instability in stock cultures of Erwinia

M. Alexeyev malexeyev at biost1.thi.tmc.edu
Fri Jan 26 10:22:40 EST 1996


In article <01I0FLRYT9420016VI at VMGW.AGR.CA>, WHEATCROFTR at NCCCOT.AGR.CA wrote:

> Subj:     RE: Instability in stock cultures of Erwinia
> 
> In article <4e3sr0$q90 at news.orst.edu>, hanusj at ava.bcc.orst.edu (Joe Hanus)
> wrote:
> 
> > A number of plant pathologists associated with Microbial Germplasm
> > Database (http://mgd.cordley.orst.edu) have posed this question.  If
> > anyone can help, I'll forward to the interested parties.
> >  > These individuals are having difficulty with stored glycerol stocks of
> > softrot Erwinia, specifically Ecc and Eca.  Each researcher uses their
> > strains for plant experiments once or so a year and the strains remain
> > stored the rest of the year.  This year, after culturing, the strains
> > were very weakly pectolytic on CVP and caused some, but less than
> > before, maceration in tubers.  I've asked about tuber age and culture
> > contamination, but my sleuthing didn't come up with anything that was
> > obviously awry.
> >  > Has anyone else encountered this problem?  If so, how common is it to
> > observe loss or reduction of pectolytic activity on CVP and maceration
> > of potato tubers of Ecc or Eca cultures stored in glycerol at below
> > -20 (usually at -70)?  Does anyone know why it happens?  Are there
> > other ways to store Ecc or Eca such that pectolytic activity is
> > preserved?
> 
>   
> M. Alexeyev REPLIED
> 
> >I have encountered similar problems when screening Erwinia and
> >Enterobacter cultures for the presence of restriction endonucleases.
> >Several strains that had RE activity have lost it after 1 month storage
in a stab at
> >room temp. I would be curious to learn the reason.
> 
> I wonder if the activity of endogenous mutagens is induced
> under these storage conditions or upon release from them.
> I would be interested in looking for active insertion elements in these
strains.
>  
> Roger Wheatcroft
> Agriculture Canada, Ottawa.
>  

Well, insertion of transposon or IS element or loss of the plasmid does
provide a sort of explanation. However, what is probability of random
inactivation of RM system by IS element and if it is not random then why?
Same goes for the loss of the plasmid. Why was it stable for several years
under nonselective conditions and then all of the sudden eliminated it
with 100% efficiency (the only difference was microaerobic conditions
employed and somewhat different medium)?  

Even more interesting things have happened with Bacillus sp. that would
produce RE when grown in protein hydrolysate based medium and not in LB
and then after few passages started to produce RE when grown in LB. 

Finally (and you probably wouldn't beleive it), I observed even decrease
in the amount of enzyme synthesised accompanied with apparent change in
specificity (no, it wasn't due to partial digest or star activity).
Unfortunately, I didn't have an opportunity to look closer into that.

M. Alexeyev.


























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