The Pseudomonas putida Crc global regulator controls the expression of genes from several chromosomal catabolic pathways for aromatic compounds

Abstract

The Crc protein is involved in the repression of several catabolic pathways for the assimilation of some sugars, nitrogenated compounds, and hydrocarbons in Pseudomonas putida and Pseudomonas aeruginosa when other preferred carbon sources are present in the culture medium (catabolic repression). Crc appears to be a component of a signal transduction pathway modulating carbon metabolism in pseudomonads, although its mode of action is unknown. To better understand the role of Crc, the proteome profile of two otherwise isogenic P. putida strains containing either a wild-type or an inactivated crc allele was compared. The results showed that Crc is involved in the catabolic repression of the hpd and hmgA genes from the homogentisate pathway, one of the central catabolic pathways for aromatic compounds that is used to assimilate intermediates derived from the oxidation of phenylalanine, tyrosine, and several aromatic hydrocarbons. This led us to analyze whether Crc also regulates the expression of the other central catabolic pathways for aromatic compounds present in P. putida. It was found that genes required to assimilate benzoate through the catechol pathway (benA and catBCA) and 4-OH-benzoate through the protocatechuate pathway (pobA and pcaHG) are also negatively modulated by Crc. However, the pathway for phenylacetate appeared to be unaffected by Crc. These results expand the influence of Crc to pathways used to assimilate several aromatic compounds, which highlights its importance as a master regulator of carbon metabolism in P. putida.

FIG. 1.

Proteomic analysis of the effect of Crc in P. putida cells growing exponentially in LB medium. Total proteins obtained from strains PBS4 (wild type for crc) and PBS4C1 (contains an inactivated crc allele), exponentially growing in LB medium, were resolved by 2-D electrophoresis. Proteins were first separated according to their isoelectric point by IEF and then resolved through a 12.5% (wt/vol) denaturing polyacrylamide gel. Protein spots were revealed by silver staining. The figure shows areas of the 2-D gels containing spots whose intensity reproducibly decreased or increased by inactivation of crc in at least three independent assays. These proteins were excised from the gel and identified by mass spectrometry; the results are indicated in Table 1. Panels on the left correspond to strain PBS4 (crc⁺) while those on the right correspond to strain PBS4C1 (crc::tet).

FIG. 2.

Catabolic pathways for aromatic compounds identified in P. putida KT2440: effect of Crc. The pathways are those reported in reference . The key intermediate compounds of the homogentisate, catechol, protocatechuate, and phenylacetate pathways are highlighted. Genes whose expression is repressed by Crc as shown in this report are indicated. The scheme was modified from reference with permission.

FIG. 3.

RT-PCR analysis of the effect of Crc on the expression of the homogentisate, benzoate, protocatechuate, and phenylacetate pathways. Serial dilutions of total RNA isolated from strains PBS4 or PBS4C1 growing exponentially in LB medium or in LB medium supplemented with 5 mM benzoate (LB+Ben), 4-hydroxybenzoate (LB + 4-OH-Ben), or 5 mM phenylacetate (LB+PA) were subjected to RT-PCR with primers designed to detect the mRNA from the hpd, hmgA, mai, benA, catA, catB, catC, pobA, pcaH, pcaG, phaE, or phaA gene. Expression of the npt gene, specifying resistance to kanamycin (present in the chromosome of PBS4 and PBS4C1), was analyzed as a control to ensure both the linearity of the response and that the amounts of RNA from the two strains were comparable. Control reaction mixtures to which no RT was added yielded no amplification product (data not shown). wt, wild type.

FIG. 4.

Utilization of benzoate, 4-hydroxybenzoate or phenylacetate by cells of strains PBS4 and PBS4C1 grown in LB medium. Cells were grown in LB medium in the presence of 5 mM benzoate, 5 mM 4-hydroxybenzoate, or 5 mM phenylacetate. At a turbidity of 0.8 (A₆₀₀), cells were collected and resuspended in LB medium containing the corresponding aromatic compound at a concentration of 5 mM. Incubation was continued with aeration at 30°C. At different times, samples were collected and the amount of the aromatic compound remaining in the culture supernatant was determined by HPLC. Values correspond to the averages of the results from two independent assays in which the amount of the indicated compound at each time point was measured at least three times.