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In order to better understand bacterial depolymerization of lignin, a new analytical approach was proposed using Pseudomonas putida KT2440 as delignifying bacterium and Escherichia coli as non-delignifying control. Two different types of lignins, technical Kraft lignin and synthetic dehydrogenopolymer (DHP), were submitted to a bioconversion kinetic study over 7 days. The concomitant analysis of the supernatant acid-precipitable lignin fraction and water-soluble extractives by high-performance size-exclusion chromatography (HPSEC) and gas chromatography – mass spectrometry (GC–MS) highlighted the specific action of P. putida towards these substrates, with the transitory formation of phenolic metabolites (dihydroferulic acid for Kraft lignins and dimers for DHP) and the prevention of Kraft lignin self-assemblying. In both cases lignin apparent depolymerization followed by repolymerization was observed. The analysis of the bacterial pellets indicated the time-increasing content of lignins associated to bacterial cells, which could account for the apparent structural changes observed with E. coli in the supernatant.

This combined computational–experimental work describes with atomistic resolution the interfacial interaction between the silk and hydroxyapatite and the potential implications for the development of osteoinductive silk biomaterials.

We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS′) accounts for the succinate-dependent CCR control. In vitro assays with purified AccS′ revealed its autophosphorylation, phosphotransfer from AccS′∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS′ may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS′ autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS′ dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability.

The properties of native spider silk vary within and across species due to the presence of different genes containing conserved repetitive core domains encoding a variety of silk proteins. Previous studies seeking to understand the function and material properties of these domains focused primarily on the analysis of dragline silk proteins, MaSp1 and MaSp2. Our work seeks to broaden the mechanical properties of silk-based biomaterials by establishing two libraries containing genes from the repetitive core region of the native Latrodectus hesperus silk genome (Library A: genes masp1, masp2, tusp1, acsp1; Library B: genes acsp1, pysp1, misp1, flag). The expressed and purified proteins were analyzed through Fourier Transform Infrared Spectrometry (FTIR). Some of these new proteins revealed a higher portion of β-sheet content in recombinant proteins produced from gene constructs containing a combination of masp1/masp2 and acsp1/tusp1 genes than recombinant proteins which consisted solely of dragline silk genes (Library A). A higher portion of β-turn and random coil content was identified in recombinant proteins from pysp1 and flag genes (Library B). Mechanical characterization of selected proteins purified from Library A and Library B formed into films was assessed by Atomic Force Microscopy (AFM) and suggested Library A recombinant proteins had higher elastic moduli when compared to Library B recombinant proteins. Both libraries had higher elastic moduli when compared to native spider silk proteins. The preliminary approach demonstrated here suggests that repetitive core regions of the aforementioned genes can be used as building blocks for new silk-based biomaterials with varying mechanical properties., National Institute of Health (NIH U01 EB014976), Air Force Office of Scientific Research (AFOSR FA9550-14-1-0015)

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.

Silk is a promising material for biomedical applications, and much research is focused on how application-specific, mechanical properties of silk can be designed synthetically through proper amino acid sequences and processing parameters. This protocol describes an iterative process between research disciplines that combines simulation, genetic synthesis, and fiber analysis to better design silk fibers with specific mechanical properties. Computational methods are used to assess the protein polymer structure as it forms an interconnected fiber network through shearing and how this process affects fiber mechanical properties. Model outcomes are validated experimentally with the genetic design of protein polymers that match the simulation structures, fiber fabrication from these polymers, and mechanical testing of these fibers. Through iterative feedback between computation, genetic synthesis, and fiber mechanical testing, this protocol will enable a priori prediction capability of recombinant material mechanical properties via insights from the resulting molecular architecture of the fiber network based entirely on the initial protein monomer composition. This style of protocol may be applied to other fields where a research team seeks to design a biomaterial with biomedical application-specific properties. This protocol highlights when and how the three research groups (simulation, synthesis, and engineering) should be interacting to arrive at the most effective method for predictive design of their material. Keywords: computational modeling; genetic synthesis; recombinant silk; spinning, National Institutes of Health (U.S.) (Grant U01 EB014976-05)

Integrative and conjugative elements (ICE) play a major role in aerobic degradation of aromatic compounds, but they have not yet been shown to be involved in anaerobic degradation. We have characterized here the ICEXTD element which endows to the beta-proteobacterium Azoarcus sp. CIB with the ability to utilize aromatic hydrocarbons. The core region of ICEXTD , which shows a remarkable synteny with that of ICEclc-like elements, allows its own intracellular and intercellular mobility. ICEXTD integrates at the tRNAGly of the host chromosome, but it can also excise to produce a ready to transfer circular form. The adaptation modules of ICEXTD represent a unique combination of gene clusters for aerobic (tod genes) and anaerobic (bss-bbs and mbd genes) degradation of certain aromatic hydrocarbons, e.g., toluene, m-xylene and cumene. Transfer of ICEXTD to other Azoarcus strains, e.g., A. evansii, confers them the ability to degrade aromatic hydrocarbons both aerobically and anaerobically. Interestingly, ICEXTD allows Cupriavidus pinatubonensis, a bacterium unable to degrade anaerobically aromatic compounds, to grow with m-xylene under anoxic conditions. Thus, ICEXTD constitutes the first mobile genetic element able to expand the catabolic abilities of certain bacteria for the removal of aromatic hydrocarbons either in the presence or absence of oxygen.

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 towards 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.

[EN]Biodegradation of aromatic compounds requires that bacteria adjust their cellular programs to cope with the dual character of such compounds, i.e., on the one side they are an important carbon and energy sources but, on the other side, they behave as stressors due to their toxic nature. In this thesis, we have used Azoarcus sp. CIB, a b- proteobacterium that is able to mineralize both aerobically and anaerobically a wide range of aromatic compounds, as model system. A detailed analysis of the bss-bbs cluster, encoding the peripheral route for the anaerobic degradation of toluene, has revealed the presence of a unique gene, tolR, that has no homologs in other organisms. The TolR protein shows an atypical modular architecture which corresponds to a new hybrid two-component system (HTCS), where the sensor histidine-kinase and the response regulator components are fused together in a single polypeptide. This work has demonstrated that TolR is the first HTCS that responds to aromatic hydrocarbons and whose response regulator has an enzymatic activity. The molecular mechanism of action of TolR involves the detection of aromatic hydrocarbons (inducers) through the PAS sensor domain. Then, an autophosphorylation and intramolecular phosphotransfer from the autoquinase domain to the receiver domain triggers the c-di-GMP phosphodiesterase activity of the efector domain. TolR activation in anaerobic conditions involves decreased c-di-GMP levels, that lead to an effective anaerobic chemotaxis towards aromatic hydrocarbons, and contribute to the cell resistance response after exposure to toxic concentrations of aromatic hydrocarbons. This is the first time that c-di GMP was shown to be involved in the bacterial adaptation to aromatic hydrocarbons. Transcriptomic studies that compare the wild-type Azoarcus sp. CIB strain and the tolR mutant Azoarcus sp. CIBdtolR strain, confirm that TolR is involved in a signaling network that controls the morphological, metabolic and stress r, [ES]La biodegradación bacteriana de compuestos aromáticos requiere que las células ajusten sus programas celulares para hacer frente al carácter dual de estos compuestos, ya que, si bien son una fuente de carbono y energía para los microorganismos, muchos de ellos son también un importante factor de estrés debido a su carácter tóxico. En esta tesis se ha utilizado como sistema modelo de estudio la b- proteobacteria Azoarcus sp. CIB, capaz de mineralizar tanto aeróbica como anaeróbicamente numerosos compuestos aromáticos. El análisis detallado de su ruta periférica de degradación anaeróbica de tolueno y m-xileno (genes bbs-bss), ha revelado la presencia de un gen, denominado tolR, que no tiene homólogos en otros organismos. Dicho gen codifica la proteína TolR que presenta una arquitectura modular atípica, y que se corresponde con la de un nuevo miembro de los sistemas reguladores de dos componentes híbridos (HTCSs), donde el componente histidín quinasa sensora y el componente regulador de respuesta aparecen fusionados en un único polipéptido. Este trabajo ha demostrado que la proteína TolR es el primer HTCS que responde a hidrocarburos aromáticos y cuyo dominio efector es una actividad enzimática. Su mecanismo molecular de actuación implica la detección, a través de su dominio sensor PAS, de hidrocarburos aromáticos que actúan como inductores, lo que activa una cascada de autofosforilación y transferencia intramolecular del grupo fosfato desde el dominio autoquinasa al dominio receiver, logrando así la activación de la región efectora con actividad fosfodiesterasa de di-GMPc. La presumible disminución de los niveles de di-GMPc en Azoarcus sp. CIB favorece la quimiotaxis anaeróbica hacia los hidrocarburos aromáticos, y participa en la respuesta de resistencia tras la exposición a concentraciones tóxicas de dichos hidrocarburos, siendo la primera vez que se demuestra la participación del di-GMPc en la adaptación bacteriana a los hidrocarburos aromáticos. Los resultados