RpoS controls the expression and the transport of the AlgE1-7 epimerases in Azotobacter vinelandii

Abstract

Azotobacter vinelandii produces differentiated cells, called cysts, surrounded by two alginate layers, which are necessary for their desiccation resistance. This alginate contains variable proportions of guluronate residues, resulting from the activity of seven extracytoplasmic epimerases, AlgE1-7. These enzymes are exported by a system secretion encoded by the eexDEF operon; mutants lacking the AlgE1-7 epimerases, the EexDEF or the RpoS sigma factor produce alginate, but are unable to form desiccation resistant cysts. Herein, we found that RpoS was required for full transcription of the algE1-7 and eexDEF genes. We found that the AlgE1-7 protein levels were diminished in the rpoS mutant strain. In addition, the alginate produced in the absence of RpoS was more viscous in the presence of proteases, a phenotype similar to that of the eexD mutant. Primer extension analysis located two promoters for the eexDEF operon, one of them was RpoS-dependent. Thus, during encysting conditions, RpoS coordinates the expression of both the AlgE1-7 epimerases and the EexDEF protein complex responsible for their transport.

Figure 1.

Western blot analysis to detect the AlgE1-7 epimerases. Cell surface protein extracts of the wild-type AEIV (1), AErpoS/pSMrpoS (2), AErpoS (3) and AErpoS/pBBR1MCS-2 (4) strains were used. The proteins were separated on a 4%–12% gradient SDS-PAGE gel. The anti-AlgE4 antibody (Høidal 2000) was used as primary antibody.

Figure 2.

Visualization of the viscosity in the RA1 culture medium after growing the wild-type strain AEIV and its rpoS mutant derivative AErpoS, without (left) or with added proteases (right).

Figure 3.

mRNA relative levels of eexD, algE1 and algE1-6 genes in the wild-type strain (black bars) and in its isogenic mutant AErpoS (grey bars) determined by qRT-PCR. The data are presented as fold changes of mRNA levels of each gene in the rpoS mutant strain relative to that of the parental strain, AEIV. Error bars represent standard deviations of three independent experiments.

Figure 4.

(A) Identification of the transcriptional start sites of eexD by primer extension analysis using total RNA isolated from strain AEIV (lane 1) and from strain AErpoS (lane 2) in vegetative growing conditions. Arrows indicate the start sites of transcription. (B) Sequence of the regulatory region of eexD. The two transcription initiation sites (+1) are indicated along with the -10 and -35 regions of the identified promoters (P1eexD and P2eexD). The ATG translational start codon is show in italics.