Evidence for a Putative Regulatory System Consisting of an ECF σE-Type Factor, LIC_12757, and a FecR-like σ Factor Regulator, LIC_12756, in the Pathogenic Spirochaetes Leptospira interrogans
Abstract
1. Introduction
2. Results
2.1. Analysis of LIC_12756 Amino Acid Sequence
2.2. Detection of LIC_12757 and LIC_12756 Interactions—In Vivo BACTH Assay
2.3. Detection of LIC_12757 and LIC_12756 Interactions—Study of LIC_12757–LIC_12756 Interactions In Vitro
2.4. Effect of LIC_12756 on LIC_12757 Activity
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Growth Media, Plasmids, and Genomic DNA
4.2. LIC_12756 Sequence Analyses—Bioinformatic Tools and Analyses
4.3. Plasmid Construction and DNA Manipulations
4.4. Detection of LIC_12757–LIC_12756 Interactions
4.4.1. BACTH Assay—Screening and β-Galactosidase Activity Assays
4.4.2. Affinity Pull-Down Assay and MS Analysis
4.5. In Vivo LIC_12757 Promoter Activity Assay
4.6. SDS-PAGE and Western Blot Analysis
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CDM | N-terminal cytoplasmic domain |
CL | cell lysate |
ECF | extracytoplasmic function |
ECR | C-terminal non-cytoplasmic domain |
RNAP | RNA polymerase core enzyme |
TM helix | transmembrane helical domain |
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E. coli Strain/Plasmid | Genotype | Reference/Source |
---|---|---|
DH5α | supE44, hsdR1, recA1, endA1, gyrA1, gyrA96, thi-1, relA1 | laboratory stock |
DHM1 | F−, cya-854, recA1, endA1, gyrA96 (Nalr), thi1, hsdR17, spoT1, rfbD1, glnV44(AS) | Euromedex (France) |
MG1655 | F−, lambda- ilvG-, rfb-50, rph-1 | laboratory stock |
XL1-Blue | recA1, endA1, gyrA96, thi-1 hsdR17, supE44, relA1, lac [F’proAB lacIq Z∆M15Tn10 (Tetr)] | laboratory stock |
pJET1.2/blunt | PCR cloning vector carrying an ampicillin resistance gene | ThermoFisher Scientific, Poznań, Poland |
pKT25 | pSU40 derivative that encodes the T25 fragment (corresponding to the first 224 amino acid residues of bacterial cyclase adenylate, CyaA), the production of which is controlled by a lac promoter; this plasmid contains a kanamycin resistance selectable marker | Euromedex (France) |
pUT18C | pUC19 derivative that encodes the T18 fragment (corresponding to the amino acid residues 225 to 399 of CyaA), the production of which is controlled by a lac promoter; this plasmid contains an ampicillin resistance selectable marker | Euromedex (France) |
pKT25-zip | pKT25 derivative encoding a fusion protein in which the leucine zipper of GCN4 is fused with the T25 fragment of CyaA; it is used together with pUT18C-zip as a positive control for complementation assay (BACTH assay) | Euromedex (France) |
pUT18C-zip | pUT18C derivative encoding a fusion protein in which the leucine zipper GCN4 is fused with the T18 fragment of CyaA; used in complementation assay (BACTH assay) | Euromedex (France) |
pAC-LIC12757 | pAC7 derivative containing the L. interrogans LIC_12757 gene under the control of a pBAD promoter and a chloramphenicol resistance gene; used for luciferase activity assay | Kędzierska-Mieszkowska et al., 2019 [44] |
prLIC12757luxAB | pGB2 derivative containing the V. harveyi luxAB genes and a promoter region of the L. interrogans LIC_12757 gene (used for luciferase activity assay) | Kędzierska-Mieszkowska et al., 2024 [31] |
pKT25-LIC12757 | pKT25 derivative encoding a fusion protein T25-LIC_12757 (BACTH assay) | this study |
pTU18C-LIC12756 | pUT18C derivative encoding a fusion protein T18-LIC_12756 (BACTH assay) | this study |
Oligonucleotide | Sequence (5′ to 3′) | Purpose |
---|---|---|
pF12757PstI | CTGCAGGGATGGCCAAAATTCCGAAAG | cloning of LIC_12757 into pJET1.2/blunt → pKT25 |
prev12757KpnI | GGTACCCGTATACTCTCAAAGTCGAAATTC | cloning of LIC_12757 into pJET1.2/blunt → pKT25 |
pF12756PstI | CTGCAGGATGGAACCTAATTCAGATTC | cloning of LIC_12756 into pJET1.2/blunt → pUT18C |
prev12756KpnI | GGTACCCGGTTCTTTGGTTGAACAAGTTC | cloning of LIC_12756 into pJET1.2/blunt → pUT18C |
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Kędzierska-Mieszkowska, S.; Kędzierska, B.; Pardyak, L.; Arent, Z. Evidence for a Putative Regulatory System Consisting of an ECF σE-Type Factor, LIC_12757, and a FecR-like σ Factor Regulator, LIC_12756, in the Pathogenic Spirochaetes Leptospira interrogans. Int. J. Mol. Sci. 2025, 26, 4994. https://doi.org/10.3390/ijms26114994
Kędzierska-Mieszkowska S, Kędzierska B, Pardyak L, Arent Z. Evidence for a Putative Regulatory System Consisting of an ECF σE-Type Factor, LIC_12757, and a FecR-like σ Factor Regulator, LIC_12756, in the Pathogenic Spirochaetes Leptospira interrogans. International Journal of Molecular Sciences. 2025; 26(11):4994. https://doi.org/10.3390/ijms26114994
Chicago/Turabian StyleKędzierska-Mieszkowska, Sabina, Barbara Kędzierska, Laura Pardyak, and Zbigniew Arent. 2025. "Evidence for a Putative Regulatory System Consisting of an ECF σE-Type Factor, LIC_12757, and a FecR-like σ Factor Regulator, LIC_12756, in the Pathogenic Spirochaetes Leptospira interrogans" International Journal of Molecular Sciences 26, no. 11: 4994. https://doi.org/10.3390/ijms26114994
APA StyleKędzierska-Mieszkowska, S., Kędzierska, B., Pardyak, L., & Arent, Z. (2025). Evidence for a Putative Regulatory System Consisting of an ECF σE-Type Factor, LIC_12757, and a FecR-like σ Factor Regulator, LIC_12756, in the Pathogenic Spirochaetes Leptospira interrogans. International Journal of Molecular Sciences, 26(11), 4994. https://doi.org/10.3390/ijms26114994