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Pseudomonas sp. strain M1 is able to mineralize highly hydrophobic and recalcitrant compounds, such as benzene, phenol, and their methylated/halogenated derivatives, as well as the backbone of several monoterpenes. The ability to use such a spectrum of compounds as the sole carbon source is, most probably, associated with a genetic background evolved under different environmental constraints. The outstanding performance of strain M1 regarding ␤-myrcene catabolism was elucidated in this work, with a focus on the biocatalytical potential of the ␤-myrceneassociated core code, comprised in a 28-kb genomic island (GI), predicted to be organized in 8 transcriptional units. Functional characterization of this locus with promoter probes and analytical approaches validated the genetic organization predicted in silico and associated the ␤-myrcene-induced promoter activity to the production of ␤-myrcene derivatives. Notably, by using a whole-genome mutagenesis strategy, different genotypes of the 28-kb GI were generated, resulting in the identification of a novel putative ␤-myrcene hydroxylase, responsible for the initial oxidation of ␤-myrcene into myrcen-8-ol, and a sensor-like regulatory protein, whose inactivation abolished the myr ϩ trait of M1 cells. Moreover, it was demonstrated that the range of monoterpene substrates of the M1 enzymatic repertoire, besides ␤-myrcene, also includes other acyclic (e.g., ␤-linalool) and cyclic [e.g., R-(ϩ)-limonene and (Ϫ)-␤-pinene] molecules. Our findings are the cornerstone for following metabolic engineering approaches and hint at a major role of the 28-kb GI in the biotransformation of a broad monoterpene backbone spectrum for its future biotechnological applications. IMPORTANCE Information regarding microbial systems able to biotransform monoterpenes, especially ␤-myrcene, is limited and focused mainly on nonsystematic metabolite identification. Complete and detailed knowledge at the genetic, protein, metabolite, and regulatory levels is essential in order to set a model organism or a catabolic system as a biotechnology tool. Moreover, molecular characterization of reported systems is scarce, almost nonexistent, limiting advances in the development of optimized cell factories with strategies based on the new generation of metabolic engineering platforms. This study provides new insights into the intricate molecular functionalities associated with ␤-myrcene catabolism in Pseudomonas, envisaging the production of a molecular knowledge base about the underlying catalytic and regulatory mechanisms of plant-derived volatile catabolic pathways.KEYWORDS Pseudomonas, biotransformation, monoterpenes, genomic island, promoter probes, ␤-myrcene hydroxylase

Pseudomonas sp. M1 is able to mineralize several unusual substrates of natural and xenobiotic origin, contributing to its competence to thrive in different ecological niches. In this work, the genome of M1 strain was resequenced by Illumina MiSeq to refine the quality of a published draft by resolving the majority of repeat-rich regions. In silico genome analysis led to the prediction of metabolic pathways involved in biotransformation of several unusual substrates (e.g., plant-derived volatiles), providing clues on the genomic complement required for such biodegrading/biotransformation functionalities. Pseudomonas sp. M1 exhibits a particular sensory and biotransformation/biocatalysis potential toward β-myrcene, a terpene vastly used in industries worldwide. Therefore, the genomic responsiveness of M1 strain toward β-myrcene was investigated, using an RNA sequencing approach. M1 cells challenged with β-myrcene(compared with cells grown in lactate) undergo an extensive alteration of the transcriptome expression profile, including 1,873 genes evidencing at least 1.5-fold of altered expression (627 upregulated and 1,246 downregulated), toward β-myrcene-imposed molecular adaptation and cellular specialization. A thorough data analysis identified a novel 28-kb genomic island, whose expression was strongly stimulated in β-myrcene-supplemented medium, that is essential for β-myrcene catabolism. This island includes β-myrcene-induced genes whose products are putatively involved in 1) substrate sensing, 2) gene expression regulation, and 3) β-myrcene oxidation and bioconversion of β-myrcene derivatives into central metabolism intermediates. In general, this locus does not show high homology with sequences available in databases and seems to have evolved through the assembly of several functional blocks acquired from different bacteria, probably, at different evolutionary stages.

The drilling, processing and transportation of oil are the main sources of pollution in water and soil. The current work analyzes the microbial diversity and aromatic compounds degradation potential in the metagenomes of communities in the wastewater treatment plant (WWTP) of a crude oil refinery. By focusing on the degradation of phenol, we observed the involvement of diverse indigenous microbial communities at different steps of the WWTP. The anaerobic bacterial and archaeal genera were replaced by aerobic and facultative anaerobic bacteria through the biological treatment processes. The phyla Proteobacteria, Bacteroidetes and Planctomycetes were dominating at different stages of the treatment. Most of the established protein sequences of the phenol degradation key enzymes belonged to bacteria from the class Alphaproteobacteria. From 35 isolated strains, 14 were able to grow on aromatic compounds, whereas several phenolic compound-degrading strains also degraded aliphatic hydrocarbons. Two strains, Acinetobacter venetianus ICP1 and Pseudomonas oleovorans ICTN13, were able to degrade various aromatic and aliphatic pollutants and were further characterized by whole genome sequencing and cultivation experiments in the presence of phenol to ascertain their metabolic capacity in phenol degradation. When grown alone, the intermediates of catechol degradation, the meta or ortho pathways, accumulated into the growth environment of these strains. In the mixed cultures of the strains ICP1 and ICTN13, phenol was degraded via cooperation, in which the strain ICP1 was responsible for the adherence of cells and ICTN13 diminished the accumulation of toxic intermediates.

Climate changes comprise increasing global temperature and water cycle deregulation (precipitation storms and long dry seasons). Many affected ecosystems are located within the Mediterranean basin, where cork oak (Quercus suber L.) is one of the most important forest ecosystems. Despite cork oak tolerance to drought, the decrease of water availability and increase of temperature is causing a serious decline of cork oak populations. In the present work, the bacterial community of cork oak soils was assessed by metabarcoding using Illumina Miseq. Soils from seven independent cork oak forests were collected along a climate gradient. In all forest soils, Proteobacteria and Actinobacteria were the richest and more abundant bacteria. Acidobacteria also presented a high relative abundance, and Chloroflexi was a rich phylum. The soil bacterial community diversity and composition was strongly affected by the climatic region where cork oak resides and specific bacterial taxa were differently affected by precipitation and temperature. Accordingly, cork oak bacterial communities clustered into three distinct groups, related with humid, sub-humid and arid/semi-arid climates. Driest and warmer forests presented more diverse bacterial communities than humid and coolest forests. However, driest climates presented more homogenous bacterial communities among forests than humid climates. Climate (mainly precipitation) revealed to be the strongest driver leading to significant variations of bacterial community profiles. The most impacted bacterial taxa by climatic variables were Proteobacteria, in particular Gammaproteobacteria and Deltaproteobacteria, Chloroflexi, and Firmicutes. Humid forests presented mainly Acidobacteria as good indicators of climate, whereas Actinobacteria members were better indicators for arid forests (mainly Gaiellales and Frankiales). Some indicator species for different climate conditions were members of the bacterial core of cork oak stands (7% of the total bacterial community). Taken together, different microbiomes were selected by the climate conditions in cork oak stands along a climate gradient and might provide the key to forest sustainability in times of global warming. challenge to cork oak forests (reviewed by Reis et al., 2017; Maghnia et al., 2019). An adaptation of cork oak populations to drier and warmer conditions is expectable (Varela et al., 2015), but the decline of existing populations have been described all over Mediterranean basin (reviewed by Reis et al., 2017). From the huge diversity of microbes present in cork oak soils, fungal communities are those more related with water transfer and increasing water availability to plants. This is mostly due to the ability of ectomycorrhizal fungi to become associated to cork oak roots, promoting water and nutrients transfer (Reis et al., 2017). However, bacteria also play an important ecological role on forest soils. Besides being important for decomposition of organic matter, N fixation, mineral weathering and consequently the release of inorg...

The composition of mammalian microbiota has been related with the host health status. In this study, we assessed the oral microbiome of 3 cetacean species most commonly found stranded in Iberian Atlantic waters (Delphinus delphis, Stenella coeruleoalba and Phocoena phocoena), using 16S rDNA-amplicon metabarcoding. All oral microbiomes were dominated by Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria bacteria, which were also predominant in the oral cavity of Tursiops truncatus. A Constrained Canonical Analysis (CCA) showed that the major factors shaping the composition of 38 oral microbiomes (p-value < 0.05) were: (i) animal species and (ii) age class, segregating adults and juveniles. The correlation analysis also grouped the microbiomes by animal stranding location and health status. Similar discriminatory patterns were detected using the data from a previous study on Tursiops truncatus, indicating that this correlation approach may facilitate data comparisons between different studies on several cetacean species. This study identified a total of 15 bacterial genera and 27 OTUs discriminating between the observed CCA groups, which can be further explored as microbiota fingerprints to develop (i) specific diagnostic assays for cetacean population conservation and (ii) bio-monitoring approaches to assess the health of marine ecosystems from the Iberian Atlantic basin, using cetaceans as bioindicators.

Monoterpenes are plant secondary metabolites, widely used in industrial processes as precursors of important aroma compounds, such as vanillin and (−)-menthol. However, the physicochemical properties of monoterpenes make difficult their conventional conversion into value-added aromas. Biocatalysis, either by using whole cells or enzymes, may overcome such drawbacks in terms of purity of the final product, ecological and economic constraints of the current catalysis processes or extraction from plant material. In particular, the ability of oxidative enzymes (e.g., oxygenases) to modify the monoterpene backbone, with high regio- and stereo-selectivity, is attractive for the production of “natural” aromas for the flavor and fragrances industries. We review the research efforts carried out in the molecular analysis of bacterial monoterpene catabolic pathways and biochemical characterization of the respective key oxidative enzymes, with particular focus on the most relevant precursors, β-pinene, limonene and β-myrcene. The presented overview of the current state of art demonstrates that the specialized enzymatic repertoires of monoterpene-catabolizing bacteria are expanding the toolbox towards the tailored and sustainable biotechnological production of values-added aroma compounds (e.g., isonovalal, α-terpineol, and carvone isomers) whose implementation must be supported by the current advances in systems biology and metabolic engineering approaches.

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