Metabolic deficiences revealed in the biotechnologically important model bacterium Escherichia coli BL21(DE3)

Abstract

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²⁺ (Ni²⁺-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO₄²⁻ ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO₄²⁻ were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO₄²⁻ and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

Figure 1. An overview of anaerobic hydrogen metabolism and nitrate respiration metabolism in E. coli.

The metabolism of pyruvate under anaerobic conditions is shown in the upper portion of the Figure. The cellular locations of the three main [NiFe]-hydrogenases, the three molybdoselenium formate dehydrogenases and the principle nitrate reductase are shown, as are the transport systems for nickel and molybdate. The metal ion requirement and regulation with respect to the global regulator FNR are also indicated by arrows.

Figure 2. Hydrogenase 1 and 2 activity-staining after native-PAGE.

Aliquots (25 µg of protein) of crude extracts derived from MC4100 (wild type), PB1000 (Δfnr) and BL21(DE3) after anaerobic growth in TGYEP with or without supplementation of 500 µM nickel(II)-chloride (Ni), 15 mM formate (F) or 0.3 mM citrulline addition of plasmid-coded fnr (p1fnr, pCH21) and hypF (pAF1) were applied to 7.5% (w/v) native-PAGE. On the right hand the migration positions of Hyd-1 and Hyd-2 are given. The band designated with an asterisk is due to a side-reaction of FDH-O/FDH-N and this activity is hydrogenase-independent.

Figure 3. Western blot analysis of anaerobic enzymes in BL21(DE3).

25 µg Polypeptides in crude extracts derived from MC4100, PB1000 (Δfnr), BL21(DE3) with and without supplementation of 500 µM nickel(II)-chloride (Ni), 15 mM formate (F), 1 mM sodium-molybdate (MoO) or addition of plasmid encoded fnr (p1fnr, p10fnr, p13fnr, pCH21) and modE (p7modE) after anaerobic growth in TGYEP, pH 6.5 were separated by 10% (w/v) SDS-PAGE and transferred to nitrocellulose membranes. The samples were treated with antiserum raised against A: FNR, B: Hyd-2 (the upper arrows represents precursor and the lower arrow mature form of the Hyd-2 large subunit), C: PflB (the arrows mark the two different migrating forms typical for active protein after contact with oxygen), D: HycG (the Hyd-3 small subunit). The lane indicating the negative control contains PB1000 (Δfnr), DHP-F2 (ΔhypF), RM220 (ΔpflAB) and CP971 (ΔhycAI), from top to bottom, respectively. The asterisks signify unidentified cross-reacting species. On the right hand are given the sizes of the respective molecular mass marker (Prestained PageRuler, Fermentas).

Figure 4. Western blot analysis of the large subunits of NAR and FDH-N.

Crude extracts (25 µg protein) of MC4100, FM460 (ΔselC), PB1000 and BL21(DE3) bearing plasmids p13fnr (fnr ⁺), pCH21 (fnr ⁺), p7modE (modE ⁺) or supplemented with 500 µM nickel(II)-chloride (Ni), 15 mM formate (F) or 1 mM molybdate (MoO), when indicated were separated on 10% (w/v) SDS-PAGE after anaerobic growth in TGYEP, pH 6.5 with 100 mM potassium-nitrate. and treated with antiserum raised against A: Nar or B: FdnG.

Figure 5. The activity of FDH-O in BL21(DE3) is restored with high concentrations of molybdate.

Crude extracts (25 µg of protein) of the various strains indicated were separated in non-denaturing PAGE and stained specifically for FDH-O activity as described in the methods section. The arrow indicates the position of the active FDH-O enzyme.