A uniform cloning platform for mycobacterial genetics and protein production

Abstract

Molecular research on mycobacteria relies on a multitude of tools for the genetic manipulation of these clinically important bacteria. However, a uniform set of vectors allowing for standardized cloning procedures is not available. Here, we developed a versatile series of mycobacterial vectors for gene deletion, complementation and protein production and purification. The vectors are compatible with fragment exchange (FX) cloning, a recently developed high-throughput cloning principle taking advantage of the type IIS restriction enzyme SapI and its capacity to generate sticky trinucleotide ends outside of its recognition sequence. FX cloning allows for the efficient cloning into an entry vector and the facile transfer of the sequenced insert into a variety of destination vectors. We generated a set of mycobacterial expression vectors spanning a wide range of expression strengths, tagging variants and selection markers to rapidly screen for the optimal expression construct in order to purify membrane proteins from the model organism Mycobacterium smegmatis. Further, we generated a series of suicide vectors containing two counterselection markers and used them to delete twenty genes encoding for potential drug efflux pumps in M. smegmatis. The vectors will further facilitate genetic and biochemical research on various mycobacterial species.

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

“…M. smegmatis mc 2 155 deletion mutants were generated as previously described (Arnold et al, ). In brief, upstream and downstream flanking regions of genes of interested were amplified from genomic DNA using primers listed in Table S7, cloned into pINIT, and sequenced, followed by transfer of the flanking regions into pKO (Apr resistance).…”

“…To construct complementation plasmids, we used the FX-cloning strategy (Geertsma & Dutzler, 2011) with the backbone pFLAG (Arnold et al, 2018). In this system, the cloned genes are expressed constitutively from the medium strength tetracycline promoter (P tet ) and stably integrated into the attB site of the genome.…”

“…M. smegmatis mc 2 155 deletion mutants were generated as previously described (Arnold et al, 2018). In brief, upstream and downstream flanking regions of genes of interested were amplified from genomic DNA using primers listed in Table S7, cloned into pINIT, and sequenced, followed by transfer of the flanking regions into pKO (Apr resistance (Ratledge & Ewing, 1996), dispersed by sonication, diluted, and plated (about 2 × 10 7 CFU per well) onto solidified minimal medium in 24-well plates, supplemented with MBT/cMBT, FeCl 3 or ethanol (control) as described above.…”

DistinctMycobacterium marinumphosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol

“…Another set of complementation plasmids was generated by means of the FX‐cloning strategy (Geertsma & Dutzler, ) using the backbone pFLAG (Arnold et al, ), which expresses the cloned gene constitutively from the medium strength tetracycline promoter. The genes are stably integrated into the attB site of the genome.…”

A guide to Mycobacterium mutagenesis

“…Recently, Arnold et al . implemented this dual counterselection in their optimized suicide vectors (the pKO series), that are part of a uniform cloning platform (based on fragment exchange using the type IIS restriction enzyme SapI) to facilitate mycobacterial genetics (also includes vectors for gene complementation, conditional knockdowns and protein production).…”