Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds.

Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.

New perspectives on the anaerobic degradation of BTEX: Mechanisms, pathways, and intermediates.

Aromatic hydrocarbons like benzene, toluene, xylene, and ethylbenzene (BTEX) can escape into the environment from oil and gas operations and manufacturing industries posing significant health risks to humans and wildlife. Unlike conventional clean-up methods used, biological approaches such as bioremediation can provide a more energy and labour-efficient and environmentally friendly option for sensitive areas such as nature reserves and cities, protecting biodiversity and public health. BTEX contamination is often concentrated in the subsurface of these locations where oxygen is rapidly depleted, and biodegradation relies on anaerobic processes. Thus, it is critical to understand the anaerobic biodegradation characteristics as it has not been explored to a major extent. This review presents novel insights into the degradation mechanisms under anaerobic conditions and presents a detailed description and interconnection between them. BTEX degradation can follow four activation mechanisms: hydroxylation, carboxylation, methylation, and fumarate addition. Hydroxylation is one of the mechanisms that explains the transformation of benzene into phenol, toluene into benzyl alcohol or p-cresol, and ethylbenzene into 1-phenylethanol. Carboxylation to benzoate is thought to be the primary mechanism of degradation for benzene. Despite being poorly understood, benzene methylation has been also reported. Moreover, fumarate addition is the most widely reported mechanism, present in toluene, ethylbenzene, and xylene degradation. Further research efforts are required to better elucidate new and current alternative catabolic pathways. Likewise, a comprehensive analysis of the enzymes involved as well as the development of advance tools such as omic tools can reveal bottlenecks degradation steps and create more effective on-site strategies to address BTEX pollution.

Sulfonamide Per- and Polyfluoroalkyl Substances Can Impact Microorganisms Used in Aromatic Hydrocarbon and Trichloroethene Bioremediation.

Per- and polyfluoroalkyl substances (PFASs) from aqueous film forming foams (AFFFs) can hinder bioremediation of co-contaminants such as trichloroethene (TCE) and benzene, toluene, ethylbenzene, and xylene (BTEX). Anaerobic dechlorination can require bioaugmentation of Dehalococcoides, and for BTEX, oxygen is often sparged to stimulate in situ aerobic biodegradation. We tested PFAS inhibition to TCE and BTEX bioremediation by exposing an anaerobic TCE-dechlorinating coculture, an aerobic BTEX-degrading enrichment culture, and an anaerobic toluene-degrading enrichment culture to n-dimethyl perfluorohexane sulfonamido amine (AmPr-FHxSA), perfluorohexane sulfonamide (FHxSA), perfluorohexanesulfonic acid (PFHxS), or nonfluorinated surfactant sodium dodecyl sulfate (SDS). The anaerobic TCE-dechlorinating coculture was resistant to individual PFAS exposures but was inhibited by >1000× diluted AFFF. FHxSA and AmPr-FHxSA inhibited the aerobic BTEX-degrading enrichment. The anaerobic toluene-degrading enrichment was not inhibited by AFFF or individual PFASs. Increases in amino acids in the anaerobic TCE-dechlorinating coculture compared to the control indicated stress response, whereas the BTEX culture exhibited lower concentrations of all amino acids upon exposure to most surfactants (both fluorinated and nonfluorinated) compared to the control. These data suggest the main mechanisms of microbial toxicity are related to interactions with cell membrane synthesis as well as protein stress signaling.

Biodegradation of aliphatic and aromatic hydrocarbons by bacteria isolated from Bahregan area.

Environmental pollution with aromatic and aliphatic hydrocarbons caused by oil and petrochemical industries has very toxic and carcinogenic effects on living organisms and should be removed from the environment. In this research, after analyzing the oil sludge of the Bahregan area, it was found that most aliphatic paraffin compounds are related to octadecane, most liquid aliphatic compounds are related to hexadecane, and most aromatic compounds are related to naphthalene, phenanthrene, fluoranthene, and anthracene. Then, we investigated the ability of native bacteria from this area, such as Thalassospira, Chromohalobacter, and a bacterial consortium, to biodegrade the dominant aromatic and aliphatic hydrocarbons found in oil sludge. The results of Gas Chromatography-Mass Spectrometry analysis showed that among the tested hydrocarbon sources, Thalassospira can completely remove octadecane and hexadecane, and Chromohalobacter can reduce hexadecane from 15.9 to 9.9%. The bacterial consortium can completely remove octadecane and reduce hexadecane from 15.9 to 5.1%, toluene from 25.6 to 0.6%, and phenanthrene from 12.93 to 6%. According to the obtained results, the bacterial consortium effectively plays a role in the biodegradation of aromatic and aliphatic hydrocarbons, making it a viable solution for treating hydrocarbon pollutants in various environments.

Efficient bio-remediation of multiple aromatic hydrocarbons using different types of thermotolerant, ring-cleaving dioxygenases derived from Aeribacillus pallidus HB-1.

As toxic contaminants, aromatic compounds are widespread in most environmental matrices, and bioenzymatic catalysis plays a critical role in the degradation of xenobiotics. Here, a thermophillic aromatic hydrocarbon degrader Aeribacillus pallidus HB-1 was found. Bioinformatic analysis of the HB-1 genome revealed two ring-cleaving extradiol dioxygenases (EDOs), among which, EDO-0418 was assigned to a new subfamily of type I.1 EDOs and exhibited a broad substrate specificity, particularly towards biarylic substrate. Both EDOs exhibited optimal activities at elevated temperatures (55 and 65 °C, respectively) and showed remarkable thermostability, pH stability, metal ion resistance and tolerance to chemical reagents. Most importantly, simulated wastewater bioreactor experiments demonstrated efficient and uniform degradation performance of mixed aromatic substrates under harsh environments by the two enzymes combined for potential industrial applications. The unveiling of two thermostable dioxygenases with broad substrate specificities and stress tolerance provides a novel approach for highly efficient environmental bioremediation using composite enzyme systems.

Bacterial crude oil and polyaromatic hydrocarbon degraders from Kazakh oil fields as barley growth support.

Bacterial strains of the genera Arthrobacter, Bacillus, Dietzia, Kocuria, and Micrococcus were isolated from oil-contaminated soils of the Balgimbaev, Dossor, and Zaburunye oil fields in Kazakhstan. They were selected from 1376 isolated strains based on their unique ability to use crude oil and polyaromatic hydrocarbons (PAHs) as sole source of carbon and energy in growth experiments. The isolated strains degraded a wide range of aliphatic and aromatic components from crude oil to generate a total of 170 acid metabolites. Eight metabolites were detected during the degradation of anthracene and of phenanthrene, two of which led to the description of a new degradation pathway. The selected bacterial strains Arthrobacter bussei/agilis SBUG 2290, Bacillus atrophaeus SBUG 2291, Bacillus subtilis SBUG 2285, Dietzia kunjamensis SBUG 2289, Kocuria rosea SBUG 2287, Kocuria polaris SBUG 2288, and Micrococcus luteus SBUG 2286 promoted the growth of barley shoots and roots in oil-contaminated soil, demonstrating the enormous potential of isolatable and cultivable soil bacteria in soil remediation. KEY POINTS: • Special powerful bacterial strains as potential crude oil and PAH degraders. • Growth on crude oil or PAHs as sole source of carbon and energy. • Bacterial support of barley growth as resource for soil remediation.

Natural polysaccharide polymer network for sustained nutrient release to stimulate the activity of aromatic hydrocarbon-degrading indigenous microflora present in groundwater.

Aromatic hydrocarbons (AHs) are known to contaminate groundwater with low indigenous microorganism populations and limited nutrient substrates for degradation reactions, resulting in weak natural remediation abilities of groundwater ecosystems. In this study, we aimed to utilize the principles of AH degradation by microorganisms to identify effective nutrients and optimize nutrient substrate allocation through actual surveys of AH-contaminated sites and microcosm experiments. Building on this, using biostimulation and controlled-release technology, we developed a natural polysaccharide-based encapsulated targeted bionutrient (SA-H-CS) that is characterized by easy uptake, good stability, controllable slow-release migration, and longevity to stimulate indigenous microflora in groundwater to efficiently degrade AHs. Results showed that SA-H-CS is a simple overall dispersion system, and nutrient components diffuse readily through the polymer network. The crosslinking of SA and CS resulted in a more compact structure of the synthesized SA-H-CS, effectively encapsulating the nutrient components and extending their active duration to >20 days. SA-H-CS improved the degradation efficiency of AHs and prompted microorganisms to maintain a high degradation rate (i.e., above 80 %) even in the presence of high concentrations of AHs, particularly naphthalene and O-xylene. Under SA-H-CS stimulation, microorganisms grew rapidly, and the diversity and total number of species of microflora increased significantly, with a notable increase in the proportion of Actinobacteria in the microbial community primarily due to the increased abundance of Arthrobacter, Rhodococcus, and Microbacterium, which are capable of degrading AHs. Concurrently, there was a notable enhancement in the metabolic function of the indigenous microbial communities responsible for AH degradation. SA-H-CS injection facilitated the delivery of nutrient components into the underground environment, improved the conversion ability of inorganic electron donors/receptors in the indigenous microbial community system, and strengthened the co-metabolism mechanism among microorganisms, achieving the goal of efficient AH degradation.

Insights into the susceptibility of Pseudomonas putida to industrially relevant aromatic hydrocarbons that it can synthesize from sugars.

Pseudomonas putida DOT-T1E is a highly solvent tolerant strain for which many genetic tools have been developed. The strain represents a promising candidate host for the synthesis of aromatic compounds-opening a path towards a green alternative to petrol-derived chemicals. We have engineered this strain to produce phenylalanine, which can then be used as a raw material for the synthesis of styrene via trans-cinnamic acid. To understand the response of this strain to the bioproducts of interest, we have analyzed the in-depth physiological and genetic response of the strain to these compounds. We found that in response to the exposure to the toxic compounds that the strain can produce, the cell launches a multifactorial response to enhance membrane impermeabilization. This process occurs via the activation of a cis to trans isomerase that converts cis unsaturated fatty acids to their corresponding trans isomers. In addition, the bacterial cells initiate a stress response program that involves the synthesis of a number of chaperones and ROS removing enzymes, such as peroxidases and superoxide dismutases. The strain also responds by enhancing the metabolism of glucose through the specific induction of the glucose phosphorylative pathway, Entner-Doudoroff enzymes, Krebs cycle enzymes and Nuo. In step with these changes, the cells induce two efflux pumps to extrude the toxic chemicals. Through analyzing a wide collection of efflux pump mutants, we found that the most relevant pump is TtgGHI, which is controlled by the TtgV regulator.

Combined Omics Approach Reveals Key Differences between Aerobic and Microaerobic Xylene-Degrading Enrichment Bacterial Communities: Rhodoferax─A Hitherto Unknown Player Emerges from the Microbial Dark Matter.

Among monoaromatic hydrocarbons, xylenes, especially the ortho and para isomers, are the least biodegradable compounds in oxygen-limited subsurface environments. Although much knowledge has been gained regarding the anaerobic degradation of xylene isomers in the past 2 decades, the diversity of those bacteria which are able to degrade them under microaerobic conditions is still unknown. To overcome this limitation, aerobic and microaerobic xylene-degrading enrichment cultures were established using groundwater taken from a xylene-contaminated site, and the associated bacterial communities were investigated using a polyphasic approach. Our results show that the xylene-degrading bacterial communities were distinctly different between aerobic and microaerobic enrichment conditions. Although members of the genus Pseudomonas were the most dominant in both types of enrichments, the Rhodoferax and Azovibrio lineages were only abundant under microaerobic conditions, while Sphingobium entirely replaced them under aerobic conditions. Analysis of a metagenome-assembled genome of a Rhodoferax-related bacterium revealed aromatic hydrocarbon-degrading ability by identifying two catechol 2,3-dioxygenases in the genome. Moreover, phylogenetic analysis indicated that both enzymes belonged to a newly defined subfamily of type I.2 extradiol dioxygenases (EDOs). Aerobic and microaerobic xylene-degradation experiments were conducted on strains Sphingobium sp. AS12 and Pseudomonas sp. MAP12, isolated from the aerobic and microaerobic enrichments, respectively. The obtained results, together with the whole-genome sequence data of the strains, confirmed the observation that members of the genus Sphingobium are excellent aromatic hydrocarbon degraders but effective only under clear aerobic conditions. Overall, it was concluded that the observed differences between the bacterial communities of aerobic and microaerobic xylene-degrading enrichments were driven primarily by (i) the method of aromatic ring activation (monooxygenation vs dioxygenation), (ii) the type of EDO enzymes, and (iii) the ability of degraders to respire utilizing nitrate.

Mixed polyaromatic hydrocarbon degradation by halotolerant bacterial strains from marine environment and its metabolic pathway.

Accidents involving diesel oil spills are prevalent in sea- and coastal regions. Polycyclic aromatic hydrocarbons (PAHs) can be adsorbed in soil and constitute a persistent contaminant due to their poor water solubility and complex breakdown. PAHs pollution is a pervasive environmental concern that poses serious risks to human life and ecosystems. Thus, it is the need of the hour to degrade and decontaminate the toxic pollutant to save the environment. Among all the available techniques, microbial degradation of the PAHs is proving to be greatly beneficial and effective. Bioremediation overcomes the drawbacks of most physicochemical procedures by eliminating numerous organic pollutants at a lower cost in ambient circumstances and has therefore become a prominent remedial option for pollutant removal, including PAHs. In the present study, we have studied the degradation of Low molecular Weight and High Molecular Weight PAH in combination by bacterial strains isolated from a marine environment. Optimum pH, temperature, carbon, and nitrogen sources, NaCl concentrations were found for efficient degradation using the isolated bacterial strains. At 250 mg/L concentration of the PAH mixture an 89.5% degradation was observed. Vibrio algiolytcus strains were found to be potent halotolerant bacteria to degrade complex PAH into less toxic simple molecules. GC-MS and FTIR data were used to probe the pathway of degradation of PAH.

Polyaromatic Hydrocarbon Specific Ring Hydroxylating Dioxygenases: Diversity, Structure, Function, and Protein Engineering.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitously present in the environment. These compounds have demonstrated both mutagenic and carcinogenic properties. In the past few decades, scientists have constantly been looking for a possible route to their biological degradation. Bacterial ring hydroxylating dioxygenases (RHDs) implicated in the polycyclic aromatic hydrocarbon degradation comprise a large family of enzymes. RHD catalyzes the stereospecific oxidation of PAHs by incorporating molecular oxygen into inert aromatic nuclei. These biocatalysts hold the potential to completely transform and mineralize toxic forms of these compounds into non-toxic forms. RHDsmediated oxygenation produces cis-dihydrodiols, a chiral compound used in pharmaceutical industries. The Molecular investigation of 16S rRNA and key functional genes involved in pollutant degradation have revealed the dominant occurrence of phylum proteobacteria and actinobacteria in hydrocarbonpolluted environments. The present review is aimed at narrating the diversity, distribution, structural and functional characteristics of RHDs. The review further highlights key amino acids participating in RHDs catalysis. It also discusses the robustness of protein engineering methods in improving the structural and functional activity of the ring hydroxylating dioxygenases.

Insights into the syntrophic microbial electrochemical oxidation of toluene: a combined chemical, electrochemical, taxonomical, functional gene-based, and metaproteomic approach.

Biodegradation of aromatic hydrocarbons in anoxic contaminated environments is typically limited by the lack of bioavailable electron acceptors. Microbial electrochemical technologies (METs) are able to provide a virtually inexhaustible electron acceptor in the form of a solid electrode. Recently, we provided first experimental evidence for the syntrophic degradation of toluene in a continuous-flow bioelectrochemical reactor known as the "bioelectric well". Herein, we further analyzed the structure and function of the electroactive toluene-degrading microbiome using a suite of chemical, electrochemical, phylogenetic, proteomic, and functional gene-based analyses. The bioelectric well removed 83 ± 7 % of the toluene from the influent with a coulombic efficiency of 84 %. Cyclic voltammetry allowed to identify the formal potentials of four putative electron transfer sites, which ranged from -0.2 V to +0.1 V vs. SHE, consistent with outer membrane c-type cytochromes and pili of electroactive Geobacter species. The biofilm colonizing the surface of the anode was indeed highly enriched in Geobacter species. On the other hand, the planktonic communities thriving in the bulk of the reactor harbored aromatic hydrocarbons degraders and fermentative propionate-producing microorganisms, as revealed by phylogenetic and proteomic analyses. Most likely, propionate, acetate or other VFAs produced in the bulk liquid from the degradation of toluene were utilized as substrates by the electroactive biofilm. Interestingly, key-functional genes related to the degradation of toluene were found both in the biofilm and in the planktonic communities. Taken as a whole, the herein reported results highlight the importance of applying a comprehensive suite of techniques to unravel the complex cooperative metabolisms occurring in METs.

Fine particulate matter induces heart defects via AHR/ROS-mediated endoplasmic reticulum stress.

Accumulating body of evidence indicates that exposure to fine particulate matter (PM2.5) is closely associated with congenital heart disease in the offspring, but the underlying molecular mechanisms remain to be elucidated. We previously reported that extractable organic matter (EOM) from PM2.5 induces reactive oxygen species (ROS) overproduction by activating aromatic hydrocarbon receptor (AHR), leading to heart defects in zebrafish embryos. We hypothesized that endoplasmic reticulum (ER) stress might be elicited by the excessive ROS production and thereby contribute to the cardiac developmental toxicity of PM2.5. In this study, we examined the effects of EOM on endoplasmic reticulum (ER) stress, apoptosis, and Wnt signal pathway in zebrafish embryos, and explored their roles in EOM-induced heart defects. Our results showed that 4-Phenylbutyric acid (4-PBA), a pharmaceutical inhibitor of ER stress, significantly attenuated the EOM-elevated heart malformation rates. Moreover, EOM upregulated the expression levels of ER stress marker genes including CHOP and PDI in the heart of zebrafish embryos, which were counteracted by genetic or pharmaceutical inhibition of AHR activity. The ROS scavenger N-Acetyl-l-cysteine (NAC) also abolished the EOM-induced ER stress. We further demonstrated that both 4-PBA and CHOP genetic knockdown rescued the PM2.5-induced ROS overproduction, apoptosis and suppression of Wnt signaling. In conclusion, our results indicate that PM2.5 induces AHR/ROS-mediated ER stress, which leads to apoptosis and Wnt signaling inhibition, ultimately resulting in heart defects.

Engineered microbes as effective tools for the remediation of polyaromatic aromatic hydrocarbons and heavy metals.

Heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) have become a major concern to human health and the environment due to rapid industrialization and urbanization. Traditional treatment measures for removing toxic substances from the environment have largely failed, and thus development and advancement in newer remediation techniques are of utmost importance. Rising environmental pollution with HMs and PAHs prompted the research on microbes and the development of genetically engineered microbes (GEMs) for reducing pollution via the bioremediation process. The enzymes produced from a variety of microbes can effectively treat a range of pollutants, but evolutionary trends revealed that various emerging pollutants are resistant to microbial or enzymatic degradation. Naturally, existing microbes can be engineered using various techniques including, gene engineering, directed evolution, protein engineering, media engineering, strain engineering, cell wall modifications, rationale hybrid design, and encapsulation or immobilization process. The immobilization of microbes and enzymes using a variety of nanomaterials, membranes, and supports with high specificity toward the emerging pollutants is also an effective strategy to capture and treat the pollutants. The current review focuses on successful bioremediation techniques and approaches that make use of GEMs or engineered enzymes. Such engineered microbes are more potent than natural strains and have greater degradative capacities, as well as rapid adaptation to various pollutants as substrates or co-metabolizers. The future for the implementation of genetic engineering to produce such organisms for the benefit of the environment andpublic health is indeed long and valuable.

Current status, challenges and prospects for lignin valorization by using Rhodococcus sp.

Lignin represents the most abundant renewable aromatics in nature, which has complicated and heterogeneous structure. The rapid development of biotransformation technology has brought new opportunities to achieve the complete lignin valorization. Especially, Rhodococcus sp. possesses excellent capabilities to metabolize aromatic hydrocarbons degraded from lignin. Furthermore, it can convert these toxic compounds into high value added bioproducts, such as microbial lipids, polyhydroxyalkanoate and carotenoid et al. Accordingly, this review will discuss the potentials of Rhodococcus sp. as a cell factory for lignin biotransformation, including phenol tolerance, lignin depolymerization and lignin-derived aromatic hydrocarbon metabolism. The detailed metabolic mechanism for lignin biotransformation and bioproducts spectrum of Rhodococcus sp. will be comprehensively discussed. The available molecular tools for the conversion of lignin by Rhodococcus sp. will be reviewed, and the possible direction for lignin biotransformation in the future will also be proposed.

The structure-function relationship of bacterial transcriptional regulators as a target for enhanced biodegradation of aromatic hydrocarbons.

The sheer persistence and dissemination of xenobiotic aromatic hydrocarbons contaminants demand sustainable solutions for degradation. Therefore, major pathways of microbial catabolism of aromatic hydrocarbons under aerobic conditions are reviewed and analysed to elicit enhanced biodegradation of aromatic hydrocarbons, via the structure-function relationship of bacterial transcriptional regulators. The initial step of the catabolism occurs via the incorporation of molecular oxygen into the aromatic ring by a multicomponent aromatic ring-hydroxylating-dioxygenase (RHD) enzyme system or monooxygenase system forming different central intermediates such as catechols, protocatechuates, gentisates, and (hydroxy)benzoquinols. The central or lower pathways involve the ring cleavage of central intermediates to tricarboxylic acids. These metabolic pathways are tightly regulated, where the inducer or substrate-specific transcriptional regulation of aromatic catabolic pathways depend on the specific regulatory proteins that acts on a specific promoter in response to a respective inducer signal. These regulatory systems have been grouped according to the regulatory proteins and their families, and identified based on their conserved motifs and their modes of DNA binding. Different regulators from protein families like AraC/XylS, LysR, XylR/NtrC, IclR, etc. have been identified, that are involved in aromatic hydrocarbon regulation. These regulatory proteins have different structures and have different mechanisms of regulation. The proteins of the XylS/AraC family have two domains structure: a highly conserved C-terminus that contains two HTH motifs and the N-terminus end containing the regulatory domain. The LysR type regulatory proteins (LTTRs) act as tetramers that have a helix-turn-helix (HTH) domain at the N terminus and a regulatory binding domain at the C terminus. The IclR regulatory proteins also have a helix-turn-helix DNA binding motif in the N-terminus domain-like LTTRs but include an effector binding motif in the C-terminus domain that is also involved in subunit multimerization. In contrast, the XylR-like regulatory proteins have three domain structures; one for effector sensing, another for ATP binding and hydrolysis, and a domain for DNA binding which contains an HTH motif. This review describes in depth and critical assessment of the aerobic bacterial degradation pathways of aromatic hydrocarbon pollutants with state of art information, underscores areas that are viable and others that require further development, with particular reference to metabolic engineering and synthetic biology applications.

Current research on simultaneous oxidation of aliphatic and aromatic hydrocarbons by bacteria of genus Pseudomonas.

One of the most frequently used methods for elimination of oil pollution is the use of biological preparations based on oil-degrading microorganisms. Such microorganisms often relate to bacteria of the genus Pseudomonas. Pseudomonads are ubiquitous microorganisms that often have the ability to oxidize various pollutants, including oil hydrocarbons. To date, individual biochemical pathways of hydrocarbon degradation and the organization of the corresponding genes have been studied in detail. Almost all studies of this kind have been performed on degraders of individual hydrocarbons belonging to a single particular class. Microorganisms capable of simultaneous degradation of aliphatic and aromatic hydrocarbons are very poorly studied. Most of the works on such objects have been devoted only to phenotype characteristic and some to genetic studies. To identify the patterns of interaction of several metabolic systems depending on the growth conditions, the most promising are such approaches as transcriptomics and proteomics, which make it possible to obtain a comprehensive assessment of changes in the expression of hundreds of genes and proteins at the same time. This review summarizes the existing data on bacteria of the genus Pseudomonas capable of the simultaneous oxidation of hydrocarbons of different classes (alkanes, monoaromatics, and polyaromatics) and presents the most important results obtained in the studies on the biodegradation of hydrocarbons by representatives of this genus using methods of transcriptomic and proteomic analyses.

Pinisolibacter aquiterrae sp. nov., a novel aromatic hydrocarbon-degrading bacterium isolated from benzene-, and xylene-degrading enrichment cultures, and emended description of the genus Pinisolibacter.

Two Gram-reaction-negative strains, designated as B13T and MA2-2, were isolated from two different aromatic hydrocarbon-degrading enrichment cultures and characterized using a polyphasic approach to determine their taxonomic position. The two strains had identical 16S rRNA gene sequences and were most closely related to Pinisolibacter ravus E9T (97.36 %) and Siculibacillus lacustris SA-279T (96.33 %). Cells were facultatively aerobic rods and motile with a single polar flagellum. The strains were able to degrade ethylbenzene as sole source of carbon and energy. The assembled genome of strain B13T had a total length of 4.91 Mb and the DNA G+C content was 68.8 mol%. The predominant fatty acids (>5 % of the total) of strains B13T and MA2-2 were C18 : 1 ω7c/C18 : 1 ω6c, C16 : 1 ω7c/C16 : 1 ω6c and C16 : 0. The major ubiquinone of strain B13T was Q10, while the major polar lipids were phosphatidyl-N-methylethanolamine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and a phospholipid. Based on phenotypic characteristics and phylogenetic data, it is concluded that strains B13T and MA2-2 are members of the genus Pinisolibacter and represent a novel species for which the name Pinisolibacter aquiterrae sp. nov. is proposed. The type strain of the species is strain B13T (=LMG 32346T=NCAIM B.02665T).

Functional roles of multiple Ton complex genes in a Sphingobium degrader of lignin-derived aromatic compounds.

TonB-dependent transporters (TBDTs) mediate outer membrane transport of nutrients using the energy derived from proton motive force transmitted from the TonB-ExbB-ExbD complex localized in the inner membrane. Recently, we discovered ddvT encoding a TBDT responsible for the uptake of a 5,5-type lignin-derived dimer in Sphingobium sp. strain SYK-6. Furthermore, overexpression of ddvT in an SYK-6-derivative strain enhanced its uptake capacity, improving the rate of platform chemical production. Thus, understanding the uptake system of lignin-derived aromatics is fundamental for microbial conversion-based lignin valorization. Here we examined whether multiple tonB-, exbB-, and exbD-like genes in SYK-6 contribute to the outer membrane transport of lignin-derived aromatics. The disruption of tonB2-6 and exbB3 did not reduce the capacity of SYK-6 to convert or grow on lignin-derived aromatics. In contrast, the introduction of the tonB1-exbB1-exbD1-exbD2 operon genes into SYK-6, which could not be disrupted, promoted the conversion of ß-O-4-, ß-5-, ß-1-, ß-ß-, and 5,5-type dimers and monomers, such as ferulate, vanillate, syringate, and protocatechuate. These results suggest that TonB-dependent uptake involving the tonB1 operon genes is responsible for the outer membrane transport of the above aromatics. Additionally, exbB2/tolQ and exbD3/tolR were suggested to constitute the Tol-Pal system that maintains the outer membrane integrity.

Genomic Aromatic Compound Degradation Potential of Novel Paraburkholderia Species: Paraburkholderia domus sp. nov., Paraburkholderia haematera sp. nov. and Paraburkholderia nemoris sp. nov.

We performed a taxonomic and comparative genomics analysis of 67 novel Paraburkholderia isolates from forest soil. Phylogenetic analysis of the recA gene revealed that these isolates formed a coherent lineage within the genus Paraburkholderia that also included Paraburkholderiaaspalathi, Paraburkholderiamadseniana, Paraburkholderiasediminicola, Paraburkholderiacaffeinilytica, Paraburkholderiasolitsugae and Paraburkholderiaelongata and four unidentified soil isolates from earlier studies. A phylogenomic analysis, along with orthoANIu and digital DNA-DNA hybridization calculations revealed that they represented four different species including three novel species and P. aspalathi. Functional genome annotation of the strains revealed several pathways for aromatic compound degradation and the presence of mono- and dioxygenases involved in the degradation of the lignin-derived compounds ferulic acid and p-coumaric acid. This co-occurrence of multiple Paraburkholderia strains and species with the capacity to degrade aromatic compounds in pristine forest soil is likely caused by the abundant presence of aromatic compounds in decomposing plant litter and may highlight a diversity in micro-habitats or be indicative of synergistic relationships. We propose to classify the isolates representing novel species as Paraburkholderia domus with LMG 31832T (=CECT 30334) as the type strain, Paraburkholderia nemoris with LMG 31836T (=CECT 30335) as the type strain and Paraburkholderia haematera with LMG 31837T (=CECT 30336) as the type strain and provide an emended description of Paraburkholderia sediminicola Lim et al. 2008.