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
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ABSTRACT |
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INTRODUCTION |
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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.
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METHODS |
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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 ml1, 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 BglIIHindIII 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
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 mll). 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
Sp/Sm at the relevant sites was verified by PCR (Nishijyo et al., 2001
).
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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 min1. Protein concentrations were determined using a Protein Assay Kit (Bio-Rad), with BSA as the standard.
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RESULTS |
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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
interposon carrying a transcriptional terminator (Fellay et al., 1987
) on PA286, gpuP or gpuA (Fig. 2c
). Plasmids pYI1047 (gpuA : :
Sp/Sm), pYI1057 (gpuP : :
Sp/Sm) and pYI1060 (PA286 : :
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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DISCUSSION |
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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 helixturnhelix 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). Helixturnhelix motifs have been located in both GpuR (aa 2848) and GbuR (aa 2241) at the N termini, using a helixturnhelix program (Dodd & Egan, 1990
). These motifs are relatively well conserved, sharing 50 % identical residues. The possible coinducer-binding domains (aa 96177 of GpuR, and aa 90171 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
subunit of the RNA polymerase holoenzyme (McFall et al., 1998
). The core 10 residues (F/LSGT/GYIGYIP) of the C-terminal domain (corresponding aa 241250 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 (36 bp) and a loop (57 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 [
G 26·3 kcal mol1 (110·04 kJ mol1)] 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.
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ACKNOWLEDGEMENTS |
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Received 10 June 2005;
revised 25 August 2005;
accepted 2 September 2005.
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