Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates

被引:125
作者
Lefebre, MD
Valvano, MA
机构
[1] Univ Western Ontario, Dept Microbiol & Immunol, London, ON N6A 5C1, Canada
[2] Univ Western Ontario, Dept Med, London, ON N6A 5C1, Canada
关键词
D O I
10.1128/AEM.68.12.5956-5964.2002
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P-BAD promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy, following the infection of Acanthamoeba polyphaga.
引用
收藏
页码:5956 / 5964
页数:9
相关论文
共 41 条
[1]   A genetic analysis system of Burkholderia cepacia: Construction of mobilizable transposons and a cloning vector [J].
Abe, M ;
Tsuda, M ;
Kimoto, M ;
Inouye, S ;
Nakazawa, A ;
Nakazawa, T .
GENE, 1996, 174 (02) :191-194
[2]   Conserved aspartic acids are essential for the enzymic activity of the WecA protein initiating the biosynthesis of O-specific lipopolysaccharide and enterobacterial common antigen in Escherichia coli [J].
Amer, AO ;
Valvano, MA .
MICROBIOLOGY-SGM, 2002, 148 :571-582
[3]   ISOLATION AND MOLECULAR CHARACTERIZATION OF A NOVEL BROAD-HOST-RANGE PLASMID FROM BORDETELLA-BRONCHISEPTICA WITH SEQUENCE SIMILARITIES TO PLASMIDS FROM GRAM-POSITIVE ORGANISMS [J].
ANTOINE, R ;
LOCHT, C .
MOLECULAR MICROBIOLOGY, 1992, 6 (13) :1785-1799
[4]   Characterization of a free-living maize-rhizosphere population of Burkholderia cepacia:: effect of seed treatment on disease suppression and growth promotion of maize [J].
Bevivino, A ;
Sarrocco, S ;
Dalmastri, C ;
Tabacchioni, S ;
Cantale, C ;
Chiarini, L .
FEMS MICROBIOLOGY ECOLOGY, 1998, 27 (03) :225-237
[5]   Burkholderia cepacia complex bacteria from clinical and environmental sources in Italy:: Genomovar status and distribution of traits related to virulence and transmissibility [J].
Bevivino, A ;
Dalmastri, C ;
Tabacchioni, S ;
Chiarini, L ;
Belli, ML ;
Piana, S ;
Materazzo, A ;
Vandamme, P ;
Manno, G .
JOURNAL OF CLINICAL MICROBIOLOGY, 2002, 40 (03) :846-851
[6]   Green fluorescent protein as a marker for Pseudomonas spp. [J].
Bloemberg, GV ;
OToole, GA ;
Lugtenberg, BJJ ;
Kolter, R .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1997, 63 (11) :4543-4551
[7]  
BURKHOLDER WH, 1950, PHYTOPATHOLOGY, V40, P115
[8]   Invasion of respiratory epithelial cells by Burkholderia (Pseudomonas) cepacia [J].
Burns, JL ;
Jonas, M ;
Chi, EY ;
Clark, DK ;
Berger, A ;
Griffith, A .
INFECTION AND IMMUNITY, 1996, 64 (10) :4054-4059
[9]   Taxonomy and identification of the Burkholderia cepacia complex [J].
Coenye, T ;
Vandamme, P ;
Govan, JRW ;
Lipuma, JJ .
JOURNAL OF CLINICAL MICROBIOLOGY, 2001, 39 (10) :3427-3436
[10]   NONCHROMOSOMAL ANTIBIOTIC RESISTANCE IN BACTERIA - GENETIC TRANSFORMATION OF ESCHERICHIA-COLI BY R-FACTOR DNA [J].
COHEN, SN ;
CHANG, ACY ;
HSU, L .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1972, 69 (08) :2110-&