A Novel Putrescine Utilization Pathway Involves γ-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 γ-glutamylation of putrescine; 3) oxidization of γ-glutamylputrescine; 4) dehydrogenation of γ-glutamyl-γ-aminobutyraldehyde; 5) hydrolysis of the γ-glutamyl linkage of γ-glutamyl-γ-aminobutyrate; and 6) transamination of γ-aminobutyrate to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate. 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 γ-glutamylation of putrescine; 3) oxidization of γ-glutamylputrescine; 4) dehydrogenation of γ-glutamyl-γ-aminobutyraldehyde; 5) hydrolysis of the γ-glutamyl linkage of γ-glutamyl-γ-aminobutyrate; and 6) transamination of γ-aminobutyrate to form the final product of this pathway, succinate semialdehyde, which is the precursor of succinate. γ-Glutamyl compounds are widely found in both prokaryotic and eukaryotic cells. For example, glutathione (γ-glutamylcysteinylglycine) is a very important antioxidant (1Mannervik B. Carlberg I. Larson K. Dolphin D. Rozanne P. Avramovic O. Coenzymes and Cofactors. 3. Wiley-Interscience, New York1989: 475-516Google Scholar) for living cells. We have studied bacterial γ-glutamyltranspeptidase (GGT) 1The abbreviations used are: GGT, γ-glutamyltranspeptidase; γ-Glu-Put, γ-glutamylputrescine; γ-Glu-GABA, γ-glutamyl-γ-aminobutyrate; GABA, γ-aminobutyrate; γ-GpNA, γ-glutamyl-p-nitroanilide; IPA, isopropylamine; HPLC, high pressure liquid chromatography; MS, mass spectrometry; GabT, GABA aminotransferase; GabD, succinic semialdehyde dehydrogenase. (2Suzuki H. Kumagai H. Tochikura T. J. Bacteriol. 1986; 168: 1325-1331Crossref PubMed Google Scholar, 3Minami H. Suzuki H. Kumagai H. Enzyme Microb. Technol. 2003; 32: 431-438Crossref Scopus (64) Google Scholar) that catalyzes the hydrolysis of the γ-glutamyl linkage of glutathione (4Suzuki H. Hashimoto W. Kumagai H. J. Bacteriol. 1993; 175: 6038-6040Crossref PubMed Google Scholar). During the course of our study on Escherichia coli GGT, we unexpectedly found that the γ-glutamyl linkage of γ-glutamyl-p-nitroanilide (γ-GpNA) was cleaved by the cell-free 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 γ-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 (5Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3236) Google Scholar). Also in E. coli, putrescine plays important roles in cell proliferation and normal cell growth (5Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3236) Google Scholar). 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 (6Boyle S.M. Markham G.D. Hafner E.W. Wright J.M. Tabor H. Tabor C.W. Gene (Amst.). 1984; 30: 129-136Crossref PubMed Scopus (54) Google Scholar). 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) (7Tabor C.W. Tabor H. Xie Q.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6040-6044Crossref PubMed Scopus (35) Google Scholar). Putrescine has been reported to be converted to γ-aminobutyraldehyde by putrescine transaminase and then to be oxidized to γ-aminobutyrate (GABA) by aminobutyraldehyde dehydrogenase (8Shaibe E. Metzer E. Halpern Y.S. J. Bacteriol. 1985; 163: 933-937Crossref PubMed Google Scholar). 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) (9Metzer E. Levitz R. Halpern Y.S. J. Bacteriol. 1979; 137: 1111-1118Crossref PubMed Google Scholar). Recently, it was reported that the ΔgabT strain grew in minimal medium containing putrescine as a sole nitrogen source but not in minimal medium containing GABA instead of putrescine (10Schneider B.L. Ruback S. Kiupakis A.K. Kasbarian H. Pybus C. Reitzer L. J. Bacteriol. 2002; 184: 6976-6986Crossref PubMed Scopus (60) Google Scholar). 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 (11Kashiwagi K. Miyamoto S. Suzuki F. Kobayashi H. Igarashi K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4529-4533Crossref PubMed Scopus (120) Google Scholar), and that putrescine outside of the cell is imported by PotFGHI, the ABC transporter (12Pistocchi R. Kashiwagi K. Miyamoto S. Nukui E. Sadakata Y. Kobayashi H. Igarashi K. J. Biol. Chem. 1993; 268: 146-152Abstract Full Text PDF PubMed Google Scholar), 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 γ-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. 2The relationship between gene designations and their products is summarized in Table II. Media and Reagents—Strains were grown in M9-tryptone (M9 minimal medium (13Miller J.H. A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 263-274Google Scholar) except that 1% Bacto-tryptone was used instead of 0.2% glucose) with high aeration (140 rpm) at 37 °C in the determination of enzyme activities, accumulation of amine or amino acids in cells, or overexpression of proteins. To determine the putrescine utilization of various strains, W-Put plates (W salts minimal medium (14Bueno R. Pahel G. Magasanik B. J. Bacteriol. 1985; 164: 816-822Crossref PubMed Google Scholar) containing 0.4% glucose as a sole carbon source and 0.2% putrescine as a sole nitrogen source (10Schneider B.L. Ruback S. Kiupakis A.K. Kasbarian H. Pybus C. Reitzer L. J. Bacteriol. 2002; 184: 6976-6986Crossref PubMed Scopus (60) Google Scholar)) were used. 1,4-14CPutrescine dihydrochloride (107 mCi/mmol) was purchased from Amersham Biosciences. Bacterial Strains and Plasmids—P1 transduction and DNA manipulation and transformation were performed by the standard methods (13Miller J.H. A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 263-274Google Scholar, 15Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001: 1.1-2.110Google Scholar). A DNA fragment of Kohara clone 257 (16Kohara Y. Akiyama K. Isono K. Cell. 1987; 50: 495-508Abstract Full Text PDF PubMed Scopus (1110) Google Scholar) 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 (17Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11304) Google Scholar) or as described previously (18Russell C.B. Thaler D.S. Dahlquist F.W. J. Bacteriol. 1989; 171: 2609-2613Crossref PubMed Google Scholar).Table IStrains and plasmids used in this studyStrain or plasmidCharacteristic or sequenceSource or Ref.Strains KJ107SH639 ΔgabT::FRTThis study KJ109SH639 ΔgoaG::kan+ ΔgabT::FRTThis study SH639F- Δggt-22Suzuki H. Kumagai H. Tochikura T. J. Bacteriol. 1986; 168: 1325-1331Crossref PubMed Google Scholar SH703F- Δggt-2 zhg::Tn1030Suzuki H. Kumagai H. Tochikura T. J. Bacteriol. 1987; 169: 3926-3931Crossref PubMed Google Scholar SK111SH703 ΔycjLC::kan+;This study SK112pBR-ycjL/SK111This study SK121pBR322-ycjLC/SK111This study SK187SH639 ΔgoaG::kan+This study SK189SH639 Δ(ordL-goaG)::kan+This study SK247SH639 ΔycjLC::FRT Δ(aldH-ordL-goaG)::FRTThis study SK263pACYC184/SK247This study SK279pACYC-aldH-ordL/SK247This study SK283pACYC-ordL/SK247This study SK285pACYC-aldH/SK247This study SK303pBelo-ycjLC-aldH-ordL-goaG/SK247This study SK306pBeloBAC11/SK247This study SO23SH639 ΔycjJ::tet+This study SO24pBR-ycjJ/SO23This study SO58SH639 ΔycjKLC::cat+FRT Δ(aldH-ordL-goaG):: kan+This study SO60pmal-c2X/SO58This study SO61pmal-c2X-ycjK/SO58This study SO66pQE-80L-ordL/SK247This study SO67pQE-80L/SK247This studyPlasmids pACYC184p15A replicon cat+ tet+New England Biolabs pACYC-aldHp15A replicon cat+ aldH+This study pACYC-aldH-ordLp15A replicon cat+ aldH+-ordL+This study pACYC-aldH-ordL-goaGp15A replicon cat+ aldH+-ordL+-goaG+This study pACYC-ordLp15A replicon cat+ ordL+; after aldH-ordL were cloned, the DNA sequence encodes aldH were looped outThis study pBeloBAC11Mini-F replicon cat+New England Biolabs pBelo-ycjLC-aldH-ordL-goaGMini-F replicon cat+ ycjL+ ycjC+ aldH+ ordL+ goaG+This study pBR322ColE1 replicon rop+ bla+ tet+New England Biolabs pBR-ycjJColE1 replicon rop+ bla+ tet+ ycjJ+This study pBR-ycjLColE1 replicon rop+ bla+ tet+ ycjL+This study pBR-ycjLCColE1 replicon rop+ bla+ ycjL+C+This study pmal-c2XColE1 replicon f1 IG rop+ bla+ lacIq Φ(malE-′lacZα) (Hyb) (tacp);(malE without signal peptide)New England Biolabs pmal-c2X-ycjKColE1 replicon f1 IG rop+ bla+ lacIq Φ(malE-ycjK) (Hyb) (tacp);(malE without signal peptide)This study pQE-80LColE1 replicon bla+ lacIq T5p-(His)6Qiagen pQE-80L-ordLColE1 replicon bla+ lacIq T5p-(His)6-ordL+This study Open table in a new tab Analysis of γ-Glutamyl Compounds, Amino Acids, and Amines—The γ-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 (19Benson J.R. Hare P.E. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 619-622Crossref PubMed Scopus (794) Google Scholar) as described previously (20Suzuki H. Izuka S. Minami H. Miyakawa N. Ishihara S. Kumagai H. Appl. Environ. Microbiol. 2003; 69: 6399-6404Crossref PubMed Scopus (55) Google Scholar), 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 γ-Glu-Put and γ-Glu-GABA, which were synthesized as described below. In our HPLC system, γ-Glu-GABA, glutamate, GABA, and γ-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 γ-GpNA hydrolysis activity of YcjL and GGT was as described previously (2Suzuki H. Kumagai H. Tochikura T. J. Bacteriol. 1986; 168: 1325-1331Crossref PubMed Google Scholar). γ-Glu-Put activity was by measuring the γ-Glu-Put synthesized from and putrescine. The reaction containing mm glutamate, mm putrescine mm 60 mm and 200 mm buffer (pH was at 37 γ-Glu-Put activity was by measuring the in γ-Glu-Put by The reaction containing mm γ-Glu-Put and mm buffer (pH was at 37 activity was by measuring the from The reaction containing various of and mm buffer (pH was at 37 GABA aminotransferase activity was by measuring the formed from GABA and The reaction containing mm GABA, mm and mm buffer (pH was at 37 trichloroacetic acid (final concentration, 10%) the and the were with of the γ-GpNA hydrolysis YcjL was from the cell-free extract of by and column using and During the the was in buffer mm (pH and mm the the enzyme was in buffer and the The enzyme was to previously with buffer C. the YcjL was eluted with in buffer from the In the using the enzyme was buffer in buffer and to the column with buffer D. The column was with buffer and YcjL was eluted at the of a gradient formed between buffer in buffer and buffer in buffer In the using the enzyme was by the and to the column previously with buffer C. The was to the column previously with buffer C. YcjL was eluted with a gradient formed between buffer and in buffer C. YcjL was eluted when the concentration of was was after induction by and from the cell-free extract by and an column England according to the by the Amino amino acid sequence of YcjL was by as described previously H. Kumagai H. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). and γ-Glu-Put and was in and then with a The mass of the γ-Glu-Put was measured by (model Japan) using as a γ-Glu-Put was by using as a at a to of and then the of were measured by mass (model and of γ-Glu-Put and was by using from the cell-free extract of the was identified by MS, and γ-Glu-Put was synthesized from putrescine and using and by The was by was by using from the cell-free extract of The was identified by The was by was performed as described previously T. T. Suzuki H. Kumagai H. J. Bacteriol. PubMed Scopus Google Scholar, T. Suzuki H. T. Kumagai H. J. Bacteriol. 2002; 184: PubMed Scopus Google Scholar) using except that M9-tryptone medium was used instead of A γ-GpNA of GGT-deficient of E. is an substrate widely used to GGT activity A. PubMed Google Scholar). We found a novel γ-GpNA hydrolysis activity in the of E. coli strain grown at high aeration of in a at To the responsible for this the relevant was purified, and its amino acid sequence was by the The amino acid sequence was with in a and the relevant gene was found to be ycjL whose was not completely the γ-GpNA hydrolysis and the responsible for the YcjL to and to and that YcjL is a novel enzyme that catalyzes the hydrolysis of the γ-glutamyl for of ycjL and Genes was not to determine the physiological role of YcjL the on minimal medium as as the strain. genes to with ycjL in E. coli cells is of ycjL, and these genes are to an has a and the γ-GpNA hydrolysis activity of strain was that of The that is a of is of ycjL in a and its is to with that of ycjL The of these genes, ycjL, and that To the of the products of this gene a gene cluster for was for in the and were found in The of of them was not known, but the of one of the in has been reported J.R. T. A. A. T. Appl. Environ. Microbiol. 2002; PubMed Scopus Google Scholar) to the the functions in and at the and both with which is at of with which is to be an hydrolysis activity for the γ-glutamyl linkage to In the pathway encoded by the is by and by in cells. is oxidized by and the is by to and we that amine is imported the E. coli cell by and its amino group is by the amine of is oxidized by the γ-glutamyl linkage of amine is to and oxidized amine by The oxidized amine be converted to the the In this the is important to of the and we for it of the we found to encode is a of the This been but its not be detected by G. P. J. Bacteriol. PubMed Google Scholar). we that the gene products of the and the (ycj gene cluster) in the and of it was to this amine to the physiological role of the ycj gene pathway for the of by J.R. T. A. A. T. Appl. Environ. Microbiol. 2002; PubMed Scopus Google Scholar). pathway for the of putrescine by E. IPA, isopropylamine; putrescine; γ-Glu-Put, γ-Glu-GABA, γ-glutamyl-γ-aminobutyrate; GABA, the was In the cell-free extract of this we found a This was eluted at in our HPLC It was and to MS, and The by was The mass of the and the of the by its to be To that catalyzes the reaction of γ-Glu-Put in and were grown in and the cell-free extract was to The strain with the encoding γ-Glu-Put the strain with the To γ-Glu-Put by in was and with glutamate, and and the was at 37 The of γ-Glu-Put was by HPLC and was not without and that is γ-Glu-Put and the products of the ycj gene cluster catabolize putrescine. Putrescine is reported to be after in cells N. Biochem. J. PubMed Scopus Google Scholar). to that report N. Biochem. J. PubMed Scopus Google Scholar), we that putrescine is in E. coli after γ-glutamylation instead of in cells. The novel putrescine utilization pathway of E. coli is summarized in and the γ-Glu-Put was to be oxidized to by and then to by To these in strains, aldH+ ordL+ and aldH+ ordL+ were of these strains were and by γ-Glu-Put The addition of aldH+ to this strain not the the addition of ordL+ to the of γ-Glu-Put both ordL+ and aldH+ were to a new that eluted at in our HPLC system was This was and to The that the new is The was to GABA and by GGT which is to the γ-glutamyl γ-Glu-Put with the addition of the cell-free extract of but not with that of that γ-Glu-Put is to by the sequential and YcjL the physiological role of YcjL in strains were and is and of YcjL in the catabolic pathway The cell-free extract of γ-GpNA hydrolysis activity with which the of activity as the strain. This be to that the of ycjL and that the of the product of which is of YcjL in the pathway, not ycjL catalyzes the γ-glutamylation of putrescine to form the γ-glutamyl linkage of γ-Glu-Put, YcjL catalyzes the hydrolysis of the γ-glutamyl linkage of γ-Glu-Put is a substrate for YcjL γ-Glu-GABA, is for the putrescine To determine which is the and of YcjL for γ-Glu-Put and were The for γ-Glu-Put was mm and that for was The for γ-Glu-Put was and that for was The for γ-Glu-Put was and that for was the for was that for The that γ-Glu-Put is the substrate of The YcjL activity measured as γ-GpNA hydrolysis activity was when E. coli was grown in the W-Put medium in the salts minimal medium containing 0.2% instead of putrescine. that YcjL is the GABA putrescine was reported to be oxidized to GABA (8Shaibe E. Metzer E. Halpern Y.S. J. Bacteriol. 1985; 163: 933-937Crossref PubMed Google Scholar) and be to succinate by and (9Metzer E. Levitz R. Halpern Y.S. J. Bacteriol. 1979; 137: 1111-1118Crossref PubMed Google Scholar). It was reported in another study on the that encodes the GABA pathway in E. coli that the strain ΔgabT not in salts minimal medium containing 0.2% GABA as a sole nitrogen this strain grew in W-Put medium (10Schneider B.L. Ruback S. Kiupakis A.K. Kasbarian H. Pybus C. Reitzer L. J. Bacteriol. 2002; 184: 6976-6986Crossref PubMed Scopus (60) Google Scholar). the cell-free extract of ΔgabT grown in W-Put medium GABA aminotransferase that GabT, is another GABA aminotransferase that is by putrescine. We that this aminotransferase was encoded by in the ycj gene cluster. The cell-free extract of and grown in M9-tryptone with mm putrescine and GABA aminotransferase with that both and are important in the of In the cell-free of the accumulation of GABA was with the pathway in a Putrescine of is a gene, whose product is to be an amine transporter from its amino acid the described the substrate of is to be putrescine. To that putrescine is imported by a transporter using putrescine was The putrescine activity of was with that of imported putrescine The that is a novel putrescine In putrescine import activity was very the strain is (12Pistocchi R. Kashiwagi K. Miyamoto S. Nukui E. Sadakata Y. Kobayashi H. Igarashi K. J. Biol. Chem. 1993; 268: 146-152Abstract Full Text PDF PubMed Google Scholar). The that our growth conditions for E. coli, the putrescine is A of be and of ycj Gene the of Putrescine in E. that the products of the ycj gene cluster an important role in the of various of the ycj gene cluster were disrupted or and the and were grown on W-Put and were not to on the W-Put and were that is the putrescine when E. coli on medium containing putrescine as a sole nitrogen and not on the W-Put aldH+ ordL+ and that the catabolic pathway in is the one when E. coli in the medium containing putrescine as the sole source of not on W-Put and The that both and are important in the of The enzymes for other in the catabolic pathway. This is with the from the of the GABA aminotransferase activity of and The of of the novel pathway we putrescine is and then but putrescine been to be to γ-aminobutyraldehyde without and then to GABA (8Shaibe E. Metzer E. Halpern Y.S. J. Bacteriol. 1985; 163: 933-937Crossref PubMed Google Scholar). However, γ-aminobutyraldehyde is and its amino group and group form (8Shaibe E. Metzer E. Halpern Y.S. J. Bacteriol. 1985; 163: 933-937Crossref PubMed Google Scholar). of putrescine its by the amino group in and by of the ycj Gene is a transcriptional regulator conditions J. Bacteriol. PubMed Google Scholar). All of the of the ycj gene cluster are to be from and were was to the of the ycj gene is of of these in a study using E. S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar), of the ycj gene cluster was as a However, the addition of putrescine to the medium as the sole nitrogen source the of ycjL not This that a new system or example, involved in the to the nitrogen the of the ycj gene cluster. The addition of glucose to the M9-tryptone medium the of ycjL not However, is of of these the of ycjL was by the addition of and succinate to the M9-tryptone that the of the of ycjL is of YcjL was in the phase not when the nutrient in the medium is that the products of the ycj gene cluster are when E. coli conditions of and the products of the ycj gene cluster catabolize putrescine both as the carbon source and as the nitrogen We not the to the growth of E. coli on medium containing putrescine as a sole carbon source E. coli not on that In the of ycjL the other of the ycj gene cluster) is in the presence of and by the addition of another carbon or nitrogen source and is by nutrient and by the addition of putrescine to the A of the of the ycj gene cluster be New Gene for the ycj Gene the after putrescine utilization pathway for the ycj gene cluster. ycjL, and be and of ycj gene cluster and their new gene gene gene Open table in a new tab We and of for of We are to of for and mass of γ-Glu-Put and We are to and for for of