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Counterselectable marker

Glen Tamura gtamura at u.washington.edu
Mon Aug 30 12:37:32 EST 1999

I am currently developing a counterselectable marker for use in Gram
Positive organisms. It works very well so far, and with remarkably little
breakthrough. I have successfully used it to create a double crossover
mutation with minimal effort. 

If anyone is interested in using this counterselectable marker, please let
me know. It should render making allelic exchanges relatively painless. 

The following is a copy of an abstract from the ASM meeting in Chicago in
June. Figures are not included. If you want a copy, give me your FAX
number and I will send it to you. 

Glen Tamura
gtamura at u.washington.edu

Development of a counter-selectable marker for use in Group B Streptococci
Tamura GS, and Przekwas JP.  University of Washington and Childrens
Hospital and Regional Medical Center, Seattle, Washington, 98105. 
	A counter-selectable marker is very useful in a variety of
situations, including cloning and allelic exchange experiments. No useful
counter-selectable marker is available for use in Group B Streptococci. We
have isolated a mutant, COH1-F1, which contains an insertion of the
transposon Tn917 into the glnQ gene. This mutant shows a >95% decrease in
its ability to transport glutamine when compared to the wild type strain
(COH1). In several other organisms, the glutamine analog
g-glutamyl-hydrazide (GGH) is toxic only when a functional glutamine
transport system is present. We hypothesized that this might provide the
basis for a useful counter-selectable marker system in GBS. We have
developed a solid media which contains GGH. When >106 COH1 were plated on
this media, no colonies were isolated after 24 hrs of incubation, while
COH1 plated on identical media lacking GGH resulted in a lawn of growth.
In contrast, growth of the glutamine transport mutant COH1-F1 was
identical on media containing and lacking GGH. We propose to develop a
complementation construct containing the glnQ gene which we hypothesize
will act as a counter-selectable marker on media containing GGH. 
Introduction. Selectable markers, such as antibiotic resistance genes,
allow survival of the host organism when challenged by a substrate. In
contrast, counter-selectable markers are genes which are lethal to the
host organism when challenged by a substrate. Counterselectable markers
can be used in a variety of situations including the following: 
1) Selecton of clones with inserts. After introduction of a multiple
cloning site into the counterselectable marker gene, disruption of the
counter-selectable marker gene by insertion of foreign DNA can allow for
selection of clones containing inserts in molecular cloning experiments. 
2) Creation of double-crossover directed mutations. Single cross-over
mutations are relatively easily created using a variety of techniques,
including suicide vectors and temperature sensitive vectors. However,
creation of a double-crossover mutation is often difficult because of a
lack of an ability to select for the absence of plasmid sequences. Rare
double-crossover events must be laboriously screened for using
replica-plating. Use of a counter-selectable marker allows for selection
for mutants containing double-crossovers by selecting for the marker being
exchanged into the gene to be disrupted, and counterselection against the
marker on the plasmid. 
	A number of counterselectable markers have been developed for a
variety of organisms. However, to date, no such counterselectable marker
system has been developed for use with streptococci. 
Figure 1: COH1-F1, a virulence mutant of GBS, carries a Tn917 mutation in
glnQ. The mutant COH1-F1 is a Tn917 transposon mutant of COH1, a wild type
GBS isolate. COH1-F1 was isolated on the basis of reduced adherence to
fibronectin. Nucleotide sequence analysis of the region surrounding the
Tn917 insertion revealed that Tn917 had inserted in an operon highly
homologous to the glutamine transport operon of E. coli. The genes and
their putativbe functions are shown. glnH and glnP are fused in GBS. 
Figure 2: COH1-F1 has a defect in glutamine transport. We hypothesized
that since COH1-F1 had a mutation in a gene highly homologous to glnQ,
that COH1-F1 would show a significant decrease in its ability to transport
glutamine. We therefore tested the ability of 
COH1 and COH1-F1 to take up 3H-glutamine. COH1-F1 had a 
significant decrease in its ability to transport glutamine, demonstrating
that glnQ is a glutamine transport gene in GBS. 
Figure 3: COH1-F1 is resistant to the anti-metabolite  GGH. Previous data
had shown that multiple organisms are highly sensitive to GGH, an analog
of glutamine. Furthermore, organisms often lose that resistance when
various glutamine transport genes are mutated. We hypothesized that wild
type GBS, such as COH1, would be sensitive to GGH, while COH1-F1 would be
resistant. We were able to demonstrate that under certain conditions,
differential sensitivity to GGH between COH1 and COH1-F1 could be
Figure 4: Map of pAG200: glnQ in pDC123. We hypothesized that
complementation of glnQ  in COH1-F1 would restore glutamine transport, and
therefore confer sensitivity to GGH upon COH1-F1. To test this hypothesis,
we created the construct pAG200, which contains glnQ in the shuttle vector
pDC123. glnQ was placed under the control of a pair of strong
constituitive promoters. This construct was used to transform COH1-F1. 
Figure 5: pAG200 confers sensitivity to GGH in the background of COH1-F1.
We then plated  COH1-F1, and COH1-F1:pAG200 on media containing
chloramphenicol and/or g-glutamyl hydrazide. As shown, no growth was seen
when COH1-F1:pAG200 was grown in the presence of GGH with chloramphenicol
(the latter was to prevent plasmid loss). These results confirm that
pAG200 confers sensitivity to GGH.
Figures 7, 8: GGH was successfully used to select for a double-crossover
mutation in glnQ in a single step. To test whether GGH could be used to
select for rare double crossover events, we created a plasmid with the
glnQ gene interrupted by an erythromycin resistance gene on the
temperature sensitive vector pVE6007. We transformed COH1 with this
plasmid. Single crossover events do not result in disruption of the glnQ
gene, while double crossover events would result in disruption of the glnQ
gene. We hypothesized that growth on GGH would therefore select for double
crossover events, while strains with an intact glnQ gene, including both
the parental strain and the progeny of single crossover events, would not
grow in the presence of GGH. We therefore grew up COH1:pAG101 at 30C, a
temperature permissive of plasmid replication, and then cured the culture
of the plasmid at the non-permissive temperature (37C). This culture was
plated on GGH. Individual clones which grew in the presence of GGH were
then picked, and checked for the presence of the doublecrossover event by
PCR using primers flanking the glnQ gene. Wild-type GBS should give an
850bp product with the primers selected. Those with a plasmid integration
(single crossover event) should give a very large product containing the
entire plasmid (>5kb). Those with a double crossover event should give a
product containing most of the glnQ gene along with the inserted erm gene,
for a total of 1650bp. We were able to isolate three clones which gave the
expected product for a double crossover event. 

1) COH1-F1, a Group B streptococcal strain with a mutation in the
glutamine transport gene glnQ, is resistant to GGH, while COH1, the
parental wild type strain, is not. 
2) pAG200, a plasmid containing glnQ, complements glnQ in trans, and
confers sensitivity to GGH
3) These vectors may be useful as counterselectable markers when used in
the background of COH1-F1
4) GGH has been successfully used to select for rare double-crossover
5) In general, GGH selection cannot be used in a wild type background

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