Saccharomyces cerevisiae -Based Molecular Tool Kit for Manipulation of Genes from Gram-Negative Bacteria

Abstract

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.Cloning using Saccharomyces cerevisiae homologous recombination is a powerful technique in which PCR primers with stretches of homologous DNA are used to target recombination of an amplicon with a vector (9,21,25,27). Cloning of an amplicon with yeast recombination into an intact vector can be done without restriction enzymes, but this requires the use of a selectable or counterselectable marker. The formation of a gap or double-stranded break in the vector by restriction enzyme digestion allows selective cloning of unmarked amplicons (29). An amplicon can seamlessly replace any part of the DNA in a vector with no need for enzyme sites at the junctions of the recombined pieces of DNA, as long as the yeast replication machinery is not replaced and the ends of the amplicons (or other DNA with homologous ends) to be added to a vector are on either side of the digest site (gap) in the vector. Therefore, multiple unmarked pieces of DNA can efficiently be assembled together in a single step using this method. In addition to fusions, directed mutations, restriction sites, and other changes can be designed into the primers. This technique is particularly useful for building vectors and complex constructs.The basic concept of cloning with gap repair is as follows. A vector able to replicate in yeast with a selective marker, such as URA3, is digested (gapped) with a restriction enzyme. Primers that amplify the region of DNA that one wants to insert into the vector are designed, containing sequences of homology to the yeast-replicating plasmid (30 to 40 bp in length). One or more targeted amplicons and the gapped vector are simultaneously introduced into yeast cells. A subset of these cells recombines the amplicon(s) and vector via Saccharomyces cerevisiae native recombination enzymes, generating stable circularized plasmids. Linearized or gapped vectors are not stable in yeast, while vectors that circularize...

Electron-shuttling antibiotics structure bacterial communities by modulating cellular levels of c-di-GMP

“…Deletion, complementation, point mutation, Histag, and constitutive expression plasmids were generated by modifying allelicreplacement vector pMQ30 (7.5-kb mobilizable vector, oriT, sacB, Gm R ) using yeast gap-repair cloning (55). All strains that were generated with these plasmids are listed in SI Appendix, Table S1 and the primers used to construct the plasmids are listed in SI Appendix, Table S2.…”

The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

“…Deletion plasmids were generated using the yeast gap repair method as described previously (18,62). The same approach was used to complement the transposon insertion mutant mexH:: tn, with the modification that plasmid pLD1574, instead of pMQ30, was used to replace the disrupted allele with the wild-type sequence.…”

LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0–1

“…E. coli S17-1 -pir was used for maintenance and conjugal transfer of plasmids. Yeast strain InvSc1 (Invitrogen) was cultured as described (30). Gentamycin (10 g ml Ϫ1 for E. coli, 30 g ml Ϫ1 for Pseudomonas) and kanamycin (30 g ml Ϫ1 ) were used where appropriate.…”