Expression and functional characterization of gfpmut3.1 and its unstable variants in Staphylococcus epidermidis.
Reporter gene systems are an invaluable tool for investigation of gene transcription activity in eukaryotes and prokaryotes. In order to analyze the temporal and spatial resolution of gene expression patterns in situ and for quantitatively investigating gene expression, the green fluorescent protein (GFP) appears to be especially useful. GFP has been broadly used in various bacterial species, however, there is only limited knowledge about key biological properties in S. epidermidis. Here, the crucial influence of different ribosomal binding sites (RBS) on gfpmut3.1 translation initiation in S. epidermidis 1457 is demonstrated. Only by using the RBS of the delta-hemolysin promoter, after 24 hours a strong fluorescence signal was obtained. The half-life of GFPmut3.1 in S. epidermidis 1457 was significantly shorter than in E. coli (7 h vs. 24 h). GFPmut3.1 derivatives with shorter half-lives (GFP(AAV) and GFP(ASV)) did not reach sufficient quantitative protein levels, and the resulting low fluorescence limits their use as reporter genes in S. epidermidis. This work provides fundamental insights into gfpmut3.1 expression in S. epidermidis and describes the crucial determinants of its biological behavior in this species. In general, this study underlines the need to accurately characterize key biological properties of this transcription marker in gram-positive hosts.
Franke
G C
GC
Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Dobinsky
S
S
Mack
D
D
Wang
C-J
CJ
Sobottka
I
I
Christner
M
M
Knobloch
J K-M
JK
Horstkotte
M A
MA
Aepfelbacher
M
M
Rohde
H
H
D019677:Artificial Gene Fusion / Q000379:methods*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006207:Half-Life; D006460:Hemolysin Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D012270:Ribosomes / Q000502:physiology; D013212:Staphylococcus epidermidis / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D049452:Green Fluorescent Proteins; C025994:delta hemolysin protein, Staphylococcus aureus