Study of enhancement and inhibition phenomena and genes relating to degradation of petroleum polycyclic aromatic hydrocarbons in isolated bacteria

Polycyclic aromatic hydrocarbons (PAHs) are xenobiotic compounds, which being degraded by chemical, physical or biological methods. The latter is the safest and the cheapest one. Two bacterial strains ASU-01 and ASU-016 were isolated from different Egyptian petroleum contaminated sites. They were genetically identified based on the analysis of the nucleotide sequences of the 16S ribosomal PNA gene and the phylogenetic tree as Enterobacter hormaechei and Pseudomonas pseudoalcaligenes respectively. When pyrene as high molecular weight (HMW)-PAH was added as a sole carbon source, both strains could degrade it with efficiency 77.7 and 83.7% within 15 days of incubation, respectively. However, when it was mixed with low molecular weight (LMW)-PAHs, two opposite phenomena appeared. The first one was enhancement, which occurred with ASU-01. This strain shifted pyrene efficiency to 98.5%. The second phenomenon was inhibition occurred with ASU-016 which completely retarded pyrene degradation. Naphthalene dioxygenase (nahAc), and catechol dioxygenases (C12O and C23O) genes were detected in the two strains based on PCR. The detected genes were confirmed by determining the different specific activities of their translated protein (enzymes) on different PAHs. The maximum values of biosurfacatant production activity and cell-surface and percentage of cell-surface hydrophobicity (CSH) were detected during the exponential phase. These latter factors increased the bioavailability and consequently, the assimilation of PAHs.     

Heavy metal ions affecting the removal of polycyclic aromatic hydrocarbons by fungi with heavy-metal resistance

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) is very common in contaminated environments. It is of paramount importance and great challenge to exploit a bioremediation to remove PAHs in these environments with combined pollution. We approached this question by probing the influence of HMs coexisting with PAHs on the removal of PAHs by Acremonium sp. P0997 possessing metal resistance. A removal capability for naphthalene, fluorene, phenanthrene, anthracene, and fluoranthenepresentalone (98.6, 99.3, 89.9, 60.4, and 70 %, respectively) and in a mixture (96.9, 71.8, 67.0, 85.0, and 87.9 %, respectively) was achieved in mineral culture inoculated with Acremonium sp. P0997, and this strain also displayed high resistance to the individual HMs (Mn²⁺, Fe²⁺, Zn²⁺, Cu²⁺, Al³⁺, and Pb²⁺). The removal of individual PAHs existing in a mixture was differently affected by the separately tested HMs. Cu²⁺enhanced the partition process of anthracene to dead or alive mycelia and the contribution of the biosorption by this strain but imposed a little negative influence on the contribution of biodegradation to the total removal of anthracene individually in a culture. However, Mn²⁺ had an inhibitory effect on the partition process of anthracene to dead or alive mycelia and decreased the contributions of both biosorption and biodegradation to the total anthracene removal. This work showcased the value of fungi in bioremediation for the environments with combined pollution, and the findings have major implications for the bioremediation of organic pollutants in metal-organic mixed contaminated sites.     

Anaerobic biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by a facultative anaerobe Pseudomonas sp. JP1

Polycyclic aromatic hydrocarbons (PAHs) are harmful persistent organic pollutants, while the high-molecular-weight (HMW) PAHs are even more detrimental to the environment and human health. However, microbial anaerobic degradation of HMW PAHs has rarely been reported. One facultative anaerobe Pseudomonas sp. JP1 was isolated from Shantou Bay, Shantou, China, which could degrade a variety of HMW PAHs. After 40 days cultivation with strain JP1, anaerobic biodegradation rate of benzo[a]pyrene (BaP), fluoranthene, and phenanthrene was 30, 47, and 5 %, respectively. Consumption of nitrate as the electron acceptor was confirmed by N-(1-naphthyl) ethylenediamine spectrophotometry. Supplementation of sodium sulfite, maltose, or glycine, and in a salinity of 0–20 ‰ significantly stimulated anaerobic degradation of BaP. Lastly, the anaerobic degradation metabolites of BaP by strain JP1 were investigated using GC/MS, and the degradation pathway was proposed. This study is helpful for further studies on the mechanism of anaerobic biodegradation of PAHs.     

Comparison of the substrate specificity of two ring-hydroxylating dioxygenases from Sphingomonas sp. VKM B-2434 to polycyclic aromatic hydrocarbons

The genes of two ring-hydroxylating dioxygenases (RHDs) of Sphingomonas sp. VKM B-2434 were cloned and expressed in Escherichia coli. The relative values of the RHD specificity constants were estimated for six polycyclic aromatic hydrocarbons (PAHs) based on the kinetics of PAH mixture conversion by the recombinant strains. The substrate specificity profiles of the enzymes were found to be very different. Dioxygenase ArhA was the most specific to acenaphthylene and showed a low specificity to fluoranthene. Dioxygenase PhnA was the most specific to anthracene and phenanthrene and showed a considerable specificity to fluoranthene. Knockout derivatives of Sphingomonas sp. VKM B-2434 lacking ArhA, PhnA, and both dioxygenases were constructed. PAH degradation by the single-knockout mutants was in agreement with the substrate specificity of the RHD remaining intact. Double-knockout mutant lacking both enzymes was unable to oxidize PAHs. A mutant form of dioxygenase ArhA with altered substrate specificity was described.     

Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil

Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p < 0.001) increases in the abundance of the GP PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial community in soil. The current study provides information on the response of soil bacterial, archaeal and fungal communities during the degradation of three priority pollutants and contributes to a knowledge base that can inform the development of effective bioremediation strategies for contaminated sites.     

Application of response surface methodology for rapid chrysene biodegradation by newly isolated marine-derived fungus Cochliobolus lunatus strain CHR4D

For the first time, Cochliobolus lunatus strain CHR4D, a marine-derived ascomycete fungus isolated from historically contaminated crude oil polluted shoreline of Alang-Sosiya ship-breaking yard, at Bhavnagar coast, Gujarat has been reported showing the rapid and enhanced biodegradation of chrysene, a four ringed high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH). Mineral Salt Broth (MSB) components such as ammonium tartrate and glucose along with chrysene, pH and trace metal solution have been successfully optimized by Response Surface Methodology (RSM) using central composite design (CCD). A validated, two-step optimization protocol has yielded a substantial 93.10% chrysene degradation on the 4^th day, against unoptimized 56.37% degradation on the 14^th day. The results depict 1.65 fold increase in chrysene degradation and 1.40 fold increase in biomass with a considerable decrement in time. Based on the successful laboratory experiments, C. lunatus strain CHR4D can thus be predicted as a potential candidate for mycoremediation of HMW PAHs impacted environments.     

Assessment of microbial communities associated with fermentative–methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20)

A controlled field experiment was conducted to assess the potential for fermentative–methanogenic biostimulation (by ammonium-acetate injection) to enhance biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) as well as polycyclic aromatic hydrocarbons (PAHs) in groundwater contaminated with biodiesel B20 (20:80 v/v soybean biodiesel and diesel). Changes in microbial community structure were assessed by pyrosequencing 16S rRNA analyses. BTEX and PAH removal began 0.7 year following the release, concomitantly with the increase in the relative abundance of Desulfitobacterium and Geobacter spp. (from 5 to 52.7 % and 15.8 to 37.3 % of total Bacteria 16S rRNA, respectively), which are known to anaerobically degrade hydrocarbons. The accumulation of anaerobic metabolites acetate and hydrogen that could hinder the thermodynamic feasibility of BTEX and PAH biotransformations under fermentative/methanogenic conditions was apparently alleviated by the growing predominance of Methanosarcina. This suggests the importance of microbial population shifts that enrich microorganisms capable of interacting syntrophically to enhance the feasibility of fermentative–methanogenic bioremediation of biodiesel blend releases.     

Naphthalene oxygenase fromCorynebacterium renale: Characterisation and mechanism of oxygenation

The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe²⁺ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H₂O₂ was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H₂O₂ was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction     

Mutations in microRNA Binding Sites of CEP Genes Involved in Cancer

The CEP genes play a pivotal role in the replication of the cell. CEP family proteins form the major constituents of the centrosome and play a prominent role in centriole biogenesis and in cell replication. Alteration in CEP genes will result in disruption of cell cycle that may in turn cause cancer. In our study, we found that 16 of the CEP genes are a potential target to miRNA that binds to complementary sequences in 3′untranslated regions (UTR) of mRNA and stop them from translation. Single nucleotide polymorphisms (SNPs) occurring naturally in such miRNA binding site can alter the miRNA: mRNA interaction and can significantly alter gene expression. We developed a systematic computational pipeline that integrates data from well-established databases, followed stringent selection criteria and identified a panel of 44 high-confidence SNPs that may impair miRNA target sites in the 3′UTR of 16 genes. Further we performed expression analysis to shed light on the potential tissues that might be affected by mutation, enrichment analysis to find the metabolic functions of the gene, and network analysis to highlight the important interactions of CEP genes with other genes to provide insight that complex network will be disturbed upon mutation. In this study, we explored and prioritised the SNPs in CEP gene which could act as a potential target in centrosome-associated human disease. Our analysis would provide a thoughtful insight to wet lab researches to understand the expression pattern of CEP genes and binding phenomenon of mRNA and miRNA upon mutation, which is responsible for inhibition of translation process at genomic levels.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

cDNA library construction from meristematic tissue of finger millet panicle

A protocol to obtain full-length cDNA using a SuperScript® Full-Length cDNA Library Construction Kit II (Invitrogen, United States) was developed, and a high quality cDNA library of meristem tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was constructed. The titer of the constructed cDNA library was 3.01 × 10⁵ CFU/mL, the average length of the insert was approximately 1070 base pairs, and the average efficiency of insertion of cDNA fragments was 99.5%. The sequencing of randomly selected clones created cDNA library was carried out. The cDNA sequences of clones were identified by BLAST search. The cDNA library analysis and selective sequencing indicate good functionality and full size of cDNA inserts of the clones. The constructed cDNA library from meristematic tissue of finger millet panicle is a good and reliable source for isolation and identification of key genes of metabolism and development of meristem as well for creation of new genetic markers for genetic research and molecular selection.     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

Immunotoxic effect of Benzo[α]Pyrene and chrysene in juvenile white shrimp Litopenaeus vannamei

The effects of benzo(a)pyrene (Bap) (0.03, 0.3 and 3 μg L^?1) and chrysene (CHR) (0.3, 2.1 and 14.7 μg L^?1) on the function of the immune system of juvenile white shrimp Litopenaeus vannamei were determined under laboratory conditions. This included the total hemocyte count (THC) in the hemolymph, phagocytic activityand pro-phenoloxidase (pro-PO) activity of the hemocyte, phenoloxidase (PO) activity, α₂-macroglobulin (α₂-M) activity, bacteriolytic activity and antibacterial activity in the hemolymph. The results showed that BaP and CHR could inhibit the immune function of L. vannamei significantly under high concentration BaP and CHR exposure. The results of this study indicated that the immunotoxicity of PAHs in a descending order was BaP>CHR. Moreover, the results indicated the THC in hemolymph, pro-PO activity and phagocytic activity of hemocyte, and bacteriolytic activity in hemolymphcould be used as potentially suitable biomarkersfor early warning indication of PAHs toxicity, this could provide useful information for toxic risk assessment of environmental pollutants.     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.