H-NS family protein MvaU downregulates phenazine-1-carboxylic acid (PCA) biosynthesis via binding to an AT-rich region within the promoter of the phz2 gene cluster in the rhizobacterium Pseudomonas strain PA1201

Abstract

Histone-like nucleoid-structuring (H-NS) proteins are key regulators in gene expression silencing and in nucleoid compaction. The H-NS family member proteins MvaU in Pseudomonas aeruginosa are thought to bind the same AT-rich regions of chromosomes and function to coordinate the control of a common set of genes. Here, we explored the molecular mechanism by which MvaU controls PCA biosynthesis in P. aeruginosa PA1201. We present evidence suggesting that MvaU is self-regulated. Deletion of mvaU significantly increased PCA production, and PCA production sharply decreased when mvaU was over-expressed. MvaU transcriptionally repressed phz2 cluster expression and consequently reduced PCA biosynthesis. β-galactosidase assays confirmed that base pairing near the −35 box is required when MvaU regulates PCA production in PA1201. Electrophoretic mobility shift assays (EMSA) and additional point mutation analysis demonstrated that MvaU directly bound to an AT-rich motif within the promoter of the phz2 cluster. Chromatin immunoprecipitation (ChIP) analysis also indicated that MvaU directly bound to the P5 region of the phz2 cluster promoter. MvaU repression of PCA biosynthesis was independent of QscR and OxyR in PA1201 and neither PCA or H 2 O 2 were the environmental signals that induced mvaU expression. These findings detail a new MvaU-dependent regulatory pathway of PCA biosynthesis in PA1201 and provide a foundation to increase PCA fermentation titer by genetic engineering.

Unraveling the regulation of pyocyanin synthesis by RsmA through MvaU and RpoS in Pseudomonas aeruginosa ID4365

“…This is not the case for the phz2 operon in the ID4365 strain, since transcription is affected when rsmA is inactivated, suggesting that this regulation is indirect. In recent work, it was found that MvaU represses the transcription of the phz2 operon by binding to the phzA2 promoter [19]; thus, we explored the possibility that the regulation of the phz2 operon by RsmA in ID4365 strain was through MvaU. We found that the expression of mvaU increases in the IDrsmA strain compared with the wild‐type strain.…”

“…It has been reported that RsaL activates the transcription of the phz2 operon; however, previously we determined that the ID4365 strain is a natural lasR mutant, and therefore, rsaL is not expressed in this strain [33]. Recently, it was described that MvaU, a histone‐like protein, is able to bind to the phz2 promoter and negatively control its transcription [19]. Therefore, we searched for the binding site for MvaU in the promoter of phzA2 in the ID4365 strain.…”

“…Pyocyanin is naturally produced by P. aeruginosa and its synthesis and regulation have been widely studied in the PAO1 and PA14 strains, two clinical isolates [3,11,12,17]. Regulation of pyocyanin production involves different systems and regulators, including the three quorum-sensing systems Las, Rhl, and Pqs, the regulators RsaL, MvaU, the alternative sigma factor RpoS and the posttranscriptional regulatory system Rsm [10,16,19,20,30].…”

“…The Pqs system also regulates pyocyanin production but particularly through PqsE, which acts along with RhlR to activate pyocyanin genes [13][14][15]. On the other hand, negative regulators of pyocyanin synthesis include RsaL, RpoS, and MvaU [16][17][18][19]. The first one acts as a negative regulator on the expression of the phz1 operon binding directly to the promoter of phzA1, but it indirectly activates the expression of the phz2 operon [17,20].…”

“…Inactivation of mvaU increases PCA synthesis, the precursor of pyocyanin. It was demonstrated that this regulator can bind to the promoter of phz2 operon negatively regulating its expression [19].…”