Bioprospection of the bacterial ß-myrcene-biotransforming trait in the rhizosphere.

The biocatalysis of ß-myrcene into value-added compounds, with enhanced organoleptic/therapeutic properties, may be performed by resorting to specialized enzymatic machinery of ß-myrcene-biotransforming bacteria. Few ß-myrcene-biotransforming bacteria have been studied, limiting the diversity of genetic modules/catabolic pathways available for biotechnological research. In our model Pseudomonas sp. strain M1, the ß-myrcene catabolic core-code was identified in a 28-kb genomic island (GI). The lack of close homologs of this ß-myrcene-associated genetic code prompted a bioprospection of cork oak and eucalyptus rhizospheres, from 4 geographic locations in Portugal, to evaluate the environmental diversity and dissemination of the ß-myrcene-biotransforming genetic trait (Myr+). Soil microbiomes were enriched in ß-myrcene-supplemented cultures, from which ß-myrcene-biotransforming bacteria were isolated, belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia classes. From a panel of representative Myr+ isolates that included 7 bacterial genera, the production of ß-myrcene derivatives previously reported in strain M1 was detected in Pseudomonas spp., Cupriavidus sp., Sphingobacterium sp., and Variovorax sp. A comparative genomics analysis against the genome of strain M1 found the M1-GI code in 11 new Pseudomonas genomes. Full nucleotide conservation of the ß-myrcene core-code was observed throughout a 76-kb locus in strain M1 and all 11 Pseudomonas spp., resembling the structure of an integrative and conjugative element (ICE), despite being isolated from different niches. Furthermore, the characterization of isolates not harboring the Myr+-related 76-kb locus suggested that they may biotransform ß-myrcene via alternative catabolic loci, being thereby a novel source of enzymes and biomolecule catalogue for biotechnological exploitation. KEY POINTS: • The isolation of 150 Myr+ bacteria hints the ubiquity of such trait in the rhizosphere. • The Myr+ trait is spread across different bacterial taxonomic classes. • The core-code for the Myr+ trait was detected in a novel ICE, only found in Pseudomonas spp.

Current Advances in the Bacterial Toolbox for the Biotechnological Production of Monoterpene-Based Aroma Compounds.

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, ß-pinene, limonene and ß-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.

Microbial Metabolic Potential of Phenol Degradation in Wastewater Treatment Plant of Crude Oil Refinery: Analysis of Metagenomes and Characterization of Isolates.

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.

New insights into the immunoproteome of B. cenocepacia J2315 using serum samples from cystic fibrosis patients.

Bacteria of the Burkholderia cepacia complex (Bcc) are ubiquitous multidrug resistant organisms and opportunistic pathogens capable of causing life threatening lung infections among cystic fibrosis (CF) patients. No effective therapies are currently available to eradicate Bcc bacteria from CF patients, as these organisms are inherently resistant to the majority of clinically available antimicrobials. An immunoproteomics approach was used to identify Bcc proteins that stimulate the humoral immune response of the CF host, using bacterial cells grown under conditions mimicking the CF lung environment and serum samples from CF patients with a clinical record of Bcc infection. 24 proteins of the Bcc strain B. cenocepacia J2315 were identified as immunoreactive, 19 here reported as immunogenic for the first time. Ten proteins were predicted as extracytoplasmic, 9 of them being conserved in Bcc genomes. The immunogenic Bcc extracytoplasmic proteins are potential targets for development of novel therapeutic strategies and diagnostic tools to protect patients against the onset of chronic Bcc lung infections.

Seasonal bacterial community dynamics in a crude oil refinery wastewater treatment plant.

The biological treatment of oil refinery effluents in wastewater treatment plants (WWTPs) relies on specialized bacteria contributing to remove organic load, nitrogen, sulfur, and phosphorus compounds. Knowledge about bacterial dynamics in WWTPs and how they affect the performance of the wastewater treatment is limited, particularly in tropical countries. The bacterial communities from three compartments of an oil refinery WWTP in Uran, India, were assessed using 16S-metabarcoding, in winter and monsoon seasons, upstream (from the surge pond) and downstream the biotower (clarifier and guard pond), to understand the effects of seasonal variations in WWTP's efficiency. The organic load and ammonia levels of the treated wastewater increased by 3- and 9-fold in the monsoon time-point. A decreased abundance and diversity of 47 genera (325 OTUs) comprising ammonia and nitrite oxidizing bacteria (AOB, NOB, denitrifiers) was observed in the monsoon season downstream the biotower, whereas 23 OTUs of Sulfurospirillum, Desulfovibrio, and Bacillus, putatively performing dissimilatory nitrate reduction to ammonia (DNRA), were 3-fold more abundant in the same compartments (DNRA/denitrifiers winter ratio < 0.5 vs. monsoon ratio around 3). The total abundance of reported sulfate- and sulfite-reducing bacteria also increased 250- and 500-fold downstream the biotower, in the monsoon time-point. Bacteria performing DNRA may thus outcompete denitrification in this WWTP, limiting the biodegradation process. The alterations detected in bacterial populations involved in the removal of nitrogen and sulfur species evidenced a reduced quality of the released wastewater and may be good candidates for the following monitoring strategies and optimization of the wastewater treatment.

Microbiota fingerprints within the oral cavity of cetaceans as indicators for population biomonitoring.

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.

Identification of genomic loci associated with genotypic and phenotypic variation among Pseudomonas aeruginosa clinical isolates from pneumonia.

In this work, a genotype-phenotype survey of a highly diversified Pseudomonas aeruginosa collection was conducted, aiming to detail pathogen-associated scenarios that clinicians face nowadays. Genetic relation based on RAPD-PCR of 705 isolates, retrieved from 424 patients and several clinical contexts, reported an almost isolate-specific molecular-pattern. Pneumonia-associated isolates HB13 and HB15, clustered in the same RAPD-PCR group, were selected to evaluate the genomic background underlying their contrasting antibiotic resistance and virulence. The HB13 genome harbors antibiotic-inactivating enzymes-coding genes (e.g. aac(3)-Ia, arr, blaVIM-2) and single-nucleotide variations (SNVs) in antibiotic targets, likely accounting for its pan-resistance, whereas HB15 susceptibility correlated to predicted dysfunctional alleles. Isolate HB13 showed the unprecedented rhl-cluster absence and variations in other pathogen competitiveness contributors. Conversely, HB15 genome comprises exoenzyme-coding genes and SNVs linked to increased virulence. Secretome analysis identified signatures features with unknown function as potential novel pathogenic (e.g. a MATE-protein in HB13, a protease in HB15) and antibiotic resistance (a HlyD-like secretion protein in HB13) determinants. Detection of active prophages, proteases (including protease IV and alkaline metalloproteinase), a porin and a peptidase in HB15 highlights the secreted arsenal likely essential for its virulent behavior. The presented phenotype-genome association will contribute to the current knowledge on Pseudomonas aeruginosa pathogenomics.

Functional Characterization of a 28-Kilobase Catabolic Island from Pseudomonas sp. Strain M1 Involved in Biotransformation of ß-Myrcene and Related Plant-Derived Volatiles.

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 ß-myrcene catabolism was elucidated in this work, with a focus on the biocatalytical potential of the ß-myrcene-associated 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 ß-myrcene-induced promoter activity to the production of ß-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 ß-myrcene hydroxylase, responsible for the initial oxidation of ß-myrcene into myrcen-8-ol, and a sensor-like regulatory protein, whose inactivation abolished the myr+ trait of M1 cells. Moreover, it was demonstrated that the range of monoterpene substrates of the M1 enzymatic repertoire, besides ß-myrcene, also includes other acyclic (e.g., ß-linalool) and cyclic [e.g., R-(+)-limonene and (-)-ß-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 ß-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 ß-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.

The microbiome of a striped dolphin (Stenella coeruleoalba) stranded in Portugal.

Infectious diseases with epizootic consequences have not been fully studied in marine mammals. Presently, the unprecedented depth of sequencing, made available by high-throughput approaches, allows detailed comparisons of the microbiome in health and disease. This is the first report of the striped dolphin microbiome in different body sites. Samples from one striped female edematous dolphin were acquired from a variety of body niches, including the blowhole, oral cavity, oral mucosa, tongue, stomach, intestines and genital mucosa. Detailed 16S rRNA analysis of over half a million sequences identified 235 OTUs. Beta diversity analyses indicated that microbial communities vary in structure and cluster by sample origin. Pathogenic, Gram-negative, facultative and obligate anaerobic taxa were significantly detected, including Cetobacterium, Fusobacterium and Ureaplasma. Phocoenobacter and Arcobacter dominated the oral-type samples, while Cardiobacteriaceae and Vibrio were associated with the blowhole and Photobacterium were abundant in the gut. We report for the first time the association of Epulopiscium with a marine mammal gut. The striped dolphin microbiota shows variation in structure and diversity according to the organ type. The high dominance of Gram-negative anaerobic pathogens evidences a cetacean microbiome affected by human-related bacteria.

Deciphering the genome repertoire of Pseudomonas sp. M1 toward ß-myrcene biotransformation.

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 ß-myrcene, a terpene vastly used in industries worldwide. Therefore, the genomic responsiveness of M1 strain toward ß-myrcene was investigated, using an RNA sequencing approach. M1 cells challenged with ß-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 ß-myrcene-imposed molecular adaptation and cellular specialization. A thorough data analysis identified a novel 28-kb genomic island, whose expression was strongly stimulated in ß-myrcene-supplemented medium, that is essential for ß-myrcene catabolism. This island includes ß-myrcene-induced genes whose products are putatively involved in 1) substrate sensing, 2) gene expression regulation, and 3) ß-myrcene oxidation and bioconversion of ß-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.

Towards the Description of the Genome Catalogue of Pseudomonas sp. Strain M1.

Pseudomonas sp. strain M1 is a soil isolate with remarkable biotechnological potential. The genome of Pseudomonas sp. M1 was sequenced using both 454 and Illumina technologies. A customized genome assembly pipeline was used to reconstruct its genome sequence to a single scaffold.