Anaerobic Growth of Bacillus mojavensis and Bacillus subtilis Requires Deoxyribonucleosides or DNA

Abstract

Bacillus mojavensis strains JF-2 (ATCC 39307), ROB2, and ABO21191T and Bacillus subtilis strains 168 (ATCC 23857) and ATCC 12332 required four deoxyribonucleosides or DNA for growth under strict anaerobic conditions. Bacillus licheniformis strains L89-11 and L87-11, Bacillus sonorensis strain TG8-8, and Bacillus cereus (ATCC 14579) did not require DNA for anaerobic growth. The requirement for the deoxyribonucleosides or DNA did not occur under aerobic growth conditions. The addition of a mixture of five nucleic acid bases, four ribonucleotides, or four ribonucleosides to the basal medium did not replace the requirement of B. mojavensis JF-2 for the four deoxyribonucleosides. However, the addition of salmon sperm DNA, herring sperm DNA, Escherichia coli DNA, or synthetic DNA (single or double stranded) to the basal medium supported anaerobic growth. The addition of four deoxyribonucleosides to the basal medium allowed aerobic growth of B. mojavensis JF-2 in the presence of hydroxyurea. B. mojavensis did not grow in DNA-supplemented basal medium that lacked sucrose as the energy source. These data provide strong evidence that externally supplied deoxyribonucleosides can be used to maintain a balanced deoxyribonucleotide pool for DNA synthesis and suggest that ribonucleotide reductases may not be essential to the bacterial cell cycle nor are they necessarily part of a minimal bacterial genome.Ribonucleotide reductases make deoxyribonucleotides from ribonucleotides regardless of whether the nucleotide is made de novo or by the salvage pathways (25,26,31). The de novo pathways make nucleotides from metabolic precursors generated in central metabolic pathways. The salvage pathways use exogenously supplied bases and nucleosides or recycle endogenously produced bases and nucleosides (23). Ribonucleotide reductases are considered essential to all organisms to regulate and maintain the deoxyribonucleotide pool necessary for DNA synthesis (9,18,25,29,32,33). Recently, the minimal essential genes of Bacillus subtilis were narrowed to only 192 "essential" genes out of 4,100 genes (19) and included the class I ribonucleotide reductase genes, nrdE and nrdF. In 1952, Lactobacillus acidophilus R-26 was reported to require at least one externally supplied deoxyribonucleoside and to lack ribonucleotide reductase activity (14). This finding suggests that ribonucleotide reductase activity may not be required for all microorganisms.In order to use deoxyribonucleosides as DNA precursors, an organism must be able to phosphorylate the deoxyribonucleoside to a deoxyribonucleotide. Escherichia coli and Salmonella enterica serovar Typhimurium do not have the deoxyribonucleoside kinases necessary for converting deoxyribonucleosides into deoxyribonucleotides and therefore cannot utilize exogenously supplied deoxyribonucleosides as sole DNA precursors (25). However, B. subtilis and lactobacilli have all of the kinases necessary to phosphorylate the deoxyribonucleosides to deoxyribonucleotides and could potentially use them as sole DNA precurs...

The Bacillus subtilis nrdEF Genes, Encoding a Class Ib Ribonucleotide Reductase, Are Essential for Aerobic and Anaerobic Growth

“…In contrast to the observation made by several groups that investigated the anaerobic metabolism of B. subtilis, recently it was found that anaerobic growth of Bacillus mojavensis and B. subtilis 168 requires externally supplied deoxyribonucleosides or DNA (7). This finding and the presence of only the oxygendependent class I RNR encoded by the B. subtilis nrdEF genes gave rise to the question of how B. subtilis is able to grow under anaerobic conditions.…”

“…␤-Galactosidase activity was determined as described previously (10) and was expressed in Miller units (27). (7). In contrast to this observation, several other investigations during the last decade elucidated the exact nature of anaerobic nitrate respiratory and fermentative growth (4,11,12,24,29,32).…”

Identification of Virulence Determinants for Endocarditis in Streptococcus sanguinis by Signature-Tagged Mutagenesis

“…The moderate but significant reduction of virulence seen for the 6-26 mutant could suggest either a spatial or temporal gradient of oxygen within the vegetation or a partial ability to overcome the loss of anaerobic RNR activity. Some Bacillus species that apparently possess only class I RNRs can use external DNA as a source of deoxyribonucleotides under anaerobic conditions, thereby overcoming the requirement for anaerobic ribonucleotide reduction (19). Given that S. sanguinis is also naturally competent and that the biofilm of the vegetation may represent a site of increased natural competence (39), DNA uptake may have aided the survival of the RNR mutants in the CI assays.…”

Ecology and genomics of Bacillus subtilis

“…This suggested that B. subtilis should be able to grow anaerobically using nitrate instead of oxygen as an electron acceptor. Anaerobic growth of B. subtilis in the presence of nitrate has since been demonstrated experimentally 25 . The discovery that B. subtilis can indeed grow anaerobically further supports the idea that vegetative life within the mostly anaerobic GI tract of animals is feasible.…”