The transition from a planktonic (free-swimming) existence to growth attached to a surface in a biofilm occurs in response to environmental factors, including the availability of nutrients. We show that the catabolite repression control (Crc) protein, which plays a role in the regulation of carbon metabolism, is necessary for biofilm formation in Pseudomonas aeruginosa. Using phase-contrast microscopy, we found that a crc mutant only makes a dispersed monolayer of cells on a plastic surface but does not develop the dense monolayer punctuated by microcolonies typical of the wild-type strain. This is a phenotype identical to that observed in mutants defective in type IV pilus biogenesis. Consistent with this observation, crc mutants are defective in type IV pilus-mediated twitching motility. We show that this defect in type IV pilus function is due (at least in part) to a decrease in pilA (pilin) transcription. We propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.

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Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the li-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc-mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc-mutants but exhibited wild-type inducible expression.Pseudomonas aeruginosa utilizes a variety of carbohydrates whose initial catabolism by specific transport systems coupled to enzymes leads to the formation of the central metabolite, 6-phosphogluconate (9). Products from the central pathway are then oxidized by the constitutively expressed tricarboxylic acid (TCA) cycle (Fig. 1). The carbohydrate transport systems and the central pathway enzymes are inducible, and their expression is subject to strong repression when acetate or TCA cycle intermediates are present in the growth medium with the sugar (7, 13). The repression of carbohydrate catabolic pathways by these organic acids enables P. aeruginosa to preferentially utilize TCA cycle intermediates before carbohydrates. Thus, when growth medium includes a limited amount of succinate plus an excess of sugar, P. aeruginosa cultures exhibit a diauxic growth response curve that reflects catabolite repression control of inducible catabolic genes (11).Other inducible catabolic pathways not involved in carbohydrate metabolism are also repressed by growth in the presence of TCA cycle intermediates. Components of these other pathways include amidase (24, 25), aklylsulfatase (2, 3), histidase (17), urocanase (18), protocatechuate 3,4-dioxygenase (27), and choline transport (22).Unlike the glucose effect on catabolite repression found in Escherichia coli and related facultative anaerobes, catabolite repression control of inducible catabolic pathways in P. aeruginosa and Pseudomonas putida does not appear to involve a cyclic-AMP-mediated mechanism (17, 23). While the general phenomenon of catabolite repression in Pseudomonas spp. has been well documented, nothing is known about the molecular basis of this central metabolic control.This report describes the first isolation and characterization of P. aeruginosa mutants that are defective in catabolite repression control of independently inducible regulatory units that encode pathways for the utilization of both carbohydrates and noncarbohydrates.

Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.Pseudomonads play an important role in nature because of their ability to metabolize natural and manufactured organic chemicals. Many of these compounds are environmental pollutants, such as benzene, toluene, xylene, ethylbenzene, styrene, and chlorobenzoates (18), and their removal has been named bioremediation. Although the enzymic pathways responsible for degradation of these pollutants may be effective when the target compound is the sole growth-supporting substrate, in nature these compounds are present as mixtures, and some substrates may be degraded preferentially. Catabolite repression control refers to the ability of an organism to preferentially metabolize one carbon source over another when both are present in the growth medium. Because of the importance of pseudomonads to bioremediation efforts, understanding the control of catabolite repression is important so that more efficient, genetically modified organisms can be utilized in the removal of these environmental pollutants.The molecular mechanisms of catabolite repression control have been extensively characterized in enteric bacteria, where glucose is the preferred carbon source. In these organisms, enzymes of the phosphoenolpyruvate-dependent phosphotransferase system mediate catabolite repression control by regulation of cyclic AMP (cAMP) concentration via adenylate cyclase activity (22). The strongest repressing substrates in Pseudomonas spp. are acetate, tricarboxylic acid cycle intermediates, and glucose (4, 10, 26). Unlike Escherichia coli, in Pseudomonas species adenylate cyclase activity, cAMP phosphodiesterase activity, and cAMP pools do not fluctuate with carbon source, nor does the addition of cAMP relieve repression of catabolite responsive pathways (21, 25). In addition, only one phosphotransferase system (fructose) has been identified in Pseudomonas (5), suggesting that PTS components are not involved in catabolite repression control in pseudomo...