Blas Blázquez

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.

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.

Identification and analysis of a glutaryl‐CoA dehydrogenase‐encoding gene and its cognate transcriptional regulator from Azoarcus sp. CIB

In this work, the gcdH gene from the denitrifying beta-proteobacterium Azoarcus sp. CIB was shown to encode a glutaryl-CoA dehydrogenase, which is essential for the anaerobic catabolism of many aromatic compounds and some alicyclic and dicarboxylic acids. The primary structure of the GcdH protein is highly conserved in many organisms. The divergently transcribed gcdR gene, encoding a LysR-type transcriptional regulator, accounts for the glutaconate/glutarate-specific activation of the Pg promoter driving expression of gcdH. The Azoarcus sp. CIBdgcdH mutant strain harbouring a disrupted gcdH gene was used as host to identify heterologous gcdH genes, such as that from Pseudomonas putida KT2440. Moreover, the expression of gcdH from P. putida can be efficiently controlled by the GcdR activator in Azoarcus sp. CIB, demonstrating the existence of cross-talk between GcdR regulators and gcdH promoters from members of different phylogenetic subgroups of proteobacteria.

Bacterial Degradation of Benzoate CROSS-REGULATION BETWEEN AEROBIC AND ANAEROBIC PATHWAYS

Background: The specific transcriptional regulation of the box pathway for aerobic benzoate degradation is unknown. Results: The BoxR/benzoyl-CoA couple controls the induction of the box genes. Conclusion: BoxR is the regulator of the box pathway in bacteria. Significance: There is cross-regulation between anaerobic and aerobic benzoate degradation pathways. We have studied for the first time the transcriptional regulatory circuit that controls the expression of the box genes encoding the aerobic hybrid pathway used to assimilate benzoate via coenzyme A (CoA) derivatives in bacteria. The promoters responsible for the expression of the box cluster in the β-proteobacterium Azoarcus sp., their cognate transcriptional repressor, the BoxR protein, and the inducer molecule (benzoyl-CoA) have been characterized. The BoxR protein shows a significant sequence identity to the BzdR transcriptional repressor that controls the bzd genes involved in the anaerobic degradation of benzoate. Because the boxR gene is present in all box clusters so far identified in bacteria, the BoxR/benzoyl-CoA regulatory system appears to be a widespread strategy to control this aerobic hybrid pathway. Interestingly, the paralogous BoxR and BzdR regulators act synergistically to control the expression of the box and bzd genes. This cross-regulation between anaerobic and aerobic pathways for the catabolism of aromatic compounds has never been shown before, and it may reflect a biological strategy to increase the cell fitness in organisms that survive in environments subject to changing oxygen concentrations.

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.

PL3 Amidase, a Tailor-made Lysin Constructed by Domain Shuffling with Potent Killing Activity against Pneumococci and Related Species

The emergence and spread of antibiotic-resistant bacteria is pushing the need of alternative treatments. In this context, phage therapy is already a reality to successfully fight certain multiresistant bacteria. Among different phage gene products, murein hydrolases responsible of phage progeny liberation (also called lysins or endolysins) are weapons that target specific peptidoglycan bonds, leading to lysis and death of susceptible bacteria when added from the outside. In the pneumococcal system, all but one phage murein hydrolases reported to date share a choline-binding domain that recognizes cell walls containing choline residues in the (lipo)teichoic acids. Some purified pneumococcal or phage murein hydrolases, as well as several chimeric proteins combining natural catalytic and cell wall-binding domains (CBDs) have been used as effective antimicrobials. In this work we have constructed a novel chimeric N-acetylmuramoyl-L-alanine amidase (PL3) by fusing the catalytic domain of the Pal amidase (a phage-coded endolysin) to the CBD of the LytA amidase, the major pneumococcal autolysin. The physicochemical properties of PL3 and the bacteriolytic effect against several pneumococci (including 48 multiresistant representative strain) and related species, like Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis, have been studied. Results have shown that low doses of PL3, in the range of 0.5–5 μg/ml, are enough to practically sterilize all choline-containing strains tested. Moreover, a single 20-μg dose of PL3 fully protected zebrafish embryos from infection by S. pneumoniae D39 strain. Importantly, PL3 keeps 95% enzymatic activity after 4 weeks at 37°C and can be lyophilized without losing activity, demonstrating a remarkable robustness. Such stability, together with a prominent efficacy against a narrow spectrum of human pathogens, confers to PL3 the characteristic to be an effective therapeutic. In addition, our results demonstrate that the structure/function-based domain shuffling approach is a successful method to construct tailor-made endolysins with higher bactericidal activities than their parental enzymes.

Degradation of cyclic diguanosine monophosphate by a hybrid two-component protein protects Azoarcus sp. strain CIB from toluene toxicity

Significance Some bacteria have a remarkable ability to grow on toxic aromatic hydrocarbons under anaerobic conditions. Although bacteria have evolved cellular mechanisms to cope with hydrocarbon toxicity, it was unclear if signal transduction systems might exist to coordinate a protective response. Here we describe a hybrid two-component system, the TolR protein, from the bacterium Azoarcus sp. strain CIB that on binding toluene and other aromatic hydrocarbons degrades intracellular cyclic diguanosine monophosphate (c-di-GMP). A drop in c-di-GMP protects cells from toluene toxicity. C-di-GMP, found universally in bacteria, is involved in diverse functions including biofilm formation and virulence. Our finding of hydrocarbon sensing and c-di-GMP degradation by TolR for purposes of modulating hydrocarbon toxicity describes an additional role for c-di-GMP in bacterial physiology. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the β-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per–Arnt–Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolRSK) and an independent response regulator (TolRRR). This classic two-component system (CTCS) operated less efficiently than TolR, suggesting that TolR was evolved as a HTCS to optimize signal transduction. Our results suggest that TolR enables Azoarcus sp. CIB to adapt to toxic aromatic hydrocarbons under anaerobic conditions by modulating cellular levels of c-di-GMP. This is an additional role for c-di-GMP in bacterial physiology.

Anaerobic pathways for the catabolism of aromatic compounds

Removal of aromatic compounds, which mostly derive from the polymer lignin, fossil fuel reservoirs, and industrial activities, is very important both for a balanced global carbon budget and to protect natural ecosystems and human health from the toxic effect of some of these environmental pollutants. Whereas the aerobic catabolism of aromatic compounds has been extensively studied for many decades, the anaerobic catabolism is a more recently discovered and so far poorly characterized microbial capacity despite the fact that anoxic conditions dominate in many natural habitats and contaminated sites. The anaerobic catabolism of aromatic compounds by some specialized bacteria becomes, thus, crucial for the biogeochemical cycles and for the sustainable development of the biosphere. Moreover, anaerobic degradation of aromatic compounds involves various intriguing biochemically unprecedented reactions that are also of great biotechnological potential as alternatives to the current synthesis processes and for the anaerobic valorization of aromatic compounds to produce biofuels, biopolymers, and commodity chemicals. In this chapter we summarize the major degradation pathways and the associated cellular responses when bacteria grow anaerobically in the presence of aromatic compounds. The unexplored potential and some biotechnological applications of the anaerobic catabolism of aromatic compounds are also discussed.

Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and m-Xylene in Azoarcus sp. CIB

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 consists of an initial activation catalyzed by a benzylsuccinate synthase (encoded by bss genes), and a subsequent modified β-oxidation of benzylsuccinate to benzoyl-CoA and succinyl-CoA (encoded by bbs genes). We have shown here that the bss and bbs genes, which are located within an integrative and conjugative element, are essential for anaerobic degradation of toluene but also for m-xylene oxidation in the denitrifying beta-proteobacterium Azoarcus sp. CIB. New insights into the transcriptional organization and regulation of a complete gene cluster for anaerobic catabolism of toluene/m-xylene in a single bacterial strain are presented. The bss and bbs genes are transcriptionally coupled into two large convergent catabolic operons driven by the PbssD and PbbsA promoters, respectively, whose expression is inducible when cells grow anaerobically in toluene or m-xylene. An adjacent tdiSR operon driven by the PtdiS promoter encodes a putative two-component regulatory system. TdiR behaves as a transcriptional activator of the PbssD, PbbsA, and PtdiS promoters, being benzylsuccinate/(3-methyl)benzylsuccinate, rather than toluene/m-xylene, the inducers 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 an overimposed regulation that depends on certain environmental factors, such as the presence/absence of oxygen or the availability of preferred carbon sources (catabolite repression). This work paves the way for future strategies toward the reliable assessment of microbial activity in toluene/m-xylene contaminated environments.