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Title: Physiological and genetic studies of 2,4-dichlorophenoxyacetate dissimilation by Burkholderia cepacia strain 2a
Author: Lin, Qiaoyi
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2012
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A chemo-heterotrophic bacterium Burkholderia cepacia strain 2a was isolated from garden soil in Wales by Dr. A. R. W. Smith (personal communication), and it has been the subject for the biochemical and genetic studies on 2,4- dichlorophenoxyacetate (2,4-D) metabolism. The genes involved in 2,4-D metabolism were located on the plasmid pIJB1 in a composite transposon Tn5530. This plasmid was sequenced in this project for the evolutionary study of this group of plasmid. The in-silico analysis of the 99,001 bp DNA sequence of pIJB1 revealed 93 open reading frames. Over 52 % of the sequence was comprised of well conserved IncPI backbone genes; the rest of the plasmid was composed of accessory genes for 2,4-D and malonate degradation on the Tn5530 transposon, and for mercury resistance on a Tn5058-like transposon. Phylogenetic analysis against twenty-two IncP-I plasmids revealed that pIJBI has the highest evolutionary relationship with the IncPI-1) plasmids pAKD4 (from Pseudomonas putida UWCI), and pEST40II (from Achromobacter xylosoxidans ssp. denitrificans EST4002). The loss of the 2,4-D metabolising phenotype during growth in non-selective (succinate-minimal salts) medium was confirmed to be due to homologous recombination of two identical ISI071 ::IS1471 elements flanking the Tn5530 transposon. Loss of the ability of strain 2a to utilise 2,4-D was followed through seven sequential subcultures in succinateMS liquid medium at different phases of growth. A cumulative 9 % of the viable cells in the seventh sub-culture was observed to have lost the 2,4-D phenotype. The functionality of the mer resistance gene operon on pIJB 1 was investigated by observing the level of inhibition different mercury compounds have on the growth of strain 2a on 4 mM succinate-MSM agar. Preliminary observation revealed that strain 2a possesses broad-spectrum resistance to both organic and inorganic mercury compounds. Strain 2a was tested for its ability to utilise a panel of the chlorinated phenoxyalkanoates to elucidate the specificity of the 2,4-D degradation pathway. Strain 2a was unable to grow on a range of substituted phenoxyalkanoates. Preliminary evidence showed that the organism cleaved the ether linkage in several of these compounds to produce the corresponding phenols, and strain 2a was observed to utilise glyoxylate, the other product of the ether-cleavage reaction; but was unable to utilise most of the phenols, due to the narrower substrate specificity of the second enzyme of the 2,4-D pathway, 2,4-dichlorophenol monooxygenase. Succinate exerted carbon catabolite repression on 2,4-D metabolism when both carbon substrates were presented in minimal salts medium (MSM) to strain 2a together. In this medium, the duration of the lag period between succinate exhaustion and the onset of 2,4-D metabolism was influenced by the previous history of the inoculum, grown in 2,4-D MSM, provided for the culture. Cells taken at mid-log phase for inoculation reproducibly produced longer lag periods (5 h) than those taken at early or late stationary phase (3 h and 3.7 h respectively). This was attributed to the different levels of poly-β-hydroxybutyrate (Hill) accumulated during growth in succinate; the greater the accumulation inside cells, the longer the lag was sustained. However, the causal link between PHB accumulation during succinate metabolism in diauxic growth and the state of the cells taken from different growth phases in 2,4-D-MSM for inoculation was unclear. The constancy of ATP levels around the period of the inter-growth lag phase suggested that PHB utilisation may have sustained cellular energy demands and thereby delayed the onset of2,4-D utilisation. The presence of the tfdK gene within the 2,4-D dissimilatory operon on pIJBl prompted the investigation into involvement of the encoded product in 2,4-D metabolism in strain 2a In-silico comparison of the tfdK amino acid sequence with its analogue in Achromobacter xylosoxidans ssp denitrificans strain EST4002 (pEST40II) and in Cupriavidus necator strain JMP134 (pJP4) revealed 100 % and 76 % identity respectively. The chemotactic role of tfdK was implicated by testing for swarming on soft-agar containing 2,4-D in JMP134 and EST4002, but little sign of swarming was observed with strain 2a The tfdK gene expression level in the three organisms, determined using qPCR, revealed that this phenotypic difference was possibly due to a diminished level of expression of the gene in stain 2a compared with the levels in the other two strains.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available