The Azotobacter vinelandii AlgU regulon during vegetative growth and encysting conditions: A proteomic approach

Abstract

In the Pseduomonadacea family, the extracytoplasmic function sigma factor AlgU is crucial to withstand adverse conditions. Azotobacter vinelandii, a closed relative of Pseudomonas aeruginosa, has been a model for cellular differentiation in Gram-negative bacteria since it forms desiccation-resistant cysts. Previous work demonstrated the essential role of AlgU to withstand oxidative stress and on A. vinelandii differentiation, particularly for the positive control of alginate production. In this study, the AlgU regulon was dissected by a proteomic approach under vegetative growing conditions and upon encystment induction. Our results revealed several molecular targets that explained the requirement of this sigma factor during oxidative stress and extended its role in alginate production. Furthermore, we demonstrate that AlgU was necessary to produce alkyl resorcinols, a type of aromatic lipids that conform the cell membrane of the differentiated cell. AlgU was also found to positively regulate stress resistance proteins such as OsmC, LEA-1, or proteins involved in trehalose synthesis. A position-specific scoring-matrix (PSSM) was generated based on the consensus sequence recognized by AlgU in P. aeruginosa, which allowed the identification of direct AlgU targets in the A. vinelandii genome. This work further expands our knowledge about the function of the ECF sigma factor AlgU in A. vinelandii and contributes to explains its key regulatory role under adverse conditions.

Fig 1. Activity of the AlgU sigma factor in A. vinelandii.

A transcriptional PalgC-gusA fusion was used to estimate the activity of AlgU in the background of the wild-type strain AEIV (solid line) or in the algU mutant (dotted line). Cells were cultured in minimum Burk’s medium supplemented with sucrose (vegetative growth) or n-butanol (encystment-inducing conditions) as the sole carbon source for the indicated time. The bars of standard deviation from three independent experiments are shown.

Fig 2. KEGG pathway enrichment of proteins of the AlgU regulon in A. vinelandii.

The analysis was conducted for the differentially expressed proteins in the absence of the sigma factor AlgU under vegetative (A) or encystment-induced (B) conditions. Positive and negative axes represent the numbers of up- or downregulated proteins, respectively.

Fig 3. The production of ARs is impaired in the absence of AlgU.

A. Staining with Fast Blue B of ARs produced by strain AEIV (WT) and by its derivative algU mutant under encysting conditions. Cells were grown on Burk’s-butanol for 48 h before staining. B. ARs quantification under vegetative growth. The wild-type strains AEIV, ATCC 9046 and ATCC 12518, and the AEIV derivative mutant algU were cultivated in Burk’s-sucrose medium for 24 h prior to ARs extraction. The bars of standard deviation from three independent experiments are shown. The asterisks denote statistical significance (unpaired Student’s t-test, *P<0.05; **P<0.01) when compared to the wild-type strain AEIV.

Fig 4. Sequence logo for AlgU DNA binding motifs.

A PSSM was used to identify AlgU binding motifs in the genome of P. aeruginosa (A) or A. vinelandii (B) (see Materials and Methods section for details). The predicted AlgU binding motifs are shown with 16 (upper panels) or 17 (lower panels) bp spacers, between the -10 and -35 boxes. The relative sizes of the letters represent their frequency in the sequences.