Genome analysis and physiological comparison of Alicycliphilus denitrificans strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.This research was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO), project number 08053, the graduate school WIMEK (Wageningen Institute for Environment and Climate Research, which is part of SENSE Research School for Socio-Economic and Natural Sciences of the Environment, www.wimek-new.wur.nl and www.sense.nl), SKB (Dutch Centre for Soil Quality Management and Knowledge Transfer, www.skbodem.nl) and the Consolider project CSD-2007-00055. The research was incorporated in the TRIAS (TRIpartite Approaches 469 toward Soil systems processes) program (http://www.nwo.nl/en/research-and-results/programmes/alw/trias-tripartite-approach-to-soil-system-processes/index. html). Flávia Talarico Saia was supported by a FAPESP (the State of São Paulo Research Foundation) scholarship (2006-01997/5). The work conducted by the DOE JGI is supported by the Office of Science of the United States Department of Energy under contract number DE-AC02-05CH11231. Alfons Stams acknowledges support by an ERC (European Research Counsil) advanced grant (project 323009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Combined Carbon and Hydrogen Isotope Fractionation Investigations for Elucidating Benzene Biodegradation Pathways

Recently, combined carbon and hydrogen isotope fractionation investigations have emerged as a powerful tool for the characterization of reaction mechanisms relevant for the removal of organic pollutants. Here, we applied this approach in order to differentiate benzene biodegradation pathways under oxic and anoxic conditions in laboratory experiments. Carbon and hydrogen isotope fractionation of benzene was studied with four different aerobic strains using a monooxygenase or a dioxygenase for the initial benzene attack, a facultative anaerobic chlorate-reducing strain as well as a sulfate-reducing mixed culture. Carbon and hydrogen enrichment factors (εC, εH) varied for the specific pathways and degradation conditions, respectively, so that from the individual enrichment factors only limited information could be obtained for the identification of benzene biodegradation pathways. However, using the slope derived from hydrogen vs carbon isotope discriminations or the ratio of hydrogen to carbon enrichment factors (Λ = ΔH/ΔC ≈ εH/εC), benzene degradation mechanisms could be distinguished. Although experimentally determined Λ values partially overlapped, ranges could be determined for different benzene biodegradation pathways. Specific Λ values were 17 for anaerobic degradation. Moreover, variations in Λ values suggest that more than one reaction mechanism exists for monohydroxylation as well as for anaerobic benzene degradation under nitrate-reducing, sulfate-reducing, or methanogenic conditions. Our results show that the combined carbon and hydrogen isotope fractionation approach has potential to elucidate biodegradation pathways of pollutants in field and laboratory microcosm studies

Bidirectional BLAST analysis of the genomes of <i>A. denitrificans</i> strains K601^T and BC.

<p>The amount of protein sequences present only in strains K601^T (left) and BC (right) and in both strains (center) is shown in the VENN diagram. 172 protein sequences of strain K601^T and 154 of strain BC could not be assigned (for instance duplicates of sequences).</p

General features of the genomes of <i>A. denitrificans</i> strains BC and K601^T.

<p>General features of the genomes of <i>A. denitrificans</i> strains BC and K601^T.</p

Gene cluster for chlorate reduction in <i>A. denitrificans</i> strain BC (<i>Aden</i>) compared to <i>I. dechloratans</i> (<i>Idec</i>).

<p>The gene cluster for chlorate reduction comprises of chlorite dismutase (<i>cld</i>), chlorate reductase subunit A, B, C and D (<i>clrA, clrB, clrC, clrD</i>), and in <i>I. dechloratans</i> it also includes an insertion sequence (<i>ISIde1</i>). The numbers represent the location of nucleotide differences (in red) of strain BC compared to <i>I. dechloratans</i> counted from the first nucleotide of each gene. The scale bar represents 500 bp. Sequences for the chlorate reduction gene cluster of <i>I. dechloratans</i> were obtained from the EMBL nucleotide sequence database (accession numbers AJ296077 and AJ566363).</p

Overview of substrate range of <i>A. denitrificans</i> strains BC and K601^T.

<p>+: growth, −: no growth, n.d.: not determined,</p>a<p>previous data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066971#pone.0066971-Mechichi1" target="_blank">[6]</a>.</p

Organization of the multicomponent benzene/phenol monooxygenase cluster (B1–B6) and catechol dioxygenases (C23O) of <i>A. denitrificans</i> strains BC and K601^T.

<p>In this gene cluster a gene coding for a transcriptional regulator (R) and a gene coding for a ferredoxin (Fe) were also found. Both strains BC and K601^T have highly similar gene clusters (99%) with differences only in subunit B2 and B4. The numbers represent the location of the nucleotide differences (in red) of strain BC compared to K601^T counted from the first nucleotide of each gene. The scale bar represents 500 bp.</p

Main metabolic pathways of <i>A. denitrificans</i>.

<p>Pathways are indicated using arrows. Black arrows indicate pathways of both strain BC and K601^T, red arrows indicate pathways of strain BC, and blue arrows pathways of strain K601^T. Red gene numbers indicate genes of strain BC (geneID is Alide_red gene number) and blue gene numbers genes of strain K601^T (geneID is Alide2_blue gene number).</p

Degradation of benzene (1), toluene (2) and acetate (3) with chlorate (a), nitrate (b) or oxygen (c) as electron acceptor by <i>A. denitrificans</i> strain BC.

<p>Benzene, toluene and acetate degradation is indicated with diamonds. Benzene and toluene concentrations are outlined on a secondary y-axis while acetate and electron acceptor concentrations are indicated on the primary y-axis. Chlorate, nitrate and oxygen consumption is depicted with squares. Chloride production when chlorate is used as electron acceptor is indicated with triangles and no electron acceptor consumption is shown when no significant difference could be observed because of presence of the electron acceptor in abundance.</p