The global regulators GacA and sigma(S) form part of a cascade that controls alginate production in Azotobacter vinelandii

Abstract

Transcription of the Azotobacter vinelandii algD gene, which encodes GDP-mannose dehydrogenase (the rate-limiting enzyme of alginate synthesis), starts from three sites: p1, p2, and p3. The sensor kinase GacS, a member of the two-component regulatory system, is required for transcription of algD from its three sites during the stationary phase. Here we show that algD is expressed constitutively throughout the growth cycle from the p2 and p3 sites and that transcription from p1 started at the transition between the exponential growth phase and stationary phase. We constructed A. vinelandii strains that carried mutations in gacA encoding the cognate response regulator of GacS and in rpoS coding for the stationary-phase sigma(S) factor. The gacA mutation impaired alginate production and transcription of algD from its three promoters. Transcription of rpoS was also abolished by the gacA mutation. The rpoS mutation impaired transcription of algD from the p1 promoter and increased it from the p2 sigma(E) promoter. The results of this study provide evidence for the predominant role of GacA in a regulatory cascade controlling alginate production and gene expression during the stationary phase in A. vinelandii.

FIG. 1

(A) Physical map of the A. vinelandii chromosomal gacA-uvr region and plasmids constructed in this study. Arrows indicate direction of transcription. Antibiotic resistance cassette is represented by the inverted triangle. Vector sequences are represented by black bars. (B) Physical map of insertional inactivation of the gacA gene in A. vinelandii ATCC 9046. Southern blot hybridization of total genomic DNA digested with SalI endonuclease, with the 0.8-kb SalI fragment as probe. Lane 1, ATCC 9046; lane 2, JM3. Abbreviations: S, SalI; C, ClaI; St, StuI.

FIG. 2

Growth and primer extension analysis of algD. (A) Growth of ATCC 9046 (solid circles) and JM3 strains (empty circles) in Burk's sucrose medium. (B) Primer extension at 8 (lane 1), 24 (lane 2), and 48 h (lane 3) incubation in Burk's sucrose medium. (C) Hybridization of a sample of the RNA (10 μg) used as template for the primer extension with a probe specific for 16S rRNA over 8, 24, and 48 h (4).

FIG. 3

Primer extension analysis of algD transcription from p1 and p2 in ATCC 9046 and CNS59 strains after 8 and 24 h of growth on Burk's sucrose medium.

FIG. 4

Northern analysis of rpoS RNA isolated from ATCC 9046 (lanes 1 and 3) and JM3 (lanes 2 and 4) after 8 and 48 h of incubation in Burk's sucrose medium.

FIG. 5

Model for regulation of algD expression by the global regulators GacA and ς^S.