The mRNA specifying the fdnG signal peptide coding region can be folded into a stem-loop structure. The position of the initiation codon is indicated in bold type; the Shine-Dalgarno ribosome-binding sequence is underlined. Numbering is shown relative to the first base of the start codon, which is designated +1. (A) Portion of the DNA from plasmid pVJS2248 (24) showing the first part of the fdnG signal peptide-coding region. To aid clarity, the amino acids are numbered with subscripts. The amino acids of the twin arginine motif are doubly underlined. The clone carries a 487-bp insert that covers the entire fdnG promoter region and fdnG sequence as far as an engineered BamHI site in the DNA after codon 44 (24). (B) The mRNA covering the region shown in panel A was folded using the program mfold 3 (25), which was accessed through the world-wide web (http://www.bioinfo.rpi.edu/applications/mfold). The codon substitutions used during this study are V₃P (GTC→CCC), R₅K (CGC→AAG), R₅S (CGC→AGC), R₅Rhigh (CGC→CGT), R₅Rlow (CGC→AGG), R₆K (AGA→AAG), R₆Rhigh (AGA→CGT), F₉L (TTT→CTG), K₁₀E (AAA→GAA), I₁₁V (ATC→GTC), I₁₁I (ATC→ATA), I₁₁T (ATC→ACT), C₁₂A (TGC→GCC), A₁₃S (GCG→AGC), G₁₄K (GGC→AAA), and R₆K/I₁₁T (AGA→AAG and ATC→ACT).

The effects of mutations in the stem and loop regions of the fdnG mRNA on expression of fdnG-lacZ. Constructs were transformed into strain MC4100-P (4), which carries the pcnB allele, providing more careful control of plasmid copy number (14, 15). (A) Constructs carrying mutations in the codons forming the first arm of the stem; (B) constructs carrying mutations in codons that fall in the loop region; (C) constructs carrying mutations in codons forming the second arm of the stem; (D) constructs carrying either individual single codon substitutions or a combined double-codon substitution that is predicted to have a compensatory effect. All strains were cultured anaerobically in Cohen and Rickenberg medium (7) supplemented with 0.2% glucose and 0.4% nitrate. β-Galactosidase enzyme activity is shown in Miller units (17), and the bars represent standard errors of the means (n = 3 to 4). (E) Correlation between stem-loop stability (measured as ΔG) and β-galactosidase enzyme activity. Free energies for each codon mutation were calculated with the program mfold3 (25) using refined energy parameters (16) and with the mRNA sequence covering codons 1 to 17 inclusive. WT, wild type.

The stem-loop structure regulates FDH-N activity in vivo. FDH-N (A) and NAR (B) enzyme assays were performed with crude cell extracts derived from strains MC4100, NRS9 (as MC4100; FdnG R₅R [CGC→CGT]), and CMP1 (as MC4100; FdnG R₅R [CGC→AGG]) or the same strains carrying plasmid pUt-AGA/AGG (22). Cells were grown anaerobically in Luria-Bertani medium supplemented with 0.2% glucose and 0.4% potassium nitrate, crude extracts were prepared, and enzyme activities were assayed exactly as described previously (21). Assays were performed in triplicate with results differing by not more than 15% of the mean. (A) FDH-N enzyme activity (measured as the rate of phenazine methosulfate-linked formate dehydrogenase activity) (9). An activity level of 100% is that measured from the crude cell extract of the wild-type (WT) strain and corresponds to 33 nmol of formate oxidized/min/mg of protein. (C) NAR enzyme activity, measured according to the method of Jones and Garland (12). An activity level of 100% is that measured in the crude cell extract of the wild-type strain and corresponds to 598 nmol of nitrate reduced/min/mg of protein. (C) Detection of FdnG polypeptide in whole cells after Western blot analysis with anti-FdnG antibodies (24). Equivalent quantities of cells were loaded in each lane. The fold overexpression of FdnG polypeptide relative to that detected in the wild-type sample is shown above each lane.