Complex and flexible catabolism in Aromatoleum aromaticum pCyN1.

Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil-rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant-derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C-H-bond cleavage at the methyl groups of p-cymene/4-ethyltoluene, toluene and p-cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium-wide (one module shared by p-cymene, 4-ethyltoluene, α-phellandrene, α-terpinene, γ-terpinene and limonene) to broad (central benzoyl-CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP-dependent (class I) benzoyl-CoA reductase and four different ß-oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low-affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability.

Aromatoleum gen. nov., a novel genus accommodating the phylogenetic lineage including Azoarcus evansii and related species, and proposal of Aromatoleum aromaticum sp. nov., Aromatoleum petrolei sp. nov., Aromatoleum bremense sp. nov., Aromatoleum toluolicum sp. nov. and Aromatoleum diolicum sp. nov.

Comparative 16S rRNA gene sequence analysis and major physiological differences indicate two distinct sublineages within the genus Azoarcus: the Azoarcus evansii lineage, comprising Azoarcusevansii (type strain KB740T=DSM 6898T=CIP 109473T=NBRC 107771T), Azoarcusbuckelii (type strain U120T=DSM 14744T=LMG 26916T), Azoarcusanaerobius (type strain LuFRes1T=DSM 12081T=LMG 30943T), Azoarcustolulyticus (type strain Tol-4T=ATCC 51758T=CIP 109470T), Azoarcustoluvorans (type strain Td21T=ATCC 700604T=DSM 15124T) and Azoarcustoluclasticus (type strain MF63T=ATCC 700605T), and the Azoarcus indigens lineage, comprising Azoarcusindigens (type strain VB32T=ATCC 51398T=LMG 9092T), Azoarcus communis (type strain SWub3T=ATCC 51397T=LMG 9095T) and Azoarcusolearius (type strain DQS-4T=BCRC 80407T=KCTC 23918T=LMG 26893T). Az. evansii lineage members have remarkable anaerobic degradation capacities encompassing a multitude of alkylbenzenes, aromatic compounds and monoterpenes, often involving novel biochemical reactions. In contrast, Az. indigens lineage members are diazotrophic endophytes lacking these catabolic capacities. It is proposed that species of the Az. evansii lineage should be classified in a novel genus, Aromatoleum gen. nov. Finally, based on the literature and new growth, DNA-DNA hybridization and proteomic data, the following five new species are proposed: Aromatoleum aromaticum sp. nov. (type strain EbN1T=DSM 19018T=LMG 30748T and strain pCyN1=DSM 19016=LMG 31004), Aromatoleum petrolei sp. nov. (type strain ToN1T=DSM 19019T=LMG 30746T), Aromatoleumbremense sp. nov. (type strain PbN1T=DSM 19017T=LMG 31005T), Aromatoleum toluolicum sp. nov. (type strain TT=DSM 19020T=LMG 30751T) and Aromatoleum diolicum sp. nov. (type strain 22LinT=DSM 15408T=LMG 30750T).

Characterization of an archaeal virus-host system reveals massive genomic rearrangements in a laboratory strain.

Halophilic archaea (haloarchaea) are known to exhibit multiple chromosomes, with one main chromosome and one or several smaller secondary chromosomes or megaplasmids. Halorubrum lacusprofundi, a model organism for studying cold adaptation, exhibits one secondary chromosome and one megaplasmid that include a large arsenal of virus defense mechanisms. We isolated a virus (Halorubrum tailed virus DL1, HRTV-DL1) infecting Hrr. lacusprofundi, and present an in-depth characterization of the virus and its interactions with Hrr. lacusprofundi. While studying virus-host interactions between Hrr. lacusprofundi and HRTV-DL1, we uncover that the strain in use (ACAM34_UNSW) lost the entire megaplasmid and about 38% of the secondary chromosome. The loss included the majority of virus defense mechanisms, making the strain sensitive to HRTV-DL1 infection, while the type strain (ACAM34_DSMZ) appears to prevent virus replication. Comparing infection of the type strain ACAM34_DSMZ with infection of the laboratory derived strain ACAM34_UNSW allowed us to identify host responses to virus infection that were only activated in ACAM34_UNSW upon the loss of virus defense mechanisms. We identify one of two S-layer proteins as primary receptor for HRTV-DL1 and conclude that the presence of two different S-layer proteins in one strain provides a strong advantage in the arms race with viruses. Additionally, we identify archaeal homologs to eukaryotic proteins potentially being involved in the defense against virus infection.

Systems Biology of Aromatic Compound Catabolism in Facultative Anaerobic Aromatoleum aromaticum EbN1T.

Members of the genus Aromatoleum thrive in diverse habitats and use a broad range of recalcitrant organic molecules coupled to denitrification or O2 respiration. To gain a holistic understanding of the model organism A. aromaticum EbN1T, we studied its catabolic network dynamics in response to 3-(4-hydroxyphenyl)propanoate, phenylalanine, 3-hydroxybenzoate, benzoate, and acetate utilized under nitrate-reducing versus oxic conditions. Integrated multi-omics (transcriptome, proteome, and metabolome) covered most of the catabolic network (199 genes) and allowed for the refining of knowledge of the degradation modules studied. Their substrate-dependent regulation showed differing degrees of specificity, ranging from high with 3-(4-hydroxyphenyl)propanoate to mostly relaxed with benzoate. For benzoate, the transcript and protein formation were essentially constitutive, contrasted by that of anoxia-specific versus oxia-specific metabolite profiles. The matrix factorization of transcriptomic data revealed that the anaerobic modules accounted for most of the variance across the degradation network. The respiration network appeared to be constitutive, both on the transcript and protein levels, except for nitrate reductase (with narGHI expression occurring only under nitrate-reducing conditions). The anoxia/nitrate-dependent transcription of denitrification genes is apparently controlled by three FNR-type regulators as well as by NarXL (all constitutively formed). The resequencing and functional reannotation of the genome fostered a genome-scale metabolic model, which is comprised of 655 enzyme-catalyzed reactions and 731 distinct metabolites. The model predictions for growth rates and biomass yields agreed well with experimental stoichiometric data, except for 3-(4-hydroxyphenyl)propanoate, with which 4-hydroxybenzoate was exported. Taken together, the combination of multi-omics, growth physiology, and a metabolic model advanced our knowledge of an environmentally relevant microorganism that differs significantly from other bacterial model strains. IMPORTANCE Aromatic compounds are abundant constituents not only of natural organic matter but also of bulk industrial chemicals and fuel components of environmental concern. Considering the widespread occurrence of redox gradients in the biosphere, facultative anaerobic degradation specialists can be assumed to play a prominent role in the natural mineralization of organic matter and in bioremediation at contaminated sites. Surprisingly, differential multi-omics profiling of the A. aromaticum EbN1T studied here revealed relaxed regulatory stringency across its four main physiological modi operandi (i.e., O2-independent and O2-dependent degradation reactions versus denitrification and O2 respiration). Combining multi-omics analyses with a genome-scale metabolic model aligned with measured growth performances establishes A. aromaticum EbN1T as a systems-biology model organism and provides unprecedented insights into how this bacterium functions on a holistic level. Moreover, this experimental platform invites future studies on eco-systems and synthetic biology of the environmentally relevant betaproteobacterial Aromatoleum/Azoarcus/Thauera cluster.