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

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.


Supplementary Methods
Nucleic acid procedures. The A. vinelandii genome sequence 1 was used for designing the primers used for PCR amplifications. The high fidelity Phusion DNA polymerase (Thermo Scientific) was used for all PCR amplifications using chromosomal DNA from strain AEIV as template.
pJGD. Plasmid pMSD27, which carries the algD gene 2 was partially digested with ScaI enzyme disrupting algD in the codon 256; ends were refilled with Klenow enzyme and a Km r cassette, excised with EcoRV endonuclease from plasmid pBSL99 3 , was ligated producing plasmid pJGD (algD::Km).
pJGEY2. cbrA ORF was PCR amplified using oligonucleotides F-1(cbrA) and R-1(cbrA) and was cloned into pMOS blue vector (GE Healthcare) producing plasmid pCN48. An Sp r cassette derived from plasmid pHP45Ω 4 was inserted into the unique StuI site disrupting the cbrA gene. The resulting plasmid, named pJGEY2 (cbrA::Sp). pAH03. A fragment of 1.9 kb was PCR amplified using primers gluP-F and gluP-R and cloned into plasmid pCR2.1-TOPO (Invitrogen) generating plasmid pAH01. The EcoRI gluP fragment of 1.945 kb, derived from plasmid pAH01, was subcloned into vector pBluescript KS + (Stratagene) producing plasmid pAH02. pAH02 was excised with StyI releasing a gluP internal fragment of 600 pb. The StyI cohesive ends were made blunt with the Klenow enzyme and a SmaI fragment carrying a Sp r cassette released from plasmid pHP45Ω 4 was then ligated, generating plasmid pAH03 (gluP::Sp). pEY05. scrX was previously shown to be dispensable for vegetative growth 5 . A region of 3.5 kb containing the gene scrX was PCR amplified using primers scrX-F and scrX-R. The resulting product was sub-cloned into vector pCR2.1-TOPO (Invitrogen) generating plasmid pEY01. An inverse PCR was conducted to delete the regulatory and the structural region of scrX, by using primers scrXInvF and scrXInv-R. The resultant product was digested with enzyme StuI and ligated to a 5 kb fragment containing the crcZ promoter, followed by the gusA gene and a Tc r cassette, generating plasmid pEY05 (PcrcZ-gusA).
The 5 kb fragment was obtained by PCR amplification using primers pJGgusF and pJGgus-R and plasmid pEY02 as DNA template. Plasmid pEY02 is a pCN154 derivative (Tc r ), used for the construction of chromosomal transcriptional fusions with gusA in A. vinelandii strain UW16 6 , and carries an XbaI-EcoRI fragment of 500 bp comprising the regulatory region of crcZ, amplified by PCR using primers pcrcZFXb and pcrcZRRI. pGJ112. A fragment of 246 bp, carrying the crcY regulatory region, was amplified by PCR using primers crcY-Xb-F and crcY-Pst-R, which include restriction sites for XbaI and PstI endonucleases, respectively. This fragment was subcloned into vector pUMATcgusAT 7 previously cut with XbaI and PstI enzymes generating plasmid pGJ112 (PcrcY-gusA).
Expression and purification of A. vinelandii Crc protein: The Crc protein was purified with no His-tag using the Impact-CN intein system (New England Biolabs), as described 8 .
To construct a plasmid specifying the Crc-intein fusion, the crc gene was PCR amplified using AEIV chromosomal DNA as template and primers crc-NdeFw and crc-XhoRv, containing a restriction site for NdeI and XhoI enzymes, respectively. The resulting fragment was cloned between the NdeI-XhoI sites of the P T7 expression vector pTYB1 (New England Biolabs), generating plasmid pEQEcrc. The amplified DNA segment was sequenced to assure the absence of undesired mutations. Plasmid pEQEcrc was transformed into the E. coli EC6779, an Hfq-null derivative of the E. coli T7 expression strain BL21(DE3) 9 , and overproduction of the Crc-intein fusion was induced by addition of 0.5 mM IPTG to cells grown to mid-log phase (A 600 of 0.6). After 4 h at 37°C, cells were collected and disrupted by sonication in 20 mM Tris-HCl, pH 8, 500 mM NaCl, 0.1% Triton X-100, 50 μM PMSF. After eliminating cell debris by centrifugation for 30 min at 20 000 × g at 4°C, the supernatant was loaded onto a chitin column. The column was extensively washed with wash buffer (250 mM NaCl, 50 mM Tris-HCl, pH 7.5). The Crc- This fragment, having a NdeI site overlapping the ATG initiation codon and a XhoI downstream of the termination codon, was sub-cloned between these sites of the T7 expression vector pET22b (Novagen) to generate pET22::Hfq. Purification of A. vinelandii Hfq protein was performed using expression plasmid pET22::Hfq. This plasmid was introduced into EC6779 strain. Hfq-His protein was purified from crude cell lysates of a culture grown to OD 600 of 0.6 prior to overnight induction with 1 mM IPTG by Ni 2+affinity chromatography using a buffer containing 20 mM Tris-HCl, pH 8.0; 300 mM NaCl, 5 mM imidazole and a protease inhibitor cocktail. The Hfq-His protein was Quantitative real time reverse transcription (qRT-PCR). The cells were collected by centrifugation, and the total RNA was extracted as described 10 . Genomic DNA contamination in the total RNA samples was removed with DNase I (Thermo Scientific).
The RNA concentration was measured by 260/280 nm ratio absorbance. RNA integrity was analyzed by agarose gel electrophoresis. The absence of DNA was verified by RT-PCR using primers for rpoS. cDNA was synthesized using the Revert Aid TM H First Strand cDNA Synthesis kit (Thermo Fisher Scientific) and a mixture of the specific DNA primers.
The primers were designed using the Primer3 program (http://bioinfo.ut.ee/primer3/) with an optimal lenght of 20 bases, and a melting temperature of 60 o C. The cDNA generated was used as template for qRT-PCR assays performed with a Light Cycler 480 II instrument (Roche), using the Maxima TM SYBR Green/ROX qPCR Master Mix (2X) kit (Thermo Scientific). Each primer set was validated by verifying specific single product amplification by melting-curve analyses. Then, the efficiency of PCR was assessed by developing standard curves for each amplicon using dilution series of the cDNA corresponding to the reference sample. cDNAs derived from the experimental and reference samples were amplified using quantities within the linear range of the standard curve. Amplification conditions were 10 min at 95° C, and a two-step cycle at 95° C for 15 s and 60° C for 60 s for a total of 40 cycles. The size of all amplimers was from 95 -110 bp. Three biological replicates (independent cell cultures) were performed with three technical replicates for each one generating similar results.     Figure S1. Predicted secondary structure of the A. vinelandii small RNA CrcZ. It was conducted using the RNAfold algorithm (http:/ rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).

Supplementary Tables
The predicted A-rich Hfq-binding motifs 1 to 6 are indicated.  The predicted A-rich Hfq-binding motifs are indicated by red numbers.