Expression of the Pseudomonas putida OCT plasmid alkane degradation pathway is modulated by two different global control signals: evidence from continuous cultures

Abstract

Expression of the genes of the alkane degradation pathway encoded in the Pseudomonas putida OCT plasmid are subject to negative and dominant global control depending on the carbon source used and on the physiological status of the cell. We investigated the signals responsible for this control in chemostat cultures under conditions of nutrient or oxygen limitation. Our results show that this global control is not related to the growth rate and responds to two different signals. One signal is the concentration of the carbon source that generates the repressing effect (true catabolite repression control). The second signal is influenced by the level of expression of the cytochome o ubiquinol oxidase, which in turn depends on factors such as oxygen availability or the carbon source used. Since under carbon limitation conditions the first signal is relieved but the second signal is not, we propose that modulation mediated by the cytochrome o ubiquinol oxidase is not classical catabolite repression control but rather a more general physiological control mechanism. The two signals have an additive, but independent, effect, inhibiting induction of the alkane degradation pathway.

FIG. 1.

Regulation of the genes encoding the 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. When no alkanes are present, alkS is expressed at low levels from promoter PalkS1; AlkS negatively modulates the expression of this promoter, resulting in low constant expression of the gene. When alkanes become available, AlkS activates transcription from the PalkB and PalkS2 promoters, generating a positive amplification loop for alkS expression. Induction of these two promoters by alkanes is negatively modulated by a dominant global control when cells are grown in the presence of alternative carbon sources, such as some organic acids (succinate or pyruvate) or amino acids.

FIG. 2.

Activities of promoters PalkB and PalkS2 in cells growing under different nutrient limitation conditions. (A) Cells were grown in chemostats with succinate as the carbon source and either at the maximal growth rate (conditions of no limitation) or at a D of 0.05 h⁻¹ by limiting the concentration of either succinate (carbon limitation), SO₄²⁻ (sulfur limitation), or NH₄⁺ (nitrogen limitation). The nonmetabolizable compound DCPK (0.05%, vol/vol) was included in the culture medium to induce transcription from the PalkB and PalkS2 promoters. After cultures reached a steady state, samples were taken, and β-galactosidase levels were measured. The values are averages from two independent assays with duplicate samples (standard deviations, less than 15%). (B) Cells were grown as described above, but samples were processed to obtain total RNA. The activities of promoters PalkB and PalkS2 were measured by real-time reverse transcription-PCR. RNA levels were normalized relative to the amount of total RNA. The values are averages from two independent assays with triplicate samples (standard deviations, less than 20%). NL, nonlimited; ScL, succinate limited; O₂L, oxygen limited (samples from the assay whose results are shown in Fig. 3); wt, wild type.

FIG. 3.

Effect of oxygen availability on the activity of the PalkB promoter. P. putida strains PBS4 (wild type [wt]), PBS4C1 (crc), and PBS4B1 (cyoB) were grown to the stationary phase in a fermentor by using 20 mM succinate as the carbon source in the presence of the inducer DCPK at a concentration of 0.05% (vol/vol). Culture medium containing 10 mM succinate and 0.05% (vol/vol) DCPK was then added to the fermentor, and the D was adjusted to 0.05 h⁻¹. The culture was allowed to stabilize until a steady state was reached, as characterized by turbidity values (A₅₄₀) of about 0.4. The oxygen supply was then decreased from 100 to 40% saturation. PalkB activity was monitored by measuring β-galactosidase activity.

FIG. 4.

Expression of the cyoA gene under different growth conditions. Cells grown under carbon limitation conditions (10 mM succinate) and in the presence of either high or low oxygen concentrations (100 and 40% saturation, as described in the legend to Fig. 3) were collected, and total RNA was purified. Expression of the cyoA promoter was determined by S1 nuclease protection assays. A similar analysis was performed with cells growing logarithmically in batch cultures by using either succinate (Scc) or citrate (Cit) at a nonlimiting concentration as the carbon source.