Dataset Information

Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance.

ABSTRACT: During encystment of Azotobacter vinelandii, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the membrane phospholipids and are also components of the layers covering the cyst. In this study, A. vinelandii strains unable to synthesize ARs were isolated after mini-Tn5 mutagenesis. Cloning and nucleotide sequencing of the affected loci revealed the presence of the transposons within the arsA gene of the previously reported arsABCD gene cluster, which encodes a type I fatty acid synthase. A mutant strain (SW-A) carrying an arsA mutation allowing transcription of arsBCD was constructed and shown to be unable to produce ARs, indicating that the ArsA protein is essential for the synthesis of these phenolic lipids. Transcription of arsA was induced 200-fold in cells undergoing encystment, but only 14-fold in aged cultures of A. vinelandii, in accordance with AR synthesis and cyst formation percentages under the two conditions. Although it was previously reported that the inactivation of arsB abolishes AR synthesis and results in a failure in encystment, the arsA mutants were able to form cysts resistant to desiccation. These data indicate that ARs play a structural role in the exine layer of the cysts, but they are not essential for either cyst formation or for desiccation resistance.

SUBMITTER: Segura D

PROVIDER: S-EPMC2681798 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance.

Segura Daniel D Vite Odon O Romero Yanet Y Moreno Soledad S Castañeda Miguel M Espín Guadalupe G

Journal of bacteriology 20090306 9

During encystment of Azotobacter vinelandii, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the membrane phospholipids and are also components of the layers covering the cyst. In this study, A. vinelandii strains unable to synthesize ARs were isolated after mini-Tn5 mutagenesis. Cloning and nucleotide sequencing of the affected loci revealed the presence of the transposons within the arsA gene of the previously reported arsABCD ...[more]

PMID: 19270099

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Project description:The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCE A. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Project description:The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting rnf1 gene expression as an additional requirement for ammonium excretion. These ammonium-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. IMPORTANCE There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.

Dataset Information

Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance.

Publications

Isolation and characterization of Azotobacter vinelandii mutants impaired in alkylresorcinol synthesis: alkylresorcinols are not essential for cyst desiccation resistance.

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