Identification and characterization of a gene that controls colony morphology and auto-aggregation in Escherichia coli K12.

Many Escherichia coli K12 strains undergo switching between two forms which differ in a number of surface properties including colony morphology and the ability to auto-aggregate. This paper describes the identification of a gene which appears to play a part in controlling this switching phenomenon. This gene has been designated mor and is located at 89 minutes on the E. coli chromosome map between the argECBH operon and the trmA gene. By manipulation of this gene it is possible to overcome the switching of surface properties and fix a strain in one form or the other. The mor gene has been cloned and its DNA sequence determined. The putative protein sequence shows a high level of homology with four regulatory genes, the ilvY, cysB and lysR genes from E. coli and the metR gene from Salmonella typhimurium. It has also been shown that the mor gene is autoregulated at the transcriptional level.

Analysis of transcription from the trfA promoter of broad host range plasmid RK2 in Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa.

Reverse transcriptase mapping has been used to analyze transcription from the trfA promoter of broad host range plasmid RK2. The results show that trfA operon mRNA has the same 5' end in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli. The strengths of wild-type and mutant trfA promoters, which differ by defined base substitutions, have been compared and the positions of their transcriptional start sites determined. While these base substitutions do not alter the transcriptional start site, they do have marked effects on promoter strength which are broadly similar in each of the host species. A single base pair substitution, which lies in the region corresponding to the E. coli promoter consensus, brings about a large reduction in gene expression while the introduction of a second mutation, at a locus outside this region, has no further effect on promoter strength. The results indicate that these Pseudomonas species possess an RNA polymerase which recognizes the same region of the trfA promoter as that utilized by E. coli RNA polymerase. Within the limits of these observations it is clear that the trfA operon is transcribed from a single promoter which can function efficiently in diverse species, a property which may be important for its broad host range.

Analysis of the vegetative replication origin of broad-host-range plasmid RK2 by transposon mutagenesis.

A range of Tn1723 transposon mutants of the oriV region of broad-host-range plasmid RK2 have been isolated, and the internal EcoRI fragment of the transposon has been deleted from each to reduce the insertion size from 9.6 kb (Tn1723) to 35 bp (delta Tn1723). Sequencing from the delta Tn1723-derived EcoRI site has allowed the precise mapping of these insertions to various points dispersed through the origin region. Using these mutants we have determined which regions of oriV RK2 are of functional importance to plasmid establishment following transformation of the host species Escherichia coli, Pseudomonas putida, and P. aeruginosa. Insertions into an A/T-rich region, and a region containing five direct repeat sequences prevented successful transformation of each host species tested, but the continuity of sequences adjacent to the five repeats were essential only in E. coli and P. putida. The establishment and maintenance in E. coli of a mini-RK2 replicon was found to be inhibited by transcription from an inducible promoter positioned to read into oriV RK2 against the direction of replication. Assays of transcription emerging from Tn1723 demonstrated significant levels from one end of the transposon only. Four mutants with insertions downstream of oriV RK2 were unable to become established in E. coli, and contained Tn1723 in the orientation which would supply transcription toward the oriV RK2 region. These results demonstrate both that the sequence requirements for oriV RK2 function differ between host bacterial species, and that origin function may be further influenced by the genetic environment in which it lies.

Use of a modified Escherichia coli trpR gene to obtain tight regulation of high-copy-number expression vectors.

It has been found that with high-copy-number vectors utilising the Escherichia coli trp promoter the amount of repressor protein produced from the single chromosomally located trpR gene is inadequate for tight repression to be obtained. An attempt has been made to overcome this problem by inserting the trpR gene in cis into the expression vector. This proved unsuccessful because transcription from the trp promoter of such a plasmid could not be induced with 3,beta-indole acrylic acid, probably because the trpR gene is autogenously regulated. However, it was found that when the natural trpR promoter was replaced with a relatively weak constitutive promoter a useful self-repressible vector could be formed. A modified trpR gene of this type has been used to obtain tightly controlled expression of human interferon-beta (IFN-beta) from a vector having a copy number of 400. Tight regulation is particularly important in this case as IFN-beta is highly toxic to the E. coli cell.