Expression of the sRNAs CrcZ and CrcY modulate the strength of carbon catabolite repression under diazotrophic or non-diazotrophic growing conditions in Azotobacter vinelandii.

Marcela Martínez-Valenzuela,Soledad Moreno,Josefina Guzmán,Cinthia Núñez,Guadalupe Espín,Carlos Leonel Ahumada-Manuel

doi:10.1371/journal.pone.0208975

Marcela Martínez-Valenzuela, Soledad Moreno + Show 4 more

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https://doi.org/10.1371/journal.pone.0208975

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Abstract

Azotobacter vinelandii is a nitrogen-fixing bacterium of the Pseudomonadaceae family that prefers the use of organic acids rather than carbohydrates. Thus, in a mixture of acetate-glucose, glucose is consumed only after acetate is exhausted. In a previous work, we investigated the molecular basis of this carbon catabolite repression (CCR) process under diazotrophic conditions. In the presence of acetate, Crc-Hfq inhibited translation of the gluP mRNA, encoding the glucose transporter in A. vinelandii. Herein, we investigated the regulation in the expression of the small non-coding RNAs (sRNAs) crcZ and crcY, which are known to antagonize the repressing activity of Hfq-Crc. Our results indicated higher expression levels of the sRNAs crcZ and crcY under low CCR conditions (i.e. glucose), in relation to the strong one (acetate one). In addition, we also explored the process of CCR in the presence of ammonium. Our results revealed that CCR also occurs under non-diazotrophic conditions as we detected a hierarchy in the utilization of the supplied carbon sources, which was consistent with the higher expression level of the crcZ/Y sRNAs during glucose catabolism. Analysis of the promoters driving transcription of crcZ and crcY confirmed that they were RpoN-dependent but we also detected a processed form of CrcZ (CrcZ*) in the RpoN-deficient strain derived from a cbrB-crcZ co-transcript. CrcZ* was functional and sufficient to allow the assimilation of acetate.

Highlights

Azotobacter vinelandii is a gamma Proteobacterium member of the Pseudomonadaceae family
These strains are Expression of the sRNAs CrcZ and CrcY during carbon catabolite repression in Azotobacter vinelandii derivatives of the wild type strain AEIV, carrying transcriptional fusions of the crcZ and crcY promoters (PcrcZ and PcrcY, respectively) with the gusA reporter gene [5]. They were cultured in BAG medium, and the activity of ß-glucuronidase was measured along the growth curve
As anticipated, during the first 10 h, cell growth occurred at the expense of acetate; thereafter, glucose assimilation started and growth continued until it reached a concentration of approximately 140μg of protein mL-1 (Fig 1A)

Summary

Introduction

Azotobacter vinelandii is a gamma Proteobacterium member of the Pseudomonadaceae family. The genus Azotobacter is characterized by its ability to develop a differentiation process under adverse growth conditions. Expression of the sRNAs CrcZ and CrcY during carbon catabolite repression in Azotobacter vinelandii role in study design, data collection and analysis, decision to publish or preparation of the manuscript

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