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Transcriptional Study of the RsmZ-sRNAs and Their Relationship to the Biosynthesis of Alginate and Alkylresorcinols in Azotobacter vinelandii

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Abstract

The GacS/A system in Azotobacter vinelandii regulates alginate and alkylresorcinols production through RsmZ1, a small regulatory RNA (sRNA) that releases the translational repression of the algD and arpR mRNAs caused by the RsmA protein. In the Pseudomonadaceae family, the Rsm-sRNAs are grouped into three families: RsmX, RmsY and RsmZ. Besides RsmZ1, A. vinelandii has six other isoforms belonging to the RsmZ family and another one to the RsmY. Environmental signals controlling rsmsRNAs genes in A. vinelandii are unknown. In this work, we present a transcriptional study of the A. vinelandii rsmZ1–7-sRNAs genes, whose transcriptional profiles showed a differential expression pattern, but all of them exhibited their maximal expression at the stationary growth phase. Furthermore, we found that succinate promoted higher expression levels of all the rsmZ1–7 genes compared to glycolytic carbon sources. Single mutants of the rsmZ-sRNAs family were constructed and their impact on alginate production was assessed. We did not observe correlation between the alginate phenotype of each rsmZ-sRNA mutant and the expression level of the corresponding sRNA, which suggests the existence of additional factors affecting their impact on alginate production. Similar results were found in the regulation exerted by the RsmZ-sRNAs on alkylresorcinol synthesis.

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Funding

Funding was provided by Benemérita Universidad Autónoma de Puebla (Grant No. Apoyo extraordinario a proyectos de investigación VIEP2016-17).

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  1. Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Apdo. Postal 1622, 72000, Puebla, Puebla, México

    Liliana López-Pliego, Liliana García-Ramírez, Emma Aurora Cruz-Gómez, Patricia Domínguez-Ojeda, Alejandra López-Pastrana, Luis Ernesto Fuentes-Ramírez & Miguel Castañeda

  2. Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo Postal 510-3, 62250, Cuernavaca, Morelos, México

    Cinthia Núñez

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  1. Liliana López-Pliego
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Electronic supplementary material

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12033_2018_102_MOESM1_ESM.eps

Supplementary Fig. S1. General diagram of the experimental strategy to construct strain EZ3, carrying a ΔrsmZ3::Gm mutation. The rsmZ3 gene in plasmid pGEMrsmZ3 is indicated by a red square. A similar procedure was followed to construct deletion mutants in genes rsmZ4-7, in some cases using different resistance cassettes. Figures are not at scale. (EPS 2261 KB)

12033_2018_102_MOESM2_ESM.eps

Supplementary Fig. S2. Genomic context and location of the rsmZ1-7 genes in A. vinelandii strain DJ. The rsmZ genes are represented in red; the flanking genes in green and the protein CDS are in cyan. Visualization of the sequences was generated used the software Artemis (Carver T. et al (2012). Bioinformatics 28(4), 464-469). (EPS 2487 KB)

12033_2018_102_MOESM3_ESM.eps

Supplementary Fig. S3. Alignment of the A. vinelandii rsmZ1-7 genes and their regulatory regions. Predicted transcriptional start sites (+1) and the -10 region of the putative promoters are indicated. Location of putative GacA binding boxes and the predicted IHF (yellow) and H-NS (green) recognition sites are indicated. RNAse E cleavage sites are marked in grey squares. (EPS 2699 KB)

12033_2018_102_MOESM4_ESM.eps

Supplementary Fig. S4. Effect of glycolytic or gluconeogenic carbon sources on the activity of a constitutively-expressed promoter. Strain EgyrAT, carrying a transcriptional fusion of the gyrA promoter with the gusA reporter gene (PgyrA-gusA) was grown in liquid Burk’s medium amended with the indicated carbon sources. Cells were collected after 48 h and the activity of ß-glucuronidase was measured. Bars of standard deviation from three independent experiments (biological replicates) are shown. (EPS 674 KB)

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López-Pliego, L., García-Ramírez, L., Cruz-Gómez, E.A. et al. Transcriptional Study of the RsmZ-sRNAs and Their Relationship to the Biosynthesis of Alginate and Alkylresorcinols in Azotobacter vinelandii. Mol Biotechnol 60, 670–680 (2018). https://doi.org/10.1007/s12033-018-0102-7

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