Excretion of the egl gene product of Pseudomonas solanacearum

Abstract

Pseudomonas solanacearum is an important phytopathogen which excretes a variety of extracellular enzymes. Pulse-chase experiments showed that one of these enzymes, a 0-1,4-endoglucanase (EGL) encoded by the egl gene, is synthesized as a higher-molecular-weight precursor polypeptide (pEGL) which is subsequently excreted into the extracellular medium as a 43-kilodalton mature protein. Si nuclease transcript mapping and DNA sequence analysis were used to identify the transcription start site and the possible translation start site of egl. Pulse-chase experiments and comparison of the putative NH2-terminal amino acid sequence of pEGL with the actual NH2-terminal amino acid sequence of mature excreted EGL suggested that pEGL has a 45-residue leader sequence preceding the N terminus of EGL which is proteolytically cleaved during export to the extracellular environment. The first 20 residues of the leader sequence resembled a typical lipoprotein signal peptide. The excretion of EGL by P. solanacearum apparently requires a membrane potential since it was blocked by carbonyl cyanide m-chlorophenyl hydrazone.

Role of the two-component leader sequence and mature amino acid sequences in extracellular export of endoglucanase EGL from Pseudomonas solanacearum

“…We previously reported the DNA sequence of the 5' end of egl and the locations of its transcription start site and translation initiation codon (21).…”

“…DNA sequence and pulse-chase experiments suggested that the egl gene encoding a 43-kDa extracellular endoglucanase (mEGL) of Pseudomonas solanacearum is translated as precursor with an unusual 45-residue, two-part signal sequence (20,21). Sequence comparison suggests that the first 19 residues are a lipoprotein signal peptide.…”

“…Medium for pulse-labeling of P. solanacearum with [35S]methionine was described previously (21). For analysis of active Egl-PhoA fusion proteins, E. coli cells containing fusion plasmids were grown in Luria broth (27); P. solanacearum cells were grown in a solution of 1% Bacto-Peptone, 0.1% yeast extract, 0.1% Casamino Acids, and 0.5% glucose.…”

“…EGL was assayed with 1% carboxymethyl cellulose substrate as described previously (21). AP activity was measured by the release of p-nitrophenol from p-nitrophenyl phosphate (13).…”

“…AP activity was measured by the release of p-nitrophenol from p-nitrophenyl phosphate (13). Glucose-6-phosphate dehydrogenase, P-NADH oxidase, and ,-lactamase were assayed as described previously (20,21).…”