Alkaline phosphatase and a cellulase reporter protein are not exported from the cytoplasm when fused to large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein.
Using a gene fusion approach, hybrid proteins were created by linking alkaline phosphatase (PhoA) or a cellulase reporter (delta CenA) to four large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein (RsaA; 1026 amino acids). Three of the sites (amino acids 189, 220, 315) were selected on the basis of TnphoA experiments that suggested the first 250-350 amino acids of RsaA could mediate export of PhoA from the cytoplasm while the fourth lay only 21 amino acids from the C-terminus. Expression of all fusions except rsaA(315):delta cenA and rsaA(315):phoA was toxic to C. crescentus. None of the gene fusions were toxic when expressed by Escherichia coli DH5 alpha, where all the hybrid proteins accumulated as inclusion bodies. The toxicity of hybrid proteins encoding 189, 220, and 1005 RsaA-derived amino acids was related to the nature of the hybrid protein itself because truncated RsaA peptides lacking their reporter domains were nontoxic. Further study of RsaA(delta C21) showed that this and presumably other truncated RsaA derivatives were neither secreted nor prone to intracellular accumulation. Although C. crescentus tolerated the expression of rsaA(315):delta cenA and rsaA(315):phoA, the encoded hybrid proteins were not exported in significant quantities from the cytoplasm. These results extend and confirm earlier work that large portions of the S-layer protein N-terminus cannot mediate export of passenger proteins from the cytoplasm and that the entire native S-layer protein may be required to properly interact with the RsaA secretion machinery.
Bingle
W H
WH
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
Smit
J
J
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D017930:Genes, Reporter; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000633:toxicity; D017384:Sequence Deletion / Q000502:physiology
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D000469:Alkaline Phosphatase; D002480:Cellulase