Protocols to identify or purify a bacterial beta-hemolysin from blood agar? - (Oct/08/2014 )

Good morning forum scientists,

 

I am working with a species of Gram-negative bacteria which has both an alpha and beta hemolytic phenotype (complete clearing of blood below and around colonies.)

 

I would very much like to isolate and identify which protein/s are responsible for the beta-hemolytic phenotype and to characterize the system involved in its production/processing.

 

I am wondering if anyone has experience in Beta-hemolysins from bacteria? 

 

I am considering native-page and zymograms with blood/erytrocytes (if even possible?), mass spec on isolated proteins from the clear agar zones themselves? But perhaps this is the wrong way to go about this. In liquid culture less beta-hemolysin is produced.

 

If someone had protocols for characterisation etc of these B-H's that would be tremendously helpful.

 

A genomic approach has identified some homologs to hemolysins and I am in the process of cloning for expression. However, approaching this at the protein level is also advised.

 

Thanks in advance

 

Chris

I think my questions were too general.

 

Let me try again,

 

1) Is there a way to differentiate between alpha and beta hemolysis other than appearance on a blood agar plate?

 

    I think a liquid RBC/hemolysis assay will show both.

 

 

2) A bacteria secretes a protein into an agar plate and it's activity is stable over time (zones of activity continue to increase). How would someone isolate the protein from the agar to use in other assays?

 

I use "protein" in my second question as someone might have protocols for isolation of non-hemolysin proteins from agar that could be used here.

 

Thanks!

 

Chris

1) No idea, but I would guess you could do a liquid broth and use a spec to see which compounds are absorbing.

2) You could take some agar and tryptic digest, then run result through a mass spec.

Hi Bob1

 

Thanks for replying,

 

1) Unfortunately the liquid culture seems to decrease the beta hemolytic phenotype (when cell-sonicates from liquid and solid media are spotted to blood agar)

 

2) If I took the agar that is cleared and digested with trypsin (I am not sure of the purpose of this here? maybe an agarose-digesting enzyme instead?) would any purification be required for mass spec? As a comparison would non beta-homolyzed agar be required?

 

Thanks for the advice again

 

Chris

The tryptic digest is so that the proteins (assuming they are proteins) are digested into peptides that can be identified by mass spec. Usually this is done with MALDI-TOF-TOF or orbitrap. If you were thinking of going down this path, you would need to talk to a mass spec facility about sample processing and analysis.