Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16

Abstract

The pehX gene encoding extracellular exo-poly-alpha-D-galacturonosidase (exoPG; EC 3.2.1.82) was isolated from a genomic library of the pectate lyase-deficient Erwinia chrysanthemi mutant UM1005 (a Nalr Kanr delta pelABCE derivative of EC16) by immunoscreening 2,800 Escherichia coli HB101 transformants with an antibody against exoPG protein. The cloned pehX gene was expressed highly from its own promoter in E. coli, and most of the enzyme was localized in the periplasm. The nucleotide sequence of pehX revealed the presence of an amino-terminal signal peptide and an open reading frame encoding a preprotein of 64,608 daltons. The cloned pehX gene was insertionally inactivated with TnphoA and used to mutate the chromosomal pehX gene of E. chrysanthemi AC4150 (Nalr) and CUCPB5006 (Nalr Kans delta pelABCE) by marker exchange mutagenesis. Analysis of the resulting mutants, CUCPB5008 (Pel+ Peh-) and CUCPB5009 (Pel- Peh-), indicated that exoPG can contribute significantly to bacterial utilization of polygalacturonate and the induction of pectate lyase in the presence of extracellular pectic polymers. CUCPB5009 retained a slight ability to pit polygalacturonate semisolid agar and macerated chrysanthemum pith tissues when large numbers of bacteria were inoculated.

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane

“…This hypothesis is supported by two observations: (i) when genes encoding E. chrysanthemi pectic enzymes and cellulases are expressed in E. coli, the proteins are found in the periplasm with their signal peptides removed (2,3,7,15,27,31,40), and (ii) mutations in the out genes result in the accumulation of these extracellular proteins in the periplasm of E. chrysanthemi, again with their signal peptides removed (1,26,50). Since (25).…”

“…The second type is exemplified by the secretion of Klebsiella pneumoniae pullulanase and E. chrysanthemi pectic enzymes and cellulases. Secreted proteins of this group possess amino-terminal signal peptides which apparently function in E. coli in facilitating translocation of the proteins across the bacterial inner membrane (2,7,15,27,40,41,45). Extracellular secretion of these proteins, however, requires the function of accessory secretion genes, such as the out genes of E. chrysanthemi and Erwinia carotovora (1,26,37,50), the xcp genes of Pseudomonas aerguinosa (14), and the pul genes of K. pneumoniae (12).…”

A Domain-Centric Analysis of Oomycete Plant Pathogen Genomes Reveals Unique Protein Organization

“…5; Supplemental Table S4). In total, 33 differentially expressed genes during plant infection in P. infestans encode proteins that contain GH domains, including GH-17 (IPR000490) in endo-1,3-b-glucosidase and GH-81 (IPR005200) in b-1,3-glucanases as well as several members of GH-28 (IPR000743), a domain involved in soft rotting of host tissues and described in both fungal and bacterial plant pathogens (He and Collmer, 1990;Ruttkowski et al, 1990). Twenty-eight P. infestans genes coding for domains involved in transmembrane transport are differentially expressed during plant infection (Supplemental Table S4).…”

Recent advances in the molecular biology ofLeifsonia xylisubsp.xyli, causal organism of ratoon stunting disease

“…It is possible to do random mutagenesis on L. xyli subsp. xyli, but site-directed mutagenesis techniques, such as marker exchange (He and Collmer 1990) have not been successful. Random mutagenesis is too slow; it took over a year to generate the 700 random mutants with Tn4431 (Brumbley et al 2002), whereas marker exchange should allow the knock out of specific genes selected from the genome database almost on a monthly basis: the time it takes to grow L. xyli subsp.…”