pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes

Abstract

Background: Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a simplification--along with an optimization--of the currently unwieldy pool of genetic tools.

Results: The functional DNA sequences present in the natural bacterial transposon Tn5 have been methodically edited and refactored for the production of a multi-purpose genetic tool named pBAM1, which allows a range of manipulations in the genome of gram-negative bacteria. This all-synthetic construct enhances the power of mini-transposon vectors for either de-construction or re-construction of phenotypes á la carte by incorporating features inspired in systems engineering: modularity, re-usability, minimization, and compatibility with other genetic tools. pBAM1 bears an streamlined, restriction site-freed and narrow-host range replication frame bearing the sequences of R6K oriV, oriT and an ampicillin resistance marker. These go along with a business module that contains a host-independent and hyperactive transposition platform for in vivo or in vitro insertion of desired DNA into the genome of the target bacterium. All functional sequences were standardized for a straightforward replacement by equivalent counterparts, if required. pBAM1 can be delivered into recipient cells by either mating or electroporation, producing transposon insertion frequencies of 1.8 × 10(-3) and 1.02 × 10(-7), respectively in the soil bacterium Pseudomonas putida. Analyses of the resulting clones revealed a 100% of unique transposition events and virtually no-cointegration of the donor plasmid within the target genome.

Conclusions: This work reports the design and performance of an all-synthetic mini-transposon vector. The power of the new system for both identification of new functions or for the construction of desired phenotypes is shown in a genetic survey of hyper-expressed proteins and regulatory elements that influence the expression of the σ54-dependent Pu promoter of P. putida.

Figure 1

pBAM1 plasmid map. Functional elements of the plasmid include relevant restriction sites, antibiotic markers (Ap, ampicillin, Km, kanamycin), transposase (tnpA), origin of replication (R6K), the origin of transfer region (oriT), mosaic element O (ME-O), and mosaic element I (ME-I), as shown.

Figure 2

Structural organization of standard mini-transposon modules. (A) Mini-Tn5 Km. Details of relevant restriction enzymes within the module are shown. The fusion of ME-I and ME-O sequences with the plasmid DNA backbone generated PvuII restriction sites that bracket the mobile segment. The red arrow indicates the position of the promoter of the Km resistance gene. MCS: multiple-cloning-site. (B) mini-Tn5GFPKm. Schematic representation of the main features of this version of the mini-transposon engineered in the pBAM1 backbone, containing the GFP gene lacking leading sequences and thus able to produce protein fusions upon chromosomal insertions in the right direction and frame. The Km resistance cassette is identical to that of the mini-Tn5Km of pBAM1.

Figure 3

Testing mini-transposon insertions in P. putida MAD1 and re Regulatory phenotypes brought about by insertions of the mini-Tn5Km of pBAM1 in P. putida MAD1. (A) Representation of the reporter module born by the P. putida MAD1 strain. Pu is induced by XylR in the presence of m-xylene vapours. (B) Schematic representation and approximate location of mini-Tn5Km insertions within xylR and lacZ in P. putida MAD1. (C) The reference condition is that of the clones of the non-mutagenized strain exposed to m-xylene and grown on a plate with X-gal for several days, which results in an intense blue colour exacerbated in the centre of the colony. (D) The other pictures represent the variety of the blue/white patterns obtained throughout the P. putida MAD1 mutagenesis experiment. The pictures were obtained with a Leica MZ FLIII stereomicroscope with an Olympus DP70 camera. See Table S3 of Additional File 1 for more details.

Figure 4

Subcellular localization of high-fluorescence GFP fusions generated by mutagenesis of P. putida with mini-Tn5GFPKm. Cultures of the cells under examination were grown until stationary phase in LB medium and prepared for epifluorescence microscopy as explained in Materials and Methods. The upper panel shows examples of GFP fused to cytosolic proteins, as indicated, whereas in lower panel contains GFP fusions in three different membrane-associated proteins. Table S4 of Additional File 1 provides more details of the GFP fusions generated.