HIGH-LEVEL EXPRESSION VECTORS FOR CAULOBACTER-CRESCENTUS INCORPORATING THE TRANSCRIPTION TRANSLATION INITIATION REGIONS OF THE PARACRYSTALLINE SURFACE-LAYER-PROTEIN GENE

A number of plasmid vectors were constructed for high-level gene expression in the dimorphic gram-negative bacteriumCaulobacter crescentus. These vectors incorporate the transcription and translation initiation regions of theC. crescentus CB15ArsaA gene, which codes for the abundantly synthesized protein comprising the bacterium's paracrystalline surface layer. The expression vectors are based on the broad-host-range IncQ plasmid RSF1010 (R300B) and incorporate thersaA promoter and transcription start site. Some vectors also contain translation initiation information; these can result in the addition of as little as a single glycine residue to the protein encoded by the cloned segment. The vectors can be introduced intoC. crescentus by electroporation at high frequency (ranging up to 106-107 electroporants/μg DNA with surface-layer-deficient mutantC. crescentus CB2A) or conjugal transfer. They range in size from 10 to 12 kb, specify either chloramphenicol or kanamycin resistance, and possess the restriction sitesEcoRI, BamHI, KpnI, andSstI for cloning genes downstream of thersaA gene sequences. For a number of the vectors, the complete nucleotide sequence is known. A comparison was made between the expression of an endoglucanase gene from these plasmids inC. crescentus CB2A and CB15A and similar constructions under the control oflacZα transcription and translation initiation signals carried on a pUC9 vector in anEscherichia coli host. The two expression systems compared favorably; cell lysates prepared fromC. crescentus CB2A exhibited 40% of the endoglucanase activity of similarly prepared lysates fromE. coli JM101. Lysates prepared fromC. crescentus CB15A exhibited only 8% of the endoglucanase activity ofE. coli lysates. © 1990 Academic Press, Inc. All rights reserved.