Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and M-Xylene in Azoarcus Sp
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fmicb-09-00506 March 20, 2018 Time: 16:29 # 1 ORIGINAL RESEARCH published: 22 March 2018 doi: 10.3389/fmicb.2018.00506 Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and m-Xylene in Azoarcus sp. CIB Blas Blázquez†, Manuel Carmona and Eduardo Díaz* Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas–Consejo Superior de Investigaciones Científicas, Madrid, Spain Alkylbenzenes, such as toluene and m-xylene, are an important class of contaminant hydrocarbons that are widespread and tend to accumulate in subsurface anoxic environments. The peripheral pathway for the anaerobic oxidation of toluene in bacteria Edited by: consists of an initial activation catalyzed by a benzylsuccinate synthase (encoded by Kian Mau Goh, bss genes), and a subsequent modified b-oxidation of benzylsuccinate to benzoyl- Universiti Teknologi Malaysia, CoA and succinyl-CoA (encoded by bbs genes). We have shown here that the bss Malaysia and bbs genes, which are located within an integrative and conjugative element, are Reviewed by: Maximino Manzanera, essential for anaerobic degradation of toluene but also for m-xylene oxidation in the University of Granada, Spain denitrifying beta-proteobacterium Azoarcus sp. CIB. New insights into the transcriptional Victor Ladero, Instituto de Productos Lácteos organization and regulation of a complete gene cluster for anaerobic catabolism of de Asturias (CSIC), Spain toluene/m-xylene in a single bacterial strain are presented. The bss and bbs genes *Correspondence: are transcriptionally coupled into two large convergent catabolic operons driven by Eduardo Díaz the PbssD and PbbsA promoters, respectively, whose expression is inducible when [email protected] cells grow anaerobically in toluene or m-xylene. An adjacent tdiSR operon driven †Present address: Blas Blázquez, by the PtdiS promoter encodes a putative two-component regulatory system. TdiR Systems Biology Programme, Centro behaves as a transcriptional activator of the PbssD, PbbsA, and PtdiS promoters, being Nacional de Biotecnología–Consejo Superior de Investigaciones benzylsuccinate/(3-methyl)benzylsuccinate, rather than toluene/m-xylene, the inducers Científicas, Madrid, Spain that may trigger the TdiS-mediated activation of TdiR. In addition to the TdiSR-based specific control, the expression of the bss and bbs genes in Azoarcus sp. CIB is under Specialty section: This article was submitted to an overimposed regulation that depends on certain environmental factors, such as the Microbiotechnology, Ecotoxicology presence/absence of oxygen or the availability of preferred carbon sources (catabolite and Bioremediation, repression). This work paves the way for future strategies toward the reliable assessment a section of the journal Frontiers in Microbiology of microbial activity in toluene/m-xylene contaminated environments. Received: 08 January 2018 Keywords: toluene, m-xylene, Azoarcus, anaerobic degradation, tdiSR, catabolite repression, biomarker Accepted: 06 March 2018 Published: 22 March 2018 Citation: INTRODUCTION Blázquez B, Carmona M and Díaz E (2018) Transcriptional Regulation Aromatic compounds such as alkylbenzenes, e.g., toluene, xylenes, ethylbenzene and benzene, of the Peripheral Pathway for the Anaerobic Catabolism are an important class of contaminants that are prominently placed amongst the US Agency for of Toluene and m-Xylene in Azoarcus Toxic Substances and Disease Registry’s list of priority pollutants because of their carcinogenic sp. CIB. Front. Microbiol. 9:506. and/or neurotoxic effects to humans. Since alkylbenzenes are relatively soluble and mobile in doi: 10.3389/fmicb.2018.00506 water, they are widespread and tend to accumulate in hostile environments, e.g., subsurface Frontiers in Microbiology| www.frontiersin.org 1 March 2018| Volume 9| Article 506 fmicb-09-00506 March 20, 2018 Time: 16:29 # 2 Blázquez et al. Toluene/m-Xylene Anaerobic Degradation anoxic environments, where they are of major concern the transcriptional organization of the bss clusters differs between to groundwater quality and ecosystem health. Therefore, species and even between strains of the same species (Coschigano, the anaerobic degradation of alkylbenzenes is an important 2000; Achong et al., 2001; Kube et al., 2004). practical aspect of bioremediation and becomes crucial for the Further anaerobic degradation of toluene consists of a biogeochemical cycles (Kummel et al., 2013; Rabus et al., 2016; modified beta-oxidation of (R)-benzylsuccinate to benzoyl-CoA Lueders, 2017). Despite the aerobic degradation of alkylbenzenes and succinyl-CoA (Figure 1A). All enzymes of this pathway has been extensively studied, the microbial catabolism of these are encoded by the bbs gene cluster (Figure 2)(Leuthner and aromatic hydrocarbons under anaerobic conditions is much less Heider, 2000; Leutwein and Heider, 2001, 2002; Kube et al., known, and novel pathways and enzymes have been recently 2004). Like in the bss gene cluster, the bbs cluster contains genes, described, some of which are of potential biotechnological e.g., bbsI and bbsJ, of unknown function (Carmona et al., 2009). interest (Rabus et al., 2016; Lueders, 2017). Toluene has been Although the gene organization of the bbsABCDEFJGH cluster is used widely as a model compound for studying anaerobic usually conserved except in some delta-proteobacteria (Figure 2) alkylbenzene degradation. Toluene can be degraded coupled to (Kube et al., 2004; Butler et al., 2007; Chaurasia et al., 2015), the anaerobic respiration with nitrate, sulfate, iron (III), manganese transcriptional organization of a bbs cluster has not been studied (IV) or carbonate serving as terminal electron acceptors (Foght, so far in any bacteria. 2008; Carmona et al., 2009; Rabus et al., 2016). Geobacter Upstream of the bss cluster in most denitrifying bacteria metallireducens GS-15 was the first pure bacterial culture there are two genes, tdiS and tdiR (toluene-degradationi nducer) described to be able to degrade toluene in anaerobic conditions (Figure 2) that were proposed to encode the sensor histidine (Lovley et al., 1989). Several isolates capable of anaerobic toluene kinase and the cognate response regulator, respectively, of degradation have been described since then, including both a two-component regulatory system likely involved in the facultative anaerobes, e.g., beta-proteobacteria of the Azoarcus, transcriptional control of the catabolic bss and bbs genes “Aromatoleum,” Thauera, Georgfuchsia, Herminiimonas genera, (Coschigano, 2000; Achong et al., 2001; Hermuth et al., 2002; and alpha-proteobacteria of the Magnetospirillum genus, and Kube et al., 2004; Kühner et al., 2005). Extracts of Escherichia coli obligate anaerobes, e.g., delta-proteobacteria of the Geobacter, cells that expressed the tdiR gene from T. aromatica K172 were Desulfobacula and Desulfobacterium genera, and some clostridial able to retard the migration of a DNA probe that contained the strains (Wöhlbrand et al., 2013; Bozinovski et al., 2014; Kim et al., bssD promoter, supporting the notion that TdiR likely behaves 2014; Strijkstra et al., 2014; Rabus et al., 2016; Lueders, 2017; as a transcriptional regulator of the bss genes (Leuthner and Meyer-Cifuentes et al., 2017). Most of the studies on anaerobic Heider, 1998). Nevertheless, a clear experimental demonstration toluene degradation have been made on the denitrifying bacteria that TdiS and TdiR control the expression of both bss and bbs Thauera aromatica K172 and T1 strains, Azoarcus sp. strain T genes in beta-proteobacteria is still missing. and “Aromatoleum aromaticum” EbN1 strain (Coschigano and Unlike toluene degradation, anaerobic degradation of xylenes Young, 1997; Leuthner et al., 1998; Krieger et al., 1999; Achong (meta-, ortho-, and para-xylene) has been much less studied. et al., 2001; Hermuth et al., 2002; Leutwein and Heider, 2002; The first reaction of the m-xylene degradation pathway is Chakraborty and Coates, 2004; Coschigano and Bishop, 2004; performed by the BSS enzyme that catalyzes the addition of Kube et al., 2004; Kühner et al., 2005). fumarate to the methyl group to form (3-methyl)benzylsuccinate The first step in the anaerobic catabolism of toluene is the (Figure 1A)(Krieger et al., 1999; Verfürth et al., 2004). The (3- addition of the methyl group of toluene to fumarate to form (R)- methyl)benzylsuccinate was also detected as a key intermediate benzylsuccinate by the strictly anoxicb enzylsuccinates ynthase of the fumarate addition pathway in xylene-degrading sulfate- (BSS), a glycyl radical enzyme (Figure 1A). The genes bssA, bssB reducing cultures (Morasch et al., 2004; Herrmann et al., and bssC code for the a (BssA), b (BssB) and g (BssC) subunits of 2009). On the other hand, it has been proposed that the bbs the heterohexameric (abg)2 BSS (Leuthner et al., 1998; Krieger gene products might be responsible of the b-oxidation of (3- et al., 2001; Bhandare et al., 2006; Funk et al., 2014; Heider methyl)benzylsuccinate to 3-methylbenzoyl-CoA and succinyl- et al., 2016). Conversion to the active, radical-containing form CoA in a similar way than in the catabolism of toluene of BSS depends on an activating enzyme (BssD) that belongs to (Figure 1A)(Krieger et al., 1999; Bozinovski et al., 2014), the family of S-adenosyl-methionine radical enzymes and that although there is not yet a genetic demonstration that bbs genes is encoded by a gene (bssD) which is closely associated with are responsible for anaerobic m-xylene degradation.