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Violacein and the Role It Plays in Quorum Sensing in Chromobacterium violaceum. Presented by: Antoinette Gaston, Master’s Level Graduate Student Advisor’s: Anthony Ejiofor and Terrance Johnson, Department of Biological Sciences Tennessee State University 4 th Annual NASA Research Symposium.

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violacein and the role it plays in quorum sensing in chromobacterium violaceum

Violacein and the Role It Plays in Quorum Sensing in Chromobacterium violaceum

Presented by: Antoinette Gaston, Master’s Level Graduate Student

Advisor’s: Anthony Ejiofor and Terrance Johnson, Department of Biological Sciences Tennessee State University

4th Annual NASA Research Symposium

historic overview
Historic Overview
  • Copper Hill and Ducktown are two cities that are located in the southeastern region of Tennessee
  • Located in Polk County this area was once a thriving portion of the breathtaking Cherokee National Forest
historic overview1
Historic Overview
  • The destruction of this scenic community began in the 1850’s
  • The area suffered heavy metal leaching into the water supply and the surrounding soil for a period of nearly 130 years
  • Large amounts of leaching lead to acid mine drainage which left the land denuded
now let s talk purple
Now, Let’s Talk Purple
  • The microorganisms in question was isolated from soil/water samples obtained from a pool located downstream on the McPherson branch and a sample spot upstream on the

McPherson branch

purple continued
Purple (continued)
  • Organisms were isolated from soil/water sample
  • Isolated organisms were then characterized according to basic microbiological techniques (morphology, Gram reaction, colony size/shape, etc.)
literature search
Literature Search
  • Information from the search lead me to a Genera of purple pigment producing microorganism (Chromobacterium)
background
Background
  • Purple pigment producing bacteria belong to either the Chromobacterium, Janthinobacterium or the Iodobacteria generas
  • The pigment produced is called violacein and can be isolated from the bacteria, characterized and used as an antimicrobial agent, a fungicide, an insecticide and an anti-cancer agent
  • The bacteria can produce polyesters which can be used to make polyhdroxyalkanoates (PHA’s) which are an alternative to plastics make by petrochemicals
  • The bacteria produce cyanide which has been noted to be useful in the harvesting of gold
background continued
Background (continued)
  • The bacteria are gram negative coccobacilli or small rods and are frequently found in soil and water (usually found in the water and the soil along the bank of the water)
  • The separation between the classes is dependent on the area in which it is found (tropical vs. subtropical vs. temperate)
  • The genome for this organism has been determined to be in the region of 4.75 x 106 bp
  • Some of the genes from this genome have been cloned and characterized
  • Current studies are being done to determine the optimum culture conditions for the production of the metabolites that would prove beneficial to the pharmacological and biotechnological fields
my interest
My Interest
  • To identify the isolated organism via analysis of its’ Carbon Utilization Pattern (CUP)
  • Evaluate the effects of violacein on colony size
approach
Approach
  • Isolate organism from soil/water samples
  • Culture organism from isolation source to culturable media source
  • Subculture organism on reduced nutrient media
  • Determine morphology, gram reaction, acid fast and oxidase reactions
  • Identify organism based on metabolic fingerprint
  • Determine optimum culture conditions of organism
isolation of organism from soil water sample
Isolation of Organism from Soil/Water sample
  • Isolation of the microorganism from soil/water samples is very difficult because they exist in small numbers
  • The remedy for this is to use sterile rice grains and subculture on reduced strength nutrient agar
sub culturing
Sub-culturing
  • This was done on 3 different reduced strength agar and tryptic soy agar (control)

*Keeble-Cross Medium

*LB

*1/2 L

*Tryptic Soy

results of basic microbiology analysis
Results of Basic Microbiology Analysis

Sample 14 Sample 15

-small rod -small rod

-Gram (-) -Gram (-)

-Acid fast (no) -Acid fast (no)

-spores (no) -spores (no)

-motile (yes) -motile (yes)

-oxidase (neg) -oxidase (neg)

metabolic fingerprinting
Metabolic Fingerprinting
  • This process involved the observation of the organisms carbon utilization patterns
  • Test used to perform this was the BioLog microbial ID/Characterization analysis
biolog
BioLog
  • A microbial identification system that identifies and characterizes organisms based on the metaboism of the organism rather than metabolic by-products
  • The system has 526 aerobic Gram (-) organisms in the database
  • Performs 95 test simultaneously and uses the characteristic reaction pattern (metabolic fingerprint) to identify the organism
  • The possible number of patterns that can arise from a single MicroPlate is > 4 x 1028
  • Accuracy of BioLog GN2 Microplate in comparison to Molecular Methods is:
      • BioLogMolecular Method

80% 72% Fermenters

88% 100% Nonfermenters

85% 89% Overall

biolog results
BioLog Results
  • Metabolic patterns of the organisms identified both organisms to be Chromobacterium violaceum
    • Sample 14 PROB 100 SIM 0.61
    • Sample 15 PROB 96 SIM 0.58
biolog results cont
BioLog Results (cont.)
  • In biochemical identification of C. violaceum the following substrates are metabolised: glucose, trehalose and N-acetylglucosamine
quorum sensing
Quorum Sensing
  • What is Quorum Sensing?

-- Intercellular signaling

between bacterial cells.

  • Possible triggers
    • Stress Adaptation response to scarcity of nutrients, elevated temperatures ~>40°C, high levels of radiation and elevated concentrations of toxic agents (even heavy metal contaminations
quorum sensing cont
Quorum Sensing (cont.)
  • Signaling compounds in Gram(-) bacteria are dependent on the secretion of non essential amino acids known as acyl homoserine lactones (aka autoinducers)
colony size
Colony Size
  • Density of colony regulates the expression of violacein pigment
  • Isolate 14 demonstrated a hastened secretion of violacein ~ 24 hrs after inoculation
  • Colony size was reduced in inoculums that had higher cell populations
conclusion
Conclusion
  • The notable change in colony size due to the number of organisms present coupled with the noted timetable for secretion of acyl homoserine lactone (AHL) demonstrated the ability of C. violaceum to control colony density.
ongoing studies
Ongoing Studies
  • Extraction and Characterization of violacein pigment
  • Genetic sequencing
acknowledgments
Acknowledgments
  • GOD
  • Dr. Terrance Johnson
  • Dr. Anthony Ejiofor
  • Dr. Todd Gary, Center of Excellence
  • Dr. Montanez Wade, Center of Excellence
  • Laboratory colleagues: Jim Farmer, Sarah Roland, Roderick Rolle, and Karen Burke
  • Glen Springs Holdings Inc.
  • Tennessee State University’s EARDA and Title III programs
  • NASA funding by means of the STARS program
  • Dr. Martin, United States Department of Agriculture