Spontaneous Spectinomycin resistance ?

[Peter M. Muriana]

In article <CvGJv9.I7 at usenet.ucs.indiana.edu>, jgraham at bronze.ucs.indiana.edu (the End) writes:
>Hello,
>
>I am seeing a relatively high rate of acquisition of spectinomycin 
>resistance in the E. coli strain I am working with (CSH20 from Miller's
>old kit). It appears that all cells in a cuture are somewhat resistant,
>and that 60 or so colonies will form from approximately 100 ul of 
>competent cells spread on a plate.
>
>
>Anyone else see this ? Any relevant knowledge of mechanisms ?
>
>Thanks much,
>
>E-mail copies of all posts,
>
>Jim
>J. E. Graham 
>Biology and Chemistry Departments 
>Indiana University Bloomington 

Jim,
  You didn't mention the concentration of antibiotic (abc) or of the
cells plated, but I would say that 60 or so colonies would not be unusual
for antibiotics like streptomycin, spectinomycin, rifamycin, etc, where 
sensitivity is the result of abc binding to a site on the ribosome or RNAP; 
a mutation at the gene level could easily remove the binding site and be 
experienced at a frequency of 10E-6 to 10E-8. In my lab we generally 
obtain abc resistant derivatives of wild type strains (mostly Gram +'s, but
sometimes Gram -'s) by plating 100 ul of overnight culture on 25-100 ug/ml
of the above abc's (for Gram +'s we'll go higher, 100-500 ug/ml) and most 
often have anywhere from 20-100 resistant 
colonies arising.  We generally will repeat on a second abc so we can do a 
double abc selection to insure we are not isolating resistant derivatives 
since the frequency is somewhat high against only one of these abc's.

Regards, Peter
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*  Peter M. Muriana, Ph.D.		Phone	= (317)-494-8284	*
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