Identification and characterization of three novel EsaI/EsaR quorum‐sensing controlled stewartan exopolysaccharide biosynthetic genes in <i>Pantoea stewartii</i> ssp<i>. stewartii</i>

Abstract

Pantoea stewartii ssp. stewartii (P. stewartii), formerly Erwinia stewartii, is a Gram-negative bacterium that causes Stewart's wilt disease in susceptible maize cultivars by colonizing the xylem as cell wall-adherent biofilms (Braun, 1982; Koutsoudis et al., 2006). This mode of growth requires the production of large amounts of stewartan exopolysaccharide (EPS), which impedes the flow of xylem sap, leading to plant wilt. Stewartan EPS is a high-molecular-weight heteropolysaccharide and represents the primary virulence factor in P. stewartii (Bradshaw-Rouse et al., 1981; Jumel et al., 1997). Mutants unable to secrete EPS adhere strongly to surfaces, tend to generate compact, amorphous biofilms and fail to spread beyond the site of infection in the xylem vessels (Koutsoudis et al., 2006). Stewartan EPS is an anionic polymer composed largely of heptasaccharide repeat units that contain galactose, glucose and glucuronic acid in a 3:3:1 ratio (Fig. 1) (Nimtz et al., 1996a; Yang et al., 1996). Its chemical structure is related to that of amylovoran, a polysaccharide and virulence determinant in Erwinia amylovora (Nimtz et al., 1996b). E. amylovora is also a xylem-dwelling pathogen and causes Fireblight disease in rosaceous plants (Geider, 2000). Sequence homology and partial genetic and biochemical verification indicate that both polymer repeat units are assembled on a polyisoprenoid lipid carrier and translocated via the Wzx membrane-associated polysaccharide specific transport (PST) protein across the inner membrane. A predicted inner membrane-localized Wzy polymerase is thought to facilitate oligomerization of the repeat units, suggesting that the mechanism of stewartan and amylovoran synthesis is related to colanic acid synthesis in Escherichia coli (Coplin et al., 1996; Reeves et al., 1996; Geider, 2000; Whitfield, 2006). All three biosynthetic pathways are under the control of the Rcs phosphorelay signal transduction system (Torres-Cabassa et al., 1987). Structure of the stewartan, amylovoran and glucoamylovoran repeating units. The stewartan and amylovoran repeating units consist of Gal, d-galactopyranose residue; Glc, d-glucopyranose; and GlcA, d-glucopyranuronic acid; Pyr, Pyruvyl (Nimtz et al., 1996a,b; and our data). Text in parentheses indicate the anomeric configuration of the residues and their respective linkages. The subscript percentage values indicate the fraction of β(1,6) linked Glc (VI) residues at the branching Gal (I) (90% for stewartan and 70% for amylovoran and gluco-amylovoran). Grey boxes highlight the arbitrary designation of the residues used in Table 2 and in the text following the abbreviation for the polysaccharide; i.e. S-VI refers to stewartan residue VI. Gluco-amylovoran EPS is produced by Eam Ea273 expressing the P. stewartii wce-II gene cluster from plasmid pAUC45. Residue GA-VII is typical of stewartan and a key feature of the heteropolymer. All three exopolymers contain roughly 1000 subunits (our data and Nimtz et al., 1996a,b). The Gal A-V residues of amylovoran can be modified with 2-, 3- or 2,3-linked O-acetyl groups (Ac) (Nimtz et al., 1996a). Whether the GA-V residue of glucoamylovoran is also modified with O-acetyl groups is not known. In P. stewartii, the biosynthesis of stewartan EPS is cell density-dependent governed by the EsaI/EsaR cell–cell signalling or quorum-sensing (QS) system (von Bodman and Farrand, 1995; von Bodman et al., 1998). The EsaR regulator dimerizes and binds target DNA in the absence of inducing levels of the acyl-homoserine lactone (AHL) signal, and loses DNA binding affinity in its presence (Minogue et al., 2002). EsaR inhibits EPS synthesis by repressing the expression of rcsA at low cell density (Carlier and von Bodman, 2006). The rcsA gene encodes an important regulatory component of the Rcs environmental signal sensing phosphorelay system (Majdalani and Gottesman, 2005). At high cell density, inducing levels of AHL, neutralize EsaR DNA binding at the rcsA promoter allowing expression of RcsA to levels required for formation of the RcsA/RcsB activation complex. This complex is necessary for the stimulated expression of the stewartan biosynthetic cps gene cluster. We have adopted the gene designation wce in place of cps following the suggested nomenclature for various bacterial polysaccharide biosynthetic genes (Reeves et al., 1996). The primary gene cluster for stewartan EPS synthesis, now termed wce-I, is structurally and functionally related to the ams gene cluster of E. amylovora (Coplin et al., 1996; Geider, 2000). For example, it is possible to complement specific wce-I mutants with corresponding genes of the ams gene cluster and vice versa (Bernhard et al., 1996). In our attempt to more fully understand the regulated functions involved in stewartan EPS synthesis, we realized that additional functions, not contained within the primary wce-I gene system, must exist elsewhere in the P. stewartii genome. First, it seemed unlikely that biosynthesis of the hexasaccharidic subunits of amylovoran and the heptasaccharidic subunits of stewartan involve the same number of glycosyl-transferases (GTs). Second, ams mutants complemented with P. stewartii wce-I genes produced EPS lacking some characteristic features of stewartan, suggesting that the wce-I gene system alone is insufficient for complete stewartan synthesis (Bernhard et al., 1996). In this study, we identified and characterized the previously unrecognized bicistronic wce-II and monocistronic wce-III loci, thereby extending the inventory of stewartan EPS biosynthetic genes in P. stewartii. Each of these gene systems encodes a putative GT with specific functional roles in the priming or completion of the stewartan repeat units. Interestingly, the wce-II and wce-III gene systems are also controlled by EsaI/EsaR QS and the Rcs signal transduction system. These findings underscore the significance of the co-ordinated regulation of stewartan synthesis in response to cell density and environmental cues. Discrepancies in the number of genes involved in stewartan synthesis encoded by the wce-I gene cluster (formerly cps) (Fig. 2) and the number of proposed glycosidic linkages in the backbone of stewartan (Fig. 1) prompted us to scan the P. stewartii genome for putative additional GT genes with a potential role in EPS synthesis. We were interested in P. stewartii gene sequences annotated in the ASAP annotation database (Glasner et al., 2003) as GT with Gene Ontology numbers GO:0008194 (UDP-glycosyltransferase activity), GO:0016757 (transferase activity, transferring glycosyl groups) and GO:0016758 (transferase activity, transferring hexosyl groups). Two potential loci emerged that were not evidently linked to lipopolysaccharide, peptidoglycan or enterobacterial common antigen synthesis. One of these loci, designated wce-II, comprised two open reading frames, which we named wceO and wzx2. The predicted product of wceO (ASAP: ACV-0283221) is a 340-amino-acid protein with a predicted molecular weight of 39 kDa. It is a putative GT of the CAZy (carbohydrate active enzymes) GT2 family (Campbell et al., 1997; Coutinho et al., 2003). The protein sequence is 21% identical and 31% similar to the inverting GT WbbE of Salmonella enterica serovar Borreze (GenBank: AAC98401.1), which is an experimentally confirmed N-acetylmannosamine transferase enzyme (Keenleyside et al., 2001). WceO is 24% identical and 35% similar to the nucleotide-diphospho-sugar transferase SpsA (GenBank: CAB15817.1) that is involved in Bacillus subtilis spore coat formation. The crystal structure of this enzyme has been published (Charnock and Davies, 1999). Alignment and domain structural analyses of WceO, WbbE and SpsA identify key signatures found in this class of non-processive GT2 proteins (Keenleyside et al., 2001). The greatest degree of conservation is in the N-terminal Domain A that features invariant residues throughout; but in particular two highly conserved aspartate (D) residues (motifs 1 and 2 shown in Fig. S1) that are predicted to play a role in activated donor substrate (Mn2+-UDP-galactose) binding and catalysis (Charnock and Davies, 1999; Keenleyside et al., 2001; Fulton et al., 2008). The C-terminal acceptor-binding Domain B exhibits lower overall conservation, but features an ED(H) motif that aligns with the conserved WbbE ED(Y) and SpsA TDD motifs (Fig. S1). Recent structural analysis of the MAP2569c GT2 of Mycobacterium shows that the catalytic Domain B accommodates the acceptor substrate hydrogen bonded to conserved threonine and arginine residues within this region. This structure also shows the spatial proximity of the donor and acceptor substrates (Fulton et al., 2008). Genetic organization of the stewartan biosynthetic genes. The wce-I gene cluster comprises 12 genes from wceG to wzx and is linked to the galFE locus. The wce-II locus encodes the wceO and wzx2 genes. The third locus involved in stewartan biosynthesis is the monogenic wce-III locus. Putative GT genes required for stewartan subunit biosynthesis are depicted in light grey. Putative genes specifying stewartan polymerization and export functions are shown in black. Previous experimental work and sequence analysis presented here suggest that synthesis of stewartan subunits begins with the addition of a galactose residue to a lipid carrier by the products of the wceG1 (Geider, 2000) and wceG2 (this study) genes. Subsequent addition of sugar residues presumably involves genes wceB, wceM, wceN, wceK and wceO, resulting in a heptameric repeat unit (galactose : glucose : glucuronic acid 3:3:1) (Nimtz, 1996a; Geider, 2000). Flippase or PTS functions encoded by wzx1 and wzx2 are predicted to transfer the lipid-linked repeat units to the periplasm. The gene product of wceL was originally described as a GT, but secondary structure analysis indicates that this gene is likely to encode a stewartan-specific Wzy EPS polymerase (S.B. von Bodman, unpub. information) (Whitfield, 2006). Finally, the predicted products of wza, wzb and wzc show high sequence homology to various EPS export machineries (Geider, 2000; Whitfield, 2006). The precise function of the wceF and wceJ genes in stewartan EPS synthesis remains to be clarified. The wzx2 gene (ASAP: ACV-0283220) encodes a putative PST protein (flippase) involved in polysaccharide repeat translocation across the bacterial inner membrane. The predicted Wzx2 protein shares relatively weak amino acid sequence homology with RbfX of E. coli K12 (GenBank: NP416541) (26% sequence identity and 46% similarity) and only 18% identity and 28% similarity with the predicted amino acid sequence of the P. stewartii wce-I-encoded wzx1 gene. However, like most PST proteins, the wce-I encoded Wzx1 and the predicted Wzx2 product possess 10 transmembrane domains, six of which are located within a loosely conserved Pfam Polysacch_synt domain (Marolda et al., 2004) (http://pfam.sanger.ac.uk/family?acc=PF01943) (Fig. S2). The third stewartan biosynthetic locus, called wce-III, contains one gene, which we designated wceG2 (ASAP: ACV-0283377). The predicted WceG2 protein is 59% identical to WceG1, which is encoded by the first gene of the wce-I operon. WceG2 also shares 62% amino sequence identity with the well-characterized WbaP undecaprenyl-phosphate UDP-galactose phosphotransferase of S. enterica (GenBank: AAC27321 and CAA40130). This protein transfers galactose phosphate residue to the undecaprenyl phosphate lipid carrier in the first committed step of EPS and O-antigen synthesis (http://pfam.sanger.ac.uk/family?acc=PF02397) (Liu et al., 1993; Saldías et al., 2008). Significantly, the predicted protein products of WceG1, WceG2 and WbaP display a highly conserved overall membrane topology with five predicted transmembrane domains as detailed by Saldías et al. (2008). To verify a role for wce-II and wce-III in stewartan polysaccharide synthesis, we created non-polar deletion mutants and evaluated their impact on stewartan production using a quantitative stewartan-specific I-ELISA immunodetection assay. The wce-II mutant strains, Pnss22 (wceO::GmR) and Pnss23 (wzx2::GmR.) were grown separately in stewartan-inducing medium to mid-exponential phase. As summarized in Table Pnss22 (wceO::GmR) and Pnss23 were for stewartan EPS synthesis the of WceO and Wzx2 in stewartan with the respective genes in from and stewartan production to levels In the wce-III mutant produced stewartan with the that WceG2 to stewartan synthesis, but is not of the wceG1 and wceG2 undecaprenyl-phosphate UDP-galactose phosphotransferase genes was by expressing wceG1 from a promoter on plasmid in the wceG2 deletion mutant which levels of stewartan EPS production These data indicate that wceG1 and wceG2 have identical functions both of which to stewartan EPS synthesis in biochemical of bacterial GT is to substrate and of the lipid-linked For this we used a genetic and biochemical to the function of WceO by the structural and functional of the P. stewartii stewartan and E. amylovora amylovoran biosynthetic We from that the expression of the ams gene cluster in P. stewartii wce-I structural mutants to the production of a polysaccharide with a characteristic amylovoran but lacking the and of amylovoran (Bernhard et al., 1996). the polysaccharide a glucose residue typical of stewartan in place of the on the galactose residue of amylovoran (Fig. 1) (Bernhard et al., 1996). This feature the presence of a GT for the addition of glucose residues to the polymer repeat units that must be located the primary EPS biosynthetic locus. The genes located within the wce-II gene cluster were the most likely to this functional To verify this we wceO and wzx2 on plasmid in E. amylovora which amylovoran EPS (Fig. 1) (Nimtz et al., 1996b). The of the two of EPS from of E. amylovora Ea273 with and were by acid and by impact This analysis that the EPS produced by E. amylovora Ea273 contains more glucose with a glucose : galactose ratio of of the ratio of amylovoran This sugar to glucose and galactose residues polymer repeat unit of the glucose and galactose residues repeat unit of As by Nimtz et al. amylovoran contains a glucose residue at the branching galactose in 70% of the repeat units (Fig. We that the additional glucose residue in is linked to the galactose as in stewartan of this from the analysis of the EPS as summarized in Table shows a of galactose characteristic of the of the amylovoran galactose Fig. 1) and a in galactose residues similar to Interestingly, the ratio of to galactose residues is in that the branching galactose residue in the backbone remains in 70% of the subunits similar to These data suggest that the wce-II encodes a β(1,6) that is for the addition of glucose residues to both stewartan and repeat units. Stewartan EPS is the primary factor of virulence in the P. wilt of (Bradshaw-Rouse et al., 1981; et al., and We in wce-II and wce-III or the infection on virulence As summarized in Table with Pnss22 (wceO::GmR) and Pnss23 were largely as In the EPS production by on the degree and of with The that EPS production not be required for the of the maize at under infection We previously that the wce-I which encodes the primary stewartan biosynthetic functions, is regulated by QS via control of The stewartan EPS and to be in that to the cell density or the amounts of EPS (von Bodman et al., 1998). The of two additional stewartan EPS biosynthetic loci, wce-II and wce-III, the these gene systems were also controlled by the EsaI/EsaR and Rcs regulatory As summarized in Table analyses using from of P. stewartii and grown under inducing and that genes wza, wceB, wzx wceO and wzx2 and wceG2 were in response to inducing in the signal mutant P. stewartii However, of these genes was in the rcsA mutant et al., 2005). these data show that stewartan EPS production requires the cell of the stewartan biosynthetic gene system. Stewartan EPS is an virulence factor of P. stewartii (Bradshaw-Rouse et al., 1981; et al., a role of the two previously wce-II and wce-III gene systems in the biosynthesis of stewartan three loci are governed by the EsaI/EsaR QS system via control of the RcsA which with the activation complex required for wce expression (Torres-Cabassa et al., et al., 2005). These are with our that stewartan synthesis the the to adhere to and biofilms both in and in the xylem of the plant (Koutsoudis et al., 2006). The wce-II gene system is for stewartan synthesis as by a highly immunodetection assay. mutants of wceO wzx2 both and stewartan synthesis These are also of wce-II in the E. amylovora Ea273 in the production of a EPS polymer that exhibits key structural to both stewartan and amylovoran analysis shows that features a Glc residue to the GA-V Gal residue in the of repeat units of the with of the characteristic of Gluco-amylovoran a stewartan We that the GA-VII Glc residue is in the same anomeric configuration as stewartan of the GT2 family tend to an inverting catalytic resulting in the addition of a glycosyl residue from to the polymer in the configuration et al., 2003). Interestingly, the by the E. amylovora Ea273 expressing the P. stewartii wce-II locus the partial 70% glucose at the branching galactose typical of This that the stewartan WceO not to the transfer of additional glucose residues to the branching galactose in the E. amylovora The for this is that P. stewartii WceO for the branching galactose residue of amylovoran, which is linked to a galactose stewartan is linked to a glucose residue (Fig. In this it is important to that the E. amylovora genome sequence a gene system, remains but be specific for amylovoran repeat unit synthesis. The gene of the wce-II locus, is a wzx PST gene also to as a PST proteins facilitate the transport of undecaprenyl-phosphate lipid-linked across the membrane (Liu et al., 1996; et al., 1999). of PST proteins are highly but to loosely to for O-antigen or EPS transport et al., 1997). of the primary structural these proteins have highly related domain as shown in the Fig. It is that PST proteins involved in O-antigen biosynthetic systems display for the and structure of the O-antigen polysaccharide et al., 1999; et al., et al. suggested that the of the PST proteins not beyond the first sugar residue linked to the undecaprenyl phosphate lipid suggest a high degree of on the that the wzx2 mutant is unable to amounts of stewartan EPS in the presence of a functional wzx1 gene encoded by the wce-I operon. In a wzx1 mutant exhibits a unpub. which a but not role for translocation of lipid-linked stewartan repeat units. It is possible that Wzx2 is specific for complete heptameric repeating units Wzx1 have or for the repeating units that a glucose at the branching These the in overall polymer synthesis by the wzx1 mutant in this as of a to a potential role for the stewartan polymer composed of heptasaccharidic and hexasaccharidic repeating units. The functions have a high degree of homology and transmembrane topology to WbaP in S. enterica et al., 2008). The are likely to the transfer of to the undecaprenyl-phosphate lipid carrier from UDP-galactose as a first step in the synthesis of stewartan repeat units (Liu et al., 1993; and 1993; Geider, 2000). The synthesis of both the and hexasaccharidic repeat units with a lipid-linked galactose, which that and WceG2 are functionally This functional the of a in the wceG1 gene et al., Reeves et al., 1996). The high sequence homology the predicted wceG1 and wceG2 genes and strongly to a gene are the potential from a We that wceG1 in the wceG2 mutant this to EPS synthesis gene synthesis of at the of EPS synthesis for of the wce-I which have However, EPS synthesis not the potential of the wceG2 mutant However, here it is important to that the virulence are and most not by the of Stewart's wilt. stewartan production a under infection but which are with large numbers of and are summarized in Table The E. coli used as and et al., for transfer of plasmid P. stewartii and E. amylovora E. coli were grown at on or in presence of The P. stewartii were grown at in in presence of of acid on medium with glucose and or E. amylovora were grown at in or in medium with glucose and DNA and were by as previously described (von Bodman et al., 1995; 1998). DNA was using and to from S1). The P. stewartii genome sequence data were by the of at genome annotation is the ASAP database at 2008). sequence were using the The was used to topology the and conserved domain used the Pfam The was created by the from the plasmid the backbone of plasmid et al., was in E. coli et al., Pnss22 and Pnss23 were created using a modified of the described by and the of the wceO and wzx2 genes were by using in Table A from plasmid et al., was the of the gene of using a with in Table The resulting products the by DNA was the using the The were the using the resulting in and The were E. coli and the were on to and were by For DNA wceO, wzx or both were by using in Table The products were the using to to and The were E. coli and P. stewartii. was from P. stewartii grown to mid-exponential in medium with or 10 as previously described (Carlier et al., 2006). was using and by the using a and the and of corresponding to from target genes under for target genes were using the and are in Table were using the values the of target in a by from a The gene as an for This gene is not regulated by QS or the Rcs regulatory system (Torres-Cabassa et al., et al., 2005). The ratio of target gene to was for and used to the expression of the target genes under QS inducing and Stewartan EPS was from 1 of P. stewartii grown in medium to phase. EPS was from with of and by at for in a and were by at for 2 was in and with 1 at for 1 A of 2 of as by the acid et al., was by on a were and of stewartan EPS were to for production in Stewartan specific were by stewartan to the mutant P. stewartii to Stewartan was in using an and was to the groups of glucuronic acid of stewartan with amounts of to The resulting was was to by in for 1 at binding were with for 1 at EPS or were with a of in at One of was to a in and at for 1 The were with of a of to in was to the and following Stewartan were on substrate and at in a were on a with amounts of stewartan to the of stewartan in a Stewartan EPS was from P. stewartii and E. amylovora by with by in We found that this step to the stewartan of stewartan and of from and were by with of were with and the were with and For EPS were first in 2 at for 2 analysis was by of et al., 2004) on an with a and an and of were with that of For analysis EPS was and in for at under were by the of and using in by using and EPS were in 2 with and was from the analysis of the by were grown in a of and in a controlled at 70% light and light were with of bacterial in roughly 1 were at 1 the plants were for was 10 on the following (von Bodman et al., 1 2 and We for mutant and and for their the EPS biochemical and structural This was by the the and the of The is not for the or of by the be to the corresponding for the