External loops at the C terminus of Erwinia chrysanthemi pectate lyase C are required for species-specific secretion through the out type II pathway.
The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pell. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.
Lindeberg
M
M
Department of Plant Pathology, Cornell University, Ithaca, New York 14853-4203, USA.
Boyd
C M
CM
Keen
N T
NT
Collmer
A
A
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D016974:Dickeya chrysanthemi / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016973:Pectobacterium carotovorum / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics; D013045:Species Specificity
D004269:DNA, Bacterial; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D011133:Polysaccharide-Lyases; C023911:pectate lyase