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.

FIG. 1.

Physical map of the A. vinelandii ars chromosomal region. The fragments contained in the corresponding plasmids are illustrated. The arrows represent genes. Restriction sites relevant for gene disruption are shown. Triangles represent insertions of either antibiotic resistance cassettes or the mTn5SSgusA40 transposon.

FIG. 2.

Staining of alkylresorcinols produced by A. vinelandii. (A) Staining of A. vinelandii mutant OV8 (1), SW136 (2), and mutant OV11 (3), grown on petri dishes containing Burk-sucrose medium (vegetative growth) or Burk-butanol medium (encystment induction). (B) Light microscopy (bright field) of Fast Blue B-stained vegetative cells and cysts of A. vinelandii SW136 and OV11. In all cases the cells were grown for 5 days.

FIG. 3.

Effects of different arsA gene insertions on alkylresocinol synthesis and on the expression of arsB. (A) Alkylresorcinol staining of SW136 and different asrA mutants. The cells were induced to encyst on Burk-butanol medium for 5 days. (B) Effects of different arsA insertions on the expression of arsB, measured by real-time reverse transcription-PCR. The levels of the arsB transcripts were measured under encystment-inducing conditions and were normalized according to the level of the gyrA mRNA. The data are presented as fold changes of mRNA levels of OV11, SW-A, and SW-AP mutant strains relative to those of the parental strain (SW136). These data represent the means of triplicates, and the error bars represent the standard deviations.

FIG. 4.

Expression of arsA, AR synthesis, and encystment in two different media, Burk-sucrose medium (vegetative growth; open symbols) or Burk-butanol (encystment induction medium; closed symbols). (A) β-Glucoronidase activity of strain OV11 containing an arsA::Tn5-gusA reporter fusion. (B) Accumulation of alkylresorcinols over time in A. vinelandii SW136. (C) Percentages of encystment of strains SW136 and OV11, measured as desiccation resistance for 5 days. The inocula were incubated for 24 h on liquid Burk-sucrose, washed with Burk's medium with no carbon source, and transferred to plates with the corresponding medium (at time zero). These data are the means of triplicates, and the error bars represent the standard deviations. One unit of β-glucoronidase activity corresponds to 1 nmol of substrate 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid hydrolyzed per min per mg of protein.

FIG. 5.

Electron micrographs of A. vinelandii cysts of strains SW136 and OV11 (algU⁺ arsA mutant) 5 days after induction on Burk-butanol medium.