A conserved and seemingly redundant Escherichia coli biotin biosynthesis gene expressed only during anaerobic growth

Abstract

Biotin is an essential metabolic cofactor and de novo biotin biosynthetic pathways are widespread in microorganisms and plants. Biotin synthetic genes are generally found clustered into bio operons to facilitate tight regulation since biotin synthesis is a metabolically expensive process. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of biotin biosynthesis, the formation of 7,8-diaminononanoate (DAPA). In Escherichia coli, DTBS is encoded by the bio operon gene bioD. Several studies have reported transcriptional activation of ynfK a gene of unknown function, under anaerobic conditions. Alignments of YnfK with BioD have led to suggestions that YnfK has DTBS activity. We report that YnfK is a functional DTBS, although an enzyme of poor activity that is poorly expressed. Supplementation of growth medium with DAPA or substitution of BioD active site residues for the corresponding YnfK residues greatly improved the DTBS activity of YnfK. We confirmed that FNR activates transcriptional level of ynfK during anaerobic growth and identified the FNR binding site of ynfK. The ynfK gene is well conserved in γ-proteobacteria.

Keywords: Escherichia coli; anaerobic growth; biotin synthesis; dethiobiotin synthetase.

Fig 1.

The reaction scheme of the BioD dethiobiotin synthetase (Krell and Eisenberg, 1970) (Käck et al., 1998). It remains unclear if BioD catalyzes formation of the N7 carbamate or selects the N7 carbamate from the mixture of N7 carbamate, N8 carbamate and the dicarbamate that spontaneously forms in solution (Gibson et al., 1995).

Fig. 2.. Sequence alignment BioD and YnfK of E. coli.

Unweighted sequence alignments was performed using T-Coffee with the default settings and displayed using Jalview. Positions having 30% or greater are highlighted. Residues where residue substitution were made are indicated by arrows. The sites denoted are those of BioD.

Fig. 3. Anaerobic growth of E. coli mutant strains.

A. The strains were streaked on M9 minimal medium containing 2 nM biotin, starved by avidin addition (avidin is a very robust protein that binds biotin very tightly with a Kd ~ 10⁻¹⁵M) and then restreaked on M9 biotin-free minimal medium and incubated at 37°C under anaerobic conditions. Panels A and B show anaerobic growth of E. coli DTBS null mutant strains and anaerobic growth of E. coli mutant strains with DAPA supplementation, respectively. The black hazy color is cleaved S-Gal (3,4-cyclohexenoesculetin β-D-galactopyranoside, a substrate for β-galactosidase, that unlike the commonly used X-Gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside), is chromogenic under anerobic conditions. S-Gal was added to facilitate detection of faint growth.

Fig. 4.. Complementation of the ΔynfK ΔbioD strain with plasmids encoding YnfK derivatives having BioD active site residues.

The proteins were expressed from the PbioB300 promoter in vector pBAD33. The biotin-free plates were incubated under anaerobic conditions for 20 h and contained S-Gal. A more detailed analysis of growth rates is given in Fig. S3.

Fig. 5.. EGS cross-linking analysis of the oligomerization state of BioD, YnfK, and the YnfK residue substitution proteins.

M: Protein standard markers. The dimer and monomer (mono) are indicated by arrows. A. EGS cross-linking analysis of BioD and YnfK. Increased YnfK crosslinking in the presence of DAPA is also shown. B. EGS cross-linking analysis of YnfK and the YnfK active site substitution proteins. The protein standards (in KDa) are 95, 72, 55 (heavy band), 43, 34, 26, and 17. Note that the YnfK crosslinking of gel A in the absence of DAPA could not be quantitated by scanning due to overloading. We therefore repeated the YnfK crosslinking with 2 mM EGS and loaded increasing amounts on a single gel (Fig S4) and scanned several lanes in the linear range with a GE Typhoon FLA7000 scanner in the fluorescent shadow mode and found that only 41% of YnfK was crosslinked.

Fig. 6.. FNR directly activates YnfK transcription.

A. Anaerobic expression of ynfK-lacZY fusions strain grown anaerobically. The strains were grown in defined medium supplemented with 500 μM IPTG for 6 at 37°C. The values are given above the bars and the results are the average of three independent experiments (the error bars denote standard error of the mean). The strain background was a ΔlacZY derivative of E. coli MG1655 and the fusion was ynfK196-lacZY carried by plasmid pBAD33. The wild type (wt) and Δfnr strains also carried the empty pQE2 vector whereas the FNR and FNRD154A proteins were expressed from the pQE2 vector. B. Electrophoretic mobility shift assays of DNA binding by FNR. EMSA showed that FNR binds to a site upstream of gene ynfK. A C. acetobutylicum bioY fragment was used as negative control (Song et al., 2021). Gel shifts are indicated by the arrows. C. Sequence alignment of E. coli FNR binding site. Unweighted sequence alignments were performed using T-Coffee with the default settings and displayed using Jalview. Positions having 50% or greater conservation with the consensus sequence are highlighted. E. coli FNR, FNR consensus binding site; E. coli ynfK FNR, FNR binding site of E. coli ynfK. The 3’ end of the ynfK binding site is 85 bp upstream of the coding sequence.

Fig. 7.. The AlphaFold model of the YnfK structure superimposed on the 0.95Å structure of BioD using ChimeraX.

The YnfK model is shown in blue and the BioD structure(Sandalova et al., 1999) is shown in yellow. The single residue mutations are shown in pink. The root mean square deviation (RMSD) between 201 pruned atom pairs is 0.734Å (across all 219 pairs:1.610). The magnesium ion is shown in green. ATP: adenosine-5-triphosphate. DNN: 7,8-diamino-nonanoic acid. Based on the modeling, Ser13 and T114 are directly involved in substrate binding, whereas I184Y is located in the dimerization interface (note that AlphaFold predictions are currently restricted to monomers). A parallel, independent analysis by Dr. Yuanyuan Hu of this laboratory gave a Cα carbons RMSD of 0.629Å over 80% of the shorter BioD residues. The RMSD for all carbon atoms was 0.646Å over 1079 C atoms indicating that not only the Cα carbons but a significant number of the sidechains could be superimposed. The few discrepancies in the superimpositions are segments of the model that were predicted with low or very low confidence (e.g. the carboxy terminus).

Fig. 8.. Phylogeny of DTBS enzymes.

The phylogenetic tree was constructed using the default setting of Neighbor Joining, Blosum62 in Jalview. Each bacterial species is given followed by the UniProt code.