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>, coyne at writes:
>In article <wakarchu.1.0 at>, wakarchu at (Dr. Warren Wakarchuk) writes:
>> In article <1992Sep1.152134.202619 at> coyne at writes:
>>>From: coyne at
>>>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
>> 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
>> wakarchu at
>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
>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

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

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