Interplay between the catabolite repression control protein Crc, Hfq and RNA in Hfq-dependent translational regulation in Pseudomonas aeruginosa

Abstract In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) act as post-transcriptional regulators during carbon catabolite repression (CCR). In this regard Crc is required for full-fledged Hfq-mediated translational repression of catabolic genes. RNAseq based transcriptome analyses revealed a significant overlap between the Crc and Hfq regulons, which in conjunction with genetic data supported a concerted action of both proteins. Biochemical and biophysical approaches further suggest that Crc and Hfq form an assembly in the presence of RNAs containing A-rich motifs, and that Crc interacts with both, Hfq and RNA. Through these interactions, Crc enhances the stability of Hfq/Crc/RNA complexes, which can explain its facilitating role in Hfq-mediated translational repression. Hence, these studies revealed for the first time insights into how an interacting protein can modulate Hfq function. Moreover, Crc is shown to interfere with binding of a regulatory RNA to Hfq, which bears implications for riboregulation. These results are discussed in terms of a working model, wherein Crc prioritizes the function of Hfq toward utilization of favored carbon sources.


Plasmids pUT18Chfq and pUT18Ccrc. For construction of N-terminal fusions
between the T18 domain of CyaA (corresponds to cyaA nt +673 to nt +1197 with regard to the A (+1) of the start codon) with PAO1 Hfq and Crc, a 299-bp fragment of hfq (nt -1 to nt +298 with regard to the A (+1) of the start codon) and a 847-bp fragment of crc (nt -1 to nt +846 with regard to the A (+1) of the start codon) were amplified by PCR using oligonucleotides X120 (5´-TTT TTT TCT GCA GCA TGT CAA AAG GGC ATT CGC TAC-3´) and F120 (see above) for hfq and W120 (5´-TTT TTT TCT GCA GTA TGC GGA TCA TCA GTG TGA AC-3´) and U117 (see above) for crc and chromosomal DNA of PAO1. The respective PCR fragment were cleaved with EcoRI and PstI and ligated into the corresponding sites of plasmid pUT18C.
Plasmid pET26bII-Crc. A 777 bp fragment of crc (nt +1 to nt +777 with regard to the A (+1) of the start codon) was amplified by PCR using oligonucleotides 5´-ATA TAC ATA TGC GGA TCA TCA GTG TGA ACG-3´ and 5´-TAT ATC TCG AGG ATG CTC AAC TGC CAG TCG-3´ and chromosomal DNA of PAO1. The PCR fragment was then cleaved with NdeI and XhoI and ligated into the corresponding sites of plasmid pET26bII. Plasmid pME9679. To construct a PAO1 in frame chromosomal deletion mutant in the crcZ gene the following procedure was used. First, two PCR products were obtained using primer pairs U1 (5´-ACG TGG ATC CAC CGC GAC CTG AAA ACC C-3´)/V1 (5´-CGG TGG GTC GGC GGA GGG CAC-3´) and W1 (5´-GTG CCC TCC GCC GAC CCA CCG ACT TGG GGG GGA GCT TCG G-3´)/X1 (5´-ACG TGA ATT CGG CGC GGA CCT GC-3´) and chromosomal DNA of PAO1, respectively. The annealed 666-bp upstream and 659-bp downstream fragments were used as a template for a second overlapping PCR with primers U1 and X1 (V1 and W1 contain a complementary sequences). The resulting fragment -with a 408-bp deletion in crcZ, which spans the promoter region of crcZ and the majority of the crcZ 12% SDS gels. The fractions containing Hfq, Crc and RNA were selected for the SEC-MAL experiment. 0.5 mg/ml BSA was used as a control.
Isolation of PAO1ΔcrcZ revertants. The strains PAO1ΔcrcZ (PAO6679) and PAO1ΔcrcZ ab (PAO6713) containing either a promoter deletion or an entire deletion of the crcZ gene were pre-incubated in BSM medium supplemented with 40 mM succinate. The cells were washed in saline and approximately 10 8 cells were spread on plates containing 40 mM succinate, acetamide, mannitol, L-histidine and D/L-alanine, respectively. The plates were incubated at 37°C until single colonies appeared. The regions encompassing the hfq (corresponded to nt -563 to nt +318 according to A (+1) of the start codon) and crc (corresponded to nt -135 to nt + 971) genes of the revertants were amplified by PCR using primer pairs Q15 (5'-TTT TTT TTT TGG ATC CTC GGC GGG GTG TCG-3') and Q85 (5'-CCC TTC CAG ATG CAC CAG-3') for hfq and U3 (5'-GCT GGT GGT GAT CGG CTT C-3') and T3 (5'-GCA GAA CCC CGC GCT CG-3') for crc, respectively. The resulting PCR fragments were sequenced (barcode economy run, Microsynth) and compared with the wt sequence using Align Sequences Nucleotide BLAST (NCBI).
Qualitative determination of substrate utilization. Overnight cultures of PAO1, PAO1Δhfq, PAO1Δcrc and PAO1ΔcrcZ grown in BSM amended with 40 mM succinate were washed and diluted in 0.9% (wt/vol) NaCl to an OD 600 of 0.045. 150 µl of the dilutions was used to inoculate each well of the GN2 MicroPlate TM (Biolog). The microplate was incubated at 35°C and rotated at 500 rpm in a THERMOstar+ apparatus (BMG Labtech). Each of the 96 wells of the plates contains a carbon source in addition to tetrazolium salts, which are reduced during respiration of the carbon source. This allows a growth independent visual screening of the carbon sources that were respired. After 24 and 48 hours changes in colour were determined visually.
Determination of the intracellular Crc concentration. PAO1 was grown in BSM medium supplemented with 40 mM succinate to an OD 600 of 2.0. The colony forming units (CFU)/ml were determined by plating serial dilutions on LB plates. To determine the Crc concentration three samples withdrawn from three individual cultures (corresponding to 50µl culture of PAO1 at an OD 600 of 2.0). The samples were centrifuged and resuspended in protein loading buffer. Either sample was separated on a 12% SDS-polyacrylamide gel together with 0.5, 1 and 2 pmol of purified Crc protein. The proteins were then electro-blotted to a nitrocellulose membrane. The blot was blocked with 5% dry milk in TBS buffer, followed by probing with rabbit anti-Crc (Pineda) antibody. The antibody-antigen complexes were visualized with alkaline-phosphatase conjugated secondary antibodies (Sigma) using the chromogenic substrates nitro blue tetrazolium chloride (NBT) and 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and analysed by ImageQuant software. The Crc concentrations were determined with the aid of defined amounts of Crc protein loaded onto the gel. The Crc concentration(s) were then normalized to the CFU/ml and multiplied with the Avogadro constant resulting in the amount of Crc molecules/cell.

Volume (ml) Molar mass (g/mol)
10.5 11.5 11.0 2x10 5 3x10 5    . CCR is alleviated in PAO1ΔcrcZ revertants. Single colonies of the strains PAO1, PAO1ΔcrcZ, PAO1ΔcrcZ sup34 , PAO1ΔcrcZ sup2b7 , PAO1ΔcrcZ sup29 , PAO1ΔcrcZ supA , PAO1ΔcrcZ supE and PAO1ΔcrcZ supG , respectively, were inoculated in 100 µl of BSM medium supplemented with either 40 mM succinate, histidine, alanine, acetamide or mannitol (the colour code of the bars are shown at the right). The strains were incubated at 37°C under shaking. After 24 h the OD 600 was measured with an iMark Tm Microplate reader (Biorad). The results represent data from two independent experiments and are shown as mean ± standard deviation.  and T18 of adenylate cyclase, respectively, were constructed as described in Text S1. The E. coli strain BTH101 was co-transformed with plasmids encoding the respective fusion proteins as indicated below the orange bars. White bar, background production of β-galactosidase in E. coli BTH101(pUT18, pKT25) harbouring the parental plasmids. Black bar, co-synthesis of T25-Crc and Hfq-T18 results in reconstitution of the cyclase activity. The results of three independent experiments were averaged and are shown as mean ± standard deviation.    Figure S9. Determination of the intracellular Crc concentration. Strain PAO1 was grown to an OD 600 of 2.0 in BSM medium supplemented with 40 mM succinate (= 3.2 ± 0.3 10 9 CFU/ml). The Crc concentration was determined in triplicate samples of PAO1 cell lysates corresponding to 50 µl of culture (lanes 1-3) using quantitative western-blotting with Crc specific antibodies. Lanes 4-6, 0.5, 1 and 2 pmol of purified Crc protein were loaded, respectively. The Crc concentration per cell was determined as described in Text S1.