Catabolite repression control of pyocyanin biosynthesis at an intersection of primary and secondary metabolism in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the catabolite repression control (Crc) protein repressed the formation of the blue pigment pyocyanin in response to a preferred carbon source (succinate) by interacting with phzM mRNA, which encodes a key enzyme in pyocyanin biosynthesis. Crc bound to an extended imperfect recognition sequence that was interrupted by the AUG translation initiation codon.     

Molecular call and response: the physiology of bacterial small RNAs

The vital role of bacterial small RNAs (sRNAs) in cellular regulation is now well-established. Although many diverse mechanisms by which sRNAs bring about changes in gene expression have been thoroughly described, comparatively less is known about their biological roles and effects on cell physiology. Nevertheless, for some sRNAs, insight has been gained into the intricate regulatory interplay that is required to sense external environmental and internal metabolic cues and turn them into physiological outcomes. Here, we review examples of regulation by selected sRNAs, emphasizing signals and regulators required for sRNA expression, sRNA regulatory targets, and the resulting consequences for the cell. We highlight sRNAs involved in regulation of the processes of iron homeostasis (RyhB, PrrF, and FsrA) and carbon metabolism (Spot 42, CyaR, and SgrS).     

Translational control and target recognition by Escherichia coli small RNAs in vivo

Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.     

The nucleotide sequence of the Pseudomonas aeruginosa pyrE-crc-rph region and the purification of the crc gene product.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.     

Isolation and phenotypic characterization of Pseudomonas aeruginosa pseudorevertants containing suppressors of the catabolite repression control-defective crc-10 allele

The amiE gene encodes an aliphatic amidase capable of converting fluoroacetamide to the toxic compound fluoroacetate and is one of many genes whose expression is subject to catabolite repression control in Pseudomonas aeruginosa. The protein product of the crc gene, Crc, is required for repression of amiE and most other genes subject to catabolite repression control in this bacterium. When grown in a carbon source such as succinate, wild-type P. aeruginosa is insensitive to fluoroacetamide (due to repression of amiE expression). In contrast, mutants harboring the crc-10 null allele cannot grow in the presence of fluoroacetamide (due to lack of repression of amiE). Selection for succinate-dependent, fluoroacetamide-resistant derivatives of the crc-10 mutant yielded three independent pseudorevertants containing suppressors that restored a degree of catabolite repression control. Synthesis of Crc protein was not reestablished in these pseudorevertants. All three suppressors of crc-10 were extragenic, and all three also suppressed a Delta crc::tetA allele. In each of the three pseudorevertants, catabolite repression control of amidase expression was restored. Catabolite repression control of mannitol dehydrogenase production was also restored in two of the three isolates. None of the suppressors restored repression of glucose-6-phosphate dehydrogenase or pyocyanin production.