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Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway.
Mol Microbiol. 1996 Apr; 20(1):175-90.MM

Abstract

The type II or Sec-dependent secretion system is used by diverse Gram-negative bacteria for secretion of extracellular proteins. Of the 12-15 proteins involved in secretion, the requirement for many has not been demonstrated and little is known about their functions in the secretion process. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete extra-cellular pectate lyases (Pels) using the type II or Out pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed genes from the other species, suggesting the presence of species-specific recognition factors in the Out systems of the two Erwinia species. We previously reported the isolation of a cosmid clone, pCPP2OO6, from E. chrysanthemi EC16, which enables Escherichia coil to secrete heterologously expressed E. chrysanthemi Pels. Sequencing in a region required for secretion revealed the presence of 12 genes, outC-M and outO. We report here the construction of functionally non-polar mutations in each gene in the outC-M operon and outS and outB using a polA(ts) strain of E. coli to facilitate homologous recombination between out genes carrying deletions and their wild-type copies on pCPP2006. By testing for complementation of each deletion with wild-type out genes from E. chrysanthemi EC16 and E. carotovora SCRI193 we have demonstrated that: (i) each out gene is required for secretion of E. chrysanthemi PelE from E. coli with the exception of outH; (ii) each mutation can be complemented by its homologue from E. carotovora, except for outC and outD; (iii) outC and outD from E. carotovora do not confer secretion of Pel1 on the E. chrysanthemi Out system; and (iv) Pel1 secretion can be conferred on the E. chrysanthemi Out system by the presence of outC-M, S and B from E. carotovora. The data suggest that OutC and OutD are gatekeepers of the Out system involved in recognition of Pels targeted for secretion but that OutC and OutD from E. carotovora cannot be successfully assembled into the E. chrysanthemi Out system.

Authors+Show Affiliations

Department of Plant Pathology, Cornell University, Ithaca, New York 14853, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8861215

Citation

Lindeberg, M, et al. "Complementation of Deletion Mutations in a Cloned Functional Cluster of Erwinia Chrysanthemi Out Genes With Erwinia Carotovora Out Homologues Reveals OutC and OutD as Candidate Gatekeepers of Species-specific Secretion of Proteins Via the Type II Pathway." Molecular Microbiology, vol. 20, no. 1, 1996, pp. 175-90.
Lindeberg M, Salmond GP, Collmer A. Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. Mol Microbiol. 1996;20(1):175-90.
Lindeberg, M., Salmond, G. P., & Collmer, A. (1996). Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. Molecular Microbiology, 20(1), 175-90.
Lindeberg M, Salmond GP, Collmer A. Complementation of Deletion Mutations in a Cloned Functional Cluster of Erwinia Chrysanthemi Out Genes With Erwinia Carotovora Out Homologues Reveals OutC and OutD as Candidate Gatekeepers of Species-specific Secretion of Proteins Via the Type II Pathway. Mol Microbiol. 1996;20(1):175-90. PubMed PMID: 8861215.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species-specific secretion of proteins via the type II pathway. AU - Lindeberg,M, AU - Salmond,G P, AU - Collmer,A, PY - 1996/4/1/pubmed PY - 1996/4/1/medline PY - 1996/4/1/entrez SP - 175 EP - 90 JF - Molecular microbiology JO - Mol Microbiol VL - 20 IS - 1 N2 - The type II or Sec-dependent secretion system is used by diverse Gram-negative bacteria for secretion of extracellular proteins. Of the 12-15 proteins involved in secretion, the requirement for many has not been demonstrated and little is known about their functions in the secretion process. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete extra-cellular pectate lyases (Pels) using the type II or Out pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed genes from the other species, suggesting the presence of species-specific recognition factors in the Out systems of the two Erwinia species. We previously reported the isolation of a cosmid clone, pCPP2OO6, from E. chrysanthemi EC16, which enables Escherichia coil to secrete heterologously expressed E. chrysanthemi Pels. Sequencing in a region required for secretion revealed the presence of 12 genes, outC-M and outO. We report here the construction of functionally non-polar mutations in each gene in the outC-M operon and outS and outB using a polA(ts) strain of E. coli to facilitate homologous recombination between out genes carrying deletions and their wild-type copies on pCPP2006. By testing for complementation of each deletion with wild-type out genes from E. chrysanthemi EC16 and E. carotovora SCRI193 we have demonstrated that: (i) each out gene is required for secretion of E. chrysanthemi PelE from E. coli with the exception of outH; (ii) each mutation can be complemented by its homologue from E. carotovora, except for outC and outD; (iii) outC and outD from E. carotovora do not confer secretion of Pel1 on the E. chrysanthemi Out system; and (iv) Pel1 secretion can be conferred on the E. chrysanthemi Out system by the presence of outC-M, S and B from E. carotovora. The data suggest that OutC and OutD are gatekeepers of the Out system involved in recognition of Pels targeted for secretion but that OutC and OutD from E. carotovora cannot be successfully assembled into the E. chrysanthemi Out system. SN - 0950-382X UR - https://www.unboundmedicine.com/medline/citation/8861215/Complementation_of_deletion_mutations_in_a_cloned_functional_cluster_of_Erwinia_chrysanthemi_out_genes_with_Erwinia_carotovora_out_homologues_reveals_OutC_and_OutD_as_candidate_gatekeepers_of_species_specific_secretion_of_proteins_via_the_type_II_pathway_ L2 - https://onlinelibrary.wiley.com/resolve/openurl?genre=article&sid=nlm:pubmed&issn=0950-382X&date=1996&volume=20&issue=1&spage=175 DB - PRIME DP - Unbound Medicine ER -