A Plasmid-Transposon Hybrid Mutagenesis System Effective in a Broad Range of Enterobacteria

Abstract

Random transposon mutagenesis is a powerful technique used to generate libraries of genetic insertions in many different bacterial strains. Here we develop a system facilitating random transposon mutagenesis in a range of different Gram-negative bacterial strains, including Pectobacterium atrosepticum, Citrobacter rodentium, Serratia sp. ATCC39006, Serratia plymuthica, Dickeya dadantii, and many more. Transposon mutagenesis was optimized in each of these strains and three studies are presented to show the efficacy of this system. Firstly, the important agricultural pathogen D. dadantii was mutagenized. Two mutants that showed reduced protease production and one mutant producing the previously cryptic pigment, indigoidine, were identified and characterized. Secondly, the enterobacterium, Serratia sp. ATCC39006 was mutagenized and mutants incapable of producing gas vesicles, proteinaceous intracellular organelles, were identified. One of these contained a β-galactosidase transcriptional fusion within the gene gvpA1, essential for gas vesicle production. Finally, the system was used to mutate the biosynthetic gene clusters of the antifungal, anti-oomycete and anticancer polyketide, oocydin A, in the plant-associated enterobacterium, Dickeya solani MK10. The mutagenesis system was developed to allow easy identification of transposon insertion sites by sequencing, after facile generation of a replicon encompassing the transposon and adjacent DNA, post-excision. Furthermore, the system can also create transcriptional fusions with either β-galactosidase or β-glucuronidase as reporters, and exploits a variety of drug resistance markers so that multiple selectable fusions can be generated in a single strain. This system of various transposons has wide utility and can be combined in many different ways.

Environmental potassium regulates bacterial flotation, antibiotic production and turgor pressure in Serratia through the TrkH transporter

“…Gene expression in uidA and lacZ fusion strains was assessed as previously reported (Ramsay et al, ; Monson et al, ). Enzymatic activity was quantified using a Gemini XPS plate reader following the parameters described previously (Ramsay et al, ).…”

“…This refraction leads to a distinctive colony opacity in gas ‘vacuolated’ bacteria, and the phase bright structures (GVs) can be seen in individual cells when observed under phase contrast microscopy (PCM) (Ramsay et al, ). These features have enabled facile screening for GV‐defective colonies (which appear translucent) in mutagenesis experiments (Ramsay et al, ; Monson et al, ; Lee et al ., ). External pressure changes can cause GV collapse and a consequent loss of buoyancy and light refraction in cells.…”

The LacI–Family Transcription Factor, RbsR, Is a Pleiotropic Regulator of Motility, Virulence, Siderophore and Antibiotic Production, Gas Vesicle Morphogenesis and Flotation in Serratia

“…Transposon mutagenesis of S39006 was carried out as described previously (Monson et al, 2015 ). Briefly, the plasmid pKRCPN1 was delivered to the recipient by conjugation with E. coli β2163.…”

Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006

“…Cultures were grown for 14 h, and 100 µl samples were removed and frozen at À80 C. They were subsequently processed as described previously (Monson et al, 2015).…”