Chambers Catherine E, Lutter Erika I, Visser Michelle B, Law Peggy P Y, Sokol Pamela A
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada.
BMC Microbiol. 2006 Dec 22;6:104. doi: 10.1186/1471-2180-6-104.
The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs.
Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR.
Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system.
洋葱伯克霍尔德菌新型cepacia CepIR群体感应系统已被证明可正向和负向调节参与铁载体产生、蛋白酶表达、运动性、生物膜形成和毒力的基因。在本研究中，采用了两种方法来鉴定受CepIR群体感应系统调控的基因。转座子诱变用于在cepI突变体中创建lacZ启动子融合体，并在N-酰基高丝氨酸内酯存在的情况下筛选差异表达。采用生物信息学方法在洋葱伯克霍尔德菌J2315基因组中筛选CepR结合位点基序。
通过转座子诱变鉴定出四个正向调节基因和两个负向调节基因，包括可能参与铁转运和毒力的基因。所选CepR调节基因的启动子区域和cepI启动子的定点诱变用于预测CepR结合的共有cep框序列。cep框的第一代共有序列用于鉴定基因组序列中的假定cep框。选择了八个潜在的CepR调节基因并分析了它们启动子的表达。八个中的六个被证明受CepR调节。从这六个基因的启动子与cepI、zmpA的启动子以及通过转座子诱变鉴定的两个CepR调节基因的启动子组合中创建了第二代基序。用新基序搜索洋葱伯克霍尔德菌J2315基因组，在65个启动子区域中鉴定出55个可能受CepR调节的cep框。
利用转座子诱变和生物信息学，已确定十二个新基因的表达受CepIR群体感应系统调控。基于十个CepR调节基因的预测cep框，开发了一个cep框共有序列。这个共有cep框已导致鉴定出超过50个可能受CepIR群体感应系统调控的新基因。
