{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T23:08:48Z","timestamp":1783465728391,"version":"3.55.0"},"reference-count":45,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,2]],"date-time":"2026-02-02T00:00:00Z","timestamp":1769990400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32470193"],"award-info":[{"award-number":["32470193"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32170177"],"award-info":[{"award-number":["32170177"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Nankai University Tianjin Applied and Fundamental Research Project","award":["22JCZDJC00410"],"award-info":[{"award-number":["22JCZDJC00410"]}]}],"content-domain":{"domain":["www.mdpi.com"],"crossmark-restriction":true},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Bacterial pathogens exploit host-derived nutrients to coordinate metabolism and virulence determinants to optimize fitness in vivo. In Pseudomonas aeruginosa, GlnK is a central regulator of nitrogen metabolism. It senses the intracellular nitrogen status by integrating 2-oxoglutarate (2-OG) and glutamine signals, which in turn triggers its uridylylation and conformational changes. This reversible post-translational modification modulates its interaction with target proteins, thereby precisely regulating carbon-nitrogen metabolic homeostasis and enabling adaptive nitrogen metabolism in response to host-derived nutrient cues. In this study, we found that glnK is upregulated during infection in a mouse pneumonia model. By growing bacteria in mouse bronchoalveolar lavage fluid (BALF), we demonstrated that the expression of glnK is activated by the NtrB-NtrC two-component regulatory system in response to the host nutrient environment. Mutation of glnK impairs bacterial virulence. Transcriptomic analysis revealed downregulation of the type III secretion system (T3SS) genes in the glnK mutant. Further studies revealed a role of GlnK in maintaining the homeostasis of the NtrB-NtrC system through a negative feedback mechanism, which is required for the expression of the T3SS genes. Collectively, these findings reveal a role of GlnK in interconnecting carbon\u2013nitrogen balance and the T3SS in response to the host environment.<\/jats:p>","DOI":"10.3390\/microorganisms14020339","type":"journal-article","created":{"date-parts":[[2026,2,2]],"date-time":"2026-02-02T12:49:44Z","timestamp":1770036584000},"page":"339","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["GlnK Regulates the Type III Secretion System by Modulating NtrB-NtrC Homeostasis in Pseudomonas aeruginosa"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7826-7096","authenticated-orcid":false,"given":"Xiaomeng","family":"Sun","sequence":"first","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qitong","family":"Du","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiming","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuetao","family":"Gong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongxin","family":"Jin","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6447-6978","authenticated-orcid":false,"given":"Shouguang","family":"Jin","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9017-0091","authenticated-orcid":false,"given":"Weihui","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/S1473-3099(18)30482-1","article-title":"Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): A randomised, controlled, double-blind phase 1\/2 trial","volume":"19","author":"Jault","year":"2019","journal-title":"Lancet Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Pouget, C., Dunyach-Remy, C., Magnan, C., Pantel, A., Sotto, A., and Lavigne, J.P. (2022). Polymicrobial Biofilm Organization of Staphylococcus aureus and Pseudomonas aeruginosa in a Chronic Wound Environment. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231810761"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"5579","DOI":"10.1038\/s41467-025-60598-4","article-title":"Phage therapy with nebulized cocktail BX004-A for chronic Pseudomonas aeruginosa infections in cystic fibrosis: A randomized first-in-human trial","volume":"16","author":"Weiner","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Scribani Rossi, C., Barrientos-Moreno, L., Paone, A., Cutruzzola, F., Paiardini, A., Espinosa-Urgel, M., and Rinaldo, S. (2022). Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23084386"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"126900","DOI":"10.1016\/j.micres.2021.126900","article-title":"Roles of virulence regulator ToxR in viable but non-culturable formation by controlling reactive oxygen species resistance in pathogen Vibrio alginolyticus","volume":"254","author":"Zhou","year":"2022","journal-title":"Microbiol. Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1038\/ismej.2008.47","article-title":"Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae","volume":"2","author":"Matz","year":"2008","journal-title":"ISME J."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Reuven, A.D., Katzenell, S., Mwaura, B.W., and Bliska, J.B. (2025). ExoS effector in Pseudomonas aeruginosa Hyperactive Type III secretion system mutant promotes enhanced Plasma Membrane Rupture in Neutrophils. PLoS Pathog., 21.","DOI":"10.1371\/journal.ppat.1013021"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2564","DOI":"10.1128\/JB.00106-12","article-title":"The distal ExsA-binding site in Pseudomonas aeruginosa type III secretion system promoters is the primary determinant for promoter-specific properties","volume":"194","author":"Brutinel","year":"2012","journal-title":"J. Bacteriol."},{"key":"ref_9","first-page":"e00209-19","article-title":"Fitting Pieces into the Puzzle of Pseudomonas aeruginosa Type III Secretion System Gene Expression","volume":"201","author":"Djapgne","year":"2019","journal-title":"J. Bacteriol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e0323922","DOI":"10.1128\/spectrum.03239-22","article-title":"Pseudomonas aeruginosa Citrate Synthase GltA Influences Antibiotic Tolerance and the Type III Secretion System through the Stringent Response","volume":"11","author":"Chen","year":"2023","journal-title":"Microbiol. Spectr."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3334","DOI":"10.1128\/IAI.00090-06","article-title":"Transcriptional induction of the Pseudomonas aeruginosa type III secretion system by low Ca2+ and host cell contact proceeds through two distinct signaling pathways","volume":"74","author":"Dasgupta","year":"2006","journal-title":"Infect. Immun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"9930","DOI":"10.1073\/pnas.0504405102","article-title":"A secreted regulatory protein couples transcription to the secretory activity of the Pseudomonas aeruginosa type III secretion system","volume":"102","author":"Urbanowski","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"6654","DOI":"10.1128\/JB.00902-09","article-title":"Mechanism of transcriptional activation by Pseudomonas aeruginosa ExsA","volume":"191","author":"Vakulskas","year":"2009","journal-title":"J. Bacteriol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Arif, S.J., Hoffman, K.M., Flynn, J.M., Wiggen, T.D., Lucas, S.K., Villarreal, A.R., Gilbertsen, A.J., Dunitz, J.M., and Hunter, R.C. (2025). Host- and microbial-mediated mucin degradation differentially shape Pseudomonas aeruginosa physiology and gene expression. PLoS Pathog., 21.","DOI":"10.1101\/2025.06.18.660398"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5967","DOI":"10.1093\/nar\/gkaa377","article-title":"PvrA is a novel regulator that contributes to Pseudomonas aeruginosa pathogenesis by controlling bacterial utilization of long chain fatty acids","volume":"48","author":"Pan","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2691","DOI":"10.1093\/nar\/gkad059","article-title":"Regulatory and structural mechanisms of PvrA-mediated regulation of the PQS quorum-sensing system and PHA biosynthesis in Pseudomonas aeruginosa","volume":"51","author":"Pan","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1038\/nrmicro.2018.9","article-title":"The microbial nitrogen-cycling network","volume":"16","author":"Kuypers","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1128\/MMBR.00025-13","article-title":"Nitrogen assimilation in Escherichia coli: Putting molecular data into a systems perspective","volume":"77","author":"Westerhoff","year":"2013","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"6264","DOI":"10.1016\/S0021-9258(19)41060-0","article-title":"Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme","volume":"250","author":"Adler","year":"1975","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e00229-21","DOI":"10.1128\/mBio.00229-21","article-title":"The Novel P(II)-Interacting Protein PirA Controls Flux into the Cyanobacterial Ornithine-Ammonia Cycle","volume":"12","author":"Bolay","year":"2021","journal-title":"mBio"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2219","DOI":"10.1016\/j.bpj.2017.04.012","article-title":"GlnK Facilitates the Dynamic Regulation of Bacterial Nitrogen Assimilation","volume":"112","author":"Gosztolai","year":"2017","journal-title":"Biophys. J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"8530","DOI":"10.1074\/jbc.M312399200","article-title":"Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB-GlnK complex formation","volume":"279","author":"Javelle","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"826","DOI":"10.1002\/mbo3.120","article-title":"Nitrogen regulation of protein-protein interactions and transcript levels of GlnK PII regulator and AmtB ammonium transporter homologs in Archaea","volume":"2","author":"Lange","year":"2013","journal-title":"MicrobiologyOpen"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e00817-20","DOI":"10.1128\/mSystems.00817-20","article-title":"The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense","volume":"5","author":"Gerhardt","year":"2020","journal-title":"mSystems"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e00039-20","DOI":"10.1128\/AEM.00039-20","article-title":"PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-l-Glutamate Kinase by l-Arginine in Corynebacterium glutamicum","volume":"86","author":"Xu","year":"2020","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e0019822","DOI":"10.1128\/mmbr.00198-22","article-title":"How Bacterial Pathogens Coordinate Appetite with Virulence","volume":"87","author":"Pokorzynski","year":"2023","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1038\/nprot.2006.24","article-title":"Mini-Tn7 insertion in bacteria with single attTn7 sites: Example Pseudomonas aeruginosa","volume":"1","author":"Choi","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1090","DOI":"10.1080\/21505594.2020.1809327","article-title":"Surfactant phospholipids act as molecular switches for premature induction of quorum sensing-dependent virulence in Pseudomonas aeruginosa","volume":"11","author":"Kuang","year":"2020","journal-title":"Virulence"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"6756","DOI":"10.1093\/nar\/gkab506","article-title":"Acetylation of the CspA family protein CspC controls the type III secretion system through translational regulation of exsA in Pseudomonas aeruginosa","volume":"49","author":"Li","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Qu, J., Yin, L., Qin, S., Sun, X., Gong, X., Li, S., Pan, X., Jin, Y., Cheng, Z., and Jin, S. (2025). Identification of the Pseudomonas aeruginosa AgtR-CspC-RsaL pathway that controls Las quorum sensing in response to metabolic perturbation and Staphylococcus aureus. PLoS Pathog., 21.","DOI":"10.1371\/journal.ppat.1013054"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"5232","DOI":"10.1016\/j.febslet.2006.08.054","article-title":"Interactions between PII proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense","volume":"580","author":"Huergo","year":"2006","journal-title":"FEBS Lett."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"6123","DOI":"10.1128\/JB.00744-09","article-title":"NtrC-dependent regulatory network for nitrogen assimilation in Pseudomonas putida","volume":"191","author":"Hervas","year":"2009","journal-title":"J. Bacteriol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e00881-13","DOI":"10.1128\/mBio.00881-13","article-title":"Nitrogen and carbon status are integrated at the transcriptional level by the nitrogen regulator NtrC in vivo","volume":"4","author":"Schumacher","year":"2013","journal-title":"mBio"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Alford, M.A., Baquir, B., An, A., Choi, K.G., and Hancock, R.E.W. (2021). NtrBC Selectively Regulates Host-Pathogen Interactions, Virulence, and Ciprofloxacin Susceptibility of Pseudomonas aeruginosa. Front. Cell Infect. Microbiol., 11.","DOI":"10.3389\/fcimb.2021.694789"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Gil-Gil, T., Cuesta, T., Hernando-Amado, S., Reales-Calderon, J.A., Corona, F., Linares, J.F., and Martinez, J.L. (2023). Virulence and Metabolism Crosstalk: Impaired Activity of the Type Three Secretion System (T3SS) in a Pseudomonas aeruginosa Crc-Defective Mutant. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241512304"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1383","DOI":"10.1128\/IAI.72.3.1383-1390.2004","article-title":"Effect of metabolic imbalance on expression of type III secretion genes in Pseudomonas aeruginosa","volume":"72","author":"Rietsch","year":"2004","journal-title":"Infect. Immun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"6670","DOI":"10.1021\/bi049474r","article-title":"Crystal structure of the C-terminal domain of the two-component system transmitter protein nitrogen regulator II (NRII.; NtrB), regulator of nitrogen assimilation in Escherichia coli","volume":"43","author":"Song","year":"2004","journal-title":"Biochemistry"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"28288","DOI":"10.1016\/S0021-9258(18)46926-8","article-title":"Reversible uridylylation of the Escherichia coli PII signal transduction protein regulates its ability to stimulate the dephosphorylation of the transcription factor nitrogen regulator I (NRI or NtrC)","volume":"269","author":"Atkinson","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"301","DOI":"10.1046\/j.1365-2958.1999.01349.x","article-title":"Characterization of the GlnK protein of Escherichia coli","volume":"32","author":"Atkinson","year":"1999","journal-title":"Mol. Microbiol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Yurist-Doutsch, S., Arrieta, M.C., Tupin, A., Valdez, Y., Antunes, L.C., Yen, R., and Finlay, B.B. (2016). Nutrient Deprivation Affects Salmonella Invasion and Its Interaction with the Gastrointestinal Microbiota. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0159676"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1002\/mbo3.54","article-title":"The global regulator Crc plays a multifaceted role in modulation of type III secretion system in Pseudomonas aeruginosa","volume":"2","author":"Dong","year":"2013","journal-title":"MicrobiologyOpen"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"7075","DOI":"10.1093\/nar\/gkab510","article-title":"Stabilization of Hfq-mediated translational repression by the co-repressor Crc in Pseudomonas aeruginosa","volume":"49","author":"Malecka","year":"2021","journal-title":"Nucleic Acids Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1470","DOI":"10.1093\/nar\/gkx1245","article-title":"Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa","volume":"46","author":"Sonnleitner","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"21866","DOI":"10.1073\/pnas.0910308106","article-title":"Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa","volume":"106","author":"Sonnleitner","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1046\/j.1365-2958.2001.02435.x","article-title":"The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa","volume":"40","author":"Nishijyo","year":"2001","journal-title":"Mol. Microbiol."}],"updated-by":[{"DOI":"10.3390\/microorganisms14071485","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2026,2,2]],"date-time":"2026-02-02T00:00:00Z","timestamp":1769990400000}}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/2\/339\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,7]],"date-time":"2026-07-07T11:22:57Z","timestamp":1783423377000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/2\/339"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,2,2]]},"references-count":45,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["microorganisms14020339"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14020339","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,2,2]]}}}