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Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density.

ABSTRACT: We have identified a gene that acts in trans to activate the expression of the phenazine biosynthetic genes in the biological control organism Pseudomonas aureofaciens 30-84. This gene, phzR (phenazine regulator), is located upstream of and divergently transcribed from the phenazine biosynthetic genes. Thus, the phenazine biosynthetic locus consists of at least two divergently transcribed operons. A functional phzR gene is required for phenazine production. The nucleotide sequence of phzR revealed an open reading frame of 723 nucleotides encoding a protein of ca. 27 kDa. The predicted amino acid sequence of PhzR has homology with other bacterial positive transcriptional activators, including LasR of Pseudomonas aeruginosa, LuxR of Vibrio fischerii, and TraR of Agrobacterium tumefaciens. The addition of cell-free supernatants from late-exponential-phase cultures of strain 30-84 resulted in expression of a genomic phzB:lacZ reporter strain at a lower cell density than normal, indicating the possible presence of an autoinducer. These results indicate that PhzR is a member of a two-component sensor-regulator family with known or predicted carboxy-terminal DNA-binding domains which regulates gene expression in response to environmental and cell density signals.

SUBMITTER: Pierson LS

PROVIDER: S-EPMC205594 | biostudies-other | 1994 Jul

REPOSITORIES: biostudies-other

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Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density.

Pierson L S LS Keppenne V D VD Wood D W DW

Journal of bacteriology 19940701 13

We have identified a gene that acts in trans to activate the expression of the phenazine biosynthetic genes in the biological control organism Pseudomonas aureofaciens 30-84. This gene, phzR (phenazine regulator), is located upstream of and divergently transcribed from the phenazine biosynthetic genes. Thus, the phenazine biosynthetic locus consists of at least two divergently transcribed operons. A functional phzR gene is required for phenazine production. The nucleotide sequence of phzR reveal ...[more]

PMID: 8021179

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Project description:The GacS/GacA two-component regulatory system activates the production of secondary metabolites including phenazines crucial for biological control activity in Pseudomonas chlororaphis 30-84. To better understand the role of the Gac system on phenazine regulation, transcriptomic analyses were conducted by comparing the wild-type strain to a gacA mutant. RNA-seq analysis identified 771 genes under GacA control, including many novel genes. Consistent with previous findings, phenazine biosynthetic genes were significantly downregulated in a gacA mutant. The transcript abundances of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS, and pip also were reduced. Moreover, the transcript abundance of three noncoding RNAs (ncRNAs) including rsmX, rsmY, and rsmZ was significantly decreased by gacA mutation consistent with the presence of consensus GacA-binding sites associated with their promoters. Our results also demonstrated that constitutive expression of rsmZ from a non-gac regulated promoter resulted in complete restoration of N-acyl-homoserine lactone (AHL) and phenazine production as well as the expression of other gac-dependent secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production also was investigated. Overexpression of rsmE, but not rsmA, resulted in decreased AHL and phenazine production in P. chlororaphis, and only a mutation in rsmE bypassed the requirement for GacA in phenazine gene expression. In contrast, constitutive expression of the phzI/phzR quorum sensing system did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac on the phenazine biosynthetic genes. On the basis of these results, we propose a model to illustrate the hierarchic role of phenazine regulators modulated by Gac in the control of phenazine production. The transcriptomic analysis also was used to identify additional genes regulated by GacA that may contribute to the biological control capability of strain 30-84.

Project description:Phenazines are bacterial secondary metabolites and play important roles in the antagonistic activity of the biological control strain P. chlororaphis 30-84 against take-all disease of wheat. The expression of the P. chlororaphis 30-84 phenazine biosynthetic operon (phzXYFABCD) is dependent on the PhzR/PhzI quorum sensing system located immediately upstream of the biosynthetic operon as well as other regulatory systems including Gac/Rsm. Bioinformatic analysis of the sequence between the divergently oriented phzR and phzX promoters identified features within the 5'-untranslated region (5'-UTR) of phzX that are conserved only among 2OHPCA producing Pseudomonas. The conserved sequence features are potentially capable of producing secondary structures that negatively modulate one or both promoters. Transcriptional and translational fusion assays revealed that deletion of 90-bp of sequence at the 5'-UTR of phzX led to up to 4-fold greater expression of the reporters with the deletion compared to the controls, which indicated this sequence negatively modulates phenazine gene expression both transcriptionally and translationally. This 90-bp sequence was deleted from the P. chlororaphis 30-84 chromosome, resulting in 30-84Enh, which produces significantly more phenazine than the wild-type while retaining quorum sensing control. The transcriptional expression of phzR/phzI and amount of AHL signal produced by 30-84Enh also were significantly greater than for the wild-type, suggesting this 90-bp sequence also negatively affects expression of the quorum sensing genes. In addition, deletion of the 90-bp partially relieved RsmE-mediated translational repression, indicating a role for Gac/RsmE interaction. Compared to the wild-type, enhanced phenazine production by 30-84Enh resulted in improvement in fungal inhibition, biofilm formation, extracellular DNA release and suppression of take-all disease of wheat in soil without negative consequences on growth or rhizosphere persistence. This work provides greater insight into the regulation of phenazine biosynthesis with potential applications for improved biological control.

Dataset Information

Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density.

Publications

Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density.

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