High school biology, independent study/science fair, introductory undergraduate microbiology, advanced college level microbiology.
May 5, 2010
Many functions in bacteria are regulated by a phenomenon variously called autoinduction or quorum sensing (QS). In QS, the bacteria constantly produce small highly diffusible molecules (often but not always acyl-homoserine actones [acyl-HSLs]), which cross the plasma membrane to the external environment. At low levels these molecules produce no evident effect. As population density increases or as diffusion is constrained, as in a biofilm for example, the concentration of A-HSL rises. At some threshold level the A-HSLs bind reversibly to specific m-RNA synthetase enzymes and these commence transcription of suites of genes previously inactive.
When first described as the mechanisms responsible for controlling bioluminescence Vibrio fisherii, autoinduction was thought a quaint if interesting phenomenon1. This view changed rapidly as A-HSLs were identified as the effector molecules2 and were shown to be functioning in a great number of additional bacterial systems including virulence3, motility4, pigment production in some bacteria5, and the develoment of normal biofilm architecture6, among many others.
It was not until 1997 that McClean et al.7 demonstrated the presence of A-HSLs in naturally occurring biofilms growing on limestone pebbles taken from the San Marcos river in Texas. There initial reporter system for A-HSLs was a strain of Agrobacterium tumefaciens bearing a lactose operon and lacking the ability to make its own A-HSLs. When a source of A-HSL of the appropriate type (N-oxo(octanoyl) HSL) was placed near the reporter strain on medium containing X-gal, a synthetic β-galactosidase substrate, the foreigh A-HSL induced the lac operon to synthesize β-galactosidase which in turn cleaved the X-gal turning the A. tumefaciens blue.
Although this represented a clever method of detecting naturally occurring A-HSLs, this system had some limitations as a teaching tool. The Lac operon was bourn on a plasmid that was unstable and had to be maintained by the inclusion of two antibiotics in the maintenance medium. The requirement for X-gal in the medium was an additional complication. Fortunately at the same time, another set of investigators had developed another reporter system using violacein production in Chromobacterium violaceum5.
C. violaceum strain CV026 fails to produce pigment due to the inability to produce the normal autoinducer N-hexanoyl HSL. Although normally non-pigmented, this strain responds to the presence of exogenous HSL by producing normal amounts of the violet pigment. C. violaceum strain 31532 over-produces the N-hexanoyl HSL, but fails to respond to it and is thus is also non-pigmented while the wild-type strain 12472 both produces and responds to N-hexanoyl HSL by producing violacein.
|ATCC 12472||Wild Type (pigmented)||Produces N-hexanoyl-HSL (C6-HSL) and responds to same.|
|CV 026||Reporter Strain (non-pigmented)||Fails to produce C6-HSL, but does respond to it.|
|ATCC 31532||C6-HSL Producer (non-pigmented)||Over produces C6-HSL, but does not respond to it.|
These strains may be obtained from Robert McClean, Biology Department Texas State, San Marcos.
Safety Note: Chromobacterium violaceum is listed as a BSL 2 pathogen, in the same category with other organisms such as Klebsiella pneumoniae, Proteus vulgaris, Salmonella—all species, and Staphylococcus aureus. Instructors should consider this when deciding whether or not to adopt this exercise.
Ideally this exercise should follow or be carried out in conjunction with a discussion of quorum sensing in the classroom. Students should be familiar with the basics of molecular biology, including transcription and translation.
The exercise described here can be used with entire classes in a manner similar to the original demonstration1, or it may be used as a jumping off point for students engaged in independent research. The exercise should reinforce the students understanding of gene induction, transcription, translation. The student may also observe that quorum sensing occurs in natural habitats and is not only a laboratory phenomenon. The techniques learned will provide the tools for students to conduct their own investigations in QS.
|1 culture||Chromobacterium violaceum Strain CV026 (reporter strain).|
|1 culture||Chromobacterium violaceum Strain 31532 (over-producer of N-hexanoyl HSL).|
|1 culture||Chromobacterium violaceum Strain 12472 (wildtype).|
|As Necessary||3 plates of LB or R2A medium per student.|
|As Necessary||Source of biofilms (pebbles from streams, soil, aquarium samples, etc.).|
|1||Zip-loc plastic bag for collection samples|
|1||Sterile glass Petri dish|
|1||Disinfectant for lab bench|
Both students and instructors will be able to evaluate the effectiveness of the QS detection system by the response of the reporter strain in the control plate (A). A positive response in the “killed” plate (C) probably indicates an error in autoclaving or the student used the wrong strain as the “reporter”.
This assay system can be used as the starting point for any number of student generated research protocols.
This material is based upon work supported by the National Science Foundation under Grant No. 0618744. Developed in collaboration with Dr. John Lennox, Penn State Altoona. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
©2002-2008 Center for Biofilm Engineering, http://www.biofilm.montana.edu