Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.     

Plant Innate Immunity Induced by Flagellin Suppresses the Hypersensitive Response in Non-Host Plants Elicited by Pseudomonas syringae pv. averrhoi

A new pathogen, Pseudomonas syringae pv. averrhoi (Pav), which causes bacterial spot disease on carambola was identified in Taiwan in 1997. Many strains of this pathovar have been isolated from different locations and several varieties of hosts. Some of these strains, such as HL1, are nonmotile and elicit a strong hypersensitive response (HR) in nonhost tobacco leaves, while other strains, such as PA5, are motile and elicit a weak HR. Based on the image from a transmission electron microscope, the results showed that HL1 is flagellum-deficient and PA5 has normal flagella. Here we cloned and analyzed the fliC gene and glycosylation island from Pav HL1 and PA5. The amino acid sequences of FliC from HL1 and PA5 are identical to P. s. pvs. tabaci (Pta), glycinea and phaseolicola and share very high similarity with other pathovars of P. syringae. In contrast to the flagellin mutant PtaΔfliC, PA5ΔfliC grows as well as wild type in the host plant, but it elicits stronger HR than wild type does in non-host plants. Furthermore, the purified Pav flagellin, but not the divergent flagellin from Agrobacterium tumefaciens, is able to impair the HR induced by PA5ΔfliC. PA5Δfgt1 possessing nonglycosylated flagella behaved as its wild type in both bacterial growth in host and HR elicitation. Flagellin was infiltrated into tobacco leaves either simultaneously with flagellum-deficient HL1 or prior to the inoculation of wild type HL1, and both treatments impaired the HR induced by HL1. Moreover, the HR elicited by PA5 and PA5ΔfliC was enhanced by the addition of cycloheximide, suggesting that the flagellin is one of the PAMPs (pathogen-associated molecular patterns) contributed to induce the PAMP-triggered immunity (PTI). Taken together, the results shown in this study reveal that flagellin in Pav is capable of suppressing HR via PTI induction during an incompatible interaction.     

Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

The Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A₂₃₀ assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. ΔhrpZ::nptII or hrpW::ΩSp^r P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.     

Occurrence of Pseudomonas syringae pv. Tomato race 1 in Italy on Pto Gene-Bearing Tomato Plants

Bacterial speck symptoms on leaves of the tomato cultivar Erminia Fl (Petoseed), heterozygous for the Pto resistant gene, were observed in June 1995 in Northern Italy. Using individual-lesion isolation, 8 bacterial isolates were obtained from the affected leaves, which were identified as Pseudomonas syringae pv. tomato by biochemical, physiological, nutritional and pathogenicity tests. When the differential cultivar Ontario 7710, homozygous for the Pto gene, was inoculated with the bacterial isolates, 4 of them provoked the typical bacterial speck symptoms and therefore belonged to race 1. This is the first occurrence of P. syringae pv. tomato race 1 in Italy and the first time this race has been isolated on a Pto gene-bearing tomato cultivar grown in open field.     

Phylogenetic Analysis of Pseudomonas syringae Pathovars Suggests the Horizontal Gene Transfer of argK and the Evolutionary Stability of hrp Gene Cluster

Pseudomonas syringae are differentiated into approximately 50 pathovars with different plant pathogenicities and host specificities. To understand its pathogenicity differentiation and the evolutionary mechanisms of pathogenicity-related genes, phylogenetic analyses were conducted using 56 strains belonging to 19 pathovars. gyrB and rpoD were adopted as the index genes to determine the course of bacterial genome evolution, and hrpL and hrpS were selected as the representatives of the pathogenicity-related genes located on the genome (chromosome). Based on these data, NJ, MP, and ML phylogenetic trees were constructed, and thus 3 trees for each gene and 12 gene trees in total were obtained, all of which showed three distinct monophyletic groups: Groups 1, 2 and 3. The observation that the same set of OTUs constitute each group in all four genes suggests that these genes had not experienced any intergroup horizontal gene transfer within P. syringae but have been stable on and evolved along with the P. syringae genome. These four index genes were then compared with another pathogenicity-related gene, argK (the phaseolotoxin-resistant ornithine carbamoyltransferase gene, which exists within the argK–tox gene cluster). All 13 strains of pv. phaseolicola and pv. actinidiae used had been confirmed to produce phaseolotoxin and to have argK, whose sequences were completely identical, without a single synonymous substitution among the strains used (Sawada et al. 1997a). On the other hand, argK were not present on the genomes of the other 43 strains used other than pv. actinidiae and pv. phaseolicola. Thus, the productivity of phaseolotoxin and the possession of the argK gene were shown at only two points on the phylogenetic tree: Group 1 (pv. actinidiae) and Group 3 (pv. phaseolicola). A t test between these two pathovars for the synonymous distances of argK and the tandemly combined sequence of the four index genes showed a high significance, suggesting that the argK gene (or argK–tox gene cluster) experienced horizontal gene transfer and expanded its distribution over two pathovars after the pathovars had separated, thus showing a base substitution pattern extremely different from that of the noncluster region of the genome. Received: 18 January 1999 / Accepted: 25 May 1999     

Effects of Environmental and Nutritional Factors on Production of the Polyketide Phytotoxin Coronatine by Pseudomonas syringae pv. Glycinea

Pseudomonas syringae pv. glycinea PG4180 produces the polyketide phytotoxin coronatine. The effects of environmental, nutritional, and host factors on growth and coronatine production by PG4180 were examined by varying the components of a defined basal medium which contained the following nutrients per liter: glucose (10 g), NH₄Cl (1 g), MgSO₄ · 7H₂O (0.2 g), KH₂PO₄ (4.1 g), K₂HPO₄ · 3H₂O (3.6 g), and FeCl₃ (2 μM). Bacterial growth was recorded as dry weight, and coronatine production was measured by high-performance liquid chromatography. Both growth and the quantity of coronatine synthesized were significantly affected by carbon source, nutrient levels (glucose, NH₄Cl, phosphate, Mg, and SO₄), amino acid supplements, and the presence of complex carbon and nitrogen sources. The yield of coronatine generally declined when conditions were varied from those in the basal medium. Coronatine production and growth were not affected when the pH was adjusted from 6.5 to 7.8. Increases in the osmolarity of the basal medium significantly decreased coronatine production without affecting growth. The addition of plant extracts, plant-derived secondary metabolites, or zinc did not affect growth or coronatine production, while the addition of millimolar levels of KNO₃ or micromolar levels of FeCl₃ significantly enhanced coronatine production. The yield of coronatine was maximized after a 7-day incubation at 18°C and 280 rpm. The results of the present study were used to formulate a medium which allowed for enhanced coronatine production in nearly all strains of P. syringae tested. A rapid method for extracting coronatine from small volumes of culture supernatant was also developed.     

Differential Implication of Glutathione, Glutathione-Metabolizing Enzymes and Ascorbate in Tomato Resistance to Pseudomonas syringae

We studied the response of glutathione‐ and ascorbate‐related antioxidant systems of the two tomato cultivars to Pseudomonas syringae pv. tomato infection. In the inoculated susceptible A 100 cultivar a substantial decrease in reduced glutathione (GSH) content, oxidised glutathione accumulation and GSH redox ratio decline as well as glutathione peroxidase activity increase were found. The enhanced glutathione reductase activity was insufficient to keep the glutathione pool reduced. A transiently increased dehydroascorbic acid (DHA) content and ascorbic acid (AA) redox ratio decrease together with ascorbate peroxidase activity suppression were observed. Adversely to the progressive reduction in GSH pool size, AA content tended to increase but the changes were more modest than those of GSH. By contrast, in interaction with the resistant Ontario cultivar the glutathione pool homeostasis was maintained throughout P. syringae attack and no significant effect on the ascorbate pool was observed. Moreover, in the resistant interaction there was a significantly higher constitutive and pathogen‐induced glutathione‐S‐transferase (GST) activity. The relationship between GST activity and DHA content found in this study indicates that this enzyme could also act as dehydroascorbate reductase. These results reflect the differential involvement of GSH and AA in tomato‐P. syringae interaction and, in favour of the former, they clearly indicate the role of GSH and GSH‐utilizing enzymes in resistance to P. syringae. The maintenance of glutathione pool homeostasis and GST induction appear to contribute to tissue inaccessibility to bacterial attack.     

Identification and Characterization of Pseudomonas syringae pv. syringae,a Causative Bacterium of Apple Canker in Korea

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of −0.7°C, indicating an INA⁺ bacterium. The ice-nucleating temperature was −4.7°C for a non-treated control (sterilized distilled water), whereas it was −9.6°C for an INA⁻ bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.     

Synthesis of a D-rhamnose branched tetrasaccharide, repeating unit of the O-chain from Pseudomonas syringae pv. Syringae (cerasi) 435

The first synthesis of a d-rhamnose branched tetrasaccharide, corresponding to the repeating unit of the O-chain from Pseudomonas syringae pv. cerasi 435, as methyl glycoside is reported. The approach used is based on the synthesis of an opportune building-block, that is the methyl 3-O-allyl-4-O-benzoyl-alpha-D-rhamnopyranoside, which was then converted into both a glycosyl acceptor and two different protected glycosyl trichloroacetimidate donors. Successive couplings of these three compounds afforded the target oligosaccharide. The reported synthesis is also useful to perform the oligomerization of the repeating unit.     

Pathovars of Pseudomonas syringae Causing Bacterial Brown Spot and Halo Blight in Phaseolus vulgaris L. Are Distinguishable by Ribotyping

Ribotyping was evaluated as a method to differentiate between Pseudomonas syringae pv. phaseolicola and pv. syringae strains causing bacterial brown spot and halo blight diseases in Phaseolus vulgaris L. Ribotyping, with restriction enzymes BglI and SalI and using the Escherichia coli rrnB operon as the probe, differentiated 11 and 14 ribotypes, respectively, and a combination of data from both procedures yielded 19 combined ribotypes. Cluster analysis of the combined ribotypes differentiated the pathovars phaseolicola and syringae, as well as different clonal lineages within these pathovars. The potential of ribotyping to screen for correlations between lineages and factors such as geographical region and/or bean varieties is also reported.     

Characterization of the argA gene required for arginine biosynthesis and syringomycin production by Pseudomonas syringae pv. syringae

Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.     

Characterization of the argA Gene Required for Arginine Biosynthesis and Syringomycin Production by Pseudomonas syringae pv. syringae

Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.     

水稻条斑病菌hrcC、hpa3和hrpE基因表达不依赖hrpG和hrpX基因调控

摘要：【目的】决定水稻条斑病菌（Xanthomonas oryzae pv. oryzicola）在非寄主植物上激发过敏反应（hypersensitive response, HR）和在寄主水稻上致病性（pathogenicity）的hrp基因簇是受hrpG和hrpX基因调控的,但还不清楚hrpG和hrpX基因是否共同决定着所有hrp基因的表达。【方法】本文通过基因敲除方式获得了水稻条斑病菌的hrpG和hrpX基因的双突变体。【结果】烟草和水稻上测定结果显示,双突变体与单突变体一样,均在烟草上失去HR激发能力和丧失在水稻上的致病性;相应地,功能互补后双突变体恢复至野生表型。细菌在水稻悬浮细胞、hrp诱导培养基XOM3和营养丰富的培养基NB中生长后的RT-PCR结果显示,NB中hrp基因低水平表达,XOM3和水稻细胞能够高水平诱导hrp基因表达。无论何种生长条件,hrpG单突变体中hrcC、hrcT、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcJ和hrpG基因不表达;hrpX单突变体中hpa2、hrcC、hpa3、hrpE和hrpG基因表达,而hpa1、hrcT、hpaB和hrcJ基因不表达;hrpG和hrpX双突变体中hrcC、hpa3和hrpE基因表达,而hpa1、hpa2、hpaB、hrcT、hrcJ和hrpG基因不表达。【结论】这提示,水稻条斑病菌的hrcC、hrpE和hpa3基因不受hrpG和hrpX基因单独或同时调控,而hrcT基因受HrpG调控。由此推测,水稻条斑病菌III型分泌系统关键组份的表达有可能通过另外的信号途径进行调控,这为进一步分析III型分泌途经的形成提供了线索。     

Characterization of the transcriptional activators SalA and SyrF, Which are required for syringomycin and syringopeptin production by Pseudomonas syringae pv. syringae

Production of the phytotoxins syringomycin and syringopeptin by Pseudomonas syringae pv. syringae is controlled by the regulatory genes salA and syrF. Analysis with 70-mer oligonucleotide microarrays established that the syr-syp genes responsible for synthesis and secretion of syringomycin and syringopeptin belong to the SyrF regulon. Vector pMEKm12 was successfully used to express both SalA and SyrF proteins fused to a maltose-binding protein (MBP) in Escherichia coli and P. syringae pv. syringae. Both the MBP-SalA and MBP-SyrF fusion proteins were purified by maltose affinity chromatography. Gel shift analysis revealed that the purified MBP-SyrF, but not the MBP-SalA fusion protein, bound to a 262-bp fragment of the syrB1 promoter region containing the syr-syp box. Purified MBP-SalA caused a shift of a 324-bp band containing the putative syrF promoter. Gel filtration analysis and cross-linking experiments indicated that both SalA and SyrF form homodimers in vitro. Overexpression of the N-terminal regions of SalA and SyrF resulted in decreased syringomycin production by strain B301D and reduced levels of beta-glucuronidase activities of the sypA::uidA and syrB1::uidA reporters by 59% to 74%. The effect of SalA on the expression of the syr-syp genes is mediated by SyrF, which activates the syr-syp genes by directly binding to the promoter regions. Both SalA and SyrF resemble other LuxR family proteins in dimerization and interaction with promoter regions of target genes.     

Use of Arabidopsis thaliana and Pseudomonas syringae in the Study of Plant Disease Resistance and Tolerance

The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae is being developed as a model experimental system for plant pathology research. Race-specific ("gene-for-gene") resistance has been demonstrated for this interaction, and pathogen genes that determine avirulence have been isolated and characterized. Because certain lines of both Arabidopsis and soybean are resistant to bacteria carrying the avirulence genes avrRpt2 and avrB, extremely similar pathogen recognition mechanisms are apparently present in these two plant species. Isogenic bacterial strains that differ by the presence of single avirulence genes are being used to analyze plant resistance. Plant resistance genes have been identified in crosses between resistant and susceptible lines. The extensive map-based cloning tools available in Arabidopsis are being used to isolate these resistance genes. In a related project, ethylene-insensitive Arabidopsis mutants are being used to examine the role of ethylene in disease development. Ethylene apparently mediates symptom formation in susceptible plants and is not required for resistance, suggesting possible strategies for enhancement of disease tolerance in crops.     

Characterization and mutational analysis of three allelic lsc genes encoding levansucrase in Pseudomonas syringae

In the plant pathogen Pseudomonas syringae pv. glycinea PG4180 and other bacterial species, synthesis of the exopolysaccharide levan is catalyzed by the extracellular enzyme levansucrase. The results of Southern blotting and PCR analysis indicated the presence of three levansucrase-encoding genes in strain PG4180: lscA, lscB, and lscC. In this study, lscB and lscC were cloned from a genomic library of strain PG4180. Sequence analysis of the two lsc genes showed that they were virtually identical to each other and highly similar to the previously characterized lscA gene. lscA and lscC had a chromosomal location, whereas lscB resided on an indigenous plasmid of PG4180. Mutants with impaired expression of individual lsc genes and double mutants were generated by marker exchange mutagenesis. Determination of levansucrase activities in these mutants revealed that the lscB gene product was secreted but not that of lscA or lscC. Our results indicated that lscB and lscC but not lscA contributed to periplasmic levan synthesis of PG4180. The lscB lscC double mutant was completely defective in levan formation and could be complemented by either lscB or lscC. Our data suggested a compartment-specific localization of two lsc gene products, with LscB being the secreted, extracellular enzyme and LscC being the predominantly periplasmic levansucrase. Results of Western blot analyses indicated that lscA was not expressed and that lscA was not associated with levansucrase activities in any particular protein fraction. LscA could be detected in PG4180 only when transcribed from the vector-borne P(lac) promoter. PCR screening in various P. syringae strains with primers derived from the three characterized lsc genes demonstrated the presence of multiple Lsc isoenzymes in other P. syringae pathovars.