Sodium Cloride And Sucrose Rescued Cupriavidus Necator H16 Δsecb Mutant When Grown In Nutrient Rich Media
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Date
2016-03
Authors
Sim, Xuan Yi
Journal Title
Journal ISSN
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Publisher
Universiti Sains Malaysia
Abstract
SecB is one of the key players in the Sec protein-translocation system
ubiquitous to Gram-negative bacteria. In this study the function of SecB was studied
by studying an unmarked Cupriavidus necator H16 ΔsecB mutant. The mutant was
constructed through allelic exchange utilizing the pDM4 suicidal vector. In order to
confirm that the phenotypic change is due to the mutated gene, a complementation
mutant was also constructed, in which vector pBBR1MCS2 harbouring a secB gene
was introduced into the C. necator H16 ΔsecB mutant via conjugation. The wild type,
C. necator H16 ΔsecB mutant and the complementation mutant were characterised
using Biolog Phenotype Microarray. The result shows that the complementation
mutant exhibits the wild type phenotype, indicating successful complementation of
the unmarked secB deletion. The C. necator H16 ΔsecB mutant did not show any
significant difference when grown on most substrate. The only exception was that
the C. necator H16 ΔsecB mutant cannot grow on nutrient rich and Bloomfield
medium unless supplemented with either sucrose (10%) or NaCl (0.5 – 1.0%). The
result also showed that KCl was unable to rescue growth, unlike sucrose and NaCl.
The inability of the mutant to grow in rich media (without sucrose or NaCl
supplementation) is unlikely due to polar effect of secB deletion in gspA as
previously proposed, because unlike E. coli, the stop codon of secB and gpsA in
C. necator do not overlap. The result also showed that the deletion of secB did not
affect the PHB production of C. necator. In Pseudomonas sp. deletion of secB led to
a 50% increase in PHA production.
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Keywords
Mutant