Markerless gene editing in Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae , the gonococcus, is a pathogen of major public health concern, but sophisticated approaches to gene manipulation are limited for this species. For example, there are few methods for generating markerless mutations, which allow the generation of precise point mutations and deletions without introducing additional DNA sequence. Markerless mutations are central to studying pathogenesis, the spread of antimicrobial resistance (AMR) and for vaccine development. Here we describe the use of galK as a counter-selectable marker that can be used for markerless mutagenesis in N. gonorrhoeae . galK encodes galactokinase, an enzyme that metabolizes galactose in bacteria that can utilize it as a sole carbon source. GalK can also phosphorylate a galactose analogue, 2-deoxy-galactose (2-DOG), into a toxic, non-metabolisable intermediate, 2-deoxy-galactose-1-phosphate. We utilized this property of GalK to develop a markerless approach for mutagenesis in N. gonorrhoeae . We successfully deleted both chromosomally and plasmid-encoded genes, that are important for gonococcal vaccine development and studies of AMR spread. We designed a positive-negative selection cassette, based on an antibiotic resistance marker and galK, that efficiently rendered N. gonorrhoeae susceptible to growth on 2-DOG. We then adapted the galK-based counter-selection and the use of 2-DOG for markerless mutagenesis, and applied biochemical and phenotypic analyses to confirm the absence of target genes. We show that our markerless mutagenesis method for N. gonorrhoeae has a high success rate, and should be a valuable gene editing tool in the future.

A Toolbox forNeisseria meningitidis: Gene Editing, Complementation and Labelling

“…We then fused this construction to the one proposed by Jones et al . (Jones et al, 2022) where the galK gene was under the control of an opaB promoter. Expressed galK phosphorylates 2-DOG and turns it into a non-metabolizable intermediate, conferring sensitivity to 2-DOG.…”

“…The mutants obtained growth comparable to that of the wild type or the strain carrying the APG cassette. Finally, the use of a cassette combining two susceptibility genes allows easy and efficient selection in N. meningitidis , while reducing the concentration of 2- DOG from 6% (Jones et al, 2022) to 1%, thus reducing costs.…”

“…This variant renders bacteria sensitive to the phenylalanine analogue p-chloro-phenylalanine (Cl-Phe) due to Ala > Gly mutation (Kast, 1994). We then fused this construction to the one proposed by Jones et al (Jones et al, 2022) where the galK gene was under the control of an opaB promoter. Expressed galK phosphorylates 2-DOG and turns it into a non-metabolizable intermediate, conferring sensitivity to 2-DOG.…”

“…PCR-based construction of interruption or deletion mutants using antibiotic resistance cassettes has led the community to neglect the development of molecular strategies and tools until recently, although the use of antibiotic resistance cassettes limits the construction of strains with multiple mutations. Strategies for constructing markerless mutants in Neisseria species have only been published in the last two years (Jones et al, 2022;Nyongesa et al, 2022;Seow et al, 2023). The work by Nyongesa et al is based on the first selection of streptomycin-resistant strains while the work by Seow et al is based on the cotransformation of a resistance cassette and that of the DNA of interest.…”

Evolution, persistence, and host adaption of a gonococcal AMR plasmid that emerged in the pre-antibiotic era

“…To define which TA systems encoded in the GL region contribute to plasmid stability, we next deleted or inactivated the toxins individually (pCtΔ vapD , pCt: ζ1 K115A , pCtΔ ζ2 ) or in combination (pCtΔ vapD : ζ1 and pCtΔTA, Fig 4C ). trbC , which encodes the conjugative pilus, was removed from all plasmids to prevent pConj re-acquisition by conjugation [ 39 ]. No pConj loss was detected after growth for 30–40 generations following deletion of vapD or ζ2 .…”