phz1 contributes much more to phenazine-1-carboxylic acid biosynthesis than phz2 in Pseudomonas aeruginosa rpoS mutant.

Pseudomonas aeruginosa PAO1, a common opportunistic bacterial pathogen, contains two phenazine-biosynthetic operons, phz1 (phzA1 B1 C1 D1 E1 F1 G1 ) and phz2 (phzA2 B2 C2 D2 E2 F2 G2 ). Each of two operons can independently encode a set of enzymes involving in the biosynthesis of phenazine-1-carboxylic acid. As a global transcriptional regulator, RpoS mediates a lot of genes involving secondary metabolites biosynthesis in many bacteria. In an other previous study, it was reported that RpoS deficiency caused overproduction of pyocyanin, a derivative of phenazine-1-carboxylic acid in P. aeruginosa PAO1. But it is not known how RpoS mediates the expression of each of two phz operons and modulates phenazine-1-carboxylic acid biosynthesis in detail. In this study, by deleting the rpoS gene in the mutant PNΔphz1 and the mutant PNΔphz2, we found that the phz1 operon contributes much more to phenazine-1-carboxylic acid biosynthesis than the phz2 operon in the absence of RpoS. With the construction of the translational and transcriptional fusion vectors with the truncated lacZ reporter gene, we demonstrated that RpoS negatively regulates the expression of phz1 and positively controls the expression of phz2, and the regulation of phenazine-1-carboxylic acid biosynthesis mediated by RopS occurs at the posttranscriptional level, not at the transcriptional level. Obviously, two copies of phz operons and their differential expression mediated by RpoS might help P. aeruginosa adapt to its diverse environments and establish infection in its hosts.

[Positive regulation in expression of the phenazine-producing operon phz2 mediated by pip in Pseudomonas aeruginosa PAO1].

UNLABELLED: Pseudomonas aeruginosa PAO1, an opportunistic pathogenic bacterium, produces phenazine and its derivatives which play a critical role in pathogen-host interaction during its infection. In a biological control strain P. chlororaphis PCL1391, Pip positively regulates PCN production. OBJECTIVE: Our aim is to identify the function and regulation of an ORF of PA0243 (the homolog of Pip) in Pseudomonas aeruginosa PAO1. METHODS: We first cloned the fragment of the pip gene from the chromosomal DNA of P. aeruginosa PAO1 and constructed the pip-defect mutant PA-PG with the insertion of gentamycin resistance cassette (aacC1). With construction and introduction of pME10P (containing the whole pip gene region) , complementation of the pip was then carried out. With creation of the mutants PA-PD-Z1G and PA-PG-Z2K, phenazine-1-carboxylic acid and pyocyanin were measured in GA medium in relative mutants, respectively. RESULTS: In GA medium, production of phenazine-1-carboxylic acid and pyocyanin in the mutant PA-PG decreased dramatically in comparison with that produced in the wild type strain PAO1. The amounts of phenazine-1-carboxylic acid and pyocyanin, however, were recovered with complementation of the derivative PA-PG bearing pME10P. The production of phenazine-1-carboxylic acid and pyocyanin in mutant PA-PG-Z2K were same to those in parental strain PA-Z2K. Phenazine-1-carboxylic acid and pyocyanin produced by the mutant PA-PD-Z1G were lower than those in the original strain PA-Z1G. CONCLUSION: With these results, it is suggested that Pip exerts positively regulation in phenazine biosynthesis by specifically modulating expression of the phz2 operon, not by mediating expression of the phzl operon in P. aeruginosa PAO1.

vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05.

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant G05ΔphzΔprn::lacZ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant G05Δvfr and G05ΔphzΔprn::lacZΔvfr. By quantifying ß-galactosidase activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant G05Δvfr, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.