Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression

被引:13
作者
Triccas, JA
Pinto, R
Britton, WJ
机构
[1] Centenary Inst Canc Med & Cell Biol, Newtown, NSW 2042, Australia
[2] Univ Sydney, Dept Med, Sydney, NSW 2006, Australia
基金
英国医学研究理事会;
关键词
mycobacteria; green fluorescent protein; gene expression; destabilized;
D O I
10.1016/S0923-2508(02)01327-X
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
The green fluorescent protein (GFP) is a useful reporter for the study of gene expression and protein localisation within living cells. The stability of GFP permits its intracellular accumulation and detection, but renders it less useful for assessing transient changes in gene expression. We have developed a destabilized form of GFP for monitoring gene expression in mycobacteria. By fusing to the C-terminal end of GFP an 11 amino acid peptide encoded by the E. coli ssrA gene, we have developed a form of GFP that exhibits gradual, time-dependant degradation within the fast-growing species Mycobacterium smegmatis. This unstable variant of GFP detected transient changes in the activity of the stress-induced Mycobacterium tuberculosis sigE promoter; by contrast, unmodified GFP only detected a delayed 'switch-on' of this promoter upon exposure to acid stress. Both forms of the protein displayed equivalent stability in the slow-growing species Mycobacterium bovis bacille Calmette-Guerin (BCG), suggesting differing recognition of the ssrA-encoded peptides in slow- and fast-growing mycobacteria. This system will facilitate studies exploring dynamic changes in mycobacterial gene expression. (C) 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.
引用
收藏
页码:379 / 383
页数:5
相关论文
共 23 条
[1]  
Andersen JB, 1998, APPL ENVIRON MICROB, V64, P2240
[2]   The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein [J].
Barker, LP ;
Brooks, DM ;
Small, PLC .
MOLECULAR MICROBIOLOGY, 1998, 29 (05) :1167-1177
[3]   Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552′phoA in vitro transposition system [J].
Braunstein, M ;
Griffin, TJ ;
Kriakov, JI ;
Friedman, ST ;
Grindley, NDF ;
Jacobs, WR .
JOURNAL OF BACTERIOLOGY, 2000, 182 (10) :2732-2740
[4]   FACS-optimized mutants of the green fluorescent protein (GFP) [J].
Cormack, BP ;
Valdivia, RH ;
Falkow, S .
GENE, 1996, 173 (01) :33-38
[5]   UNDERSTANDING, IMPROVING AND USING GREEN FLUORESCENT PROTEINS [J].
CUBITT, AB ;
HEIM, R ;
ADAMS, SR ;
BOYD, AE ;
GROSS, LA ;
TSIEN, RY .
TRENDS IN BIOCHEMICAL SCIENCES, 1995, 20 (11) :448-455
[6]   GREEN FLUORESCENT PROTEIN AS A MARKER FOR GENE-EXPRESSION AND CELL BIOLOGY OF MYCOBACTERIAL INTERACTIONS WITH MACROPHAGES [J].
DHANDAYUTHAPANI, S ;
VIA, LE ;
THOMAS, CA ;
HOROWITZ, PM ;
DERETIC, D ;
DERETIC, V .
MOLECULAR MICROBIOLOGY, 1995, 17 (05) :901-912
[7]   Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS) [J].
Graham, JE ;
Clark-Curtiss, JE .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (20) :11554-11559
[8]   Sequence determinants of C-terminal substrate recognition by the Tsp protease [J].
Keiler, KC ;
Sauer, RT .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (05) :2589-2593
[9]   Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA [J].
Keiler, KC ;
Waller, PRH ;
Sauer, RT .
SCIENCE, 1996, 271 (5251) :990-993
[10]   GREEN FLUORESCENT PROTEIN AS A NEW EXPRESSION MARKER IN MYCOBACTERIA [J].
KREMER, L ;
BAULARD, A ;
ESTAQUIER, J ;
POULAINGODEFROY, O ;
LOCHT, C .
MOLECULAR MICROBIOLOGY, 1995, 17 (05) :913-922