<i>Alteromonas</i> As a Key Agent of Polycyclic Aromatic Hydrocarbon Biodegradation in Crude Oil-Contaminated Coastal Sediment

Following the 2007 oil spill in South Korean tidal flats, we sought to identify microbial players influencing the environmental fate of released polycyclic aromatic hydrocarbons (PAHs). Two years of monitoring showed that PAH concentrations in sediments declined substantially. Enrichment cultures were established using seawater and modified minimal media containing naphthalene as sole carbon source. The enriched microbial community was characterized by 16S rRNA-based DGGE profiling; sequencing selected bands indicated <i>Alteromonas</i> (among others) were active. <i>Alteromonas</i> sp. SN2 was isolated and was able to degrade naphthalene, phenanthrene, anthracene, and pyrene in laboratory-incubated microcosm assays. PCR-based analysis of DNA extracted from the sediments revealed naphthalene dioxygenase (NDO) genes of only two bacterial groups: <i>Alteromonas</i> and <i>Cycloclasticus</i>, having gentisate and catechol metabolic pathways, respectively. However, reverse transcriptase PCR-based analysis of field-fixed mRNA revealed <i>in situ</i> expression of only the <i>Alteromonas</i>-associated NDO genes; in laboratory microcosms these NDO genes were markedly induced by naphthalene addition. Analysis by GC/MS showed that naphthalene in tidal-flat samples was metabolized predominantly via the gentisate pathway; this signature metabolite was detected (0.04 μM) in contaminated field sediment. A quantitative PCR-based two-year data set monitoring <i>Alteromonas</i>-specific 16S rRNA genes and NDO transcripts in sea-tidal flat field samples showed that the abundance of bacteria related to strain SN2 during the winter season was 20-fold higher than in the summer season. Based on the above data, we conclude that strain SN2 and its relatives are site natives--key players in PAH degradation and adapted to winter conditions in these contaminated sea-tidal-flat sediments

Comparison of the gene content of strains SN2, AltDE, and ATCC 27126.

<p>(a) A Venn diagram of shared and specific CDS genes in each strain. Percentages of COG categories in the three <i>Alteromonas</i> species. (b) All orthologous genes and (c) specific orthologous genes between strains SN2 and AltDE and (d) between strains SN2 and ATCC 27126. The alphabetic code for the column charts is as follows: C, energy production and conversion; D, cell division and chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme metabolism; I, lipid metabolism; J, translation, ribosomal structure, and biogenesis; K, transcription; L, DNA replication, recombination, and repair; M, cell envelope biogenesis, outer membrane; N, cell motility and secretion; O, posttranslational modification, protein turnover, and chaperones; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknown; T, signal transduction mechanisms; U, intracellular trafficking, secretion, and vesicular transport; V, defense mechanisms. Asterisks appear when a difference between treatments was at least 20%.</p

A circular map representing the genome of <i>Alteromonas</i> sp. SN2.

<p>Forward strand and reverse strand CDSs (blue) are depicted on the outermost two circles of the map, respectively, and RNA genes (tRNA: red; rRNA: violet; others: gray) are also shown on the same circles. The third circle represents the BLASTN comparison of the strain AltDE genome against the strain SN2 genome (dark red indicates highly homologous CDSs). G+C content (black) and GC skews (GC skew+: green, GC skew-: violet) are drawn on the fourth and fifth circles, respectively.</p

General features of the whole genomes of three <i>Alteromonas</i> strains^a.

a<p>The genome analysis was carried out at JGI Integrated Microbial Genomes (<a href="http://img.jgi.doe.gov/" target="_blank">http://img.jgi.doe.gov/</a>).</p>b<p>Numbers of total protein coding genes.</p>c<p>ANI, Average nucleotide identity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035784#pone.0035784-Konstantinidis1" target="_blank">[34]</a>.</p

Correspondence analysis of codon usage among three <i>Alteromonas</i> species genomes.

<p>Eleven types of codon use are shown for <i>Alteromonas</i> strains SN2, AltDE, and ATCC 27126</p><p>Abbreviations: T3,C3,A3 and G3 indicate the frequencies of bases at position 3 of each codon.</p><p>GC, GC content in coding genes (G+C)</p><p>GC3s, GC of silent 3^rd codon position</p><p>Fop, frequency of optimal codon index</p><p>CBI, codon bias index</p><p>CAI, codon adaptation index</p><p>L_sym, number of synonymous codons</p><p>L_nsym, number of non-synonymous codons</p

Growth (optical density) of three <i>Alteromonas</i> strains (SN2, AltDE and ATCC 27126) in marine broth at eight different temperatures ranging from 5 to 40°C.

<p>Closed circles indicate the growth rate for strain SN2, open circles for strain AltDE and closed triangles for strain ATCC 27126. Data show averages of three replicate tubes.</p

Relative synonymous codon usage (RSCU) in three <i>Alteromonas</i> strains (SN2, AltDE, and ATCC 27126).

<p>RSCU values were calculated by summing the values for all of the genes. Correspondence analysis of codon usage as shown was carried out using the web-based codonw 1.4.4 program (Rice <i>et al</i>., 2000). Codons of amino acids on X axis were arranged based on the ascending RSCU values of strain SN2.</p

Features of binned contigs for genomes of thaumarchaeota, epsilon- and gammaproteobacteria (≥ 5 Kb contigs).

<p>ND, not detected.</p

Principal component analysis of oligonucleotide frequencies in assembled contigs from two archaeal enrichment cultures

<p>(A) AR culture, and (B) SJ culture. Reference genomes are shown as larger circles. The total number of contigs for each group (<i>Gammaproteobacteria</i>, <i>Epsilonproteobacteria,</i> and <i>Thaumarchaeota</i>), total length, mean length, and GC content range are also indicated. The contig types and published genomes are as follows: orange, <i>Gammaproteobacteria</i>; yellow, <i>Thaumarchaeota</i>; green, <i>Epsilonproteobacteria</i>; light green, assembled contigs including viral coding sequences; gray, not identified; red, <i>Ca</i>. “Cenarchaum symbiosum” A (CsymA); fuchsia, <i>Ca</i>. “C. symbiosum” B (CsymB); lime, <i>Nitrosopumilus maritimus</i> SCM1 (Nmar); blue, <i>Ca</i>. “Nitrosoarchaeum koreensis” MY1 (MY1); cyan, <i>Ca</i>. “Nitrosoarchaeum limnia” (Nlim); violet, <i>Ca</i>. “Nitrososphaera gargensis” (Ngar); teal, <i>Sulfurovum</i> sp. NBC37-1 (Sul); and purple, <i>Thiomicrospira crunogena</i> XCL-2 (Tcr).</p

Comparison of the <i>Ca</i>. “Nitrosopumilus sediminis” AR2 genomic region containing genes for urea utilization with those of <i>Ca</i>. “Cenarchaeum symbiosum” and environmental metagenomes.

<p><i>Ca</i>. “N. sediminis” AR2 genome is central, with the <i>Ca</i>. “C. symbiosum”, <i>Ca</i>. “Nitrososphaera gargensis”, and environmental metagenomic regions above and below, respectively. Homologous genes are connected with shaded regions, and the shaded color indicates the percent identity as determined by TBLASTX.</p