Pseudomonas aeruginosa PAO1 genes for 3-guanidinopropionate and 4-guanidinobutyrate utilization may be derived from a common ancestor

Yuji Nakada1 and Yoshifumi Itoh2

1 Department of Nursing, Faculty of Nursing and Rehabilitation, Aino University, Higashiohda 4-5-4, Ibaraki, Osaka 567-0012, Japan
2 Akita Research Institute of Food and Brewing, Sanuki 4-26, Akita 010-1623, Japan

Correspondence
Yoshifumi Itoh
yosifumi{at}arif.pref.akita.jp


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pseudomonas aeruginosa PAO1 utilizes 3-guanidinopropionate (3-GP) and 4-guanidinobutyrate (4-GB), which differ in one methylene group only, via distinct enzymes: guanidinopropionase (EC 3.5.3.17; the gpuA product) and guanidinobutyrase (EC 3.5.3.7; the gbuA product). The authors cloned and characterized the contiguous gpuPAR genes (in that order) responsible for 3-GP utilization, and compared the deduced sequences of their putative protein products, and the potential regulatory mechanisms of gpuPA, with those of the corresponding gbu genes encoding the 4-GB catabolic system. GpuA and GpuR have similarity to GbuA (49 % identity) and GbuR (a transcription activator of gbuA; 37 % identity), respectively. GpuP resembles PA1418 (58 % identity), which is a putative membrane protein encoded by a potential gene downstream of gbuA. These features of the GpuR and GpuP sequences, and the impaired growth of gpuR and gpuP knockout mutants on 3-GP, support the notion that GpuR and GpuP direct the 3-GP-inducible expression of gpuA, and the uptake of 3-GP, respectively. Northern blots of mRNA from 3-GP-induced PAO1 cells revealed three transcripts of gpuA, gpuP, and gpuP and gpuA together, suggesting that gpuP and gpuA each have a 3-GP-responsible promoter, and that some transcription from the gpuP promoter is terminated after gpuP, or proceeds into gpuA. Knockout of gpuR abolished 3-GP-dependent synthesis of the transcripts, confirming that GpuR activates transcription from these promoters, with 3-GP as a specific co-inducer. The sequence conservation between the three functional pairs of the Gpu and Gbu proteins, and the absence of gpuAPR in closely related species, imply that the triad gpu genes have co-ordinately evolved from origins common to the gbu counterparts, to establish an independent catabolic system of 3-GP in P. aeruginosa.


Abbreviations: ABS, activation binding site; ADH, arginine dehydrogenase; 4-GB, 4-guanidinobutyrate; 3-GP, 3-guanidinopropionate; RBS, recognition binding site


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Unlike many pseudomonads that utilize 4-guanidinobutyrate (4-GB), but not 3-guanidinopropionate (3-GP), as the carbon and nitrogen source, Pseudomonas aeruginosa can use either (Tricot et al., 1990; Nakada & Itoh, 2002; Yorifuji et al., 1983). The catabolic route of 4-GB in Pseudomonas putida and P. aeruginosa is integrated into the arginine dehydrogenase (ADH) pathway of arginine catabolism (Chou & Rodwell, 1972; Cunin et al., 1986; Haas et al., 1990; Jann et al., 1988; Vanderbilt et al., 1975) (Fig. 1). This pathway of P. putida starts with the oxidative deamination of L-arginine by adh (also called L-arginine oxidase) to 2-ketoarginine, which is decarboxylated to 4-guanidinobutyraldehyde. The oxidation product of 4-guanidinobutyraldehyde, 4-GB, is converted by guanidinobutyrase (EC 3.5.3.7; the gbuA product) into 4-aminobutyrate, before being channelled into the tricarboxylic acid cycle (Fig. 1). P. aeruginosa PAO1 also has all of the ADH pathway enzymes, except for ADH itself. Racemase and D-arginine dehydrogenase are thought to convert L-arginine to 2-ketoarginine via the D-isomer in this bacterium (Fig. 1). Exogenous 2-ketoarginine and its downstream intermediates, including 4-GB, are effective carbon and nitrogen sources for this strain (Haas et al., 1990; Jann et al., 1988; Nakada & Itoh, 2002).



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Fig. 1. Catabolism of 3-GP and 4-GB in P. aeruginosa PAO1. Only relevant intermediates and genes are presented. The gpuA and gbuA genes encode guanidinopropionase and guanidinobutyrase, respectively.

 
The P. aeruginosa PAO1 GbuA has been purified and characterized in some detail (Nakada & Itoh, 2002; Yorifuji & Sugai, 1978). This enzyme belongs to the arginase/agmatinase family of proteins, and is highly specific to 4-GB. Guanidinopropionase (EC 3.5.3.17) has also been purified from this strain (Yorifuji & Sugai, 1978; Yorifuji et al., 1982, 1983). We refer to the gene for this enzyme as gpuA. GpuA catalyses the specific hydrolysis of 3-GP into {beta}-alanine and urea (Yorifuji & Sugai, 1978); the former product can be used as a source of carbon and nitrogen, and as a precursor for the synthesis of fatty acids, amino acids and other metabolites, after conversion into acetyl-CoA (Fig. 1). The strict substrate specificity of both GbuA and GpuA has allowed the isolation of gbuA9005 (defective in 4-GB utilization) and gpu-9018 (defective in 3-GP utilization) mutants (Haas et al., 1984). We cloned and characterized gbuA, along with the cognate gbuR regulator gene, which encodes a regulatory protein that directs the inducible expression of gbuA in the presence of exogenous 4-GB (Nakada & Itoh, 2002). The gpu-9018 locus has been mapped at 18 min of the chromosome, but not yet cloned (Haas et al., 1984; Holloway et al., 1994). Although GbuA and GpuA use different substrates with high specificity, both enzymes act on a scissile guanidino-bond, and they have a Mn2+ requirement for activity, a high optimum pH, and similarly sized native forms and subunits (Nakada & Itoh, 2002; Yorifuji et al., 1982). Furthermore, peptide maps of the enzymes have suggested some similarity in their amino acid sequences (Yorifuji et al., 1983).

To examine the molecular structure of GpuA, and the regulatory mechanism underlying the specific induction of GpuA synthesis by exogenous 3-GP, we cloned the gpu-9018 locus, and identified three genes (gpuPAR) that are required for 3-GP utilization. Structural and functional analyses of these genes established that gpuA is an allele of gpu-9018, that gpuP encodes a 3-GP transport protein having homology to PA1418 (a gene downstream of gbuA), and that gpuR specifies a transcription activator of gpuPA. The striking sequence similarity between the Gpu and Gbu counterparts implies that the gpu and gbu gene trios have evolved from common triad ancestors.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids and media.
P. aeruginosa PAO strains and Escherichia coli (Table 1) were cultured in nutrient yeast broth (NYB) or Luria–Bertani (LB) medium, respectively, supplemented with antibiotics when necessary (Haas et al., 1977; Hoang et al., 1998; Sambrook et al., 1989). The P. aeruginosa PAO strains were grown in minimal medium P (MMP; Haas et al., 1977) containing the indicated carbon and nitrogen sources at a concentration of 20 mM for strain construction, growth experiments and enzyme assays.


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Table 1. Strains and plasmids

 
DNA techniques.
DNA purification, restriction enzyme analysis, and other DNA techniques were carried out as described (Nakada & Itoh, 2002; Nishijyo et al., 2001; Sambrook et al., 1989). Nucleotide sequences on both strands were determined using an ABI310 DNA sequencer (Perkin-Elmer) and ABI Big-dye Terminator Cycle sequencing kits (Perkin-Elmer).

Cloning the gpu-9018 locus.
A plasmid library of Sau3AI fragments of the PAO1 chromosomal DNA was constructed in E. coli XL-1 Blue by shotgun cloning into the mobilizable shuttle vector pNIC6011 (Nishijyo et al., 2001), and library plasmids were then transferred into strain PAO4173 (gpu-9018) by conjugation using the helper E. coli HB101/pRK2013 (Comai et al., 1983), followed by selection of 3-GP-utilizing (Gpu+) transconjugants on MMP agar containing 125 µg carbenicillin ml–1, and 20 mM 3-GP as the carbon and nitrogen source. After restriction analysis, and sequencing the plasmid inserts in several Gpu+ transconjugants, we further investigated plasmid pYJ104 carrying a 4·5 kb gpu-9018 fragment.

Plasmid and strain construction.
To localize gpu-9018 on the chromosomal DNA region cloned in plasmid pYJ104 by complementation tests, we constructed deletion and insertion derivatives as follows. Removal of the 1·0 kb BglII–HindIII fragment carrying portions of PA289 and PA290 yielded plasmid pYI1035 (Fig. 2b). Insertion of a gentamicin-resistant (Gm) cassette from plasmid pPS858 (Hoang et al., 1998) into the PvuII site of PA288 as a SmaI fragment, and into the BamHI site of PA287, as a BamHI fragment, created plasmids pYI1041 and pYI1043, respectively (Fig. 2b). Similarly, insertion of an {Omega}Sp/Sm interposon (Fellay et al., 1987) into the same sites of PA288 and PA287 resulted in plasmids pYI1047 and pYI1057, respectively (Fig. 2c). Insertion of the interposon into the BglII site in the 3' region of PA286 generated plasmid pYI1060 (Fig. 2c). To construct knockout mutants of PA287, PA288 and PA289, appropriate DNA regions carrying the genes of interest were cloned into the suicide plasmid pEX18Ap (mob+ sacB) (Hoang et al., 1998), followed by the insertion of a Gm cassette (Hoang et al., 1998) into the BamHI, PvuII and SmaI sites of PA287, PA288 and PA289 on the resultant plasmids, respectively. These constructs were then conjugated into strain PAO1 (Nishijyo et al., 2001), and GmR transconjugants harbouring the plasmid sequences integrated at the corresponding chromosome locus by recombination were selected on LB agar containing gentamicin (100 µg ml–l). Subsequent selection on LB containing 5 % (w/v) sucrose, which prevents the growth of cells having the pEX18Ap (sacB) sequence (Hoang et al., 1998), yielded strains PAO4520 (PA289=gpuR : : Gm), PAO4522 (PA288=gpuA : : Gm) and PAO4524 (PA287=gpuP : : Gm). Correct insertion of the Gm cassette and {Omega}Sp/Sm at the relevant sites was verified by PCR (Nishijyo et al., 2001).



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Fig. 2. Gene organization of (a) the gpuPAR locus, and knockout mutants of the gpu genes, (b) plasmids used in complementation tests, and (c) plasmids used in analysis of gpuPAR transcription in vivo. (a) Arrows below the map indicate transcripts of gpuPAR genes. (b) Plasmids were derived from plasmid pYJ104 by deletion or by insertion of a Gm cassette (Hoang et al., 1998) at the indicated restriction sites. Complementation tests were carried out on MMP agar (Haas et al., 1977) supplemented with 20 mM 3-GP as the source of carbon and nitrogen. +, Growth of single colony within 18 h; –, no growth at 48 h. (c) Mutant plasmids were constructed by insertion of a {Omega}Sp/Sm interposon carrying a transcription terminator (Fellay et al., 1987) at the indicated restriction sites of the target genes. Abbreviations for restriction sites: Ba, BamHI; Bg, BglII; E, EcoRI; H, HindIII; Pv, PvuII; Sm, SmaI; Su, Sau3AI (only sites used in construction of pYJ104 are indicated); X, XhoI.

 
Northern blotting, primer extension and RT-PCR.
Total RNA was isolated from PAO1 cells exponentially growing (OD600 0·3) in MMP containing the indicated carbon and nitrogen sources at 20 mM, as described (Nishijyo et al., 2001). Samples of RNA (50 µg) in 10 % (w/v) glyoxal were resolved, together with RNA markers (Toyobo Biochemicals), on 1·0 % (w/v) agarose HS (Nippon Gene), and blotted onto Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) using a GenVac Blotter (Pharmacia LKB). Transcripts of gpuAPR blotted on the membranes were hybridized with a gpuA probe (a PvuII–BstEII fragment of 421 bp; nt +380 to +800), or a gpuP probe (a 921 bp EcoO109I–SmaI fragment; nt –81 to +840) labelled with [{alpha}-32P]dCTP (220 TBq mmol–1; Amersham Pharmacia Biotech). The hybridized DNA fragments on the membranes were detected by exposure to X-ray film. In primer extension experiments, RNA samples (20 µg) were annealed with oligonucleotides (5'-GCGCATGAAGGTCGGGATACCGGCGAAG-3', complementary to the region at nt +48 to +75 of gpuA; and 5'-CGAGGAAATCGCTCTGGCTCTTCGCCTTG-3', complementary to the region at nt +72 to +100 of gpuP), and end-labelled with [{gamma}-32P]ATP (220 TBq mmol–1; Amersham Pharmacia Biotech) using polynucleotide kinase (Takara Bio). A complementary strand was synthesized using avian reverse transcriptase (RAV-2; Takara Bio) in the presence of deoxyribonucleotides. Synthesized cDNAs were then resolved on 6 % (w/v) denatured polyacrylamide gels, along with sequence ladders generated using the BcaBEST sequencing kit (Takara Bio), the 32P-end-labelled oligonucleotide primers, and plasmid pYI104 as the template. Radioactive DNA fragments on the gels were visualized on X-ray film. We used a BcaBEST RNA PCR kit (Takara Bio) to confirm read-through of the transcription from the gpuP promoter into gpuA, and to determine relative amounts of gpuA and gpuP transcripts by RT-PCR. cDNAs of gpuP and gpuA mRNA regions were synthesized using the above total RNA samples, and oligonucleotides complementary to +933 to +957 of gpuA (primer A1), and +664 to +692 of gpuP (primer P1), respectively, under the conditions recommended by the supplier; primer A1 forms both gpuA and gpuPA cDNAs (gpuA/PA cDNA), and primer P1 forms gpuP cDNAs. Amplification of various regions by PCR (denaturing at 94 °C for 30 s, annealing at 57 °C for 30 s, and elongation at 72 °C for 1 min; 28 cycles) proceeded using the following cDNAs and oligonucleotide pairs: gpuA/PA cDNA with primers A1 and A2 (nt +273 to +302 of gpuA) for gpuA cDNA; gpuP cDNA with primers P1 and P2 (corresponding to nt –37 to –9 of gpuP) for gpuP cDNA; and gpuA/PA cDNA with primers PA1 (nt +992 to +1020 of gpuP) and PA2 (complementary to +312 to +340 of gpuA) for the intergenic region between gpuP and gpuA. The PCR products were resolved on 1·0 % agarose gels by electrophoresis, and stained with ethidium bromide.

Enzyme assays.
We prepared extracts from cells growing exponentially (OD600 0·5) in MMP containing L-glutamate (a control), 3-GP, L-glutamate and 3-GP, or 4-GB, as sole carbon and nitrogen sources (each at 20 mM), as described (Nakada & Itoh, 2002). Guanidinopropionase was assayed (in triplicate) under the same conditions as guanidinobutyrase (Nakada & Itoh, 2002), except the pH of the glycine buffer was 9·0 instead of 9·5. The reaction product, urea, was measured according to Chou & Rodwell (1972). One enzyme unit was defined as the amount of enzyme required to form 1 µmol product min–1. Protein concentrations were determined using a Protein Assay Kit (Bio-Rad), with BSA as the standard.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and structure of the gpuAPR genes
We cloned a 4·5 kb Sau3AI fragment carrying the gpu-9018 locus into plasmid pYJ104 (Fig. 2b) by functional complementation of the 3-GP-utilization (Gpu+) phenotype of strain PAO4173 (gpu-9018), as described in Methods. Nucleotide sequencing revealed that this insert corresponded to nt 321 848 to 326 320 of the PAO1 genome, which contain three complete and two truncated genes of suggested or unknown function (http://www.pseudomonas.com) (Fig. 2a, b). The first gene was the 3' portion of PA286, encoding a probable fatty acid desaturase with 85 % similarity to the stearoyl-CoA desaturase of Azotobacter vinelandii (Mylona et al., 1996). The second gene (PA287) encoded a possible transport protein (461 aa) of Mr 48 461 with 12 potential transmembrane helices, and 46 % similarity to the PanF sodium/pantothenate symporter of E. coli (Jackowski & Alix, 1990). The product of this gene had 58 % identity to the product of PA1418 located downstream of gbuA (Nakada & Itoh, 2002). The third gene (PA288) specified a putative amidinohydrolase (371 aa, Mr 38 859) that shares 57 % similarity with E. coli agmatinase (the speB product), a putrescine biosynthetic enzyme (Glansdorff, 1996), and hence called speB1 (http://www.pseudomonas.com). The fourth gene (PA289) specified a putative regulatory protein (297 aa, Mr 33 385) homologous (<40 % identity) to the LysR family of proteins (Schell, 1993), including GbuR (37 % identity). The fifth, and last, truncated gene (PA290) on the insert encoded a polypeptide (232 aa) of Mr 36 387 that was homologous to the PleD protein of Caulobacter crescentus (Hecht & Newton, 1995). Knockout of PA287 and PA289, as well as PA288 (speB1), resulted in impaired growth on 3-GP (see below), suggesting that these genes are important for 3-GP utilization. We therefore designated PA287, PA288 and PA289 as gpuP, gpuA and gpuR, respectively (Fig. 2a).

Identification of gpu-9018 as gpuA
To localize gpu-9018 on the cloned DNA fragment, we constructed deletion and insertion derivatives from plasmid pYJ104, and tested their ability to restore the Gpu+ phenotype of strain PAO4173 (gpu-9018). Plasmid pYI1035, having a deletion in the 3' portion of gpuR, restored the Gpu+ phenotype of the mutant, whereas plasmid pYI1041, having an insertion of a Gm cassette in gpuA, did not (Fig. 2b). Moreover, inactivation of gpuA on the chromosome by insertion of the Gm cassette, as in strain PAO4522 (Fig. 2b), abolished GpuA synthesis, resulting in the Gpu phenotype (see below). These results, and the similarity of the molecular mass between the deduced GpuA (Mr 38 869) and purified guanidinopropionase (35 kDa) (Yorifuji et al., 1982), supported the notion that gpuA is an allele of gpu-9018, and specifies guanidinopropionase. We accordingly renamed gpu-9018 as gpuA9018. Sequencing of gpuA9018 identified a transversion of T at nt 168 to G, which caused an alteration of His at amino acid position 56 to Gln.

Functions of gpuP and gpuR in 3-GP utilization
Strain PAO1 did not form a measurable amount [<0·1 units (mg protein)–1] of GpuA during growth in MMP containing either L-glutamate or 4-GB. When 3-GP was present, this strain produced the enzyme in quantities of up to 1·4±0·1 units (mg protein)–1. When presented together with 3-GP, L-glutamate minimally affected GpuA synthesis [1·2±0·1 units (mg protein)–1]. To determine whether gpuP and gpuR are involved in 3-GP utilization, and affect GpuA synthesis, we constructed knockout mutants PAO4524 (gpuP : : Gm) and PAO4520 (gpuR : : Gm) by inserting a Gm cassette (Hoang et al., 1998) into the BamHI site of gpuP, and into the SmaI site of gpuR, respectively, on the chromosome of strain PAO1 (Fig. 2a). Strains PAO4524 (gpuP : : Gm) and PAO4520 (gpuR : : Gm) did not grow on MMP agar containing 3-GP as the sole source of carbon and nitrogen. The amounts of GpuA in these knockout mutants cultured in MMP containing L-glutamate and 3-GP were negligible [<0·1 units (mg protein)–1]. The observed Gpu phenotype of the mutants thus appeared to correlate with the inability to express GpuA, probably as a consequence of impaired uptake of the inducer 3-GP (in strain PAO4524), and of the absence of the GpuR transcription activator for the enzyme gene (in strain PAO4520).

Transcription units of the gpuPAR genes
The gpuPAR genes are all located in the same orientation, with intergenic spaces for promoters: 230 bp between PA286 and gpuP, 56 bp between gpuP and gpuA, and 53 bp between gpuA and gpuR (Fig. 2a). We initially analysed transcription units of gpuPAR in vivo using plasmids with an inserted {Omega} interposon carrying a transcriptional terminator (Fellay et al., 1987) on PA286, gpuP or gpuA (Fig. 2c). Plasmids pYI1047 (gpuA : : {Omega}Sp/Sm), pYI1057 (gpuP : : {Omega}Sp/Sm) and pYI1060 (PA286 : : {Omega}Sp/Sm) (Fig. 2c) each restored the Gpu+ phenotype of strains PAO4520 (gpuR : : Gm), PAO4522 (gpuA : : Gm) and PAO4524 (gpuP : : Gm), respectively, indicating that the gpuPAR genes each have their own promoter (Fig. 2a).

Transcription from the predicted gpuA (PgpuA), gpuP (PgpuP) and gpuR (PgpuR) promoters was further investigated by Northern blotting, using RNA samples prepared from PAO1 cells cultured in MMP containing a non-inducible substrate, glutamate or 4-GB, or the inducible substrate 3-GP, and from PAO4520 (gpuR : : Gm) cells cultured in MMP containing both L-glutamate and 3-GP (inducible conditions). No gpuP and gpuA transcripts were detected in RNA samples from cells grown in L-glutamate or 4-GB medium (Fig. 3, lanes 1, 2, 4 and 5), or from the gpuR mutant cells (data not shown). In contrast, 1300 and 2400 nt transcripts were detected with a gpuA probe (+380 to +800 of gpuA) in the RNA sample from the cells with induced 3-GP (Fig. 3, lane 3) at a ratio of 7·6 : 1. Probing with a gpuP sequence (nt –81 to +840 of gpuP) detected 2400 and 1500 nt transcripts in the same RNA sample (Fig. 3, lane 6). These results support the notion that PgpuP and PgpuA exist, and suggest that some transcription from PgpuP proceeds into gpuA.



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Fig. 3. Northern blots of gpuPA transcripts. RNA samples prepared from PAO1 cells grown in MMP plus 20 mM glutamate (lanes 1 and 4), MMP plus 20 mM 4-GB (lanes 2 and 5), and MMP plus 20 mM 3-GP (lanes 3 and 6), were resolved on agarose gels and blotted onto Hybond-N+ nylon membranes. Transcripts were detected using 32P-labelled probes, gpuA (nt +380 to +800 from translation initiation codon) (lanes 1–3) and gpuP (nt –81 to +840) (lanes 4–6). Numbers left and right of the gel indicate sizes of RNA markers and transcripts, respectively.

 
We quantified the gpuP and gpuA transcripts, and investigated the possible read-through of transcription from PgpuP into gpuA by RT-PCR. We synthesized cDNAs for gpuA, gpuP and gpuPA mRNAs using as templates total RNAs extracted from PAO1 cells induced or uninduced with 3-GP, and oligonucleotides complementary to appropriate sites of the genes as primers (see Methods). Specific regions of the cDNAs were subsequently amplified by PCR using the cDNA samples and pairs of oligonucleotides corresponding to the sites of interest. Primers A1 and A2 amplified 685 bp fragments of gpuA (nt +273 to +957) from the gpuA/PA cDNAs (Fig. 4, lane 2), and primers P1 and P2 synthesized 729 bp fragments of gpuP (nt –37 to +692) from the gpuP cDNA (Fig. 4, lane 4). Fragments were not amplified when the cDNAs were synthesized from the uninduced cells (Fig. 4, lanes 1, 3 and 5). The ratio of the amplified gpuA and gpuP fragments was about 7 : 1, in accordance with the relative amounts of transcripts generated from PgpuP and PgluA (Fig. 3). A 789 bp fragment (nt +992 of gpuP to +340 of gpuA; Fig. 4, lane 6) that included the 56 bp intergenic spacer between the two genes was amplified by PCR from the gpuA/PA cDNA samples using primers PA1 and PA2, confirming that some transcription from PgpuP proceeds into gpuA.



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Fig. 4. RT-PCR analyses of gpuA and gpuP transcripts and intergenic region. cDNAs for gpuA and gpuPA mRNAs (gpuA/PA cDNAs upstream from nt +957 of gpuA), and for gpuP mRNA (gpuP cDNA upstream from nt +692 of gpuP), were synthesized using total RNA extracted from PAO1 cells cultured in MMP containing L-glutamate or 3-GP, and primer A1 or primer P1, respectively. The cDNAs and oligonucleotide primers used in PCR were: lanes 1 and 2, gpuA/PA cDNAs and primers A1 and A2; lanes 3 and 4, gpuP cDNA and primers P1 and P2; lanes 5 and 6, gpuA/PA cDNAs and primers PA1 and PA2. Lanes 1, 3 and 5, cDNAs synthesized with RNA samples from uninduced cells; lanes 2, 4 and 6, cDNAs from cells with induced 3-GP. Primer sequences: primer A1, complementary to nt +933 to +957 of gpuA; primer A2, nt +273 to +302 of gpuA; primer P1, complementary to nt +664 to +692 of gpuP; primer P2, nt –37 to –9 of gpuP; primer PA1, nt +992 to +1020 of gpuP; primer PA2, complementary to nt +312 to +340 of gpuA. M, EcoRI and HindIII digests of {lambda} DNA as size markers.

 
Identification of the PgpuA and PgpuP promoters
To localize the gpuA, gpuP and gpuR promoters, we determined the 5' ends of their transcripts by primer extension using RNA samples from PAO1 cells cultured in MMP with L-glutamate or 3-GP. The cDNAs of these genes synthesized from the uninduced cells grown in L-glutamate were not detected. On the other hand, an RNA sample from the 3-GP-induced cells yielded two cDNAs for the gpuA transcript, and one for the gpuP transcript (Fig. 5a, and b). However, a cDNA for the gpuR transcript was not detected (data not shown), indicating that the expression level of this regulatory gene was very low. A comparison with sequence ladders localized the 5' ends of the gpuA transcripts at 296 and 299 bp upstream from the translation initiation ATG codon of gpuA (Fig. 5a), allowing identification of the –35 (5'-TGGTCA-3') and –10 (5'-GAGCAT-5') sequences of PgpuA at around 1080 nt of the gpuP coding region (1386 nt). The 5' end of the gpuP transcript was located 53 bp upstream of the translation initiation ATG codon of this gene (Fig. 5b), positioning the –35 (5'-TTCAGC-3') and –10 (5'-TATCTT-3') sequences of PgpuP at appropriate distances from the 5' end. The –10 and –35 sequences of these promoters resembled those for the {sigma}70 RNA polymerase holoenzyme.



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Fig. 5. Primer extension analysis of gpuA (a) and gpuP (b) transcripts. Complementary strands were synthesized using 32P-end-labelled oligonucleotides (corresponding to nt +48 to +75 of gpuA, and to nt +72 to +100 of gpuP) as primers and RNA samples from PAO1 cells grown in MMP plus 20 mM glutamate (lane 1), or MMP plus 20 mM 3-GP (lane 2), and then resolved on 6 % (w/v) denatured polyacrylamide gels, together with sequence ladders (G, A, T and C). Asterisks indicate 5' ends of gpuA and gpuP transcripts.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Peptide mapping has revealed some similarity between the GpuA and GbuA sequences (Yorifuji et al., 1983) that can now be examined at the amino acid level. These enzymes of the arginase/agmatinase family share 49 % identity and 68 % similarity in terms of amino acids. Because of their similarity of about 57 % to E. coli agmatinase (the speB product) involved in putrescine synthesis (Glansdorff, 1996), the genes of GpuA and GbuA have been annotated as speB1 and speB2, respectively (http://www.pseudomonas.com). The absence of any other speB homologues in P. aeruginosa PAO1 indicates that this strain has no speB. In fact, this strain recruits agmatine deiminase (the aguA product) and N-carbamoylputrescine amidohydrolase (the aguB product), which were discovered as the catabolic enzymes of agmatine (Nakada et al., 2001) in the biosynthetic conversion of agmatine to putrescine (Nakada & Itoh, 2003). A detailed phylogenetic analysis (Sekowska et al., 2000) has divided the arginase/agmatinase family of proteins into arginases, agmatinases, and a third group of plant and bacterial enzymes, including the SpeB1 and SpeB2 of P. aeruginosa PAO1. Our previous identification of SpeB2 as GbuA assumed that the third group consists of amidinohydrolases that act upon 4-GB or other guanidino acids (Nakada & Itoh, 2002). The present identification of SpeB1 as GpuA supports this assumption.

The PA1418 product has apparent similarity (58 % identity) to GpuP, and this gene is located near gbuAR (Nakada & Itoh, 2002), implying its involvement in 4-GB uptake. However, PA1418 knockout minimally affects the Gbu+ phenotype (Nakada & Itoh, 2002). Thus, P. aeruginosa PAO1 must have a 4-GB transport gene that is not linked to gbuAR. If PA1418 encodes a 4-GB transport protein, a second transport gene would hinder the effect of the PA1418 mutation on 4-GB utilization. Therefore, a possible role of PA1418 in 4-GB transport cannot be ruled out at this stage. To clarify the role of PA1418 in 4-GB import, the second transport gene should be identified.

Proteins of the LysR family, to which GpuR and GbuR belong, have a helix–turn–helix DNA-binding motif of about 20 aa at the N terminus, a coinducer recognition domain at the centre, and a C-terminal domain important for both DNA binding and the coinducer response (Schell, 1993). Helix–turn–helix motifs have been located in both GpuR (aa 28–48) and GbuR (aa 22–41) at the N termini, using a helix–turn–helix program (Dodd & Egan, 1990). These motifs are relatively well conserved, sharing 50 % identical residues. The possible coinducer-binding domains (aa 96–177 of GpuR, and aa 90–171 of GbuR) are slightly more homologous (42 % identity) than the overall amino acid identity (37 %) of the proteins. However, frequent substitutions must have arisen to match the coinducer-binding domains with specific coinducers that differ in one methylene group only. Upon interaction with a coinducer, LysR proteins bind to two regulatory sites around the –70 (recognition binding site; RBS) and –35 (activation binding site; ABS) regions of promoters, allowing the regulatory proteins to interact with the {alpha} subunit of the RNA polymerase holoenzyme (McFall et al., 1998). The core 10 residues (F/LSGT/GYIGYIP) of the C-terminal domain (corresponding aa 241–250 of GpuR) are very similar between GpuR and GbuR. The RBS and ABS motifs for the LysR family proteins have characteristic hairpin structures with a stem (3–6 bp) and a loop (5–7 bp) (Schell, 1993). Possible RBS and ABS motifs can be found around the –80 and –35 regions, respectively, of the gpuA and gpuP promoters and of the gbuA promoter (Nakada & Itoh 2002). Binding experiments are warranted to demonstrate the functions of these putative motifs in the inducible expression of the genes for GpuR and GbuR.

The cellular concentrations of GpuP and GpuA required to accomplish their roles appear to be controlled at the level of transcription (Figs 3 and 4). About half of the transcription from the gpuP promoter terminates after gpuP (Figs 2a and 3), probably due to the terminator-like structure of the 9 bp stem and 3 bp loop [{Delta}G –26·3 kcal mol–1 (–110·04 kJ mol–1)] located 8 bp downstream of the translation termination codon, but the other half proceeds into gpuA over the termination signal for transcription (Figs 2a and 3). Primer extension experiments localized the –35 and –10 sequence of the gpuA promoter within the gpuP-coding region, and transcription from this promoter starts before the termination signal. However, because of the powerful transcriptional activity of the gpuA promoter, and the weak attenuation of transcription by the terminator, transcription from the gpuP promoter can proceed into gpuA beyond the termination signal.

The most striking features of the 3-GP and 4-GB utilization components are that GpuP, GpuA and GpuR have significant homology to PA1418, GbuA and GbuR, respectively. Although a role for PA1418 in 4-GB uptake remains to be demonstrated, the key triad genes for 3-GP and 4-GB utilization (i.e. transport, catabolism and gene regulation) appear to have been derived from a common set of origins via gene duplication, and subsequent co-evolution to co-ordinately develop specificity to the relevant compounds. The absence of a 3-GP catabolic system in other closely related Pseudomonas species (Yorifuji et al., 1983), and the highest similarity between GpuA and GbuA among the orthologous group of the arginase/agmatinase proteins (http://www.ncbi.nih.gov/COG), imply that such genetic events occurred after the divergence of P. aeruginosa species, thus establishing independent and efficient catabolic 3-GP and 4-GB systems in this species. Alternatively, Pseudomonas species other than P. aeruginosa have lost the 3-GP utilization system during or after divergence from an ancestor.


   ACKNOWLEDGEMENTS
 
We are grateful to N. Gotoh for providing strain PAO4173, and to D. Haas, S. Heeb, U. Schnider and H. Schweizer for gifts of plasmids. We also thank K. Kimura for technical assistance. This study was supported by a grant-in-aid for scientific research (B) from Japan Society for the Promotion of Science (to Y. I., 14360060).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 10 June 2005; revised 25 August 2005; accepted 2 September 2005.



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