Transcriptional organization and regulation of the L-idonic acid pathway (GntII system) in Escherichia coli

Abstract

The genetic organization of the idn genes that encode the pathway for L-idonate catabolism was characterized. The monocistronic idnK gene is transcribed divergently from the idnDOTR genes, which were shown to form an operon. The 215-bp regulatory region between the idnK and idnD genes contains promoters in opposite orientation with transcription start sites that mapped to positions -26 and -29 with respect to the start codons. The regulatory region also contains a single putative IdnR/GntR binding site centered between the two promoters, a CRP binding site upstream of idnD, and an UP element upstream of idnK. The genes of the L-idonate pathway were shown to be under catabolite repression control. Analysis of idnD- and idnK-lacZ fusions in a nonpolar idnD mutant that is unable to interconvert L-idonate and 5-ketogluconate indicated that either compound could induce the pathway. The L-idonate pathway was first characterized as a subsidiary pathway for D-gluconate catabolism (GntII), which is induced by D-gluconate in a GntI (primary gluconate system) mutant. Here we showed that the idnK and idnD operons are induced by D-gluconate in a GntI system mutant, presumably by endogenous formation of 5-ketogluconate from D-gluconate. Thus, the regulation of the GntII system is appropriate for this pathway, which is primarily involved in L-idonate catabolism; the GntII system can be induced by D-gluconate under conditions that block the GntI system.

FIG. 1.

Primer extension of the transcription start sites for idnD and idnK. (A) Extension of the idnD transcript (PD1). (B) Extension of the idnK transcript (PK1). Lanes: PE, primer extension products; G, A, T, and C, corresponding sequence ladders.

FIG. 2.

Northern blot analysis of the idnK, idnD, idnO, idnT, and IdnR transcripts in E. coli W1485. Total RNA was isolated from late-log-phase cultures grown on MOPS minimal medium containing the carbohydrate listed above each lane. A total of 5 μg of RNA was loaded per lane. Estimated transcript sizes (in kilobases) are shown to the right of each blot and were determined from an RNA Millennium Marker (Ambion, Inc.) run with each independent RNA gel (data not shown). Hybridizations were carried out with 300-nucleotide probes specific for the gene encoding the protein indicated under each blot.

FIG. 3.

RT analysis of the idnDOTR and idnK transcripts. (A) A 1.5% agarose gel showing the RT-PCR products with template RNA isolated from cells grown on MOPS compete medium containing 0.2% l-idonate. Lanes 2 to 12 correspond to regions 2 to 12 in the schematic representation (B). The RT-PCR products shown in lanes 2 to 12 were generated with primer pairs that flanked the corresponding regions depicted in the schematic. The length of each predicted RT-PCR product is shown in the schematic (in base pairs). Lanes: 1, 1-kb DNA ladder; 15, 100-bp DNA ladder; 13, control PCR product obtained from E. coli W1485 genomic DNA with a primer set that generated a 620-bp DNA fragment; 14, control PCR product obtained from total RNA with the same primer set as in lane 13. The values on the left and right are sizes in base pairs.

FIG. 4.

Northern blot analysis of a GntI system mutant. idnD (A) and idnK (B) transcription is shown. Total RNA was isolated from late-log-phase cultures of E. coli NP202 (W1485 ΔgntRKU) grown on LB medium containing the carbohydrate listed above each lane. An aliquot of 5 μg of RNA was loaded per lane, and the bars and corresponding values to the left of each blot show the locations and sizes (in kilobases) of RNA standards.

FIG. 5.

2-D PAGE of extracted proteins from cells grown in MOPS minimal medium containing 0.2% d-glucose (A) or l-idonate (B). The two modified forms of IdnD and IdnO were identified by MS/MS as described in the text and are indicated by arrows 1 and 2 and arrows 3 and 4, respectively.