Inactivation of cytochrome o ubiquinol oxidase relieves catabolic repression of the Pseudomonas putida GPo1 alkane degradation pathway

Abstract

Expression of the alkane degradation pathway encoded by the OCT plasmid of Pseudomonas putida GPo1 is regulated by two control systems. One relies on the transcriptional regulator AlkS, which activates expression of the pathway in the presence of alkanes. The other, which is a dominant global regulation control, represses the expression of the pathway genes when a preferred carbon source is present in the growth medium in addition to alkanes. This catabolite repression control occurs through a poorly characterized mechanism that ultimately regulates transcription from the two AlkS-activated promoters of the pathway. To identify the factors involved, a screening method was developed to isolate mutants without this control. Several isolates were obtained, all of which contained mutations that mapped to genes encoding cytochrome o ubiquinol oxidase, the main terminal oxidase of the electron transport chain under highly aerobic conditions. Elimination of this terminal oxidase led to a decrease in the catabolic repression observed both in rich Luria-Bertani medium and in a defined medium containing lactate or succinate as the carbon source. This suggests that catabolic repression could monitor the physiological or metabolic status by using information from the electron transport chain or from the redox state of the cell. Since inactivation of the crc gene also reduces catabolic repression in rich medium (although not that observed in a defined medium), a strain was generated lacking both the Crc function and the cytochrome o terminal oxidase. The two mutations had an additive effect in relieving catabolic repression in rich medium. This suggests that crc and cyo belong to different regulation pathways, both contributing to catabolic repression.

FIG. 1.

P. putida GPo1 alkane degradation pathway. The genes are grouped into two clusters, alkBFGHJKL and alkST, both of which are regulated by the AlkS protein. In the absence of alkanes, AlkS is expressed from promoter PalkS1; AlkS acts as a repressor of this promoter, allowing for low expression levels. This promoter is recognized by σ^S-RNA polymerase, being active only in the stationary phase of growth. In the presence of alkanes, AlkS activates expression from the PalkB and PalkS2 promoters, generating a positive amplification loop on alkS expression. Activation of these two promoters by alkanes is strongly repressed by catabolic repression when cells grow exponentially in rich LB medium. Growth in a minimal medium containing some organic acids (lactate or succinate) as a carbon source generates a milder catabolic repression effect.

FIG. 2.

Insertion points of mini-Tn5Sm in the P. putida catabolic repression mutants isolated. The upper scheme shows the genes encoding the P. putida cyo complex (28) and the positions of the mini-Tn5Sm insertions found in mutant strains RCM4, RCM18, RCM2A, RCM2B, RCM3A, RCM6A, RCM22A, RCM117A, and RCM137A (indicated by arrows). The cyoA, cyoB, cyoC, and cyoD genes encode subunits II, I, III, and IV of the oxidase complex, respectively; cyoE encodes the heme o synthase. The bottom scheme represents the electron transport chain. NDH, NADH dehydrogenase; LDH, lactate dehydrogenase; SDH, succinate dehydrogenase; UQ, oxidized ubiquinone; UQH₂, reduced ubiquinone; cyo, cytochrome o ubiquinol oxidase.

FIG. 3.

Effect of the cyoB mutation on induction of the PalkB promoter in cells growing with different carbon sources. P. putida strains PBS4 and PBS4B1 (PBS4 with a knockout mutation at the cyoB gene) were grown in duplicate flasks either in LB medium (B) or in minimal salts medium containing either citrate (Cit) (A), succinate (Scc) (C), or lactate (Lac) (D) as the carbon source. At an A₆₀₀ of 0.08, the nonmetabolizable inducer DCPK was added to one of the flasks; the other was left as a noninduced control. Aliquots were taken at different times, and the β-galactosidase activity was measured. The plots show the values observed for induced cultures (noninduced cultures had very low β-galactosidase activities [30 to 100 Miller units] and are not represented). The values shown correspond to three to six independent assays, all of which are represented on the same plot.

FIG. 4.

Effect of the cyoB mutation on expression of the PalkB and PalkS2 promoters. Strains PBS4 and PBS4B1 were grown in LB medium supplemented with the inducer DCPK (0.05% [vol/vol]). At an A₆₀₀ of 0.8, cells were collected and the total RNA was obtained. The levels of mRNA originated at the PalkB and PalkS2 promoters were measured by S1 nuclease protection assays in the presence of a large excess of the probe. Promoter PalkS1 is inactive under these conditions (7) and gave no signal (results not shown). The cDNA resistant to S1 nuclease was resolved in a denaturing polyacrylamide gel, in parallel with a DNA size ladder obtained by chemical sequencing of the ssDNA used as probe.

FIG. 5.

Effect of the simultaneous inactivation of the crc and cyo genes on catabolic repression of the PalkB promoter. P. putida strains PBS4, PBS4B1 (PBS4 with a knockout mutation at the cyoB gene), PBS4C1 (PBS4 with a knockout mutation at the crc gene), and PBS4BC1 (PBS4 with knockout mutations at the cyoB and crc genes) were grown in duplicate flasks in LB medium. At an A₆₀₀ of 0.08, the nonmetabolizable inducer DCPK was added to one of the flasks, whereas the other flask was left as a noninduced control. Aliquots were taken at different times, and the β-galactosidase activity was measured. The levels of β-galactosidase are represented as a function of cell growth. The plot shows the values observed for induced cultures (noninduced cultures had very low β-galactosidase activities [30 to 90 Miller units] and are not represented). The values shown correspond to several independent assays, all represented on the same plot.