Glucose uptake in Azotobacter vinelandii occurs through a GluP transporter that is under the control of the CbrA/CbrB and Hfq-Crc systems

Abstract

Azotobacter vinelandii, a strict aerobic, nitrogen fixing bacterium in the Pseudomonadaceae family, exhibits a preferential use of acetate over glucose as a carbon source. In this study, we show that GluP (Avin04150), annotated as an H⁺-coupled glucose-galactose symporter, is the glucose transporter in A. vinelandii. This protein, which is widely distributed in bacteria and archaea, is uncommon in Pseudomonas species. We found that expression of gluP was under catabolite repression control thorugh the CbrA/CbrB and Crc/Hfq regulatory systems, which were functionally conserved between A. vinelandii and Pseudomonas species. While the histidine kinase CbrA was essential for glucose utilization, over-expression of the Crc protein arrested cell growth when glucose was the sole carbon source. Crc and Hfq proteins from either A. vinelandii or P. putida could form a stable complex with an RNA A-rich Hfq-binding motif present in the leader region of gluP mRNA. Moreover, in P. putida, the gluP A-rich Hfq-binding motif was functional and promoted translational inhibition of a lacZ reporter gene. The fact that gluP is not widely distributed in the Pseudomonas genus but is under control of the CbrA/CbrB and Crc/Hfq systems demonstrates the relevance of these systems in regulating metabolism in the Pseudomonadaceae family.

Figure 1

Genome context and nucleotide sequence of the A. vinelandii CrcZ sRNA. Terminators are indicated by small stem-loops. The six CrcZ A-rich Hfq-binding motifs are indicated by red boxes. The −12 and −24 regions of the predicted RpoN promoter and putative sequences recognized by CbrB (black boxes) are indicated. The predicted −10 and −35 regions of an RpoD promoter are underlined. Inverted repeats at the end of CrcZ are denoted by solid arrows.

Figure 2

The HK CbrA is necessary for glucose utilization. (a) Growth kinetics of the reference strain AEalgD (algD::Km) (closed symbols) and its derivative mutant AH1 (cbrA::miniTn5) (open symbols) in liquid Burk’s minimal medium amended with 50 mM glucose (dashed lines) or 2% sucrose (solid lines). (b) Growth kinetics (circles), and acetate (triangles) or glucose (diamonds) consumption during the culture of the reference strain AEalgD (algD::Km) (closed symbols) or its derivative mutant AH1 (cbrA::miniTn5) (open symbols) in Burk’s minimal medium supplemented with both 30 mM glucose and 30 mM acetate as carbon sources. (c) gluP mRNA levels determined by qRT-PCR analysis in strain AEalgD (white columns) and in mutant AH1 (grey columns) in the diauxic growth of panel (b). Total RNA was extracted from cells at the indicated times. The bars of standard deviation from three independent experiments are shown. Significant differences were analysed by t-test. Statistical significance is indicated (***p < 0.001).

Figure 3

Effect of the HK CbrA on crcZ and crcY gene expression. Expression of crcZ and crcY genes was assessed by PcrcZ-gusA (a) and PcrcY-gusA (b) transcriptional fusions, respectively. These transcriptional fusions were tested in wild type and cbrA genetic backgrounds. The strains used were: AE-Zgus (wild type; black bars) and CbrA-Zgus (cbrA::Sp; white bars) (a); AE-Ygus (wild type; black bars) and CbrA-Ygus (cbrA::Sp; white bars) (b). Cultures were developed in minimal Burk’s-sucrose medium. Aliquots were taken at the indicated times and ß-glucuronidase activity was measured. The bars of standard deviation from three independent experiments are shown. Significant differences were analysed by t-test. Statistical significance is indicated (*p < 0.05, **p < 0.01 or ***p < 0.001).

Figure 4

Hfq-Crc proteins form a stable ribonucleoprotein complex with CrcZ. Ribonucleoprotein complexes formed in the presence of the sRNA CrcZ and increasing concentrations of Hfq (0, 0.02, 0.05, 0.1, 0.2, 0.5 and 1 µM) (a) or in the presence of both Crc and Hfq (b and c). In (d) Crc was substituted by 1 µM of bovine serum albumin (BSA). Crc and Hfq were added at the indicated concentrations (expressed as monomers and hexamers respectively). RNA and protein-RNA complexes were resolved in a non-denaturing polyacrylamide gel. The position of free RNA, and of the ribonucleoprotein (RNP) complexes detected, is indicated.

Figure 5

Crc over-expression diminishes growth on glucose. (a) Growth kinetics of the wild type A. vinelandii AEIV strain, harbouring the empty vector pSRK-Km (■) or the pSRK-crc (crc ⁺) vector, in the presence (●) or absence (○) of 1 mM IPTG. Cultures were developed in Burk’s minimal medium supplemented with 30 mM glucose (BG) as the sole carbon source and 1.5 µgml⁻¹ of kanamycin as a selection marker. 25 ml of Burk’s medium supplemented with 30 mM sucrose were cultured for 18 h; cells were harvested by centrifugation, washed with phosphate buffer 10 mM pH 7.2 and resuspended in the same solution. 400 µg of these cells were used to inoculate 50 ml of BG medium and samples were collected at the indicated times for protein quantification. Cell growth was estimated by determining protein concentration since the production of the exo-polysaccharide alginate by the AEIV strain prevents assessment of growth by optical density. The results represent the averages of the results of three independent experiments, and error bars depict standard deviations. (b) Relative expression levels of gluP mRNA, quantitated by qRT-PCR analysis, in cells of the wild type AEIV strain carrying the vector pSRK-crc in the absence (white colums) or presence (grey columns) of 1 mM IPTG. The growth conditions were as in panel (a). Total RNA was extracted from cells at the indicated times. The bars of standard deviation from three independent experiments are shown. Significant differences were analysed by t-test. Statistical significance is indicated (*p < 0.05 or ***p < 0.001).

Figure 6

The gene gluP of A. vinelandii encodes a glucose transporter. (a) Genomic context of the A. vinelandii gluP gene. (b) Growth of the A. vinelandii glup::Sp mutant AHI30 and its derivative carrying the pSRK-gluP plasmid (gluP ⁺) on plates of solid Burk’s medium supplemented with 2% sucrose (BS) or 50 mM glucose (BG). Where indicated, 1 mM IPTG was added to induce transcription of gluP from the lac promoter. (c) Growth kinetic, measured as cell biomass (g l⁻¹) (squares) and glucose consumption (circles) by the E. coli WHIPC mutant (closed symbols) or its derivative carrying the pSRK-gluP (gluP ⁺) plasmid (open symbols), in the presence of 0.1 mM IPTG. Cultures were developed in M9 mineral medium supplemented with 2.5 g l⁻¹ of glucose.

Figure 7

Hfq-Crc proteins form a stable ribonucleoprotein complex with the A-rich Hfq-binding RNA motif of gluP. (a) Binding of the A. vinelandii Crc and Hfq proteins to an RNA oligonucleotide containing the A-rich Hfq-binding motif present at the translation initiation region from gluP. (b) Molar ratio of the A. vinelandii Crc and Hfq proteins needed to form a ribonucleoprotein complex with the RNA containing the A-rich Hfq-binding gluP motif. As a control an RNA oligonucleotide lacking an A-rich motif was used (c). (d) Binding of the P. putida (Pp) Crc and Hfq proteins to the RNA A-rich Hfq binding motif of gluP. RNA and protein-RNA complexes were resolved in a non-denaturing polyacrilamide gel. The concentration of Crc (expressed as monomers) and Hfq (expressed as hexamers) is indicated. Arrows point to the position of free RNA and of the ribonucleoprotein complex (RNP). (e) Sequence of the gluP mRNA leader region. The underlined sequence corresponds to the RNA oligonucleotide used in the band-shift assays, which contains the A-rich motif. The AUG translation initiation codon is in bold face. The sequence of the oligonucleotide used as control in panel (c), named DmpR 6C, is also shown.

Figure 8

Effect of the Crc protein on the expression of a gluP’-lacZ translational fusion in P. putida. The strains used were KT2440 (wild type) and KTCRC (crc::tet) carrying plasmid pEQ424P (gluP’-lacZ). Cells were grown in LB medium and where indicated, expression of the gluP’-lacZ translational fusion was induced from the Ptrc promoter by addition of 0.5 mM IPTG. (a) Growth kinetic (measured as the turbidity at 600 nm) of each strain. (b) Activity of ß-galactosidase as a function of cell growth. (c) ß-galactosidase activity values of the gluP’-lacZ translational fusion observed at a turbidity of 0.6 (mid-exponential phase) for KT2440 or KTCRC strain. Three independent assays were performed, and a representative one is shown. Significant difference was analyzed by t-test. Statistical significance is indicated (**p < 0.01).