Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola

Abstract

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae, Erwinia amylovora, and other bacterial species. The lsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene in Escherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3. 0-kb PstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to the E. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring the lsc gene. Expression of lsc was found to be dependent on the vector-based Plac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.

FIG. 1

Structure of the EPS levan as a β-(2,6) polyfructan.

FIG. 2

Restriction analysis of various PstI-digested genomic library clones following agarose gel electrophoresis. The 3.0-kb fragment conferring levan synthesis on E. coli is marked with an arrow. In plasmid pSKL3Sm (lane 6) this fragment is increased in size due to the insertion of an antibiotic resistance cassette. Lanes: 1, pCK2; 2, pCK6; 3, pCK9; 4, pCK10; 5, pSKL3; 6, pSKL3Sm.

FIG. 3

Restriction map of the 3.0-kb PstI fragment from P. syringae pv. glycinea PG4180 inserted in plasmid pSKL3 and location of nested deletion clones (top) conferring a levan⁺ or levan⁻ phenotype on E. coli grown on LB agar plates containing 5% sucrose (bottom). The lsc gene (shaded arrow), the orientation of the vector-based P_lac promoter (small arrow), and the inserted Sm-Sp antibiotic resistance cassette are indicated. The photograph shows E. coli(pBluescript) (A), E. coli(pSKL3) (B), E. coli(pDK-B4) (C), and E. coli(pDK-A6). The restriction enzymes used are indicated as follows: Ba, BamHI; Bs, BstEII; Cl, ClaI; Dr, DraI; F, FspI; M, MluI; N, NaeI; P, PstI; Pv, PvuI; Sc, ScaI; and Sp, SphI.

FIG. 4

Nucleotide sequence and predicted amino acid sequence of the lsc gene from P. syringae pv. glycinea PG4180. Nucleotides and amino acid residues are numbered on the left and right, respectively. The putative ribosome binding site (SD, underlined), the DraI and BstEII restriction sites (underlined) used for insertion of the Sm^r-Sp^r cassette, the stop codon (∗), and the putative transcriptional terminator sequences (horizontal arrows) are indicated. Triangles mark the sequence ends of deletion clones used in this study. Primer binding sites used for PCR screening of the lsc gene are indicated by dotted arrows. Shaded boxes mark the conserved regions in levansucrases of gram-negative bacteria (2). SD, Shine-Dalgarno sequence.

FIG. 5

Dendrogram illustrating the sequence relationship of levansucrases from gram-negative and gram-positive bacteria, including P. syringae pv. glycinea (Lsc-Psg), P. syringae pv. phaseolicola (Lsc-Psp), E. amylovora (Lsc-Eam), Z. mobilis (LevU-Zm), A. diazotrophicus (LsdA-Ad), B. amyloliquifaciens (SacB-Ba), Bacillus stearothermophilus (SurB-Bst), B. subtilis (SacB-Bs), and S. mutans (SacB-Sm). The branch points represent distance coefficients calculated by the GROWTREE program. Distance values range from 0 to 100, with a distance value of 0 representing complete identity between two sequences.

FIG. 6

PCR detection of genes homologous to lsc from P. syringae pv. glycinea PG4180 with the primers LSCF and LSCR derived from the N- and C-terminal regions of lsc. Genomic DNAs from various bacterial strains were used as templates. Lanes: 1, E. coli(pSKL3); 2, P. syringae pv. glycinea PG4180; 3, P. syringae pv. phaseolicola NCPPB 1321; 4, P. syringae pv. morsprunorum Pm7; 5, P. syringae pv. lachrymans GSPB82a; 6, P. syringae pv. pisi GSPB104; 7, P. agarici GSPB2305; 8, P. syringae pv. atropurpurea MAFF301313; 9, P. syringae pv. tomato DSM50315; 10, P. syringae pv. pisi GSPB1477; 11, P. syringae pv. syringae FF5; 12, Pseudomonas putida GSPB1498; 13, X. campestris DSM1050. The arrow indicates the 1.25-kb PCR product typical of the lsc gene.

FIG. 7

Southern hybridization analysis of total SalI-digested genomic DNA from various pseudomonads hybridized with a digoxigenin-labeled probe specific to the PCR-amplified lsc gene of P. syringae pv. glycinea PG4180. The PCR product used as a DNA probe is marked by a black arrow. Molecular size markers are outlined to the right. Lanes: 1, PCR product of the lsc gene from PG4180; 2, P. viridiflava GSPB1686; 3, P. cichorii GSPB2097; 4, P. fuscovaginae GSPB2309; 5, P. marginalis GSPB92; 6, P. syringae pv. maculicola GSPB2145; 7, P. syringae pv. savastanoi GSPB2264; 8, P. fluorescens GSPB1714; 9, P. syringae pv. syringae Pss 61; 10, P. syringae pv. atropurpurea MAFF301313; 11, P. syringae pv. pisi GSPB104; 12, P. syringae pv. glycinea PG4180; 13, P. syringae pv. phaseolicola GSPB796; 14, P. syringae pv. lachrymans GSPB82a; 15, P. syringae pv. tomato DSM50312.