RpoN-dependent adaptation of burkholderia xenovorans LB400 for biodegradation and bioremediation of dibenzofuran / Noor Faizul Hadry Nordin
Alternative sigma subunit-54 (RpoN) forms holoenzyme complex when associated with core RNA Polymerase (RNAP) to specifically recognise and initiate transcription of specific sets of genes in response to environmental stimuli. RpoN has important role in many major adaptive responses in bacteria...
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| Summary: | Alternative sigma subunit-54 (RpoN) forms holoenzyme complex when associated with core
RNA Polymerase (RNAP) to specifically recognise and initiate transcription of specific sets of
genes in response to environmental stimuli. RpoN has important role in many major adaptive
responses in bacteria and is involved in various physiological responses such as pathogenesis,
quorum sensing and bioremediation. The main focus of this study is to gain insight into the role
of alternative sigma factor-54 (RpoN) of Burkholderia xenovorans LB400 in degradation of
dibenzofuran via biphenyl degradation pathway. Additionally, this study also investigated the
ability of Burkholderia cenocepacia J2315 in utilisation of dibenzofuran. The single knockout
mutants of rpoN genes were established using pKNOCK suicide vector series resulting two rpoN
mutants of Burkholderia xenovorans LB400; NRPLB [(rpoN1 mutant) and NRP2LB (rpoN2
mutant)] and one rpoN mutant of Burkholderia cenocepacia J2315 [NRPJ (rpoN mutant)]. The
physiological and metabolic responses analyses were conducted to differentiate the single-gene
knockout mutants from their wildtype strains; Burkholderia xenovorans LB400 and
Burkholderia cenocepacia J2315. The physiological response analysis demonstrated that the
ability of the mutants NRPLB and NRP2LB to form biofilm were not affected with inactivation
of rpoN genes. However, the biofilm formation in NRPJ was reduced indicating the involvement
of rpoN gene in formation of biofilm in Burkholderia cenocepacia J2315. Inactivation of rpoN2
gene does not affect motility of NRP2LB (rpoN2 mutant). However, inactivation of rpoN1 gene
significantly reduced motility of NRPLB (rpoN1 mutant). Metabolic response analysis shows
that rpoN genes play an important role in utilisation of nitrogenous compound even though the
effects are depending on the species of the nitrogen. The altered nitrogen utilisation profile when
using ammonium, histidine, asparagines, nitrate, glutamine and alanine as sole nitrogen source in single-gene knockout mutants indicate that rpoN genes of Burkholderia xenovorans LB400 and
Burkholderia cenocepacia J2315 are active and functional for nitrogen utilisation. The ability of
Burkholderia xenovorans LB400 and Burkholderia cenocepacia J2315 in degrading orthosubstituted PCBs such as dibenzofuran was also determined. Degradation studies of
dibenzofuran showed significant differences between wildtype Burkholderia xenovorans LB400,
Burkholderia cenocepacia J2315 and their single-gene knockout mutants. Degradation rate was
found higher in NRP2LB (rpoN2 mutant) compared to wildtype Burkholderia xenovorans
LB400 but reduced significantly in NRPLB (rpoN1 mutant). This result was supported by gene
expression analysis where RpoN-dependent bphA gene that encodes for biphenyl dioxygenase
was highly expressed in NRP2LB (rpoN2 mutant) thus enhanced the degradation of
dibenzofuran via biphenyl degradation pathway. This result indicates the important role of rpoN1
gene in Burkholderia xenovorans LB400 in degradation of dibenzofuran. Simple phytotoxicity
assay showed that byproducts from degradation of dibenzofuran by wildtype Burkholderia
xenovorans, NRPLB (rpoN1 mutant) and NRP2LB (rpoN2 mutant) is less toxic towards the test
species compared to dibenzofuran. Furthermore, the degradation byproducts from NRP2LB
(rpoN2 mutant) was able to enhanced the growth of Sorghum saccharatum compared to control
(water). |
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