José Luis Dader García

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.

Genomic Insights in the Metabolism of Aromatic Compounds in Pseudomonas

Pseudomonads are ubiquitous γ-proteobacteria with a remarkable degree of physiological and genetic adaptability. Members of the genus Pseudomonas are found in large numbers in different natural environments (soil, freshwater, marine) as well as in association with plants and animals. These bacteria are involved in important metabolic activities in the environment, being element cycling and degradation of biogenic and xenobiotic pollutants some of their major tasks56, 64, 90, 96. The metabolic versatility of Pseudomonas strains has been used for biotechnological applications, mainly to degrade waste (bioremediation) and to synthesize specialty chemicals (biocatalysis)69, 99.

Atlantic salmon (Salmo salar L.) serum vitellogenin neutralises infectivity of infectious pancreatic necrosis virus (IPNV).

Vitellogenin is a phosphoglycoprotein which represents the main precursor of the egg yolk in teleost fish. This reproductive protein was also demonstrated to play an important role in innate immunity by acting as a pattern recognition molecule capable of binding to bacteria, fungi and enhancing macrophage phagocytosis. The presented results demonstrate that, egg homogenate, ovarian fluid and serum of mature female Atlantic salmon have high neutralising ability for infectious pancreatic necrosis virus (IPNV). Vitellogenin from mature female Atlantic salmon serum, purified by immuno-affinity on a column matrix coated with monoclonal anti-Atlantic salmon vitellogenin antibody, was able to neutralise between 9.1 x 10(4) and 3.09 x 10(5) TCID(50) IPNV mg(-1) of protein. To the authors knowledge, this is the first time that the neutralising activity of vitellogenin on a teleost virus has been demonstrated. The results may explain why IPNV is difficult to detect by culture methods in ovarian fluid and egg homogenates from carrier mature females and suggest a possible means of vertical transmission via the egg.

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.

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.

3-Hydroxyphenylpropionate and Phenylpropionate Are Synergistic Activators of the MhpR Transcriptional Regulator from Escherichia coli

The degradation of the aromatic compound phenylpropionate (PP) in Escherichia coli K-12 requires the activation of two different catabolic pathways coded by the hca and the mhp gene clusters involved in the mineralization of PP and 3-hydroxyphenylpropionate (3HPP), respectively. The compound 3-(2,3-dihydroxyphenyl)propionate (DHPP) is a common intermediate of both pathways which must be cleaved by the MhpB dioxygenase before entering into the primary cell metabolism. Therefore, the degradation of PP has to be controlled by both its specific regulator (HcaR) but also by the MhpR regulator of the mhp cluster. We have demonstrated that 3HPP and DHPP are the true and best activators of MhpR, whereas PP only induces no response. However, in vivo and in vitro transcription experiments have demonstrated that PP activates the MhpR regulator synergistically with the true inducers, representing the first case of such a peculiar synergistic effect described for a bacterial regulator. The three compounds enhanced the interaction of MhpR with its DNA operator in electrophoretic mobility shift assays. Inducer binding to MhpR is detected by circular dichroism and fluorescence spectroscopies. Fluorescence quenching measurements have revealed that the true inducers (3HPP and DHPP) and PP bind with similar affinities and independently to MhpR. This type of dual-metabolite synergy provides great potential for a rapid modulation of gene expression and represents an important feature of transcriptional control. The mhp regulatory system is an example of the high complexity achievable in prokaryotes.

Deciphering the transcriptional regulation of cholesterol catabolic pathway in mycobacteria: Identification of the inducer of KstR repressor

Background: KstR represses expression of numerous genes responsible for cholesterol catabolism in Mycobacterium. Results: 3-Oxo-4-cholestenoic acid is identified as the inducer molecule of M. smegmatis KstR repressor. Conclusion: Oxidation of C3 and C26 of cholesterol is required to activate the system. Significance: The finding of the KstR inducer molecule represents new insights in developing new targets to fight against M. tuberculosis. Cholesterol degradation plays a prominent role in Mycobacterium tuberculosis infection; therefore, to develop new tools to combat this disease, we need to decipher the components comprising and regulating the corresponding pathway. A TetR-like repressor (KstR) regulates the upper part of this complex catabolic pathway, but the induction mechanism remains unknown. Using a biophysical approach, we have discovered that the inducer molecule of KstR in M. smegmatis mc2155 is not cholesterol but 3-oxo-4-cholestenoic acid, one of the first metabolic intermediates. Binding this compound induces dramatic conformational changes in KstR that promote the KstR-DNA interaction to be released from the operator, retaining its dimeric state. Our findings suggest a regulatory model common to all cholesterol degrading bacteria in which the first steps of the pathway are critical to its mineralization and explain the high redundancy of the enzymes involved in these initial steps.

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.

Regulation of adenylate cyclase from brain membranes of the insect Ceratitis capitata

Abstract 1. 1. The regulatory properties of cations, guanine nucleotides and neurotransmitters on adenylate cyclase from brain membranes of the insect Ceratitis capitata have been studied. 2. 2. The Mg 2+ -basal enzyme activity was enhanced by GTP (200%) and GppNHp (600%). 3. 3. The stimulatory effect of neurotransmitters was dependent on the presence of either GTP or GppNHp. The maximum increase of the Mg 2+ -GTP-basal activity was achieved by octopamine followed by catecolamines, whereas serotonin and dopamine did not exert any effect on the enzyme. However, serotonin and dopamine stimulated the enzyme activity in the presence of GppNHp. 4. 4. Adrenaline, noradrenaline, serotonin and dopamine did not show any additive behaviour on the regulatory properties of octopamine in the Mg 2+ -GppNHp system. 5. 5. The cation influence on the basal activity followed a different order whether the system was in the presence of GppNHp or not. 6. 6. The activation order was Mn 2+ > Mg 2+ > Co 2+ in the absence of the analog, whereas it was Co 2+ > Mg 2+ > Mn 2+ when the cations regulated the enzyme system in the presence of GppNHp; the simultaneous presence of octopamine modifies the relative regulatory influence of cations and the nucleotide, conforming that the activity of cations are not restricted to the quelation of the substrate. 7. 7. The F − −Me 2+ -basal activity was depressed by GppNHp. The lack of additivity between F − and the guanine nucleotide analog suggests that the effects of both components are mediated by a common regulatory subunit.

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons.

A number of pharmaceutical steroid synthons are currently produced through the microbial side‐chain cleavage of natural sterols as an alternative to multi‐step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc2155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc2155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (KshAB) and a ketosteroid Δ1‐dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4‐androstadiene‐3,17‐dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039‐5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4‐androstene‐3,17‐dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc2155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.