Quorum-sensing mutations affect attachment and stability of Burkholderia cenocepacia biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

Synthesis of multiple N-acylhomoserine lactones is wide-spread among the members of the Burkholderia cepacia complex

Seventy strains of the Burkholderia cepacia complex, which currently comprises six genomic species, were tested for their ability to produce N-acylhomoserine lactone (AHL) signal molecules. Using thin layer chromatography in conjunction with a range of AHL biosensors, we show that most strains primarily produce two AHLs, namely N-octanoylhomoserine lactone (C8-HSL) and N-hexanoylhomoserine lactone (C6-HSL). Furthermore, some strains belonging to B. vietnamiensis (genomovar V) produce additional long chain AHL molecules with acyl chains ranging from C10 to C14. For B. vietnamiensis R-921 the structure of the most abundant long chain AHL was confirmed as N-decanoylhomoserine lactone (C10-HSL) by liquid chromatography-mass spectrometry (LC-MS) in combination with total chemical synthesis. Interestingly, a number of strains, most notably all representatives of B. multivorans (genomovar II), did not produce AHLs at least under the growth conditions used in this study. All strains were also screened for the production of extracellular lipase, chitinase, protease, and siderophores. However, no correlation between the AHL production and the synthesis of these exoproducts was apparent. Southern blot analysis showed that all the B. cepacia complex strains investigated, including the AHL-negative strains, possess genes homologous to the C8-HSL synthase cepI and to cepR, which encodes the cognate receptor protein. The nucleotide sequence of the cepI and cepR genes from one representative strain from each of the six genomovars was determined. Furthermore, the cepI genes from the different genomovars were expressed in Escherichia coli and it is demonstrated that all genes encode functional proteins that direct the synthesis of C8-HSL and C6-HSL. Given that cepI from the B. multivorans strain encodes a functional AHL synthase, yet detectable levels of AHLs were not produced by the wild-type, this suggests that additional regulatory functions may be present in members of this genomovar that negatively affect expression of cepI.     

Immuno-capture differential display method (IDDM) for the detection of environmentally induced promoters in rhizobacteria

A rapid immunological method for trapping and selection of functionally regulated prokaryotic promoters is described. The method is based on application of a novel mini-Tn5 derived promoter probe (pUTTKZY-promoterless lacZY as a reporter and kanamycin resistance) to mutagenise a plant growth promoting fluorescent pseudomonad, Pseudomonas fluorescens 54/96. The transposon allows selection of operon fusion mutants (lacZY(+)) directly on media containing lactose as a sole carbon source as well as selection for kanamycin and lacZ (beta-galactosidase) expression on X-gal indicator media. We have extended the technique to target the surface expression of the induced lactose permease gene (lacY) from mutagenised libraries and the immuno-capture of bacteria with magnetic beads and anti-LacY monospecifc antisera. The benefits of the lacZY reporter are that a library can be rapidly generated and screened in vitro to isolate non-expressed mutants for further in situ screening. Here we demonstrate the development and utility of the technique and its potential as a differential display method for the isolation of promoters that direct regulated gene expression in the phytosphere, or under other imposed conditions.