The GacS Sensor Kinase Regulates Alginate and Poly-β-Hydroxybutyrate Production in Azotobacter vinelandii

Abstract

Azotobacter vinelandii produces two polymers: the extracellular polysaccharide alginate and the intracellular polyester poly-␤-hydroxybutyrate (PHB). A cosmid clone (pSMU588) from an A. vinelandii gene library diminished alginate production by A. vinelandii mucoid strain ATCC 9046. The nucleotide sequence and predicted amino acid sequence of the locus responsible for the mucoidy suppression revealed 65% identity to Pseudomonas GacS, a transmembrane sensor kinase of the two-component regulators, whose cognate response regulator, GacA, is a global activator regulating several products and virulence factors. Plasmid pMC15, harboring gacS, and a strain carrying a gacS nonpolar mutation were constructed. Either pMC15 or the gacS mutation significantly reduced alginate production and transcription of algD, the gene coding for the key enzyme GDP-mannose dehydrogenase of the alginate biosynthetic pathway. We found that the gacS mutation also reduced PHB accumulation and impaired encystment. Taken together, these data indicate that in A. vinelandii the gacSA global system regulates polymer synthesis.Azotobacter vinelandii is a nitrogen-fixing soil bacterium that undergoes differentiation to form desiccation-resistant cysts and produces two polymers of industrial importance: alginate and poly-␤-hydroxybutyrate (PHB). Both polymers are involved in the encystment process; alginate is a component of the cyst capsule (28), and PHB accumulation correlates with the frequency of cyst formation (30).A. vinelandii has been shown to possess an alginate biosynthetic gene cluster (2,16,20,25,32), organized in three operons, one of which transcribes algD encoding a GDP-mannose dehydrogenase, which converts GDP-mannose to GDP-mannuronic acid, the substrate for alginate polymerization. The algUmucABCD cluster controls alginate production. AlgU is a E homolog (19). The mucA and mucB genes code for negative regulators of AlgU activity. In strain ATCC 9046, transcription of the algD gene is initiated at three sites, one of which is AlgU dependent (2). AlgU activity was shown to be involved in the encystment process independent of its role in alginate synthesis (23).Three enzymes are involved in PHB biosynthesis in A. vinelandii: a ␤-ketothiolase, an acetoacetyl-coenzyme A reductase and a PHB synthetase (18). PHB synthesis in A. vinelandii was shown to be regulated at the level of the ␤-ketothiolase activity (18). The genes encoding the enzymes participating in PHB synthesis in A. vinelandii have not been reported.Members of our group have previously reported the identification of cosmid pSMU588 from a gene bank of nonmucoid strain UW136, which reduced alginate production in A. vinelandii ATCC 9046 (19). The characterization of pSMU588 reported here allowed us to identify a regulatory gene homologous to Pseudomonas gacS, coding for a sensor kinase of the two-component regulatory systems. This study also provides evidence for GacS playing a role as a regulator of alginate and PHB synthesis in A. vinelandii. a Alginate production was determin...

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

“…6a) and calculations made with  software, as described by Espinosa-Urgel & Tormo (1993), show that the sequence upstream of P1 has intrinsic DNA curvature (data not shown). In support of this hypothesis, it has been shown that algD transcription and alginate formation in A. vinelandii is regulated by GacSA, a sensor kinase that influences RpoS levels in the cell (Castaneda et al, 2000). Similarly, alginate formation in P. aeruginosa is greatly reduced in an RpoS mutant (Suh et al, 1999).…”

Production of polyhydroxybutyrate and alginate from glycerol byAzotobacter vinelandiiunder nitrogen-free conditions

“…16 The regulatory mechanisms for the production of these biopolymers have been analyzed. [16][17][18][19][20][21][22][23][24][25] Thus, the biopolymers are expected to be produced from excess and/or unused resource by A. vinelandii.…”

Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

“…These are known under two different names, PhaR and AniA (11,12,28,29,46). In addition, two different types of transcriptional regulators have been reported elsewhere to be involved in PHB production in Azotobacter vinelandii (4,45).…”