{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,9]],"date-time":"2026-10-09T02:13:19Z","timestamp":1791511999469,"version":"4.3.4"},"reference-count":39,"publisher":"American Society for Microbiology","issue":"7","license":[{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T00:00:00Z","timestamp":1783382400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01-AI109025"],"award-info":[{"award-number":["R01-AI109025"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01-AI153414"],"award-info":[{"award-number":["R01-AI153414"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["T32-GM007231"],"award-info":[{"award-number":["T32-GM007231"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,7,7]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Transposon mutagenesis coupled with deep sequencing (Tn-seq) is currently being deployed in microbial eukaryotes, including the opportunistic yeast pathogen\n                      <jats:italic toggle=\"yes\">Candida glabrata<\/jats:italic>\n                      , for functional genomics research. This method depends on the generation of highly diverse pools of transposon insertion mutants to cover all genes while minimizing the presence of markers and remnants of engineering. Up to now, pools of\n                      <jats:italic toggle=\"yes\">Hermes<\/jats:italic>\n                      transposon insertion mutants in\n                      <jats:italic toggle=\"yes\">C. glabrata<\/jats:italic>\n                      were generated in uracil-requiring\n                      <jats:italic toggle=\"yes\">ura3\u2206<\/jats:italic>\n                      auxotrophs, limiting their use in nutrient-restricted environments, such as those of the host. Indeed, we found that\n                      <jats:italic toggle=\"yes\">ura3\u2206<\/jats:italic>\n                      mutants were outcompeted by\n                      <jats:italic toggle=\"yes\">URA3+<\/jats:italic>\n                      prototrophs during colonization of the mouse gastrointestinal tract. To avoid using auxotrophs in Tn-seq experiments, a new scheme was developed for generating prototrophic pools of\n                      <jats:italic toggle=\"yes\">Hermes<\/jats:italic>\n                      insertion mutants. The scheme involved introducing a recessive cycloheximide resistance mutation in the chromosomal\n                      <jats:italic toggle=\"yes\">RPL28<\/jats:italic>\n                      gene, which did not alter fitness during mouse colonization. When implemented in several different strains of\n                      <jats:italic toggle=\"yes\">C. glabrata<\/jats:italic>\n                      , high insertion densities were obtained, and differences in subtelomeric chromatin compaction were observed that correlated with natural variation in the silencing gene,\n                      <jats:italic toggle=\"yes\">SIR3<\/jats:italic>\n                      . However, all the strains lacked insertions in the\n                      <jats:italic toggle=\"yes\">PDR1<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">CDR1<\/jats:italic>\n                      genes, which are necessary for resistance to cycloheximide and other antifungals. We directly tested the effect of\n                      <jats:italic toggle=\"yes\">pdr1\u2206<\/jats:italic>\n                      mutants and found that they exhibited moderate fitness defects in the gastrointestinal tract of mice even in the absence of antifungals. Thus, the new scheme easily generates high-quality pools of insertion mutants in prototrophic\n                      <jats:italic toggle=\"yes\">C. glabrata<\/jats:italic>\n                      with only minor and knowable limitations.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Treatment of fungal infections may be improved by a deeper understanding of the genetic mechanisms of colonization within host organisms. Current approaches to deep genetic sequencing in eukaryotic microbes often involve engineered components that have significant biases, minimize microbial complexity, or alter the normal\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      fitness of opportunistic fungal pathogens. This study designs a new method for developing transposon insertion mutants in\n                      <jats:italic toggle=\"yes\">Candida glabrata<\/jats:italic>\n                      that does not innately introduce altered fitness in mouse models of gastrointestinal tract infection. This scheme is also portable across strains and possibly even fungal species. The findings show that the new method can be used in this pathogenic yeast to yield highly complex pools and reliably identify genetic components of colonization in mouse models of infection.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00848-26","type":"journal-article","created":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T22:39:21Z","timestamp":1780094361000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Facile generation of\n                    <i>Hermes<\/i>\n                    insertion mutants in prototrophic\n                    <i>Candida glabrata<\/i>\n                    for use in nutrient-limited environments"],"prefix":"10.1128","volume":"14","author":[{"given":"Anna M.","family":"Zaeske","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/00za53h95","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of 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