Rhodococcus opacus

What is the Research Question?

Armando VitalRivera High School

Brownsville ISDDr. Kung-Hui (Bella) Chu

Assistant Professor, Department of Civil Engineering (Environmental Engineering)

Texas A&M University

TYPE OF ENGINEERING

Civil Engineering LabEnvironmental research is conducted in the

CVLB lab.Working with two of Dr. Chu’s PhD students,

Myung Hee Kim and Do Gyun Lee

Center for Phage Technology (CPT) labBiology research is conducted in the Center for

Phage TechnologyWorking with Dr. Jason Gill and Dr. Ry Young

BACKGROUND OF RESEARCH PROJECT

Dr. Kung-Hui Chu WorkBiodegradation and bioremediation of priority

pollutants and emerging contaminantsMolecular quantification of microbial risk in

waterOptimization of bioenergy productionApplication of bioretention for stormwater runoff

managementAdvancing knowledge on microbial ecology of

nitrogen and carbon cycles

RELEVANCE OF THE RESEARCH

Research QuestionA tale of two phages: Is phage DNA sequence

highly conserved over time and space?

Significance: Phage Genome Evolution

THE LAB WORK

Techniques used in

molecular biology for working

with the phages.Applied microbiology, virology, and

environmental engineering.

RESEARCH OBJECTIVE

Isolate and characterize phages that infect Rhodococcus opacus.

Collect activated sludge and soil samples that may contain phage.

EXPLORATION

What do we know about these phages?A phage, Ropa 4, was isolated in Germany over

20 years agoThree R. opacus phages were recently isolated in

Chu’s laboratory The genomes of all four phages were sequenced

at the CPT and found to be almost identical

SURPRISE DISCOVERY!

The DNA of the newly isolated phage is almost 100% identical to that of the phage isolated from Germany.Signifies a new finding in “Phage Genome

Evolution”

Approach: E3 teachers will repeat our work at other laboratory in order to rule out any cross contamination during isolation phages in Dr. Chu’s lab. 

BACKGROUND ON PHAGES

Bacteriophages are viruses

that infect bacterial cells. Phages cannot reproduce on their own.The phage hijacks the cell’s machinery to

reproduce progeny.Phages are specific for their host bacteria.YouTube - T4 Virus infecting a bacteria.

Rhodococcus opacus

Rhodococcus opacus is a

specific bacteriaBelongs to the family Actinomycetes,

related to MycobacteriumR. opacus is a rod, nonmotile,

mycobacteriumThis bacteria was used as the host to

isolate phage in Chu’s laboratory

Impact of the Research

Potentially open a new research direction in phage genomics.Some Rhodococcus opacus species are

pathogens. A better understanding of phages specific to R.

opacus can enhance the development of phage therapy.

Some R. opacus are foaming bacteria in biological wastewater treatment processes.Phage treatment might be possible to minimize

common sludge bulking problem.

THE CHALLENGE FOR SCIENCE

The cell wall of the bacteria is difficult disrupt.

First step is to see which phages can actually infect R. opacus

Rhodococcus-specific phages can lyse the cell.

LYSIS PROCESS

Lysis via a 3 component

system in MycobacteriaA holin protein opens a pore in the cytoplasmic

membraneThe creation of the pore triggers the release of

endolysinAn esterase enzyme is also released that

degrades the outer mycolic acid cell wall layer

Prepare 4 litters of R2A broth

(media/nutrients)

Prepare broth with 1.5% Agar (solid)

Research Activities Phage Titration

Prepare broth with 0.8% Agar

(gel)

Prepare petri dishes

Add R. opacus to molten agar

Pour bacterial lawn to petri dishes to achieve an even

growth of bacteria

Collect soil samples

Collect phage from soil

Apply phage spots to bacterial lawn

Proceed to full-plate titration of phage.

RESEARCH ACTIVITIES

Learn laboratory aseptic techniques when working with BL1 agents.Use ethanol as sanitizer Use flame to allow clean air to move upwards

preventing bacteria from falling on the bench.

Prepare soft agar overlay platesPrepare media of R2A with 1.5% Agar

concentration and 0.8% agar concentration.

Spot Titration of phage

Pour a bacterial lawnLabel bottom surface of plate with phage

and lawn strain.In aseptic environment, transfer host

culture into molten agar and vortex.Pour contents of tube onto agar surface.Allow agar to solidify.

Spot Titration of Phage

Apply phage spots to lawn.Aspirate phage dilution and deposit over

corresponding label marked on plate.Allow plate to dry near a flame for 15 minIncubate plates, inverted, at optimal growth

temperature until plaques appear in the lawn.

Individual plaques may be counted to estimate the concentration of phage.

EXPERIMENTS

Collecting soil samplesPreparing media (broth/plates)Enriching samples for phageGrowing liquid cultures of bacteriaPlating out phageCollecting plaques of phage on petri

dishes

DATA TO BE GENERATED

Data that will help characterize the phages from the soil samples collected from a sewer facility and close to a gasoline station.

The model used will be comparing the characteristics of newly found phages to the phage currently available.

Future Implications

Is the high similarity of phage

sequences unique to Rhodococcus species?

Does the high similarity of phage sequences present concern G+ pathogens?

What is the implications of high conservation of phage sequences?

SUMMARY

Learn how to grow R. opacus bacteria.Collect soil samples from a sewer facility

and close to a gasoline station.Isolate, and characterize newly found

phages.Compare newly found phages to the

phage found in Germany.

CLASSROOM APPLICATION

Combined lesson plan with Biology and Algebra 1 classes.

Analyze and evaluate phage growth rate using functions and scatter plots.

ACKNOWLEDGMENTS

TAMU E3 ProgramNational Science FoundationNuclear Power InstituteTexas Workforce CommissionDr. Kung-Hui (Bella) ChuThe Center for Phage TechnologyDr. Jason Gill and Dr. Ry YoungMyung Hee Kim and Do Gyun LeeAndy Hernandez (partner)

THANK YOU!