Regulation of the carnitine pathway in Escherichia coli: investigation of the cai-fix divergent promoter region

Abstract

The divergent structural operons caiTABCDE and fixABCX of Escherichia coli are required for anaerobic carnitine metabolism. Transcriptional monocopy lacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS. Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an fnr mutant, corresponding to its role as the primary target for anaerobiosis. Transcript analysis identified two divergent transcription start points initiating 289 bp apart. DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region. Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at -41.5 of the cai transcriptional start site, plays a direct role in the sole cai activation. In contrast, mutation in CRP site 2, positioned at -69.5 of the fix promoter, caused only a threefold reduction in fix expression. Thus, the role of the third CRP site, located at -126.5 of fix, might be to reinforce the action of site 2. A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription. This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.

FIG. 1

Growth phase regulation of the cai and fix operons. Strain NM522 harboring either plasmid pAB20 (caiT-lacZ) (A) or plasmid pAB30 (fixA-lacZ) (B) was grown anaerobically at 30°C in LB supplemented with 1 μM sodium molybdate, 1 μM sodium selenite, and 20 mM dl-carnitine. Cell samples were collected for measurement of optical density (OD) and for determination of β-galactosidase specific activity.

FIG. 2

Effects of deletions in the cai-fix intergenic region on in vivo expression of the caiT-lacZ and fixA-lacZ fusions. (A) Schematic representation of the cai-fix divergent promoter region as previously suggested by Eichler et al. (9, 10). The first open reading frames of the fix and cai operons initiate at positions 324 and 792, respectively. Curved arrows indicate putative transcriptional start sites. Two proposed binding sites for the ς⁵⁴-associated RNA polymerase are indicated by open diamonds, and one possible −10 box is represented by an open rectangle. Two remarkable inverted repeats, which might have a regulatory role, are designated 1 and 2. A perfect consensus sequence for the binding of CRP is shown by an oval. (B) Deletion analysis of the cai-fix intercistronic region. The arrows show the extension and orientation of fragments linked to the lacZ reporter gene on monocopy plasmid pJEL250. The coordinates at the top correspond to the numbering of nucleotides in accordance with the previously published sequence (9) and give useful restriction sites. The table on the right shows the names of the lacZ fusion plasmids listed in Table 1 and the relevant β-galactosidase activities. Strains NM522 (wild type [wt]) and MAM102 (rpoN), carrying the lacZ fusion plasmids, were grown anaerobically at 30°C in TYEP medium in the presence of 20 mM dl-carnitine. β-Galactosidase activities were expressed as nanomoles of o-nitrophenol produced per milligram (dry weight) of bacteria per minute. Each value represents the average of four independent experiments. nd, not determined. (C) Schematic representation of the cai-fix divergent promoter region as deduced from the complete study reported in this paper (see also Fig. 5).

FIG. 3

Identification of the cai and fix transcriptional start sites. Primer extension analyses of the fix (A) and cai (B) operon transcripts were carried out with RNA isolated from cells grown under anaerobic conditions in the presence of 20 mM dl-carnitine. The first four lanes A, C, G, and T, give the DNA sequence of plasmid pCTK generated by using the same primer. (A) Lanes: 1, MAM102 (rpoN)/pCTK; 2, NM522; 3, NM522/pCTK. (B) Lanes: 1, MAM102 (rpoN)/pCTK; 2, NM522/pCTK; 3, NM522. The −10 region and the transcription start point (+1) of the cai and fix operons are indicated on the right. (C) S1 nuclease mapping of the 5′ end of the cai transcript. RNA was isolated as described for primer extension assays. The TGCA sequence is the same as in panel B and serves as a size marker. Lanes: 1, MAM102 (rpoN)/pCTK; 2, NM522/pCTK; 3, probe without S1 nuclease digestion. The sizes of the detected bands are on the left. The start site (+1) of the cai operon and coordinates (in parentheses) relative to Fig. 2 are on the right.

FIG. 4

Specific binding of CRP in the cai-fix regulatory region. (A) Gel mobility shift assay of the cai-fix intercistronic region with purified CRP. The α-³²P-end-labeled 396-bp BspHI-HpaI DNA insert of plasmid pCTB was incubated in the presence of 1 μg of poly(dI-dC)-(dI-dC) without CRP (lane 1) or with 6.17 nM (lane 2), 123 nM (lane 3), or 1.23 μM (lane 4) CRP. The reactions were carried out in the presence of 50 μM cAMP. Free (F) and CRP-bound (B1, B2, and B3) DNA bands are indicated. (B and C) DNase I footprinting of the CRP-DNA interactions in the cai and fix promoter region. Labeling was performed on both strands independently, from either the BglII end of the 507-bp EcoRV-BglII fragment corresponding to the coding strand of cai (B) or the BspHI end of the 396-bp BspHI-HpaI fragment corresponding to the coding strand of fix (C). Protected regions are indicated by vertical brackets and marked 1 through 3. The CRP concentrations are as follows: lane 1, 0; lane 2, 6.17 nM; lane 3, 123 nM; lane 4, 1.23 μM. The values to the left of panels B and C are coordinates relative to the transcription start sites of cai and fix, respectively.

FIG. 5

Locations of the sequences protected by CRP from cleavage by DNase I. The protected sequences, termed CRP1 to CRP3, are represented by horizontal brackets. The locations of the transcription initiation sites of fixA and caiT are indicated by arrows designated +1. The putative −10 and −35 boxes are underlined (for fixA) or overlined (for caiT), and two inverted repeats are highlighted by converging dashed arrows. The start codons of fixA and caiT are in boldface. Useful restriction sites are also noted.