A Novel Putrescine Utilization Pathway Involves (cid:1) -Glutamylated Intermediates of Escherichia coli K-12*

A novel bacterial putrescine utilization pathway was discovered. Seven genes, the functions of whose products were not known, are involved in this novel pathway. Five of them encode enzymes that catabolize putrescine; one encodes a putrescine importer, and the other encodes a transcriptional regulator. This novel pathway involves six sequential steps as follows: 1) import of putrescine; 2) ATP-dependent (cid:1) -glutamylation of putrescine; 3) oxidization of (cid:1) -glutamylputrescine; 4) de-hydrogenation of (cid:1) -glutamyl- (cid:1) -aminobutyraldehyde; 5) hydrolysis of the (cid:1) -glutamyl linkage of (cid:1) -glutamyl- (cid:1) -aminobutyrate; and 6) transamination of (cid:1) -aminobu-tyrate to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate. (cid:1) -Glutamyl compounds are widely found in both prokaryotic and eukaryotic cells. For example, glutathione ( (cid:1) -glutamylcystei-nylglycine) is a very important antioxidant (1) for living cells. Bacterial Strains and Plasmids— P1 transduction and DNA manip-ulation and transformation were performed by the standard methods (13, 15). A DNA fragment of Kohara clone 257 (16) containing the ycj gene cluster was cloned onto high copy number plasmids. The bacterial Chemical) from the cell-free extract of SK247. After the structure was identified by NMR, MS, and MS/MS spectroscopic analysis, (cid:1) -Glu-Put was enzymatically synthesized from putres- cine and glutamate using MalE-YcjK and purified by Dowex 50W-X8. The purity was authenticated by HPLC. (cid:1) -Glu-GABA was purified by anion exchange chromatography using Dowex 1-X8 (Dow Chemical) from the cell-free extract of SK279. The structure was identified by NMR. The purity was checked by HPLC. pression and that the deletion of goaG , the product of which is located downstream of YcjL in the metabolic pathway, did not influence ycjL expression. YcjK catalyzes the (cid:1) -glutamylation of putrescine to form the (cid:1) -glutamyl linkage of (cid:1) -Glu-Put, whereas YcjL catalyzes the hydrolysis of the (cid:1) -glutamyl linkage of (cid:1) -Glu-GABA. If (cid:1) -Glu-Put is a better substrate for YcjL than (cid:1) -Glu-GABA, there is no good reason for the (cid:1) -glutamy-late putrescine consuming ATP. To determine which is the better substrate, K m and k cat values of YcjL for (cid:1) -Glu-Put and (cid:1) -Glu-GABA were determined. The K m for (cid:1) -Glu-Put was 18.5 m M and that for (cid:1) -Glu-GABA was 2.93 m M . The k cat for (cid:1) -Glu-Put was 434 min (cid:3) 1 and that for (cid:1) -Glu-GABA was 2490 min (cid:3) 1 . The k cat / K m for (cid:1) -Glu-Put was 23.5 and that for (cid:1) -Glu-GABA was 850, i.e. the k cat / K m for (cid:1) -Glu-GABA was 36 times higher than that for (cid:1) -Glu-Put. The results showed that (cid:1) -Glu-GABA rather than (cid:1) -Glu-Put is the natural substrate of YcjL. The YcjL activity measured as (cid:1) -G p NA hydrolysis activity was 16 times higher when E. coli was grown in the W-Put medium rather than in the W salts minimal medium containing 0.2% NH 4 Cl instead of putrescine.

␥-Glutamyl compounds are widely found in both prokaryotic and eukaryotic cells. For example, glutathione (␥-glutamylcysteinylglycine) is a very important antioxidant (1) for living cells.
We have studied bacterial ␥-glutamyltranspeptidase (GGT) 1 (2, 3) that catalyzes the hydrolysis of the ␥-glutamyl linkage of glutathione (4). During the course of our study on Escherichia coli GGT, we unexpectedly found that the ␥-glutamyl linkage of ␥-glutamyl-p-nitroanilide (␥-GpNA) was cleaved by the cellfree extract of a GGT-deficient strain. The enzyme that is responsible for this reaction was purified, and the relevant gene, ycjL, was identified. Further study of ycjL and neighboring genes (ycj gene cluster) revealed that the products of the ycj gene cluster constitute a novel putrescine-utilizing pathway in which ␥-Glu-Put is involved.
Putrescine, the substrate of this pathway, is one of polyamines that are found in a wide range of organisms from bacteria to plants and animals especially when these cells are proliferating or in stressful conditions (5). Also in E. coli, putrescine plays important roles in cell proliferation and normal cell growth (5). Putrescine is synthesized from ornithine by ornithine decarboxylase (SpeC) or from arginine by the sequential actions of arginine decarboxylase (SpeA) and agmatinase (SpeB) in E. coli cells (6). Putrescine is converted to spermidine, another polyamine, by the addition of an aminopropyl group derived from decarboxylated S-adenosylmethionine through the activity of spermidine synthase (SpeE) (7).
Putrescine has been reported to be converted to ␥-aminobutyraldehyde by putrescine transaminase and then to be oxidized to ␥-aminobutyrate (GABA) by aminobutyraldehyde dehydrogenase (8). However, neither of the genes encoding this pathway has been identified yet. GABA is converted to succinate semialdehyde by GABA aminotransferase (GabT) and then is oxidized to succinate by succinate semialdehyde dehydrogenase (GabD) (9). Recently, it was reported that the ⌬gabT strain grew in minimal medium containing putrescine as a sole nitrogen source but not in minimal medium containing GABA instead of putrescine (10). This unexpected growth in the presence of putrescine likely resulted from the specific induction of a gab-independent enzyme by putrescine. It was reported that an excess amount of putrescine is exported by PotE, putrescine ornithine antiporter (11), and that putrescine outside of the cell is imported by PotFGHI, the ABC transporter (12), according to the environment in which E. coli lives.
In this paper we report on a completely new catabolic pathway for extracellular putrescine. This pathway involves the ␥-glutamylation of putrescine and plays an important physiological role to utilize putrescine in nutrient starvation. All the enzymes, a transporter, and a regulator that constitute this pathway are encoded by the ycj gene cluster. 2
Bacterial Strains and Plasmids-P1 transduction and DNA manipulation and transformation were performed by the standard methods (13,15). A DNA fragment of Kohara clone 257 (16) containing the ycj gene cluster was cloned onto high copy number plasmids. The bacterial strains and plasmids used in this study are summarized in Table I. Genes were disrupted according to the method of Datsenko and Wanner (17) or as described previously (18).
Analysis of ␥-Glutamyl Compounds, Amino Acids, and Amines-The ␥-glutamyl compounds, amino acids, and amines in the samples were measured by using an HPLC system (model LC-9A; Shimadzu, Kyoto, Japan) equipped with a Shim-pack Amino-Na column (Shimadzu), with gradient elution at 60°C at a flow rate of 0.6 ml/min. The gradient of the mobile phase was formed with buffer A (66.6 mM tri-sodium citrate dehydrate, 1% perchloric acid, 7% ethanol (pH 3.2)) and buffer B (200 mM tri-sodium citrate dehydrate, 200 mM boric acid, 0.12 N NaOH (pH 10)). The concentration of buffer B was kept at 0% until 9 min. It was linearly increased to 7% from 9 to 13 min, to 8% from 13 to 17.2 min, and then to 11%. o-Phthalaldehyde was used as the detection reagent (19) as described previously (20), and the fluorescence was detected with a fluorescence detector (model RF-535; Shimadzu) as the absorbance at 450 nm, with excitation at 348 nm. Standard compounds were purchased from Nacalai Tesque, Kyoto, Japan, and Sigma, except for ␥-Glu-Put and ␥-Glu-GABA, which were synthesized as described below. In our HPLC system, ␥-Glu-GABA, glutamate, GABA, and ␥-Glu-Put was eluted at 8, 11, 26, and 29 min, respectively. Before measuring amino acids from the cell-free extract, the extract was previously treated with trichloroacetic acid (final concentration, 10%) to precipitate its proteins.
Assays for Enzymes-The ␥-GpNA hydrolysis activity of YcjL and GGT was assayed as described previously (2). ␥-Glu-Put synthetase activity was determined by measuring the ␥-Glu-Put synthesized from glutamate and putrescine. The reaction mixture containing 50 mM sodium glutamate, 50 mM putrescine dihydrochloride, 15 mM ATP, 60 mM MgCl 2 , and 200 mM imidazole chloride buffer (pH 7.0) was incu-bated at 37°C. ␥-Glu-Put oxidase activity was assayed by measuring the decrease in ␥-Glu-Put by HPLC. The reaction mixture containing 1 mM ␥-Glu-Put and 10 mM potassium phosphate buffer (pH 7.0) was incubated at 37°C. ␥-Glu-GABA hydrolase activity was determined by measuring the glutamate released from ␥-Glu-GABA. The reaction mixture containing various concentrations of ␥-Glu-GABA and 50 mM imidazole-HCl buffer (pH 8.7) was incubated at 37°C. GABA aminotransferase activity was determined by measuring the glutamate formed from GABA and ␣-ketoglutarate. The reaction mixture containing 10 mM GABA, 10 mM ␣-ketoglutarate monosodium salt, and 100 mM potassium phosphate buffer (pH 7.0) was incubated at 37°C. Adding trichloroacetic acid (final concentration, 10%) terminated the reactions, and the filtrates were analyzed with HPLC.
Purification of Proteins-Following the ␥-GpNA hydrolysis activity, YcjL was purified from the cell-free extract of SH639 (⌬ggt-2) by ammonium sulfate precipitation (30 -50%) and column chromatographies using DEAE-Cellulofine A-500m (Chisso, Tokyo, Japan), phenyl-Sepharose CL-4B (Amersham Biosciences), Cellulofine GC-700m (Chisso), and MiniQ (Amersham Biosciences). During the purification, the protein was basically dissolved in buffer C (50 mM Tris-HCl (pH 8.7) and 5 mM ␤-mercaptoethanol). After the ammonium sulfate precipitation, the enzyme was dissolved in buffer C and dialyzed against the same buffer. The dialyzed enzyme solution was applied to DEAE-Cellulofine A-500m previously equilibrated with buffer C. Without washing the column, YcjL was eluted with 0.2 M NaCl in buffer C from the column. In the purification step using phenyl-Sepharose CL4B, the enzyme solution was dialyzed against buffer D (0.8 M ammonium sulfate in buffer C) and applied to the column equilibrated with buffer D. The column was washed with buffer D, and YcjL was eluted at the end of a liner gradient formed between buffer D (0.8 M ammonium sulfate in buffer C) and This study pQE-80L ColE1 replicon bla ϩ lacI q T5 p -(His) 6 Qiagen pQE-80L-ordL ColE1 replicon bla ϩ lacI q T5 p -(His) 6 -ordL ϩ This study buffer E (40% ethylene glycol in buffer C). In the purification step using Cellulofine GC-700, the enzyme solution was concentrated by the ammonium sulfate precipitation and applied to the column previously equilibrated with buffer C. The active fraction was applied to the MiniQ column previously equilibrated with buffer C. YcjL was eluted with a linear gradient formed between buffer C and 0.5 M NaCl in buffer C. YcjL was eluted when the concentration of NaCl was 0.15 M. MalE-YcjK was overexpressed after induction by isopropyl-␤-D-thiogalactopyranoside and purified from the cell-free extract by ammonium sulfate precipitation (30 -50%) and an amylose column (New England Biolabs) chromatography according to the protocol recommended by the manufacturer. N-terminal Amino Acid Sequence-The N-terminal amino acid sequence of YcjL was determined by Edman degradation as described previously (21).
Nuclear Magnetic Resonance Spectrometry and Mass Spectrometry-Purified ␥-Glu-Put and ␥-Glu-GABA was dissolved in D 2 O and then analyzed with a Bruker 500 MHz spectrometer. The mass of the purified ␥-Glu-Put was measured by fast atom bombardment (FAB ϩ )-mass spectrometry (model JEOLJMS600; JEOL, Tokyo, Japan) using glycerol as a matrix. Purified ␥-Glu-Put was fragmented by using N 2 as a collision gas (at 5 millitorrs) at a collision energy (RO2 to RO1) of 10 -11.5 eV, and then the masses of fragments were measured by ion spray mass spectrometry (model API-3000; PE Sciex).
Synthesis and Purification of ␥-Glu-Put and ␥-Glu-GABA-␥-Glu-Put was originally purified by cation exchange chromatography using Dowex 50W-X8 (Dow Chemical) from the cell-free extract of SK247. After the structure was identified by NMR, MS, and MS/MS spectroscopic analysis, ␥-Glu-Put was enzymatically synthesized from putrescine and glutamate using MalE-YcjK and purified by Dowex 50W-X8. The purity was authenticated by HPLC. ␥-Glu-GABA was purified by anion exchange chromatography using Dowex 1-X8 (Dow Chemical) from the cell-free extract of SK279. The structure was identified by NMR. The purity was checked by HPLC.
Transport Assay-The transport assay was performed as described previously (22,23) using [ 14 C]putrescine dihydrochloride, except that M9-tryptone medium was used instead of M63 medium.

RESULTS
A Novel ␥-GpNA Hydrolysis Activity of GGT-deficient Strain of E. coli-␥-GpNA is an artificial substrate widely used to measure GGT activity (24). We found a novel ␥-GpNA hydrolysis activity in the cytosol of E. coli strain SH639 (⌬ggt-2) grown at high aeration (60 ml of culture in a 300-ml Erlenmeyer flask shaken at 140 rpm). To identify the protein responsible for this activity, the relevant protein was purified, and its N-terminal amino acid sequence was determined by the Edman degradation method. The amino acid sequence obtained, MEN-IMNNPVIG, was compared with sequences in a data base, Colibri (genolist.pasteur.fr/Colibri/), and the relevant gene was found to be ycjL whose function was not known. Strain SK111 (⌬ycjLC) completely lost the ␥-GpNA hydrolysis activity, and SK112 (pBR-ycjL/SK111) overproduced the protein responsible for the activity. Purified YcjL hydrolyzed glutamine to glutamate and theanine (␥-glutamylethylamide) to glutamate and ethylamine. These results suggested that YcjL is a novel enzyme that catalyzes the hydrolysis of the ␥-glutamyl linkage.
Hypothesis for Physiological Roles of ycjL and Genes Around ycjL-It was not easy to determine the physiological role of YcjL because the ⌬ycjL mutant could grow on M9-glucose minimal medium as well as the wild-type strain. Several genes seem to function with ycjL in E. coli cells (Fig. 1). ycjC is located downstream of ycjL, and these two genes are suggested to compose an operon. YcjC has a helix-turn-helix motif, and the ␥-GpNA hydrolysis activity of strain SK112 (pBR-ycjL/⌬ycjLC) was 400 times that of SK121 (pBR-ycjLC/⌬ycjLC). The results indicate that YcjC is a repressor of ycjL. ycjK is located upstream of ycjL in a reverse orientation, and its transcription is suggested to overlap with that of ycjL (Fig. 1). The disposition of these three genes, ycjK, ycjL, and ycjC, suggested that they functioned together. To presume the function of the products of this gene cluster, a homologous gene cluster for ycjKLC was searched for in the data base GenomeNet (www.genome.ad.jp/), and several were found in bacteria. The function of most of them was not known, but the function of one of them, the ipuABCDEFGH operon in Pseudomonas sp., has been reported (25) (Fig. 2A). According to the report, the operon functions in isopropylamine (IPA) degradation. YcjK and IpuC showed 29.5% identity at the protein level, and both had homology with glutamine synthetase. YcjJ, which is located at downstream of ycjK (Fig. 1), showed 19.5% identity with IpuG, which is thought to be an IPA transporter. Furthermore, IpuF had hydrolysis activity for the ␥-glutamyl linkage similar to YcjL. In the degradation pathway encoded by the ipu operon, IPA is taken up by IpuG and ␥-glutamylated by IpuC in Pseudomonas sp. cells. Subsequently, ␥-glutamyl-IPA is oxidized by IpuD, and the product, ␥-glutamyl-alaninol, is hydrolyzed by IpuF to glutamate and alaninol ( Fig. 2A). Here we hypothesized that some amine is first imported into the E. coli cell by YcjJ, and its amino group is ␥-glutamylated by YcjK. Subsequently, the amine part of ␥-glutamyl-amine is oxidized by some oxidoreductase. Finally, the ␥-glutamyl linkage of ␥-glutamyloxidized amine is hydrolyzed to glutamate and oxidized amine by YcjL. The oxidized amine can probably be converted to the next catabolite more easily than the original amine. In this hypothesis, the oxidoreductase is important to make sense of the ␥-glutamylation, and we searched for it near ycjJKLC. Downstream of the ycjLC operon, we found ordL predicted to encode oxidoreductase. ordL is a member of the aldH-ordL-goaG operon. This operon had already been reported, but its mRNA could not be detected by Northern analysis (26). Therefore, we hypothesized that the gene products of ycjJ, ycjK, the ycjLC operon, and the aldH-ordL-goaG operon (ycj gene cluster) work together in the uptake and degradation of some amine. Therefore, it was essential to identify this amine to elucidate the physiological role of the ycj gene cluster.
YcjK Is ␥-Glu-put Synthetase-To accumulate the ␥-glutamyl-amine, SK247 (⌬ycjLC ⌬(aldH-ordL-goaG); ycjJ ϩ ycjK ϩ ycjL Ϫ ycjC Ϫ aldH Ϫ ordL Ϫ goaG Ϫ ) was constructed. In the cellfree extract of this strain, we found a much accumulated compound. This compound was eluted at 28 min in our HPLC system. It was purified and subjected to NMR, MS, and MS/MS spectroscopic analysis. The chemical structure estimated by NMR was ␥-Glu-Put. The mass of the molecular ion (m/z 218) and the fragmentation pattern of the compound by MS/MS indicate its structure to be ␥-Glu-Put. To confirm that YcjK   FIG. 1. Organization of the ycj and aldH-ordL-goaG loci. The relative location and putative transcriptional orientation of genes at these loci are represented by arrows. ycjJ p , ycjK p , ycjLC p , and (aldH-ordL-goaG) p represent the locations of putative promoters.
Putrescine is reported to be degraded after acetylation in mammalian cells (27). Referring to that report (27), we hypothesized that putrescine is degraded in E. coli after ␥-glutamylation instead of acetylation in mammalian cells. The entire novel putrescine utilization pathway of E. coli proposed here is summarized in Fig. 2B.
YcjL Is ␥-Glu-GABA Hydrolase-To elucidate the physiological role of YcjL in vivo, two strains were constructed, SK189 (⌬(ordL-goaG)) and SK187 (⌬goaG). OrdL is located upstream, and GoaG downstream, of YcjL in the hypothetical catabolic pathway (see Fig. 2B). The cell-free extract of SK189 (⌬(ordL-goaG)) exhibited only 23% ␥-GpNA hydrolysis activity compared with SK187 (⌬goaG) which exhibited the same level of activity as the wild-type strain. This result can be interpreted to mean that the metabolite upstream of YcjL, ␥-glutamyl-␥aminobutyraldehyde, and/or ␥-Glu-GABA could induce ycjL ex- pression and that the deletion of goaG, the product of which is located downstream of YcjL in the metabolic pathway, did not influence ycjL expression. YcjK catalyzes the ␥-glutamylation of putrescine to form the ␥-glutamyl linkage of ␥-Glu-Put, whereas YcjL catalyzes the hydrolysis of the ␥-glutamyl linkage of ␥-Glu-GABA. If ␥-Glu-Put is a better substrate for YcjL than ␥-Glu-GABA, there is no good reason for the ␥-glutamylate putrescine consuming ATP. To determine which is the better substrate, K m and k cat values of YcjL for ␥-Glu-Put and ␥-Glu-GABA were determined. The K m for ␥-Glu-Put was 18.5 mM and that for ␥-Glu-GABA was 2.93 mM. The k cat for ␥-Glu-Put was 434 min Ϫ1 and that for ␥-Glu-GABA was 2490 min Ϫ1 . The k cat /K m for ␥-Glu-Put was 23.5 and that for ␥-Glu-GABA was 850, i.e. the k cat /K m for ␥-Glu-GABA was 36 times higher than that for ␥-Glu-Put. The results showed that ␥-Glu-GABA rather than ␥-Glu-Put is the natural substrate of YcjL. The YcjL activity measured as ␥-GpNA hydrolysis activity was 16 times higher when E. coli was grown in the W-Put medium rather than in the W salts minimal medium containing 0.2% NH 4 Cl instead of putrescine. These results indicated that YcjL is ␥-Glu-GABA hydrolase.
GoaG Is the Second GABA Aminotransferase-Previously, putrescine was reported to be oxidized directly to GABA (8) and be degraded to succinate by GabT and GabD (9). It was reported in another study on the gabDTPC operon that encodes the GABA degradation pathway in E. coli that the strain ⌬gabT did not grow in W salts minimal medium containing 0.2% GABA as a sole nitrogen source, whereas this strain grew in W-Put medium (10). Moreover, the cell-free extract of ⌬gabT grown in W-Put medium exhibited significant GABA aminotransferase activity. These results suggested that besides GabT, there is another GABA aminotransferase that is induced by putrescine. We suspected that this second aminotransferase was encoded by goaG in the ycj gene cluster. The cell-free extract of KJ107 (⌬gabT), SK187 (⌬goaG), and KJ109 (⌬gabT ⌬goaG) grown in M9-tryptone supplemented with 5 mM putrescine exhibited 68, 35, and 1.3% GABA aminotransferase activity, respectively, compared with SH639 (gabT ϩ goaG ϩ ). These results indicate that both GabT and GoaG are important in the catabolism of GABA. In the cell-free extracts of the ⌬goaG strain, accumulation of GABA was observed. These results agreed well with the putrescine-catabolizing pathway postulated in Fig. 2B.
YcjJ Is a Novel Putrescine Importer-Downstream of ycjK, there is a gene, ycjJ, whose product is suggested to be an amine transporter from its amino acid sequence. From the results described so far, the substrate of YcjJ is expected to be putrescine.
To show that putrescine is imported by YcjJ, a transporter assay using [ 14 C] putrescine was performed. The putrescine transport activity of SO23 (⌬ycjJ) was reduced compared with that of SH639 (ycjJ ϩ ), whereas SO24 (pBR-ycjJ/SO23) imported putrescine over 24 times more effectively than SH639 (ycjJ ϩ ) (Fig. 4). The result indicated that YcjJ is a novel putrescine importer. In SO23 (⌬ycjJ), putrescine import activity was very much diminished, although the strain is potF ϩ G ϩ H ϩ I ϩ (12). The result indicates that under our growth conditions for E. coli, the putrescine importer mainly expressed is YcjJ. A more detailed analysis of YcjJ will be published elsewhere.
Disruption and Complementation of ycj Gene Cluster Influence the Utilization of Putrescine in E. coli-To show that the products of the ycj gene cluster play an important role in the catabolism of putrescine, various parts of the ycj gene cluster were disrupted or complemented, and the mutants and transformants were grown on W-Put medium. Whereas SO23 (⌬ycjJ) and SO25 (pBR322/SO23) were not able to grow on the W-Put plates, SH639 (ycjJ ϩ ) and SO24 (pBR-ycjJ/SO23) were (Fig. 5A). These results suggested that YcjJ is the main putrescine importer when E. coli grows on medium containing putrescine as a sole nitrogen source. Whereas SK247 (⌬(ycjLC-aldH-ordL-goaG)) and SK306 (pBeloBAC11/SK247) could not grow on the W-Put plates, SH639 (ycjL ϩ C ϩ aldH ϩ ordL ϩ goaG ϩ ) and SK303 (pBelo-ycjLC-aldH-ordL-goaG/SK247) could (Fig. 5B). These results suggested that the catabolic pathway shown in Fig. 2B is the main one when E. coli grows in the medium containing putrescine as the sole source of nitrogen. KJ109 (⌬gabT ⌬goaG) did not grow on W-Put plates, whereas KJ107 (⌬gabT), SK187 (⌬goaG), and SH639 (goaG ϩ gabT ϩ ) did (Fig. 5C). The result suggested that both GoaG and GabT are important in the catabolism of GABA. The two enzymes can substitute for each other in the catabolic pathway. This is consistent with the results from the assay of the GABA aminotransferase activity of SK187 (⌬goaG), KJ107 (⌬gabT), and KJ109 (⌬goaG ⌬gabT).

DISCUSSION
The Significance of ␥-Glutamylation of Putrescine-In the novel pathway we proposed, putrescine is first ␥-glutamylated and then catabolized (Fig. 2B), but previously, putrescine had been thought to be degraded directly to ␥-aminobutyraldehyde without any modification and then to GABA (8). However, ␥-aminobutyraldehyde is unstable, and its amino group and aldehyde group form cyclic ⌬ 1 -pyrroline nonenzymatically (8). ␥-Glutamylation of putrescine may facilitate its catabolism by masking the amino group in advance and by preventing cyclization.
Regulation of the ycj Gene Cluster-Nac is a 70 -dependent transcriptional regulator under nitrogen-limited conditions (28). All of the members of the ycj gene cluster are expected to be transcribed from 70 -dependent promoters, and many results obtained so far were under nitrogen-limited conditions. Therefore, Nac was expected to control the expression of the ycj gene cluster; however, there is no Nac-binding site upstream of any of these genes. Furthermore, in a previous study using microarrays (29), no member of the ycj gene cluster was recognized as a Nac-controlled gene. However, the addition of putrescine to the medium as the sole nitrogen source (W-put medium) induced the expression of ycjL (data not shown). This result suggested that a new system or protein (for example, YcjC) involved in the response to the nitrogen starvation controls the expression of the ycj gene cluster. The addition of glucose to the M9-tryptone medium repressed the expression of ycjL (data not shown). However, there is no CRP-binding site upstream of any of these genes. Furthermore, the expression of ycjL was also repressed by the addition of glycerol and succinate to the M9-tryptone medium. These results suggested that the regulation of the expression of ycjL is independent of CRP. YcjL was highly expressed in the early stationary phase (data not shown) when the nutrient in the medium is drying up. These results suggested that the products of the ycj gene cluster are expressed when E. coli grows under conditions of starvation, and the products of the ycj gene cluster catabolize putrescine both as the carbon source and as the nitrogen source. We could not perform the experiment to detect the growth of E. coli on medium containing putrescine as a sole carbon source because wild-type E. coli could not grow on that medium.
In summary, the expression of ycjL (and maybe the other members of the ycj gene cluster) is repressed in the presence of ycjC and by the addition of another carbon or nitrogen source more easily utilized than putrescine, and is induced by nutrient starvation and by the addition of putrescine to the medium. A more detailed analysis of the regulation of the ycj gene cluster will be published elsewhere.
Acknowledgments-We thank Dr. Michihiko Kataoka and Dr. Sakayu Shimizu, Graduate School of Agriculture, Kyoto University, for protein sequencing of PuuD. We are grateful to Dr. Kazuhiro Irie, FIG. 5. Physiological role of ycj gene products in E. coli cells grown in minimal medium containing putrescine as a sole nitrogen source. A, strains relevant to ycjJ were streaked on plates of W salts minimal medium containing 0.2% putrescine as a sole nitrogen source (W-Put plates). The vector used was pBR322. "p" in the figure indicates the plasmid vector. B, strains relevant to the (ycjLC-aldH-ordL-goaG) gene cluster were streaked on W-Put plates. The vector used was pBeloBAC11. C, strains relevant to goaG and gabT were streaked on W-Put plates. Graduate School of Agriculture, Kyoto University, for NMR and mass spectrometry analyses of ␥-Glu-Put and ␥-Glu-GABA. We are also indebted to Dr. Tatsuo Kurihara and Dr. Nobuyoshi Esaki, Institute for Chemical Research, Kyoto University, for MS/MS spectrometry analysis of ␥-Glu-Put.