The Bacillus subtilis nrdEF genes, encoding a class Ib ribonucleotide reductase, are essential for aerobic and anaerobic growth

Abstract

Ribonucleotide reductases (RNRs) are essential for the biosynthesis of the deoxyribonucleoside triphosphates of DNA. Recently, it was proposed that externally supplied deoxyribonucleosides or DNA is required for the growth of Bacillus subtilis under strict anaerobic conditions (M. J. Folmsbee, M. J. McInerney, and D. P. Nagle, Appl. Environ. Microbiol. 70:5252-5257, 2004). Cultivation of B. subtilis on minimal medium in the presence of oxygen indicators in combination with oxygen electrode measurements and viable cell counting demonstrated that growth occurred under strict anaerobic conditions in the absence of externally supplied deoxyribonucleosides. The nrdEF genes encode the only obvious RNR in B. subtilis. A temperature-sensitive nrdE mutant failed to grow under aerobic and anaerobic conditions, indicating that this oxygen-dependent class I RNR has an essential role under both growth conditions. Aerobic growth and anaerobic growth of the nrdE mutant were rescued by addition of deoxynucleotides. The nrd locus consists of an nrdI-nrdE-nrdF-ymaB operon. The 5' end of the corresponding mRNA revealed transcriptional start sites 45 and 48 bp upstream of the translational start of nrdI. Anaerobic transcription of the operon was found to be dependent on the presence of intact genes for the ResDE two-component redox regulatory system. Two potential ResD binding sites were identified approximately 62 bp (site A) and 50 bp (site B) upstream of the transcriptional start sites by a bioinformatic approach. Only mutation of site B eliminated nrd expression. Aerobic transcription was ResDE independent but required additional promoter elements localized between 88 and 275 bp upstream of the transcriptional start.

FIG. 1.

Mutation of nrdEF genes eliminates anaerobic growth of B. subtilis. Strains were grown under strict anaerobic conditions in minimal medium supplemented with 10 mM nitrate. B. subtilis JH642 wild-type cells (dotted line) and the temperature-sensitive nrdE mutant ts-A13 (solid line) were grown at 45°C, the nonpermissive temperature of the mutant strain. Mutant strain PB1679 was rescued by addition of 100 μg ml⁻¹ deoxynucleotides and grew like wild-type cells (dashed line). OD₅₇₈, optical density at 578 nm.

FIG. 2.

Ribonucleotide reductase genes of B. subtilis are transcribed in an nrdIEF ymaB operon. (A) Ten micrograms of total RNA was separated on a 1% denaturing agarose gel and analyzed by Northern blotting. An nrdE-specific RNA probe was used for hybridization. Two transcripts were detected in RNA from aerobically grown cells (lanes A) and fermentatively grown cells (lanes F); the position of a major ca. 3,400-nucleotide transcript whose size corresponded to the size of an nrdIEF transcript and the position of a minor 4,100-nucleotide transcript corresponding to an nrdIEF ymaB operon are indicated by arrows. The size standards were ethidium bromide-stained 16S and 23S rRNA species and RNA molecular weight marker no. 1 (Roche Diagnostics GmbH, Mannheim, Germany) (lane M). nt, nucleotide. (B) Schematic representation of the nrdIEF ymaB region. The coding regions of the genes are indicated by arrows. The proposed transcriptional start point and termination signals are indicated by a small arrow and hairpin structures, respectively. The transcripts determined are indicated by dashed lines. The numbering corresponds to the numbering of the genomic sequence. (C) Determination of the transcription start site of nrdIEF by primer extension analysis. Total RNA was isolated from JH642 cells grown aerobically (lane 1) and under fermentative conditions (lane 2) and was analyzed. The same primer used for the primer extension analysis was used for sequencing reactions (lanes G, A, T, and C). The arrows indicate the positions of primer extension products, and asterisks indicate the 5′ end of the nrdIEF mRNA in the sequence. (D) Promoter sequences of the nrdI promoter fused to the lacZ reporter gene. The 5′ ends of the promoter fragments are labeled −275 and −87. The arrows indicate the locations and orientations of potential ResD binding sites A and B, and the bases changed for mutagenesis of the binding site are indicated by boldface type. Transcriptional start sites are indicated by arrows. The sequence of the −10 region is in italics. A potential ribosome binding site (RBS) is underlined. The start codon of NrdI is indicated by boldface type.