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

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.

Fig 1. Transcriptional regulation of crcZ and crcY in diazotrophic conditions.

A. Growth kinetic (circles) and acetate (triangles) or glucose (squares) consumption of the A. vinelandii wild type strain AEIV cultured in Burk’s minimum medium supplemented with 30 mM acetate and 30 mM glucose (BAG medium). B. Activity of the promoters for crcZ and crcY. Strains AE-Zgus and AE-Ygus, carrying PcrcZ-gusA (circles) and PcrcY-gusA (triangles) transcriptional fusions, respectively, were cultured in 25 mL of Burk’s medium amended with 30 mM acetate for 12 h; Afterward, the same amount of cell culture (corresponding to 200 μg of protein) was used to inoculate 50 ml of BAG medium. Cells were harvested along the growth curve and the activity of ß-glucuronidase (ß-Gluc) was determined. C. Quantification of CrcZ, CrcY and gluP transcripts by qRT-PCR analysis. The total RNA was extracted from cells growing in diauxic BAG medium at the expense of acetate (5h; gray bars) or glucose (20 h; black bars). The bars of standard deviation from three independent experiments are shown. In panel A these bars are not visible since they are smaller than the symbols used. Significant differences were analyzed by t-test. Statistical significance is indicated (**p<0.01).

Fig 2. Quantification of the CrcZ and CrcY levels.

Relative levels of the CrcZ and CrcY sRNAs, measured by qRT-PCR in cells of the A. vinelandii AEIV strain growing exponentially at the expense of acetate (gray bars) or glucose (black bars) in BAG medium in the absence (diazotrophy) or presence (non-diazotrophy) of 15 mM NH₄Cl. The values are expressed as RNA copies relative to those of gyrA mRNA (internal standard).

Fig 3. Transcriptional regulation of CrcZ and CrcY in non-diazotrophic conditions.

A. Growth kinetic (circles) and acetate (triangles) or glucose (squares) consumption of the A. vinelandii wild type strain AEIV cultured in Burk’s minimum medium supplemented with 30 mM acetate, 30 mM glucose and 15 mM NH₄Cl (BAG-N medium). B. Activity of the crcZ and crcY promoters. Strains AE-Zgus and AE-Ygus, carrying PcrcZ-gusA (circles) and PcrcY-gusA (triangles) transcriptional fusions, respectively, were cultured in 25 mL of Burk’s medium amended with 30 mM acetate for 12 h; then, 50 ml of BAG medium was inoculated with the same amounts of cells (corresponding to 200 μg of protein). Cells were harvested along the growth curve and the activity of ß-glucuronidase (ß-Gluc) was determined. C. Quantification of CrcZ, CrcY and gluP transcripts by qRT-PCR analysis. The total RNA was extracted from cells growing in diauxic BAG-N medium at the expense of acetate (5h; gray bars) or glucose (15 h; black bars). The bars of standard deviation from three independent experiments are shown. In panel A these bars are not visible since they are smaller than the symbols used. Significant differences were analyzed by t-test. Statistical significance is indicated (**p<0.01 or ***p<0.0001).

Fig 4. Transcriptional regulation of CrcZ and CrcY sRNAs.

A. DNA sequence of the regulatory region of crcZ. The transcriptional initiation site is indicated (+1), along with the predicted σ⁷⁰ and σ⁵⁴ promoters. The putative sequences recognized by CbrB are indicated. B. Identification of the transcription initiation site of crcZ. The primer extension analysis was conducted with RNA extracted from cells grown in BAG (lanes 1 and 2) or BAG-N (lanes 3 and 4) medium, during growth at the expense of acetate (lanes 1 and 3) or glucose (lanes 2 and 4). A primer complementary to crcZ was used and its sequence is indicated in panel A. The cDNA obtained was resolved in a denaturing poly-acrylamide gel, side by side with DNA sequence ladders obtained by chemical sequencing of crcZ. The transcriptional initiation site is indicated by an arrow. A second signal, corresponding to a transcript 13 nt longer than the primary one, is also indicated (*). C. Quantification of CrcZ and CrcY transcripts by qRT-PCR analysis in the wild type train AEIV (wt) and in its derivative cbrA::Sp mutant EQR02 (cbrA), grown in diauxic BAG medium at the expense of acetate (5h) or glucose (20 h). The bars of standard deviation from three independent experiments are shown. Significant differences were analyzed by t-test. Statistical significance is indicated (***p<0.001).

Fig 5. Transcriptional regulation of CrcZ and CrcY by RpoN.

A. Growth kinetic (circles) and acetate (triangles) or glucose (squares) consumption by the AErpoN (rpoN::Gm) strain cultivated in BAG-N medium. B. The activity of PcrcZ and PcrcY is RpoN-dependent. Strains AE-Zgus (PcrcZ-gusA) (wt) and AE-Ygus (PcrcY-gusA) (wt), and their respective rpoN::Gm derivatives (rpoN) were cultured in 25 mL of Burk’s medium amended with 30 mM acetate for 12 h, afterward 200 μg of protein derived from these pre-inoculums were used to inoculate 50 ml of BAG-N medium. Cells were harvested under acetate growing conditions (5h) and the activity of ß-glucuronidase (ß-Gluc) was determined. C. Quantification of CrcZ and CrcY transcripts by qRT-PCR analysis in the wild type strain AEIV (black bars) or in AErpoN strain (white bars). The total RNA was extracted from cells growing in diauxic BAG-N medium at the expense of acetate (5h). The bars of standard deviation from three independent experiments are shown. Significant differences were analyzed by t-test. Statistical significance is indicated (**p<0.01 or ***p<0.0001).

Fig 6. Identification of a cbrB-crcZ co-transcript in A. vinelandii.

A. Representation of the cbrB-crcZ locus in A. vinelandii. The positions of the promoters is indicated, along with the oligonucleotides used for the reverse transcription-PCR (RT-PCR) assay of panel B. B. Identification of crcZ transcripts by RT-PCR. The wild-type strain AEIV was cultured in BAG medium with or without ammonium (NH₄⁺), under acetate (Ac) or glucose (Gl) consumption. The total RNA was purified and used to generate cDNA using an oligonucleotide annealing within crcZ (Zrv1). The cDNA was PCR amplified with primer pairs Zfw2/Zrv1 or Zfw1/Zrv1, generating products of 330 and 100 bp, respectively. Control reactions using genomic DNA (lanes 1 and 7) or RT-PCR reactions in the absence of cDNA (lanes 2 and 8) are shown. M, DNA Molecular Weight Marker. C. The activity of the cbrB promoter (PcbrB) is RpoN-independent. Strain JG513 (PcbrB-gusA; wt) and its rpoN^- derivative AErpoNBgus, were cultured in BAG-N medium. Cells were harvested under acetate (5h) or glucose growing conditions (15 h), and the activity of ß-glucuronidase (ß-Gluc) was determined. D. Sensitivity of crcZ transcripts from the wild type strain AEIV (wt) or its isogenic rpoN^- mutant to the TEX enzyme. crcZ RT-PCR reactions were performed as in panel B, using primers pair Zfw1/Zrv1, and total RNA extracted from cells grown in BAG-N medium for 5h. When indicated, prior to the generation of the cDNA, RNA samples were treated with TEX. The amount of cDNA in nanograms used as a template for the PCR reaction in each experimental condition is indicated at the top. RT-PCR reactions using 20 nanograms of cDNA derived from the gyrA mRNA was used as an internal control, using primer pair gyrAfw/gyrArev (100 bp amplicon). Control reactions using genomic DNA (C+) or RT-PCR reactions in the absence of cDNA (C-) are shown. M, DNA molecular weight marker. The assay was repeated twice obtaining essentially the same results.

Fig 7. Growth of A. vinelandii strains on solid Burk’s medium.

The wild type strain AEIV (1) and its isogenic RpoN- (2) and CbrB-null mutants (3) were cultured on plates of minimum Burk’s medium amended with 30 mM of acetate, fumarate or malate. The plates were incubated at 30°C /48 h.

Fig 8. Model for the regulation of CCR in A. vinelandii, in diauxic glucose-acetate growth.

A. The two-component system CbrA/CbrB is necessary for the expression of crcZ and crcY sRNAs genes from their RpoN-dependent promoters. A variant of CrcZ (CrcZ*) is produced from the processing of a cbrB-crcZ co-transcript, which is expressed from PcbrB. CrcZ, CrcZ* and CrcY sRNAs antagonize the translational repressing effect of Hfq-Crc on target genes. The phosphate groups at the 5’ end of crcY, crcZ and crcZ* transcripts are represented by yellow circles B. In the presence of both, acetate and glucose, translation of the gluP mRNA, encoding the glucose transporter, is inhibited by Hfq-Crc. C. This repression is alleviated as a consequence of the activation of the two-componente system CbrA/CbrB once the acetate is consumed, resulting in higher levels of CrcZ and CrcY, which sequester Hfq-Crc. For simplicity, other Hfq-Crc targets during glucose CCR are not shown [5]. Only three out of the six A-rich Hfq binding motifs of CrcZ/Y are represented to bind Hfq-Crc.