extracellular glycanases of rhizobium leguminosarum are ... · extracellular glycanases of...

JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Mar. 2000, p. 1304–1312 Vol. 182, No. 5

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Extracellular Glycanases of Rhizobium leguminosarumAre Activated on the Cell Surface by anExopolysaccharide-Related Component

ANGELES ZORREGUIETA, CHRISTINE FINNIE,† AND J. ALLAN DOWNIE*

John Innes Centre, Norwich, NR4 7UH, United Kingdom

Received 13 September 1999/Accepted 7 December 1999

Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopo-lysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wallcellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies ofR. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB,CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMCdegradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutantstrains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless itis in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no suchdiffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but theseenzymes were inactive unless they came into contact with an EPS1 strain, indicating that EPS is required foractivation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction,indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer ofPlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on alawn of R. leguminosarum. This indicates that the surface of A. tumefaciens is inappropriate to activate CMCdegradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secretedglycanases by surface components may occur in other species.

Nodule morphogenesis on the roots of leguminous plantscan be induced by chemical signaling molecules made by rhi-zobia. These morphogenetic signals (Nod factors) are oli-gomers of (usually four or five) b,1-4-linked N-acetylglu-cosamine residues carrying an N-linked fatty acyl group on theterminal (nonreducing) sugar (8, 11). In several legumes, nod-ule morphogenesis can be induced by the purified signals in theabsence of any bacteria (8, 11). For a successful symbiosis to beestablished between rhizobia and legumes, it is also necessaryfor the bacteria to invade the plant root. Invasion usuallyoccurs via infection threads, which are intracellular tunnelsmade as a result of plant cell walls laid down within the cyto-plasm of root hair epidermal and cortical cells. Although Nodfactors induce the production of “cytoplasmic bridges,” intra-cellular structures thought to be precursors of the infectionthreads, the presence of bacteria is necessary for developmentof infection threads (46). Invasion is a clonal event, and therhizobia grow at the tip of the infection thread; it is this growth(rather than bacterial migration) that enables the bacteria toreach the growing nodule meristem (16). The bacteria arereleased into nodule cells where they differentiate and inducethose genes required for nitrogen fixation.

Selectivity during this infection process is important becauseit is crucial for the plant to exclude other potential invasivebacteria. Therefore, there are several checks and balances re-

quired to ensure that invasion is limited to the appropriatebacteria. It is evident that Nod factors play a role in recognitionduring invasion since bacterial mutants that make Nod factors,lacking host specific modifications, are unable to infect nor-mally (1). There are several other factors that are necessary forinfection. In some rhizobia there is clear evidence for a role forthe secreted signaling protein NodO (13, 49) that forms cation-selective pores in membranes and has been proposed to act onthe plant plasma membrane (42). It appears that at least insome situations NodO plays a role in the establishment ofinfection events (20). NodO is secreted via a type I proteinsecretion system encoded by prsD and prsE (14, 15), which alsosecretes enzymes involved in processing of the bacterial exopo-lysaccharide (EPS). The EPS is essential for invasion. Rhizo-bial mutants lacking EPS cannot invade (29). In some casesaddition a low-molecular-weight fraction of an EPS-derivedoligosaccharide can restore infection to EPS-deficient mutants,suggesting a signaling process (2, 10, 18). The role of EPS ininfection is not well understood, although it does appear it maybe required for maturation and elongation of infection threads(7). One proposal is that there may be a role for specific EPSfragments in suppressing plant defense responses during infec-tion (34).

Low-molecular-weight EPS is produced in Rhizobium me-liloti by two processes, a specific biosynthetic route and cleav-age of a higher-molecular-weight form (19). A type I secretionsystem (encoded by the prsDE genes) is required for the se-cretion of the EPS-cleaving enzymes ExoK and ExsH (50), andPlyA and PlyB of R. leguminosarum are secreted similarly (14,15). The mature EPS of R. meliloti is not cleaved by the ExoKand ExsH glycanases; these enzymes cleave nascent EPS chains(52), and the absence of the succinyl group decreased the

* Corresponding author. Mailing address: John Innes Centre, Nor-wich Research Park, Colney, Norwich, NR4 7UH, United Kingdom.Phone: 1603-452571. Fax: 1603-456844. E-mail: [email protected].

† Present address: Department of Plant Biology, Royal Veterinaryand Agricultural University, 1871 Frederiksberg C, Copenhagen, Den-mark.

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susceptibility of succinoglycan to cleavage (51). It is not knownif modification of the R. leguminosarum EPS confers resistanceto cleavage by PlyA and/or PlyB.

The PlyA and PlyB glycanases from R. leguminosarum arenot specific for the EPS polymer but can also degrade carboxy-methyl cellulose (CMC). These enzymes appeared to remaincell bound (14, 15) because degradation of CMC or EPS in-corporated into agar plates only occurred directly below thecolony and there was no halo of degradation beyond the col-ony, as is usually seen with many other enzymes secreted viatype I secretion systems. Several groups have analyzed cellu-lases produced by rhizobia because many of the observations ofinfection appear to involve degradation of the plant cell wall(5, 21, 30, 39, 45). However, there was little or no cellulaseactivity in a cell-free culture supernatant from R. leguminosa-rum bv. trifolii (24, 33). These observations, together with theabsence of a halo of CMC degradation around colonies of R.leguminosarum bv. viciae grown on CMC-containing plates,suggested that the CMC-degrading activity secreted via type Isecretion system remains cell bound (15). In this study wedemonstrate that CMC-degrading activity is released from thecell surface but is inactive. Activation only occurs when contactis made with the bacteria. This activation is specific for the cellsurface and the enzymes secreted by R. leguminosarum cannotbe activated by the surfaces of other species of rhizobia.

MATERIALS AND METHODS

Microbiological methods. Rhizobium spp. were grown at 28°C in TY medium(3) with appropriate antibiotics at the following concentrations (mg/ml): strep-tomycin, 400; kanamycin, 20; spectinomycin, 20; tetracycline, 10; lividomycin, 5.For polysaccharide preparation, bacteria were grown in Y medium (41) contain-ing mannitol (0.2% [wt/vol]). Culture optical densities were measured at 600 nmwith an MSE Spectro-Plus spectrophotometer. The bacterial strains and plas-mids used are described in Table 1. Plasmids were transferred to Rhizobium spp.by triparental mating with a helper plasmid.

Construction of EPS mutants. Plasmid pIJ7298 carries the polysaccharidesynthesis genes pssFCDE linked to the protein secretion genes prsDE and theglycanase gene plyA (15). The EPS mutant strains A507 and A517 were gener-ated by first mutagenizing pIJ7298 with Tn5. Several Tn5-containing derivativesof pIJ7298 were transferred to 8401/pRL1JI, and the Tn5 insertions were re-combined into the genome by homologous recombination selecting for markerexchange as described previously (40). Two of the Tn5 mutations caused anEPS2 phenotype when they were marker exchanged to make strains A507 andA517. The mutations in A507 and A517 were mapped 1.8 and 8.6 kb upstreamof the start of pssF in what is thought to be a large cluster of genes required forEPS synthesis. To generate a mutant lacking both the prsDE secretion genes andthe pss gene region, a deletion mutant was constructed. pIJ7298 carrying theprsDE and pssFCDE genes (15) was digested with HindIII. This results in thedeletion of about 20 kb of DNA including prsDE, pssFCDE, and other uniden-tified EPS biosynthetic genes (see above) and leaves about 2 kb of DNA up-stream of prsD and about 6 kb of DNA downstream of the pss gene cluster. The3.4-kb HindIII fragment from Tn5 carrying nptII was cloned into the deletedderivative of pIJ7298 to form pIJ7471. The deletion allele was recombined intothe genome of 8401/pRL1JI by marker exchange by using plasmid pPH1JI toselect for recombinants essentially as described previously (40). A kanamycin-resistant, tetracycline-sensitive mutant was selected and called A550. This strain

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Relevant characteristics Reference or source

StrainsR. leguminosarum bv. viciae isogenic strains

8401 R. leguminosarum lacking pSym, Strr 288401/pRL1JI Derivative of 8401 carrying the symbiotic plasmid pRL1JI 12A168 8401/pRL1JI pssA1::Tn5 4A412 8401/pRL1JI prsD1::Tn5 14A507 8401/pRL1JI pss5::Tn5 This workA517 8401/pRL1JI pss6::Tn5 This workA550 8401/pRL1JI DplyA-prs-pss::nptII This workA600 8401/pRL1JI plyB1::Tn5 15A638 8401/pRL1JI plyA3::Spcr 15A640 8401/pRL1JI plyA3::Spcr plyB1::Tn5 15

Other strains1021 R. meliloti S. Long3855 R. leguminosarum bv. viciae 25ANU843 R. leguminosarum bv. trifolii 9ANU2840 Tn5-induced EPS2 mutant of ANU280 (Smr Rifr derivative

of NGR234)6

C58C1 A. tumefaciens 47CE3 R. etli 35CIAT899 R. tropici 32NZP2213 R. loti DSIR Culture CollectionRCR5 R. leguminosarum bv. trifolii 22Rhizobium sp. strain NGR234 Broad-host-range Rhizobium sp. 44USDA193 S. fredii USDA-ARS National

Rhizobium CultureCollection

VF39 R. leguminosarum bv. viciae 37

PlasmidspIJ7298 pLAFR1 cosmid carrying prsDE and plyA 14pIJ7471 HindIII deletion of pIJ7298 with the kanamycin resistance

gene from Tn5 on a 3.4-kb HindIII fragmentThis work

pIJ7709 plyB cloned in pIJ1891 15pIJ7871 plyA cloned behind a vector promoter in pKT230 15pRGC1 pLAFR1 cosmid carrying the egl endoglycanase gene from

A. caulinodans17

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is EPS2 and was confirmed to be defective for protein secretion by testing for thesecretion of NodO as described previously (14). The mutant behaved as ex-pected, in that it was complemented for protein secretion by pIJ7298, but not bythe HindIII deleted derivative of it (pIJ7471) used to make the mutant.

Plate assays. CMC was incorporated into the Y–0.2% mannitol agar plates at0.1%. Colonies were grown for 3 days at 28°C and washed off with water. Theplates were then flooded with 0.1% (wt/vol) Congo red in water (43) for 15 min,washed for 10 min with 1 M NaCl, and then washed for 5 min with 5% aceticacid. Degradation of CMC was observed as clearings (reduction of staining). Toprepare a CMC plate containing a bacterial lawn, a sterile cotton tipped rod wasdipped in a liquid suspension containing about 107 CFU/ml and used to spreada layer on the agar. To test the effect of a bacterial lawn on the ability of coloniesto degrade CMC, 10 ml of a bacterial suspension (ca. 107 CFU/ml) was loaded ona plate to form a homogeneous colony. The plates were then incubated at 28°Cfor 4 days. For detection of EPS degradation activity, EPS was precipitated froma 5 days of culture of 8401/pRL1JI with 3 volumes of ethanol and then redis-solved in sterile deionized water. The EPS was incorporated into Y-agar platesat about 2 mg/ml. Assays of EPS degradation by colonies and colonies grown onlawns was as described above for CMC degradation except that the plates wereincubated for 2 days and the wash with 1 M NaCl was omitted.

RESULTS

Identification of diffusible glycanase activity. R. leguminosa-rum bv. viciae produces two glycanases that are secreted in aprsD-dependent way. They were called PlyA and PlyB and aresomewhat unusual because, although they are fully secreted,they appeared to remain attached to the bacterial cell surface.Generally, when bacterial proteins are secreted via a type Isecretion system, the activity of the secreted protein (e.g., he-molysins, proteases, or lipases) can be detected beyond thearea of growth of colonies grown on the appropriate indicatoragar medium. This results in the formation of a “halo” ofclearing of an appropriate substrate such as the lysis of redblood cells by hemolysin or degradation of milk proteins bysecreted proteases.

Although PlyA and PlyB are secreted by a typical type Isecretion system (14, 15) and cleave the substrate CMC, thezone of CMC degradation with the wild-type strain 8401/pRL1JI does not extend beyond the edge of the colony (Fig.1a). The protein secretion (prsD) mutant A412 does not inducesuch CMC degradation because neither PlyA or PlyB (themajor enzymes that cleave CMC) are secreted. However, whenwe grew a colony of the prsD mutant (A412) in close proximityto the wild-type parent (8401/pRL1JI) a small zone of CMCcleavage could be detected below that part of the mutantcolony which was closest to the colony of the wild type (Fig.1a). Initially, we assumed that the partial zone of clearingbelow A412 was caused by cell lysis of A412, resulting in therelease of PlyA and PlyB; this could, for example, have beencaused by secretion of a bacteriocin or some other factor from8401/pRL1JI. However, strain 8401 (lacking the indigenousplasmid pRL1JI, which carries the gene encoding bacteriocin),also activated CMC degradation below the colony of A412(Table 2), demonstrating that it was not the bacteriocin thatcaused this effect.

To determine if the induced degradation was related torelease of PlyA or PlyB, we carried out a similar test using aplyA plyB double mutant (A640) grown adjacent to 8401/pRL1JI. We saw enhanced degradation below the colony ofA640 in the zone closest to the colony of 8401/pRL1JI (Fig.1b). This demonstrates that the cleavage of CMC induced by8401/pRL1JI under the adjacent colony cannot be due to re-lease of PlyA or PlyB following cell lysis. An alternative expla-nation is that PlyA and/or PlyB secreted by the wild-type straincould be present as a halo around the 8401/pRL1JI colony butthat CMC degradation is not activated unless the secretedglycanase is in direct contact with the colony. Such a modelcould account for the lack of CMC degradation in the zonesbetween the colonies (Fig. 1a and b).

If such a model holds true, it should be possible to demon-strate the presence of a halo of CMC degradation beyond theedge of the colony by activating the secreted enzyme. Wedevised an assay to test this; the prsD secretion mutant (A412)was inoculated as a lawn onto the CMC agar, and then strain8401/pRL1JI was grown as a colony on the surface of the agar.This resulted in a clear halo of CMC degradation (Fig. 1c).With colonies of the prsD mutant A412 and the plyA plyBdouble mutant A640 grown on a lawn of A412, there was nota halo of CMC degradation typical of that seen with 8401/pRL1JI (Fig. 1c and d). When the lawn of A412 (prsD) wasreplaced with a lawn of A640 (plyA plyB), we also observedclear evidence of a halo of degradation around colonies of8401/pRL1JI but not A412 or A640 (data not shown). These

FIG. 1. Activation of glycanase by cells of mutants defective for extracellularglycanase production. In panels a and b, colonies of strain 8401/pRL1JI weregrown adjacent to the protein secretion mutant A412 (prsD) or the glycanasemutant A640 (plyA plyB); the cells were grown for 3 days on Y medium con-taining CMC. In panels c and d, these strains were grown on the same mediumthat had been seeded with a lawn of A412 and incubated for 4 days. After growth,the cells were washed off, and the plates were stained with Congo red; theunstained regions correspond to areas where the CMC has been degraded.

TABLE 2. Induction of a clearing on neighboring colonies

Donor IndicatorCMC degradation inducedbelow adjacent indicator

colony

8401/pRL1JI A412 (prsD) 18401 A412 (prsD) 18401/pRL1JI A640 (plyA plyB) 1A168 (pssA) A412 (prsD) 1A550 (Dprs Dpss) A412 (prsD) 28401/pRL1JI A168 (pssA) 2

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observations indicate that the halo around 8401/pRL1JI is dueto the secretion of PlyA and/or PlyB.

Whereas there is very little CMC degradation below colo-nies of A640 or A412 grown alone, some CMC degradation isseen when these colonies are grown on a lawn of A412 (Fig. 1cand d) or A640 (data not shown), and this was somewhatenhanced at the perimeter of the colony. We are not sure as towhy such an effect is seen only when these mutants are grownon a lawn of A412. It may be related to the fact that A412 itselfcan induce some CMC degradation as seen in the center of thecolony of A412 in Fig. 1a. This CMC degradation may be dueto lysis of some cells in the A412 lawn, resulting in the releaseof glycanases. Alternatively, this degradation could be due tothe presence of another glycanase that has not yet been iden-tified, but which accumulates as colonies get older. Even withthe double mutant A640 we observe some CMC degradation(see, for example, Fig. 6a) which increases with time of incu-bation.

EPS mutants do not activate CMC degradation. One of thesubstrates of PlyA and PlyB is the EPS, and we thought itpossible that the EPS might activate PlyA and/or PlyB to de-grade CMC. A simple prediction of such a model is that mu-tants defective for EPS production should be defective forCMC degradation even under the zone of colony growth. Wetested several different EPS-deficient mutants for their abilityto cleave CMC. As shown (Fig. 2), they are all significantlyaffected in their ability to degrade CMC. The mutant (A168),which was most severely affected for EPS production, carries amutation in the pssA gene that encodes the first glycosyl trans-ferase required for EPS biosynthesis (4, 23, 48). As shown (Fig.2), this mutant has a very low background of CMC degradationsimilar to that seen with the secretion mutant A412. Other EPSmutants affected in EPS formation are also affected for CMCdegradation (Fig. 2), although the pssA mutant seemed to beslightly more defective for CMC degradation than the othermutants. These observations suggest that the EPS plays a rolein activation of CMC degradation by the secreted glycanases.However, this experiment does not eliminate the alternativeexplanation, that EPS mutants might be defective for secretionof PlyA and PlyB. We tested for secretion of these glycanasesby growing a colony of the EPS mutant A168 (pssA) adjacent

to a colony of the prsD secretion mutant A412. This cross-feeding resulted in activation of CMC degradation below thecolony of A412 (Fig. 3). As a control we made a mutant (A550)that is defective for both protein secretion and for EPS pro-duction; this mutant (A550) induced no degradation below theadjacent colony of A412 (Fig. 3 and Table 2).

The secretion of CMC-degrading enzymes by the EPS mu-tant A168 can also be demonstrated by growing a lawn of theprsD mutant on CMC agar and then inoculating the EPS2

mutant as a colony. In this case (Fig. 4b), the halo produced bythe pssA mutant (A168) is similar to that produced by thecontrol 8401/pRL1JI. This can be explained if the glycanase issecreted by A168 but is inactive for CMC degradation, unlessthere are EPS1 cells present (i.e., A412 in the lawn) (Fig. 4aand b). The observation that the plyA plyB double mutant,A640, does not form a diffusible halo (Fig. 1d) demonstratesthat the halo of degradation is due to PlyA and/or PlyB. Similarobservations to those in Fig. 4b were seen if the lawn wasformed by A640 (data not shown).

If the EPS defective mutant, A168, is grown as a lawn on theagar, typical CMC degradation is seen under a colony of 8401/pRL1JI, but there is no halo (Fig. 4c). The absence of a halo inthis case can be explained on the basis that A168, which formsthe lawn, produces no EPS and therefore degradation onlyoccurs adjacent to the EPS produced by 8401/pRL1JI, indicat-ing that the EPS plays a role in halo formation. Similar CMCdegradation was seen when the prsD secretion mutant A412and the plyA plyB double mutant, A640, were grown on a lawnof the EPS mutant A168 (Fig. 4c). In this assay it appears thatit is the (EPS) surface of the colonies of A412 or A640 thatactivate CMC degradation by glycanases that are produced(but are inactive) in the lawn formed by A168. In these cases,however, the zone of CMC degradation is limited to the edgeof the colony and no halo is formed. This fits with the model

FIG. 2. EPS2 mutants are defective for CMC degradation. Growth andstaining conditions were as in Fig. 1a and b. The EPS-defective mutants A168,A507, and A517 have much less CMC degradation than their parent 8401/pRL1JI and have similar or lower levels of CMC degradation than the glycanasesecretion mutant A412.

FIG. 3. The EPS-deficient mutant A168 can induce CMC degradation belowan adjacent (EPS1) colony. Growth and staining conditions were as in Fig. 1aand b. Strain A168 produces no EPS, and A550 is defective for both EPSproduction and protein secretion. A168 but not A550 induced CMC degradationbelow part of the adjacent colony of the secretion mutant A412.



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that the surface of the mutants A412 and A640 activates CMCdegradation.

The results above indicate a role for EPS in the activation ofCMC degradation. We prepared a crude EPS fraction from8401/pRL1JI and from A412 by ethanol precipitation fromculture supernatants. This EPS preparation was incorporatedinto CMC assay plates, which were then inoculated with strains8401/pRL1JI (WT), A168 (pssA), and the protein secretionmutant A412 (prsD) as a negative control. No halo of CMCdegradation was observed around 8401/pRL1JI or A168 (datanot shown), indicating that the EPS precipitated from thegrowth medium is insufficient to activate CMC degradation.This implies that either another factor is required for activa-tion of CMC degradation or that some EPS component notprecipitated is required for activation. The results of theseexperiments with EPS incorporated into plates, are consistentwith our earlier observations, which demonstrated that PlyAand PlyB degrade EPS incorporated into plates, but that thezone of degradation did not extend beyond the edge of thecolony (reference 15 and Fig. 5a). When we incorporated EPSinto agar plates and carried out EPS degradation assays in asimilar way to the CMC degradation assays described in Fig. 1cand d and Fig. 4b (with colonies inoculated on a lawn of A412or A640), we observed halos of degradation of EPS similar tothose seen with CMC (Fig. 5b and c). Also shown in Fig. 5a is

the observation that the EPS2 mutant (A168) is defective forEPS degradation, even though this mutant produced gly-canases that can be activated by the surfaces of A412 (Fig. 5b)or A640 (Fig. 5c).

PlyB is responsible for halo formation. Are both secretedglycanases, PlyA and PlyB, activated by the presence of EPS1

bacteria and diffusible through the agar? To answer thesequestions, we analyzed the ability of plyA and plyB mutants toproduce halos on CMC plates containing a lawn of A412 (Fig.6b). The plyA mutant (A638) retained activity, but the plyBmutant (A600) did not (Fig. 6), demonstrating that PlyB isresponsible for most of the diffusible activity. A limitation ofthis assay is that plyA seemed to be relatively weakly expressed(15), and so the apparent lack of a halo of PlyA activity (in theplyB mutant) could be due to insufficient expression of plyA. Tocompensate for this, we used the cloned plyA gene on pIJ7871,which confers good CMC degradation to the plyA plyB mutantA640 (Fig. 6a). A640/pIJ7871 did not induce halo formation onan A412-CMC layer (Fig. 6b), indicating that PlyA probablymostly remains cell bound. We also analyzed CMC degrada-tion below colonies of A412 (prsD) in a neighboring colonyassay by using strain A640 carrying cloned plyA (pIJ7871) orplyB (pIJ7709). The results of this assay (Fig. 6c) confirm thatPlyB is diffusible but PlyA is not. However, PlyA does seem torequire activation by an EPS-related component because noCMC degradation was observed by the EPS2 mutant A168(pssA) even when pIJ7871 was present (not shown).

Analysis of CMC degradation by LPS mutants. We consid-ered the possibility that activation of CMC degradation mayalso involve the lipopolysaccharide (LPS) surface layer. Wetested the glycanase activity of several different mutants of R.leguminosarum bv. viciae affected for LPS biosynthesis (26, 31).Of those tested, only one mutant (B659) had reduced CMCdegradation. This mutant retained the ability to secrete PlyB(and probably PlyA) because when it was tested for CMCdegradation using a lawn of A412, a clear halo of degradationcould be detected. Therefore, only this mutant behaved in asimilar way to the EPS-defective mutants. B659 is somewhatdifferent from the other LPS mutants tested, in that both itsEPS and its LPS production are affected (38). Since the otherLPS mutants retained normal levels of CMC-degrading activ-

FIG. 4. Cross-stimulation of glycanase activity among EPS2, glycanase se-cretion, and glycanase-defective mutants. Cells, as indicated, were grown onCMC-agar (a), CMC-agar seeded with a lawn of the secretion mutant A412(prsD) (b), and CMC-agar seeded with the EPS2 mutant A168 (pssA) (c).Growth and staining was as described for Fig. 1.

FIG. 5. Assays of EPS degradation by glycanase, secretion, and EPS2 mu-tants. The assays were carried out as described for CMC degradation (Fig. 1),except that EPS replaced the CMC in the agar. In panel a the colonies weregrown on EPS-agar. The degradation below 8401/pRL1JI did not extend beyondthe colony; there was a low level of degradation seen with the glycanase mutantA640 (plyA plyB) and the secretion mutant A412 (prsD), but none was seen withthe EPS2 mutant A168. In panel b the EPS-agar was seeded with a lawn of A412(prsD), and in panel c there was a lawn of A640 (plyA plyB).



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ity, we conclude that the LPS is not necessary for the activationof CMC degradation by the secreted glycanases.

PlyB is not activated by surface oligosaccharides of relatedbacteria. Agrobacterium tumefaciens is closely related to R.leguminosarum, but it cannot degrade CMC (Fig. 7a). WhenplyB (on pIJ7709) was transferred to A. tumefaciens, the colo-nies remained unable to cleave CMC (Fig. 7a). An explanationfor this lack of activity is that A. tumefaciens may not providethe appropriate factor that activates PlyB. When colonies of A.tumefaciens carrying pIJ7709 (plyB) were grown on CMC platesinoculated with a lawn of A412 (prsD), a halo of CMC degrada-tion was clearly seen (Fig. 7b). Since no such CMC degradationoccurred in the absence of either plyB (Fig. 7b) or added A412(Fig. 7a), this demonstrates that PlyB can be secreted by A.tumefaciens but is inactive unless activated by the appropriatecell surface (in this case provided by A412 but not A. tumefa-ciens). The observation that A. tumefaciens does not induceCMC degradation enabled us to test whether the surface ofthis bacterium could activate PlyB. When 8401/pRL1JI wasinoculated onto a CMC plate containing a lawn of A. tumefa-ciens no halo of CMC degradation was observed (data not

shown). This reconfirms the observation that the surface of A.tumefaciens cannot activate PlyB.

Activation and inhibition of CMC degradation by other rhi-zobia. We extended the analysis of CMC degradation to otherrhizobia. R. tropici CIAT899, R. fredii USDA193, and Rhizo-bium sp. strain NGR234 induced CMC degradation at a levelgreater than that seen with R. leguminosarum bv. viciae 8401/pRL1JI; R. leguminosarum bv. trifolii RCR5 and ANU843, R.leguminosarum bv. viciae 3855 and VF39, and R. loti NZP2213were similar to 8401/pRL1JI; R. etli CE3 had somewhat lessdegradation than 8401/pRL1JI under the growth conditionstested, and R. meliloti 1021 as previously described (50) wasunable to degrade CMC. In each case, CMC degradation waslimited to the zone below the colony, and no halo of degrada-tion was observed (Fig. 8a).

Since there is CMC degradation by several of these strains,we are in a position to test if there are diffusible enzymes thatcan be activated by the R. leguminosarum mutant A412 (prsD)that does not secrete glycanases. This was done by inoculatingeach strain onto a lawn of A412 on a CMC plate (Fig. 8b). Asexpected, R. leguminosarum bv. viciae strains 3855 and VF39produced a halo of CMC degradation, as did R. leguminosarumbv. trifolii RCR5 and ANU843. R. etli CE3 formed a weak halothat is correlated with the very low CMC degradation. Noneof the other strains (including R. tropici CIAT899, R. fredii

FIG. 6. plyB but not plyA encodes a diffusible glycanase. CMC degradationwas assayed using CMC-agar (a and c) or CMC agar seeded with a lawn of theprotein secretion mutant A412 (b). Growth and staining were as in Fig. 1. Thestrains used are the parental strain 8401/pRL1JI and its derivatives carryingmutations in plyA (A638), plyB (A600), or both plyA and plyB (A640) and A640derivatives carrying cloned plyA (on pIJ7871) or plyB (on pIJ7709). In panel c,the A640 cells carrying cloned plyA or plyB were cultured at various distancesfrom A412 to get a measure of the relative distance of cross activation by PlyB.

FIG. 7. plyB cloned in A. tumefaciens produces a glycanase that is not de-tected unless cells of R. leguminosarum are present. CMC degradation wasassayed using CMC-agar (a) or CMC-agar seeded with a lawn of the proteinsecretion mutant A412 (b). In panel a, A. tumefaciens strain induces no CMCdegradation even if it carries plyB cloned on pIJ7709, but plyB-dependent activitycan be seen in panel b, where a lawn of A412 is present. Assay conditions are asdescribed in Fig. 1.



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USDA193, Rhizobium sp. strain NGR234, and R. loti NZP2213)produced a halo of CMC degradation. The absence of halosindicates that either the CMC-degrading enzymes produced bythese strains are not released from the surface or that they arereleased but are inactive and are not activated by the surface ofR. leguminosarum. In the absence of derivatives of these vari-ous strains defective for glycanase production or secretion, it isdifficult to unambiguously determine whether the extracellularglycanase activity is only active if the appropriate cell surface ispresent. However, EPS-deficient mutants of NGR234 havebeen described (6). One such mutant (ANU2840), blocked inan early stage of EPS biosynthesis, was tested to determine ifloss of EPS production in this strain is associated with loss ofCMC degradation. No CMC degradation was observed directlybelow the colony, indicating that CMC degradation byNGR234 is activated by an EPS-related component. In con-trast, the cloned endoglycanase egl gene (on pRGC1) fromAzorhizobium caulinodans was transferred into the exopolysac-charide defective mutant of R. leguminosarum bv. viciae; a highlevel of CMC degradation was observed indicating that thisglycanase is not activated by EPS.

We also tested if the surface of R. meliloti (which induces noCMC degradation) is able to replace the surface of A412 toactivate glycanases by inoculating each strain on a CMC platecontaining a lawn of R. meliloti. In this case, no halo of CMCdegradation was observed around the colonies of all testedstrains (Fig. 8c).

During the course of analysis of CMC degradation by vari-ous strains, we observed that R. leguminosarum bv. viciae A412(prsD) strongly reduced CMC degradation below the coloniesof those strains that could degrade CMC (Fig. 8b). This is mostobvious with R. tropici CIAT899, R. fredii USDA193, and Rhi-zobium sp. strain NGR234, which normally induce very strongCMC degradation (Fig. 8a). This mirrors the observation madewith A. tumefaciens carrying PlyB (pIJ7709); this strain inducesa halo of CMC degradation (Fig. 7b) caused by secreted PlyB,but there is little CMC degradation below the colony, indicat-ing that PlyB activity may be inhibited by the surface of A.tumefaciens. A similar effect was seen with the EPS-deficientmutant (A168) of R. leguminosarum bv. viciae grown on a lawnof A412 (Fig. 4b).

DISCUSSION

Previously, Finnie et al. (15) concluded that the secretedglycanases encoded by plyA and plyB remain attached to thesurface of R. leguminosarum bv. viciae. This conclusion wasdrawn on the basis of the absence of CMC or EPS cleavagebeyond the edge of agar colonies and was consistent withreports by others (24, 33) that there is cell-associated cellulaseactivity in different rhizobia, but there are extremely low levelsof activity in the culture supernatants of strains that havecell-associated cellulase activity. The observations describedhere demonstrate that, at least for PlyB, our earlier conclu-sions are incorrect because PlyB is secreted and diffuses awayfrom the cells but is inactive unless it is in contact with the cellsurface. It is evident that some component associated with EPSbiosynthesis is necessary for activation of PlyB since the EPS-defective mutants cannot induce activation. We were unable toactivate PlyB with an ethanol-precipitated preparation of EPS,indicating that the mature EPS does not induce the activation.This result fits with our previous observation that colonies of R.leguminosarum bv. viciae grown on agar containing EPS candegrade the EPS below the colony, but there is no halo of EPSdegradation extending beyond the edge of the colony. Theseobservations are consistent with a model in which nascent EPSor an intermediate in EPS biosynthesis activates PlyB to cleaveboth CMC and EPS. Recent work with R. meliloti is pertinentto this point. The mature succinoglycan of R. meliloti is notcleaved by the secreted glycanases ExoK and ExsH (52). It wasrecently demonstrated that the levels of succinylation and acet-ylation strongly influence the susceptibility of nascent succino-glycan to glycanases (51). In light of the observations describedhere, there may be a possible alternative explanation of theobservations made with R. meliloti, namely, that the maturesuccinoglycan can be cleaved but only, for example, if theglycanase is activated by an immature succinoglycan compo-nent.

The R. leguminosarum bv. viciae plyA gene product seems tobehave somewhat differently from PlyB in that we did notobserve CMC-degrading activity beyond the edge of the colonyunder any of the situations tested. Nevertheless, PlyA behaveslike PlyB in that it is inactive in EPS-defective mutants and canbe activated by the surface of glycanase-nonsecreting strains

FIG. 8. CMC degradation by various rhizobia. CMC degradation was assayed using CMC-agar (a), CMC-agar seeded with a lawn of the protein secretion mutantA412 (b), or CMC-agar seeded with a lawn of R. meliloti 1021 (c). The strains used are R. leguminosarum bv. viciae 8401/pRL1JI, R. etli CE3, R. leguminosarum bv.trifolii RCR5 and ANU843, R. leguminosarum bv. viciae 3855 and VF39, R. tropici CIAT899, S. fredii USDA193, R. meliloti 1021, Rhizobium sp. strain NGR234, andR. loti NZP2213.



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that make EPS. PlyA and PlyB are 71% identical, but PlyAcontains an extra 50 amino acids near the C-terminal domainwhich could play a role in maintaining PlyA attachment to thecell surface.

Why should the glycanases PlyA and PlyB only be active inassociation with the rhizobial cell surface? This begs the ques-tion as to what their primary role or roles are in the life cycleof Rhizobium spp. One of the roles could be to cleave strandsof EPS, which otherwise might be so long that they couldimpede bacterial movement. The observations presented heresuggest a mechanism of limiting the degree of EPS degrada-tion, if only those enzymes adjacent to the cell surface couldcleave the mature EPS. Therefore, the EPS capsule, which mayprotect the bacteria from environmental stresses, would not begreatly degraded by glycanases released from the cell surface.

Since these glycanases have the ability to degrade CMC, theycould in theory degrade cellulose-based polymers in plant cellwalls. Could such degradative activity account in part for theobserved degradation of legume root cell walls adjacent to thegrowth of rhizobia (5, 21, 30, 39, 45)? The observations that theglycanase activation can be species specific and that similarEPS-dependent activation occurs with Rhizobium sp. strainNGR234 may suggest a role in invasion. However, the obser-vation that prsD secretion mutants of R. leguminosarum biovarsviciae and trifolii and R. meliloti form infected nodules (14, 27,50) argues against an essential role for such secreted enzymesduring the invasion process. There is of course the possibilitythat there are other glycanases secreted via a different ex-porter, and we have some indication that this may be the casesince a plyA plyB double mutant does retain some residualCMC degradation that becomes more evident after prolongedgrowth of colonies on CMC-containing plates.

A key point that we have not yet addressed is the nature ofthe factor that activates PlyA and PlyB. The EPS or somederivative of it is clearly involved, at least in part. One attrac-tive model is that it is a nascent EPS chain formed prior tomodification. The EPS of R. leguminosarum bv. viciae 8401/pRL1JI consists of a repeat unit of the b,1-4-linked residuesglc, glcA, glcA, and glc with the first glucose residue carrying ab,1-6-linked side chain of three glucose residues terminatedwith a galactose. Three of the glucose residues are acetylated,and the side chain sugars are substituted with pyruvate andb-OH-butyrate groups (36, 48). In the absence of mutantsdefective in acetylation, pyruvylation, or OH-butyrylation it isdifficult to test whether unsubstituted EPS polymers can acti-vate the glycanase activity. It is important to note that whateverEPS-related component causes activation of PlyB, it enablesthis glycanase to degrade mature EPS. This conclusion can bedrawn because when ethanol-precipitated EPS is included inthe agar there is no halo of degradation, but the presence of alawn of an EPS-producing strain (that lacks glycanases) caninduce a halo of degradation of the EPS incorporated into theagar (Fig. 5). It remains to be demonstrated which cellulases,from rhizobia or other bacteria, are activated by their cellsurfaces. We already know that the A. caulinodans endogly-canase EglI, which is secreted by the same type I secretionsystem as PlyA and PlyB, showed high CMC-degrading activitywhen expressed in an EPS mutant of R. leguminosarum, sug-gesting it is not specifically activated by an EPS component.Furthermore, it does not degrade R. leguminosarum EPS.Therefore, we believe that endoglycanases may fall into twogroups that are or are not activated by a cell surface-relatedcomponent.

ACKNOWLEDGMENTS

We thank A. Davies for help with bacterial strains and B. Rolfe andE. Gartner for kindly sending strain ANU2840. M. Dow made con-structive comments on the manuscript.

This work was supported by the BBSRC. A.Z. was supported byCONICET Argentina.

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