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.

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 P N promoter running the expression of the bzd catabolic genes was identified. Gel retardation assays and P N ::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 P N 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 threedimensional 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.

Rapid emergence of antibiotic resistance is one of the most challenging global public health concerns. In particular, vancomycin-resistant Enterococcus faecium infections have been increasing in frequency, representing 25% of enterococci infections in intensive care units. A novel class of 1,2,4-triazolo[1,5-a]pyrimidines active against E. faecium is reported herein. We used a three-component Biginelli-like heterocyclization reaction for the synthesis of a series of these derivatives based on reactions of aldehydes, β-dicarbonyl compounds, and 3-alkylthio-5-amino-1,2,4-tria-zoles. The resulting compounds were assayed for antimicrobial activity against the ESKAPE panel of bacteria, followed by investigation of their in vitro activities. These analyses identified a subset of 1,2,4-triazolo[1,5-a]pyrimidines that had good narrow-spectrum antibacterial activity against E. faecium and exhibited metabolic stability with low intrinsic clearance. Macromolecular synthesis assays revealed cell-wall biosynthesis as the target of these antibiotics.

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