NUROP Congress Paper
A Second Quorum Sensing Regulon in Burkholderia pseudomallei
Department of Biochemistry, Faculty of Science, National University of Singapore
10 Kent Ridge Road, Singapore 117597
, a Gram-negative soil bacterium, is the causative agent of
melioidosis. Quorum sensing is a mechanism responsible for the regulated expression of
virulence genes in many bacterial pathogens. The first quorum sensing regulon in B.
, Ais/Air, was recently identified in our laboratory. The report describes the
identification of a second quorum sensing regulon, RhlI/RhlR, in B. pseudomallei
. By data
mining of the recently completed B. pseudomallei
K96243 genome sequence using homologous
sequences from related bacteria, we have found several putative quorum sensing genes in B.
Gene-specific primers were designed to amplify the putative quorum sensing
. The primers included Bam
HI restriction sites to facilitate the cloning of
the PCR products into a broad host range mobilizable plasmid, pRK415. The PCR products
were sequenced to obtain the nucleotide sequences of
the rhlI and rhlR
genes B. pseudomallei
KHW, a clinical isolate. The translated amino acid sequences of the B. pseudomallei
and RhlR proteins show high homology to the RhlI and RhlR proteins, respectively, of the P.
quorum sensing system. INTRODUCTION
is the causative agent of melioidosis, a systemic and potential
life -threatening disease in humans . The bacteriums is intrinsically resistant to many antibiotics and its mechanisms of virulence are still obscure. In this study, we aim to identify quorum sensing regulons in B. pseudomallei
using a virulent locally isolated clinical strain, KHW.
Quorum sensing is a two component gene regulation system, which controls cell density-
dependent expression of diverse bacterial phenotypes. It consists of a N-acyl homoserine lactones (AHLs) autoinducer synthase, and an autoinducer regulator protein. Quorum sensing is known to regulate the production of virulence factors, motility, biofilm formation, plasmid transfer, and antibiotic resistance in several gram-negative bacterial pathogens (Lutter, et al
, 2001). The strict control of virulence factor expression may be important in preventing the microorganism from alerting its host to its presence when infecting populations are small. As such, individual bacterial cells are essentially delayed from producing virulence factors until they are in a population (quorum) that is large enough to overwhelm the host (Whitehead, et al
Our laboratory has recently identified a pair quorum sensing genes in B. pseudomallei
which we called ais
. Our studies with the ais
deletion mutant of B. pseudomallei
1 Student 2 Senior Lecturer (UROPS supervisor)
that there may be additional quorum sensing regulons in B. pseudomallei
. Moreover, in P.
, a closely related specie of B. pseudomallei
, at least two pairs of quorum sensing
genes , lasI/lasR
, are involved in the regulation of virulence (Whitehead, et al,
2001). This project describes the identification of a second pair of quorum sensing genes, rhlI/rhlR
, in B. pseudomallei
. RESULTS AND DISCUSSION
A. Bioinformatics research of the putative regions of the second pair of quorum sensing
genes of B. pseudomallei K96243 strain.
Pairwise alignment was performed using protein sequences of quorum sensing systems
families) of related bacterial species as queries in a tblastn search against the B. pseudomallei
K96243 genomic translated protein database at www.sanger.ac.uk. The results identified a region at nucleotide positions 2130000 ~ 2132100 on B. pseudomallei
chromosome 2 which has high similarity (30-50%) to quorum sensing gene families , which includes the synthase and regulator genes. The putative quorum sensing genes were named rhlI
. Open Reading Frame predictions of the putative B. pseudomallei
quorum sensing genes using Vector NTITM Suite 8 ORF analyses program, identified rhlI
coding sequence at 2131287 ~ 2131895 and rhlR
coding sequence at 2130282 ~ 2130974 on the B. pseudomallei
K96243 chromosome 2. (Fig. 1) A putative promoter sequence, gccgctcttgcaatcgttattgc atttgatattatttgcaga caatt tca was identified (2130052 ~ 2130097) using the Neural Network Promoter Prediction at http://www.fruitfly.org/seq_tools/promoter.html. (Fig. 1.)
Multiple amino acid sequences alignment of translated B. pseudomallei
K96243 RhlI with
other AHL synthase proteins, and RhlR with other AHL dependent transcriptional regulator proteins of related bacterial species showe d that there were conserved motifs within quorum sensing systems. This strongly suggests that the B. pseudomallei
sequences which we have identified are likely to be involved in quorum sensing.
Fig. 1. The physical map of putative quorum sensing genes rhlI
(orange arrow) on B.
K96243 chromosome 2 (nucleotide positions 2130000 to 2132100). Green arrows
indicate the PCR primer pairs, rhlIF1/rhlIR1 and rhlRF1/rhlRR1. The putative promoter is
indicated by the dark red arrow. B. Primers for PCR
Primer pairs, rhlIF1/rhlIR1 and rhlRF1/rhlRR1 were designed, using Vector NTITM Suite 8
Primer Design program, to inc lude Bam
HI restriction site s in the sense and antisense primers so as to facilitate the cloning of the rhlI
PCR products, respectively, into the broad host range, mobilizable vector, pRK415 (Fig. 1, Table 1). 2 nucleotides, GC, were added to the beginning of rhlRF1 and rhlIR1, to allow the cutting of the PCR product by Bam
HI. The primer stocks were dissolved with sterile distilled water and were diluted to 10 ìM before use.
Table 1. Primers for the amplification and cloning of B. pseudomallei rhlI
HI restriction site is in bold)
rhlR R1 5’- ATGAAACGGGCGGCGCTAACGGAT -3’
C. PCR amplification of the B. pseudomallei KHW rhlI and rhlR
Polymerase chain reactions were done to amplify the full-length of rhlI
their promoter region. B. pseudomallei
KHW , a local clinic virulent strain was used as template. The reagents and the optimal PCR thermal cycle for rhlI
are listed in Table 2A, 2B.
Table 1A. PCR mixture components of rhlI
1B. Optimal PCR thermal cycle for
The size s of the PCR products obtained were as expected from the rhlI
predicted (Fig.2). PCR products were extracted using BIO 101 GENECLEAN® SPIN kit according to the manufacturers instructions .
agarose gel electrophoresis of rhlR
obtained by PCR in buffer conta ining 1.0mM
(1079bp) obtained by PCR in buffer
D. Cloning of rhlI and rhlR into vector pRK415
The plasmid vector, pRK415, and purified rhlI
PCR products were each digested
HI (1u/ì L) for 2 hours at 37°C. Linearized pRK415 was treated with CIAP (calf
intestinal alkaline phosphatase) to hydrolyze 5’-phosphate groups so as to prevent self-ligation
of the vector DNA. Ligation of pRK415 and rhlI
was performed using T4 DNA ligase
(0.3u/ ì L) at 16°C overnight. The ligation products were transformed into competent E. coli
DH5áëpir by electroporation at 1.8kV. Transformants were selected on LA agar containing
25 ì g/ml tetracycline, 0.01% (w/v) X-gal and 0.2mM IPTG.
E. DNA sequencing of B. pseudomallei KHW rhlI and rhlR
DNA sequencing of B. pseudomallei
PCR products were performed
using the ABI BigDye reagents and analyzed on an ABI377 automated DNA sequencer (Perkin-Elmer). rhlIF1/rhlIR1 and rhlRF1/rhlRR1 were used as sequencing primers. Sequence of B. pseudomallei
KHW rhlI and
rhlR were analyzed using Vector NT ITM ContigExpress program. Pairwise sequence alignment of the B. pseudomallei
DNA sequences with that of B. pseudomallei
K96243 showed that they share 97% and 99% similarity, respectively. Homology search of the translated B. pseudomallei
KHW protein RhlI and RhlR against GenBank protein database using blastp identified several proteins which share high similarity (40-50%) with RhlI are N-acyl-homoserine lactone synthases, and high similarity (41-49%) with RhlR are acylhomoserine lactone dependent transcriptional regulators, respectively. The highest scores were those from Burkholderia spp.
and Pseudomonas spp.
, which are taxonomically closely related to B. pseudomallei
. This strongly suggests that the rhlI
genes which we have identified comprise a second quorum sensing system in B. pseudomallei
Barbara-Ann D. Conway, Vicnays Venu, David P. Speert
“Biofilm Formation and Acyl
Homoserine Lactone production in the Burkholderia cepacia Complex” Journal of Bacteriology
Oct. 2002, p. 5678-5685. Vol.184, No.20 E. Lutter, S. Lewenza, J.J. Dennis, M.B. Visser, P.A. Sokol
“Distribution of Quorum-Sensing
Genes in the Burkholderia cepacia
Complex” Infection and Immunity
, July 2001, p. 4661-4666 Neil A. Whitehead, Anne M.L. Barnard, Holly Slater, Natalie J.L. Simpson, george P.C.
“Quorum-sensing in Gram-negative bacteria” FEMS Microbiology reviews
Roger S. Smith, Sarah G. Harris, Richard Phipps, Barbara Iglewski “
Quorum-Sensing Molecule N-(3-Oxododecanoyl) Homoserine Lactone Contributes
to Virulence and Induces Inflammation in Vivo” Journal of Bacteriology
, Feb. 2002, p. 1132-
1139. Vol.184, No.4
August 19, 2010 Mr. John Cockburn Director Equipment Division, Office of Energy Efficiency Natural Resources Canada 930 Carling Avenue, 2nd Floor, Room 25 Ottawa, ON K1A 0Y3 Re: Canada Gazette, Part I; June 21, 2010 Dear Mr. Cockburn: The Security Industry Association (SIA) and the Canadian Security Association (CANASA) are pleased to respond to your request for comments on the Natural Res
PUBLICATIONS 1/ Articles dans des revues à comité de lecture : 86-1 Caroli F., Leplège A., Mercuel A., Urgence Psychiatrique et Troubles Somatiques, L'Encéphale , 1986, XII:335-41. 89-1 Chebili S. et Leplège A., 1989, Toxicomanie, prostitution et Sida : problèmes éthiques , Nervure , t.II, n.8, pp.55-7. 91-1 Leplège A. Pour un historique de la définition scientifique du médic