Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteriaA collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.
Carbon source-dependent inhibition of xyl operon expression of the Pseudomonas putida TOL plasmidTOL plasmid-encoded degradation of benzyl alcohol by Pseudomonas putida is inhibited by glucose and other compounds related to the main carbohydrate metabolism in Pseudomonas species. We report here that this effect is exerted at the level of expression of the xyl catabolic operons, and two xyl promoters, Pu and Ps, were identified as the primary targets of this inhibition. xyl promoter activation was also inhibited by glucose in the heterologous Escherichia coli system, apparently not however by the classical mechanism of enteric catabolite repression.
Engineering of alkyl- and haloaromatic-responsive gene expression with mini-transposons containing regulated promoters of biodegradative pathways of PseudomonasNon-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cellsNicholas P. West, John T. Fitter, Ute Jakubzik et al.|FEMS Microbiology Letters|2006 Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1-4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37 degrees C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.
CarbonSource-Dependent Inhibition ofxylOperonExpression ofthePseudomonas putida TOLPlasmidTOLplasmid-encoded degradation ofbenzyl alcohol byPseudomonas putida isinhibited byglucose andother compounds related tothemaincarbohydrate metabolism inPseudomonas species. We report herethatthiseffect isexerted atthelevel ofexpression ofthexylcatabolic operons,andtwoxylpromoters, PuandPs, wereidentified astheprimary targets ofthis inhibition. xylpromoter activation was alsoinhibited byglucose intheheterologous Escherichia coilsystem, apparently nothowever bytheclassical mechanism ofenteric catabolite repression. Microbial degradation ofaromatic compounds isan importantapproach toeliminating toxic pollutants fromtheenvironment (2,23,25).Therespective catabolic pathways have generally beenanalyzed underconditions inwhichthetarget compound ispresent asthesole growth-supporting substrate.