Characterization and Mutational Analysis of Three Allelic lsc Genes Encoding Levansucrase in Pseudomonas syringae

Abstract

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.The extracellular enzyme levansucrase (Lsc) (EC 2.4.1.10) catalyzes the following three reactions: (i) synthesis of levan from sucrose by transfructosylation while releasing glucose, (ii) hydrolysis of levan to monosaccharides of fructose, and (iii) exchange of [ 14 C]glucose in the reaction fructose-2,1-glucose plus [14 C]glucose to fructose-2,1-[ 14 C]glucose plus glucose (19). Levan is a high-molecular-weight ␤-(2,6)-polyfructan with extensive branching through ␤-(2,1) linkages. Lsc produces both linkage types.Levansucrases have been isolated from various bacteria, such as Bacillus subtilis, Streptococcus mutans, Zymomonas mobilis, Acetobacter diazotrophicus, Erwinia amylovora, and Pseudomonas syringae (2,9,18,22,34,41). In contrast to levansucrases from gram-positive bacteria, which differ widely in their biochemical characteristics, levansucrases from gramnegative bacteria are similar in their molecular mass and substrate-independent expression (16,22,23,43).The bacterial blight pathogen of soybean, Pseudomonas syringae pv. glycinea PG4180, causes formation of water-soaked lesions that develop into necrotic leaf spots surrounded by chlorotic halos. Like other exopolysaccharides (EPS), levan could have particular functions before or du...

A Type II Protein Secretory Pathway Required for Levansucrase Secretion by Gluconacetobacter diazotrophicus

“…The LsdA signal peptide has the essential features of a Secdependent signal peptide. By contrast, other gram-negative bacteria, including the close relative Gluconacetobacter xylinus, secrete levansucrases by a signal-peptide-independent pathway (14,18,23,41,42,45). Some of these enzymes have been localized and are fully active in the periplasm of the native host or a recombinant E. coli strain (14,18,19,23), and the occurrence of a type II secretion pathway responsible for their transfer across the outer membrane cannot be ruled out.…”

“…Levansucrase secretion in gram-positive bacteria involves cleavage of signal-peptidecontaining precursors in Bacillus subtilis (43), Bacillus amyloliquefaciens (46), Geobacillus stearothermophilus (24), Paenibacillus polymyxa (7), Streptococcus salivarius (34), Actinomyces naeslundii (6), Arthrobacter nicotinovorans (36), and Lactobacillus reuteri (49). However, with the exception of LsdA, the other gram-negative levansucrases characterized so far are secreted by a signal-peptide-independent pathway; this occurs in Zymomonas mobilis (41), Erwinia amylovora (14), Rahnella aquatilis (42), Pseudomonas syringae (18,23), and Gluconacetobacter xylinus (45). LsdA is synthesized as a precursor with a 30-residue N-terminal signal peptide, which is cleaved off during transport to the periplasm, where the enzyme adopts its final conformation.…”

Sensor Kinases RetS and LadS RegulatePseudomonas syringaeType VI Secretion and Virulence Factors

“…Levan is a highmolecular-weight ␤-(2,6)-polyfructan that is thought to function as an extracellular storage compound metabolized by P. syringae during periods of nutrient deprivation (44). Levan synthesis is catalyzed by the periplasmic enzyme levansucrase, which is encoded by lscC (47). Alginate has been implicated in the virulence of P. syringae because it is involved in both epiphytic fitness and the dissemination of the bacterium in planta (36,85).…”

The algT Gene of Pseudomonas syringae pv. glycinea andNew Insights into the Transcriptional Organization of the algT-muc Gene Cluster

“…EPS have multiple functions, including water absorption, accumulation of minerals and nutrients, and protection from hydrophobic and toxic macromolecules (14). P. syringae produces at least two EPS: (i) levan, a ␤-(2,6) polyfructan with extensive branching through ␤-(1,4) linkages; and (ii) alginate, a copolymer of O-acetylated ␤-(1,4)-linked Dmannuronic acid and its C-5 epimer, L-guluronic acid (25,28,38,49). Previous studies on EPS produced by P. syringae indicated that alginate was the major exopolysaccharide produced in planta (17).…”