non-specific cleavage of X-gal

Michael Benedik bchs1b at Elroy.UH.EDU
Wed Sep 9 23:21:54 EST 1992


In article <1992Sep9.110653.202722 at uctvax.uct.ac.za>, coyne at uctvax.uct.ac.za writes:
>In article <wakarchu.1.0 at biologysx.lan.nrc.ca>, wakarchu at biologysx.lan.nrc.ca (Dr. Warren Wakarchuk) writes:
>> In article <1992Sep1.152134.202619 at uctvax.uct.ac.za> coyne at uctvax.uct.ac.za writes:
>>>From: coyne at uctvax.uct.ac.za
>>>Subject: X-GAL CLEAVAGE BY V. CHOLERAE
>>>Date: 1 Sep 92 15:21:33 +0200
>> 
>>>Dear Netters,
>> 
>>>I am attempting to isolate transposon generated lacZ operon fusions in 
>>>Vibrio cholerae by cleavage of X-gal to generate blue colonies.  According to
>>>Bergeys, V. cholerae is lactose negative.  However, I have found that
>>>V. cholerae is able to cleave X-gal to generate blue colour which is a major 
>>>problem with respect to my strategy.  I would greatly appreciate it if anyone 
>>>could supply me with an answer to the following question:
>> 
>>>   Does V. choerae possesses a beta-galactosidase enzyme?
>>>   (The bacterium was scored as lac- as a function of growth on lactose).
>> 
>>>You may post the answer e-mail it directly to me.
>> 
>>>Many thanks in advance,
>> 
>>>Vernon Coyne
>>>vernon at micro.uct.ac.za
>>> 
>> Your problem is likely that another glycosidase like Beta-glucosidase is 
>> cleaving the X-gal.  Most gram negative strains have such enzymes and the 
>> specificity is rather "loose".  E. coli Beta-galactosidase does cleave other 
>> glucosides as well.  You might have to look for the enzyme itself, not its 
>> activity.
>> 
>> Warren Wakarchuk
>> IBS NRCC
>> wakarchu at biologysx.lan.nrc.ca
>
>First let me thank Warren for his response to my last posting.  Warren's reply
>has prompted the following question:  
>
>If X-gal is such a sensitive substrate for detecting beta-galactosidase
>activity, and if most gram negative bacteria possess other glycosidases which
>are capable of cleaving X-gal, is it ever possible to get truly "white" 
>colonies where lacZ is NOT expressed from an upstream promoter (as in operon
>fusions) in a lac- gram negative strain grown on media containing X-gal?  The
>answer to this question is important to our current research as we are
>attempting to generate lacZ operon fusions using Tn5 in order to identify 
>chromosomal genes which respond to specific stresses.
>
>I would be most grateful for any information or suggestions regarding this
>problem.
>
>Vernon Coyne 
>University of Cape Town


In my limited experience I have found that it is entirely strain dependent.
For example in my lab I have some strains of Serratia marcescens which
give blue colonies and some which don;t, although both are Lac- by virtue
of being unable to grow on lactose or produce B-galactosidase as detected
by a standard assay with ONPG.
I have two suggestions: (1) you could try to isolate a mutants strain
of your Vibreo which is white on Xgal. This should be relatively easy
to do after a round of chemical mutagenesis, assuming of course that there
is only a single enzyme which is producing your background. Alternatively
you could try some different "wild type" strains of Vibreo to see how
they work. (2) Switch to a different reporter gene. Derivatives of Tn5 have
been made with PhoA, Lux, Cat and I am sure some other reporters as well.
Although not as convenient as LacZ, they might still allow you to do the
experiment. 

	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU



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