A second quorum-sensing system regulates cell surface properties but not phenazine antibiotic production in Pseudomonas aureofaciens.
The root-associated biological control bacterium Pseudomonas aureofaciens 30-84 produces a range of exoproducts, including protease and phenazines. Phenazine antibiotic biosynthesis by phzXYFABCD is regulated in part by the PhzR-PhzI quorum-sensing system. Mutants defective in phzR or phzI produce very low levels of phenazines but wild-type levels of exoprotease. In the present study, a second genomic region of strain 30-84 was identified that, when present in trans, increased beta-galactosidase activity in a genomic phzB::lacZ reporter and partially restored phenazine production to a phzR mutant. Sequence analysis identified two adjacent genes, csaR and csaI, that encode members of the LuxR-LuxI family of regulatory proteins. No putative promoter region is present upstream of the csaI start codon and no lux box-like element was found in either the csaR promoter or the 30-bp intergenic region between csaR and csaI. Both the PhzR-PhzI and CsaR-CsaI systems are regulated by the GacS-GacA two-component regulatory system. In contrast to the multicopy effects of csaR and csaI in trans, a genomic csaR mutant (30-84R2) and a csaI mutant (30-84I2) did not exhibit altered phenazine production in vitro or in situ, indicating that the CsaR-CsaI system is not involved in phenazine regulation in strain 30-84. Both mutants also produced wild-type levels of protease. However, disruption of both csaI and phzI or both csaR and phzR eliminated both phenazine and protease production completely. Thus, the two quorum-sensing systems do not interact for phenazine regulation but do interact for protease regulation. Additionally, the CsaI N-acylhomoserine lactone (AHL) signal was not recognized by the phenazine AHL reporter 30-84I/Z but was recognized by the AHL reporters Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136(pCF240). Inactivation of csaR resulted in a smooth mucoid colony phenotype and formation of cell aggregates in broth, suggesting that CsaR is involved in regulating biosynthesis of cell surface components. Strain 30-84I/I2 exhibited mucoid colony and clumping phenotypes similar to those of 30-84R2. Both phenotypes were reversed by complementation with csaR-csaI or by the addition of the CsaI AHL signal. Both quorum-sensing systems play a role in colonization by strain 30-84. Whereas loss of PhzR resulted in a 6.6-fold decrease in colonization by strain 30-84 on wheat roots in natural soil, a phzR csaR double mutant resulted in a 47-fold decrease. These data suggest that gene(s) regulated by the CsaR-CsaI system also plays a role in the rhizosphere competence of P. aureofaciens 30-84.
Zhang
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Department of Plant Pathology, University of Arizona, Tucson, Arizona 85721, USA.
Pierson
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LS
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020689:Exopeptidases / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D010619:Phenazines / Q000378:metabolism*; D018517:Plant Roots / Q000382:microbiology; D011549:Pseudomonas / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D013499:Surface Properties; D015534:Trans-Activators; D014908:Triticum / Q000382:microbiology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C439292:CsaI protein, Pseudomonas chlororaphis; C439293:CsaR protein, Pseudomonas chlororaphis; D010619:Phenazines; D015534:Trans-Activators; C088386:homoserine lactone; D020689:Exopeptidases; D015107:4-Butyrolactone