THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 hy The American Society for Biochemistry end Molecular Biology, Inc.

Vol. 266, No. 25, Issue of September 5, pp. 16854-16858,1991 Printed in U.S.A.

N-Acetylglutamic Acid: An Extracellular nod Signal of Rhizobium trifolii ANUS43 That Induces Root Hair Branching and Nodule-like Primordia in White Clover Roots*

(Received for publication, March 18, 1991)

Saleela Philip-Hollingsworth$, Rawle I. Hollingsworth8, and Frank B. DazzoST From the Departments of $Microbiology and §Biochemistry and Chemistry, Michigan State University, East Lansing, Michigan 48824-1 101

An extracellular metabolite purified from Rhizo- bium trifolii ANUS43 was established as N-acetylglu- tamic acid (GluNAc) by ‘H NMR and Fourier transform IR spectroscopy, gas chromatography/mass spectrom- etry of its methylated product, and organic synthesis. TLC analyses indicated that extracellular accumula- tion of GluNAc by R. trifolii ANUS43 grown in defined BIII culture medium was dependent on induction of its bacterial nodulation (nod) genes and the positive reg- ulatory gene nodD on its symbiotic plasmid. ‘H NMR analyses showed less GluNAc in fractionated culture supernatants of nodL and nodM mutant derivatives of R. trifolii ANU843. GluNAc induced three morpholog- ical responses on axenic roots of white clover seedlings: ( i ) root hair branching; ( i i ) tip swelling followed by resumed elongation of root hairs; and (iii) a slight increase in foci of cortical cell divisions, which devel- oped into nodule-like primordia. These biological ac- tivities of extracellular GluNAc from R. trifolii ANUS43 were confirmed with authentic standards of GluNAc. These results indicate that extracellular ac- cumulation of N-acetylglutamic acid is linked to fla- vone-dependent metabolism involving nodD, nodL, and nodM in R. trifolii ANUS43. This constitutes the first report on the structure of a nod-dependent extra- cellular signal from R. trifolii that can affect root hair and nodule development in white clover and whose biological activity on this host has been confirmed with authentic standards.

Rhizobium excretes metabolites that induce a diversity of morphological changes on root hairs of its leguminous host. These morphological responses include enhanced prolifera- tion, elongation, and various deformations (helices, branches, bulbs, and curling at hair tips) (7, 30, 36, 37). Some types of root hair deformation can be induced by cell-free extracts of culture fluid or washed suspensions of rhizobia cultured with- out flavone inducers of nodulation ( n o d ) genes on their sym-

* This work was supported by United States Department of Agri- culture-CRGO Grant 90-37120-5554, the Michigan State University- National Science Foundation Center for Microbial Ecology Grant STC-8809640, Department of Energy Grant DE-FG02-89ER14029, and a postdoctoral fellowship (to S. P.-H.) from the College of Natural Sciences (Michigan State University). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

n To whom correspondence should be addressed. Tel.: 517-353- 8649; Fax: 517-353-8953.

biotic plasmid (pSym)’ (3, 6, 12, 28, 30, 36, 37). Culture supernatants of rhizobia grown with appropriate flavone or root exudate to enhance expression of pSym nod genes gen- erally exhibit more exaggerated ability to deform root hairs (1, 2, 13, 15). These culture supernatants can also elicit other morphogenic responses in host plants, including thickening and shortening of host roots (3, 33), reduced root elongation (13), and mitosis in non-host cells grown in vitro (26). There is also evidence that nodule initiation can result from diffu- sible substances excreted by Rhizobium during their interac- tion with host roots (4, 15, 16, 19, 20, 22).

Recently, Rhizobium meliloti has been reported to excrete a sulfated glucosamine tetrasaccharide bearing three N-acetyl groups and an N-C16 bisunsaturated fatty acid that induces root hair branching and nodule-like structures on its specific host, alfalfa (18,19). Production of this metabolite is depend- ent upon nodH, a pSym nod gene of R. meliloti essential for alfalfa-specific nodulation. This constitutes the first report on the structure of a biologically active molecule excreted from R. meliloti that can be linked to the function of a pSym nod gene. Such extracellular metabolites are referred to as nod signals.

We are studying extracellular metabolites from Rhizobium leguminosarum biovar trifolii (hereafter called R. trifolii) that can induce the morphological changes that occur during in- fection and nodulation of its homologous host, white clover. Our results to date have shown that the cell-free culture supernatant from wild-type R. trifolii ANU843 contains sev- eral bacterial factors capable of affecting development of the root nodule symbiosis with white clover (15,16). Extracellular accumulation of some of these metabolites is flavone-inde- pendent, whereas others are significantly enhanced by fla- vones that activate pSym nod expression. In this study, we purified one of these metabolites from the culture supernatant fluid of R. trifolii ANU843, established its structure as N- acetylglutamic acid (GluNAc), and demonstrated the flavone dependence and certain pSym nod requirements for its extra- cellular accumulation. We have also documented several mor- phological changes in axenic white clover roots induced by this metabolite.

MATERIALS AND METHODS AND RESULTS’

DISCUSSION

In this study, we have purified and characterized a low molecular weight, biologically active metabolite from the cul-

l The abbreviations used are: pSym, symbiotic plasmid; GluNAc, N-acetylglutamic acid; BF-5, bacterial factor 5; FTIR, Fourier trans- form infrared; GC/MS, gas chromatography/mass spectrometry; HPLC, high pressure liquid chromatography.

Portions of this paper (including “Materials and Methods,” “Re-

16854

GluNAc and R. trifolii- White Clover Interactions 16855

FIG. 6. Morphological changes in axenic roots of Dutch white clover (A-C and E-H) and Salina strawberry clover ( D ) induced by application of N-acetylglutamic acid. Seedlings were treated with Fahraeus medium only (A), BF-5 isolated from the culture supernatant of R. trifolii ANU843 (R-U and G), or N-acetyl- glutamic acid from a commercial source ( E and H ) or from organic synthesis (F). Note the branchings and other root hair deformations in B-Fand the round nodule-like cortical meristems (arrowheads) in C-H. Bar scale is 100 pm.

FIG. 7. Root hair deformations on Dutch white clover in- oculated with R. trifoli i ANU843. Seedlings were pretreated with Fahraeus medium ( A ) or BF-5 ( R ) before inoculation. Bar scale is 100 pm.

ture supernatant fluid of wild-type R. trifolii ANU843 grown in defined BIII medium (5). The metabolite was assigned the trivial name bacterial factor 5 (BF-5). The structure of BF-5 was established as N-acetylglutamic acid by proton NMR and Fourier transform IR spectroscopy, combined gas chromatog- raphy/mass spectrometry of the methylated derivative, and organic synthesis. BF-5 induced some of the morphological changes that occur in white clover during infection and no- dulation by this nitrogen-fixing bacterium. This is the first report on the structure of a nod-dependent extracellular signal from R. trifolii capable of affecting development of root hairs and nodules in white clover and whose biological activity has been confirmed with authentic standards.

Our interest in BF-5 began with the finding that its extra- cellular accumulation by R. trifolii ANU843 required growth in the presence of a flavone (e.g. 7, 4'-dihydroxyflavone) capable of inducing nod genes and, in addition, the positive regulatory nodD gene. Together, the flavone and nodD control expression of other pSym nod genes encoding common and

sults," Figs. 1-5, Tables 1 and 2, and "Acknowledgments") are pre- sented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

host-specific functions required for infection and nodulation of clover roots by R. trifolii (9, 17, 23, 24). These results suggested that extracellular accumulation of GluNAc was linked to the cascade of metabolic functions encoded by certain pSym nod gene(s) in R. trifolii ANU843.

In Escherichia coli, GluNAc is an intracellular intermediate in the pathway leading to biosynthesis of arginine, pyrimi- dines, and polyamines (14). Its synthesis from glutamate and acetyl-coA in this pathway is catalyzed by N-acetylglutamate synthase (14). We considered the possibility that nodM and nodL functions may affect the accumulation of GluNAc for the following reasons. (i) Both are pSym nod genes requiring nodD and flavone for induction (9, 17, 24, 29, 35); (ii) it has been proposed that nodM is related to genes encoding certain amidotransferases (11,29) that use glutamine as amino donor and glutamate as by-product (14,21); and (iii) nodL is related to a gene encoding an acetyltransferase using acetyl-coA as donor (10). To test this possibility, we analyzed GluNAc in fractionated culture supernatants of nodL and nodM mutant derivatives of R. trifolii ANU843 grown with 7,4'-dihydroxy- flavone. Our proton NMR analyses indicated significantly diminished amounts of GluNAc in equivalent fractions from both nodL and nodM mutant derivatives. However, since the primary functions have not yet been established for purified protein products of these nod genes, it is uncertain whether the negative effects of these mutations on extracellular accu- mulation of GluNAc are direct or indirect.

The unexpected accumulation of extracellular GluNAc in abnormal amounts may be a result of overflow from normal pathways upon induction of pSym nod genes. Its excretion is consistent with selection against assimilating toxic levels in the cytoplasm or with a function outside the cell, or both. Such secondary metabolites are called "shunt metabolites" (34).

GluNAc induced three morphological responses on axenic white clover seedling roots: (i) root hair branching; (ii) tip swelling followed by resumed elongation of root hairs; and (iii) a slight increase in foci of cortical cell divisions, which developed into nodule-like primordia. This biological activity of BF-5 was confirmed on white clover with commercially available and chemically synthesized GluNAc. We view these morphological responses as a redirection of wall growth in root hairs and a localized stimulation of cell division in cortical cells. GluNAc also induced root hair branching on strawberry clover (another host species), but none of these morphological responses on alfalfa or birds-foot trefoil (non- host legumes). Thus, legumes are not equally responsive to BF-5, and this may vary even among species in the homolo- gous clover cross-inoculation group. Root hair branching and induction of foci of cortical cell divisions on white clover were more significantly enhanced by GluNAc than by L-G~u, L- Gln, L-Arg, or ammonium ion at equimolar concentrations. This implies a chemical specificity (especially the importance of acetylation) in the biological activity of BF-5 on white clover.

We conclude that N-acetylglutamic acid contributes to the diversity of extracellular signals from R. trifolii that trigger morphological responses in its clover host during infection and nodulation processes. One of the major challenges in plant-microbe research over the next few years will be to construct an acceptable model that accommodates this and all other data linking pSym nod functions in development of the nitrogen-fixing Rhizobium-legume symbiosis.

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SUPPLEMENTARY MATERIAL TO

N-ACETYLGLUTAMIC ACID: AN EXTRACELLULAR m D SIGNAL OF RHIZOBIUM T R l F O L i I

CLOVER ROTS

S a l e e l a P h i I I p - H O I I i n g s w 0 r t h . R w I e I . H o i l i n g s w o r t h . mnd F r a n k 8. Daezo

D e p a r t m e n t s of M l e r o b i o l o g y a n d B 1 0 e h e m i s t r y . M l e h l g a n State U n t v e r s i t y . E a s t L a n s i n g , M l e h i g a n 48824 U.S.A.

ANU843 WHICH INDUCES Ra3T HAIR BRANCHING AND NODULE-LIKE PRIMORDIA I N WHITE

M T E R I A L S a n d METHODS

GluNAc and R. trifolii- White Clover Interactions 16857

i n f r e q u e n c y ( T a b l e 2 ) and cxsggera led i n mo tpho loqy (Fxg . 7A-m) i f s e e d l i n g s were p r e l r e t l l r d w i t h BF-5 before i n O F U l e l i O n w i t h R . l r i t o l i i ANU843 cells. T h i s d i l l e r e n e e i n f r e q u e n c y of deformed roe1 hair: e n i n o e v l a t e d s e e d l i n g s was c l o s e 10 t h e I n e r e a r e i n f I c q u c m y i n d u c e d on a x e n i c S c e d l I n g r b y RF-5 alone (Table I ) . P r e i n o c u l a t i o n lreslrnenl w ! l h BF-5 d i d not a f f e e l t h e

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