María Teresa Zamarro

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

SUMMARY Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.

The bzd Gene Cluster, Coding for Anaerobic Benzoate Catabolism, in Azoarcus sp. Strain CIB

We report here that the bzd genes for anaerobic benzoate degradation in Azoarcus sp. strain CIB are organized as two transcriptional units, i.e., a benzoate-inducible catabolic operon, bzdNOPQMSTUVWXYZA, and a gene, bzdR, encoding a putative transcriptional regulator. The last gene of the catabolic operon, bzdA, has been expressed in Escherichia coli and encodes the benzoate-coenzyme A (CoA) ligase that catalyzes the first step in the benzoate degradation pathway. The BzdA enzyme is able to activate a wider range of aromatic compounds than that reported for other previously characterized benzoate-CoA ligases. The reduction of benzoyl-CoA to a nonaromatic cyclic intermediate is carried out by a benzoyl-CoA reductase (bzdNOPQ gene products) detected in Azoarcus sp. strain CIB extracts. The bzdW, bzdX, and bzdY gene products show significant similarity to the hydratase, dehydrogenase, and ring-cleavage hydrolase that act sequentially on the product of the benzoyl-CoA reductase in the benzoate catabolic pathway of Thauera aromatica. Benzoate-CoA ligase assays and transcriptional analyses based on lacZ-reporter fusions revealed that benzoate degradation in Azoarcus sp. strain CIB is subject to carbon catabolite repression by some organic acids, indicating the existence of a physiological control that connects the expression of the bzd genes to the metabolic status of the cell.

BzdR, a Repressor That Controls the Anaerobic Catabolism of Benzoate in Azoarcus sp. CIB, Is the First Member of a New Subfamily of Transcriptional Regulators

In this work, we have studied the transcriptional regulation of the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB. The transcription start site of the PN promoter running the expression of the bzd catabolic genes was identified. Gel retardation assays and PN::lacZ translational fusion experiments performed both in Azoarcus sp. CIB and Escherichia coli cells have shown that bzdR encodes a specific repressor that controls the inducible expression of the adjacent bzd catabolic operon, being the first intermediate of the catabolic pathway (i.e. benzoyl-CoA, the actual inducer molecule). This is the first report of a transcriptional repressor and a CoA-derived aromatic inducer controlling gene expression in the anaerobic catabolism of aromatic compounds. DNase I footprinting experiments revealed that BzdR protected three regions (operators) at the PN promoter. The three operators contain direct repetitions of a TGCA sequence that forms part of longer palindromic structures. In agreement with the repressor role of BzdR, operator region I spans the transcription initiation site as well as the -10 sequence for recognition of the RNA polymerase. Primary sequence analyses of BzdR showed an unusual modular organization with an N-terminal region homologous to members of the HTH-XRE family of transcriptional regulators and a C-terminal region similar to shikimate kinases. A three-dimensional model of the N-terminal and C-terminal regions of BzdR, generated by comparison with the crystal structures of the SinR regulator from Bacillus subtilis and the shikimate kinase I protein from E. coli, strongly suggests that they contain the helix-turn-helix DNA-binding motif and the benzoyl-CoA binding groove, respectively. The BzdR protein constitutes, therefore, the prototype of a new subfamily of transcriptional regulators.

Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway

The mbd cluster encoding genes of the 3-methylbenzoyl-CoA pathway involved in the anaerobic catabolism of 3-methylbenzoate and m-xylene was characterized for the first time in the denitrifying β-Proteobacterium Azoarcus sp. CIB. The mbdA gene product was identified as a 3-methylbenzoate-CoA ligase required for 3-methylbenzoate activation; its substrate spectrum was unique in activating all three methylbenzoate isomers. An inducible 3-methylbenzoyl-CoA reductase (mbdONQP gene products), displaying significant amino acid sequence similarities to known class I benzoyl-CoA reductases catalysed the ATP-dependent reduction of 3-methylbenzoyl-CoA to a methyldienoyl-CoA. The mbdW gene encodes a methyldienoyl-CoA hydratase that hydrated the methyldienoyl-CoA to a methyl-6-hydroxymonoenoyl-CoA compound. The mbd cluster also contains the genes predicted to be involved in the subsequent steps of the 3-methylbenzoyl-CoA pathway as well as the electron donor system for the reductase activity. Whereas the catabolic mbd genes are organized in two divergent inducible operons, the putative mbdR regulatory gene was transcribed separately and showed constitutive expression. The efficient expression of the mbd genes required the oxygen-dependent AcpR activator, and it was subject of carbon catabolite repression by some organic acids and amino acids. Sequence analyses suggest that the mbd gene cluster was recruited by Azoarcus sp. CIB through horizontal gene transfer.

Analysis of Dibenzothiophene Desulfurization in a Recombinant Pseudomonas putida Strain

ABSTRACT Biodesulfurization was monitored in a recombinant Pseudomonas putida CECT5279 strain. DszB desulfinase activity reached a sharp maximum at the early exponential phase, but it rapidly decreased at later growth phases. A model two-step resting-cell process combining sequentially P. putida cells from the late and early exponential growth phases was designed to significantly increase biodesulfurization.

Whole-genome analysis of Azoarcus sp. strain CIB provides genetic insights to its different lifestyles and predicts novel metabolic features

The genomic features of Azoarcus sp. CIB reflect its most distinguishing phenotypes as a diazotroph, facultative anaerobe, capable of degrading either aerobically and/or anaerobically a wide range of aromatic compounds, including some toxic hydrocarbons such as toluene and m-xylene, as well as its endophytic lifestyle. The analyses of its genome have expanded the catabolic potential of strain CIB toward common natural compounds, such as certain diterpenes, that were not anticipated as carbon sources. The high number of predicted solvent efflux pumps and heavy metal resistance gene clusters has provided the first evidence for two environmentally relevant features of this bacterium that remained unknown. Genome mining has revealed several gene clusters likely involved in the endophytic lifestyle of strain CIB, opening the door to the molecular characterization of some plant growth promoting traits. Horizontal gene transfer and mobile genetic elements appear to have played a major role as a mechanism of adaptation of this bacterium to different lifestyles. This work paves the way for a systems biology-based understanding of the abilities of Azoarcus sp. CIB to integrate aerobic and anaerobic metabolism of aromatic compounds, tolerate stress conditions, and interact with plants as an endophyte of great potential for phytostimulation and phytoremediation strategies. Comparative genomics provides an Azoarcus pan genome that confirms the global metabolic flexibility of this genus, and suggests that its phylogeny should be revisited.

Oxygen-dependent regulation of the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus sp. strain CIB.

The role of oxygen in the transcriptional regulation of the PN promoter that controls the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB has been investigated. In vivo experiments using PN::lacZ translational fusions, in both Azoarcus sp. strain CIB and Escherichia coli cells, have shown an oxygen-dependent repression effect on the transcription of the bzd catabolic genes. E. coli Fnr was required for the anaerobic induction of the PN promoter, and the oxygen-dependent repression of the bzd genes could be bypassed by the expression of a constitutively active Fnr* protein. In vitro experiments revealed that Fnr binds to the PN promoter at a consensus sequence centered at position -41.5 from the transcription start site overlapping the -35 box, suggesting that PN belongs to the class II Fnr-dependent promoters. Fnr interacts with RNA polymerase (RNAP) and is strictly required for transcription initiation after formation of the RNAP-PN complex. An fnr ortholog, the acpR gene, was identified in the genome of Azoarcus sp. strain CIB. The Azoarcus sp. strain CIB acpR mutant was unable to grow anaerobically on aromatic compounds and it did not drive the expression of the PN::lacZ fusion, suggesting that AcpR is the cognate transcriptional activator of the PN promoter. Since the lack of AcpR in Azoarcus sp. strain CIB did not affect growth on nonaromatic carbon sources, AcpR can be considered a transcriptional regulator of the Fnr/Crp superfamily that has evolved to specifically control the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus.

Identification of the Geobacter metallireducens bamVW two-component system, involved in transcriptional regulation of aromatic degradation.

ABSTRACT Regulation of aromatic degradation in obligate anaerobes was studied in the Fe(III)-respiring model organism Geobacter metallireducens GS-15. A two-component system and a σ54-dependent promoter were identified that are both involved in the regulation of the gene coding for benzoate-coenzyme A ligase, catalyzing the initial step of benzoate degradation.

New insights into the BzdR-mediated transcriptional regulation of the anaerobic catabolism of benzoate in Azoarcus sp. CIB.

The expression of the bzd genes involved in the anaerobic degradation of benzoate in Azoarcus sp. CIB is controlled by the specific BzdR transcriptional repressor at the P(N) promoter. This catabolic promoter is also subject to catabolite repression by some organic acids. In vivo and in vitro experiments have shown that BzdR behaves as a repressor of the P(R) promoter by overlapping the transcription initiation site as well as the -35 and -10 boxes, benzoyl-CoA being the inducer molecule. In addition, by using a P(N) : : lacZ fusion both in Azoarcus sp. CIB and in an isogenic strain lacking the bzdR gene, we have shown that the succinate-dependent catabolite repression requires participation of the BzdR repressor.

Unraveling the Specific Regulation of the Central Pathway for Anaerobic Degradation of 3-Methylbenzoate

Background: The specific transcriptional regulation of the mbd pathway for anaerobic 3-methylbenzoate degradation is unknown. Results: The MbdR/3-methylbenzoyl-CoA couple controls the induction of the mbd genes. Conclusion: MbdR is the regulator of the mbd pathway in Azoarcus sp. CIB. Significance: This work highlights the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds. The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the β-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB1, and P3R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATACN10GTAT) have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e. 3-methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdR-dependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as the central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.