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Study of the sRNA RsmY involved in the genetic regulation of the synthesis of alginate and alkyl resorcinols in Azotobacter vinelandii

  • Liliana López-Pliego
  • Giselda Mena-Muñoz
  • Juan L. Terán-Melo
  • Luis E. Fuentes
  • Cinthia E. Núñez
  • Miguel CastañedaEmail author
Original Paper
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Abstract

Azotobacter vineladii is a Gram-negative bacterium that produces alginate and poly-hydroxybutyrate (PHB), two polymers of biotechnological interest. This bacterium has the ability to form desiccation-resistant cysts. In the cyst the membrane phospholipids are replaced with a family of phenolic lipids called alkylresorcinols (ARs). The alginate, PHB, and ARs are controlled by the GacS/A two-component system and the small regulatory RNA (sRNA) RsmZ1, belonging to the Rsm (Csr) regulatory system. The Rsm (Csr) systems usually possess two or more sRNAs, in this regard A. vinelandii is the bacterium with the highest number of rsm-sRNAs. Originally, the presence of two sRNAs of the RsmY family (RsmY1 and RsmY2) was reported, but in a subsequent work it was suggested that they conformed to a single sRNA. In this work we provide genetic evidence confirming that rsmY1 and rsmY2 constitute a single gene. Also, it was established that rsmY mutation decreased alginate and ARs production, but did not affect the PHB synthesis. Transcriptional studies showed that rsmY has its higher expression during the stationary growth phase, and in the absence of RsmZ1, rsmY increases its transcription. Interestingly, rsmY expression was influenced by the carbon source, but its expression did not correlate with alginate production.

Keywords

Azotobacter rsmY GacS/A Alginate Alkylresorcinols 
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Notes

Acknowledgements

This work was supported by VIEP-BUAP, grant 100301900-VIEP 2018–19. L. López-Pliego, G. Mena-Muñoz Huerta and JM Terán-Melo thanks CONACyT for Ph. D. and M.Sc. scholarships.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

203_2019_1769_MOESM1_ESM.pptx (227 kb)
Construction of pUMAPgyrKm. To generate the expression vector pUMAPgyrKm, first, a 2 kb fragment which contains the lacI gene, a tac promoter, a multicloning site, and in tandem the rrnBT1 and rrnBT2 transcriptional terminators were amplified by PCR. To carry out this PCR reaction was used as template the plasmid pYanni3 (Graupner and Wackernagel 2000) and the primers TacFw and TacRv, which possesses a cleavage site XhoI. The PCR product previously digested with XhoI was cloned in the single XhoI present in the pUMA vector (a) (Cocotl et al. 2014) generating the pUMAPtac plasmid (b). To generate the pUMAPgyrKm the lacI gene and the promoter tac were removed from the pUMAPtac using the restriction enzymes XhoI and EcoRI and were replaced with the constitutive promoter gyrA (Pgyr), the resulting plasmid was called pUMAPgyr (c). The Pgyr was amplified with the primers gyrAFXhoI and gyrAREcoRI using chromosomal DNA from the strain E as a template. Later the pUMAPgyr was partially digested with XhoI and a Km resistance cassette, obtained from the pBSL99 plasmid (Alexeyev et al. 1995), was ligated, and a plasmid with the resistance gene inserted near to Pgyr and in opposite direction was selected (d) (PPTX 227 kb)
203_2019_1769_MOESM2_ESM.pptx (100 kb)
Fig. S2 Sequence of the rsmYlocus in A. vinelandii. The rsmY genes (rsmY1 and rsmY2) predicted and annotated in Gene bank and Rfam databases are indicated as arrows in the figure. The new rsmY gene (rsmY1Y2) is also represented as an arrow. The GacA box and the -10 promoter elements are indicated. The asterisks show the identical nucleotides with the consensus reported for GacA binding box and the promoters of rsm sRNAs genes in Pseudomonas spp. (PPTX 99 kb)

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© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Centro de Investigaciones en Ciencias Microbiológicas, Instituto de CienciasBenemérita Universidad Autónoma de PueblaPueblaMexico
  2. 2.Departamento de Microbiología Molecular, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico

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