Functional Genomics of Anaerobic Degradation of Hydrocarbons: An Introduction
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Anaerobic biodegradation of hydrocarbons is typically studied with newly isolated microorganisms not yet accessible to genetic manipulation. Functional genomics, viz., genome-based differential proteomics in conjunction with targeted metabolite analysis, has been instrumental for discovering a multitude of novel reactions and pathways involved in the biochemically challenging process of anaerobic hydrocarbon degradation.
The rapid advancements in genome sequencing technologies subjected life sciences to fundamental methodological and conceptual expansions during the last two decades. The technological development starting with the first-generation sequencing (Sanger) proceeded via massively parallel sequencing by means of second-generation instruments (e.g., Illumina), providing huge amounts of short read length (150 bp) with high quality, to the third-generation sequencing that analyzes single DNA molecules in real time, generating long read lengths (median ~6 kbp), but with quality still in need for improvement (e.g., PacBio and MinION) (Shendure et al. 2017; Reuter et al. 2015). The second- and third-generation sequencing technologies do not only provide low cost access to high quality genome sequences of any pure culture of interest but will also open up new avenues for profiling microbial communities and for microbiome research (Kerkhof et al. 2017; Michas et al. 2017). This dramatic increase in access to sequencing data is met by open bioinformatics platforms for automated data processing and analysis, e.g., IMG/M (Chen et al. 2016) and MG-RAST (Wilke et al. 2016). Notwithstanding, manual expert annotation is still very much required in the context of newly discovered metabolic capacities or reconstruction of metabolic networks that are not covered by well-studied standard organisms. Despite the affluent information provided by genomics, these data are restricted to the current state of knowledge present in the database and only allow for a theoretical view on an organism’s metabolism, cellular properties, and potential interactions. Considering that proteins are the active entities that bring a cell to life, proteomics plays a key role in postgenomic investigations of a study organism. Owing to the immense diversity of proteins (amino acid composition, size, polarity, subcellular localization, complex formation, abundance dynamics, etc.), there is not a single methodological approach that covers a given proteome. Rather, a whole suite of methods ranging from sample preparation via (quantitative) protein/peptide separation to mass spectrometry-based analysis and bioinformatics-based identification have to be integrated according to the demands of a given project (Wöhlbrand et al. 2013). Proteomics standards are required for cross-project comparisons and particularly for metaproteomics; this challenge is particularly met by the initiatives within the framework of the Human Proteome Project (e.g., Deutsch et al. 2017). The combination of genomics and proteomics, viz., proteogenomics, is a particularly promising and growing field, as it allows refining the genome annotation and at the same time unraveling the dynamics and complexity of protein formation/abundance (e.g., Menschaert and Fenyö 2017).
The benefit of applying proteogenomics to the field of anaerobic microbial hydrocarbon degradation was early on substantiated by enabling the identification of the complete gene cluster for the anaerobic degradation of ethylbenzene in the denitrifying bacterium Aromatoleum aromaticum EbN1 (Rabus et al. 2002, 2019). The subsequently reported genome of strain EbN1 (Rabus et al. 2005) together with a comprehensive proteome profiling of substrate-adapted cells (Wöhlbrand et al. 2007) yielded new reaction/pathway discoveries and first global insights into the regulatory capacities of such an anaerobic degradation specialist. Meanwhile, several proteogenomic studies concerned with a variety of facultatively and strictly anaerobic microorganisms have further expanded our knowledge on the genes, enzymes, and pathways involved in anaerobic degradation of aromatic compounds (e.g., Heintz et al. 2009; Bergmann et al. 2011; Nobu et al. 2015).
A further instrumental approach supplementing proteogenomics in pathway discovery is targeted metabolite analysis, e.g., with isotope labeled substrates. Unequivocal identification of hitherto unknown metabolites strongly corroborates the predictions from differential proteogenomics and likewise provides initial hints on new reactions to be further elaborated by proteogenomics. This is exemplified by the identification of p-hydroxyacetophenone as a key intermediate of the newly discovered anaerobic degradation pathway for p-ethylphenol in A. aromaticum EbN1 (Wöhlbrand et al. 2008). Further integration of a global metabolomics approach will be an important leverage to eventually promote research on anaerobic degradation of hydrocarbons to the systems biology level.
This section on anaerobic degradation of hydrocarbons is subdivided into three chapters, reflecting the mode of energy generation: denitrification, sulfate reduction, and metal reduction.
The chapter on denitrifying bacteria by Rabus and Wilkes (n.d.-a) first summarizes the state of knowledge on A. aromaticum EbN1, which represents the proteogenomically best studied anaerobic degrader of aromatic compounds (including hydrocarbons), covering insights into pathways, the architecture and regulation of the catabolic network, and adaptation to habitat-relevant conditions such as solvent stress, substrate mixtures, and slow growth. The second major part of this chapter revolves around Azoarcus sp. strain CIB, which is best studied with respect to the mechanisms of transcriptional control, e.g., the bzd and mbd operons for anaerobic degradation of benzoate and 3-methylbenzoate, respectively; furthermore, a new strategy to achieve solvent tolerance involving c-di-GMP is mentioned. The chapter progresses by presenting insights into the anaerobic degradation of para-alkylated benzoate and toluenes in A. aromaticum EbN1, Thauera sp. strain pCyN2, and Magnetosprillum sp. strain pMbN1. Finally, genome-based analyses of Azoarcus sp. strain PA01, Azoarcus anaerobius and Thauera aromatica are summarized.
The chapter on sulfate-reducing bacteria by Rabus and Wilkes (n.d.-b) is first concerned with toluene-degrading Desulfobacula toluolica Tol2, which represents the aromatic compound-degrading sulfate reducer to be first comprehensively studied by integrating genomics, proteomics, and targeted metabolite analyses. A second emphasis is on naphthalene-degrading SRB, involving the pure cultures NaphS2, NaphS3, and NaphS6, as well as the intensively studied enrichment culture N47. Finally, genome-based studies on Desulfobacula sp. TS, Desulfococcus multivorans, and Desulfotomaculum gibsoniae GrollT are presented.
The chapter on metal-reducing bacteria capable of anaerobic degradation of aromatic compounds by Tremblay and Zhang (n.d.) first summarizes available cultures and then proceeds according to compound type, from benzene, via alkylbenzenes to polyaromatic hydrocarbons. A large part of the presented studies uses the iron-reducing deltaproteobacterium Geobacter metallireducens as a model system and comprises biochemical and/or functional genomic approaches. Particular emphasis is on the proposed anaerobic hydroxylation of benzene and the ATP-independent reduction of the central intermediate benzoyl-CoA.
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