The cyo operon of Pseudomonas putida is involved in carbon catabolite repression of phenol degradation

A bicistronic reporter consisting of the promoterless genes aacC1 (conferring gentamycin resistance) and lacZ fused to the catabolic promoter of the phenol degradation genes was used to identify and analyse mutants of Pseudomonas putida with altered carbon catabolite repression (CR) of phenol degradation. Out of approximately 2500 mini-Tn5 mutants analysed so far, 12 mutants that were resistant to gentamycin during growth on succinate were identified. In eight of these mutants mini-Tn5 was inserted into one of the genes of the cyo operon. The cyo operon encodes the cytochrome o ubiquinol oxidase, the terminal oxidase of the cyanide-sensitive branch of the respiratory chain. In these mutants the activity of the PphlA promoter was significantly increased during growth on succinate and reached 15-20% of that found during growth with the non-repressing carbon source pyruvate. During growth on glucose the reduction of CR was less obvious, during growth on lactate CR was unchanged. The possible significance of the cyo operon for the generation of signal(s) for carbon catabolite repression is discussed.     

Isolation and characterization of catabolite repression control mutants of Pseudomonas aeruginosa PAO.

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 11-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.     

Induction specificity and catabolite repression of the early enzymes in camphor degradation by Pseudomonas putida

The ability of bornane and substituted bornanes to induce the early enzymes for d(+)-camphor degradation and control of these enzymes by catabolite repression were studied in a strain of a Pseudomonas putida. Bornane and 20 substituted bornane compounds showed induction. Of these 21 compounds, bornane and 8 of the substituted bornanes provided induction without supporting growth. Oxygen, but not nitrogen, enhanced the inductive potency of the unsubstituted bornane ring. All bornanedione isomers caused induction, and those with substituents on each of the three consecutive carbon atoms, including the methyl group at the bridgehead carbon, showed induction without supporting growth. Although it was not possible to obtain experimental data for a case of absolute gratuitous induction by compounds not supporting growth, indirect evidence in support of gratuitous induction is presented. It is proposed that the ability of P. putida to tolerate the unusually high degree of possible gratuitous induction observed for camphor catabolism may be related to the infrequent occurrence of bicyclic ring structures in nature. Survival of an organism with a broad specificity for gratuitous induction is discussed. Glucose and succinate, but not glutamate, produced catabolite repression of the early camphor-degrading enzymes. Pathway enzymes differ in their degree of sensitivity to succinate-provoked catabolite repression. The ability of a compound to produce catabolite repression is not, however, directly related to the duration of the lag period (diauxic lag) between growth on camphor and growth on the repressing compound.     

Cyclic adenosine 3',5'-monophosphate levels in Pseudomonas putida and Pseudomonas aeruginosa during induction and carbon catabolite repression of histidase synthesis.

Inducibility of histidase (histidine ammonia-lyase, EC 4.3.1.3) in Pseudomonas putida and Pseudomonas aeruginosa was observed to be strongly affected by succinate-provoked catabolite repression, but this did not occur as a consequence of reduced intracellular cyclic adenosine 3',5'-monophosphate levels, and repression could not be alleviated by exogenously added cyclic adenosine 3,'5'-monophosphate. Milder repression of histidase by lactate was also not reversed by the addition of cyclic adenosine 3',5'-monophosphate. These results, along with data showing intracellular cyclic adenosine 3',5'-monophosphate levels remained essentially constant during growth on such diverse carbon sources as histidine, acetamide, glucose, and succinate, indicated that catabolite repression of histidase synthesis by efficient carbon sources was not mediated through variations in internal cyclic adenosine 3,'5'-monophosphate.     

Regulatory exaptation of the catabolite repression protein (Crp)-cAMP system in Pseudomonas putida

The genome of the soil bacterium Pseudomonas putida KT2440 encodes singular orthologues of genes crp (encoding the catabolite repression protein, Crp) and cyaA (adenylate cyclase) of Escherichia coli. The levels of cAMP formed by P. putida cells were below detection with a Dictyostelium biosensor in vivo. The cyaA(P. putida) gene was transcribed in vivo but failed to complement the lack of maltose consumption of a cyaA mutant of E. coli, thereby indicating that cyaA(P. putida) was poorly translated or rendered non-functional in the heterologous host. Yet, generation of cAMP by CyaA(P. putida) could be verified by expressing the cyaA(P. putida) gene in a hypersensitive E. coli strain. On the other hand, the crp(P. putida) gene restored the metabolic capacities of an equivalent crp mutant of E. coli, but not in a double crp/cyaA strain, suggesting that the ability to regulate such functions required cAMP. In order to clarify the breadth of the Crp/cAMP system in P. putida, crp and cyaA mutants were generated and passed through a battery of phenotypic tests for recognition of gross metabolic properties and stress-endurance abilities. These assays revealed that the loss of each gene led in most (but not all) cases to the same phenotypic behaviour, indicating a concerted functionality. Unexpectedly, none of the mutations affected the panel of carbon compounds that can be used by P. putida as growth substrates, the mutants being impaired only in the use of various dipeptides as N sources. Furthermore, the lack of crp or cyaA had little influence on the gross growth fingerprinting of the cells. The poor physiological profile of the Crp-cAMP system of P. putida when compared with E. coli exposes a case of regulatory exaptation, i.e. the process through which a property evolved for a particular function is co-opted for a new use.     

PanB is involved in nicotine metabolism in Pseudomonas putida

A nicotine-sensitive mutant was generated from the nicotine-degrading bacterium, Pseudomonas putida strain J5, by mini-Tn5 transposon mutagenesis. This mutant was unable to grow with nicotine as the sole carbon source but could grow with glucose. Sequence analysis showed that the Tn5 transposon inserted at the site of the ketopantoate hydroxymethyltransferase gene (panB), which had 54% identity to PanB in Escherichia coli K-12. In-frame deletion of the panB gene abolished the nicotine-degrading ability of strain J5, while complementation with panB from P. putida J5 and E. coli K-12 restored the degrading activity of the mutant to the wild-type level. These results suggest that ketopantoate hydroxymethyltransferase is a crucial enzyme in nicotine metabolism in P. putida J5.     

Genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in Pseudomonas putida TMB.

The catabolic pathway for the degradation of aromatic hydrocarbons encoded by Pseudomonas putida TMB differs from the TOL plasmid-encoded pathway as far as regulation of the upper pathway is concerned. We found, by analyzing Tn5-induced mutants and by Southern blot hybridization with appropriate probes derived from the TOL plasmid pWW0, that the catabolic genes of strain TMB were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain. The catabolic genes of TMB and pWW0 had sequence homology, as shown by Southern blot hybridization, but differed significantly in their restriction patterns. The analysis of the mutants suggests that a regulatory mechanism similar to that present in pWW0 coexists in TMB with a second mode of regulation which is epistatic on the former and that the chromosomal region carrying the catabolic genes is prone to rearrangements and deletions.     

Role of the ptsN gene product in catabolite repression of the Pseudomonas putida TOL toluene degradation pathway in chemostat cultures

The Pseudomonas putida KT2440 TOL upper pathway is repressed under nonlimiting conditions in cells growing in chemostat with succinate as a carbon source. We show that the ptsN gene product IIA(Ntr) participates in this repression. Crc, involved in yeast extract-dependent repression in batch cultures, did not influence expression when cells were growing in a chemostat with succinate at maximum rate.     

Catabolite repression and nitrogen control of allantoin-degrading enzymes in Pseudomonas aeruginosa

The formation of the allantoin-degrading enzymes allantoinase, allantoicase and ureidoglycolase in Pseudomonas aeruginosa was found to be regulated by induction, catabolite repression and nitrogen control. Induction was observed when urate, allantoin or allantoate were included in the growth medium, but not with ureidoglycolate. Tricarboxylic acid cycle intermediates exerted catabolite repression of the synthesis of the three enzymes, while pyruvate and glucose caused less repression. The operation of a nitrogen control mechanism in the regulation of the allantoin-degrading enzymes could be demonstrated with glutamine synthetase-negative mutants, which showed elevated synthesis and escape from catabolite repression when growth was limited for glutamine.     

Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa.

Mutants which are defective in catabolite repression control (CRC) of multiple independently regulated catabolic pathways have been previously described. The mutations were mapped at 11 min on the Pseudomonas aeruginosa chromosome and designated crc. This report describes the cloning of a gene which restores normal CRC to these Crc- mutants in trans. The gene expressing this CRC activity was subcloned on a 2-kb piece of DNA. When this 2-kb fragment was placed in a plasmid behind a phage T7 promoter and transcribed by T7 RNA polymerase, a soluble protein with a molecular weight (MW) of about 30,000 was produced in Escherichia coli. A soluble protein of identical size was overproduced in a Crc- mutant when it contained the 2-kb fragment on a multicopy plasmid. This protein could not be detected in the mutant containing the vector without the 2-kb insert or with no plasmid. When a 0.3-kb AccI fragment was removed from the crc gene and replaced with a kanamycin resistance cassette, the interrupted crc gene no longer restored CRC to the mutant, and the mutant containing the interrupted gene no longer overproduced the 30,000-MW protein. Pools of intracellular cyclic AMP and the activities of adenylate cyclase and phosphodiesterase were measured in mutant and wild-type strains with and without a plasmid containing the crc gene. No consistent differences between any strains were found in any case. These results provide original evidence for a 30,000-MW protein encoded by crc+ that is required for wild-type CRC in P. aeruginosa and confirms earlier reports that the mode of CRC is cyclic AMP independent in this bacterium.     

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.