Abstract The lipids poly-β-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azoto- bacter vinelandii cysts. Cysts are formed in less than 0.01% of late stationary phase cells grown on sucrose. Culturing vegetative cells in n-butanol or β-hydroxybutyrate induces encystment. After induction of encystment, PHB rapidly accumulates in large granules. Then, the cells begin the synthesis of alkylresorcinols that replace the phospho- lipids in the membranes and are components of the exine, the outer layer of the cyst envelope. Vegetative cells do not synthesize alkylresorcinols. We report here the effect of mutations in the phbBAC operon, coding for the en- zymes of the PHB biosynthetic pathway, on the synthesis of alkylresorcinols and cyst formation. The phb mutations did not impair the capacity to form mature cysts. How- ever, the cysts formed by these strains posses a thicker ex- ine layer and a higher content of alkylresorcinols than the cysts formed by the wild-type strain. A blockage of PHB synthesis caused by phb mutations resulted in the synthe- sis of alkylresorcinols and encystment even under non-in- ducing conditions. We propose that, as a consequence of the blockage in the PHB biosynthetic pathway, the acetyl- CoA and reducing power pools are increased causing the shift to lipid metabolism required for the synthesis of alkylresorcinols and cyst formation. Keywords Encystment · Polyhydroxyalkanoate · PHB synthase Introduction Azotobacter vinelandii is a nitrogen-fixing soil bacterium that produces polymers of industrial importance: alginate, poly-β-hydroxybutyrate (PHB), and the alkylresorcinols 5-n-heneicosylresorcinol, 5-n-tricosylresorcinol, and their galactoside derivatives. These compounds are related to the morphological differentiation that this bacterium un- dergoes to form desiccation-resistant cysts. A mature cyst consists of a contracted cell (central body) surrounded by a capsule made up of a thin outer layer (exine) and a thicker inner layer (intine). The polysaccharide alginate is a com- ponent of the intine and exine layers (Sadoff 1975). Alkyl- resorcinols are lipids that replace the membrane phospho- lipids during encystment and are also components of the exine (Reusch and Sadoff 1981). PHB accumulates as large granules in stationary-phase vegetative cells and is also a major component of the central body of cysts. Cysts are formed by fewer than 0.01% of cells grown on glucose at late stationary phase (Lin and Sadoff 1968). However, removing glucose or sucrose from exponential- phase cultures and replacing it with n-butanol or β-hy- droxybutyrate can induce encystment (Lin and Sadoff 1968). This induction results in accumulation of PHB and the synthesis or alkylresorcinols. Thus, a shift from carbo- hydrate to lipid metabolism results in cyst formation. In fact, the addition of a carbohydrate (glucose) to cultures undergoing encystment prevents the formation of mature cysts (Lin and Sadoff 1968). Several lines of evidence suggested that PHB could be essential for the encystment process: the product of PHB degradation, β-hydroxybutyrate, or related compounds like n-butanol or crotonate, induce encystment (Reusch and Sadoff 1981). A direct relationship between the extent of PHB accumulation and the degree of encystment has been shown (Stevenson and Socolofsky 1966). It has also been stated that, under carbon limitation, the internal PHB reserve is used as a source of carbon and energy to enable the cells to form cysts (Stevenson and Sokolofsky 1966; Wyss et al. 1969; Sadoff 1975). Daniel Segura · Tania Cruz · Guadalupe Espín Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly--hydroxybutyrate synthesis Arch Microbiol (2003) 179 : 437–443 DOI 10.1007/s00203-003-0553-4 Received: 18 February 2003 / Revised: 10 April 2003 / Accepted: 16 April 2003 / Published online: 6 May 2003 ORIGINAL PAPER D. Segura · T. Cruz · G. Espín (✉) Instituto de Biotecnología, Universidad Nacional Autónoma de México, Departamento de Microbiología Molecular, Apartado Postal 510–3, 62271 Cuernavaca, Morelos, México Fax: +52-777-3172388, e-mail: espin@ibt.unam.mx © Springer-Verlag 2003