Phylogenetic diversity, polyamine pattern and DNA base composition of members of the order Planctomycetales.

The 16S rDNA sequences of 20 novel isolates of members of the order Planctomycetales were compared to those of the type strains of described planctomycete species and 22 planctomycete isolates for which the 16S rDNA sequences had been previously determined. The novel isolates could be assigned to several phylogenetically broad groups, four of which are defined by the genera Gemmata, Isosphaera, Planctomyces and Pirellula. To evaluate polyamines as a chemotaxonomic marker within this order, the polyamine pool was determined for six planctomycete reference species and for 20 planctomycete isolates. All analysed members of the order Planctomycetales contained significant amounts of polyamines. sym-Homospermidine (HSPD) is present in all strains except Planctomyces limnophilus and related strains, which had high amounts of putrescine (PUT) as the dominant polyamine component. The distribution of PUT, HSPD and spermidine reflects the phylogenetic diversity within the Planctomycetales as closely related representatives of the phylogenetic groups defined by described species and novel isolates exhibit similar polyamine patterns. Determination of the DNA base composition revealed G + C contents of > 60 mol% for members of Gemmata and Isosphaera whereas, except for two isolates, strains which are phylogenetically associated with Planctomyces and Pirellula had G + C contents of 51-57 mol%.

Ribonucleotide reductase in Bacillus subtilis--evidence for a Mn-dependent enzyme.

The reduction of 2'-ribonucleotides to 2'-deoxyribonucleotides, a unique step in DNA formation, is catalyzed by ribonucleotide reductase (RRase), an allosterically regulated, cell cycle-dependent enzyme. This work reports a reversible impairment of DNA formation and ribonucleotide reduction upon manganese depletion in Bacillus subtilis demonstrated through in vivo labeling with necleic acid precursors and enzyme assays with ether-permeabilized cells. No deoxyadenosylcobalamin-dependent reduction of ribonucleotides was detected in the cytosol, and the properties of a partially purified enzyme fraction, i.e., sensitivity towards EDTA and hydroxyurea (HU), indicated a metal-dependent type of RRase. The enzyme was enriched by gel filtration on Superose 12 from glycerol- or fumarate-grown cells and submitted to Q-band electron paramagnetic resonance (EPR) spectroscopy for further characterization of the metal center. A distinct Mn(II) signal was obtained in both preparations characteristic of a protein-bound mangaenese in a mononuclear metal center with axial symmetry. The intensity of this Mn signal was not affected by addition of the radical scavenger HU (10 mM) but reduced in the presence of 2.5 mM EDTA. On the basis of these results, we suggest that Bacillus subtilis has a Mn-dependent ribonucleotide reductase.

A divalent metal site in the small subunit of the manganese-dependent ribonucleotide reductase of Corynebacterium ammoniagenes.

Based on its metallo-cofactor, the manganese-dependent ribonucleotide reductase (Mn-RRase) responsible for delivery of DNA precursors in the Mn-requiring Gram-positive bacterium Corynebacterium (formerly Brevibacterium) ammoniagenes ATCC 6872 is no longer considered as a simple analogue of the aerobic Fe-RRase of Escherichia coli but as the prototype of the class IV enzymes (1). Deliberate dissociation of the Mn-RRase holoenzyme and an improved sample preparation of the dimeric CA2 protein allowed further characterization of the inherent metallo-cofactor by Q-band electron paramagnetic resonance (EPR) spectroscopy. At 40 K, a distinct hyperfine sextet (I = 5/2,55Mn) pattern with a weak zero-field splitting was detected in the CA2 protein prepared from manganese-sufficient cells displaying high RRase activity as expected. This Q-band Mn(II) signal was absent in the apo-CA2 protein obtained from manganese-depleted cells devoid of this enzymatic activity. The presence of a mixed valence manganese cluster in the C. ammoniagenes RRase is excluded since no complex multiline EPR signals were detected in the CA2 protein even at very low (8 K) temperature. The observed Mn(II) spectrum indicates a protein-bound manganese which was modified in the presence of 5.7 mM p-methoxyphenol, but is insensitive toward 10 mM EDTA. Thus, the manganese appeared to be either strictly bound or buried within a hydrophobic pocket of the CA2 protein, inaccessible for EDTA.

Detection of a stable free radical in the B2 subunit of the manganese ribonucleotide reductase (Mn-RRase) of Corynebacterium ammoniagenes.

Ribonucleotide reductases catalyze the irreversible reductive formation of 2'-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Corynebacterium ammoniagenes ATCC 6872. Enrichment by 2'5'-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with SuperoseTM12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K. The EPR spectrum exhibits an easily saturable doublet of 1.8 mT splitting and a line width of 1.3 mT at g = 2.0040. The EPR signal intensity showed a clear correlation with the enzymatic activity upon long-time storage at ambient temperature (294 K) and inactivation by the specific RRase inhibitor hydroxyurea (HU). This leads to the assumption of a protein-linked radical, with functional significance, in the metal-containing 100 kDa B2 subunit of the MnRRase of Corynebacterium ammoniagenes.