Molecular analysis of the rlsA gene regulating levan production by the fireblight pathogen Erwinia amylovora

Physiological and Molecular Plant Pathology (1999) 54, 187–201 Article No. pmpp.1999.0198, available online at http :\www.idealibrary.com.on

Molecular analysis of the rlsA gene regulating levan production by the fireblight pathogen Erwinia amylovora Y. Z and K. G* Max-Planck-Institut fuW r Zellbiologie, Rosenhof, D-68526 Ladenburg, Germany (Accepted for publication January 1999)

Natural Erwinia amyloŠora can display a reduced level of levan synthesis. A gene (rlsA) responsible for the suppression of low levansucrase synthesis by E. amyloŠora was cloned by complementation of natural levan-deficient strains lacking levansucrase expression. The rlsA region was sequenced, and an open reading frame of 266 amino acids was found. Sequence analysis revealed that RlsA resembles a transcriptional activator and may be a member of the LysR family. Overexpression of rlsA from a high copy number plasmid led to a two-fold increase of levan production, but had no influence on amylovoran synthesis. A mutant created by site-directed mutagenesis of wild type strain Eal\79 had the same phenotype as natural levan-deficient strains. The rlsA gene of natural levan deficient strains (with the exception of strain PD494) could be amplified by PCR, yielding the same DNA fragments as wild type strains. The gene was absent in a strain with a large deletion affecting the hrp region of E. amyloŠora. A match of the nucleotide sequence of rlsA with a sequence database revealed its identity with an incomplete open reading frame located downstream of two recently characterised dsp genes. # 1999 Academic Press

INTRODUCTION

Erwinia amyloŠora, the causative agent of fire blight, can affect apple and pear trees as well as a range of other rosaceous ornamental plant species [34 ]. The disease has been spread from North America to other countries this century. The isolated strains are quite homogeneous in their properties and some can be distinguished by the change of a few DNA fragments seen in pulsed field gel electrophoresis analysis [37 ]. The pathogen produces two kinds of extracellular polysaccharides (EPS), amylovoran and levan, which affect bacterial virulence. Amylovoran is an acidic EPS consisting of a repeat unit containing four galactose molecules linked to a glucuronic acid residue [29 ] and is strictly required for pathogenicity, because mutants cannot cause disease symptoms on plants. Levan is a neutral polyfructan (β-2,6-D-fructofuranan) synthesised from sucrose by the enzyme levansucrase [24 ] and is thought to have a role in virulence, since mutants show retarded development of disease symptoms [23 ]. The gene for levansucrase (lsc) of E. amyloŠora has been characterised and found to encode a 46 kDa protein that has no signal peptide for secretion [23 ]. Several natural * To whom correspondence should be addressed. Abbreviations used in the text : Cm, chloramphenicol ; EPS, extracellular polysaccharide ; Km, kanamycin ; Sm, streptomycin ; StI, standard I medium ; StII, standard II medium ; Tc, tetracycline. 0885-5765\98\050187j15 $30.00\0

# 1999 Academic Press

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levan-deficient strains contain an intact lsc gene without transcription or without secretion of levansucrase [5 ]. It has been assumed that the lsc gene is down-regulated in most of the mutants, because the cloned lsc-gene on high copy number plasmids restored the strains to normal levan synthesis. In this paper, we cloned a gene which can complement low levan production of mutants deficient in transcription of the intact lsc gene.

MATERIALS AND METHODS

Bacterial strains and plasmids Bacterial strains are given in Table 1 and plasmids in Table 2. Bacteria were grown in Standard I medium (St.I, Merck AG, Darmstadt, Germany), in LB (Luria Bertani) broth or on the corresponding agar plates at 28 mC for E. amyloŠora or 37 mC for Escherichia coli. Antibiotic concentrations were 20 µg ml−" in the case of tetracycline (Tc) and 500 µg ml−" for streptomycin (Sm). E. coli strain S17-1 (with a low resistance to Sm) contains a chromosomal insertion of an RP4-derivative and does not require a helper plasmid for transfer of cosmids carrying the origin of transfer from RP4. After conjugation on a nitro-cellulose filter, transfer of the cosmid to the Sm-resistant

T 1 Bacterial strains Strains E. coli DH5α HB101 JM83-2 S17-1 E. amyloŠora CFBP1430 E8 E8Sm Eal\79 Eal\79-RA Eal\79-RB Eal\79-RL1 PD350 PD350Sm PD439 PD439Sm PD494 PD494Sm PD576 PD576Sm PD579 PD579Sm PMV6076

Properties

Source\reference

F-, endA1, hsdR17 (rk− mk+), supE44, thi, recA1, gyrA96, relA1, ∆lacZM15 recA, hsdR, hsdM, rpsL, leu, thi, pro ∆(lac-pro), ∆lacZM15, thi, fd Gene2 inserted in galK thi, pro, recA, hsdR-, hsdMj, chromosomal insertion of RP4-2-Tc :: Mu-Km :: Tn7

BRL

France, Crataegus sp., 1972 Spontaneous avirulent mutant of E9, rcsB−, lsc− Spontaneous mutant of E8, Smr Wild type (Northern Germany, 1979) Eal\79 ; rcsA :: fdA2, Kmr Eal\79, rcsB :: fdA2, Kmr Eal\79, rlsA :: pfdB1091, Cmr Levan deficient Spontaneous mutant of PD350, Smr Levan deficient Spontaneous mutant of PD439, Smr Levan deficient Spontaneous mutant of PD494, Smr Levan deficient Spontaneous mutant of PD576, Smr Levan deficient Spontaneous mutant of PD579, Smr hrp-deletion mutant of CFBP1430

[2 ] [5 ] This study [18 ] [1 ] [4 ] This study [5 ] This study [5 ] This study [5 ] This study [5 ] This study [5 ] This study [2, 37 ]

[9 ] [21 ] [32 ]

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recipient strain was screened on agar plates with Sm and Tc. For triparental mating, donor and recipient were mixed with the helper strain in equal volumes and the cells spotted on a nitro-cellulose filter placed on nutrient agar without antibiotics and incubated over night at 28 mC. T 2 Plasmids and PCR primers

Plasmid

Relevant characteristics

pBB103 pBB109

pUC19

2n6 kb EcoRI-EcoRV subclone in pBlueSK+, rlsA+, Apr 1n6 kb EcoRV-HindIII subclone in pBlueKS−, rlsA−, Apr ColE1 ori, polylinker from pUC18 with Kmr ColE1 ori, lacZh, Apr Genomic library constructed by packaging the partial Sau3A digest of chromosomal DNA of E. amyloŠora in pLA2917 0n33 kb Asp718\HindIII fragment from rlsA in pfdB14Zh, Cmr fd-ori, lacZh from pUC18, Cmr 14 kb BamHI subclone of pLS100 in pBGS18, rlsA+, Kmr 6 kb BglII-BamHI subclone of pLS100 in pBGS18, rlsA+, Kmr subclone of pGB108 with an Asp718 deletion, 1n6 kb, rlsA−, Kmr clone from pCLP with 20 kb insert, rlsA+, Tcr 12n5 kb EcoRI subclone of pLS100 in pBlueSK+, rlsA+, Apr clone from pCLP with 20 kb insert, Tcr ColE1 ori, Km :: Tn7, Tra RK2j, ∆repRK2, low resistance to Sm (20 µg ml−"), resistance to spectinomycin and trimethoprim 1n5 kb HaeIII subclone of pBB103 in pBlueSK+ (EcoRV), rlsA+, Plac−, Apr ColE1, ori, lacZh, Apr

Primers : L80 L778

5h-ATCATGGCAATAACTCC 5h-CCTTTGCCGCAGATGATT

pBGS18 pBlueSK+\KS− pCLP pfdB1091 pfdB14Zh pGB105 pGB108 pGB110 pLS100 pLS111 pLS700 pRK2013 :: Tn7 pRLS20

Source\ reference This study This study [33 ] Stratagene [2 ] This study [22 ] This study This study This study This study This study This study [19 ] This study [36 ] *1156 (c) *2

* Start position in the nucleotide sequence ; (c), in complementary strand.

Electroporation was carried out using 0n2 cm cuvettes with a BioRad gene pulser as described previously [27 ]. The transformants were plated on a selective agar and incubated for 2 days at 28 mC. DNA manipulations Plasmid DNA was extracted and purified with a Nucleobond AX-100 kit from Macherey-Nagel (Germany). Preparation of chromosomal DNA, agarose gel electrophoresis, cloning, transformation, conjugation and restriction endonuclease

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mapping were performed as described [30 ]. Electroporation of E. amyloŠora with plasmid DNA was described previously [27 ]. DNA sequencing was carried out with an A.L.F. sequencer (Pharmacia) according to the supplier’s introductions. PCR amplification was performed as described by Bereswill et al. [6 ]. The nucleotide sequences of primers L778 and L80 are listed in Table 2. Site-directed mutagenesis A DNA fragment with part of the rlsA gene, was cloned into the suicide vector pfdB14Zh (Cmr) whose replication requires gene 2 of bacteriophage fd. The created suicide plasmid pfdB1091 was introduced into E. amyloŠora by electroporation. Gene disruption mutants were selected on StI agar plates with Cm (20 µg ml−") and transferred several times to fresh agar plates to confirm the stability of the rlsA mutation. Cell growth and measurement of leŠansucrase and amyloŠoran E. amyloŠora was grown in StI or in MM2 medium [5 ] for 2 days at 28 mC. After centrifugation, the concentration of amylovoran in the supernatant was determined by the CPC assay [3 ]. To determine levansucrase activity bacteria were grown in LB medium at 28 mC for 48 h. The supernatants were mixed with an equal volume of LSbuffer containing 100 m sodium phosphate (pH 7n0), 2  sucrose plus 0n05 % NaN $ to avoid further cell growth [5 ], and incubated at 37 mC for 48 h. The turbidity was determined at 600 nm after appropriate dilution. The assays were done in triplicate giving similar results. Levan formation on agar plates was estimated by the size of dome-shaped colonies on Standard II-agar (St.II, Merck AG, Darmstadt, Germany) after 2 days incubation at 28 mC. RESULTS

Complementation of natural leŠan-deficient mutants The natural levan-deficient strains PD350, PD439, PD494, PD579, and the spontaneous mutant E8 carry the intact lsc structural gene, which is not transcribed [5 ]. E8Sm was used for complementation of levan production with a genomic library derived from strain CFBP1430 (pCLP) [2 ] by tri-parental mating with an E. coli strain carrying the helper plasmid pRK2013 :: Tn7. Transconjugants were selected for Sm- and Tcresistance and screened on StII agar with sucrose. From 250 Tc\Sm resistant colonies two colonies showed restoration of levan production to a different extent after 24 h at 28 mC on StII agar containing sucrose. The two positive clones were confirmed by the enzymatic assay for levansucrase activity (Table 3). Restriction analysis with HindIII, SalI, BamHi and PstI showed different patterns without common bands in the two complementing cosmids isolated (data not shown). They were named pLS100 and pLS700. For transfer to other levan-deficient strains, the cosmids were brought into E. coli S17-1 by transformation of CaCl -treated competent cells, and then transferred into # E. amyloŠora by conjugation. Natural levan-deficient strains responded differently to these cosmids : type I, represented by strains E8 and PD494, without transcription of lsc [5 ], were complemented by pLS100, but barely by pEA700 ; type II, with strains PD350, PD439 and PD579, also without lsc transcription, could be complemented with

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pLS100 and cosmid pLS700 ; type III, with strain PD576, which transcribed lsc, but lacked enzyme secretion, was not complemented by one of the cosmids (Table 3). Expression of levansucrase from E. coli can be seen on agar plates with sucrose by dome shaped colonies after incubation for two days [23 ]. Strain S17-1 did not show the colony morphology typical for levan formation on StII agar with sucrose when carrying one of the two cosmids thereby differing from plasmids with the lsc gene. Furthermore, PCR amplification with the primers Lsp1 and Lsp6 from the lsc gene [5 ] did not give a positive signal with isolated DNA of pLS100. Therefore, the cosmid does not carry the structural levansucrase gene of E. amyloŠora.

T 3 Complementation of leŠan production of E. amylovora with pLS100 and pLS700 Strains

Plasmid

Classification

Levan assaya

StII agarj5 % sucroseb

Eal\79 E9 E8Sm E8Sm E8Sm PD494Sm PD494Sm PD494Sm PD439Sm PD439Sm PD439Sm PD350Sm PD350Sm PD350Sm PD579Sm PD579Sm PD579Sm PD576Sm PD576Sm PD576Sm

— — — pLS100 pLS700 — pLS100 pLS700

wild type wild type type I

0n626 0n647 0n007 0n247 0n071 0n009 0n269 0n053 0n050 0n684 0n780 0n013 0n553 0n509 0n010 0n459 0n515 0 0 0

jjj jjj k jj j k jj j k jjj jjj k jj jj k jj jj k k k

type I type II

pLS100 pLS700 — pLS100 pLS700 — pLS100 pLS700 — pLS100 pLS700

type II type II type III

a Levansucrase activity in the supernatant ; b size of levan domes for colonies : jjj, large to j, small colonies ; k, without levan production.

Subcloning of pLS100 Plasmid pLS100 contains a 20 kb chromosomal insertion including a 12n5 kb EcoRI fragment, which was subcloned into pBluescript SK+, resulting in plasmid pLS111. It restored the levan production when introduced into levan deficient strain PD579Sm. The restriction map of the insert in pLS111 is shown in Fig. 1. Subclones pGB108 and pBB103 were created, which restored levan production to PD579Sm, in contrast to subclones pGB110 and pBB109 (Table 2, Fig. 1). The two restriction sites HindIII and Asp718 in pBB103, which respectively delimit the left end of pBB109 insert and the right end of pGB110 insert, are apparently located within the complementing gene. Additionally, subclones were created to sequence the fragment inserted into pBB103.

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Y. Zhang and K. Geider P H A EV Bg Ha P Ha

E

* rlsA *

1 kb

Complementation EV

B

H Sp E pLS111

+

pGB108

+

pBB103

+

pGB110

–

pRLS20

+

pBB109

–

pfdB1091 – F. 1. Restriction mapping of 12n5 kb EcoRI insert of pLS111 and the respective subclones used for sequence analysis. E : EcoRI, Bg : BglII, P : PstI, Hi : HindIII, A : Asp718 ; EV : EcoRV, Sp : SphI, B : BamHI, H : HaeIII. * position of primers L80 and L778 for PCR amplification of rlsA.

DNA sequence analysis of the rlsA gene Part of pBB 103 and derivatives of the plasmid were sequenced with the reverse and universal primers from pBGS18 and the T7 and T3 primers from pBlueSK+. The whole nucleotide sequence of the insertion of pBB103 was determined for both strands. The sequence contains only one large open reading frame (ORF266), which encodes 266 amino acids. The gene was designated rlsA for its ability to regulate the levansucrase gene (regulator of levansucrase synthesis). The putative molecular weight and isoelectric point (pI) of the RlsA are 30 kDa and 8n0, respectively. A ribosome binding site (RBS) AAGGGA is located 8 bp upstream of the putative ATG initiation codon. From the sequence, two HaeIII sites were found to flank rlsA. The 1518 bp HaeIII fragment was subcloned by insertion into pBlueSK+ at the EcoRV site, creating pRLS20 (Fig. 1). Promoter prediction by the internet program NNPP (http:\\ www.hgc.lbl.gov\projects\) revealed a possible promoter region between nucleotides 263 and 308 (Fig. 2). The inverted repeat sequences downstream of rlsA (nucleotides 1228 to 1236 and 1245 to 1253) may be a rho-independent transcription termination sequence (Fig. 2). Plasmid pRLS20 restored the levan production of several natural levan-deficient strains (Fig. 3). It implied that RlsA had an activator function lacking in natural levandeficient strains. Since the rlsA gene in pRLS20 was inserted in reverse orientation to the lac-promoter in pBlueSK+, expression of rlsA in pRLS20 supported a promoter in front of rlsA. A BLITZ search of databases (http:\\www.ebi.ac.uk\searches\blitz.html) for amino acid sequence showed homology of RlsA to the members of the LysR family of transcriptional regulators at their N-terminus [25, 31 ] (Fig. 4). Typical members of the LysR family are of similar size (250 to 350 amino acids), contain a DNA binding region (helix-turn-helix (HTH) motif) in the conserved N-terminal domain, and require an inducing compound for activation [31 ]. An alignment of the first 75 residues at the Nterminus of the deduced RlsA protein with seven members of the LysR family showed 43 % to 51 % similar to 29 % and 32 % identical residues (Table 4). Except that the CatM protein of Acinetobacter calcoaceticus is a repressor, all members mentioned above

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F. 2. Nucleotide and the deduced amino acid sequence of rlsA. Plasmid pRLS20 was used for sequencing. The putative k35, k10, j1 region of the promoter and ribosomal binding site (RBS) for the initiation of translation are indicated in bold face. The helix-turn-helix motif is doubly underlined. The restriction sites used in subcloning of rlsA are underlined. The translation start codon ATG (rlsA ) and stop codon (TGA) are marked. The potential transcription termination sequences (nucleotides 1228 to 1236 and 1245 to 1253) are indicated by arrows. Nucleotides comprising the sequence for primers L778 and L80 used in PCR assays are printed in italic letters. The nucleotide sequence of rlsA has the accession number AJ131559 in the EMBL Nucleotide Sequence Database.

are transcriptional activators. In addition, an incomplete ORF (accession no. U97504) with identity of all nucleotide residues to rlsA was found, which is adjacent to described genes of the dsp\hrp region [8 ]. A weight matrix method was used for the evaluation of the HTH DNA-binding motif [17 ]. When RlsA is scored for similarity to known helix-turn-helix DNA binding domains, a score of SD 4n18 was found for the 20-amino-acid region that precisely aligns with the HTH motif of other proteins of the LysR family members (Fig. 4). This implied that there is a 90 % probability that the region in RlsA is an HTH motif [17 ]. Like other HTH sequences identified previously in the LysR family, the HTH motif of

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Y. Zhang and K. Geider 10 9 8

Levansucrase

7 6 5 4 3 2

pD579Sm (pRLS20)

PD579Sm

PD439Sm (pRLS20)

PD439Sm

PD350Sm (pRLS20)

PD350Sm

PD494Sm (pRLS20)

PD494Sm

E8Sm (pRLS20)

0

E8Sm

1

F. 3. Levansucrase activity measured as turbidity in OD units of different natural levan'!! deficient strains and their transformants carrying pRLS20. T 4 Homology of the first 75 amino acids at N-terminus of RlsA to some members of the LysR family

Bacterium Erwinia amyloŠora Salmonella typhimurium Salmonella dublin Acinetobacter calcoaceticus Escherichia coli Ralstonia eutropha Bacillus subtilis Ralstonia solanacearum

Protein RlsA SpvR SpvR CatM TdcA CfxR YwfK PhcA

Homology (%)a Identity Similarity

32 32 32 29 32 32 29

45 45 51 45 47 47 43

Swiss-prot identification

VRPRISALDU VRPRISALTY CATMIACICA TDCAIECOLI CFXRIALCEU YWFKIBACSU PHCAIBURSO

a Homology calculation was performed with Align v. 2.0.

RlsA has the conserved alanine and valine at positions 5 and 15, respectively, and no glycine in position 5. Considering the suppression effect of pRLS20 on levan production of several deficient strains, RlsA may be a DNA binding protein to activate the transcription of lsc of E. amyloŠora.

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F. 4. Alignment of the N-terminal 75 residues of RlsA with members of the LysR family. The helix-turn-helix (HTH) is marked and the conservative residues in HTH are printed in bold. (:) : identical amino acid, (.) : conservative amino acid change.

Mutation of the rlsA gene by gene disruption In order to confirm the function of rlsA in levan-deficient strains, mutagenesis of the rlsA gene was performed in the wild type strain Ea1\79. After the 0n7 kb Asp718\HindIII internal fragment of rlsA was cloned into vector pfdB14Zh creating the suicide plasmid pfdB1091 (Fig. 1), the plasmid was introduced into strain Eal\79 by electroporation to produce rlsA mutants by gene disruption. Homologous recombination with the chromosomal rlsA copy resulted in distortion of the functional copy, creating mutant Eal\79-RL1. This strain did not produce levan on StII sucrose agar and had no detectable levansucrase activity in culture supernatants (Fig. 5). This result also showed that the rlsA gene is required for the expression of levansucrase in E. amyloŠora. Influence of the rlsA on leŠan and amyloŠoran production and Širulence of E. amylovora PMV6076 lacks at least the complete hrp- and dsp-region of CFBP1430 [2 ] and is also a levan-deficient strain. With pRLS20, it produced twice as much levansucrase as the parent strains CFBP1430 (Fig. 5). Similarly, overexpression of rlsA from pRLS20 also resulted in a twofold increase of levansucrase for strain Eal\79, which already showed good enzymatic activity in the wild type version (Fig. 5). A DNA fragment with the promoter region of the lsc gene was fused to the promoterless lacZ gene (S. Bereswill, S. Jock, and K. Geider, unpublished). The expression of β-galactosidase was strongly reduced in the levan-deficient strain PD494 in comparison to Eal\79 with normal levan synthesis. This effect implied a function of rlsA on transcription of lsc. rcsA and rcsB are positive regulators of exopolysaccharide synthesis by E. amyloŠora [4, 7, 12, 14 ]. Because overexpression of the rcsA and rcsB genes in E. amyloŠora strongly interfered with levan synthesis [4 ], the influence of rlsA on amylovoran production was also investigated. rlsA did not complement amylovoran production in the rcsB mutant E8 and the rcsA mutants Eal\79-RA and Eal-MG (data not shown). Moreover, it had no effect on the EPS production of PD350, PD439, PD494, PD579, which have low amylovoran production (data not shown). Amylovoran synthesis by Eal\79, Eal\79RL1 and Eal\79(pRLS20) did not show a significant difference. The effect of rlsA on virulence and pathogenicity of E. amyloŠora was investigated by comparison of Eal\79 and Eal\79-RL1 on young apple seedlings. Both strains produced typical symptoms. Since the seedlings were without long shoots, the progress of necrotic symptoms could not be determined. It can be expected that rlsA mutants

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Y. Zhang and K. Geider 12

10

Levansucrase

8

6

4

Ea1/79 (pRLS20)

Ea1/79-RL1

Ea1/79

PMV6076 (pRLS20)

PMV6076

0

CFBP1430

2

F. 5. Turbidity test of the levansucrase activity of E. amyloŠora strains. Assays were done with the wild type strain CFBP1430 and a mutant with a deletion in the hrp\dsp region, the wild type strain Eal\79 and the created rlsA gene disruption mutant Eal\79-RL1 in the presence or absence of plasmid pLS20 with the intact rlsA gene.

are similar to lsc mutants for reduced spread on shoots, when compared to the wild type E. amyloŠora strain [23 ]. InŠestigation of the rlsA gene in natural leŠan-deficient strains and an hrp-deletion mutant The primers L778 and L80 from the sequence of the insert in pRLS20 were used to amplify the rlsA gene in E. amyloŠora strains by PCR (Figs 1 and 2). They amplified the region as a 1n14 kb DNA fragment for all strains assayed except for PD494, PMV6076 and Eal\79-RL1 (Fig. 6). PMV6076 was indicative for the location of rlsA adjacent to the dsp\hrp region of E. amyloŠora. Considering a deletion in this chromosomal area of PMV6076 [2, 37 ], it was concluded that the rlsA gene is located within the 100 kb fragment deleted from CFBP1430. In addition, a recent BLITZ search in the EMBL Nucleotide Sequence Database revealed identity for all nucleotides comprising an

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incomplete ORF in the dsp region of E. amyloŠora (accession number U97504). The insertion of pfdB14 with rlsA-bordering fragments into the rlsA gene of Eal\79-RL1 increased the PCR fragment to a size, which is too large for amplification under the conditions applied. A lack of a PCR signal for PD494 may be explained by base changes or rearrangements in the chromosomal dsp\hrp region. M a

b

c

d

e

f

g

h

i

j

k

M

1 kb

F. 6. PCR investigation of rlsA in E. amyloŠora strains. PCR products were separated on a 0n8 % agarose gel. Lane a : H O ; b : PMV6076 ; c : CFBP1430 ; d : E9 ; e : E8 ; f : PD579 ; g : PD439 ; # h : PD350 ; i : PD494 ; j : Eal\79 ; k : Eal\79-RL1 ; M : 1 kb molecular weight marker.

DISCUSSION

The synthesis of levan by E. amyloŠora is catalysed by the extracellular enzyme levansucrase, which cleaves sucrose into glucose and fructose, and polymerises the fructose residues. Sorbitol and sucrose are the main soluble carbohydrates in perennial parts of rosaceous plants [13, 26 ]. Therefore, the levan produced by E. amyloŠora could be an important virulence factor during propagation of bacteria in plant tissue [23 ]. On the other hand, the constitutive expression of the levansucrase gene can be disadvantageous to the cells in some environmental conditions. Natural levan deficient strains from fruit orchards may support this assumption [5 ]. A defect did not affect structural gene lsc but regulation of transcription. A gene involved in lsc-regulation was cloned by complementation of natural levandeficient strains with a genomic library. Sequence analysis revealed that the gene named rlsA for regulator of levansucrase synthesis is related to the LysR family of transcriptional activators [25 ]. The LysR family contains many prokaryotic regulators, affecting very diverse genes and functions. On the other hand, LysR-type proteins have a number of common features. RlsA was identified as a member of the LysR family primarily by the homology of its N-terminus over the first 75 residues. The 20 amino acids with a helix-turn-helix (HTH) motif in RlsA are located at the same position as in other LysR proteins and may serve as the DNA-binding domain in transcriptional regulation. The low activity of the lsc-promoter fused to a reporter gene in strain PD494 in contrast to strain Eal\79 with normal levan synthesis supports this assumption. A coinducer recognition\response domain or a conserved C-terminal domain were not identified in RlsA.

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RlsA differs from typical LysR proteins by its size (266 amino acids) and the content of arginine and lysine. It is shorter than that of characterised LysR proteins, which consist of 305p20 residues. Many LysR proteins show a low lys content and a high arg content compared with those of other prokaryotic proteins [35 ]. RlsA does not fit into this pattern, because it has a lys\(argjlys) ratio of 0n71 instead of less than 0n25 for typical LysR proteins. An exception is MleR of Lactococus lactis with a ratio of 0n74 [35 ]. Most LysR genes are transcribed independently from the gene(s) that they regulate, and regulation may involve overlapping promoters like the LysR protein CysB in Pseudomonas aeruginosa [15 ]. The region up to 830 bp upstream of rlsA was also sequenced in this study (data not shown). Sequence analysis did not reveal an open reading frame even without ATG as start codon. Furthermore, the lsc gene is not localised adjacent to rlsA. LysR proteins are coinducer-responsive transcriptional activators. Sucrose, the substrate for levansucrase, does not affect levansucrase activity [23 ]. A few LysR proteins, such as NodD3, also do not require a coinducer [28 ]. Overexpression of E. amyloŠora rcsA and rcsB genes strongly reduce the expression of levansucrase [4 ]. Mutants in rcsA and rcsB produce the same amount of levansucrase as the parent strain. The effect of rcsA and rcsB overexpression on levansucrase synthesis could be due to the increase in amylovoran production which alters the cell metabolism of E. amyloŠora and may reduce secretion of levansucrase. Although some LysR proteins are global regulators, the rlsA gene did not complement rcsA mutants Eal\79-RA and Eal\79-MG or the rcsB mutant Eal\79-RB in amylovoran production. On the other hand, the rlsA mutant Eal\79-RL1 produced the same level of amylovoran as the wild type. Therefore, rlsA seems to be unrelated to the amylovoran regulation network. This conclusion agrees with characterisation of strain E8, which is defective in amylovoran and levan production. It has a nine-nucleotide deletion at the start of the rcsB gene [4 ]. Even in low copies, the intact rcsB gene complemented amylovoran synthesis but not levan production of E8. This implied different regulations for both phenotypes. In addition, low expression of the rcsB gene recovered EPS production of type I and type II levan-deficient strains but not levan production (data not shown). Except for PD494, the rlsA gene of natural levan deficient strains did not show a difference from that of wild type strain Eal\79 in amplification of rcsA. The PCR assay revealed that the rlsA gene is missing in mutated strain PMV6076. PMV6076 is a hrp and dsp mutant of CFBP1430 with a 100 kb deletion in the chromosome [37 ]. The levan-deficient mutant was complemented by rlsA. No significant difference in EPS production was observed for PMV6076, CFBP1430 and PMV6076(pRLS20). A recent search in the EMBL Nucleotide Database revealed identity with an incompletely sequenced ORF located distal of two dsp genes in the hrp region [8, 20 ] (accession no. U97504), which was also classified as an lysR homologue. The start codon of the ORF is positioned 752 bp downstream of dspF and it is transcribed in the same direction as dspF. The large intergenic space could mean transcriptional control apart from the dsp genes. On the other hand, a temperature dependent regulation of levan synthesis [5 ] may refer to participation of the alternative σ factor HrpL, which is involved in temperature dependent regulation of hrp and dsp genes. The role of rlsA in levan synthesis is supported by complementation of deficient strains with the intact gene and by creation of a mutant of wild type strain Eal\79.

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Levan-positive strains can occasionally form levan-deficient colonies on sucrose containing agar, indicating a frequent change of lsc-regulation, which could involve rlsA. On the other hand, the levan deficient-strain PD494 transiently reverted to levansynthesis [5 ]. A similar event has been observed for the phcA gene (for phenotype conversion gene) of R. solanacearum, a plant pathogen causing bacterial wilt in various plant species [10, 11 ]. PhcA is also a member of the LysR family and a global transcriptional activator for production of EPS and a variety of extracellular proteins, some of which contribute to virulence. It has been demonstrated that the phenotype conversion under a certain growth condition is attributed to insertions within the phcA gene [10, 11 ]. PCR analysis of levan deficient strains with primers from the rlsA region produced the expected size of the amplification product. No signal was obtained with DNA from strain PD494, which might differ in the corresponding nucleotide sequence. rlsA is the first gene identified to be directly related to regulation of levansucrase. Previous studies on H-NS, a general negative regulator in Gram-negative bacteria, have revealed that an hns mutant showed an increase in levansucrase expression and overexpression of the gene reduced levan and amylovoran production [1 ]. The DNA binding protein H-NS is a global regulator for E. coli [16 ] and may also interfere with the expression of several genes in E. amyloŠora. Since RlsA complements strains without detectable lsc-transcription, it seems to be a transcriptional activator and its requirement for expression of levansucrase was emphasised by site-directed creation of an rlsA mutant of Eal\79. Several conclusions can be made from this study and previous reports [5, 23 ] : (i) levansucrase is expressed by E. amyloŠora in the presence of a functional rlsA gene independent from the carbon source ; (ii) inactivation of rlsA produces the same phenotype as observed for natural levan deficient strains ; (iii) rlsA is a member of the LysR family and may function as a transcriptional activator ; (iv) rlsA is unrelated to the amylovoran regulatory system ; (v) the gene is located downstream of dspF in the dsp\hrp region of E. amyloŠora. REFERENCES 1. Aldridge PD. 1997. The environmental and genetic regulation of virulence mechanisms of Erwinia amyloŠora. Ph.D. Thesis, University of Leicester, U.K. 2. Barny MA, Guinebretiere MH, Marcais B, Coissac E, Paulin JP, Laurent J. 1990. Cloning of a large gene cluster involved in Erwinia amyloŠora CFBP1430 virulence. Molecular Microbiology 4 : 777–786. 3. Bellemann P, Bereswill S, Berger S, Geider K. 1994. Visualization of capsule formation by Erwinia amyloŠora and assays to determine amylovoran synthesis. International Journal of Biological Macromolecules 16 : 290–296. 4. Bereswill S, Geider K. 1997. Characterization of the rcsB gene from Erwinia amyloŠora and its influence on exopolysaccharide synthesis and virulence of the fire blight pathogen. Journal of Bacteriology 179 : 1354–1361. 5. Bereswill S, Jock S, Aldridge P, Janse JD, Geider K. 1997. Molecular characterization of natural Erwinia amyloŠora strains deficient in levan synthesis. Physiological and Molecular Plant Pathology 51 : 215–225. 6. Bereswill S, Pahl A, Bellemann P, Zeller W, Geider K. 1992. Sensitive and species-specific detection of Erwinia amyloŠora by polymerase chain reaction-analysis. Applied and EnŠironmental Microbiology 58 : 3522–3526. 7. Bernhard F, Poetter K, Geider K, Coplin D. 1990. The rcsA gene of Erwinia amyloŠora : Identification, nucleotide sequence, and regulation of exopolysaccharide biosynthesis. Molecular Plant-Microbe Interactions 3 : 429–437.

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