Enrichment, Isolation and phylogenetic identification of fluoranthene and nonylphenol-degrading bacteria

Published ThesisPolycyclic aromatic hydrocarbons (PAHs) and Endocrine disrupting chemicals (EDCs) are man-made chemicals that cause cancer and alter the function of endocrine systems in both humans and wildlife, respectively. PAHs and EDCs are considered as one of the priority pollutants and world-wide research is on-going to develop bioremediation strategies to remove these toxic xenobiotics from environment. Understanding indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on PAHs and EDCs has been generated from developed regions. In this direction, recent studies revealed presence of different PAHs and EDCs in South African natural resources. However, to date, study on analysis of microorganisms capability to utilize/degrade EDCs has not been reported and studies on PAHs are scares from South Africa. Soil samples collected at the different coal-fired power stations in and around Mpumalanga province, South Africa was used for enriching microorganisms. Enrichment method employed for isolating fluoranthene (as a model compound for PAHs) or nonylphenol (as a model compound for EDCs) degrading microorganisms. Identification of microorganisms was carried out using 16S rRNA gene analysis. Phylogenetic analysis of isolates was carried out using MEGA5. For each substrate, six pure and distinct bacterial cultures were successfully enriched. Pseudomonas dominated the strains enriched on nonylphenol, with 5 of the 6 isolates belonging to this genus. All four of these isolates however belong to different species. Highest diversity observed when fluoranthene was used as a carbon source. Strains of Pseudomonas, Stenotrophomonas, Cupravidus and Ochrobactrum were isolated using fluoranthene as a carbon source. Study results are the beginning of identification of microorganisms capable of degrading carcinogenic and endocrine disruptors and pave the way for exploring PAHs and EDCs degrading microorganisms from South Africa. An article on EDCs utilization organisms and their capability to degrade nonylphenol is submitted to South African Journal of Sciences. Here the details: Qhanya LB et al. (2016) Isolation and characterization of endocrine disruptor nonylphenol-using bacteria from South Africa. SAJS-2016-0287 (under review). Apart from my Masters study, I also supervised four B. Tech student projects and managed to publish an article with students. Furthermore, I also worked on few bioinformatics projects and earned co-authorship in two manuscripts listed below: Parvez M, Qhanya LB, Mthakathi NT, Kgosiemang IKR, Bamal HD, Pagadala NS, Xie T, Yang H, Chen H, Theron CW, Monyaki R, Raselemane SC, Salewe V, Mongale BL, Matowane RG, Abdalla SMH, Booi WI, van Wyk M, Olivier D, Boucher CE, Nelson DR, Tuszynski JA, Blackburn JM, Yu J-H, Mashele SS, Chen W, Syed K. (2016) Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s. Scientific Reports | 6:33099 | DOI: 10.1038/srep33099. Qhanya LB, Matowane G, Chen W, Sun Y, Letsimo EM, Parvez M, Yu J-H, Mashele SS, Syed K. (2015) Genome-wide annotation and comparative analysis of cytochrome P450 monooxygenases in basidiomycete biotrophic plant pathogens. PLoS ONE 10(11): e0142100. doi:10.1371/journal.pone.0142100. Sello MM, Jafta N, Nelson DR, Chen W, Yu J-H, Parvez M, Kgosiemang IKR, Monyaki R, Raselemane SC, Qhanya LB, Mthakathi NT, Mashele SS, Syed K. (2015) Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes. Scientific Reports 07/2015; 5. DOI:10.1038/srep11572 · (Discovered novel P450 fusion protein). In addition to the above credits, I was featured on national TV and in newspapers for discovering a novel drug target. I also presented work at both national and international (Canada) conferences

Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s

Published ArticleSince the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria -42; fungi -19; plant -28; animal -22; plant and animal -1 and common P450 family -1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study's results offer new understanding of the dynamic structural nature of P450s

Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

Published ArticleEndocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41–46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa

Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

Published ArticleCytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes

Fungal Genomes and Insights into the Evolution of the Kingdom

The kingdom Fungi comprises species that inhabit nearly all ecosystems. Fungi exist as both free-living and symbiotic unicellular and multicellular organisms with diverse morphologies. The genomes of fungi encode genes that enable them to thrive in diverse environments, invade plant and animal cells, and participate in nutrient cycling in terrestrial and aquatic ecosystems. The continuously expanding databases of fungal genome sequences have been generated by individual and large-scale efforts such as Génolevures, Broad Institute's Fungal Genome Initiative, and the 1000 Fungal Genomes Project (http://1000.fungalgenomes.org). These efforts have produced a catalog of fungal genes and genomic organization. The genomic datasets can be utilized to better understand how fungi have adapted to their lifestyles and ecological niches. Large datasets of fungal genomic and transcriptomic data have enabled the use of novel methodologies and improved the study of fungal evolution from a molecular sequence perspective. Combined with microscopes, petri dishes, and woodland forays, genome sequencing supports bioinformatics and comparative genomics approaches as important tools in the study of the biology and evolution of fungi

Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

Endocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41–46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa. Significance:  • First report of endocrine disruptor nonylphenol-using bacteria from South Africa • Six bacterial species capable of using nonylphenol as a carbon source were isolated • Results will pave the way for further exploration of endocrine disruptors degrading microbes from South Afric

Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

Endocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41–46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa. Significance:  First report of endocrine disruptor nonylphenol-using bacteria from South Africa Six bacterial species capable of using nonylphenol as a carbon source were isolated Results will pave the way for further exploration of endocrine disruptors degrading microbes from South Afric

Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

<div><p>Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (<i>Armillaria mellea</i>), Pucciniomycotina (<i>Melampsora laricis-populina</i>, <i>M</i>. <i>lini</i>, <i>Mixia osmundae</i> and <i>Puccinia graminis</i>) and Ustilaginomycotina (<i>Ustilago maydis</i>, <i>Sporisorium reilianum</i> and <i>Tilletiaria anomala</i>), revealed the presence of numerous putative P450s ranging from 267 (<i>A</i>. <i>mellea)</i> to 14 (<i>M</i>. <i>osmundae</i>). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in <i>A</i>. <i>mellea</i> when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in <i>P</i>. <i>graminis</i> and <i>M</i>. <i>laricis-populina</i>. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host/ecological niche can influence shaping the P450 content of an organism. The present study initiates our understanding of P450 family patterns in basidiomycete biotrophic plant pathogens.</p></div

Clade level classification of P450 families.

<p>Clade level classification of P450 families.</p

Comparative analysis of member P450s between biotrophic and non-biotrophic basidiomycetes belonging to Pucciniomycotina (A) and Ustilaginomycotina (B).

<p>The number in parenthesis indicates P450 family numbers. The number in parenthesis next to each species indicates the total P450 count in the particular species.</p