Archives of Microbiology

, Volume 179, Issue 6, pp 437–443 | Cite as

Encystment and alkylresorcinol production by Azotobacter vinelandii strains impaired in poly-β-hydroxybutyrate synthesis

Original Paper


The lipids poly-β-hydroxybutyrate (PHB) and alkylresorcinols are the major metabolic products of Azotobacter 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 phospholipids 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 enzymes 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. However, the cysts formed by these strains posses a thicker exine 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 synthesis of alkylresorcinols and encystment even under non-inducing 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.


Encystment Polyhydroxyalkanoate PHB synthase 


  1. Bali A, Blanco G, Hill S, Kennedy C (1992) Excretion of ammonium by a nifL of Azotobacter vinelandii fixing nitrogen. Appl. Environ Microbiol 58:1711–1718PubMedGoogle Scholar
  2. Campos M E, Martínez-Salazar J, Lloret L, Moreno S, Núñez C, Espín G, Soberón-Chávez G (1996) Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii. J Bacteriol 178:1793–1799PubMedGoogle Scholar
  3. Chohan SH, Copeland L (1998) Acetoacetyl-CoA reductase and polyhydroxybutyrate synthesis in Rhizobium (Cicer) sp. strain CC1192. Appl Environ Microbiol 64:2859–2863PubMedGoogle Scholar
  4. Fellay R, Frey J, Krisch H (1987) Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria. Gene 52:147–154PubMedGoogle Scholar
  5. Gama-Castro S, Núñez C, Segura D, Moreno S, Guzmán J, Espín G (2001) Azotobacter vinelandii aldehyde dehydrogenase regulated by sigma54: role in alcohol catabolism and encystment. J Bacteriol. 183:6169–74Google Scholar
  6. Gerngross TU, Snell KD, Peoples OP, Sinskey AJ, Csuhai E, Masamune S, Stuble J (1994) Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity. Biochemistry 33:9311–9320PubMedGoogle Scholar
  7. Kennedy C, Gamal R, Humphrey R, Ramos J, Brigle K, Dean D (1986) The nifH, nifM and nifN genes of Azotobacter vinelandii: characterization by Tn5 mutagenesis and isolation from pLARF1 gene banks. Mol Gen Genet 205:318–325Google Scholar
  8. Kozubek A, Tyman JH (1999) Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chem Rev 99:1–26PubMedGoogle Scholar
  9. Law JH, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36Google Scholar
  10. Lin LP, Sadoff HL (1968) Encystment and polymer production in Azotobacter vinelandii in the presence of β-hydroxybutyrate. J Bacteriol 95:2336–2343PubMedGoogle Scholar
  11. Lowry O H, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin-phenol reagent. J Biol Chem 193:265–272Google Scholar
  12. Maehara A, Ueda S, Nakano H, Yamane T (1999) Analysis of a polyhydroxyalkanoic acid granule-associated 16-kilodalton protein and its putative regulator in the pha locus of Paracoccus denitrificans. J Bacteriol 181:2914–2921PubMedGoogle Scholar
  13. Manchak J, Page WJ (1994) Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD. Microbiology 140:953–963Google Scholar
  14. Matsusaki H, Manji S, Taguchi K, Kato M, Fukui T, Doi Y (1998) Cloning and molecular analysis of the poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61–3. J Bacteriol 180:6459–6467PubMedGoogle Scholar
  15. Mejía-Ruíz H, Moreno S, Guzmán J, Nájera R, León R, Soberón-Chavez G, Espín G (1997) Isolation and characterization of an Azotobacter vinelandii algK mutant. FEMS Microbiol Lett 156:101–106PubMedGoogle Scholar
  16. Nowak-Thompson B, Hammer PE, Hill DS, Stafford J, Torkewitz N, Gaffney TD, Lam ST, Molnár I, Ligon JM (2003) 2,5-Dialkylresorcinol biosynthesis in Pseudomonas aurantiaca: Novel head-to-head condensation of two fatty acid-derived precursors. J Bacteriol 185(3):860–869PubMedGoogle Scholar
  17. Peralta-Gil M, Segura D, Guzmán J, Servín-González L, Espín G (2002) Expression of the Azotobacter vinelandii poly-β-hydroxybutyrate biosynthetic phbBAC operon is driven by two overlapping promoters and is dependent on the transcriptional activator PhbR. J Bacteriol. 184:5672–5677Google Scholar
  18. Pettinari J M, Vázquez GJ, Silberschmidt D, Rehm B, Steinbüchel A, Mendez BS (2001) Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8. Appl Env Microbiol 67:5331–5334CrossRefGoogle Scholar
  19. Reusch RN, Sadoff HL (1981) Lipid metabolism during encystment of Azotobacter vinelandii. J Bacteriol 145:889–895PubMedGoogle Scholar
  20. Sadoff HL (1975) Encystment and germination in Azotobacter vinelandii. Bacteriol Rev 39:516–539PubMedGoogle Scholar
  21. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  22. Sanger F, Nicklen S, Coulson A R (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedGoogle Scholar
  23. Segura D, Vargas E, Espín G (2000) β-Ketothiolase genes in Azotobacter vinelandii. Gene 260:113–120.CrossRefPubMedGoogle Scholar
  24. Senior PJ, Dawes EA (1973) The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem J. 134:225–38.Google Scholar
  25. Senior PJ, Beech GA, Ritchie GA, Dawes EA (1972) The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii. Biochem J. 128:1193–201Google Scholar
  26. Slater S, Houmiel KL, Tran M, Mitsky TA, Taylor NB, Padgette SR, Gruys KJ (1998) Multiple β-ketothiolases mediate poly(β-hydroxyalkanoate) copolymer synthesis in Ralstonia eutropha. J Bacteriol 180:1979–1987PubMedGoogle Scholar
  27. Stevenson LH, Socolofsky MD (1966) Cyst formation and poly-β-hydroxybutyric acid accumulation in Azotobacter. J Bacteriol 91:304–310PubMedGoogle Scholar
  28. Tluscik F, Kozubek A, Mejbaum-Katzenellenbogen W (1981) Alkylresorcinols in rye (Secale cereale L.) grains. VI. Colorimetric micromethod for the determination of alkylresorcinols with the use of diazonium salt, Fast Blue B. Acta Soc Bot Pol 50:645–651Google Scholar
  29. Vela GR, Rosenthal RS (1972) Effect of peptone on Azotobacter morphology. J Bacteriol 111:260–266PubMedGoogle Scholar
  30. Wieczorek, R, Pries A, Steinbuchel A, Mayer F (1995) Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus. J Bacteriol 177:2425–2435PubMedGoogle Scholar
  31. Wyss O, Smith DD, Pope LM, Sokolofsky KE (1969) Endogenous encystment of Azotobacter vinelandii. J Bacteriol 100:475–479Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMéxico

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