system of the ABC type which possibly mediates the uptake of the acidic amino acids and/or their amides. This assumption is based on the genetic organization of certain Glu-related genes in P. aeruginosa, P. putida and P. fluorescens (see Fig. 7). In P. aeruginosa PAO1, a series of eight consecutive genes (PA1342PA1335) encode proteins that all appear to be involved in the uptake and utilization of acidic amino acids. PA13421339 code for an ABC transporter that, by sequence similarity, mediates amino acid uptake. Two subsequent genes, PA1338 and PA1337, encode a
-glutamyltransferase and PGA (ansB), respectively, while PA1336 and PA1335 encode a two-component system with strong similarity to dctBD, a system controlling dicarboxylate utilization in rhizobia (Wang et al., 1989
). In P. putida KT2440, the genes for a closely related two-component system (PP10671066) are immediately adjacent to those encoding the ABC transporter mentioned above (PP10711068) while the ansB gene (PP2453) and the
-glutamyltransferase gene (PP4659) are located elsewhere. The sequence identity between the ABC transporters of the two strains (PP10711068 and PA13421339) is 85 % at the protein level while the two-component systems (PP10671066 and PA13361335) have 76 % of the amino acid residues in common. A similar arrangement of genes for an ABC transporter and a two-component system was detected in the unfinished genome data of P. fluorescens SBW25 (available at the Sanger Institute, http://www.sanger.ac.uk/Projects/P_fluorescens/). The respective open reading frames which are contained in fragment Pflu346g05.q1kb show a very high degree of sequence similarity to PP10651071. The predicted amino acid sequence of gene 2 (see Fig. 7
), which encodes a periplasmic solute-binding protein, contains the complete N-terminal sequence determined for spot Pf2 (i.e. AELTGTLKKINDXGT, see Table 2
), and the predicted sequence of gene 5 (the ATP-binding component of the ABC transporter) shows 93 % identity with the predicted sequence of PP1068.
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The outer-membrane porin D (Pp8) facilitates the uptake of amino acids and/or peptides by P. aeruginosa (Trias & Nikaido, 1990). Ochs et al. (1999)
further showed that oprD expression in this organism is strongly enhanced by amino acids (including Glu, Arg and Ala) and repressed by succinate. The arginine-mediated induction of oprD was mediated by the regulatory protein ArgR, whereas the Glu-induced expression of OprD was independent of ArgR, indicating the presence of more than a single activation mechanism.
Unlike the proteins discussed above, Pp9, a putative carboxyphosphonoenolpyruvate phosphonomutase, has no apparent relation to amino acid metabolism. The only known function of this enzyme is to catalyse a step in the biosynthesis of the antibiotic bialaphos in Streptomyces hygroscopicus (Lee et al., 1995). However, the deduced amino acid sequence of Pp9 also shows similarity to more common phosphopyruvate hydratases from intermediary metabolism (e.g. enolase; Lee et al., 1995
). Thus, the annotation of PP1389 as a PEP phosphonomutase may be erroneous. Further experiments are required to characterize the role of Pp9 in P. putida KT2440.
The proteins upregulated during growth on /glucose (Pp10Pp13) can be related to the uptake and degradation of glucose (Pp11, Pp12) or diamines, respectively (Pp10 and Pp13). The transaminase Pp10 was shown to catalyse a step in the biosynthesis of 1,3-diaminopropane by Acinetobacter baumannii (Ikai & Yamamoto, 1997
). However, other functions appear to be possible as well, for instance the synthesis of Glu from 2,4-diaminobutyrate.
The increased synthesis of a sugar uptake system during growth on glucose (spot Pp12) is not surprising, while the upregulation of putrescine uptake (Pp13, gene: potF) is more difficult to explain. Putrescine, a component of root exudates, was shown to inhibit growth of P. fluorescens WCS365 and its ability to colonize tomato roots (Kuiper et al., 2001). Sauer & Camper (2001)
, studying changes in gene expression during attachment of P. putida to surfaces, found that 15 proteins were upregulated following bacterial adhesion and 30 proteins were downregulated. The downregulated proteins include the potF gene product (Pp13) as well as PGA (Pp3/4) and other proteins involved in amino acid uptake and metabolism. Although these findings are difficult to interpret at present, they support the notion that profound changes in the metabolism of amino acids and polyamines accompany the change from free-living to sessile growth in pseudomonads.
Our present data further indicate that most of the proteins upregulated by Glu depend on the alternative sigma factor 54 (RpoN) for expression. With the possible exception of aspartase (Pp6), none of the Glu-responsive proteins was synthesized in an RpoN- mutant of strain KT2440. As a result of this diminished presence of amino-acid-metabolizing enzymes, this strain exhibits a severe growth defect in media lacking glucose and NH4Cl (Köhler et al., 1989
). It is now well established that activation of the
54RNA polymerase holoenzyme requires additional enhancer-binding proteins with ATPase activity to stimulate transcription (Buck et al., 2000
). The function of these proteins is to facilitate conversion of the closed promoter complex to an open one. Usually the enhancer proteins are so-called response regulators i.e. proteins that transmit environmental signals from a membrane-bound sensor kinase to the transcription complex (Chang & Stewart, 1998
). A set of individual genes and/or operons controlled by one and the same response regulator is called a regulon. In our opinion, the present data indicate that the P. putida proteins upregulated by acidic amino acids and their amides (and other proteins not yet identified) may all be products of a regulon responsible for their uptake and metabolism. In order to substantiate this hypothesis, it has to be demonstrated that a single response regulator binds to and enhances transcription of these genes. Experiments aiming at the identification of such a response regulator are now under way in our laboratory.
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Received 2 May 2003;
revised 14 July 2003;
accepted 14 July 2003.
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