Role of alternative sigma factor algU in encystment of Azotobacter vinelandii

Abstract

Alginate is essential for encystment in Azotobacter vinelandii. Transcription of the algD gene, which codes for GDP-mannose dehydrogenase, a key enzyme in the alginate biosynthetic pathway, is initiated at two promoters, one of which, p2, has sigmaE consensus sequences. AlgU is the A. vinelandii alternative sigmaE factor. In this study, we constructed an algU mutant (SMU88) which, as expected, is impaired in alginate production, encystment, and transcription of the algD gene from the p2 promoter. Plasmid pJMSAT1, carrying the A. vinelandii algU gene, restored alginate production and encystment to SMU88 and to strain UW136, a naturally occurring algU mutant. Plasmid pSMU865, carrying the A. vinelandii mucABCD genes coding for negative regulators of AlgU activity and previously shown to diminish alginate production in the wild-type strain, ATCC 9046, was shown here to impair encystment and transcription of the algD gene from the p2 algU-dependent promoter. Since nonencysting strain ATCC 9046/pSMU865 produced more alginate than some encysting strains, such as UW136/pJMSAT1, we propose an AlgU role in encystment, independent of the structural role that alginate plays in mature cysts.

FIG. 1

Construction of strain SMU88. (A) Schematic representation of the A. vinelandii algU region and the algU::Km mutation in plasmid pSMU88. Bar, 100 bp. (B) Southern blot hybridization of total genomic DNA digested with PstI endonuclease, with pSMU85 as a probe. Lanes: 1, ATCC 9046; 2, SMU88. Molecular sizes (in kilobases) are indicated on the right.

FIG. 2

Primer extension analysis of algD transcription in strains SMU88 and ATCC 9046, with and without plasmid pSMU865. (A) DNA sequence of the 5′ end of algD. The arrows indicate the start sites of algD transcription, the p1 and p2 promoters are indicated (overbar), and the complementary sequences where oligonucleotides algD1 and algD2 (used for primer extension analysis) were generated are underlined. The algD ATG initiation codon is shown in boldface type. (B and C) Primer extension of the algD gene with oligonucleotide algD1 in strains SMU88 (lane 1) and ATCC 9046 with (lane 2) and without (lane 3) plasmid pSMU865 (B) and with oligonucleotide algD2 in strain SMU88 (C). The algD sequence ladders (GATC) were produced with the oligonucleotides used for primer extension.

FIG. 3

Electron micrographs were produced as described previously (19). Thin sections of A. vinelandii cysts formed by strains SMU88 (A), SMU88/pDMUM13 (B), ATCC 9046 (C), and ATCC 9046/pSMU865 (D) are shown. Abbreviations: EX, exine; IN, intine; CB, central body; PHB, poly-β-hydroxybutyrate. Bars, 0.4 μm.