Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents.
OBJECTIVE
To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens.


RESULTS
The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 x 10(8) to 1 CFU ml(-1) within 1.5-10.3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 x 10(8) to 130 CFU ml(-1) was achieved within 2.6-22.4 h, respectively.


CONCLUSIONS
The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature.


CONCLUSIONS
With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.
Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996-1605, USA.
S
S
Ripp
P
P
Jegier
M
M
Birmele
C M
CM
Johnson
K A
KA
Daumer
J L
JL
Garland
G S
GS
Sayler
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000235:genetics; D048908:Aliivibrio fischeri / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000235:genetics*; D015169:Colony Count, Microbial / Q000379:methods; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D029968:Escherichia coli Proteins / Q000235:genetics; D005516:Food Microbiology; D018545:Lactuca / Q000382:microbiology; D049408:Luminescent Agents; D008164:Luminescent Proteins; D015398:Signal Transduction / Q000235:genetics; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D049408:Luminescent Agents; D008164:Luminescent Proteins; C085427:LuxI protein, Bacteria; D014157:Transcription Factors; C088386:homoserine lactone; D015107:4-Butyrolactone