Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa

Abstract

In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.

Fig. 1.

Genetic organization of the amiL-amiE region in the PAO1 chromosome. The amiL nucleotide sequence, the -35 and -10 promoter elements, and the amiE start codon (32) are shown in boldface. Convergent arrows and a stem-loop symbol indicate the ρ-independent terminator of amiL. Vertical arrows indicate the sites of lacZ fusions, followed by designations of the resulting plasmid constructs. The CA motif and the corresponding mutated sequence in pME9657 (shown above the CA motif) are boxed.

Fig. 2.

Posttranscriptional catabolite repression of aliphatic amidase. Bacteria were grown to OD₆₀₀ ≈ 1.5 in minimal medium amended with succinate (gray bars), glucose (white bars), or mannitol (black bars). Media also contained the inducer lactamide. (A) Catabolite repression of amidase was measured by determining specific enzyme activities (in micromoles of acetylhydroxamate formed per minute and OD₆₀₀) in the wild-type PAO1 using an acetyltransferase assay (33). (B) Crc-dependent catabolite repression of amiE does not involve the ami promoter. β-Galactosidase activities of a transcriptional ami-lacZ fusion carried by pME9656 were similar in the wild-type PAO1 and in the crc mutant PAO6673. (C) Crc-dependent catabolite repression of amiE occurs at the translational level. β-Galactosidase activities of a translational amiE′-′lacZ fusion carried by pME9655 varied in parallel to amidase activities. (D) Mutation of the CA motif preceding amiE (in pME9657) results in partial loss of catabolite repression, by comparison with wild-type expression (in pME9655). β-Galactosidase activity of the mutated amiE′-′lacZ fusion was measured in the wild-type PAO1/pME9657 (white bar) and compared with that of the parental amiE′-′lacZ fusion in PAO1/pME9655 (dotted bar) and in PAO6673/pME9655 (hatched bar), after growth in succinate minimal medium to an OD₆₀₀ ≈ 1.5.

Fig. 3.

Location of the crcZ gene (open arrow) between the cbrB and pcnB genes (gray arrows) in P. aeruginosa. The existence of an inversely oriented sRNA gene, P30 (open arrow with dashed lines), has been published (26). ρ-independent terminators are indicated by stem–loop structures. The deletions of the putative P30 sequence (in strain PAO6677) and of the crcZ promoter region (in PAO6679) are indicated by underlining the nucleotide sequence. The nucleotide sequence of crcZ is shown in boldface, and its σ⁵⁴-promoter (-12 and -24 boxes) is shown in boldface and boxed. The five CA motifs are boxed. A possible ρ-independent terminator of P30 is indicated by convergent arrows placed above the sequence. The 3′ end of the in vitro transcribed CrcZ′-RNA used for the band shift experiment (Fig. 6 and Fig. S6) is indicated by an asterisk.

Fig. 4.

Evidence for CrcZ sRNA. Northern blots of the wild-type PAO1 (lane 1), the crcZ-internal deletion mutant PAO6677 (lane 2), and the crcZ promoter mutant PAO6679 (lane 3) were obtained from cells grown in succinate minimal medium to OD₆₀₀ ≈ 2. Ten micrograms of total RNA was loaded onto a 12% polyacrylamide gel, and CrcZ sRNA and fragments thereof were detected with a digoxigenin (DIG)-labeled double-stranded probe (A) or a strand-specific single-stranded probe (B), respectively. The signal of 5S rRNA was used as a loading control.

Fig. 5.

The levels of CrcZ expression vary according to the carbon sources used. (A) Ten micrograms of total RNA purified from the wild-type PAO1, grown either in minimal medium with succinate (S), glucose (G), or mannitol (M) to OD₆₀₀ ≈ 1.5, was used for an estimation of CrcZ levels in a Northern blot experiment, which was performed with a DIG-labeled double-stranded probe. 5S rRNA was used as a loading control. The normalized signals of CrcZ are shown in the graph. (B) β-Galactosidase activities of a chromosomally encoded crcZ-lacZ fusion were measured in strain PAO1 grown to OD₆₀₀ ≈ 1.5 in minimal medium with succinate (gray bar), glucose (white bar), or mannitol (black bar), respectively.

Fig. 6.

CrcZ′ prevents the Crc protein from binding to amiE′ mRNA. Different amounts of Crc were added to radioactively labeled amiE′ mRNA (from left, first four lanes). To an incubation mixture containing a 50-fold molar excess of Crc over amiE′, nonlabeled CrcZ′ was added at the concentrations indicated (next three lanes), showing that CrcZ sequesters Crc from amiE′ mRNA. The same experiment was performed with nonlabeled RsmZ sRNA, where this titrating effect was not observed (last three lanes).

Fig. 7.

Model of CrcZ as an antagonist of Crc in catabolite repression. The concentration of CrcZ changes according to the carbon source. In the presence of a preferred carbon source (e.g., succinate), the level of CrcZ is low and Crc binds to catabolite repression-sensitive mRNAs such as amiE mRNA and thereby blocks ribosome binding. When a nonpreferred substrate source such as mannitol is the sole carbon source, the expression of CrcZ sRNA increases under the control of the CbrA/CbrB two-component system. This results in sequestration of Crc protein by CrcZ and allows ribosome binding and translation of the target mRNAs. With glucose as the sole carbon source, an intermediate amount of CrcZ allows partial sequestration of Crc protein, leading to moderate expression of target mRNAs.