Carbon-source-dependent expression of the PalkB promoter from the Pseudomonas oleovorans alkane degradation pathway

Abstract

Pseudomonas oleovorans GPo1 can metabolize medium-chain-length alkanes by means of an enzymatic system whose induction is regulated by the AlkS protein. In the presence of alkanes, AlkS activates the expression of promoter PalkB, from which most of the genes of the pathway are transcribed. In addition, expression of the first enzyme of the pathway, alkane hydroxylase, is known to be influenced by the carbon source present in the growth medium, indicating the existence of an additional overimposed level of regulation associating expression of the alk genes with the metabolic status of the cell. Reporter strains bearing PalkB-lacZ transcriptional fusions were constructed to analyze the influence of the carbon source on induction of the PalkB promoter by a nonmetabolizable inducer. Expression was most efficient when cells grew at the expense of citrate, decreasing significantly when the carbon source was lactate or succinate. When cells were grown in Luria-Bertani rich medium, PalkB was strongly down-regulated. This effect was partially relieved when multiple copies of the gene coding for the AlkS activator were present and was not observed when the promoter was moved to Escherichia coli, a heterologous genetic background. Possible mechanisms responsible for PalkB regulation are discussed.

FIG. 1

The alkane oxidation pathway and transcriptional fusions constructed. (A) Terminal oxidation of medium-chain-length n-alkanes by the enzymes of the pathway encoded in the OCT plasmid, and genes involved. The genes are clustered in two operons; expression from the PalkB promoter is activated by the AlkS regulator in the presence of inducers (adapted from reference 50). (B) PalkB-lacZ and PalkS-lacZ transcriptional fusions used throughout this work. The presumed binding region of the AlkS activator at PalkB (53) is indicated. The DNA segments are not drawn to scale.

FIG. 2

Induction of the PalkB promoter in culture media with different carbon sources. P. putida PBS4, harboring a PalkB-lacZ fusion and the alkS gene integrated into the chromosome, was grown in duplicate in LB medium or in minimal salts medium supplemented with citrate (Cit), succinate (Scc), or lactate (Lct). At a cell density of about 0.08, the nonmetabolizable inducer DCPK was added to one of the flasks, leaving the other one as a noninduced control. Aliquots were taken at different times, and β-galactosidase levels were measured. The plot shows the induction of PalkB observed as a function of cell density, calculated as the level of β-galactosidase detected in the presence of inducer divided by that observed in the absence of inducer. A minimum of three to five independent assays were performed for each medium; representative results are shown. Maximum β-galactosidase levels observed corresponded to about 10,000 Miller units. Basal levels in the absence of inducer were low during the exponential and early stationary phases, slowly increasing with cell density (20 to 80 Miller units), and had similar values in all growth media. Basal levels were higher in overnight cultures, which explains why induction values frequently declined in overnight cultures in minimal salts media. The value corresponding to the highest cell density was taken from overnight cultures.

FIG. 3

Analysis of the transcripts originated at the PalkB promoter in cells grown with different carbon sources. P. putida PBS4, harboring a PalkB-lacZ fusion and the alkS gene integrated into the chromosome, was grown in duplicate in LB medium or in minimal salts medium supplemented with citrate (Cit), succinate (Scc), or lactate (Lct). At a cell density of about 0.08, the nonmetabolizable inducer DCPK was added to one of the flasks, leaving the other one as a noninduced control. Aliquots were taken at different times (intervals of 15 to 60 min, depending on the growth phase), and total RNA was purified. The amount of mRNA originated at PalkB promoter region was analyzed by S1 nuclease assays in the presence of an excess of a ³²P-labeled ssDNA hybridizing to the 5′ end of the transcript (see Materials and Methods). Probe sequences protected from S1 nuclease by hybridization with transcripts originated at the PalkB region were identified in a denaturing polyacrylamide gel, and their amounts were quantified in a Bio-Rad Molecular Imager. A single band (shown in the inserts) of a size corresponding to an mRNA originated at PalkB was obtained. Plots show the amount of signal detected in induced cultures represented versus the cell density observed at each sampling time. No mRNA originated at PalkB could be detected in noninduced cultures.

FIG. 4

Induction of the PalkB promoter in spent LB medium or in minimal salts medium in the presence of Casamino Acids. P. putida PBS4, harboring a PalkB-lacZ fusion and the alkS gene integrated into the chromosome, was grown in LB medium, in spent LB medium (see text), or in minimal salts medium supplemented with either lactate plus Casamino Acids (Lct+CS) or citrate plus Casamino Acids (Cit+CS). Induction of PalkB was determined as indicated for Fig. 2; the plot shows the induction observed expressed as a function of cell density.

FIG. 5

Comparison of the growth rate of P. putida PBS4 in different media with the level of PalkB repression. The doubling times and PalkB repression values for cells grown in minimal salts medium with different carbon sources or in LB medium are represented. Unless indicated otherwise, the growth temperature was 30°C. Repression values denote the induction level of PalkB promoter observed at a cell density of 0.5 (A₆₀₀) in cells grown with the indicated carbon source, measured as indicated for Fig. 2, relative to the induction level observed when cells used citrate as the carbon source (induction in citrate divided by the induction in any other carbon source). All values correspond to the average of several independent assays (error bars are shown). Cit, citrate; Lct, lactate; Scc, succinate; Sp-LB, spent LB medium; Cit+CS, citrate plus Casamino Acids; LB, LB medium.

FIG. 6

Expression of the PalkS promoter in cultures grown at the expense of different carbon sources. P. putida PS16, harboring a PalkS-lacZ fusion integrated into the chromosome, was grown in LB or in minimal salts medium supplemented with citrate (Cit) or lactate (Lct). Levels of β-galactosidase were measured at different cell densities in each medium. The plot shows the amount of β-galactosidase observed in each case (in Miller units) as a function of cell density (A₆₀₀).

FIG. 7

Effect of increasing the number of copies of the alkS gene on the level of AlkS protein. Expression of the PalkB promoter was analyzed in strains PBS4 (PalkB-lacZ and alkS in monocopy, integrated into the chromosome; rectangles), PBS4 harboring plasmid pHCP1 (PalkB-lacZ in multicopy and alkS in monocopy; triangles), and PBS4 harboring plasmid pHCPR1 (both PalkB-lacZ and alkS in multicopy; circles). Cells were grown in duplicate in minimal salts medium with citrate as carbon source; at an optical density of about 0.08, the inducer DCPK was added to one of the flasks, leaving the other one as a noninduced control. The plot shows the levels of β-galactosidase observed (in Miller units) at different times after induction versus the cell density observed at the moment of sampling. Open symbols correspond to expression observed in the absence of inducer; filled symbols indicate expression in the presence of inducer. LC and HC indicate low copy and high copy, respectively.

FIG. 8

Effect of an increase in alkS gene dosage on PalkB modulation in different growth media. (A) P. putida PBS4 harboring plasmid pHCS1 (alkS gene in multicopy and the PalkB-lacZ fusion inserted into the chromosome) was grown in LB medium (open rectangles) or in minimal salts medium supplemented with either lactate (Lct; open triangles) or citrate (Cit; open circles), and induction of the PalkB promoter was assayed as indicated for Fig. 2. The plot shows the induction observed expressed as a function of cell density. PalkB induction in strain PBS4 (PalkB-lacZ and alkS in monocopy) grown in LB is also shown for comparison (filled rectangles). LC, in monocopy; HC, alkS in multicopy. (B) Levels of mRNA originated at the PalkB promoter in strain PBS4 harboring plasmid pHCS1 (alkS gene in multicopy and PalkB-lacZ in monocopy), grown in LB medium, determined as indicated for Fig. 3. The graph shows the mRNA levels observed in samples taken at different times after induction; no mRNA originated at PalkB was detected in noninduced cells.

FIG. 9

Induction of the PalkB promoter in LB medium when transferred to E. coli. E. coli W3110-B1, harboring the PalkB-lacZ fusion and the alkS gene integrated into the chromosome, was grown in LB medium, and induction of the PalkB promoter was assayed by measuring either β-galactosidase activity as indicated for Fig. 2 (A) or the transcripts originated at the promoter as indicated for Fig. 3 (B). The plots show either induction of the PalkB promoter as a function of cell density (A) or the amounts of transcripts originated at PalkB expressed as a function of cell density (B).