The Purine-Utilizing Bacterium <em>Clostridium acidurici</em> 9a: A Genome-Guided Metabolic Reconsideration

<div><p><em>Clostridium acidurici</em> is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia <em>C. cylindrosporum</em> and <em>C. purinilyticum</em>, <em>C. acidurici</em> serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic <em>Clostridium</em>. The genome of <em>C. acidurici</em> 9a consists of one chromosome (3,105,335 bp) and one small circular plasmid (2,913 bp). The lack of candidate genes encoding glycine reductase indicates that <em>C. acidurici</em> 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of <em>C. acidurici</em> 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as <em>C. acetobutylicum</em>, <em>C. saccharolyticum</em>, and <em>C. beijerinckii</em>. The only amino acid that can be degraded by <em>C. acidurici</em> is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by <em>C. acidurici</em> but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of <em>C. acidurici</em> towards bacitracin, acriflavine and azaleucine was experimentally confirmed.</p> </div

Metabolic pathways involed in purine utilization of <i>C. acidurici</i> 9a.

<p>The scheme represents a genome-based model of important metabolic and energy conservation steps during purine utilization of <i>C. acidurici</i> 9a. Relevant enzymes and their corresponding locus tags are shown in blue ellipses and end-products in green letters. Ferredoxin-involving steps (light green box) were modified after Vogels and van der Drift <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051662#pone.0051662-Vogels1" target="_blank">[53]</a>. Abbreviations: Fd, ferredoxin; ox, oxidized; red, reduced; THF, tetrahydrofolate; ?, unknown.</p

Growth on uric acid and glycine (A) and corresponding gene expression at different time points (B).

<p>(<b>A</b>) Growth of cells was measured over 12 h in medium containing different concentrations of uric acid (UA) with (dashed line) or without (solid line) glycine (Gly). Non-adapted cells were shifted directly from uric acid medium to uric acid with glycine whereas adapted cells were grown three times in 12 mM uric acid medium with 100 mM glycine prior to transfer and measurement. (<b>B</b>) Dependent on the addition of glycine a semi-quantitative analysis of transcription levels of genes involved in the conversion of glycine to acetate was performed. Shown are the results for adapted cells grown in 3 mM and 12 mM uric acid medium or in the same media supplemented with 100 mM glycine. Samples were taken after 1 h, 3 h, 5 h, 7 h and 9 h. DNA as template in the RT-PCR reaction served as positive control (P) and water as negative control (N). Abbreviations: <i>gcvPB</i>, glycine dehydrogenase beta subunit (Curi_c00770); <i>glyA</i>, serine hydroxymethyltransferase (Curi_c17240); <i>ackA,</i> acetate kinase (Curi_c16390); <i>por1/por2</i>, pyruvate:ferredoxin oxidoreductase (Curi_c20330/Curi_c21330); <i>fdhA</i>/<i>fdhB</i>, formate dehydrogenase subunits A; (Curi_c16670, Curi_c16640); <i>fdhF1/fdhF2</i>, formate dehydrogenase H (Curi_c29370/Curi_c29380); <i>rpoA</i>, DNA-directed RNA polymerase alpha subunit (Curi_c22350, reference).</p

Additional file 2: Data S2. of Genome sequence of Pedobacter glucosidilyticus DD6b, isolated from zooplankton Daphnia magna

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Additional file 1: of Genome sequence of Shinella sp. strain DD12, isolated from homogenized guts of starved Daphnia magna

Composition of MDS3 medium and growth conditions in phosphite assimilation tests. (PDF 64.3 kb

Venn diagram of all analyzed OTUs in fertilized and non-fertilized soils at entire and active bacterial community level.

<p>Depicted were OTUs estimated at entire community level (fertilized soil), active community level (fertilized soil), entire community level (non-fertilized soil), and active community level (non-fertilized soil) and all other possible interfaces.</p