We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.

A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria

Despite over a century of research, tuberculosis remains a leading cause of infectious death worldwide. Faced with increasing rates of drug resistance, the identification of genes that are required for the growth of this organism should provide new targets for the design of antimycobacterial agents. Here, we describe the use of transposon site hybridization (TraSH) to comprehensively identify the genes required by the causative agent, Mycobacterium tuberculosis, for optimal growth. These genes include those that can be assigned to essential pathways as well as many of unknown function. The genes important for the growth of M. tuberculosis are largely conserved in the degenerate genome of the leprosy bacillus, Mycobacterium leprae, indicating that non-essential functions have been selectively lost since this bacterium diverged from other mycobacteria. In contrast, a surprisingly high proportion of these genes lack identifiable orthologues in other bacteria, suggesting that the minimal gene set required for survival varies greatly between organisms with different evolutionary histories.

https://onlinelibrary.wiley.com/doi/epdf/10.1046/j.1365-2958.2003.03425.x

Genes required for mycobacterial growth defined by high density mutagenesis

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-) Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules Mutations at two other exo loci were not located on either megaplasmid To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others This plant phenotype was not observed to change with A tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules

Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes.

Small bacteriophage Mu transposable elements containing the lac operon structural genes were constructed to facilitate the isolation and use of Mu insertions and lac gene fusions. These mini-Mu elements have selectable genes for either ampicillin or kanamycin resistance and can be used to form both transcriptional and translational lac gene fusions. Some of the mini-Mu-lac elements constructed are deleted for the Mu A and B transposition genes and form stable insertions that cannot undergo transposition unless complemented for these functions. A procedure was developed for selecting mini-Mu insertions specifically into plasmids, including commonly used high-copy-number cloning vectors such as pBR322. Mu insertions in pBR322 were found to be distributed around the plasmid, but insertions in certain regions occurred more frequently than in others.

Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons.

Publisher Summary This chapter discusses the uses of transposons with emphasis on Tn 10. As transposable elements have become indispensable tools for bacterial genetics, many different types of specialized transposon derivatives have been constructed. The most widely used constructs are derived from insertion sequence (IS)-based elements (Tn10 and Tn5) or from bacteriophage Mu; constructs based on cointegrate-forming elements are also available. This chapter discusses the major types of transposon constructs available and describes a method to best choose the most appropriate construct for the desired application. The chapter also discusses methods used for Tnl0-derived transposon vehicles and the most recent set of useful Tnl0 vehicles..

Uses of transposons with emphasis on Tn10.

We assessed the effect of insertions of the kanamycin resistance transposon Tn5 in the lac operon of Escherichia coli on the expression of distal genes lacY and lacA (melibiose fermentation at 41 degrees C and thiogalactoside transacetylase synthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 in lacY) was strongly polar. However, approximately one-third of the insertion mutants expressed distal genes at low levels due to a promoter associated with Tn5. To localize this promoter, we (i) reversed the orientation of Tn5 at several sites and (ii) replaced wild-type Tn5 with several substitution derivatives which lack Tn5's central region. Neither alteration changed the expression of distal genes. Thus, in contrast to transposons IS2 and TnA. Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon. Our results suggest that the promoter is within 186 base pairs of the ends of Tn5. It is possible that the promoter is detected in only a fraction of insertions because it overlaps Tn5-target sequence boundary.

L Crossland

Papers

Polarity of Tn5 insertion mutations in Escherichia coli.