{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,2]],"date-time":"2026-04-02T18:54:16Z","timestamp":1775156056951,"version":"3.50.1"},"reference-count":35,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2019,12,15]],"date-time":"2019-12-15T00:00:00Z","timestamp":1576368000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"undefined  &lt;span style=&quot;color:gray;font-size:10px;&quot;&gt;undefined&lt;\/span&gt;","award":["DS-530\/L136-D241-18-1A"],"award-info":[{"award-number":["DS-530\/L136-D241-18-1A"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>There is limited information on gene expression in the pathogenic spirochaete Leptospira interrogans and genetic mechanisms controlling its virulence. Transcription is the first step in gene expression that is often determined by environmental effects, including infection-induced stresses. Alterations in the environment result in significant changes in the transcription of many genes, allowing effective adaptation of Leptospira to mammalian hosts. Thus, promoter and transcriptional start site identification are crucial for determining gene expression regulation and for the understanding of genetic regulatory mechanisms existing in Leptospira. Here, we characterized the promoter region of the L. interrogans clpB gene (clpBLi) encoding an AAA+ molecular chaperone ClpB essential for the survival of this spirochaete under thermal and oxidative stresses, and also during infection of the host. Primer extension analysis demonstrated that transcription of clpB in L. interrogans initiates at a cytidine located 41 bp upstream of the ATG initiation codon, and, to a lesser extent, at an adenine located 2 bp downstream of the identified site. Transcription of both transcripts was heat-inducible. Determination of clpBLi transcription start site, combined with promoter transcriptional activity assays using a modified two-plasmid system in E. coli, revealed that clpBLi transcription is controlled by the ECF \u03c3E factor. Of the ten L. interrogans ECF \u03c3 factors, the factor encoded by LIC_12757 (LA0876) is most likely to be the key regulator of clpB gene expression in Leptospira cells, especially under thermal stress. Furthermore, clpB expression may be mediated by ppGpp in Leptospira.<\/jats:p>","DOI":"10.3390\/ijms20246325","type":"journal-article","created":{"date-parts":[[2019,12,16]],"date-time":"2019-12-16T05:19:38Z","timestamp":1576473578000},"page":"6325","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Identification of \u03c3E-Dependent Promoter Upstream of clpB from the Pathogenic Spirochaete Leptospira interrogans by Applying an E. coli Two-Plasmid System"],"prefix":"10.3390","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5756-3013","authenticated-orcid":false,"given":"Sabina","family":"K\u0119dzierska-Mieszkowska","sequence":"first","affiliation":[{"name":"Department of General and Medical Biochemistry, University of Gda\u0144sk, Faculty of Biology, 80-308 Gda\u0144sk, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4902-3348","authenticated-orcid":false,"given":"Katarzyna","family":"Potrykus","sequence":"additional","affiliation":[{"name":"Department of Bacterial Molecular Genetics, Faculty of Biology, University of Gda\u0144sk, 80-308 Gda\u0144sk, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5997-2555","authenticated-orcid":false,"given":"Zbigniew","family":"Arent","sequence":"additional","affiliation":[{"name":"University Centre of Veterinary Medicine, University of Agriculture in Krakow, 30-059 Krakow, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joanna","family":"Krajewska","sequence":"additional","affiliation":[{"name":"Department of General and Medical Biochemistry, University of Gda\u0144sk, Faculty of Biology, 80-308 Gda\u0144sk, Poland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,12,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Costa, F., Hagan, J.E., Calcagno, J., Kane, M., Torgerson, P., Martinez-Silveira, M.S., Stein, C., Abela-Ridder, B., and Ko, A.I. (2015). Global morbidity and mortality of leptospirosis: A systematic review. PLoS Negl. Trop. Dis., 9.","DOI":"10.1371\/journal.pntd.0003898"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/2046-0481-65-8","article-title":"Seroprevalence of Leptospira Hardjo in the irish suckler cattle population","volume":"65","author":"Ryan","year":"2012","journal-title":"Ir. Vet. J."},{"key":"ref_3","first-page":"369","article-title":"New data concerning diagnosis and control of swine leptospirosis","volume":"87","author":"Pejsak","year":"2012","journal-title":"\u017bycie Weterynaryjne"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1136\/vr.101239","article-title":"Seroprevalence study of leptospirosis in horses in northern Poland","volume":"172","author":"Arent","year":"2013","journal-title":"Vet. Rec."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1148","DOI":"10.1017\/S0950268812002087","article-title":"Emergence of novel Leptospira serovars: A need for adjusting vaccination policies for dogs?","volume":"141","author":"Arent","year":"2013","journal-title":"Epidemiol. Infect."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"582","DOI":"10.1136\/vr.101969","article-title":"Isolation of leptospires from genital tract of sheep","volume":"173","author":"Arent","year":"2013","journal-title":"Vet. Rec."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1016\/j.vetmic.2014.06.015","article-title":"Pathogenesis of leptospirosis: Cellular and molecular aspects","volume":"172","author":"Adler","year":"2014","journal-title":"Vet. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1434","DOI":"10.1038\/s41598-018-38329-1","article-title":"Inferring pathogen-host interactions between Leptospira interrogans and Homo sapiens using network theory","volume":"9","author":"Kumar","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Fouts, D.E., Matthias, M.A., Adhikarla, H., Adler, B., Amorim-Santos, L., Berg, D.E., Bulach, D., Buschiazzo, A., Chang, Y.-F., and Galloway, R.L. (2016). What makes a bacterial species pathogenic?: Comparative genomic analysis of the genus Leptospira. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004403"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fcimb.2017.00010","article-title":"Genome-wide transcriptional start site mapping and sRNA identification in the pathogen Leptospira interrogans","volume":"7","author":"Zhukova","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2133","DOI":"10.1093\/nar\/gky010","article-title":"A sigma factor toolbox for orthogonal gene expression in Escherichia coli","volume":"46","author":"Bervoets","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1590\/S0100-879X2004000400003","article-title":"Genome features of Leptospira interrogans serovar Copenhageni","volume":"37","author":"Nascimento","year":"2004","journal-title":"Braz. J. Med. Biol. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5848","DOI":"10.1128\/IAI.00755-06","article-title":"Effects of temperature on gene expression patterns in Leptospira interrogans serowar Lai as assessed by whole-genome microarrays","volume":"74","author":"Lo","year":"2006","journal-title":"Infect. Immun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/S0065-2911(02)46002-X","article-title":"The extracytoplasmic function (ECF) sigma factors","volume":"46","author":"Helmann","year":"2002","journal-title":"Adv. Microb. Physiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3711","DOI":"10.1128\/IAI.05168-11","article-title":"Inactivation of clpB in the pathogen Leptospira interrogans reduces virulence and resistance to stress conditions","volume":"79","author":"Lourdault","year":"2011","journal-title":"Infect. Immun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1186\/s12866-016-0774-8","article-title":"Immunoreactivity of the AAA+ chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals","volume":"16","author":"Krajewska","year":"2016","journal-title":"BMC Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Krajewska, J., Arent, Z., Zolkiewski, M., and K\u0119dzierska-Mieszkowska, S. (2018). Isolation and identification of putative protein substrate of the AAA+ chaperone chaperone ClpB from pathogenic spirochaete Leptospira interrogans. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19041234"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4254","DOI":"10.1128\/jb.173.14.4254-4262.1991","article-title":"ClpB is the Escherichia coli heat shock protein F84.1","volume":"173","author":"Squires","year":"1991","journal-title":"J. Bacteriol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4488","DOI":"10.1093\/nar\/28.22.4488","article-title":"Transcription in Mycoplasma pneumonia","volume":"28","author":"Weiner","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"20170","DOI":"10.1016\/S0021-9258(20)80709-1","article-title":"Site-directed mutagenesis of the dual translational initiation sites of the clpB gene of Escherichia coli and characterization of its gene products","volume":"268","author":"Park","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1016\/j.jmb.2009.11.059","article-title":"Synergistic cooperation between two ClpB isoforms in aggregate reactivation","volume":"396","author":"Nagy","year":"2010","journal-title":"J. Mol. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e02068-1","DOI":"10.1128\/AEM.02068-18","article-title":"Cis-Acting determinant limiting expression of sphingomyelinase gene sph2 in Leptospira interrogans, identified with a gfp reporter plasmid","volume":"84","author":"Matsunaga","year":"2018","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/S0167-7012(01)00237-8","article-title":"A two-plasmid system for identification of promoters recognized by RNA polymerase containing extracytoplasmic stress response \u03c3E in Escherichia coli","volume":"45","author":"Rezuchova","year":"2001","journal-title":"J. Microbiol. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0378-1097(03)00480-4","article-title":"New members of the Escherichia coli \u03c3E regulon identified by a two-plasmid system","volume":"225","author":"Rezuchova","year":"2003","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Krajewska, J., Modrak-W\u00f3jcik, A., Arent, Z., Wi\u0119ckowski, D., Zolkiewski, M., Bzowska, A., and K\u0119dzierska-Mieszkowska, S. (2017). Characterization of the molecular chaperone ClpB from the pathogenic spirochaete Leptospira interrogans. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0181118"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1128\/MMBR.69.4.527-543.2005","article-title":"Alternative sigma factors and their roles in bacterial virulence","volume":"69","author":"Kazmierczak","year":"2005","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"4627","DOI":"10.1128\/JB.01981-05","article-title":"Growth phase-Dependent regulation of the extracytoplasmatic stress factor, \u03c3E, by guanosine 3\u2019, 5\u2019-bispyrophosphate (ppGpp)","volume":"188","author":"Costanzo","year":"2016","journal-title":"J. Bacteriol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1111\/j.1365-2958.2007.06072.x","article-title":"ppGpp and DskA likely regulate the activity of the extracytoplasmatic stress factor \u03c3E in Escherichia coli by both direct and indirect mechanisms","volume":"67","author":"Costanzo","year":"2008","journal-title":"Mol. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"7279","DOI":"10.1128\/JB.01047-09","article-title":"Promoter strength properties of the complete sigma E regulon of Escherichia coli and Salmonella enterica","volume":"191","author":"Mutalik","year":"2009","journal-title":"J. Bacteriol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1146\/annurev.micro.62.081307.162903","article-title":"(p)ppGpp: Still magical?","volume":"62","author":"Potrykus","year":"2008","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1128\/MMBR.00046-09","article-title":"ppGpp conjures bacterial virulence","volume":"74","author":"Dalebroux","year":"2010","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1111\/1574-6968.12279","article-title":"Characterization of a bifunctional enzyme with (p)ppGpp-hydrolase\/synthase activity in Leptospira interrogans","volume":"348","author":"He","year":"2013","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1128\/JB.06238-11","article-title":"Effects on growth by changes of the balance between GreA, GreB, and DksA suggest mutual competition and functional redundancy in Escherichia coli","volume":"194","author":"Vinella","year":"2012","journal-title":"J. Bacteriol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1016\/0378-1119(84)90207-5","article-title":"A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors","volume":"31","author":"Churchward","year":"1984","journal-title":"Gene"},{"key":"ref_35","unstructured":"Harlow, E., and Lane, D. (1988). Antibodies: A Laboratory Manual, CSH."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/24\/6325\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:42:32Z","timestamp":1760190152000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/20\/24\/6325"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,12,15]]},"references-count":35,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2019,12]]}},"alternative-id":["ijms20246325"],"URL":"https:\/\/doi.org\/10.3390\/ijms20246325","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,12,15]]}}}