We examined the properties of mutants of E. coli which are defective with respect to nitrate reductase activity. chlE::Mu cts and chlG::Mu cts mutants were all chlorate resistant, and the strains that we examined all synthesized nitrate reductase apoenzyme. We concluded that the chlE and chlG loci, like the chlA, chlB, and chlD loci, are involved in the synthesis or insertion of molybdenum cofactor. We identified at least four distinct phenotypic classes of chlC::TnlO mutants, all of which were fully or partially sensitive to chlorate. Two of these classes may represent lesions in the structural genes for nitrate reductase subunits A and C. Two other classes may be altered in the regulation of the expression of nitrate reductase or other anaerobic enzymes. We propose the mnemonic nar for naming individual genes within the chlC locus.

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...

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.The genus Pseudomonas is noteworthy for its diversity in habitat and physiology. Some Pseudomonas strains are able to use over 100 organic compounds as the sole or principal source of carbon. Pseudomonas aeruginosa can utilize at least 80 different organic compounds (41). A mechanism of catabolite repression control exists in these organisms (18) which prevents them from wasting energy maintaining the enzymes for all these catabolic pathways and ensures the preferential utilization of the most efficient source of carbon and energy. Such a regulatory mechanism has also been identified in the enteric bacteria (20) and in Bacillus spp. (6).In the enteric bacteria, the molecular mechanism of catabolite repression control involves a catabolite activator protein (Cap) which, when bound to cyclic AMP (cAMP), interacts with promoter regions of regulated genes to facilitate the binding of RNA polymerase, thereby initiating transcription (20). In the presence of glucose, the cAMP pool is lowered, Cap is not bound, and the regulated genes are not transcribed (i.e., they are repressed). The effect of glucose on cAMP pools is mediated by components of the phosphoenolpyruvate phosphotransferase system which also serve to activate adenylate cyclase (33). Since the initial identification of these regulatory components, catabolite repression has proven to be a global mechanism in the enteric bacteria, affecting at least 28 separate promoters which regulate biosynthetic as well as catabolic operons (7). It also has been found to act as a negative regulator as well as a positive regulator (1,7,23,24).In Bacillus spp. catabolite repression is not mediated by glucose, nor is cAMP involved (6,37,40). Genetic evidence has implicated the catabolite control protein (CcpA), a member of the GalR family of repressor proteins (6), Hpr, a component of the phosphoenolpyruvate phosphotransferase system (9), and a cis-acting DNA sequence (CRE) (15,48). Recent work has shown that HPr specifically phosphorylated at Ser-46 forms a complex wit...

Vfr of Pseudomonas aeruginosa is 91 % similar to the cAMP receptor protein (CRP) of Escherichia coli. Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P. aeruginosa as CRP did for E. coli. This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P. aeruginosa. In a vfr101 ::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain. In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101 ::aacC1 mutant. Thus, a mutation in vfr affected production of at least 60 proteins in P. aeruginosa. In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed. To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (K d ) for binding to cAMP were determined. The K d values were 16 µM for Vfr and 04 µM for CRP, suggesting that these proteins have a similar affinity for cAMP. Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E. coli. This study presents evidence that Vfr binds to the E. coli lac promoter and that this binding requires the presence of cAMP. Finally, the possible involvement of Vfr in catabolite repression control in P. aeruginosa was investigated. It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants. This implied that catabolite repression control was not affected by the vfr null mutation. In support of this, the cloned vfr gene failed to complement a mutation in the P. aeruginosa crc gene. Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P. aeruginosa, Vfr is not required for catabolite repression control in this bacterium.