Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants
The frequency of the induction by nttrosoguanidine of glyphosate resistant mutants was compared for recipient and donor, as well as for lysogenic and non-lysogenic E. coli cells. It was found that integration of viral genomes and also larger replicons such as F-factor into host chromosome increased...
Gespeichert in:
| Veröffentlicht in: | Биополимеры и клетка |
|---|---|
| Datum: | 1997 |
| 1. Verfasser: | |
| Format: | Artikel |
| Sprache: | Englisch |
| Veröffentlicht: |
Інститут молекулярної біології і генетики НАН України
1997
|
| Schlagworte: | |
| Online Zugang: | https://nasplib.isofts.kiev.ua/handle/123456789/155687 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants / E. Cherepenko // Биополимеры и клетка. — 1997. — Т. 13, № 6. — С. 493-496. — Бібліогр.: 13 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859611166248009728 |
|---|---|
| author | Cherepenko, E.I. |
| author_facet | Cherepenko, E.I. |
| citation_txt | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants / E. Cherepenko // Биополимеры и клетка. — 1997. — Т. 13, № 6. — С. 493-496. — Бібліогр.: 13 назв. — англ. |
| collection | DSpace DC |
| container_title | Биополимеры и клетка |
| description | The frequency of the induction by nttrosoguanidine of glyphosate resistant mutants was compared for recipient and donor, as well as for lysogenic and non-lysogenic E. coli cells. It was found that integration of viral genomes and also larger replicons such as F-factor into host chromosome increased the level of glyphosate resistance by the factor ranging from 1.6 to 6. Mutants tolerating 0.2 rnM of the inhibitor were obtained one order of magnitude more frequently than mutants tolerating 1 mM of this inhibitor. One half of the mutants of every group were resistant not only to the analogue of glycine,but also to the analogue of lysine. An attempt to clone an insertion from a gene library of one of the mutants was attempted but failed. Study on the nature of this gene is in progress.
Вивчено частоту індукції гліфосатстійких мутантів у реципіент-донорських, а також лізогенних-нелізогенних штамах клітин Е. coli. Показано, що інтеграція вірусного, а також більшого F-реплікона підвищує частоту індукції гліфосат стійких мутантів від 1,6 до 6 разів. Мутанти, стійки до 0,2 мМ гліфосату, виникають у 10 разів частіше мутантів, стійких до 1 мМ цього аналога гліцину. Половина мутантів кожної групи стійка не лише до аналога гліцину, але й до аналога лізину. Клонувати ген гліфосатстійкості не вдалося. Досліджується домінантно-рецесивна природа цього гена.
Изучена частота индукции глифосатустойчивых мутантов в реципиент-донорских, а также лизогенных-нелизогенных штаммах. Показано, что интеграция вирусного и более крупного F-репликона увеличивает частоту индукции gly -мутантов от 1,6 до 6 раз. Мутанты, устойчивые к 0,2 мМ глифосата, возникают в 10 раз чаще таковых, устойчивых к 1 мМ этого аналога глицина. Половина мутантов каждой группы устойчива не только к аналогу глицина, но и к аналогу лизина Клонировать ген глифосатустойчивости не удалось. Изучается доминантно-рецессивная природа этого гена.
|
| first_indexed | 2025-11-28T12:16:37Z |
| format | Article |
| fulltext |
ISSN 0233-7657. Биополимеры и клетка. 1997. Т. 13. № 6
ГЕНОМ И ЕГО РЕГУЛЯЦИЯ
Genetic mechanisms of the resistance
of Escherichia coli to amino acid antimetabolites.
2. Study of the frequency of induction and
properties of glyphosate resistant mutants
E. Cherepenko
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
150 Zabolotnogo str., Kiev, 252143
The frequency of the induction by nitrosoguanidine of glyphosate resistant mutants was compared for
recipient and donor, as well as for lysogenic and non-lysogenic E. coli cells. 11 was found that integration
of viral genomes and also larger replicons such as F-factor into host chromosome increased the level of
glyphosate resistance by the factor ranging from J.6 to 6. Mutants tolerating 0.2 rnM of the inhibitor were
obtained one order of magnitude more frequently than mutants tolerating 1 mM of this inhibitor. One half
of the mutants of every group were resistant not only to the analogue of glycine,but also to the analogue
of lysine. An attempt to clone an insertion from a gene library of one of the mutants was attempted but
failed. Study on the nature of this gene is in progress.
Introduction. Antimetabolites are extensively used as
drugs and pesticides and are widely studied as «lead»
compounds in «drug dcsign» programs [1 ] (for rev.
see [2, 3]). However in practice cellular drug re
sistance developing in different taxonomic groups
makes the use of such inhibitors less efficient. What
is genetic and molecular basis of such a resistance?
To investigate this the well-studied mechanisms of
glyphosate inhibition may be used as a good ex
perimental model.
The broad-spectrum non-selective herbicide gly
phosate N- [phosphonomethyl 3-glycine specifically in
hibits the 5-enolpyruvylshikimate-3-phosphate syn
thase (EPSPS), which catalyses the sixth step in the
shikimate biosynthetic pathway the occurence of
which is restricted to bacteria, fungi and plants [4].
Mutational changes rendering this enzyme insensitive
to the inhibitor, as well as target site overproduction
due to gene amplification (or specific promoter chan
ges) have been shown to lead to the glyphosate
resistance (for rev. see [5]). However these events as
well as reduced inhibitor uptake, increase in its
© E. C H E R E P E N K O , 1997
degradation are rare events [6—8 ], whereas the
probability of glyphosate-resistant (glyr) colonies ap
pearance is 2 orders of magnitude higher in E. coli
[6]. We have recently shown that most of the glyr
clones are single gene mutants which are mapped at
4 different loci of the E. coli chromosome map [9].
We wondered if integration of different replicons into
the host chromosome have an effect on the probability
of the appropriate gene mutability. To answer this
question the induction probability of gly r mutants in
recipient and donor as well as lysogenic and non-
lysogenic E. coli cells was measured.Also the nature
of the mutants obtained was studied.
Materials and Methods- Chemicals. N-methyl-N-
nitro-nitrosoguanidine (NG) and S-2-amino-ethyl-L-
cysteine were supplied by «Sigma» (Germany). As a
source of glyphosate the commercial herbicide Roun
d u p 0 0 with the isopropylamine salt of glyphosate as
an active ingredient was used.
Bacterial Strains and Growth Media. The E. coli
strains employed and their genotypes are listed in
Table 1. As basal salt media, M9 and LB medium
were used [10].
Mutagenesis and Genetic Methods. For muta
genesis, nitrosoguanidine treatment was employed as
493
C H E R E P E N K O E.
Table !
Strains used
Relevant genotype Prophage
1 F A B n 5 7 thr-1, thi-J, lacy-1, mtl-1, xylJ5, galK2, proA2,
argE3, str31} tsx33, sup37, Іеиб, ara, his4C
2 F~ A B u 5 7 The same
3 Hfr С Prototrophic
4 Hfr B2625 The same
5 Hfr Рз B346 cys, gal а, ~ Г / , Т/
6 F" D H 5 « supE44yDlacU169(lp8olacZDM15),hsdR17,
recAly endAJ, gyrA96, thiJ, relA
7 F~ HB-101 supE44t hsdS20(rB~tnB~)recAJ3, ara!4, proA2,
lacYl, galK2, rpsl20y xyl!5t rntU
None
^ i m m 4 3 4
A
None
None
None
None
Dr. V. Lantzov, Inst, of Nucl.
Res. (Russia)
Lysogenized in this work
Dr. V. Lantzov, Inst, of Nucl.
Res. (Russia)
Inst. Industr. Microbe Genet,
and Selection (Russia)
Ibid
Ibid
Ibid
Table 2
Average number* of glyr mutants per 10b of mutagenized E. coli cells appearing after NG treatment
*Not less than 3 experiments; **because Roundup (300 g/1 of isopropylammonium salt of glyphosate) was used, 1 dose was determined as
the minimal quantity required to suppress growth of all untreated cells in control.
described by Miller [10]. Gly r mutants obtained in
primary selection experiments were studied by a
passage on increasing concentration of glyphosate
alone or a mixture of glyphosate and AEC
(200//g/ml).
Construction of glyr mutant genomic DNA library
and complementation. DNA isolated from this mutant
was partially digested with Sau3A and fractionated on
a 0.8 % agarose gel. Fragments smaller than 3 kb (A
DNA HindiII markers) were recovered from the gel
using the glassmilk procedure (BiolOl, Inc). Ana
logously molecules of BamHl digested, dephospho-
rylated Bluescript SK+ vector (from «Stratagene»,
USA) were recovered from the gel. The fragments
from the two sources were ligated in a ratio of 1:1
(total 40 ng of DNA used) at 4 °С overnight [11 ] and
were used to transform cells of competent E. coli
strain DH5a (from Bethesda Res. Lab). Diluted
aliquots of the transformed ceils were plated on LB
agar containing ampicillin at 50 jug/ml and X-gal and
IPTG thus allowing to determine the percent of
plasmids having DNA inserts [11].
The remainder of the transformants were ino
culated into 3 ml of LB supplemented with ampicillin
and incubated overnight at 37 °С. A plasmid library
was generated by harvesting the plasmids by alkaline
lysis [11]. The library obtained was used to trans
form both DH5a and HB-101 cells made competent
following the method of Hanahan 1121. The trans
formants were plated on M9 medium containing
different concentrations of glyphosate and all auxo
t roph ic additions required.
Results and Discussion. The frequency of the
induction of gly r mutants by NG for different E. coli
cells represented by recipient — donor and lysogenic-
nonlysogenic cells are shown in Table 2.
494
G E N E T I C MECHANISMS O F T H E R E S I S T A N C E OF E. coli
Table 3
Number of mutants tolerating increasing doses of glyphosate and AEC
From the results shown in Table 2 it can be
concluded that integration of different replicons into
the host chromosome slightly increased the proba
bility of a gly r mutation. The increase was more
pronounced in the strain with both F-factor and A
replicons integrated not far from each other (about 10
min on the E. coli chromosome map). As was shown
earlier, the mutations obtained in this strain map at
4 different loci [9].
On the basis of their tolerance to increasing doses
of the inhibitor, mutations obtained could be divided
into 2 groups, i. e. those tolerating low doses and
those tolerating higher doses. The frequency of the
first group is one order of magnitude higher than the
other. The frequency of mutants resistant to both
glyphosate and AEC is evident from Table 3.
The results in Table 3 demonstrate that almost
half of the mutants tolerating both low and higher
doses of glyphosate also tolerated a toxic analogue
lysine and thus were multiple-resistant.
Thus, gly r mutants appearing after NG treatment
constitute 4 different groups tolerating different doses
; 2
Fig. 1. Partia digestion of g ly r mutant genomic DNA with Sau3A
restriction endonuclease lanes: / — IDNA Hind III markers; 2—4 —
dynamics of a mutant DNA (7 /ug) hydrolysis for 20, 40 and 60 min
appropriately with 4 units of the enzyme
Fig. 2. Demonstration of the size of DNA fragments used for gene
library construction lanes: 7 — ADNA Hindlll markers; 2—4 — glyx
mutant genomic DNA (10 /ug) hydrolysed with Sau3A
Fig. 3. Purification of Bluescript SK vector DNA using glassmilk
procedure (B io l01 , Inc.)
495
C H E R E P E N K O E.
of glyphosate and cross-resistant to AEC. How many
genes are involved in this phenotype and what is their
nature?
Because mdr genes in eukaryotes are responsible
for multidrugresistance based on drug efflux out of
the cell (for rev. see [13]), and as such a gene should
be dominant over its wild type allele the cloning of
multiresistant gly r mutant DNA was tried. This mu
tant DNA was partially digested with Sau3A (Fig.
1), fragments of ~ 2 kb recovered from the gel (Fig.
2) and ligated to the vector (Fig. 3) (see Materials
and Methods). Thus, approximately 80 % of the
transformants produced due to the ligation product
were of white color on the indicator plates and the
number of inserts obtained was about 10 000. Using
this l ibrary, DH5a a n d HB-101 cells were
transformed and after washing with M9 medium
plated on selective medium containing 1 dose of
glyphosate. A couple of white colonies were obtained
in both cases but none of them were highly efficient
in retransformation experiments and consequently the
gene was not cloned. It this gene codes for a recessive
trait another approach should be used [9] and this
work is now in progress.
Acknowledgements. I appreciate the helpful dis
cussions with professor Nikolaus Amrhein (ETH,
Zurich). I thank professor Sydney Craig (UNC,
Chapel Hill) for letting me use his lab to construct the
gene library. The excellent technical assistance of
Oxana Karpenko is also gratefully acknowledged.
О. Й. Черепепко
Генетичні механізми стійкості клітин Escherichia coli
до амінокислотних антиметаболітів. 2. Вивчення частоти
індукції та властивостей гліфосатстійких мутантів
Резюме
Вивчено частоту індукції гліфосатстійких мутантів у ре-
ципіент-донорських, а також лізогенних-нелізогенних штамах
клітин Е. coli. Показано, що інтеграція вірусного, а також
більшого F-реплікона підвищує частоту індукції гліфосат
стійких мутантів від 1,6 до 6 разів. Мутанти, стійки до
0,2 мМ гліфосату, виникають у 10 разів частіше мутантів,
стійких до 1 мМ цього аналога гліцину. Половина мутантів
кожної групи стійка не лише до аналога гліцину, але й до
аналога лізину. Клонувати ген гліфосатстійкості не вдалося.
Досліджується домінантно-рецесивна природа цього гена.
Е. И. Черепенко
Генетические механизмы устойчивости клеток Escfierichia coli к
аминокислотным антиметаболитам. 2. Изучение частоты
индукции и свойства глифосатустойчивых мутантов
Резюме
Изучена частота индукции глифосатустойчивых мутантов в
реципиент-донорских, а также лизогенных-нелизогенных
штаммах. Показано, что интеграция вирусного и более круп
ного F-репликона увеличивает частоту индукции gly -мутан
тов от 1,6 до 6 раз. Мутанты, устойчивые к 0,2 мМ
глифосата, возникают в 10 раз чаще таковых, устойчивых к
1 мМ этого аналога глицина. Половина мутантов каждой
группы устойчива не только к аналогу глицина, но и к аналогу
лизина Клонировать ген глифосатустойчивости не удалось.
Изучается доминантно-рецессивная природа этого гена.
REFERENCES
1. Eakin A., Nieves-Alicea R., Tosado-Acevedo R. et al Com
parative compliment selection in bacteria enables screening for
lead compounds targeted to a purine salvage enzyme of
parasites / / Antimicrob. Agents and Chemother.—1995.—
3 9 . — P . 620—625
2. Craig S. Purine salvage enzymes as targets for the chemo-
theurapeutic treatment of parasitic diseases / / Biopolymers and
Cel l .—1994 .—10, N 6 .—P. 6 5 — 7 1 .
3. Bugg Ch., Carson W., Montgomery J. Drugs by design / / Sci.
Amer .—1993 .—269 .—P. 9 2 — 9 8 .
4. Steinrucken H., Amrhein N. The herbicide glyphosate is a
potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate
synthase / / Biochem. and Biophys. Res. Communs.—1980.—
9 4 . — P . 1207—1212 .
5. Kishore C , Shah D. Aminoacid biosynthesis inhibitors as
herbicides / / Ann. Rev. B iochem.—1988 .—57.—P. 627—663 .
6. Comai L., Sen JL, Stalker D. An altered aroA gene product
confers resistance to the herbicide glyphosate / / Science.—
1983 .—221 .—P. 3 7 0 — 3 7 1 .
7. Amrhein N., Johaning D., Schab J. et al. Biochemical basis for
glyphosate-tolerance in a bacterium and a plant tissue culture
/ / F E B S . — 1 9 8 3 . — 1 5 7 . — P . 191 — 196.
8. Cherepenko E. Gene amplication and herbicide-tolerance / /
Biopolymers and Ce l l .—1993 .—9, N 3 .—P. 3—16.
9. Cherepenko E., Karpenko O., Maliuta S. Genetic mechanisms
of Escherichia coli resistance to amino acid antimetabolites / /
Ib id .—1994.—10, N 4 .—P. 7 9 — 8 2 .
10. Miller J. Experiments in Molecular Genetics.—New York: Cold
Spring Harbor Lab., 1972.
11. Sambrook J., Fritsch E., Maniatis T. Molecular cloning. A
Laboratory Manual.—New York: Cold Spring Harbor Lab.,
1989.
12. Hanahan D. Studies in transformation of Escherichia coli with
plasmids / / J. Мої. B io l .—1983 .—166 .—P. 557—580 .
13. Endicott J., Ling V. The biochemistry of P-glycoprotein-
mediated multidrug resistance / / Annu. Rev. Biochem.—
1989 .—58 .—P. 137—149.
Received 24.02.97
496
|
| id | nasplib_isofts_kiev_ua-123456789-155687 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-11-28T12:16:37Z |
| publishDate | 1997 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Cherepenko, E.I. 2019-06-17T10:25:19Z 2019-06-17T10:25:19Z 1997 Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants / E. Cherepenko // Биополимеры и клетка. — 1997. — Т. 13, № 6. — С. 493-496. — Бібліогр.: 13 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0004AC https://nasplib.isofts.kiev.ua/handle/123456789/155687 The frequency of the induction by nttrosoguanidine of glyphosate resistant mutants was compared for recipient and donor, as well as for lysogenic and non-lysogenic E. coli cells. It was found that integration of viral genomes and also larger replicons such as F-factor into host chromosome increased the level of glyphosate resistance by the factor ranging from 1.6 to 6. Mutants tolerating 0.2 rnM of the inhibitor were obtained one order of magnitude more frequently than mutants tolerating 1 mM of this inhibitor. One half of the mutants of every group were resistant not only to the analogue of glycine,but also to the analogue of lysine. An attempt to clone an insertion from a gene library of one of the mutants was attempted but failed. Study on the nature of this gene is in progress. Вивчено частоту індукції гліфосатстійких мутантів у реципіент-донорських, а також лізогенних-нелізогенних штамах клітин Е. coli. Показано, що інтеграція вірусного, а також більшого F-реплікона підвищує частоту індукції гліфосат стійких мутантів від 1,6 до 6 разів. Мутанти, стійки до 0,2 мМ гліфосату, виникають у 10 разів частіше мутантів, стійких до 1 мМ цього аналога гліцину. Половина мутантів кожної групи стійка не лише до аналога гліцину, але й до аналога лізину. Клонувати ген гліфосатстійкості не вдалося. Досліджується домінантно-рецесивна природа цього гена. Изучена частота индукции глифосатустойчивых мутантов в реципиент-донорских, а также лизогенных-нелизогенных штаммах. Показано, что интеграция вирусного и более крупного F-репликона увеличивает частоту индукции gly -мутантов от 1,6 до 6 раз. Мутанты, устойчивые к 0,2 мМ глифосата, возникают в 10 раз чаще таковых, устойчивых к 1 мМ этого аналога глицина. Половина мутантов каждой группы устойчива не только к аналогу глицина, но и к аналогу лизина Клонировать ген глифосатустойчивости не удалось. Изучается доминантно-рецессивная природа этого гена. I appreciate the helpful discussions with professor Nikolaus Amrhein (ETH, Zurich). I thank professor Sydney Craig (UNC, Chapel Hill) for letting me use his lab to construct the gene library. The excellent technical assistance of Oxana Karpenko is also gratefully acknowledged. en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка Геном и его регуляция Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants Генетичні механізми стійкості клітин Escherichia coli до амінокислотних антиметаболітів. 2. Вивчення частоти індукції та властивостей гліфосатстійких мутантів Генетические механизмы устойчивости клеток Escherichia coli к аминокислотным антиметаболитам. 2. Изучение частоты индукции и свойства глифосатустойчивых мутантов Article published earlier |
| spellingShingle | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants Cherepenko, E.I. Геном и его регуляция |
| title | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants |
| title_alt | Генетичні механізми стійкості клітин Escherichia coli до амінокислотних антиметаболітів. 2. Вивчення частоти індукції та властивостей гліфосатстійких мутантів Генетические механизмы устойчивости клеток Escherichia coli к аминокислотным антиметаболитам. 2. Изучение частоты индукции и свойства глифосатустойчивых мутантов |
| title_full | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants |
| title_fullStr | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants |
| title_full_unstemmed | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants |
| title_short | Genetic mechanisms of the resistance of Escherichia coli to amino acid antimetabolites. 2. Study of the frequency of induction and properties of glyphosate resistant mutants |
| title_sort | genetic mechanisms of the resistance of escherichia coli to amino acid antimetabolites. 2. study of the frequency of induction and properties of glyphosate resistant mutants |
| topic | Геном и его регуляция |
| topic_facet | Геном и его регуляция |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/155687 |
| work_keys_str_mv | AT cherepenkoei geneticmechanismsoftheresistanceofescherichiacolitoaminoacidantimetabolites2studyofthefrequencyofinductionandpropertiesofglyphosateresistantmutants AT cherepenkoei genetičnímehanízmistíikostíklítinescherichiacolidoamínokislotnihantimetabolítív2vivčennâčastotiíndukcíítavlastivosteiglífosatstíikihmutantív AT cherepenkoei genetičeskiemehanizmyustoičivostikletokescherichiacolikaminokislotnymantimetabolitam2izučeniečastotyindukciiisvoistvaglifosatustoičivyhmutantov |