Substrate-Dependent Regulation of Anaerobic Degradation Pathways for Toluene and Ethylbenzene in a Denitrifying Bacterium, Strain EbN1

Abstract

Anaerobic biodegradation of toluene and ethylbenzene is of environmental concern and biochemical interest due to toxicity and novel reactions, respectively. The denitrifying strain EbN1 is unique in anaerobically degrading both alkylbenzenes via different pathways which converge at benzoyl coenzyme A. The organization of genes involved in both pathways was only recently determined for strain EbN1. In the present study, global expression analysis (DNA microarray and proteomics) indicated involvement of several thus-far-unknown proteins in the degradation of both alkylbenzenes. For example, orf68 and orf57, framing the ebd operon, are implicated in ethylbenzene degradation, and the ebA1932 and ebA1936 genes, located 7.2 kb upstream of the bbs operon, are implicated in toluene degradation. In addition, expression studies were now possible on the level of the complete pathways. Growth experiments demonstrated that degradative capacities for toluene and ethylbenzene could be simultaneously induced, regardless of the substrate used for adaptation. Regulation was studied at the RNA (real-time reverse transcription-PCR and DNA microarray) and protein (two-dimensionaldifference gel electrophoresis) level by using cells adapted to anaerobic growth with benzoate, toluene, ethylbenzene, or a mixture of toluene and ethylbenzene. Expression of the two toluene-related operons (bss and bbs) was specifically induced in toluene-adapted cells. In contrast, genes involved in anaerobic ethylbenzene degradation were induced in ethylbenzene-and toluene-adapted cells, suggesting that toluene may act as a gratuitous inducer. In agreement with the predicted sequential regulation of the ethylbenzene pathway, Ebd proteins (encoding subunits of ethylbenzene dehydrogenase) were formed in ethylbenzene-but not in acetophenone-adapted cells, while Apc proteins (subunits of predicted acetophenone carboxylase) were formed under both conditions.

Solvent Stress Response of the Denitrifying Bacterium “ Aromatoleum aromaticum ” Strain EbN1

“…Proteins involved in the anaerobic degradation of ethylbenzene, toluene, p-cresol, and phenol have been identified in previous proteomic studies with strain EbN1 (28,54). Here, 11 and 12 2D electrophoresis-separated protein spots could be correlated with ethylbenzene and toluene degradation pathways, respectively (see Table S2 in the supplemental material).…”

“…Notably, constitutive formation of SodB and KatA in anaerobically growing strain EbN1 seems likely, since the corresponding protein spots were detected on almost all 2D electrophoresis gels in this and previous studies (28,54).…”

“…In addition, strain EbN1 also utilizes p-cresol, phenol, and some other polar aromatic compounds under anoxic conditions. Recently, the complete genome sequence of strain EbN1 was determined, allowing detailed reconstruction of its metabolic network and in-depth proteomic analysis of substrate-dependent regulation of individual degradation pathways (28,35,54). Strain EbN1 represents a promising model organism for in situ processes, since toluene-amended microcosms from oil-contaminated and pristine sites displayed rapid consumption of nitrate (31) and contained high numbers of bacteria related to the phylogenetic cluster harboring strain EbN1 (20,31).…”

Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2

“…Cooperation between an SCPx thiolase and a DUF35 protein has also been reported for PhlB and PhlC, which are involved in acetylation of an aromatic polyketide in P. putida (Bangera & Thomashow, 1999). (Kube et al, 2004;Kü hner et al, 2005) and Thauera aromatica (Leuthner & Heider, 2000).…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…16), but the relevance of these genes in anaerobic toluene catabolism and whether they belong to the bss operon are still unknown (202). In the bss cluster of G. (203), Azoarcus sp. strain T (accession number AY032676) (1), T. aromatica K172 (accession numbers AJ001848 and AF173961) (155,216), T. aromatica T1 (accession numbers U57900 and AF113168) (68,72), Thauera sp.…”