Two Pathway-Specific Transcriptional Regulators, PltR and PltZ, Coordinate Autoinduction of Pyoluteorin in Pseudomonas protegens Pf-5
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Cultural Conditions
2.2. Construction of GFP Transcriptional Reporters and Expression Constructs
2.3. Construction of Pf-5 Mutants
2.4. Assays for Monitoring GFP Activity of Reporter Constructs
2.5. Protein Purification and In Vitro Activity Assays
3. Results
3.1. PltR Is Required But Not Sufficient for the Autoinduction of Pyoluteorin
3.2. PltZ Is an Autorepressor and Directly Represses Expression of Linked Transporter Genes
3.3. Pyoluteorin Interacts with PltZ and Relieves PltZ-Mediated Transcriptional Repression of pltIJKNOP Transporter Genes
3.4. PltZ Is Involved in the Autoinduction of Pyoluteorin
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Strains, Plasmids, or Primers | Genotype, Relevant Characteristics or Sequences | Reference or Source |
---|---|---|
Strains # | ||
P. protegens | ||
LK099 | Wild-type strain Pf-5. | [26] |
JL4805 | Pf-5 derivative strain contains an in-frame deletion of pltA in the chromosome. | [27] |
LK530 | Pf-5 derivative strain contains an in-frame deletion of pltA and pltR in the chromosome. This mutant was generated by deleting pltR in JL4805. | This study |
LK419 | Pf-5 derivative strain contains an in-frame deletion of pltA and pltZ in the chromosome. This mutant was generated by deleting pltZ in JL4805. | This study |
P. fluorescens SBW25 | SBW25 is a member of the P. fluorescens group that does not contain plt gene cluster. | [28] |
E. coli | ||
S17-1 | recA pro hsdR−M+ RP4 2-Tc::Mu-Km::Tn7 Smr Tpr | [29] |
BL21 (DE3) | ompT hsdSB (rB−mB−) gal dcm | NEB |
Plasmids | ||
pEX18Tc | Gene replacement vector with MCS from pUC18, sacB+ Tcr | [30] |
pEX18Tc-ΔpltR | pEX18Tc containing pltR with a 925-bp in-frame deletion. | [24] |
pEX18Tc-ΔpltZ | pEX18Tc containing pltZ with a 621-bp in-frame deletion. | This study |
pPROBE-NT | pBBR1, containing a promoterless gfp, Kmr | [31] |
pL-gfp | Called ppltL-gfp previously. Contains the intergenic region between pltR and pltL including pltL promoter fused with a promoterless gfp. | [32] |
pRL-gfp | Contains pltR and the intergenic region between pltR and pltL including pltL promoter fused with a promoterless gfp. | [24] |
pI-gfp | Contains the intergenic region between pltI and pltZ including pltI promoter fused with a promoterless gfp. | This study |
pZ-gfp | Contains the intergenic region between pltI and pltZ including pltZ promoter fused with a promoterless gfp. | This study |
pME6010 | pACYC177-pVS1 shuttle vector, Tcr | [33] |
PME6010-pltZ | pME6010 with a 697-bp XhoI-KpnI PCR fragment amplified from the genomic DNA of Pf-5, containing a constitutively expressed pltZ gene. | This study |
Primers * | oligonucleotide sequences (5′to 3′) | |
pltZ-F2 | ACTCGAGAAAATAACAGATACTGGCCATG | |
pltZ-R2 | ATAGGTACCGACTATTGGGCAATGGC | |
pltI-F1 | ATAGGATCCAGCAGACGGAATTTGGGC | |
pltI-R1 | TATGGTACCCCTACAATCAACTGCTTCTTC | |
pltI-F4 | TGGCGAATGTCGACGTTGC | |
pltI-R4 | TATGAATTCCTCGTGGTCTGAGCGG | |
pltZ 5′primer | GTCATACATATGAAGCAACCCCCCGCTC | |
pltZ 3′primer | ATGATCTCGAGCCGACTATTGGGCAATG | |
pltZ UpF-Xba | CTCCTCTCTAGATCAAAACGTGCCTTGGACTG | |
pltZ UpR | ACTATTGGGCAATGATGTTCCTCGTGGTCTGAG | |
pltZ DnF | ACGAGGAACATCATTGCCCAATAGTCGGCTCA | |
pltZ DnR-Xba | CACACCTCTAGAACATCCCCTGCTGTTTCTTC |
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Yan, Q.; Liu, M.; Kidarsa, T.; Johnson, C.P.; Loper, J.E. Two Pathway-Specific Transcriptional Regulators, PltR and PltZ, Coordinate Autoinduction of Pyoluteorin in Pseudomonas protegens Pf-5. Microorganisms 2021, 9, 1489. https://doi.org/10.3390/microorganisms9071489
Yan Q, Liu M, Kidarsa T, Johnson CP, Loper JE. Two Pathway-Specific Transcriptional Regulators, PltR and PltZ, Coordinate Autoinduction of Pyoluteorin in Pseudomonas protegens Pf-5. Microorganisms. 2021; 9(7):1489. https://doi.org/10.3390/microorganisms9071489
Chicago/Turabian StyleYan, Qing, Mary Liu, Teresa Kidarsa, Colin P. Johnson, and Joyce E. Loper. 2021. "Two Pathway-Specific Transcriptional Regulators, PltR and PltZ, Coordinate Autoinduction of Pyoluteorin in Pseudomonas protegens Pf-5" Microorganisms 9, no. 7: 1489. https://doi.org/10.3390/microorganisms9071489