Isolation and molecular characterization of a fluorous-degrading bacterium, pseudomonas spp 1-1
Student thesis: Doctoral Thesis
In recent years, the industrial use of poly- and per-fluorinated compounds (PFCs) has rapidly expanded, and there have been serious concerns worldwide over the levels and adverse impact of PFCs in the environment. The recalcitrance of these contaminants has resulted in their accumulation and persistence in the environment and biota. Studies on PFCs have also demonstrated the diverse toxicities of PFCs to human health. Despite the increasing interest on microbial degradation of fluoroorganic compounds for bioremediation purposes, the degradation of fluorous (perfluorinated) compounds has so far received little attention. To date, only a few species of microbes have been shown to possess the ability to degrade polyfluorinated compounds. In this project, water and/or soil samples were collected from eight different sites in Hong Kong and mainland China that are suspected to be contaminated with various forms of fluorinated hydrocarbons. Selective enrichment for fluorous-degrading bacteria was performed under both aerobic and anaerobic conditions in nutrient medium or mineral salts medium using perfluoro-tert-butyl alcohol (PFTB) as substrate. Preliminary screens on the above water samples showed that fluoride release was observed only in bacterial mixtures harvested from the Causeway Bay typhoon shelter (in Hong Kong) under aerobic conditions using the ISE combination fluoride method. Sequencing of the16S rDNA and phylogenetic analysis of a purified PFTB-tolerant bacterial strain from the Causeway Bay waters indicated that it shares 99.86% sequence similarity with Pseudomonas spp HMPB1 and is designated here as Pseudomonas spp 1-1 (Ps. spp 1-1). Fluoride released from different PFCAs by Pseudomonas spp 1-1 was measured under both aerobic and anaerobic conditions. It was observed that only PFPeA (C5) and PFHpA (C7) were biodefluorinated by cell extracts and/or whole cells of Pseudomonas spp 1-1 cultured under aerobic conditions in rich NM medium. Defluorination assays were performed using cell extracts and a bio-defluorination activity was detected and demonstrated to catalyze the release of 1.03 μmol fluoride per hour/mg protein from 5 mM perfluoropentanoate acid (PFPeA, C5). The degradation of various PFCAs (of chain lengths that range from 8 to 13) by Pseudomonas spp 1-1 was also analyzed by means of the LC/MS/MS technique. Following a 90-day incubation of live cells of Pseudomonas spp 1-1with 10 μM perfluorononanoic acid (PFNA, C9), a 21% reduction in PFNA concentration was observed. However, no appreciable change in PFNA concentration was detected when PFNA was incubated with dead cells (autoclaved at 121 °C for 20 min) of Pseudomonas spp 1-1 or live E.coli DH5α cells at the same cell density. When 8 μM of perfluoroundecanoic acid (PFUnA, C11) was incubated with live cells of Pseudomonas spp 1-1 for a period of 90 days, LC/MS/MS analysis showed a 41% reduction in PFUnA concentration, but no appreciable reduction in PFUnA concentration was detected when PFUnA was incubated with either NM medium alone or killed cells of Pseudomonas spp 1-1 (controls). Interestingly, in addition to the 41% reduction in PFUnA (C11) concentration, an equivalent amount of PFNA (C9) was also detected in the "PFUnA + live Pseudomonas spp" mixture. The results strongly indicated the bio-defluorination of PFUnA (C11) to PFNA (C9) by live cells of Pseudomonas spp 1-1 although no other smaller molecular weight fluorous metabolites were detected by LC/MS/MS analysis. Our findings are consistent with the notion that Pseudomonas spp 1-1 has the capacity to catalyze the transformation of long chain PFCAs (e.g. C11) to shorter chain PFCAs (e.g. C9). Whole genome sequencing of Pseudomonas spp 1-1 was carried out using Ion-Torrent PGM sequencing technology. The loading density of the PGM 316 Chip was about 83% and the final library read was ~56% which was sufficient for high efficiency genome sequencing. The quality metrics of the test fragments, TF-Aand TF_D, showed high reading quality of the sequence. About 163 contigs of genomic sequence was generated using Ion-Torrent sequencing technology, which were assembled usingPseudomonas fluorescens Pf-5(NC_004129) as the reference genome because it shares the highest sequence identity with Pseudomonas spp 1-1 based on genome blast analysis. Overall, the genome size of Pseudomonas spp 1-1 was estimated to be 5.627 X 106 bp and the GC content ~61.93%. Genome phylogenetic analysis showed that Pseudomonas spp 1-1 is very closely related to various member species/strains in the genus of Pseudomonas. The assembled genome sequence was annotated using the RAST Server (Rapid Annotations using Subsystema Technology). The Pseudomonas spp 1-1 genome contains 6877 protein-coding genes and 52 RNA genes, and 51% of the genes were distributed to 541 functional subsystems which were used to reconstruct the metabolic network. Based on KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis using the SEED viewer online software, the genes of three putative metabolic pathways for halogenated hydrocarbons were identified in Pseudomonas spp 1-1 and they represented the pathways for 1,2-dichloroethane, 1,3-dichloropropene and 3-fluorobenzoate degradation. Based on bioinformatics analyses with different software programs, a total of ten different dehalogenase-like genes were identified in the Pseudomonas spp 1-1 genome. Five of the genes specify for dehalogenase-like proteins of the α/β hydrolase superfamily and the other five code for haloacid dehalogenase (HAD) proteins. Sequence alignment and prediction of the 3D structure of the five α/β hydrolase proteins showed that protein-291, protein-1704, protein-6588, and protein-5328 contain all of the conserved and functionally-important amino acid residues in the catalytic active site (in the Asp-His-Asp/Glu catalytic triad) and halide-binding site. However, protein-972 lacks the halide-binding amino acid residue which suggests this protein may not be related to defluorination function. Sequence alignment and 3D structure prediction analysis of the five haloacid dehalogenase-like proteins encoded by gene-3488, -4565, -6388, -5099 and -2266 showed that protein-3488, protein-5099 and protein-2266 contain all of the functionally-conserved and key catalytic amino acid residues such as Asp in the N-terminus (for nucleophilic attack), another Asp (for stabilization of the rotation of nucleophile) and Lys (stabilization of the substrate). However, protein-4565 lacks the Asp residue that is associated with stabilization of the rotation of nucleophile and protein-6388 lacks the Asp residue in the active site for nucleophilic attack. Whether these differences render the two proteins other functions not related to defluorination would require further studies. Expression levels of the ten dehalogenase-like genes were measured by quantitative real-time PCR (qRT-PCR) on total RNA extracted from Pseudomonas spp 1-1 cultured in nutrient medium in the presence and absence of PFNA. Expression of all ten dehalogenase-like genes were upregulated, albeit at varying levels, in nutrient medium containing 5 mM PFNA as compared to the control, which suggest that all ten genes are induced by PFNA. It is proposed that global transcriptomic analysis will provide the paradigm for mapping and annotating the primary transcriptomes that may be associated with degradation of PFCAs in Pseudomonas spp 1-1.
- Biodegradation, Pseudomonas, Fluorine compounds, Metabolism