DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum.
The pglA gene encodes a 52-kilodalton extracellular polygalacturonase (PGA) which is associated with the phytopathogenic virulence of Pseudomonas solanacearum. The nucleotide sequence of pglA and the putative amino acid sequence of the PGA protein were determined. A computer search identified a 150-residue region of PGA which was similar (41%) to the amino acid sequence of a region of the PG-2A polygalacturonase from tomato. Comparison of the amino terminus of the pglA open reading frame with the actual amino-terminal sequence of purified extracellular PGA suggested that pglA is initially translated as a higher-molecular-mass precursor with a 21-residue amino-terminal signal sequence. Localization of various pglA-phoA fusion proteins in Escherichia coli and P. solanacearum indicated that the 21-residue leader sequence directs the export of PhoA only as far as the periplasm of both bacteria. Deletion of the last 13 residues of PGA eliminated its catalytic activity, as well as its ability to be exported outside of the P. solanacearum cell. Our results suggest that PGA excretion occurs in two steps. The first step involves a signal sequence cleavage mechanism similar to that used for periplasmic proteins and results in export of PGA across the inner membrane; the second step (transit of the outer membrane) occurs by an unknown mechanism requiring sequences from the mature PGA protein and biochemical factors absent from E. coli.
Department of Microbiology, University of Georgia, Athens 30602.
J H
JH
Huang
M A
MA
Schell
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005838:Genotype; D008969:Molecular Sequence Data; D010957:Plasmids; D011133:Polysaccharide-Lyases / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014774:Virulence / Q000235:genetics
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D011133:Polysaccharide-Lyases; C023911:pectate lyase