The unphosphorylated EIIA(Ntr) protein represses the synthesis of alkylresorcinols in Azotobacter vinelandii

Abstract

Upon encystment induction, Azotobacter vinelandii produces the phenolic lipids alkylresorcinols (ARs) that are structural components of the cysts. The enzymes responsible for the ARs synthesis are encoded in the arsABCD operon, whose expression is activated by ArpR. The transcription of arpR is initiated from an RpoS dependent promoter. The nitrogen-related phosphotransferase system (PTS(Ntr)) is a global regulatory system present in Gram negative bacteria. It comprises the EI(Ntr), NPr and EIIA(Ntr) proteins encoded by ptsP, ptsO and ptsN genes respectively. These proteins participate in a phosphoryl-group transfer from phosphoenolpyruvate to protein EIIA(Ntr) via the phosphotransferases EI(Ntr) and NPr. In A. vinelandii, the non-phosphorylated form of EIIA(Ntr) was previously shown to repress the synthesis of poly-ß-hydroxybutyrate. In this work, we show that PTS(Ntr) also regulates the synthesis of ARs. In a strain that carries unphosphorylated EIIA(Ntr), the expression of arpR was reduced, while synthesis of ARs and transcription of arsA were almost abrogated. The expression of arpR from an RpoS-independent promoter in this strain restored the ARs synthesis. Taken together these results indicate that unphosphorylated EIIA(Ntr) negatively affects activation of arpR transcription by RpoS.

Fig 1. PTS^Ntr controls the ARs synthesis.

Staining (A) and quantification (B) of alkylresorcinols produced by strains of A. vinelandii in BBOH medium to 120 hours of incubation. These data are mean of three independent experiments, error bars, SD.

Fig 2. Effect of PTS^Ntr on arsA expression.

β-glucuronidase activity in UW136 wild type strain and pts mutants carrying transcriptional arsA-gusA (A), or translational arsA´-´gusA (B) gene fusions. The cells were grown for 72 h in BBOH medium at 30°C. The data represent the mean of three independent experiments. Error bars, SD.

Fig 3. Effect of PTS^Ntr on arpR expression.

β-glucuronidase activity in UW136 wild type strain and pts mutants carrying transcriptional arpR-gusA (A) or translational arpR´-´gusA (B) gene fusions. The cells were grown for 72 h in BBOH medium at 30°C. The data represent the mean of three independent experiments. Error bars, SD.

Fig 4. The unphosphorylated EIIA^Ntr negatively affects the ARs synthesis.

Effect of H68A mutation on EIIA^Ntr on ARs production (A), and activity of transcriptional arsA-gusA and arpR-gusA fusions (B). The strains UW136::pALA7 (black bars) and UW136::pALA8a (white bars) carry an EIIA^Ntr and H68A EIIA^Ntr, respectively. The cells were grown for 120 h for (A) and 72 h for (B) in BBOH medium at 30°C. The data represent the mean of three independent experiments. Error bars, SD.

Fig 5. Effect of arpR expression from RpoS-independent promoter in the strain that carries the nonphosphorylatable EIIA^Ntr H68A protein.

(A) Staining of ARs produced by UW136 and U136::pALA8a strains, transformed with plasmid PBpgyrA-arpR, carrying a constitutively expressed arpR gene or the empty plasmid pBBR1MCS-5 as negative control. (B) Quantification of ARs levels produced by the strains of the panel A. The data represent the mean of three independent experiments. Error bars, SD.

Fig 6. Model for the control of ARs synthesis by PTS^Ntr in A. vinelandii.

The EIIA^Ntr protein in its unphosphorylated form represses the arpR expression both transcriptional (RpoS activity) and posttranscriptional levels. The dashed lines and gray arrows indicate negative effect and activation, respectively. PEP: Phosphoenolpyruvate, P: Phosphoryl group.