Applied Microbiology and Biotechnology
Involvement of quorum sensing genes in biofilm development and degradation of
polycyclic aromatic hydrocarbons by a marine bacterium Pseudomonas aeruginosa
N6P6
Neelam Mangwani, Supriya Kumari and Surajit Das*
Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life
Science, National Institute of Technology, Rourkela- 769 008, Odisha, India
*For correspondence. Tel. +91661 2462684; Fax. +91661 2462022
E-mail: surajit@nitrkl.ac.in or surajit@myself.com
Supplementary file
Table S1 List of primers used for PCR and qRT-PCR.
Table S2 Correlation coefficient matrix of biofilm growth, lasI expression, rhlI expression,
EPS content and phenanthrene degradation over the period of 7 d (n=4).
Table S3 Correlation coefficient matrix of biofilm growth, lasI expression, rhlI expression,
EPS content and pyrene degradation over the period of 7 d (n=4).
Fig. S1 HPLC chromatogram of (a) standard C4-HSL (b) C4-HSL and 3OC12-HSL mixture
(c) P. aeruginosa N6P6 extract.
Fig. S2 PCR amplification of lasI (295bp) and rhlI (156bp) in P. aeruginosa N6P6.
Fig. S3 Total carbohydrate and protein content of P. aeruginosa N6P6 biofilm-EPS at
different growth phase.
Fig. S4 Detection of AHL produced by P. aeruginosa N6P6 treated with different
concentrations of tannic acid by C. violaceum CV026 (a) Control (b) 10 % DMSO (c) 0.1
mg/ml Tannic acid (d) 0.2 mg/ml Tannic acid (e) 0.3 mg/ml Tannic acid (f) 0.4 mg/ml Tannic
acid.
Fig. S5 Effect of tannic acid on biofilm formation by P. aeruginosa N6P6.
Fig. S6 (a) Inhibition of pyocyanin production by P. aeruginosa N6P6 in presence of tannic
acid (b) Growth curve of P. aeruginosa N6P6 in presence of 0.3 mg/ml tannic acid.
Fig. S7 Relative expression of lasI and rhlI genes of P. aeruginosa N6P6 in the presence of
0.3 mg/ml tannic acid as determined by qRT PCR.
Fig. S8 Effect of tannic acid (0.3 mg/ml) on phenanthrene (a) and pyrene (b) degradation
over the period of 7 days (initial concentration 100 mg/l).
Table S1
PCR
Target
Gene
lasI
Semi-quantitative
rhlI
16S
lasI
qRT-PCR
rhlI
16S
Oligonucleotide
sequence (5'−3')
Forward
CGTGCTCAAGTGTTCAAGG
Reverse
TACAGTCGGAAAAGCCCAG
Forward
TTCATCCTCCTTTAGTCTTCCC
Reverse
TTCCAGCGATTCAGAGAGC
Forward
GTCTTCGGATTGTAAAGCAC
Reverse
GCTACACAAGGAAATTCCAC
Forward
GAAATCGATGGTTATGACGC
Reverse
TACAGTCGGAAAAGCCCAG
Forward
TTCATCCTCCTTTAGTCTTCCC
Reverse
TTCCAGCGATTCAGAGAGC
Forward
GTCTTCGGATTGTAAAGCAC
Reverse
GCTACACAAGGAAATTCCAC
Amplicon size
295 bp
155 bp
284 bp
220 bp
156 bp
284 bp
Table S2
PARAMETERS
Growth
1.000
Growth
0.916*
lasI
0.717
rhlI
0.870
carbohydrate
0.856
protein
0.953**
1d
0.989***
3d
0.948**
5d
0.937*
7d
* P<0.1; **P<0.05; ***P<0.01
lasI
rhlI
Carbohydrate
Protein
1d
3d
5d
7d
1.000
0.766
0.988**
0.933*
0.863
0.909*
0.946**
0.943*
1.000
0.672
0.485
0.483
0.617
0.569
0.550
1.000
0.967**
0.854
0.882
0.949*
0.950*
1.000
0.914*
0.902
0.974**
0.980**
1.000
0.987**
0.974**
0.972**
1.000
0.976**
0.970**
1.000
1.000
1.000
Table S3
PARAMETERS Growth
1.000
Growth
0.916*
lasI
0.717
rhlI
0.870
carbohydrate
0.856
protein
0.963**
1d
0.924*
3d
0.864
5d
0.812
7d
* P<0.1; **P<0.05; ***P<0.01
lasI
rhlI
Carbohydrate
Protein
1d
3d
5d
7d
1.000
0.766
0.988**
0.933*
0.963**
0.979**
0.988***
0.969**
1.000
0.672
0.485
0.861
0.630
0.681
0.634
1.000
0.967**
0.910*
0.986**
1.000
0.994***
1.000
0.834
0.983**
0.962**
0.961**
1.000
0.922*
0.910*
0.866
1.000
0.982**
0.966**
1.000
0.995**
1.000
(a)
(b)
(c)
Fig. S1
Fig. S2
400
350
µg/mg of EPS
300
250
200
150
100
50
0
15 h
24 h
48 h
Time (h)
Carbohydrate
Fig. S3
Protein
72 h
Fig. S4
Absorbance at 595 nm
4
3
2
1
0
Control
DMSO
0.3 mg/ml
Tannic acid
Absorbance at 520 nm
Fig. S5
1
0.8
0.6
0.4
0.2
0
Control
DMSO
Fig. S6 a
0.3 mg/ml
Tannic acid
Absorbance at 595 nm
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
4
8
12
16
20
24
Time (hours)
LB
LB+DMSO
TA
Fig. S6 b
Relative expression
1.6
1.2
0.8
0.4
0
Control
lasI
Fig. S7
rhlI
Residual phenanthrene (mg/l)
120
100
80
60
40
20
0
0d
1d
48h
3d
Time (in days)
48h T
5d
7d
control
Fig. S8 a
Residual pyrene (mg/l)
120
100
80
60
40
20
0
0d
1d
48h
3d
Time (in days)
48h T
Fig. S8 b
5d
control
7d