Isolation and characterization of an Azotobacter vinelandii algK mutant

Mejıća-Ruıćz, Humberto; Moreno, Soledad; Guzmán, Josefina; Nájera, Rosa María Sánchez; León, Renato; Soberón-Chávez, Gloria; Espıćn, Guadalupe

doi:10.1111/j.1574-6968.1997.tb12712.x

Cited by 18 publications

(17 citation statements)

References 16 publications

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“…Because a mutation in arsB was shown to impair ARs synthesis (7), the question of whether the inability of the OV11 and OV8 mutants to produce ARs was due to polarity of the arsA::Tn5 mutations on arsB was raised. In A. vinelandii, the insertion of the ⍀-Km cassette from plasmid pHP45⍀-Km (6) into a gene in the same direction of its transcription produces mutations allowing transcription of the downstream genes in the same operon (16). Strains SW-A and SW-AP, which contain an asrA::⍀-Gm insertion with the cassette oriented in the same or in the opposite direction of arsA, respectively, were constructed as described in Materials and Methods.…”

Section: Resultsmentioning

confidence: 99%

“…For this staining the cells were grown for 5 days in the appropriate medium, and samples of the culture were placed on a microscope slide and stained with a solution of 0.5% Fast Blue B in 5% acetic acid for 10 min. Electron microscopy was carried out using cells grown on BBOH for 5 days, as previously reported (16).…”

Section: Methodsmentioning

confidence: 99%

“…The polysaccharide alginate is also a major component of the cyst capsule and is essential for the differentiation process, since mutations in alginate biosynthetic genes abrogate the formation of cysts resistant to desiccation (5,16). In A. vinelandii ATCC 9046 inactivation of algU, the gene coding for the sigma factor AlgU ( E ), impairs alginate synthesis and cyst formation (18), as this sigma factor is required for full expression of the alginate biosynthetic genes algD and algC (8,15,18).…”

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Isolation and Characterization of Azotobacter vinelandii Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance

et al. 2009

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Isolation and Characterization of Azotobacter vinelandii Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance

et al. 2009

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“…Staining of A. vinelandii cells was performed as described by Vela and Wyss (59). Electron microscopy was performed essentially as described by Mejía-Ruíz et al (37), except that the cells were grown in RA1 medium and the fixation with glutaraldehyde was performed for 16 h.…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

et al. 2006

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Alginate is a linear copolymer of ␤-D-mannuronic acid and its C-5-epimer, ␣-L-guluronic acid. During biosynthesis, the polymer is first made as mannuronan, and various fractions of the monomers are then epimerized to guluronic acid by mannuronan C-5-epimerases. The Azotobacter vinelandii genome encodes a family of seven extracellular such epimerases (AlgE1 to AlgE7) which display motifs characteristic for proteins secreted via a type I pathway. Putative ATPase-binding cassette regions from the genome draft sequence of the A. vinelandii OP strain and experimentally verified type I transporters from other species were compared. This analysis led to the identification of one putative A. vinelandii type I system (eexDEF). The corresponding genes were individually disrupted in A. vinelandii strain E, and Western blot analysis using polyclonal antibodies against all AlgE epimerases showed that these proteins were present in wild-type culture supernatants but absent from the eex mutant supernatants. Consistent with this, the wild-type strain and the eex mutants produced alginate with about 20% guluronic acid and almost pure mannuronan (<2% guluronic acid), respectively. The A. vinelandii wild type is able to enter a particular desiccation-tolerant resting stage designated cyst. At this stage, the cells are surrounded by a rigid coat in which alginate is a major constituent. Such a coat was formed by wild-type cells in a particular growth medium but was missing in the eex mutants. These mutants were also found to be unable to survive desiccation. The reason for this is probably that continuous stretches of guluronic acid residues are needed for alginate gel formation to take place.

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“…Alginate is essential for the encystment process. Mutations that impair alginate synthesis also impair the formation of cysts (Campos et al, 1996;Mejía-Ruíz et al, 1997;Castañeda et al, 2001). Alginate monomer composition is also important for cyst formation, as the AlgE1-AlgE7 epimerases are, in combination, essential for the formation of mature cysts (Steigedal et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii

et al. 2014

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In Azotobacter vinelandii, a cyst-forming bacterium, the alternative sigma factor RpoS is essential to the formation of cysts resistant to desiccation and to synthesis of the cyst-specific lipids, alkylresorcinols. In this study, we carried out a proteome analysis of vegetative cells and cysts of A. vinelandii strain AEIV and its rpoS mutant derivative AErpoS. This analysis allowed us to identify a small heat-shock protein, Hsp20, as one of the most abundant proteins of cysts regulated by RpoS. Inactivation of hsp20 did not affect the synthesis of alkylresorcinols or the formation of cysts with WT morphology; however, the cysts formed by the hsp20 mutant strain were unable to resist desiccation. We also demonstrated that expression of hsp20 from an RpoSindependent promoter in the AErpoS mutant strain is not enough to restore the phenotype of resistance to desiccation. These results indicate that Hsp20 is essential for the resistance to desiccation of A. vinelandii cysts, probably by preventing the aggregation of proteins caused by the lack of water. To our knowledge, this is the first report of a small heat-shock protein that is essential for desiccation resistance in bacteria.

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Isolation and characterization of an Azotobacter vinelandii algK mutant

Cited by 18 publications

References 16 publications

Isolation and Characterization of Azotobacter vinelandii Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance

Isolation and Characterization of Azotobacter vinelandii Mutants Impaired in Alkylresorcinol Synthesis: Alkylresorcinols Are Not Essential for Cyst Desiccation Resistance

Identification and Characterization of anAzotobacter vinelandiiType I Secretion System Responsible for Export of the AlgE-Type Mannuronan C-5-Epimerases

A small heat-shock protein (Hsp20) regulated by RpoS is essential for cyst desiccation resistance in Azotobacter vinelandii

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