Characterization of the genes coding for the putative sigma factor AlgU and its regulators MucA, MucB, MucC, and MucD in Azotobacter vinelandii and evaluation of their roles in alginate biosynthesis

Abstract

The study of the biosynthesis of alginate, the exopolysaccharide produced by Azotobacter vinelandii and Pseudomonas aeruginosa, has biotechnological and medical significance. We report here the identification of the A. vinelandii genes coding for the putative sigma factor AlgU and its negative regulators MucA and MucB through the suppression of the highly mucoid phenotype of an A. vinelandii strain by a plasmid encoding MucA and MucB. The sequences of the A. vinelandii algU, mucA, and mucB genes are highly homologous to those of the corresponding P. aeruginosa genes, AlgU shows 93% identity, and MucA and MucB are 64.4 and 63.9% identical, respectively. Forming part of the same operon as algU, mucA, and mucB, two additional genes (mucC and mucD) were identified and sequenced; the product of the former gene is homologous to ORF4 of Photobacterium sp. strain SS9, and that of the latter gene belongs to the HtrA serine protease family. Interestingly, the nonmucoid A. vinelandii UW136 had a 0.9-kb insertion within the algU gene. A strong correlation between AlgU activity and alginate production by A. vinelandii was also found, as reflected in the level of algD transcription.

Alginate formation in Azotobacter vinelandii UWD during stationary phase and the turnover of poly-β-hydroxybutyrate

“…The region of the algU gene expected to contain the natural insertion element was amplified by PCR, using primers WJP13 (hybridizing to the 5h end of algU) and WJP46 (hybridizing to algU downstream of the insertion element). The expected PCR products with and without the insertion element were about 1322 and 322 bp, respectively (calculated from Martinez-Salazar et al, 1996). As shown in Fig.…”

Regulation of exopolysaccharide synthesis in Rhizobium sp. strain TAL1145 involves an alternative sigma factor gene, rpoH2

“…In A. vinelandii, an alternative sigma factor gene, algU, has 93 % identity to the algT gene in P. aeruginosa. Alginate production by A. vinelandii is strongly correlated with AlgU activity, and disruption of algU resulted in nonmucoid mutant (Martinez-Salazar et al, 1996). AlgT or AlgU is functionally equivalent to the extreme heat shock sigma factor s E in E. coli and other Gram-negative bacteria.…”

Temperature-responsive genetic loci in the plant pathogen Pseudomonas syringae pv. glycinea The GenBank accession numbers for the nucleotide sequences of mutants 560, 561, 562, 563, 564, 568, 570, 574, 590, 591, 593, 596, 599, 601, 605, 608, 613, 617, 618, 626 and 632 determined in this work are AF274322–AF274342, respectively.

“…If mucD of PG4180 were part of the algT operon, one would expect that the algE-like locus would be expressed preferentially at 28 mC. Transcription of algE in P. aeruginosa and Azotobacter vinelandii was previously shown to be under the control of AlgT (Ertesvag et al, 1995 ;Martinez-Salvazar et al, 1996). Since copper ions induce alginate biosynthesis in P. syringae (Kidambi et al, 1995), we subsequently tested the alginate production of mutants 561 and 599 on MG agar plates supplemented with 250 µg cupric sulfate ml −" and compared it to the phenotype of the wild-type.…”