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Volume 171, Issue 4

The c-di-GMP effector FleQ controls alginate production by repressing transcription of in Open Access

Graphical Abstract

Graphical Abstract

In addition to activating the alginate polymerase complex, we found that the second messenger c-di-GMP is necessary for the transcriptional activation of the gene, which encodes the key enzyme for alginate biosynthesis. FleQ was identified as a transcriptional regulator that exerts a repressive effect by directly binding to the regulatory region. Moreover, c-di-GMP was found to be essential for the expression of genes encoding alginate modification enzymes (AlgE1-6), which are involved in cyst capsule formation. However, our data indicate that FleQ is not involved in this regulation.

Abstract

Production of the exopolysaccharide alginate by , member of the family, is positively controlled by the second messenger c-di-GMP. This effect was solely attributed to the role of c-di-GMP in activating the alginate polymerase complex. In this study, the role of c-di-GMP in transcription, which encodes the key enzyme for alginate synthesis, was investigated. transcription correlated with artificially high or low levels of c-di-GMP. Moreover, FleQ, one of the best-characterized c-di-GMP effectors, was found to exert a negative effect on alginate production and transcription, as both increased in a Δ mutant relative to the wild-type strain or the Δ/+ complemented strain. Electrophoretic mobility shift assays (EMSAs) confirmed that FleQ directly binds to the regulatory region of , which was consistent with the presence of two FleQ binding sites in the vicinity of the RpoS-dependent promoter. In , c-di-GMP is essential for the expression of alginate C-5 epimerases (AlgE1-6), which are necessary for structuring the envelope of differentiated cells, known as cysts. However, FleQ was not involved in this regulation. Collectively, our results support a model in which transcription is under the positive control of c-di-GMP, while FleQ may only partially mediate this effect. In contrast, our study revealed a FleQ-independent regulatory mechanism for the control of encystment.

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  • Accepted:
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Keyword(s): algD , alginate , Azotobacter vinelandii , c-di-GMP , encystment and FleQ
Funding
This study was supported by the:
  • Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (Award PAPIIT IN209521)
    • Principal Award Recipient: CinthiaNuñez
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2025-04-24
2026-02-19

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References

  1. Kennedy C, Rudnick P, MacDonald ML, Melton T. Genus III Azotobacter. In Noel NK NN, Garryty GM. eds Bergey’s Manual of Systematic Bacteriology New York: Springer; 2005 pp 384–402
     [Google Scholar]
  2. Segura D, Núñez C, Espín G. Azotobacter cyst. In Sons eWJ. eds UK: Chichester; 2020
  3. Aasfar A, Bargaz A, Yaakoubi K, Hilali A, Bennis I et al. Nitrogen fixing Azotobacter species as potential soil biological enhancers for crop nutrition and yield stability. Front Microbiol 2021; 12:628379 Epub 20210225 [View Article] [PubMed]
     [Google Scholar]
  4. Núñez C, López-Pliego L, Ahumada-Manuel CL, Castañeda M. Genetic regulation of alginate production in Azotobacter vinelandii, a bacterium of biotechnological interest: a mini-review. Front Microbiol 2022; 13:845473 Epub 20220323 [View Article] [PubMed]
     [Google Scholar]
  5. Hay ID, Ur Rehman Z, Moradali MF, Wang Y, Rehm BHA. Microbial alginate production, modification and its applications. Microb Biotechnol 2013; 6:637–650 Epub 20130819 [View Article] [PubMed]
     [Google Scholar]
  6. Urtuvia V, Maturana N, Acevedo F, Peña C, Díaz-Barrera A. Bacterial alginate production: an overview of its biosynthesis and potential industrial production. World J Microbiol Biotechnol 2017; 33:198 Epub 20171007 [View Article] [PubMed]
     [Google Scholar]
  7. Moreno S, Nájera R, Guzmán J, Soberón-Chávez G, Espín G. Role of alternative sigma factor algU in encystment of Azotobacter vinelandii. J Bacteriol 1998; 180:2766–2769 [View Article] [PubMed]
     [Google Scholar]
  8. Martínez-Salazar JM, Moreno S, Nájera R, Boucher JC, Espín G et al. 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. J Bacteriol 1996; 178:1800–1808 [View Article] [PubMed]
     [Google Scholar]
  9. Castañeda M, Sánchez J, Moreno S, Núñez C, Espín G. The global regulators GacA and sigma(S) form part of a cascade that controls alginate production in Azotobacter vinelandii. J Bacteriol 2001; 183:6787–6793 [View Article] [PubMed]
     [Google Scholar]
  10. Núñez C, León R, Guzmán J, Espín G, Soberón-Chávez G. Role of Azotobacter vinelandii mucA and mucC gene products in alginate production. J Bacteriol 2000; 182:6550–6556 [View Article] [PubMed]
     [Google Scholar]
  11. Baynham PJ, Wozniak DJ. Identification and characterization of AlgZ, an AlgT-dependent DNA-binding protein required for Pseudomonas aeruginosa algD transcription. Mol Microbiol 1996; 22:97–108 [View Article] [PubMed]
     [Google Scholar]
  12. Xu B, Soukup RJ, Jones CJ, Fishel R, Wozniak DJ. Pseudomonas aeruginosa AmrZ binds to four sites in the algD promoter, inducing DNA-AmrZ complex formation and transcriptional activation. J Bacteriol 2016; 198:2673–2681 Epub 20160909 [View Article] [PubMed]
     [Google Scholar]
  13. Leech AJ, Sprinkle A, Wood L, Wozniak DJ, Ohman DE. The NtrC family regulator AlgB, which controls alginate biosynthesis in mucoid Pseudomonas aeruginosa, binds directly to the algD promoter. J Bacteriol 2008; 190:581–589 [View Article] [PubMed]
     [Google Scholar]
  14. Mohr CD, Hibler NS, Deretic V. AlgR, a response regulator controlling mucoidy in Pseudomonas aeruginosa, binds to the FUS sites of the algD promoter located unusually far upstream from the mRNA start site. J Bacteriol 1991; 173:5136–5143 [View Article] [PubMed]
     [Google Scholar]
  15. Ahumada-Manuel CL, Martínez-Ortiz IC, Hsueh BY, Guzmán J, Waters CM et al. Increased c-di-GMP levels lead to the production of alginates of high molecular mass in Azotobacter vinelandii. J Bacteriol 2020; 202: [View Article] [PubMed]
     [Google Scholar]
  16. Martínez-Ortiz IC, Ahumada-Manuel CL, Hsueh BY, Guzmán J, Moreno S et al. Cyclic di-GMP-mediated regulation of extracellular mannuronan C-5 epimerases is essential for cyst formation in Azotobacter vinelandii. J Bacteriol 2020; 202:e00135-20 [View Article] [PubMed]
     [Google Scholar]
  17. Jenal U, Reinders A, Lori C. Cyclic di-GMP: second messenger extraordinaire. Nat Rev Microbiol 2017; 15:271–284 Epub 20170206 [View Article] [PubMed]
     [Google Scholar]
  18. Hengge R. Trigger phosphodiesterases as a novel class of c-di-GMP effector proteins. Philos Trans R Soc Lond B Biol Sci 2016; 371:20150498 [View Article] [PubMed]
     [Google Scholar]
  19. Ahumada-Manuel CL, Guzmán J, Peña C, Quiroz-Rocha E, Espín G et al. The signaling protein MucG negatively affects the production and the molecular mass of alginate in Azotobacter vinelandii. Appl Microbiol Biotechnol 2017; 101:1521–1534 [View Article] [PubMed]
     [Google Scholar]
  20. Merighi M, Lee VT, Hyodo M, Hayakawa Y, Lory S. The second messenger bis-(3’-5’)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa. Mol Microbiol 2007; 65:876–895 [View Article] [PubMed]
     [Google Scholar]
  21. Whitney JC, Whitfield GB, Marmont LS, Yip P, Neculai AM et al. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa. J Biol Chem 2015; 290:12451–12462 [View Article] [PubMed]
     [Google Scholar]
  22. Oladosu VI, Park S, Sauer K. Flip the switch: the role of FleQ in modulating the transition between the free-living and sessile mode of growth in Pseudomonas aeruginosa. J Bacteriol 2024; 206: Epub 20240304 [View Article] [PubMed]
     [Google Scholar]
  23. Matsuyama BY, Krasteva PV, Baraquet C, Harwood CS, Sondermann H et al. Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa. Proc Natl Acad Sci USA 2016; 113:E209–18 [View Article] [PubMed]
     [Google Scholar]
  24. Baraquet C, Murakami K, Parsek MR, Harwood CS. The FleQ protein from Pseudomonas aeruginosa functions as both a repressor and an activator to control gene expression from the pel operon promoter in response to c-di-GMP. Nucleic Acids Res 2012; 40:7207–7218 [View Article] [PubMed]
     [Google Scholar]
  25. Hickman JW, Harwood CS. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 2008; 69:376–389 [View Article] [PubMed]
     [Google Scholar]
  26. Baraquet C, Harwood CS. Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ. Proc Natl Acad Sci USA 2013; 110:18478–18483 [View Article] [PubMed]
     [Google Scholar]
  27. Larsen B, Haug A. Biosynthesis of alginate. 1. Composition and structure of alginate produced by Azotobacter vinelandii (Lipman). Carbohydr Res 1971; 17:287–296 [View Article] [PubMed]
     [Google Scholar]
  28. Núñez C, Peña C, Kloeckner W, Hernández-Eligio A, Bogachev AV et al. Alginate synthesis in Azotobacter vinelandii is increased by reducing the intracellular production of ubiquinone. Appl Microbiol Biotechnol 2013; 97:2503–2512 Epub 20120810 [View Article] [PubMed]
     [Google Scholar]
  29. Bali A, Blanco G, Hill S, Kennedy C. Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl Environ Microbiol 1992; 58:1711–1718 [View Article] [PubMed]
     [Google Scholar]
  30. Page WJ, Sadoff HL. Physiological factors affecting transformation of Azotobacter vinelandii. J Bacteriol 1976; 125:1080–1087 [View Article] [PubMed]
     [Google Scholar]
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–275 [PubMed]
     [Google Scholar]
  32. Knutson CA, Jeanes A. A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem 1968; 24:470–481 [View Article] [PubMed]
     [Google Scholar]
  33. Alexeyev MF, Shokolenko IN, Croughan TP. Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis. Gene 1995; 160:63–67 [View Article] [PubMed]
     [Google Scholar]
  34. Cocotl-Yañez M, Moreno S, Encarnación S, López-Pliego L, Castañeda M et al. A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii. Microbiology 2014; 160:479–487 Epub 20140102 [View Article] [PubMed]
     [Google Scholar]
  35. Campos M, Martínez-Salazar JM, Lloret L, Moreno S, Núñez C et al. Characterization of the gene coding for GDP-mannose dehydrogenase (algD) from Azotobacter vinelandii. J Bacteriol 1996; 178:1793–1799 [View Article] [PubMed]
     [Google Scholar]
  36. Barry T, Geary S, Hannify S, MacGearailt C, Shalloo M et al. Rapid mini-preparations of total RNA from bacteria. Nucleic Acids Res 1992; 20:4940 [View Article] [PubMed]
     [Google Scholar]
  37. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001; 25:402–408 [View Article] [PubMed]
     [Google Scholar]
  38. Baraquet C, Harwood CS. FleQ DNA binding consensus sequence revealed by studies of FleQ-dependent regulation of biofilm gene expression in Pseudomonas aeruginosa. J Bacteriol 2016; 198:178–186 Epub 20151019 [View Article] [PubMed]
     [Google Scholar]
  39. Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 1994; PubMed PMID:28–36 [PubMed]
     [Google Scholar]
  40. Grant CE, Bailey TL, Noble WS. FIMO: scanning for occurrences of a given motif. Bioinformatics 2011; 27:1017–1018 Epub 20110216 [View Article] [PubMed]
     [Google Scholar]
  41. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010; 26:841–842 Epub 20100128 [View Article] [PubMed]
     [Google Scholar]
  42. Bustamante VH, Martínez LC, Santana FJ, Knodler LA, Steele-Mortimer O et al. HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2. Proc Natl Acad Sci USA 2008; 105:14591–14596 [View Article] [PubMed]
     [Google Scholar]
  43. Martínez-Flores I, Pérez-Morales D, Sánchez-Pérez M, Paredes CC, Collado-Vides J et al. In silico clustering of Salmonella global gene expression data reveals novel genes co-regulated with the SPI-1 virulence genes through HilD. Sci Rep 2016; 6:37858 [View Article] [PubMed]
     [Google Scholar]
  44. Høidal HK, Glaerum Svanem BI, Gimmestad M, Valla S. Mannuronan C-5 epimerases and cellular differentiation of Azotobacter vinelandii. Environ Microbiol 2000; 2:27–38 [View Article] [PubMed]
     [Google Scholar]
  45. Moreno S, Ertesvåg H, Valla S, Núñez C, Espin G et al. RpoS controls the expression and the transport of the AlgE1-7 epimerases in Azotobacter vinelandii. FEMS Microbiol Lett 2018; 365:fny210 [View Article] [PubMed]
     [Google Scholar]
  46. Ertesvåg H. Alginate-modifying enzymes: biological roles and biotechnological uses. Front Microbiol 2015; 6:523 Epub 20150527 [View Article] [PubMed]
     [Google Scholar]
  47. Hay ID, Wang Y, Moradali MF, Rehman ZU, Rehm BHA. Genetics and regulation of bacterial alginate production. Environ Microbiol 2014; 16:2997–3011 Epub 20140218 [View Article] [PubMed]
     [Google Scholar]
  48. Liang Z, Rybtke M, Kragh KN, Johnson O, Schicketanz M et al. Transcription of the alginate operon in Pseudomonas aeruginosa is regulated by c-di-GMP. Microbiol Spectr 2022; 10:e0067522 [View Article] [PubMed]
     [Google Scholar]
/content/journal/micro/10.1099/mic.0.001556
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The c-di-GMP effector FleQ controls alginate production by repressing transcription of algD in Azotobacter vinelandii
Microbiology 171, 001556 (2025); https://doi.org/10.1099/mic.0.001556
/content/journal/micro/10.1099/mic.0.001556
/content/journal/micro/10.1099/mic.0.001556
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