Regulation of aerobic and anaerobic D-malate metabolism of Escherichia coli by the LysR-type regulator DmlR (YeaT)

Abstract

Escherichia coli K-12 is able to grow under aerobic conditions on D-malate using DctA for D-malate uptake and the D-malate dehydrogenase DmlA (formerly YeaU) for converting D-malate to pyruvate. Induction of dmlA encoding DmlA required an intact dmlR (formerly yeaT) gene, which encodes DmlR, a LysR-type transcriptional regulator. Induction of dmlA by DmlR required the presence of D-malate or L- or meso-tartrate, but only D-malate supported aerobic growth. The regulator of general C(4)-dicarboxylate metabolism (DcuS-DcuR two-component system) had some effect on dmlA expression. The anaerobic L-tartrate regulator TtdR or the oxygen sensors ArcB-ArcA and FNR did not have a major effect on dmlA expression. DmlR has a high level of sequence identity (49%) with TtdR, the L- and meso-tartrate-specific regulator of L-tartrate fermentation in E. coli. dmlA was also expressed at high levels under anaerobic conditions, and the bacteria had D-malate dehydrogenase activity. These bacteria, however, were not able to grow on D-malate since the anaerobic pathway for D-malate degradation has a predicted yield of < or = 0 ATP/mol D-malate. Slow anaerobic growth on D-malate was observed when glycerol was also provided as an electron donor, and D-malate was used in fumarate respiration. The expression of dmlR is subject to negative autoregulation. The network for regulation and coordination of the central and peripheral pathways for C(4)-dicarboxylate metabolism by the regulators DcuS-DcuR, DmlR, and TtdR is discussed.

FIG. 1.

Transcriptional regulation of dmlA-lacZ gene fusion by C₄-dicarboxylates and the regulators DmlR, TtdR, and DcuS during aerobic growth. The dmlA-lacZ gene fusion was present on plasmid pMW323, and its expression was measured in E. coli wild-type strain MC4100 and dmlR-, ttdR-, and dcuS-deficient derivatives. Bacteria were grown under aerobic conditions in eM9 medium containing gluconate (50 mM) and the effectors indicated (50 mM). The strains used were strains MC4100 (wild type) (open bars), IMW533 (dmlR) (black bars), IMW523 (ttdR) (gray bars), and IMW262 (dcuS) (striped bars).

FIG. 2.

Aerobic (A) and anaerobic (B) growth of E. coli wild-type and mutant strains on d-malate and related C₄-dicarboxylates. (A) Wild-type strain MC4100 was grown in eM9 medium with 50 mM d-malate (•), l-tartrate (▪), meso-tartrate (▴), or succinate (▾). (B) E. coli wild-type strain MC4100 (•), frdABCD mutant MI1443 (▪), fumB mutant JW4083 (▾), dmlA mutant JW1789 (▸), and dcuS mutant IMW262 (⧫) were grown anaerobically using eM9 medium with d-malate plus glycerol (20 mM each. Wild-type strain MC4100 was also grown on d-malate without glycerol (○).

FIG. 3.

Transcriptional regulation of the dmlA-lacZ fusion in response to electron acceptors and glucose (A) and mutations in fnr or arcB (B). (A) Wild-type strain MC4100 containing pMW323 (with the dmlA-lacZ gene fusion) was grown in eM9 medium containing gluconate (50 mM) for aerobic growth or in glycerol (50 mM) with DMSO (20 mM) for anaerobic growth. The following compounds were included in the individual experiments, as indicated on the x axis: H₂O (no addition), d-malate (d-mal), d-malate plus sodium nitrate (NO₃⁻), and d-malate plus glucose (gluc) (50 mM each). Growth was performed under aerobic (O₂) or anaerobic (N₂) conditions. (B) Strains MC4100 (wild type [wt]), IMW151a (fnr), and RM313 (arcA), each containing pMW323 (with the dmlA-lacZ gene fusion), were grown under anaerobic conditions in eM9 medium containing glycerol (50 mM), DMSO (20 mM), and d-malate (50 mM). The β-galactosidase activity of the bacteria was determined in the mid-exponential growth phase (OD₅₇₈, 0.5 to 0.8).

FIG. 4.

Effects of C₄-dicarboxylates and the regulators TtdR, DcuS, and DmlR on the expression of dmlR-lacZ under aerobic (A) and anaerobic (B) growth conditions. (A) Strains IMW563 (wild type) (open bars), IMW564 (ttdR) (gray bars), IMW566 (dcuS) (striped bars), and IMW565 (dmlR) (black bars) carrying a chromosomal dmlR-lacZ gene fusion were grown in eM9 medium containing gluconate and the effectors indicated on the x axis (50 mM each) under aerobic conditions. (B) Anaerobic growth of the same strains in eM9 medium containing gluconate (50 mM), glycerol (50 mM), and DMSO (20 mM) with and without d-malate (50 mM).