{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T14:01:22Z","timestamp":1790085682417,"version":"4.0.1"},"reference-count":53,"publisher":"American Society for Microbiology","license":[{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100004731","name":"Natural Science Foundation of Zhejiang Province","doi-asserted-by":"publisher","award":["LQ24C010003"],"award-info":[{"award-number":["LQ24C010003"]}],"id":[{"id":"10.13039\/501100004731","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      PA0947 is annotated as an essential hypothetical protein in\n                      <jats:italic toggle=\"yes\">Pseudomonas aeruginosa<\/jats:italic>\n                      and shares substantial sequence identity with the\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      replication regulator Hda, yet its cellular function and genetic bypass logic in\n                      <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                      have not been experimentally characterized. Here, we constructed a plasmid-based temperature-sensitive complementation strain,\n                      <jats:italic toggle=\"yes\">\u0394pa0947<\/jats:italic>\n                      (pTS-\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      ), to enable conditional depletion of\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      . The mutant grew like wild type at 30\u00b0C but failed to proliferate at 42\u00b0C, demonstrating\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      as essential for growth.\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      depletion triggered rapid filamentation, increased PicoGreen-associated fluorescence, and an elevated oriC\/ter ratio, consistent with dysregulated replication initiation. In addition,\n                      <jats:italic toggle=\"yes\">E. coli hda<\/jats:italic>\n                      did not complement\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      depletion, and overexpression of either\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      or\n                      <jats:italic toggle=\"yes\">E. coli hda<\/jats:italic>\n                      impaired growth, indicating species-dependent incompatibility and dosage sensitivity. To identify suppressors capable of bypassing\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      loss, we performed a spontaneous suppressor screen and isolated four independent suppressor mutants that grew without\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      . Genome resequencing of these suppressors revealed convergent mutations in the\n                      <jats:italic toggle=\"yes\">dnaA<\/jats:italic>\n                      locus, including upstream regulatory indels and C-terminal coding changes. Upstream mutations reduced\n                      <jats:italic toggle=\"yes\">dnaA<\/jats:italic>\n                      transcript levels by qRT-PCR, whereas coding changes compromised DnaA\u2013oriC binding in EMSA. Together, these results identify\n                      <jats:italic toggle=\"yes\">dnaA<\/jats:italic>\n                      as the convergent bypass locus for\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      essentiality and link\n                      <jats:italic toggle=\"yes\">pa0947<\/jats:italic>\n                      function to tight control of DnaA-driven initiation in\n                      <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                      .\n                    <\/jats:p>\n                    <jats:p\/>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Many bacterial essential genes remain poorly understood, limiting efforts to exploit them as antibiotic targets. PA0947 is predicted to be essential in the opportunistic pathogen\n                        <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                        , yet its essentiality and physiological role have not been experimentally defined. Using a conditional depletion strategy based on plasmid-borne, temperature-sensitive complementation, we show that PA0947 loss causes severe growth failure, filamentation, and abnormal DNA accumulation, consistent with disrupted control of chromosome duplication. Suppressor analysis identified four independent suppressors, including promoter lesions that reduced dnaA expression and coding changes that compromised DnaA\u2013oriC binding. Together, our results establish in\n                        <jats:italic toggle=\"yes\">P. aeruginosa<\/jats:italic>\n                        that PA0947 is required to keep replication initiation within a narrow activity window compatible with robust growth, and they highlight conditional depletion plus suppressor mapping as a broadly useful strategy to uncover the function and bypass logic of essential bacterial genes.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00619-26","type":"journal-article","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T13:12:41Z","timestamp":1790082761000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Analysis of the plasmid-based ts-mutant\n                    <i>\u0394pa0947<\/i>\n                    (pTS-\n                    <i>pa0947<\/i>\n                    ) reveals\n                    <i>pa0947<\/i>\n                    essentiality and\n                    <i>dnaA<\/i>\n                    -centered suppressor bypass in\n                    <i>Pseudomonas aeruginosa<\/i>"],"prefix":"10.1128","author":[{"given":"Qinghai","family":"Tang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03mys6533","id-type":"ROR","asserted-by":"publisher"}],"name":"Systems Biology Laboratory, School of Marine Science and Technology, Zhejiang Ocean University","place":["Zhoushan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huiting","family":"Zhu","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03mys6533","id-type":"ROR","asserted-by":"publisher"}],"name":"Systems Biology Laboratory, School of Marine Science and Technology, Zhejiang Ocean University","place":["Zhoushan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0885-4575","authenticated-orcid":true,"given":"Zhili","family":"Yang","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03mys6533","id-type":"ROR","asserted-by":"publisher"}],"name":"Systems Biology Laboratory, School of Marine Science and Technology, Zhejiang Ocean University","place":["Zhoushan, China"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/s1526-0550(02)00003-3"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms11040916"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(17)30753-3"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2012.08.002"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bcp.2016.12.002"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/mbio.02051-23"},{"key":"e_1_3_4_8_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1422186112"},{"key":"e_1_3_4_9_2","first-page":"73","article-title":"Annotation and curation of hypothetical proteins: prioritizing targets for experimental study","volume":"5","author":"Naveed M","year":"2018","unstructured":"Naveed M, Chaudhry Z, Ali Z, Amjad M, Zulfiqar F, NumanA. 2018. 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