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The plasmid ColD-CA23, a high copy number plasmid of 5.12 kb, contains genes for colicin D (cda), for immunity colicin D (cdi), and for a lysis function (cdl). These genes are arranged on a contiguous 2.4 kb fragment in the following sequence: cda, cdi, cdl. They are transcribed in two operons, one transcribing cda and cdl from a SOS inducible promoter, the other transcribing cdi in the opposite direction. The expression of cda and cdl is modulated by a repressor, cdr, which is encoded on the same transcript as cda and cdl. In the absence of this repressor, transcription from the SOS inducible colicin D promoter is exceptionally strong and leads to protein contents up to 50% of total cellular proteins. This autoregulative repressor is a new finding in the control mechanisms of expression of colicins. We have also identified the gene product of cdl to be a 10,000 dalton protein.

A soil isolate of a Pseudomonas sp. can utilize myo-inositol (MI) as the sole carbon source. In this strain, MI is transported through the membrane by a high-affinity transport system in which a periplasmic binding protein is involved. Mutants impaired in the transport system were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently identified by their slow growth rate at low MI concentrations. Strains with a low linear initial rate of MI uptake were analyzed. Using a broad-host-range cosmid cloning system, we have constructed a gene bank of the wild-type Pseudomonas sp. in an Escherichia coli recA-host. A rapid mating technique enabled us to screen the gene library for clones which are able to restore the active transport of MI in the mutant. An 11.5-kilobase segment containing genes involved in the MI transport has been isolated, and its restriction enzyme cleavage map has been determined.

The plasmid ColD-CA23, a high-copy-number plasmid of 5.12 kilobases, encodes colicin D, a protein of approximately 87,000 daltons which inhibits bacterial protein synthesis. Colicin D production is under the control of the Escherichia coli SOS regulatory system and is released to the growth medium via the action of the lysis gene product(s). A detailed map of the ColD plasmid was established for 10 restriction enzymes. Using in vitro insertional omega mutagenesis and in vivo insertional Tn5 mutagenesis, we localized the regions of the plasmid responsible for colicin D activity (cda), for mitomycin C-induced lysis (cdl), and for colicin D immunity (cdi). These genes were all located contiguously on a 2,400-base-pair fragment similar to a large number of other Col plasmids (A, E1, E2, E3, E8, N, and CloDF). The ColD plasmid was mobilizable by conjugative transfer by helper plasmids of the IncFII incompatibility group, but not by plasmids belonging to the groups IncI-alpha or IncP. The location of the mobilization functions was determined by deletion analysis. The plasmid needs a segment of 400 base pairs, which is located between the mob genes and the gene for autolysis, for its replication.