Comparison between wild type 1026b, DD503 and JW270
From Figs 3 and 5, we observe that
1026b Δasd and Bp82 bear the
same biofilm composition while JW270 and E264 are similar despite the
fact that JW270 was generated from the DD503 (Fig S1), a derivative of
1026b in which the genes encoding amrAB-oprA antibiotic efflux
pump has been deleted [39]. To confirm that the mutation used to
generate JW270 may be responsible for its different biofilm composition,
we repeated the assays for protein, eDNA and glucose concentration using
the wild type 1026b, DD503 and JW270.
1026b and its direct derivative DD503 have the similar protein
concentration (Fig 6a, p = 0.006), and eDNA florescence (Fig 6c, p =
0.03) at 72 hours but DD503 shows an increased polysaccharide
concentration throughout the experiment time span (Fig 6b). However,
once the wcb operon is deleted in DD503 to generate JW270, the
polysaccharide and protein concentrations drastically decreases but
shows an inverse increase in eDNA. There is a significant difference
between the concentrations of all biofilm components when DD503 and
JW270 are compared (p = 0.01-0.04). This data implies that the deletion
of the wcb cluster may be responsible for the low biofilm
polysaccharide and protein concentrations and the overcompensation of
eDNA export in JW270 to maintain a stable biofilm. To quickly confirm
our hypothesis, we performed an ELISA test using JW270 as test sample,
and 1026b Δasd and Bp82 as positive controls. B.
thailandensis E264 which lacks the CPS cluster was used as negative
control. The ELISA test was performed using an anti-CPS primary
antibody, 4B11 [52], and a goat anti-mouse alkaline phosphatase
secondary antibody. The results shows reactivity between the 4B11
antibody and 1026b Δasd and Bp82 but not towards JW270 orB. thailandensis E264 (Fig. S2). Taken together, the results
demonstrate an inverse relationship between eDNA and CPS I inBurkholderia biofilms.