{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T01:35:44Z","timestamp":1768527344871,"version":"3.49.0"},"posted":{"date-parts":[[2025,11,7]]},"group-title":"Microbiology","reference-count":51,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T00:00:00Z","timestamp":1762473600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  PlrSR, a member of the NtrYX family of two-component regulatory systems (TCSs), is required for the classical bordetellae, including the causative agent of whooping cough,\n                  <jats:italic>Bordetella pertussis<\/jats:italic>\n                  , to persist in the lower respiratory tract. The\n                  <jats:italic>plrSR<\/jats:italic>\n                  genes are in the middle of a six-gene cluster whose regulation and roles during infection were unknown.\n                  <jats:italic>rsmB<\/jats:italic>\n                  and\n                  <jats:italic>plrP<\/jats:italic>\n                  are often found 5\u2019 to\n                  <jats:italic>plrSR<\/jats:italic>\n                  homologs in \u03b2- and \u03b3- proteobacteria, while\n                  <jats:italic>trkAH<\/jats:italic>\n                  are often found 3\u2019 to\n                  <jats:italic>plrSR<\/jats:italic>\n                  homologs in \u237a-proteobacteria. We investigated these genes to determine if they have a functional link to\n                  <jats:italic>plrSR<\/jats:italic>\n                  . We found that this gene cluster does not function as an operon. Rather, it contains two internal promoters: a weaker promoter in the 3\u2019 end of\n                  <jats:italic>rsmB<\/jats:italic>\n                  and a stronger promoter in the 3\u2019 end of\n                  <jats:italic>plrS.<\/jats:italic>\n                  Additionally, our results indicate that PlrP functions as a third component of the PlrSR TCS. Genetic manipulations of\n                  <jats:italic>plrP, plrS,<\/jats:italic>\n                  and\n                  <jats:italic>plrR<\/jats:italic>\n                  indicate that PlrP is essential\n                  <jats:italic>in vitro<\/jats:italic>\n                  and inhibits PlrS phosphatase activity, likely through PlrS\u2019s PDC domain. Since our results indicate that PlrR can be phosphorylated by another unknown phosphodonor\n                  <jats:italic>in vitro<\/jats:italic>\n                  , limiting PlrS phosphatase activity ensures PlrR\u223cP is not dephosphorylated to lethally low levels. Using natural-host models, we determined that high levels of PlrR\u223cP are required for\n                  <jats:italic>in vivo<\/jats:italic>\n                  survival, and PlrP affects PlrS activity\n                  <jats:italic>in vivo<\/jats:italic>\n                  . Given that\n                  <jats:italic>plrP<\/jats:italic>\n                  homologs always colocalize with\n                  <jats:italic>ntrYX<\/jats:italic>\n                  homologs, we propose that PlrP may fulfill similar functions in other \u03b2- and \u03b3-proteobacteria that encode NtrYX- family TCSs, including nonpathogens.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    <jats:italic>Bordetella<\/jats:italic>\n                    species, including\n                    <jats:italic>B. pertussis<\/jats:italic>\n                    , the causal agent of whooping cough, cause respiratory infections in humans and other animals. Their PlrSR two component regulatory systems, members of the NtrYX family, are required for survival in the lower respiratory tract. We characterized the six-gene cluster that includes\n                    <jats:italic>plrS<\/jats:italic>\n                    and\n                    <jats:italic>plrR<\/jats:italic>\n                    , identifying one promoter within the first gene that drives expression of the second gene, which we named\n                    <jats:italic>plrP<\/jats:italic>\n                    , as well as plrS, and another promoter near the 3\u2019 end of\n                    <jats:italic>plrS<\/jats:italic>\n                    that drives expression of\n                    <jats:italic>plrR<\/jats:italic>\n                    and the downstream\n                    <jats:italic>trkAH<\/jats:italic>\n                    genes. Our data indicate that the\n                    <jats:italic>plrP<\/jats:italic>\n                    gene product is an essential third component of the PlrSR TCS, functioning to prevent PlrS from acting as a strong phosphatase\n                    <jats:italic>in vitro<\/jats:italic>\n                    . Comparative analyses suggest that PlrP homologs are present, and may function similarly, in NtrYX-family TCSs in other \u03b2- and \u03b3-proteobacteria. Our results are important because they provide insight into how bacteria sense and respond to their environment, including those they experience while causing human infection, and this understanding could inform therapeutic and vaccine development.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.11.07.687182","type":"posted-content","created":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T03:55:16Z","timestamp":1762574116000},"source":"Crossref","is-referenced-by-count":0,"title":["Genetic evidence for a periplasmic protein as a component for a subset of NtrYX two-component systems"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7775-8039","authenticated-orcid":false,"given":"Alexa R.","family":"Wolber","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5426-5539","authenticated-orcid":false,"given":"Liliana S.","family":"McKay","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard M.","family":"Johnson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zain T.","family":"Hameed","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katlyn B.","family":"Mote","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Steven M.","family":"Julio","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peggy A.","family":"Cotter","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025111008300509000_2025.11.07.687182v1.1","doi-asserted-by":"publisher","DOI":"10.1128\/aem.57.4.1202-1206.1991"},{"key":"2025111008300509000_2025.11.07.687182v1.2","unstructured":"CDC. 2025. 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