Long-Range PCR Reveals the Genetic Cargo of IncP-1 Plasmids in the Complex Microbial Community of an On-Farm Biopurification System Treating Pesticide-Contaminated Wastewater.

Promiscuous plasmids like IncP-1 plasmids play an important role in the bacterial adaptation to pollution by acquiring and distributing xenobiotic catabolic genes. However, most information comes from isolates and the role of plasmids in governing community-wide bacterial adaptation to xenobiotics and other adaptive forces is not fully understood. Current information on the contribution of IncP-1 plasmids in community adaptation is limited because methods are lacking that directly isolate and identify the plasmid borne adaptive functions in whole-community DNA. In this study, we optimized long-range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA. The DNA between the IncP-1 backbone genes trbP and traC, a main insertion site of adaptive trait determinants, is amplified and its content analyzed by high-throughput sequencing. The method was applied to DNA of an on-farm biopurification system (BPS), treating pesticide contaminated wastewater, to examine whether horizontal gene exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. The cargo recovered from BPS community DNA encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, genes with catabolic traits composed only a minor fraction of the cargo, indicating that the IncP-1 region between trbP and traC is not a major contributor to catabolic adaptation of the BPS microbiome. Instead, it contains a functionally diverse set of genes which either may assist biodegradation functions, be remnants of random gene recruitment, or confer other crucial functions for proliferation in the BPS environment. IMPORTANCE This study presents a long-range PCR for direct and cultivation-independent access to the identity of the cargo of a major insertion hot spot of adaptive genes in IncP-1 plasmids and hence a new mobilome tool for understanding the role of IncP-1 plasmids in complex communities. The method was applied to DNA of an on-farm biopurification system (BPS) treating pesticide-contaminated wastewater, aiming at new insights on whether horizontal exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. Unexpectedly, catabolic functions represented a small fraction of the cargo genes while multiple other gene functions were recovered. These results show that the cargo of the target insertion hot spot in IncP-1 plasmids in a community, not necessarily relates to the main obvious selective trait imposed on that community. Instead, these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

Understanding the Formation and Mechanism of Anticipatory Responses in Escherichia coli.

Microorganisms often live in complex habitats, where changes in the environment are predictable, providing an opportunity for microorganisms to learn, anticipate the upcoming environmental changes and prepare in advance for better survival and growth. One such environment is the mammalian intestine, where the abundance of different carbon sources is spatially distributed. In this study, we identified seven spatially distributed carbon sources in the mammalian intestine and tested whether Escherichia coli exhibits phenotypes that are consistent with an anticipatory response given their spatial order and abundance within the mammalian intestine. Through RNA-Seq and RT-PCR validation measurements, we found that there was a 67% match in the expression patterns between the measured phenotypes and what would otherwise be expected in the case of anticipatory behavior, while 83% and 0% were in agreement with the homeostatic and random response, respectively. To understand the genetic and phenotypic basis of the discrepancies between the expected and measured anticipatory responses, we thoroughly investigated the discrepancy in D-galactose treatment and the expression of maltose operon in E. coli. Here, the expected anticipatory response, based on the spatial distribution of D-galactose and D-maltose, was that D-galactose should upregulate the maltose operon, but it was the opposite in experimental validation. We performed whole genome random mutagenesis and screening and identified E. coli strains with positive expression of maltose operon in D-galactose. Targeted Sanger sequencing and mutation repair identified that the mutations in the promoter region of malT and in the coding region of the crp gene were the factors responsible for the reversion in the association. Further, to identify why positive association in the D-galactose treatment and the expression of the maltose operon did not evolve naturally, fitness measurements were performed. Fitness experiments demonstrated that the fitness of E. coli strains with a positive association in the D-galactose treatment and the expression of the maltose operon was 12% to 20% lower than that of the wild type strain.

Bacterial degradation of mixed-PAHs and expression of PAH-catabolic genes.

Polyaromatic hydrocarbons (PAHs) are hazardous organic compounds with established toxicity, carcinogenicity, and mutagenicity, ubiquitous distribution, and persistence in different environmental matrices. In the present study, degradation of the mixture of PAHs (phenanthrene, anthracene, fluorene, and pyrene) by Kocuria flava and Rhodococcus pyridinivorans was investigated. The individual strains and consortium of both degraded 55.6%, 59.5%, and 59.1% of 10 mg L-1 of mixed PAHs, respectively, within 15 days. The participation of catabolic enzymes [catechol 2,3-dioxygenase (C23O), dehydrogenase (DH), and peroxidase (POD)] was confirmed during catalytic oxidation through meta-cleavage of mixed PAHs in this study. The catabolic gene expression of naphthalene dioxygenase (NAH) and catechol 2,3-dioxygenase (C23O) during degradation was confirmed using RT-qPCR in the present study. This is the first study that shows significant gene expression of the catabolic genes during degradation of mixed PAHs by selected bacterial strains. The C23O gene showed a 6.02 log fold higher expression in Kocuria flava in comparison to Rhodococcus pyridinivorans whereas NAH gene exhibited a 7.9 log fold higher expression in Rhodococcus pyridinivorans in comparison to Kocuria flava. Hence it is likely to conclude that combination of Kocuria flava and Rhodococcus pyridinivorans can effectively remove hazardous mixture of PAHs from the contaminated environmental matrix.

The transcription factor ZmNAC126 accelerates leaf senescence downstream of the ethylene signalling pathway in maize.

Leaf senescence is an integral part of plant development, during which, nutrients are remobilized from senescent leaves to fast-growing organs. The initiation and progression dynamics of leaf senescence is therefore vital not only to the maximal accumulation of assimilates but also to the efficient remobilization of nutrients. Senescence is a finely tuned process that involves the action of a large number of transcription factors (TFs). The NAC TFs play critical roles in regulating leaf senescence in Arabidopsis, wheat, rice and tomato. Here, we identified a NAC TF, ZmNAC126 that is responsive to leaf senescence in maize. Ectopic overexpression of ZmNAC126 in Arabidopsis and maize enhanced chlorophyll degradation and promoted leaf senescence. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that ZmNAC126 could directly bind to the promoters of major chlorophyll catabolic genes in maize. Dual-luciferase assay in maize protoplasts indicated that ZmNAC126 positively regulates these chlorophyll catabolic genes in maize. Moreover, ZmNAC126 could be induced by ethylene, and ZmEIN3, a major TF of ethylene signalling, could bind to its promoter to transactivate its expression. Taken together, ZmNAC126 may play a pivotal role in regulating natural and ethylene-triggered leaf senescence in maize.

A comprehensive review of metabolic and genomic aspects of PAH-degradation.

Polyaromatic hydrocarbons (PAHs) are considered as hazardous organic priority pollutants. PAHs have immense public concern and critical environmental challenge around the globe due to their toxic, carcinogenic, and mutagenic properties, and their ubiquitous distribution, recalcitrance as well as persistence in environment. The knowledge about harmful effects of PAHs on ecosystem along with human health has resulted in an interest of researchers on degradation of these compounds. Whereas physico-chemical treatment of PAHs is cost and energy prohibitive, bioremediation i.e. degradation of PAHs using microbes is becoming an efficient and sustainable approach. Broad range of microbes including bacteria, fungi, and algae have been found to have capability to use PAHs as carbon and energy source under both aerobic and anaerobic conditions resulting in their transformation/degradation. Microbial genetic makeup containing genes encoding catabolic enzymes is responsible for PAH-degradation mechanism. The degradation capacity of microbes may be induced by exposing them to higher PAH-concentration, resulting in genetic adaptation or changes responsible for high efficiency towards removal/degradation. In last few decades, mechanism of PAH-biodegradation, catabolic gene system encoding catabolic enzymes, and genetic adaptation and regulation have been investigated in detail. This review is an attempt to overview current knowledge of microbial degradation mechanism of PAHs, its genetic regulation with application of genetic engineering to construct genetically engineered microorganisms, specific catabolic enzyme activity, and application of bioremediation for reclamation of PAH-contaminated sites. In addition, advanced molecular techniques i.e. genomic, proteomic, and metabolomic techniques are also discussed as powerful tools for elucidation of PAH-biodegradation/biotransformation mechanism in an environmental matrix.

Quantification of Naphthalene Dioxygenase (NahAC) and Catechol Dioxygenase (C23O) Catabolic Genes Produced by Phenanthrene-Degrading Pseudomonas fluorescens AH-40.

BACKGROUND: Petroleum polycyclic aromatic hydrocarbons (PAHs) are known to be toxic and carcinogenic for humans and their contamination of soils and water is of great environmental concern. Identification of the key microorganisms that play a role in pollutant degradation processes is relevant to the development of optimal in situ bioremediation strategies. OBJECTIVE: Detection of the ability of Pseudomonas fluorescens AH-40 to consume phenanthrene as a sole carbon source and determining the variation in the concentration of both nahAC and C23O catabolic genes during 15 days of the incubation period. METHODS: In the current study, a bacterial strain AH-40 was isolated from crude oil polluted soil by enrichment technique in mineral basal salts (MBS) medium supplemented with phenanthrene (PAH) as a sole carbon and energy source. The isolated strain was genetically identified based on 16S rDNA sequence analysis. The degradation of PAHs by this strain was confirmed by HPLC analysis. The detection and quantification of naphthalene dioxygenase (nahAc) and catechol 2,3-dioxygenase (C23O) genes, which play a critical role during the mineralization of PAHs in the liquid bacterial culture were achieved by quantitative PCR. RESULTS: Strain AH-40 was identified as pseudomonas fluorescens. It degraded 97% of 150 mg phenanthrene L-1 within 15 days, which is faster than previously reported pure cultures. The copy numbers of chromosomal encoding catabolic genes nahAc and C23O increased during the process of phenanthrene degradation. CONCLUSION: nahAc and C23O genes are the main marker genes for phenanthrene degradation by strain AH-40. P. fluorescence AH-40 could be recommended for bioremediation of phenanthrene contaminated site.

Biological agents for 2,4-dichlorophenoxyacetic acid herbicide degradation.

Phenoxy herbicides are the most widely used family of herbicides worldwide. The dichlorophenoxyacetic acid (2,4-D) is extensively used as a weed killer on cereal crops and pastures. This herbicide is highly water-soluble, and even after a long period of disuse, considerable amounts of both 2,4-D and its main product of degradation, 2,4 dichlorophenol (2,4-DCP), might be found in nature. Biological decomposition of pesticides is an expressive and effective way for the removal of these compounds from the environment. The role of bacteria as well as the enzymes and genes that regulate the 2,4-D degradation has been widely studied, but the 2,4-D degradation by fungi, especially regarding the ability of white-rot basidiomycetes as agent for its bioconversion, has been not extensively considered. This review discusses the current knowledge about the biochemical mechanisms of 2,4-D biodegradation, focused on the role of white-rot fungi in this process. Finally, the cultivation conditions and medium composition for the growth of 2,4-D-degrading microorganisms are also addressed.

Comprehensive Comparative Analysis of Cholesterol Catabolic Genes/Proteins in Mycobacterial Species.

In dealing with Mycobacterium tuberculosis, the causative agent of the deadliest human disease-tuberculosis (TB)-utilization of cholesterol as a carbon source indicates the possibility of using cholesterol catabolic genes/proteins as novel drug targets. However, studies on cholesterol catabolism in mycobacterial species are scarce, and the number of mycobacterial species utilizing cholesterol as a carbon source is unknown. The availability of a large number of mycobacterial species' genomic data affords an opportunity to explore and predict mycobacterial species' ability to utilize cholesterol employing in silico methods. In this study, comprehensive comparative analysis of cholesterol catabolic genes/proteins in 93 mycobacterial species was achieved by deducing a comprehensive cholesterol catabolic pathway, developing a software tool for extracting homologous protein data and using protein structure and functional data. Based on the presence of cholesterol catabolic homologous proteins proven or predicted to be either essential or specifically required for the growth of M. tuberculosis H37Rv on cholesterol, we predict that among 93 mycobacterial species, 51 species will be able to utilize cholesterol as a carbon source. This study's predictions need further experimental validation and the results should be taken as a source of information on cholesterol catabolism and genes/proteins involved in this process among mycobacterial species.

Vertical response of microbial community and degrading genes to petroleum hydrocarbon contamination in saline alkaline soil.

A column microcosm was conducted by amending crude oil into Dagang Oilfield soil to simulate the bioremediation process. The dynamic change of microbial communities and metabolic genes in vertical depth soil from 0 to 80 cm were characterized to evaluate the petroleum degradation potential of indigenous microorganism. The influence of environmental variables on the microbial responds to petroleum contamination were analyzed. Degradation extent of 42.45% of n-alkanes (C8-C40) and 34.61% of 16ΣPAH were reached after 22 weeks. Relative abundance of alkB, nah, and phe gene showed about 10-fold increment in different depth of soil layers. Result of HTS profiles demonstrated that Pseudomonas, Marinobacter and Lactococcus were the major petroleum-degrading bacteria in 0-30 and 30-60 cm depth of soils. Fusarium and Aspergillus were the dominant oil-degrading fungi in the 0-60 cm depth of soils. In 60-80 cm deep soil, anaerobic bacteria such as Bacteroidetes, Lactococcus, and Alcanivorax played important roles in petroleum degradation. Redundancy analysis (RDA) and correlation analysis demonstrated that petroleum hydrocarbons (PHs) as well as soil salinity, clay content, and anaerobic conditions were the dominant effect factors on microbial community compositions in 0-30, 30-60, and 60-80 cm depth of soils, respectively.

Response of microbial community and catabolic genes to simulated petroleum hydrocarbon spills in soils/sediments from different geographic locations.

AIMS: Study the response of microbial communities and selected petroleum hydrocarbon (PH)-degrading genes on simulated PH spills in soils/sediments from different geographic locations. METHODS AND RESULTS: A microcosm experiment was conducted by spiking mixtures of petroleum hydrocarbons (PHs) to soils/sediments collected from four different regions of China, including the Dagang Oilfield (DG), Sand of Bohai Sea (SS), Northeast China (NE) and Xiamen (XM). Changes in bacterial community and the abundance of PH-degrading genes (alkB, nah and phe) were analysed by denaturing gradient electrophoresis (DGGE) and qPCR, respectively. Degradation of alkanes and PAHs in SS and NE materials were greater (P < 0·05) than those in DG and XM. Clay content was negatively correlated with the degradation of total alkanes by 112 days and PAHs by 56 days, while total organic carbon content was negatively correlated with initial degradation of total alkanes as well as PAHs. Abundances of alkB, nah and phe genes increased 10- to 100-fold and varied by soil type over the incubation period. DGGE fingerprints identified the dominance of α-, ß- and γ-Proteobacteria (Gram -ve) and Actinobacteria (Gram +ve) bacteria associated with degradation of PHs in the materials studied. CONCLUSION: The geographic divergence resulting from the heterogeneity of physicochemical properties of soils/sediments appeared to influence the abundance of metabolic genes and community structure of microbes capable of degrading PHs. SIGNIFICANCE AND IMPACT OF THE STUDY: When developing practical in-situ bioremediation approaches for PHs contamination of soils/sediment, appropriate microbial community structures and the abundance of PH-degrading genes appear to be influenced by geographic location.

Complete nucleotide sequence of the self-transmissible TOL plasmid pD2RT provides new insight into arrangement of toluene catabolic plasmids.

In the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pD2RT of Pseudomonas migulae strain D2RT isolated from Baltic Sea water. The pD2RT is 129,894 base pairs in size with an average G+C content of 53.75%. A total of 135 open reading frames (ORFs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. ORFs encoding proteins with putative functions in stress response, transposition and site-specific recombination were also predicted. Analysis of the organization and nucleotide sequence of pD2RT backbone region revealed high degree of similarity to the draft genome sequence data of the plant-pathogenic pseudomonad Pseudomonas syringae pv. glycinea strain B076, exhibiting relatedness to pPT23A plasmid family. The pD2RT backbone is also closely related to that of pGRT1 of Pseudomonas putida strain DOT-T1E and pBVIE04 of Burkholderia vietnamiensis strain G4, both plasmids are associated with resistance to toluene. The ability of pD2RT to self-transfer by conjugation to P. putida recipient strain PaW340 was experimentally determined. Genetic organization of toluene-degrading (xyl) genes and flanking DNA segments resembles the structure of Tn1721-related class II transposon Tn4656 of TOL plasmid pWW53 of P. putida strain MT53. The complete sequence of the plasmid pD2RT extends the known range of xyl genes carriers, being the first completely sequenced TOL plasmid, which is not related to well-studied IncP plasmid groups. We also verified the functionality of the catabolic route encoded by pD2RT by monitoring the expression of the xylE gene in pD2RT bearing hosts along with bacterial strains containing TOL plasmid of IncP-9 group. The growth kinetics of plasmid-bearing strains was found to be affected by particular TOL plasmid.

Bacterial enzymatic degradation of recalcitrant organic pollutants: catabolic pathways and genetic regulations.

Contamination of soil and natural water bodies driven by increased organic pollutants remains a universal concern. Naturally, organic pollutants contain carcinogenic and toxic properties threatening all known life forms. The conventional physical and chemical methods employed to remove these organic pollutants ironically produce toxic and non-ecofriendly end-products. Whereas microbial-based degradation of organic pollutants provides an edge, they are usually cost-effective and take an eco-friendly approach towards remediation. Bacterial species, including Pseudomonas, Comamonas, Burkholderia, and Xanthomonas, have the unique genetic makeup to metabolically degrade toxic pollutants, conferring their survival in toxic environments. Several catabolic genes, such as alkB, xylE, catA, and nahAc, that encode enzymes and allow bacteria to degrade organic pollutants have been identified, characterized, and even engineered for better efficacy. Aerobic and anaerobic processes are followed by bacteria to metabolize aliphatic saturated and unsaturated hydrocarbons such as alkanes, cycloalkanes, aldehydes, and ethers. Bacteria use a variety of degrading pathways, including catechol, protocatechuate, gentisate, benzoate, and biphenyl, to remove aromatic organic contaminants such as polychlorinated biphenyls, polycyclic aromatic hydrocarbons, and pesticides from the environment. A better understanding of the principle, mechanisms, and genetics would be beneficial for improving the metabolic efficacy of bacteria to such ends. With a focus on comprehending the mechanisms involved in various catabolic pathways and the genetics of the biotransformation of these xenobiotic compounds, the present review offers insight into the various sources and types of known organic pollutants and their toxic effects on health and the environment.

Update on new trend and progress of the mechanism of polycyclic aromatic hydrocarbon biodegradation by Rhodococcus, based on the new understanding of relevant theories: a review.

Rapid industrial and societal developments have led to substantial increases in the use and exploitation of petroleum, and petroleum hydrocarbon pollution has become a serious threat to human health and the environment. Polycyclic aromatic hydrocarbons (PAHs) are primary components of petroleum hydrocarbons. In recent years, microbial remediation of PAHs pollution has been regarded as the most promising and cost-effective treatment measure because of its low cost, robust efficacy, and lack of secondary pollution. Rhodococcus bacteria are regarded as one of main microorganisms that can effectively degrade PAHs because of their wide distribution, broad degradation spectrum, and network-like evolution of degradation gene clusters. In this review, we focus on the biological characteristics of Rhodococcus; current trends in PAHs degradation based on knowledge maps; and the cellular structural, biochemical, and enzymatic basis of degradation mechanisms, along with whole genome and transcriptional regulation. These research advances provide clues for the prospects of Rhodococcus-based applications in environmental protection.

Bacterial community dynamics and associated genes in hydrocarbon contaminated soil during bioremediation using brewery spent grain.

Brewery spent grain (BSG) has previously been exploited in bioremediation. However, detailed knowledge of the associated bacterial community dynamics and changes in relevant metabolites and genes over time is limited. This study investigated the bioremediation of diesel contaminated soil amended with BSG. We observed complete degradation of three total petroleum hydrocarbon (TPH C10-C28) fractions in amended treatments as compared to one fraction in the unamended, natural attenuation treatments. The biodegradation rate constant (k) was higher in amended treatments (0.1021k) than in unamended (0.059k), and bacterial colony forming units increased significantly in amended treatments. The degradation compounds observed fitted into the elucidated diesel degradation pathways and quantitative PCR results showed that the gene copy numbers of all three associated degradation genes, alkB, catA and xylE, were significantly higher in amended treatments. High-throughput sequencing of 16S rRNA gene amplicons showed that amendment with BSG enriched autochthonous hydrocarbon degraders. Also, community shifts of the genera Acinetobacter and Pseudomonas correlated with the abundance of catabolic genes and degradation compounds observed. This study showed that these two genera are present in BSG and thus may be associated with the enhanced biodegradation observed in amended treatments. The results suggest that the combined evaluation of TPH, microbiological, metabolite and genetic analysis provides a useful holistic approach to assessing bioremediation.

Elucidating the biodegradation pathway and catabolic genes of benzophenone-3 in Rhodococcus sp. S2-17.

A new bacterium, Rhodococcus sp. S2-17, which could completely degrade an emerging organic pollutant, benzophenone-3 (BP-3), was isolated from contaminated sediment through an enrichment procedure, and its BP-3 catabolic pathway and genes were identified through metabolic intermediate and transcriptomic analyses and biochemical and genetic studies. Metabolic intermediate analysis suggested that strain S2-17 may degrade BP-3 using a catabolic pathway progressing via the intermediates BP-1, 2,4,5-trihydroxy-benzophenone, 3-hydroxy-4-benzoyl-2,4-hexadienedioic acid, 4-benzoyl-3-oxoadipic acid, 3-oxoadipic acid, and benzoic acid. A putative BP-3 catabolic gene cluster including cytochrome P450, flavin-dependent oxidoreductase, hydroxyquinol 1,2-dioxygenase, maleylacetate reductase, and α/ß hydrolase genes was identified through genomic and transcriptomic analyses. Genes encoding the cytochrome P450 complex that demethylates BP-3 to BP-1 were functionally verified through protein expression, and the functions of the other genes were also verified through knockout mutant construction and intermediate analysis. This study suggested that strain S2-17 might have acquired the ability to catabolize BP-3 by recruiting the cytochrome P450 complex and α/ß hydrolase, which hydrolyzes 4-benzoyl-3-oxoadipic acid to benzoic acid and 3-oxoadipic acid, genes, providing insights into the recruitment of genes of for the catabolism of emerging organic pollutants.

Biodegradation Potential and Putative Catabolic Genes of Culturable Bacteria from an Alpine Deciduous Forest Site.

Microbiota from Alpine forest soils are key players in carbon cycling, which can be greatly affected by climate change. The aim of this study was to evaluate the degradation potential of culturable bacterial strains isolated from an alpine deciduous forest site. Fifty-five strains were studied with regard to their phylogenetic position, growth temperature range and degradation potential for organic compounds (microtiter scale screening for lignin sulfonic acid, catechol, phenol, bisphenol A) at low (5 °C) and moderate (20 °C) temperature. Additionally, the presence of putative catabolic genes (catechol-1,2-dioxygenase, multicomponent phenol hydroxylase, protocatechuate-3,4-dioxygenase) involved in the degradation of these organic compounds was determined through PCR. The results show the importance of the Proteobacteria phylum as its representatives did show good capabilities for biodegradation and good growth at -5 °C. Overall, 82% of strains were able to use at least one of the tested organic compounds as their sole carbon source. The presence of putative catabolic genes could be shown over a broad range of strains and in relation to their degradation abilities. Subsequently performed gene sequencing indicated horizontal gene transfer for catechol-1,2-dioxygenase and protocatechuate-3,4-dioxygenase. The results show the great benefit of combining molecular and culture-based techniques.

Reappraisal of the optimal fasting time for insulin tolerance tests in mice.

OBJECTIVE: Most studies routinely use overnight or 6 h of fasting before testing metabolic glucose homeostasis in mice. Other studies used empirically shorter fasting times (<6 h). We attempted to determine the shortest fasting time required for optimal insulin responsiveness while minimizing metabolic stress. METHODS: A course of fasting for up to 24 h (0, 2, 4, 6, 12, and 24 h) was conducted in C57Bl/6J male mice. Body weight, metabolic parameters, and insulin tolerance were measured in each experimental group. The organs were collected at the same time on separate occasions and glycogen and metabolic gene expression were measured in the liver and skeletal muscle. RESULTS: Our data show that blood glucose levels do not significantly change during a 6 h fast, while plasma insulin levels decrease to similar levels between 2 h and 6 h of fasting. During overnight (12 h) and 24 h fasts, a robust decrease in blood glucose and plasma insulin was observed along with a profound depletion in liver glycogen content. Insulin tolerance was comparable between baseline and 6 h fasts while 4 h and 6 h fasts were associated with a greater depletion of liver glycogen than 2 h fasts, impacting the glucose counter-regulatory response. Fasting induced progressive weight loss that was attenuated at thermoneutrality. Fasting longer than 4 h induced major body weight loss (>5%) and significant changes in catabolic gene expression in the liver and skeletal muscle. CONCLUSION: Collectively, these data suggest that 2 h of fasting appears optimal for the assessment of insulin tolerance in mice as this duration minimizes major metabolic stress and weight loss.

Advances in monitoring of catabolic genes during bioremediation.

Biodegradation of xenobiotic compounds by microbes is exploited in the clean up of contaminated environments by bioremediation. Catabolic (or functional) genes encode for specific enzymes in catabolic pathways such as key enzymes in xenobiotic degradation pathways. By assessing the abundance or the expression of key genes in environmental samples one can get a potential measure of the degradation activity. One way of assessing the abundance and expression of specific catabolic genes is by analyzing the metagenomic DNA and RNA from environmental samples. Three major challenges in the detection and quantification of catabolic genes in bioremediation studies are 1) the accurate and sensitive quantification from environmental samples 2) the coverage of the enzymatic potential by the targeted genes 3) the validation of the correlation with actual observed degradation activities in field cases. New advances in realtime PCR, functional gene arrays and meta-transcriptomics have improved the applicability of catabolic gene assessment during bioremediation.

Seasonal changes in the abundance of bacterial genes related to dimethylsulfoniopropionate catabolism in seawater from Ofunato Bay revealed by metagenomic analysis.

Ofunato Bay is located in the northeastern Pacific Ocean area of Japan, and it has the highest biodiversity of marine organisms in the world, primarily due to tidal influences from the cold Oyashio and warm Kuroshio Currents. Our previous results from performing shotgun metagenomics indicated that Candidatus Pelagibacter ubique and Planktomarina temperata were the dominant bacteria (Reza et al., 2018a, 2018b). These bacteria are reportedly able to catabolize dimethylsulfoniopropionate (DMSP) produced from phytoplankton into dimethyl sulfide (DMS) or methanethiol (MeSH). This study was focused on seasonal changes in the abundances of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater of Ofunato Bay by BLAST+ analysis using shotgun metagenomic datasets. We found seasonal changes among the Candidatus Pelagibacter ubique strains, including those of the HTCC1062 type and the Red Sea type. A good correlation was observed between the chlorophyll a concentrations and the abundances of the catabolic genes, suggesting that the bacteria directly interact with phytoplankton in the marine material cycle system and play important roles in producing DMS and MeSH from DMSP as signaling molecules for the possible formation of the scent of the tidewater or as fish attractants.

Molecular perspectives and recent advances in microbial remediation of persistent organic pollutants.

Nutrition and pollution stress stimulate genetic adaptation in microorganisms and assist in evolution of diverse metabolic pathways for their survival on several complex organic compounds. Persistent organic pollutants (POPs) are highly lipophilic in nature and cause adverse effects to the environment and human health by biomagnification through the food chain. Diverse microorganisms, harboring numerous plasmids and catabolic genes, acclimatize to these environmentally unfavorable conditions by gene duplication, mutational drift, hypermutation, and recombination. Genetic aspects of some major POP catabolic genes such as biphenyl dioxygenase (bph), DDT 2,3-dioxygenase, and angular dioxygenase assist in degradation of biphenyl, organochlorine pesticides, and dioxins/furans, respectively. Microbial metagenome constitutes the largest genetic reservoir with miscellaneous enzymatic activities implicated in degradation. To tap the metabolic potential of microorganisms, recent techniques like sequence and function-based screening and substrate-induced gene expression are proficient in tracing out novel catabolic genes from the entire metagenome for utilization in enhanced biodegradation. The major endeavor of today's scientific world is to characterize the exact genetic mechanisms of microbes for bioremediation of these toxic compounds by excavating into the uncultured plethora. This review entails the effect of POPs on the environment and involvement of microbial catabolic genes for their removal with the advanced techniques of bioremediation.