Small Broad-Host-Range lacZ Operon Fusion Vector with Low Background Activity

The use of gene fusion technologies for transcriptional analysis of gene expression, especially of those genes whose products are difficult to assay, has been well documented (12–15). Many narrow and broad-host-range plasmid-based systems are available for constructing such gene fusions (6,7, 14). To circumvent some of the inherent problems of plasmid-based systems, which have been discussed before (3), systems have been developed for single-copy, chromosomal insertion of gene fusions (4,9,14). For some applications (e.g., deletion mapping of regulatory regions or use in bacteria for which no integration-proficient vectors are known), plasmid-based fusions are still indispensable. The most useful vectors use a promoterless β-galactosidase (β-gal) gene, lacZ, as a reporter. However, most broad-host-range lacZbased vectors available to date exhibit some potentially serious drawbacks, including (i) high levels of background expression due to transcription from plasmid-encoded promoters and/or efficient translation of low levels of lacZ message due to a strong consensus Shine-Dalgarno (SD) sequence, (ii) a lack of unique cloning sites, and (iii) large size due to the presence of unknown and usually unnecessary nucleotide sequences. To address these is sues, a new small broad-host-range transcriptional lacZ operon fusion vector was constructed and tested in Pseudomonas aeruginosa . The new lacZ vector, pTZ110 (Figure 1A), was obtained in two steps. First, a 1544-bp blunt-ended PstI-StuI fragment from pSF2 (10), containing an origin of transfer, the ori1600 broadhost-range origin of replication and its associated replication protein (16), was ligated into a BalI site located 271 nucleotides downstream of the lacZ gene of pGE593 (2) to obtain pTZ100. This operon fusion vector contains unique EcoRI, SmaI, and BamHI sites upstream of a promoterless lacZ gene. Second, pTZ110 was obtained by ligating a 26-bp oligonucleotide linker with EcoRI and BamHI overhangs between the same sites of pTZ100. The resulting operon fusion vector contains a multiple cloning site with six unique restriction enzyme cleavage sites, stop codons in the three reading frames, and a modified SD sequence, which is close to consensus but not too strong. The multiple cloning site was designed to contain blunt-end generating sites close to each end and compatible sites (e.g., BamHI and XhoI allow cloning of fragments generated with BglII and SalI, respectively). Using the pBR322-based repliBenchmarks