Genome analysis for the identification of genes involved in phenanthrene biodegradation pathway in Stenotrophomonas indicatrix CPHE1. Phenanthrene mineralization in soils assisted by integrated approaches.

Phenanthrene (PHE) is a highly toxic compound, widely present in soils. For this reason, it is essential to remove PHE from the environment. Stenotrophomonas indicatrix CPHE1 was isolated from an industrial soil contaminated by polycyclic aromatic hydrocarbons (PAHs) and was sequenced to identify the PHE degrading genes. Dioxygenase, monooxygenase, and dehydrogenase gene products annotated in S. indicatrix CPHE1 genome were clustered into different trees with reference proteins. Moreover, S. indicatrix CPHE1 whole-genome sequences were compared to genes of PAHs-degrading bacteria retrieved from databases and literature. On these basis, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis pointed out that cysteine dioxygenase (cysDO), biphenyl-2,3-diol 1,2-dioxygenase (bphC), and aldolase hydratase (phdG) were expressed only in the presence of PHE. Therefore, different techniques have been designed to improve the PHE mineralization process in five PHE artificially contaminated soils (50 mg kg-1), including biostimulation, adding a nutrient solution (NS), bioaugmentation, inoculating S. indicatrix CPHE1 which was selected for its PHE-degrading genes, and the use of 2-hydroxypropyl-ß-cyclodextrin (HPBCD) as a bioavailability enhancer. High percentages of PHE mineralization were achieved for the studied soils. Depending on the soil, different treatments resulted to be successful; in the case of a clay loam soil, the best strategy was the inoculation of S. indicatrix CPHE1 and NS (59.9% mineralized after 120 days). In sandy soils (CR and R soils) the highest percentage of mineralization was achieved in presence of HPBCD and NS (87.3% and 61.3%, respectively). However, the combination of CPHE1 strain, HPBCD, and NS showed to be the most efficient strategy for sandy and sandy loam soils (LL and ALC soils showed 35% and 74.6%, respectively). The results indicated a high degree of correlation between gene expression and the rates of mineralization.

Insights into the biodegradation of polycaprolactone through genomic analysis of two plastic-degrading Rhodococcus bacteria.

Polycaprolactone (PCL) is an aliphatic polyester often utilized as a model to investigate the biodegradation potential of bacteria and the involved catabolic enzymes. This study aims to characterize PCL biodegradative metabolic potential and correlate it to genomic traits of two plastic-degrading bacteria-Rhodococcus erythropolis D4 strain, a new isolate from plastic-rich organic waste treatment plant, and Rhodococcus opacus R7, known for its relevant biodegradative potential on polyethylene and similar compounds. After preliminary screening for bacteria capable of hydrolyzing tributyrin and PCL, the biodegradation of PCL was evaluated in R. erythropolis D4 and R. opacus R7 by measuring their growth and the release of PCL catabolism products up to 42 days. After 7 days, an increase of at least one order of magnitude of cell number was observed. GC-MS analyses of 28-day culture supernatants showed an increase in carboxylic acids in both Rhodococcus cultures. Furthermore, hydrolytic activity (~5 U mg-1) on short/medium-chain p-nitrophenyl esters was detected in their supernatant. Finally, a comparative genome analysis was performed between two Rhodococcus strains. A comparison with genes annotated in reference strains revealed hundreds of gene products putatively related to polyester biodegradation. Based on additional predictive analysis of gene products, gene expression was performed on a smaller group of genes, revealing that exposure to PCL elicits the greatest increase in transcription for a single gene in strain R7 and two genes, including that encoding a putative lipase, in strain D4. This work exhibits a multifaceted experimental approach to exploit the broad potential of Rhodococcus strains in the field of plastic biodegradation.

Genome-Based Exploration of Rhodococcus Species for Plastic-Degrading Genetic Determinants Using Bioinformatic Analysis.

Plastic polymer waste management is an increasingly prevalent issue. In this paper, Rhodococcus genomes were explored to predict new plastic-degrading enzymes based on recently discovered biodegrading enzymes for diverse plastic polymers. Bioinformatics prediction analyses were conducted using 124 gene products deriving from diverse microorganisms retrieved from databases, literature data, omic-approaches, and functional analyses. The whole results showed the plastic-degrading potential of Rhodococcus genus. Among the species with high plastic-degrading potential, R. erythropolis, R. equi, R. opacus, R. qingshengii, R. fascians, and R. rhodochrous appeared to be the most promising for possible plastic removal. A high number of genetic determinants related to polyester biodegradation were obtained from different Rhodococcus species. However, score calculation demonstrated that Rhodococcus species (especially R. pyridinivorans, R. qingshengii, and R. hoagii) likely possess PE-degrading enzymes. The results identified diverse oxidative systems, including multicopper oxidases, alkane monooxygenases, cytochrome P450 hydroxylases, para-nitrobenzylesterase, and carboxylesterase, and they could be promising reference sequences for the biodegradation of plastics with C-C backbone, plastics with heteroatoms in the main chain, and polyesters, respectively. Notably, the results of this study could be further exploited for biotechnological applications in biodegradative processes using diverse Rhodococcus strains and through catalytic reactions.

Effects of Inulin-Based Prebiotics Alone or in Combination with Probiotics on Human Gut Microbiota and Markers of Immune System: A Randomized, Double-Blind, Placebo-Controlled Study in Healthy Subjects.

The gut microbiota is implicated in diverse interactions affecting human health. The present study reports a randomized, double-blind, placebo-controlled clinical study conducted by administering a new synbiotic formulation composed of two Lactobacillus strains (L. plantarum and L. acidophilus) and one Bifidobacterium strain (B. animalis subsp. lactis) and two types of fructans (fructo-oligosaccharides with a degree of polymerization of 3-5 and inulin-type fructans with 10 DP). The effects of this synbiotic were evaluated on healthy subjects for 28 days and the maintenance of its efficacy was evaluated at the end of a follow-up period of 28 days. The synbiotic treatment contributes to higher biodiversity of the gut microbiota, increasing the community richness with respect to the group with the prebiotics alone and the placebo group. Its positive effect is also reflected in the variation of microbial community structure favoring the beneficial short-chain fatty acids bacterial producers. The amelioration of the health status of the subjects was also established by the reduction of common infectious disease symptom incidence, the stimulation of the gut immune system showing a noteworthy variation of fecal ß-defensin2 and calprotectin levels, and the modulation of the response of the respiratory tract's immune system by salivary IgA as well as total antioxidant capacity biomarkers.

Identification of a Novel Biosurfactant with Antimicrobial Activity Produced by Rhodococcus opacus R7.

Rhodococcus members excrete secondary metabolites, especially compounds which act as biosurfactants. In this work, we demonstrated the ability of Rhodococcus opacus R7 to produce a novel bioactive compound belonging to the class of biosurfactants with antimicrobial properties during the growth on naphthalene. Chemical and biochemical analyses of the isolated compound demonstrated that the biosurfactant could be classified as a hydrophobic peptide. The ESI-full mass spectrometry revealed that the isolated biosurfactant showed a molecular weight of 1292 Da and NMR spectra evidenced the composition of the following amino acid residues: Ala, Thr, Asp, Gly, Ser. Surfactant activity of the R. opacus R7 compound was quantified by the critical micelle dilution (CMD) method and the critical micelle concentration (CMC) was estimated around 20 mg L-1 with a corresponding surface tension of 48 mN m-1. Moreover, biological assays demonstrated that R. opacus R7 biosurfactant peptide exhibited antimicrobial activity against Escherichia coli ATCC 29522 and Staphylococcus aureus ATCC 6538 with the minimum inhibition growth concentration (MIC) values of 2.6 mg mL-1 and 1.7 mg mL-1, respectively. In this study for the first time, a hydrophobic peptide with both biosurfactant and antimicrobial activity was isolated from a bacterium belonging to Rhodococcus genus.

Effectiveness of Multistrain Probiotic Formulation on Common Infectious Disease Symptoms and Gut Microbiota Modulation in Flu-Vaccinated Healthy Elderly Subjects.

The decline of the immune system with aging leads elderly people to be more susceptible to infections, posing high risk for their health. Vaccination is thus important to cope with this risk, even though not always effective. As a strategy to improve protection, adjuvants are used in concomitance with vaccines, however, occasionally producing important side effects. The use of probiotics has been proposed as an alternative to adjuvants due to their efficacy in reducing the risk of common infections through the interactions with the immune system and the gut microbiota. A placebo-controlled, randomized, double-blind, clinical trial was carried out on fifty elderly subjects, vaccinated for influenza, to determine the efficacy of a probiotic mixture in reducing common infection symptoms. The incidence of symptoms was evaluated after 28 days of probiotic intake (namely, T28) and after further 28 days of follow-up (namely, T56). The number of subjects, as well as the number of days with symptoms, was remarkably reduced at T28, and even more at T56 in the probiotic group. Furthermore, the influence of probiotics on immunological parameters was investigated, showing a significant positive improvement of total antioxidant capacity and ß-defensin2 levels. Finally, faecal samples collected from participants were used to assess variations in the gut microbiota composition during the study, showing that probiotic intake enhanced the presence of genera related to a healthy status. Therefore, the collected results suggested that the treatment with the selected probiotic mixture could help in reducing common infectious disease symptom incidence through the stimulation of the immune system, improving vaccine efficacy, and modulating the composition of the resident gut microbiota by enhancing beneficial genera.

Characterization of the Biological Activities of a New Polyphenol-Rich Extract from Cinnamon Bark on a Probiotic Consortium and Its Action after Enzymatic and Microbial Fermentation on Colorectal Cell Lines.

Cinnamon polyphenols are known as health-promoting agents. However, their positive impact depends on the extraction method and their bioaccessibility after digestion. In this work, cinnamon bark polyphenols were extracted in hot water and subjected to an in vitro enzymatic digestion. After a preliminary characterization of total polyphenols and flavonoids (respectively 520.05 ± 17.43 µgGAeq/mg and 294.77 ± 19.83 µgCATeq/mg powder extract), the extract antimicrobial activity was evidenced only against Staphylococcus aureus and Bacillus subtilis displaying a minimum inhibition growth concentration value of 2 and 1.3 mg/mL, respectively, although it was lost after in vitro extract digestion. The prebiotic potential was evaluated on probiotic Lactobacillus and Bifidobacterium strains highlighting a high growth on the in vitro digested cinnamon bark extract (up to 4 × 108 CFU/mL). Thus, the produced SCFAs and other secondary metabolites were extracted from the broth cultures and determined via GC-MSD analyses. The viability of healthy and tumor colorectal cell lines (CCD841 and SW480) was assayed after the exposition at two different concentrations (23 and 46 µgGAeq/mL) of the cinnamon extract, its digested, and the secondary metabolites produced in presence of cinnamon extract or its digested, showing positive protective effects against a tumorigenic condition.

Transcriptomic analysis of Rhodococcus opacus R7 grown on polyethylene by RNA-seq.

Plastic waste management has become a global issue. Polyethylene (PE) is the most abundant synthetic plastic worldwide, and one of the most resistant to biodegradation. Indeed, few bacteria can degrade polyethylene. In this paper, the transcriptomic analysis unveiled for the first time Rhodococcus opacus R7 complex genetic system based on diverse oxidoreductases for polyethylene biodegradation. The RNA-seq allowed uncovering genes putatively involved in the first step of oxidation. In-depth investigations through preliminary bioinformatic analyses and enzymatic assays on the supernatant of R7 grown in the presence of PE confirmed the activation of genes encoding laccase-like enzymes. Moreover, the transcriptomic data allowed identifying candidate genes for the further steps of short aliphatic chain oxidation including alkB gene encoding an alkane monooxygenase, cyp450 gene encoding cytochrome P450 hydroxylase, and genes encoding membrane transporters. The PE biodegradative system was also validated by FTIR analysis on R7 cells grown on polyethylene.

Recent Trends on Biosurfactants With Antimicrobial Activity Produced by Bacteria Associated With Human Health: Different Perspectives on Their Properties, Challenges, and Potential Applications.

The attention towards the bacteria associated with human health is growing more and more, above all regarding the bacteria that inhabit the niches offered by the human body, i.e., the gastrointestinal tract, skin, vaginal environment, and lungs. Among the secondary metabolites released by microorganisms associated with human health, little consideration is given to the biosurfactants, molecules with both hydrophobic and hydrophilic nature. Their role in the complex human environment is not only the mere biosurfactant function, but they could also control the microbiota through the quorum sensing system and the antimicrobial activity. These functions protect them and, accordingly, the human body principally from microbial and fungal pathogens. Consequently, nowadays, biosurfactants are emerging as promising bioactive molecules due to their very different structures, biological functions, low toxicity, higher biodegradability, and versatility. Therefore, this review provides a comprehensive perspective of biosurfactants with antimicrobial activity produced by bacteria associated with the human body and related to everything human beings are in contact with, e.g., food, beverages, and food-waste dumping sites. For the first time, the role of an "-omic" approach is highlighted to predict gene products for biosurfactant production, and an overview of the available gene sequences is reported. Besides, antimicrobial biosurfactants' features, challenges, and potential applications in the biomedical, food, and nutraceutical industries are discussed.

Transcriptomic Analysis of Rhodococcus opacus R7 Grown on o-Xylene by RNA-Seq.

Xylenes are considered one of the most common hazardous sources of environmental contamination. The biodegradation of these compounds has been often reported, rarer the ability to oxidize the ortho-isomer. Among few o-xylene-degrading bacteria, Rhodococcus opacus R7 is well known for its capability to degrade diverse aromatic hydrocarbons and toxic compounds, including o-xylene as only carbon and energy source. This work shows for the first time the RNA-seq approach to elucidate the genetic determinants involved in the o-xylene degradation pathway in R. opacus R7. Transcriptomic data showed 542 differentially expressed genes that are associated with the oxidation of aromatic hydrocarbons and stress response, osmotic regulation and central metabolism. Gene ontology (GO) enrichment and KEGG pathway analysis confirmed significant changes in aromatic compound catabolic processes, fatty acid metabolism, beta-oxidation, TCA cycle enzymes, and biosynthesis of metabolites when cells are cultured in the presence of o-xylene. Interestingly, the most up-regulated genes belong to the akb gene cluster encoding for the ethylbenzene (Akb) dioxygenase system. Moreover, the transcriptomic approach allowed identifying candidate enzymes involved in R7 o-xylene degradation for their likely participation in the formation of the metabolites that have been previously identified. Overall, this approach supports the identification of several oxidative systems likely involved in o-xylene metabolism confirming that R. opacus R7 possesses a redundancy of sequences that converge in o-xylene degradation through R7 peculiar degradation pathway. This work advances our understanding of o-xylene metabolism in bacteria belonging to Rhodococcus genus and provides a framework of useful enzymes (molecular tools) that can be fruitfully targeted for optimized o-xylene consumption.

Genome analysis and -omics approaches provide new insights into the biodegradation potential of Rhodococcus.

The past few years observed a breakthrough of genome sequences of bacteria of Rhodococcus genus with significant biodegradation abilities. Invaluable knowledge from genome data and their functional analysis can be applied to develop and design strategies for attenuating damages caused by hydrocarbon contamination. With the advent of high-throughput -omic technologies, it is currently possible to utilize the functional properties of diverse catabolic genes, analyze an entire system at the level of molecule (DNA, RNA, protein, and metabolite), simultaneously predict and construct catabolic degradation pathways. In this review, the genes involved in the biodegradation of hydrocarbons and several emerging plasticizer compounds in Rhodococcus strains are described in detail (aliphatic, aromatics, PAH, phthalate, polyethylene, and polyisoprene). The metabolic biodegradation networks predicted from omics-derived data along with the catabolic enzymes exploited in diverse biotechnological and bioremediation applications are characterized.

Genome-based analysis for the identification of genes involved in o-xylene degradation in Rhodococcus opacus R7.

BACKGROUND: Bacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes. These bacteria, widely distributed in the environment, are known to be a powerhouse of numerous degradation functions, due to their ability to metabolize a wide range of organic molecules including aliphatic, aromatic, polycyclic aromatic compounds (PAHs), phenols, and nitriles. In accordance with their immense catabolic diversity, Rhodococcus spp. possess large and complex genomes, which contain a multiplicity of catabolic genes, a high genetic redundancy of biosynthetic pathways and a sophisticated regulatory network. The present study aimed to identify genes involved in the o-xylene degradation in R. opacus strain R7 through a genome-based approach. RESULTS: Using genome-based analysis we identified all the sequences in the R7 genome annotated as dioxygenases or monooxygenases/hydroxylases and clustered them into two different trees. The akb, phe and prm sequences were selected as genes encoding respectively for dioxygenases, phenol hydroxylases and monooxygenases and their putative involvement in o-xylene oxidation was evaluated. The involvement of the akb genes in o-xylene oxidation was demonstrated by RT-PCR/qPCR experiments after growth on o-xylene and by the selection of the R7-50 leaky mutant. Although the akb genes are specifically activated for o-xylene degradation, metabolic intermediates of the pathway suggested potential alternative oxidation steps, possibly through monooxygenation. This led us to further investigate the role of the prm and the phe genes. Results showed that these genes were transcribed in a constitutive manner, and that the activity of the Prm monooxygenase was able to transform o-xylene slowly in intermediates as 3,4-dimethylphenol and 2-methylbenzylalcohol. Moreover, the expression level of phe genes, homologous to the phe genes of Rhodococcus spp. 1CP and UPV-1 with a 90% identity, could explain their role in the further oxidation of o-xylene and R7 growth on dimethylphenols. CONCLUSIONS: These results suggest that R7 strain is able to degrade o-xylene by the Akb dioxygenase system leading to the production of the corresponding dihydrodiol. Likewise, the redundancy of sequences encoding for several monooxygenases/phenol hydroxylases, supports the involvement of other oxygenases converging in the o-xylene degradation pathway in R7 strain.

Phenotype microarray analysis may unravel genetic determinants of the stress response by Rhodococcus aetherivorans BCP1 and Rhodococcus opacus R7.

In the present study, the response of Rhodococcus aetherivorans BCP1 and Rhodococcus opacus R7 to various stress conditions and several antimicrobials was examined by PM in relation with genetic determinants, as revealed by annotation analysis of the two genomes. Comparison between metabolic activities and genetic features of BCP1 and R7 provided new insight into the environmental persistence of these two members of the genus Rhodococcus.

Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance.

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.

Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism.

Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain.