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 fo ur large N-terminal portions of the Caulobacter crescentus surface (S) -layer protein (RsaA; 1026 amino acids). Three of the sites (amino aci ds 189, 220, 315) were selected on the basis of TnphoA experiments tha t 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 f rom the C-terminus. Expression of all fusions except rsaA(315):Delta c enA and rsaA(315):phoA was toxic to C. crescentus. None of the gene fu sions were toxic when expressed by Escherichia coli DH5 alpha, where a ll the hybrid proteins accumulated as inclusion bodies. The toxicity o f hf brid proteins encoding 189, 220, and 1005 RsaA-derived amino acid s was related to the nature of the hybrid protein itself because trunc ated RsaA peptides lacking their reporter domains were nontoxic. Furth er study of RsaA(Delta C21) showed that this and presumably other trun cated RsaA derivatives were neither secreted nor prone to intracellula r accumulation. Although C. crescentus tolerated the expression of rsa A (315):Delta cenA and rsaA(315):phoA, the encoded hybrid proteins wer e not exported in significant quantities from the cytoplasm. These res ults extend and confirm earlier work that large portions of the S-laye r protein N-terminus cannot mediate export of passenger proteins from the cytoplasm and that the entire native S-layer protein may be requir ed to properly interact with the RsaA secretion machinery.