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To better understand such complexities in the context of niche bacterial populations in\n                      <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                      and other gram-negatives, here we construct and characterize a novel and versatile dual-reporter plasmid,\n                      <jats:underline>C<\/jats:underline>\n                      onstitutive\n                      <jats:underline>G<\/jats:underline>\n                      FP,\n                      <jats:underline>V<\/jats:underline>\n                      ariable\n                      <jats:underline>mS<\/jats:underline>\n                      carlet-I (pCG-VmS), whose utility proves useful for longitudinal assays of transcriptional reporter activity\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      ,\n                      <jats:italic toggle=\"yes\">ex vivo<\/jats:italic>\n                      , and in whole animals. As an added utility, we show that pCG-VmS can also be used for translational fusions to monitor bacterial cellular protein localization. We then exploit pCG-VmS to visualize\n                      <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                      T3SS expression in a human corneal cell line that identifies a distinct intracellular \u201cquiescent\u201d population, where the T3SS is never expressed. An\n                      <jats:italic toggle=\"yes\">ex vivo<\/jats:italic>\n                      murine eye infection model is then used to verify the existence of the two \u2018T3SS-on\u201d versus \u2018T3SS-off\u2019 expression profiles in infected tissues. In sum, pCG-VmS allows for novel spatiotemporal insights into\n                      <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                      protein localization and infection routes and can be tailored to other plasmid backbones amenable to different gram-negative bacteria.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        In this study, we develop a novel, well-optimized fluorescent dual-reporter system, pCG-VmS, which can be used for both transcriptional monitoring and protein localization. We demonstrate its use with a range of\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        experiments with\n                        <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                        and whole-animal imaging of squid colonization with\n                        <jats:italic toggle=\"yes\">Vibrio fischeri<\/jats:italic>\n                        . After showing pCG-VmS efficacy and versatility, we use the plasmid to simultaneously visualize the naturally occurring type-3 secretion system (T3SS) active (T3SS-on) and \u201cquiescent\u201d (T3SS-off) populations during intracellular infection of a human corneal epithelial cell line and the murine eye. Taken together, pCG-VmS is well-balanced in its optical traits and can be used for a variety of both\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        studies to help separate and further interrogate difficult niche spatiotemporal bacterial populations.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00490-26","type":"journal-article","created":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T13:01:45Z","timestamp":1780491705000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["A versatile dual-color bacterial reporter system highlights two distinct\n                    <i>Pseudomonas aeruginosa<\/i>\n                    type 3 secretion system intracellular 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