Vol. 54, No. 10

Expression of Nodulation Genes in Rhizobium leguminosarumbiovar trifolii Is Affected by Low pH and by Ca and Al Ions

ALAN E. RICHARDSON,"2 RICHARD J. SIMPSON,' MICHAEL A. DJORDJEVIC,2 AND BARRY G. ROLFE2*

Plant and Soil Sciences Section, School of Agriculture and Forestry, The University of Melbourne, Parkville, Victoria3052,' and Plant Molecular Biology, Research School of Biological Sciences, Australian National University,

P.O. Box 475, Canberra City, ACT, 2601,2 Australia

Received 11 April 1988/Accepted 25 July 1988

Early stages in the infection of leguminous plants by Rhizobium spp. are restricted at low pH and are furtherinfluenced by the presence of Ca and Al ions. In the experiments reported here, transcriptional andtranslational fusions of the Escherichia coli lacZ gene to Rhizobium leguminosarum biovar trifolii nodulation(nod) genes were used to investigate the effects of pH and of Ca and Al ions on nod gene expression. Theregulatory nodD gene in R. leguminosarum biovar trifolii was constitutively expressed at a range of pH levelsbetween 4.8 and 6.5, and expression was not affected by the addition of 22.5 ,uM Al or 1,250 ,uM Ca. Inductionof expression of nodA, nodF, and region II nodulation genes in the presence of 5 x 10-7 M 7,4'-dihydroxy-flavone was restricted at a pH of <5.7 and was extremely poor at pH 4.8. Induction of nodA expression was

further restricted by 22.5 ,uM Al over a range of pH levels but was increased in the presence of Ca. The additionof Ca, however, only slightly alleviated the Al-mediated inhibition of nodA induction. Induction of expressionof nodA was equally sensitive to low pH in three strains of R. leguminosarum biovar trifolii (ANU845, ANU815,and ANU1184), which exhibited contrasting growth abilities in solution culture at a pH of <5.0. Aluminum,however, differentially affected the induction of nodA in these three strains, with the most Al-tolerant strain forgrowth being the most Al-sensitive strain for nod gene induction. Poor induction of expression of nodulationgenes in R. leguminosarum biovar trifolii was considered to be an important factor contributing to theacid-sensitive step of legume root infection.

The formation of nitrogen-fixing nodules on roots ofleguminous plants is influenced by numerous environmentalfactors (7, 27). Both the growth of Rhizobium spp. and theinfection of legume roots are restricted by low pH and ionimbalances (e.g., Ca deficiency and Al toxicity) that are

associated with acidic soils. Nodulation of Trifolium subter-raneum L. (subterranean clover) is reduced at a pH of <5.0and is generally inhibited by a pH of <4.5, even in thepresence of high numbers of Rhizobium leguminosarumbiovar trifolii (24, 28). Nodulation of subterranean clover atlow pH is further restricted in the presence of Al (23) and isincreased in the presence of Ca (28).An acid-sensitive step in the nodulation of Medicago

sativa L. has been shown to occur within the first 12 h afterinoculation, despite the presence of high numbers of Rhizo-bium meliloti (32). The period of greatest acid sensitivitycoincides with, or occurs prior to, the curling of root hairsand occurs simultaneously with the most Ca-requiring stageof nodule formation (32, 33). An acid-sensitive step haslikewise been observed in the early stages of infection ofPisum sativum L. (26). Therefore, it is apparent that theacid-sensitive step in nodule formation coincides with thetiming of expression of nodulation genes in Rhizobium spp.(13).Nodulation of legume roots requires the coordinated

expression of at least eight genes in several of the fast-growing Rhizobium spp. Nodulation genes in these rhizobiaare located on a large indigenous symbiosis (Sym) plasmid(16, 25, 42) and are arranged into at least four separatetranscriptional units. In R. leguminosarum biovars viciaeand trifolii, these have been designated the nodABCIJ,nodD, nodFEL, and nodMN operons (11, 39, 43, 45).

* Corresponding author.

Expression of these genes in R. leguminosarum biovartrifolii is necessary for normal host-specific nodulation ofclover (12).

In culture media and in the absence of the host plant, onlynodD is constitutively expressed. The nodABC genes of R.leguminosarum biovar trifolii, which are essential for thecurling of root hair cells and the initiation of nodule forma-tion (14, 16, 46), are expressed at very low levels (20). Rapidinduction of expression of nodABC and other inducible nodgenes occurs only in the presence of a functional nodD geneand plant-derived flavonoid compounds (13, 20, 31, 36, 40,48). The principal flavone inducer that is present in cloverroot exudates has been identified as 7,4'-dihydroxyflavone(DHF), which, at low concentrations (5 x 10-7 M), in-creases expression of the nodABC operon in R. legumino-sarum biovar trifolii by up to 25-fold (13, 36). The inductionof expression of nod genes in other fast-growing rhizobia hasalso been shown to require plant-derived flavone and flava-none inducers, which are also present in root exudates ofhost plants (17, 31, 34, 40, 48).The restriction or inhibition of nodule formation under

acidic conditions may be explained by an understanding ofthe interaction between the host plant and the symbiont atthe molecular level. In a previous report (37), it was dem-onstrated that the nod gene induction activity of clover rootexudates from seedlings was reduced at low pH and that thepresence of Ca in plant growth media resulted in markedincreases in activity at a range of pHs. The effects ofconcentrations of H and Ca ions on the net activity offlavone inducers in root exudates was thus considered to bean important factor contributing to the acid sensitivity ofnodule formation. In this study we examined the effects oflow pH and the influence of Ca and Al ions on the inductionof expression of nodulation genes in R. leguminosarum

2541

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1988, p. 2541-25480099-2240/88/102541-08$02.00/0Copyright C) 1988, American Society for Microbiology

2542 RICHARDSON ET AL.

TABLE 1. Bacterial strains and plasmids used in this study and their relevant characteristics

Strain or plasmid Relevant characteristics and phenotypesa Reference or source

R. leguminosarum biovar trifoliiANU843 Wild type, Hac+ Nod' Fix', partially acid tolerant 38ANU845 pSym- ANU843, Hac- Nod- 42ANU794 Derivative of wild-type R. leguminosarum biovar trifolii TA1, Smr 15

Hac+ Nod' Fix', acid intolerantANU815 ApSym ANU794, Hac- Nod- 15ANU1173 Derivative of wild-type R. leguminosarum biovar trifolii NA3001, AIRCSb

Smr Hac+ Nod' Fix', acid tolerantANU1184 pSym- ANU1173, Hac- Nod- This study

Plasmidsc1. pRtO32::nod-932 nodD::lac fusion 202. pRtO32::nod-218 nodA::lac fusion 203. pRtO32::nod-1027 nodF::lac fusion 204. pRtO32::nod-711 region II::lac fusion 205. pRtO32::nod-1J4 nodA::lac fusion 13a Abbreviations: Hac, root hair curling; Nod, nodule formation; Fix, nitrogen fixation; Smr, streptomycin resistant; pSym-, symbiotic plasmid deleted.b Australian Inoculants Research and Control Service, Gosford, New South Wales, Australia.c Derivatives of pRtO32 (42) containing transcriptional fusions (fusions 1 to 4) or a translational fusion (fusion 5) of the E. coli lacZ gene to specific nod genes.

biovar trifolii in the presence of a known concentration ofDHF.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study are listed in Table 1. A Sym-derivative of the acid-tolerant strain ANU1173 was isolatedby heat curing the wild-type strain at 40°C (49). This strainwas designated ANU1184. Putative Sym plasmid-curedstrains were identified by their inability to nodulate Trifoliumrepens (white clover) by an agar plate method (38). Loss ofthe Sym plasmid was confirmed by plasmid visualization byusing a modified Eckhardt procedure (35) and by Southernblot DNA analysis with a nodD-specific DNA probe (A. E.Richardson, unpublished data).The plasmids used in this study (Table 1) were derivatives

of the self-transmissible plasmid pRtO32, which carries a14-kilobase-pair HindIII fragment encoding nodulation andhost specificity determinants of R. leguminosarum biovartrifolii ANU843 (42). Construction of the pRtO32 derivativescontaining transcriptional or translational fusions of theEscherichia coli lac operon (4) to specific nodulation geneshas been described previously (13, 20). Plasmids containingspecific lacZ gene fusions were transferred to Sym plasmid-cured R. leguminosarum biovar trifoiji strains by conjuga-tion by using a patch mating technique (15). Transconjugantswere selected by replica plating on modified TM minimalmedia (20) containing 200 ,ug of kanamycin sulfate ml-'.Growth of R. leguminosarum biovar trifolii at low pH and in

the presence of Al. Strains of R. leguminosarum biovartrifolii were grown at a range of pHs in liquid medium whichcontained 10 g of mannitol liter-1, 1.8 g of sodium glutamateliter-', and basal nutrients and which was based on themedium of Keyser and Munns (22). The basal nutrients were1.5 mM KCI, 300 ,uM MgSO4, 300 ,uM CaCl2, 10 ,MFe-EDTA, 5 ,uM KH2PO4, 1 ,uM MnCl2, 0.4 ,uM ZnSO4, 0.1,uM CuCl2, 0.02 ,uM Na2MoO4, and 0.001 ,uM Co(NO3)2.Vitamins were also added at concentrations equivalent tothose present in 2 g of yeast extract liter-' (5). The pH of themedium was measured with a glass-combination pH elec-trode (Ross electrode model 81-04; Orion Research Inc.,Cambridge, Mass.) and was adjusted to the various pH

levels with either HCl or NaOH before the media wereautoclaved. When necessary, filter-sterilized AlCl3 (0.01 M)was added after the media had been autoclaved, to achieve afinal concentration of 22.5 ,uM. Duplicate flasks were inoc-ulated with freshly grown cells of each R. leguminosarumbiovar trifolii strain, to give an initial cell density of -5 x 103cells ml-'. Cell densities were determined by plating 0.1 mlof 10-fold serial dilutions on BMM solid medium (1). Cul-tures were grown with orbital shaking at 28°C.

Induction assays and measurement of ,-galactosidase activ-ity. For P-galactosidase induction assays, strains of R.leguminosarum biovar trifolii containing the nod-lac genefusions were grown overnight in BMM liquid medium (1) atpH 6.5 to an early logarithmic phase of growth (opticaldensity of 0.20 to 0.25 at 600 nm). Assays were conductedwith 200 p.l of culture added to 1.2 ml of 30 mM 2-(N-morpholino)ethanesulfonic acid (MES buffer) at pH 4.8, 5.0,5.2, 5.4, 5.7, or 6.5. The final induction assay medium ateach pH contained an expected basal concentration of 42p.M Ca (i.e., that which was carried over from the BMMmedium in which the cells were grown). The Ca concentra-tions given throughout this report are those of the added Ca(i.e., 0, 125, or 1,250 ,uM). Solutions also contained Al at afinal concentration of 0, 7.5, or 22.5 p.M. The volume of theassay solution was made up to 1.6 ml with either distilledH20 or a solution of DHF (obtained from J. Redmond,Macquarie University, Macquarie, New South Wales, Aus-tralia), to give a final concentration of 5.0 x 10-7 M DHF.Cultures were incubated at 28°C for 90 min, to allow for theinduction of expression of R. leguminosarum biovar trifoliinodulation genes and, subsequently, for the expression ofthe lacZ gene and synthesis of P-galactosidase.

Following the induction period, ,-galactosidase activitywas determined in 400-,ul fractions of lysed cultures in Zbuffer (30) at pH 7.5 in the presence of o-nitrophenyl-p-D-galactopyranoside by methods described elsewhere (37).The 3-galactosidase activities of R. leguminosarum biovartrifolii cultures were calculated as described by Miller (30)and were shown either as activity both in the presence andthe absence of DHF or, alternatively, as a ratio of theactivity induced in the presence of DHF relative to theactivity in the absence of DHF.

APPL. ENVIRON. MICROBIOL.

RHIZOBIUM nod GENE EXPRESSION AT LOW pH 2543

0t

-4

5

10 ANU843

6 V -A A

2 -

0 l l lANUI 173

8

6-

4

2-

ANU7948

2

0A0 2 4 6 8

Time (days)

FIG. 1. Growth of R. leguminosarum biovar trifolii ANU843,ANU1173, and ANU794 in solution culture at pH 6.5 (U, O), 5.0 (0,0), and 4.6 (A, A) in the absence (closed symbols) and the presence

(open symbols) of 22.5 ,uM Al. Each point plotted is the geometricmean of two observations.

All solutions were sterilized by autoclaving prior to use,

except for Al stock solutions, which were sterilized bymembrane filtration (pore size, 0.45 ,um). Stock solutions ofDHF (10-' M) were made up in ethanol. Final stock solutionconcentrations of DHF (4.0 x 10-6 M) were made in steriledistilled H20 following evaporation of the ethanol.The pHs of induction cultures were measured before and

after the incubation period. The pHs of solutions during the,-galactosidase assay period were also measured to confirmthat the pH was within the optimum range for maximumactivity of the enzyme. The influence ofpH and of Ca and Alions on ,B-galactosidase activity in either sodium phosphate(100 mM) or MES (30 mM) buffer was also determined byusing purified E. coli P-galactosidase (grade VIII; SigmaChemical Co., St. Louis, Mo.).

RESULTS

Growth of R. leguminosarum biovar trifolii at low pH and inthe presence of Al. Growth of wild-type R. leguminosarumbiovar trifolii ANU843, ANU1173, and ANU794 in solutionculture at pH 6.5, 5.0, and 4.6 in the presence of 0 and 22.5puM Al is shown in Fig. 1. Strain ANU1173 was tolerant tolow pH and initiated logarithmic growth at a pH as low as

4.6. Strain ANU794 failed to increase in cell number at pH4.6 and exhibited poor growth at pH 5.0. Growth ofANU794at pH 5.0 was further inhibited by the presence of 22.5 ,uMAl, while R. leguminosarum biovar trifolii ANU1173 grew

A B C D3-

FIG. 2. Gel electrophoresis pattern of plasmid DNA isolatedfrom R. leguminosarum biovar trifolii. Lane A, ANU1184; lane B,ANU1173; lane C, ANU845; lane D, ANU843. Note the loss of thesmallest resident plasmid in strains ANU1184 and ANU845, whichare nonnodulating derivatives of wild-type strains ANU1173 andANU843, respectively.

rapidly under these conditions (Fig. 1). Strain ANU843exhibited an intermediate tolerance to growth at low pH inthe presence and absence of Al (Fig. 1).The resident Sym plasmid from the acid-tolerant strain

ANU1173 was cured (resulting in strain ANU1184) by heatcuring techniques (Fig. 2). The growth of Sym plasmid-deleted derivatives of the three wild-type R. leguminosarumbiovar trifoiji strains (ANU845, ANU1184, and ANU815)was examined at pH 6.5, 5.0, and 4.6 (data not shown) andwas the same as that shown in Fig. 1 for the respectiveparental strains. Growth at low pH of the three Sym plasmid-deleted R. leguminosarum biovar trifoiji strains containingpRtO32: :nodA2J8 (see below) was also identical to thatshown for the respective parental strains (A. E. Richardson,unpublished data). In all instances, the pH of the mediumduring the growth period did not change from the initial pH,unless substantial growth occurred (i.e., a final cell densityof >i107 cells ml-').

Effect of pH on the expression and induction of specificnodulation genes in R. leguminosarum biovar trifolii. To testthe effect of pH on the expression of specific nodulationgenes, R. leguminosarum biovar trifoiji strains carrying thelacZ-containing derivatives of pRtO32 were grown to anearly logarithmic phase of growth in BMM medium (pH 6.5)and were subsequently incubated at the various pHs in thepresence and absence of the nod gene-inducing compoundDHF. The presence of 30 mM MES buffer satisfactorilycontrolled the pH of the induction assays, and the pH did notshift by more than 0.1 pH unit from the initial pH in any ofthe treatments throughout the 90-mmn induction period. Afterinduction, the pH of cultures was readjusted to between 7.0and 7.4 by the addition of Z buffer (pH 7.5), to ensure thatoptimum P3-galactosidase activity was obtained. The activityof pure preparations of P3-galactosidase was adversely af-fected only when the pH was less than 6.8. In addition, at pH7.0, MES buffer, Ca (125 and 1,250 FM), or Al (7.5 and 22.5R.aM) did not affect the activity of purified P3-galactosidase(data not shown).

Early-log-phase cultures of ANU845 cells carrying deriv-atives of pRtO32 were subsequently exposed to a range ofpHs to measure the effect of pH on the induction ofexpression of transcriptional lacZ gene fusions to specific R.leguminosarum biovar trifoiji nodulation genes. The results

VOL. 54, 1988

2544 RICHARDSON ET AL.

2

0

3

2

0

2-

A

._

"0

0CT3la._

0

CZm

tocn~4._0

rA

2

6nodD

4

2

0

nodA

6

4

2

Z"0c)CZ

la

._ncolODC&

-

0

on

en._

nodF4 -

2 S ]_

4

2 u zregion II

4.8 5.0 5.2 5.4 5.7 6.5

pH of induction assay

FIG. 3. Expression and induction of nodulation genes in R.leguminosarum biovar trifolii at a range of pHs in the absence (solidboxes) and presence (hatched boxes) of 5 x 10-7 M DHF. Expres-sion was determined by measuring ,-galactosidase activity of tran-scriptional lacZ gene fusions to the nodD, nodA, nodF, and regionII genes introduced into R. leguminosarum biovar trifolii ANU845.The mean of two observations is plotted for each treatment.

(Fig. 3) show that the nodD gene (as measured frompRtO32::nodD932) was expressed at a similar level in boththe absence and presence of DHF and was not affected bypHs between 4.8 and 6.5.

In contrast to nodD expression, the expression of severalinducible nodulation genes (e.g., nodA) was adversely af-fected at low pH (Fig. 3). In the absence of DHF, a low butconsistent level of expression of these nod genes was ob-served at all pHs (Fig. 3). Expression of nodA (as measuredfrom pRt3O2::nodA218) was increased 11- to 14-fold in thepresence of 5 x 10-' M DHF at pH 6.5 (Fig. 3 and 4,respectively). At a lower pH, expression of nodA was alsoinduced in the presence of DHF; however, the magnitude ofthe increase was dependent on the pH of the inductionassay. At pH 4.8, for instance, only a 1.6- to 3.3-foldincrease in induction of nodA expression was observed (Fig.3 and 4). Induction of the lacZ gene fusion to the nod-711locus (region II) (which is transcribed in the same operon as

nodABC) and to the nodF locus was similarly affected over

the range of pHs tested (Fig. 3). Induction of nodF (nod-1027) and region II (nod-711) genes in the presence of DHFwas generally inhibited at a pH of <5.7.The influence of Ca and Al on the expression of nodA and

nodD in R. leguminosarum biovar trifolii at low pH. R.leguminosarum biovar trifolii ANU845 cells containing plas-mids to measure nodD or nodA expression were grown to an

early logarithmic phase of growth at pH 6.5 and thenincubated for measurement of nod gene expression at vari-

pH 6.5

pH 5.7

pH 5.2

pH 4.8

7.5 22.5 125 1250Control pM Al uM Ca

Treatment

FIG. 4. Effects of Ca and Al on the expression and induction ofnodA in R. leguminosarum biovar trifolii at pH 6.5, 5.7, 5.2, and 4.8in the absence (solid boxes) and presence (hatched boxes) of 5 x10-7 M DHF. The concentrations shown are those of the added Caand Al. All treatments including controls contained a basal concen-

tration of 42 ,uM Ca and 0 F.M Al. Expression was determined bymeasuring ,-galactosidase activity of a transcriptional lacZ genefusion to the nodA gene introduced into R. leguminosarum biovartrifolii ANU845. The mean of two observations is plotted for eachtreatment.

ous pHs in the presence and absence of Ca and Al. Expres-sion of nodD (from pRtO32: :nodD932) at pH 6.5 and 4.8 wasnot affected by the presence of either Al or Ca ions (Fig. 5).In contrast, induction of nodA expression (pRtO32::nodA218) was reduced at low pH and was further influencedby the presence of Al and Ca ions (Fig. 4). The P-galacto-sidase activity associated with the expression of nodA in theabsence of DHF was low, but it was generally similar in alltreatments (Fig. 4). The expression of nodA in the presenceof 5 x 10-7 M DHF was induced 14-, 16-, 4-, and 3-foldrelative to the level of expression in the absence of DHF atpH 6.5, 5.7, 5.2, and 4.8, respectively. In the presence of 7.5and 22.5 puM Al, induction of expression of nodA was

reduced at all pH levels (Fig. 4). The addition of Ca (125 and1,250 F.M) increased the induction of nodA expression in thepresence of DHF, but only at the lower pH levels examined(pH 5.2 and 4.8; Fig. 4).

Induction of expression of nodA in R. leguminosarum biovartrifolii strains with different abilities to grow at low pH. To testthe effect of pH on the induction of nodA expression inseveral R. leguminosarum biovar trifolii strains, plasmidspRtO32::nodA2J8 and pRtO32::nodAJ14 (which contains atranslational fusion of Mu dlI 1734 to nodA) were introducedinto strains ANU845, ANU1184, and ANU815. R. legumi-nosarum biovar trifolii strains were grown to an early

(pRtO32::nod 932)

(pRtO32::nod 218)

(pRtO32::nodlO27)

(pRtO32::nod71 1)

NeMI

Jwd

APPL. ENVIRON. MICROBIOL.

I

u

RHIZOBIUM nod GENE EXPRESSION AT LOW pH 2545

pH 6.5

c724-00

pH 4.8

0

7.5 22.5 125 1250Control juM Al gM Ca

TreatmentFIG. 5. Effects of Ca and Al on the expression of nodD in R.

leguminosarum biovar trifolii at pH 6.5 and 4.8 in the absence (solidboxes) and presence (hatched boxes) of 5 x 10-7 M DHF. Theconcentrations shown are those of the added Ca and Al. Alltreatments including controls contained a basal concentration of 42,uM Ca and 0 ,uM Al. Expression was determined by measuring,-galactosidase activity of a transcriptional lacZ gene fusion to thenodD gene introduced into R. leguminosarum biovar trifoliiANU845. The mean of two observations is plotted for each treat-ment.

logarithmic phase of growth in BMM liquid medium at pH6.5 prior to induction assay at various pHs. The results (Fig.6) show that the induction by DHF of expression of thetranscriptional lacZ gene fusion to nodA (pRtO32: :nodA218)was similarly affected at a range ofpHs in R. leguminosarumbiovar trifolii ANU1184 and ANU815 as it was for strain

6

4-1

0

10

4

2

0

6

4

2

0

4.8 5.0 5.2 5.4 5.7 6.5

pH of induction assay

FIG. 6. Expression and induction of nodA in two strains of R.leguminosarum biovar trifolii at a range of pHs in the absence (solidboxes) and presence (hatched boxes) of 5 x 1o-7 M DHF. Expres-sion was determined by measuring ,B-galactosidase activity of atranscriptional lacZ gene fusion to the nodA gene introduced into R.leguminosarum biovar trifolii ANU1184 and ANU815. The mean oftwo observations is plotted for each treatment.

ANU845 (Fig. 3). Poor induction of nodA expression in thethree R. leguminosarum biovar trifolii strains occurred at pH4.8, despite the contrasting ability of these strains to initiaterapid logarithmic growth at this pH (Fig. 1).A low pH similarly reduced the induction of expression of

the lacZ translational fusion to nodA (pRtO32::nodA114) inR. leguminosarum biovar trifolii ANU845, ANU815, andANU1184 (Table 2). At a pH of >5.4, the induction ofexpression of nodA in the three R. leguminosarum biovartrifolii strains was not markedly affected by the addition of125 or 1,250 ,uM Ca. However, at a lower pH, Ca increasedthe induction of nodA expression by a similar magnitude inall strains. In contrast, 22.5 ,uM Al restricted the induction ofnodA expression in the presence of DHF. However, theextent to which Al affected expression of nodA was depen-dent on the strain of R. leguminosarum biovar trifolii. Forinstance, in R. leguminosarum biovar trifolii ANU1184, Alrestricted induction of nodA expression at pH levels below5.4. This was evident despite the ability of this strain and itscorresponding parental strain (ANU1173) to grow rapidly inthe presence of 22.5 puM Al at a pH as low as 4.6 (Fig. 1).Similar Al-mediated inhibition of nodA expression was ob-served in ANU845 (Table 2), despite the intermediate toler-ance for growth of this strain in the presence of Al. Theinduction of nodA expression in strain ANU815, which wasthe least tolerant to Al for growth, was not affected by 22.5,uM Al. Furthermore, the reduced induction of expression ofnodA that was observed at a low pH in the presence of 22.5p,M Al (e.g., in R. leguminosarum biovar trifolii ANU845and ANU1184) was not alleviated at the lower pHs by theaddition of 1,250 ,uM Ca (Table 2).

DISCUSSION

Nodulation of subterranean clover roots in solution cul-ture has been shown to be restricted at a pH of <5.5, even inthe presence of high numbers of R. leguminosarum biovartrifolii (24, 28). These observations are consistent with theresults of the present experiments, in which induction of nodgene expression was adversely affected in R. leguminosarumbiovar trifolii at a pH of <5.7. Moreover, enhanced induc-tion of nod gene expression in the presence of Ca was alsoconsistent with observations that Ca increases nodulation ofsubterranean clover roots at a range of pHs and lowers theapparent critical pH requirement for successful nodulation(28, 29). The concentrations of Al used in these experiments(7.5 to 22.5 ,uM) have been shown to restrict or inhibit thenodulation of subterranean clover in solution culture at pH4.5 (23). Poorer induction of nod gene expression in thepresence of Al was similarly observed at a range of pHlevels. Since nod gene expression is necessary for theinitiation of the early steps of nodulation, our results areconsistent with the notion that an acid-sensitive step in theinfection of M. sativa in solution culture (i) occurs within thefirst 12 h after inoculation, (ii) occurs prior to the curling ofroot hair cells, (iii) is apparent despite inoculation withadequate numbers of R. meliloti, and (iv) occurs simulta-neously with the most Ca-requiring phase of the infectionprocess (32, 33).The wild-type strains of R. leguminosarum biovar trifolii

used throughout these experiments (ANU843, ANU794, andANU1173) exhibited clear differences in their ability to growat low pH and in the presence of Al. Despite this, theinduction of nodA expression in the presence of DHF wasequally inhibited in early-log-phase cultures of all strainsunder acidic conditions. It was also evident that tolerance

VOL. 54, 1988

2546 RICHARDSON ET AL.

TABLE 2. Effects of pH and of Ca and Al ions on the expression and induction of a translational lacZ gene fusion to the nodAgene of R. leguminosarum biovar trifolii in strains ANU845, ANU815, and ANU1184

R. leguminosarum biovar trifolii Activity in the absence Induced expressionc in the presence of 5 x 1i-7 M DHF at pH:strain and treatmenta of DHF (mean ± SD)b 4.8 5.0 5.2 5.4 5.7 6.5

ANU845 (pRtO32::nodA1J4)Control 57 + 21 6 11 17 36 37 4922.5 FM Al 55 + 13 3 3 7 13 13 49125 FM Ca 82 ± 18 11 19 29 34 40 361,250 ,uM Ca 76 ± 25 18 29 31 35 35 36125 ,uM Ca-22.5 ,uM Al 47 ± 7 4 5 7 11 23 491,250 ,M Ca-22.5 uM Al 68 ± 15 4 4 8 24 40 35

ANU815 (pRtO32::nodA114)Control 54 ± 9 10 13 18 26 34 3222.5 ,M Al 33 ± 5 13 17 19 30 35 52125 ,M Ca 41 ± 7 13 19 37 47 44 451,250 ,M Ca 53 ± 15 21 28 32 35 34 34125 F±M Ca-22.5 p.M Al 43 ± 4 7 14 21 27 41 491,250 ,uM Ca-22.5 puM Al 41 ± 3 11 17 22 32 47 45

ANU1184 (pRtO32::nodA1J4)Control 48 ± 7 11 19 25 30 33 4022.5 p.M Al 51 ± 16 2 4 6 24 20 48125 pM Ca 59 ± 24 15 22 21 33 39 341,250 ,uM Ca 47 ± 16 24 25 35 37 38 35125 ,uM Ca-22.5 ,uM Al 60 ± 11 2 3 4 20 43 321,250 pFM Ca-22.5 FM Al 47 ± 10 6 10 22 31 34 36a Concentrations shown are those of the added Ca and Al. All treatments including controls contained a basal concentration of 42 ,uM Ca and 0 ,uM Al.b Mean 0-galactosidase activity (and standard deviation) of R. leguminosarum biovar trifolii strains in each treatment determined in the absence of DHF over

the range of pHs.c Induced expression of nodA at each pH was determined by the ratio of P-galactosidase activity in the presence of DHF relative to ,3-galactosidase activity

in the absence of DHF.

for growth at low pH in laboratory media was independent ofthe Sym plasmid and that the introduction of derivatives ofpRtO32 did not affect the growth characteristics of the Symplasmid-deleted strains at low pH. Although nod gene induc-tion in these three strains was equally sensitive to low pH,the addition of Ca and Al, however, was shown to differen-tially affect the induction of expression of the nodA gene.Thus, it was apparent from these results that Al indepen-dently affected both the growth of the bacteria and theinduction of nod gene expression. It is therefore possiblethat selection of R. leguminosarum biovar trifolii for toler-ance to growth at low pH and in the presence of Al may notnecessarily select for tolerance with regard to nod geneexpression under such conditions. The experiments reportedhere, however, were performed only with lac gene fusions tothe nodulation genes of strain ANU843, which were intro-duced into the various R. leguminosarum biovar trifoliistrains. It is unknown whether genes that reside in moreacid-tolerant strains would respond similarly to environmen-tal variables.The results presented here with R. leguminosarum biovar

trifolii ANU845 showed that the regulatory nodD gene wasexpressed constitutively both in the presence and in theabsence ofDHF over a range ofpHs between 4.8 and 6.5. Incontrast to this, low pH inhibited the induction of theexpression of flavone-inducible nodulation genes in R. legu-minosarum biovar trifolii (which includes the nodABC genesthat are known to be involved in root hair curling). Poorerinduction of these genes occurred at low pH despite (i) aconstant and low level of expression over the pH range in theabsence of DHF, (ii) DHF being present at a concentrationwhich has been shown not to be limiting for induction of R.leguminosarum biovar trifolii nod genes in assays at nearneutral pH (13, 36), and (iii) cultures of R. leguminosarum

biovar trifolii being used in their most responsive phase ofgrowth for rapid induction of nod gene expression (i.e.,early-logarithmic-phase cultures [13]). Results of these ex-periments also indicated that the expression of nodD was notaffected by the presence of Ca and Al ions at a range of pHs.In contrast to this, Ca increased the level of induction of thenodA gene at a pH of <5.7, while Al adversely affected theinduction of nodA expression at all pHs.

It is known that the induction of nodulation genes infast-growing Rhizobium spp. requires the expression of thenodD gene and the presence of specific flavonoid inducers. Ithas been suggested that flavonoids interact with the carboxy-terminal region of the nodD gene product (3, 19), which theninduces expression of other nodulation genes (18, 41, 43, 44,48). The expression of these genes is essential for nodulationability. In these experiments, expression of nodD was unaf-fected at low pH and flavone was added in adequate con-centrations for nod gene induction. Despite this, induction ofnod gene expression in R. leguminosarum biovar trifolii wasrestricted at low pH. Thus, it is possible that the low pHeither reduced the activity of flavone inducer or influencedthe responsiveness of R. leguminosarum biovar trifolii to itspresence. It has been shown that the nod gene-inducingactivity of DHF is dependent on hydroxylation of the C-7position of the A ring and the C-4' position of the B ring (13).It is unlikely that hydroxylation of DHF would be affectedby the pH range used throughout these experiments, as thePKa values of the phenolic hydroxyl groups are above 9 and,thus, they would not be ionized at pH 4.0 to 7.0. As aconsequence, changes in the solubility of DHF at low pHwere also not expected (J. R. Redmond, personal communi-cation). A possibility, therefore, is that low pH restricts theuptake of DHF by R. leguminosarum biovar trifolii and thatthis process is further influenced by Ca and Al.

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RHIZOBIUM nod GENE EXPRESSION AT LOW pH 2547

Poor induction of nod gene expression in rhizobia couldinfluence subsequent nodulation of host plants in severalways. For instance, reduced expression of nodABC genesadversely affects the ability of the bacteria to curl root haircells (14, 16, 25, 46). Alternatively, expression of nodFEgenes in R. leguminosarum biovar trifolii is known to benecessary for normal host-specific nodulation of cloverspecies (12). It may also be possible that the specific attach-ment of rhizobia to root hair cells is itself the first limitingstep under acidic conditions. Attachment of rhizobia tolectins on the surface of the roots has been shown to be pHdependent (9) and, more importantly, has been shown to bemediated by the bacteria only when they are exposed to rootexudates (10). More recently, it has been shown that nodgene expression is necessary in the elicitation of the lectin-binding ability in R. leguminosarum biovar trifolii (8).

It is currently possible to identify three important factorsthat could affect the induction of nodulation gene expressionin R. leguminosarum biovar trifolii and, consequently, thatcould restrict or inhibit early stages of nodulation underacidic conditions. First, there is the inability of R. legumi-nosarum biovar trifolii to grow rapidly at low pH and in thepresence of Al (47). Low numbers of R. leguminosarumbiovar trifolii and poor colonization of acidic soils have beenshown to restrict nodulation of host plants (6; A. E. Richard-son, A. P. Henderson, G. S. James, and R. J. Simpson, SoilBiol. Biochem., in press). Rapid growth of rhizobia withinthe root environment would be necessary for successfulinfection, as rapid induction of nod gene expression occursmost readily in bacterial cultures in an early logarithmicphase of growth (13). Furthermore, because emerging roothair cells are themselves only transiently infectible forperiods as short as 6 h (2; S. Z. Huang, M. A. Djordjevic,and B. G. Rolfe, J. Plant Physiol., in press), rapid growth ofthe bacteria and the ability to respond to the flavone inducersreleased from the emerging root hair cells would be criticalto the subsequent success of infection. Second, we havereported that the net nod gene-inducing activity of exudatesfrom subterranean clover seedlings is restricted at a pH of<5.0 (37). It has also been shown that the concentration offlavone inducer released from roots of M. sativa growing inLeonard jars at near neutral pH is limiting for nodulation(21). Thus, a lower release of flavone inducers under acidicconditions would exacerbate poor induction of nodulationgene expression. Third, the sensitivity to low pH and to Al ofthe induction of nodulation gene expression in R. legumino-sarum biovar trifolii (described in this report) would furthercontribute to restricted nodulation under acidic conditions.

ACKNOWLEDGMENTSThis study was supported by grants (to B.G.R., M.A.D., and

R.J.S.) from the Wool Research Trust Fund on the recommendationof the Australian Wool Corporation and a grant (to B.G.R.) from theAustralian Meat and Livestock Research and Development Corpo-ration. Support to A.E.R. from a Commonwealth PostgraduateResearch Award is also acknowledged.We thank R. Roughley (Australian Inoculants Research and

Control Service, Gosford. New South Wales, Australia) for makingavailable to us R. leguminosarum biovar trifolii NA3001. We alsothank Elena Gartner for excellent technical assistance and JanMcIver and Anne Moten for helpful discussions.

LITERATURE CITED1. Bergersen, F. J. 1961. The growth of Rhizobium in synthetic

media. Aust. J. Biol. Sci. 14:349-360.2. Bhuvaneswari, T. V., A. A. Bhagwat, and W. D. Bauer. 1981.

Transient susceptibility of root cells in four common legumes to

nodulation by rhizobia. Plant Physiol. 68:1144-1149.3. Burn, J. E., L. Rossen, and A. W. B. Johnston. 1987. Four

classes of mutation in the nodD gene of Rhizobium legumino-sarum biovar viciae which affect its ability to autoregulate and/or to activate other nod genes in the presence of flavonoidinducers. Genes Dev. 1:456-464.

4. Castilho, B. A., P. Olfson, and M. J. Casadaban. 1984. Plasmidinsertion mutagenesis and lac gene fusion with mini-Mu bacte-riophage transposons. J. Bacteriol. 158:488-495.

5. Chakrabarti, S., M. S. Lee, and A. H. Gibson. 1981. Diversity inthe nutritional requirements of strains of various Rhizobiumspecies. Soil Biol. Biochem. 13:349-354.

6. Coventry, D. R., J. R. Hirth, T. G. Reeves, and H. R. Jones.1985. Development of populations of Rhizobium trifolii andnodulation of subterranean clover following the cropping phasein crop-pasture rotations in South-Eastern Australia. Soil Biol.Biochem. 17:17-22.

7. Dart, P. J. 1974. The infection process, p. 381-429. In A.Quispel (ed.), Frontiers of biology, vol. 33. The biology ofnitrogen fixation. North-Holland Publishing Co., Amsterdam.

8. Dazzo, F. B., R. I. Hollingsworth, J. E. Sherwood, M. Abe, E. M.Hrabak, A. E. Gardiol, H. S. Pankratz, K. B. Smith, and H.Yang. 1985. Recognition and infection of clover root hairs byRhizobium trifolii, p. 239-245. In H. J. Evans, P. J. Bottomley,and W. E. Newton (ed.), Nitrogen fixation research progress.Martinus Nijhoff, Dordrecht, The Netherlands.

9. Dazzo, F. B., and D. H. Hubbel. 1975. Cross-reactive antigensand lectin as determinants of symbiotic specificity in the Rhizo-bium-clover association. Appl. Microbiol. 30:1017-1033.

10. Dazzo, F. B., M. R. Urbano, and W. J. Brill. 1979. Transientappearance of lectin receptors on Rhizobium trifolii. Curr.Microbiol. 2:15-20.

11. Djordjevic, M. A., D. W. Gabriel, and B. G. Rolfe. 1987.Rhizobium-the refined parasite of legumes. Annu. Rev. Phy-topathol. 25:145-168.

12. Djordjevic, M. A., R. W. Innes, C. A. Wijffelman, P. R.Schofield, and B. G. Rolfe. 1986. Nodulation of specific legumesis controlled by several distinct loci in Rhizobium trifolii. PlantMol. Biol. 6:389-401.

13. Djordjevic, M. A., J. W. Redmond, M. Batley, and B. G. Rolfe.1987. Clovers secrete specific phenolic compounds which eitherstimulate or repress nod gene expression in Rhizobium trifolii.EMBO J. 6:1173-1179.

14. Djordjevic, M. A., P. R. Schofield, R. W. Ridge, N. A. Morrison,B. J. Bassam, J. Plazinski, J. M. Watson, and B. G. Rolfe. 1985.Rhizobium nodulation genes involved in root hair curling (Hac)are functionally conserved. Plant Mol. Biol. 4:147-160.

15. Djordjevic, M. A., W. Zurkowski, J. Shine, and B. G. Rolfe.1983. Sym plasmid transfer to various symbiotic mutants ofRhizobium trifolii, R. leguminosarum, and R. meliloti. J. Bac-teriol. 156:1035-1045.

16. Downie, J. A., C. D. Knight, A. W. B. Johnston, and L. Rossen.1985. Identification of genes and gene products involved in thenodulation of peas by Rhizobium leguminosarum. Mol. Gen.Genet. 198:255-262.

17. Firmin, J. L., K. E. Wilson, L. Rossen, and A. W. B. Johnston.1986. Flavonoid activation of nodulation genes in Rhizobiumreversed by other compounds present in plants. Nature(London) 324:90-92.

18. Hong, G. F., J. E. Burn, and A. W. B. Johnston. 1987. Evidencethat DNA involved in the expression of nodulation (nod) genesin Rhizobium binds to the product of the regulatory gene nodD.Nucleic Acids Res. 15:9677-9690.

19. Horvath, B., C. W. B. Bachem, J. Schell, and A. Kondorosi.1987. Host-specific regulation of nodulation genes in Rhizobiumis mediated by a plant-signal, interacting with the nodD geneproduct. EMBO J. 6:841-848.

20. Innes, R. W., P. L. Kuempel, J. Plazinski, H. Canter-Cremers,B. G. Rolfe, and M. A. Djordjevic. 1985. Plant factors induceexpression of nodulation and host-range genes in Rhizobiumtrifolii. Mol. Gen. Genet. 201:426-432.

21. Kapulnik, Y., C. M. Joseph, and D. A. Phillips. 1987. Flavonelimitations to root nodulation and symbiotic nitrogen fixation in

VOL. 54, 1988

2548 RICHARDSON ET AL.

alfalfa. Plant Physiol. 84:1193-1196.

22. Keyser, H. H., and D. N. Munns. 1979. Tolerance of rhizobia toacidity, aluminum and phosphate. Soil Sci. Soc. Am. J. 43:519-523.

23. Kim, M.-K., C. J. Asher, D. G. Edwards, and R. A. Date. 1985.Aluminium toxicity: effects on growth and nodulation of subter-ranean clover, p. 501-503. In Proceedings of the XV Interna-tional Grassland Congress. Science Council of Japan & Japa-nese Society of Grassland Science, Tochigi-ken, Japan.

24. Kim, M.-K., D. G. Edwards, R. A. Date, and C. J. Asher. 1985.Effects of pH on nodulation and growth of subterranean clovercultivars, p. 543-544. In Proceedings of the XV InternationalGrassland Congress. Science Council of Japan & JapaneseSociety of Grassland Science, Tochigi-ken, Japan.

25. Kondorosi, E., Z. Banfalvi, and A. Kondorosi. 1984. Physicaland genetic analysis of a symbiotic region ofRhizobium meliloti:identification of nodulation genes. Mol. Gen. Genet. 193:445-452.

26. Lie, T. A. 1969. The effect of low pH on different phases ofnodule formation in pea plants. Plant Soil 31:391-406.

27. Lie, T. A. 1974. Environmental effects on nodulation andsymbiotic nitrogen fixation, p. 555-582. In A. Quispel (ed.),Frontiers of biology, vol. 33. The biology of nitrogen fixation.North-Holland Publishing Co., Amsterdam.

28. Loneragan, J. F., and E. J. Dowling. 1958. The interaction ofcalcium and hydrogen ions in the nodulation of subterraneanclover. Aust. J. Agric. Res. 9:464 472.

29. Lowther, W. L., and J. F. Loneragan. 1968. Calcium andnodulation in subterranean clover (Trifolium subterraneam L.).Plant Physiol. 43:1362-1366.

30. Miller, J. 1972. Experiments in molecular genetics. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

31. Mulligan, J. T., and S. R. Long. 1985. Induction of Rhizobiummeliloti nodC expression by plant exudate requires nodD. Proc.Natl. Acad. Sci. USA 82:6609-6613.

32. Munns, D. N. 1968. Nodulation of Medicago sativa in solutionculture. I. Acid-sensitive steps. Plant Soil 28:129-146.

33. Munns, D. N. 1970. Nodulation of Medicago sativa in solutionculture. V. Calcium and pH requirements during infection. PlantSoil 32:90-102.

34. Peters, N. K., J. W. Frost, and S. R. Long. 1986. A plant flavone,luteolin, induces expression of Rhizobium meliloti nodulationgenes. Science 233:977-980.

35. Plazinski, J., Y. H. Cen, and B. G. Rolfe. 1985. General methodfor the identification of plasmid species in fast-growing soilmicroorganisms. Appl. Environ. Microbiol. 48:1001-1003.

36. Redmond, J. R., M. Batley, M. A. Djordjevic, R. W. Innes, P. L.Kuempel, and B. G. Rolfe. 1986. Flavones induce expression of

nodulation genes in Rhizobium. Nature (London) 323:632-635.37. Richardson, A. E., M. A. Djordjevic, B. G. Rolfe, and R. J.

Simpson. 1988. Effects of pH, Ca and Al on the exudation fromclover seedlings of compounds that induce the expression ofnodulation genes in Rhizobium trifolii. Plant Soil 109:37-47.

38. Rolfe, B. G., P. M. Gresshoff, and J. Shine. 1980. Rapidscreening for symbiotic mutants of Rhizobium and white clover.Plant Sci. Lett. 19:277-284.

39. Rossen, L., E. 0. Davis, and A. W. B. Johnston. 1987. Plant-induced expression of Rhizobium genes involved in host speci-ficity and early stages of nodulation. Trends Biochem. Sci. 12:430-433.

40. Rossen, L., C. A. Shearman, A. W. B. Johnston, and J. A.Downie. 1985. The nodD gene of Rhizobium leguminosarum isautoregulatory and in the presence of plant exudate induces thenodA, B, C genes. EMBO J. 4:3369-3373.

41. Rostas, K., E. Kondorosi, B. Horvath, A. Simoncsits, and A.Kondorosi. 1986. Conservation of extended promoter regions ofnodulation genes in Rhizobium. Proc. Natl. Acad. Sci. USA 83:1757-1761.

42. Schofield, P. R., R. W. Ridge, B. G. Rolfe, J. Shine, and J. M.Watson. 1984. Host-specific nodulation is encoded on a 14 kbDNA fragment in Rhizobium trifolii. Plant Mol. Biol. 3:3-11.

43. Schofield, P. R., and J. M. Watson. 1986. DNA sequence of theRhizobium trifolii nodulation genes reveals a reiterated andpotentially regulatory sequence preceding the nodABC andnodFE genes. Nucleic Acids Res. 14:2891-2905.

44. Spaink, H. P., R. J. H. Okker, C. A. Wijffelman, E. Pees, andB. J. J. Lugtenberg. 1987. Promoters in the nodulation region ofthe Rhizobium leguminosarum Sym plasmid pRLlJI. Plant Mol.Biol. 9:27-39.

45. Surin, B. P., and J. A. Downie. 1988. Characterization of theRhizobium leguminosarum genes nodLMN involved in efficienthost-specific nodulation. Mol. Microbiol. 2:173-183.

46. Torok, I., E. Kondorosi, T. Stepkowski, J. Posfai, and A.Kondorosi. 1984. Nucleotide sequence of Rhizobium melilotinodulation genes. Nucleic Acids Res. 12:9509-9524.

47. Wood, M., and J. E. Cooper. 1988. Acidity, aluminium andmultiplication of Rhizobium trifolii: effects of initial inoculumdensity and growth phase. Soil Biol. Biochem. 20:83-87.

48. Zaat, S. A., C. A. Wijffelman, H. P. Spaink, A. A. N. vanBrussel, R. J. H. Okker, and B. J. J. Lugtenberg. 1987. Induc-tion of the nodA promoter of Rhizobium leguminosarum Symplasmid pRLlJI by plant flavanones and flavones. J. Bacteriol.169:198-204.

49. Zurkowski, W., and Z. Lorkiewicz. 1979. Plasmid-mediatedcontrol of nodulation in Rhizobium trifolii. Arch. Microbiol.123:195-201.

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