{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T03:46:43Z","timestamp":1790740003215,"version":"4.1.0"},"reference-count":43,"publisher":"Microbiology Society","issue":"6","license":[{"start":{"date-parts":[[2022,6,29]],"date-time":"2022-06-29T00:00:00Z","timestamp":1656460800000},"content-version":"unspecified","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2022,6,29]]},"abstract":"<jats:p>\n                  <jats:italic>\n                     <jats:named-content content-type=\"species\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.1910\" xlink:type=\"simple\">Neisseria gonorrhoeae<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic>, 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 <jats:italic>galK<\/jats:italic> as a counter-selectable marker that can be used for markerless mutagenesis in <jats:italic>\n                     <jats:named-content content-type=\"species\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.1910\" xlink:type=\"simple\">N. gonorrhoeae<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic>. <jats:italic>galK<\/jats:italic> 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 <jats:italic>\n                     <jats:named-content content-type=\"species\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.1910\" xlink:type=\"simple\">N. gonorrhoeae<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic>. 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 <jats:italic>galK<\/jats:italic>, that efficiently rendered <jats:italic>\n                     <jats:named-content content-type=\"species\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.1910\" xlink:type=\"simple\">N. gonorrhoeae<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic> susceptible to growth on 2-DOG. We then adapted the <jats:italic>galK<\/jats:italic>-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 <jats:italic>\n                     <jats:named-content content-type=\"species\">\n                        <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"uri\" xlink:href=\"http:\/\/doi.org\/10.1601\/nm.1910\" xlink:type=\"simple\">N. gonorrhoeae<\/jats:ext-link>\n                     <\/jats:named-content>\n                  <\/jats:italic> has a high success rate, and should be a valuable gene editing tool in the future.<\/jats:p>","DOI":"10.1099\/mic.0.001201","type":"journal-article","created":{"date-parts":[[2022,6,28]],"date-time":"2022-06-28T17:30:45Z","timestamp":1656437445000},"source":"Crossref","is-referenced-by-count":14,"title":["Markerless gene editing in Neisseria gonorrhoeae"],"prefix":"10.1099","volume":"168","author":[{"given":"Rebekah A.","family":"Jones","sequence":"first","affiliation":[{"name":"Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, 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