Genome sequence of the deltaproteobacterial strain NaphS2 and analysis of differential gene expression during anaerobic growth on naphthalene

Abstract

Background: Anaerobic polycyclic hydrocarbon (PAH) degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined.

Results: Draft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds.

Conclusion: This study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster in this organism, suggests that NaphS2 degrades both compounds via parallel mechanisms. Elucidation of the key genes necessary for anaerobic naphthalene degradation may provide the ability to track naphthalene degradation through in situ transcript monitoring.

Figure 1. NaphS2 has three benzoyl-CoA reductases, with differing patterns of expression.

Two panels are depicted for each gene displayed. Red bars represent mRNA or protein up/downregulation on benzoate vs. pyruvate, blue bars represent mRNA or protein up/downregulation on naphthalene vs. benzoate. For each set of genes, the upper panel shows mRNA up/down-regulation, benzoate vs. pyruvate, naphthalene vs. benzoate, in fold change units. Stars indicate significant changes (values >2). N/A indicates that no data was available. The lower panel shows protein up/down-regulation, with the same conventions used for depicting mRNA expression except that units are Z-score difference and significance was determined by the t-test.

Figure 2. Proposed Pathways for Naphthalene and 2-Methylnaphthalene degradation.

Steps 1–3 outline proposed steps involved in degradation of 2-methylnaphthalene. One model for naphthalene degradation involves the methylation of naphthalene to 2-methylnaphthalene and its subsequent degradation by Nms and Bns in steps analogous to toluene degradation. Steps 1a, 2a, and 3 outline a proposed mechanism for naphthalene degradation in which naphthalene is carboxylated and coadenylated. Later steps common to both the methylation and carboxylation models of naphthalene degradation include reduction of naphthoyl-CoA by NcrABCD (step 3).

Figure 3. Quantitative RT-PCR analysis of transcripts during growth on pyruvate, benzoate, and naphthalene.

Quantitative RT-PCR analysis was conducted to confirm expression of genes upregulated during growth on naphthalene, using RNA from microarray experiments for genes indicated in each panel. (A) Quantitative RT-PCR of select genes using gene specific primers during the reverse transcription reaction. (B) Quantitative RT-PCR of select genes using random primers during the reverse transcription reaction.

Figure 4. NaphS2 degrades toluene.

Increase in cell density and sulfide concentration during anaerobic growth of strain NaphS2 with toluene. The growth experiment was performed in a 150 ml serum bottle containing 100 ml of medium and toluene dissolved in heptamethylnonane.

Figure 5. The bnsA-H gene cluster in NaphS2 and N47 and the homologous BbsA-H clusters from other species.

Arrangement of bnsA-H genes in strain NaphS2 compared to sulfate-reducing culture N47 and other species. The arrangement of the cluster is similar to EbN1, however genes downstream of bbsH in G. metallireducens are also present downstream of bnsH in NaphS2.