Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, February 2000, p. 437-440, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification and Characterization of the Point
Mutation Which Affects the Transcription Level of the Chromosomal
3-N-Acetyltransferase Gene of Streptomyces
griseus SS-1198
Jun
Ishikawa,*
Atsuko
Sunada,
Ritsuko
Oyama, and
Kunimoto
Hotta
Department of Bioactive Molecules, National
Institute of Infectious Diseases, Tokyo 162-8640, Japan
Received 9 April 1999/Returned for modification 18 July
1999/Accepted 4 October 1999
 |
ABSTRACT |
We determined the molecular basis for the enhanced expression of
the aac(3)-Xa gene encoding an aminoglycoside
3-N-acetyltransferase in Streptomyces griseus.
A C
T substitution was identified at the putative promoter of the
mutant gene. RNA analyses demonstrated that the substitution caused a
marked increase in the production of the gene-specific transcripts.
Therefore, it seemed very likely that the aac(3)-Xa gene
was activated by the substitution resulting in the emergence of a
stronger promoter.
| |
TEXT |
Aminoglycoside resistance genes are
frequently found on mobile genetic elements known as plasmids and
transposons, but in some cases they are found on chromosomes. The
latter case includes genes for aminoglycoside acetyltransferase (AAC)
enzymes, for example, the aac(2')-Ia gene of
Providencia stuartii (22), the aac(2')-Ib gene of Mycobacterium fortuitum
(1), the aac(2')-Ic gene of Mycobacterium
tuberculosis (2), the aac(2')-Id gene of
Mycobacterium smegmatis (2), the
aac(6')-Ic gene of Serratia marcescens
(25), the aac(6')-Ig gene of Acinetobacter
haemolyticus (19), the aac(6')-Ii gene of
Enterococcus faecium (5), and the
aac(6')-Ij gene of Acinetobacter sp. strain 13 (18). Since these chromosomal aac genes are
universally present in the respective species, it has been suggested
that chromosomal AAC enzymes have other metabolic functions in addition
to aminoglycoside acetylation. However, their primary role is still
unknown, with an exception. It has been demonstrated that AAC(2')-Ia
contributes to the O-acetylation of peptidoglycan in
P. stuartii (20).
Our studies have focused on the mechanism of aminoglycoside resistance
in actinomycetes, especially on AAC enzymes (9, 10, 13, 14).
Actinomycetes are a source of wide varieties of antibiotic resistance
genes as well as a treasure box of antibiotic-synthesizing pathways. We
have been investigating multiple resistances to aminoglycosides in
aminoglycoside-producing actinomycetes. Consequently, we demonstrated that there are aminoglycoside resistances that do not contribute to the
self resistance of aminoglycoside producers and thereby can be
designated secondary aminoglycoside resistance (13). An
aminoglycoside resistance gene in a streptomycin-producing soil
isolate, Streptomyces griseus SS-1198, is a case in point. This gene was initially discovered by the emergence of a kanamycin (Km)-resistant mutant through the protoplast regeneration treatment of
the wild-type strain SS-1198, which was unable to grow in the presence
of 5 µg of Km/ml. In previous studies, we have characterized a high
level (1,000 µg/ml) of Km resistance by the mutant SS-1198PR. Cloning
experiments revealed that the Km resistance gene, aac(3)-Xa (formerly kan), encodes an aminoglycoside
3-N-acetyltransferase, AAC(3) (12). DNA
hybridization experiments have demonstrated that the
aac(3)-Xa gene was located in the chromosome and was present
in all strains of S. griseus tested (11). On the
other hand, a wild-type allele from strain SS-1198 has also been cloned (16). Restriction analysis has shown no substantial
structural difference between wild-type and mutant gene fragments
(16), suggesting that the Km resistance phenotype of the
mutant gene was due to a point mutation of the wild-type gene. In this
paper, we identified the location of the point mutation by determining the sequence differences between them and demonstrated the marked enhancement of the aac(3)-Xa gene expression by the mutant.
Bacterial strains and plasmids.
S. griseus SS-1198 is a
soil isolate, and SS-1198PR strain is a Km-resistant mutant obtained
through protoplast regeneration treatment of SS-1198 (12,
16). Streptomyces lividans TK21 and pIJ702
(17) were used for recombinant DNA experiments as a host
strain and a vector plasmid, respectively.
Mapping the mutation.
To locate the mutation site of the
mutant aac(3)-Xa gene of strain SS-1198PR, hybrid genes were
constructed by swapping restriction fragments of the wild-type gene
with those of the mutant gene. The 1.8-kb
BglII-BamHI fragment, containing the wild-type or
mutant gene, was divided into 0.5-kb BglII-BamHI
and 1.3-kb BamHI fragments which contained mainly upstream
regions and structural genes, respectively. After the insertion of
these fragments into pIJ702 (17) with all possible
combinations, the hybrid genes were tested for their ability to confer
Km resistance to S. lividans TK21. As shown in Table
1, a high level (1,000 µg/ml) of Km
resistance was obtained only with genes containing the 0.5-kb
BglII-BamHI fragment derived from the mutant
gene. By contrast, genes containing the 0.5-kb fragment from the
wild-type gene conferred resistance to Km at concentrations as low as
50 µg/ml. Thus, the high level of Km resistance conferred by hybrid
genes depended upon the origin of the 0.5-kb
BglII-BamHI fragment, while no effect was
observed with the 1.3-kb BamHI fragment. This result
indicates that the mutation responsible for the Km resistance must be
present within the 0.5-kb BglII-BamHI fragment,
in which the putative promoter of the mutant aac(3)-Xa gene
is contained (15).
Comparing nucleotide sequences between the wild-type and mutant
genes.
To identify the precise position of the mutation, we
determined the nucleotide sequence of the 0.5-kb
BglII-BamHI fragments of the wild-type
aac(3)-Xa gene and compared it with that of the mutant gene
(15). It turned out that a C:G pair at the position of 
12
of the wild-type gene was replaced by a T:A pair in the mutant gene
(Fig. 1). It was noted that the
substitution occurred at the putative 10 sequence of the mutant
aac(3)-Xa gene (Fig. 1) (15).
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 1.
Comparison between upstream sequences of the wild-type
and mutant aac(3)-Xa genes. Single base substitution is
represented by an arrow. The BspHI site of the wild-type
sequence is overlined and the putative 10 sequence of the mutant gene
is underlined. The transcriptional starting point of the mutant gene
was treated as the +1 position (15).
|
|
We also determined the nucleotide sequence of the 1.3-kb
BamHI fragment of the wild-type gene and compared the
sequence with
that of the mutant gene. No difference between them was
observed
(data not
shown).
Further evidence for the single base substitution could be obtained
from restriction fragment length polymorphism, because
the substitution
of T for C in the mutant
aac(3)-Xa gene resulted
in the lack
of a
BspHI site in the wild-type sequence (Fig.
1).
Total
DNAs (5 µg each) from the wild-type and mutant strains were
digested
with
BspHI and
SphI, electrophoresed on 0.8%
agarose
gel, blotted onto a nylon membrane (Amersham Hybond-N+), and
hybridized
with the
32P-labeled 0.5-kb
BglII-
BamHI fragment of the mutant
aac(3)-Xa gene under the conditions described by Church and
Gilbert (
4).
For the wild-type strain, SS-1198, two bands
(2.4 and 7.9 kb)
were detected, but only one band (10.3 kb) was
observed for the
mutant strain SS-1198PR (data not shown). This result
clearly
shows the lack of the
BspHI site in the mutant
aac(3)-Xa gene.
Consequently, the substitution was confirmed
at the genomic DNA
level.
Detection of mRNA by slot blotting and reverse transcriptase
(RT)-PCR.
Since the point mutation at the corresponding region of
the 10 sequence of the mutant aac(3)-Xa gene was
confirmed, it was expected that the mutation affected transcription
level. To analyze this possibility, RNA was extracted from log phase
cells of the wild-type and mutant strains (8), and slot blot
hybridization experiments were carried out with the 0.5-kb
BglII-BamHI fragment of the aac(3)-Xa
gene. No transcripts in the wild-type cells were detectable under the
conditions used (Fig. 2, lane 1), while
the mutant clearly produced aac(3)-Xa RNA (Fig. 2, lane 2).
View larger version (72K):
[in this window]
[in a new window]
|
FIG. 2.
Slot blot hybridization. Lanes 1 and 2, RNA from the
wild-type (sensitive) S. griseus strain SS-1198 and
chromosomal mutant (resistant) of this strain, S. griseus
SS-1198PR, respectively; lane 3, S. lividans TK21 carrying
the cloned mutant gene on the plasmid pANT3-1 (12); lane 4, the plasmidless S. lividans TK21. The slot blot was probed
with the 32P-labeled 0.5-kb
BglII-BamHI fragment of the aac(3)-Xa
gene.
|
|
The wild-type sequence, CATGAT (Fig.
1), corresponding to
the putative
10 sequence of the mutant gene, falls into the CANNAT
class that has been found in other
Streptomyces 10 regions
(
3).
This led us to suspect that the wild-type gene must
produce a
small amount of transcripts. In order to demonstrate the
presence
of
aac(3)-Xa RNA in the wild-type strain, we
carried out RT-PCR,
which is a more sensitive method than filter
hybridization. Total
RNA was extracted from log phase cells with
Sepasol-RNA I (Nacalai
Tesque) according to the manufacturer's
instructions and treated
with DNase I (Takara Shuzo). Heat-denatured
RNA samples (
2 µg)
were reverse transcribed with 100 U of ReverTra
Ace (TOYOBO) and
2.5 µM KANP6 or STRP2 primer in a final volume of 20 µl at 42°C
for 30 min, 50°C for 30 min, and 55°C for 30 min.
PCR was carried
out with a 2-µl aliquot of the mixture, 1 µM
concentrations of
each primer, 2.5 U of AmpliTaq (GeneAmp PCR reagent
kit; Perkin-Elmer),
1.5 mM MgCl
2, and 10% (vol/vol)
dimethyl sulfoxide. PCR conditions
were 98°C for 3 min, 98°C for 1 min, 60°C for 1 min, 72°C for
2 min for 35 cycles, followed by
72°C for 5 min. Reaction products
were separated on a 1.5% agarose
gel.
RT-PCR experiments were performed with the KANP1 and KANP2 primers,
which were located at a mid-region of the
aac(3)-Xa gene
(Fig.
3A). RNase A-treated samples and
chromosomal DNA were also
used as negative and positive controls,
respectively, in addition
to the amplification of
aphD RNA
(
6) for monitoring the reaction.
As expected, a PCR product
of 353 bp was amplified. This result
indicates that the
aac(3)-Xa gene was expressed in the wild-type
strain.
However, it was possible that read-through products of
a gene upstream
of the
aac(3)-Xa gene would be amplified. We therefore
carried out RT-PCR experiments with a KANP3 or KANP4 primer (Fig.
3A),
whose 5' ends were located just at or 87 bp upstream from
the
transcriptional starting point of the mutant
aac(3)-Xa gene
(
15), respectively, instead of with the KANP1 primer. In
both
strains, KANP3-directed product (632 bp) was amplified but
KANP4-directed
product (719 bp) was not. Thus, it turned out that only
aac(3)-Xa gene-specific transcripts were detected in these
experiments.
The results suggest that the transcription starting point
of the
wild-type gene is the same as or in close proximity to that of
the mutant gene. This would be supported by missing promoter-like
sequences in the region-spanning primers KANP3 and KANP4.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 3.
RT-PCR analyses. (A) Schematic representation of the
locations of the primers. PCR products are indicated by bars with their
expected size. Nucleotide sequences of the primers are follows: KANP1,
5'-ACTCTGTTGGTCACCTGCGG-3'; KANP2,
5'-TGCAGCAGCGTCATCGTGTC-3'; KANP3,
5'-CCCCAGTCCGTGTTCCGG-3'; KANP4,
5'-CTGATAGTCGAGAAAGGCCC-3'; KANP6,
5'-AGCATGGAACCGACGATCAC-3'; STRP1,
5'-ACCACGACGAGGAGAGCAG-3'; STRP2,
5'-TTCCGTCAGCAGGTCGAAG-3'. (B) Amplification of
aac(3)-Xa RNA with KANP1 and KANP2 (lanes 1 to 3) and of
aphD RNA with STRP1 and STRP2 (lanes 4 and 5) in the
wild-type strain. Two micrograms of RNA was reverse transcribed with
KANP6 (lanes 1 and 2) or STRP2 primer (lane 4), and a 2-µl aliquot
was used for amplification. RNase A-treated RNA (lane 1) and 250 ng of
total DNA (lanes 3 and 5) were used as negative and positive controls,
respectively. HaeIII digestion of  X174 DNA served as the
size standard (lanes M). (C) Amplification of aac(3)-Xa RNA
with KANP1 (lanes 1 and 4), KANP3 (lanes 2 and 5) and KANP4 (lanes 3 and 6) as 5' primers in the wild-type (lanes 1 to 3) and mutant (lanes
4 to 6) strains HaeIII digestion of X174 DNA served as
the size standard (lanes M).
|
|
Concluding remarks.
We identified the single base substitution
responsible for the activation of the aac(3)-Xa gene. Slot
blot analysis demonstrated that this mutation resulted in a marked
increase in the transcription level of the aac(3)-Xa gene in
the mutant strain, compared with that in the wild-type strain. The
substitution of T for C in the mutant gene led to the formation of a
sequence, TATGAT, that is more similar to the prokaryote
[TATAAT] and Streptomyces
[TA(G/C)(G/C/A)(G/T)T] 10 consensus sequences
(3). Moreover, searching the transcription factor database
(7) showed that there was no recognition site for a
prokaryotic transcription regulator in the proximity of the mutation
site (data not shown). It is therefore suggested that the substitution
of T for C at the first position (12) of the putative 10 sequence
resulted in the emergence of a stronger promoter. However, we cannot
rule out the possibility of the mutation creating a new promoter rather
than creating a stronger promoter because the 5' ends of
aac(3)-Xa gene-specific transcripts were not identified in
the wild-type strain.
aac(3)-Xa RNA could be detected in the wild-type cells by
using RT-PCR, whereas the wild-type strain SS-1198 is sensitive
to Km.
It seems likely that the expression of the
aac(3)-Xa gene
in
the wild-type cells is too weak to contribute to its Km resistance.
In
contrast, the cloning of the wild-type gene with a high-copy-number
vector demonstrated that the wild-type gene could confer a low
level
(50 µg/ml) of Km resistance to
S. lividans, suggesting
that
the difference between resistance levels conferred by the
wild-type
gene and the mutant genes would be due not to derepression in
the heterologous host but to a gene dosage effect. However, we
cannot
rule out the presence of negative regulators, such as
aar factors which regulate the expression of the
aac(2')-Ia gene
in
P. stuartii (
21-24). We have currently found
that protoplast regeneration
treatment is not necessary for obtaining
Km-resistant mutants,
because we isolated such mutants without
protoplasting (unpublished
data). In preliminary experiments, the
majority of the mutants
have DNA amplification as a possible mechanism
for Km resistance
(
16). One such mutant was found to have
the same base substitution
described in this paper. However, several
mutants have neither
DNA amplification nor promoter mutations. These
observations may
suggest the existence of a novel regulatory factor,
such as a
repressor, for the expression of the
aac(3)-Xa
gene in
S. griseus.
Further studies to analyze this
possibility are in
progress.
The
aac(3)-Xa gene exists in all strains of
S. griseus tested (
11), suggesting that the gene has a
primary function other
than aminoglycoside modification. This would
agree with the result
that the gene was expressed in the wild-type
strain. Although
only a small amount of the gene-specific RNA was
detected in the
wild-type strain, the expression of the gene may be
controlled
by specific timing. Identification of such timing may yield
interesting
information on the regulation of the
acc(3)-Xa gene.
| |
ACKNOWLEDGMENTS |
We thank N. Summers and K. Summers for correcting the manuscript
and the late K. Kawaguchi for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan. Phone: 81 3 (5285) 1111. Fax:
81 3 (5285) 1272. E-mail: jun{at}nih.go.jp.
| |
REFERENCES |
| 1.
|
Ainsa, J. A.,
C. Martin,
B. Gicquel, and R. Gomez-Lus.
1996.
Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.
Antimicrob. Agents Chemother.
40:2350-2355[Abstract].
|
| 2.
|
Ainsa, J. A.,
E. Perez,
V. Pelicic,
F. X. Berthet,
B. Gicquel, and C. Martin.
1997.
Aminoglycoside 2'-N-acetyltransferase genes are universally present in mycobacteria: characterization of the aac(2')-Ic gene from Mycobacterium tuberculosis and the aac(2')-Id gene from Mycobacterium smegmatis.
Mol. Microbiol.
24:431-441[CrossRef][Medline].
|
| 3.
|
Bourn, W. R., and B. Babb.
1995.
Computer assisted identification and classification of streptomycete promoters.
Nucleic Acids Res.
23:3696-3703[Abstract/Free Full Text].
|
| 4.
|
Church, G. M., and W. Gilbert.
1984.
Genomic sequencing.
Proc. Natl. Acad. Sci. USA
81:1991-1995[Abstract/Free Full Text].
|
| 5.
|
Costa, Y.,
M. Galimand,
R. Leclercq,
J. Duval, and P. Courvalin.
1993.
Characterization of the chromosomal aac(6')-Ii gene specific for Enterococcus faecium.
Antimicrob. Agents Chemother.
37:1896-1903[Abstract/Free Full Text].
|
| 6.
|
Distler, J.,
A. Ebert,
K. Mansouri,
K. Pissowotzki,
M. Stockmann, and W. Piepersberg.
1992.
Gene cluster for streptomycin biosynthesis in Streptomyces griseus: nucleotide sequence of three genes and analysis of transcriptional activity.
Nucleic Acids Res.
15:8041-8056[Abstract/Free Full Text].
|
| 7.
|
Ghosh, D.
1991.
New developments of a transcription factors database.
Trends Biochem. Sci.
16:445-447[CrossRef][Medline].
|
| 8.
|
Hopwood, D. A.,
M. J. Bibb,
K. F. Chater,
T. Kieser,
C. J. Bruton,
H. M. Kieser,
D. J. Lydiate,
C. P. Smith,
J. M. Ward, and H. Schrempf.
1985.
Genetic manipulation of Streptomyces: a laboratory manual, p. 214-220.
The John Innes Foundation, Norwich, England.
|
| 9.
|
Hotta, K.,
A. Sunada,
J. Ishikawa,
S. Mizuno,
Y. Ikeda, and S. Kondo.
1998.
The novel enzymatic 3"-N-acetylation of arbekacin by an aminoglycoside 3-N-acetyltransferase of Streptomyces origin and the resulting activity.
J. Antibiot.
51:735-742[Medline].
|
| 10.
|
Hotta, K.,
C.-B. Zhu,
T. Ogata,
A. Sunada,
J. Ishikawa,
S. Mizuno,
Y. Ikeda, and S. Kondo.
1996.
Enzymatic 2'-N-acetylation of arbekacin and antibiotic activity of its product.
J. Antibiot.
49:458-464[Medline].
|
| 11.
|
Hotta, K., and J. Ishikawa.
1988.
Strain- and species-specific distribution of the streptomycin gene cluster and kan-related sequences in Streptomyces griseus.
J. Antibiot.
41:1116-1123[Medline].
|
| 12.
|
Hotta, K.,
J. Ishikawa,
M. Ichihara,
H. Naganawa, and S. Mizuno.
1988.
Mechanism of increased kanamycin-resistance generated by protoplast regeneration of Streptomyces griseus. I. Cloning of a gene segment directing a high level of an aminoglycoside 3-N-acetyltransferase activity.
J. Antibiot.
41:94-103[Medline].
|
| 13.
|
Hotta, K.,
J. Ishikawa,
T. Ogata, and S. Mizuno.
1992.
Secondary aminoglycoside resistance in aminoglycoside-producing strains of Streptomyces.
Gene
115:113-117[CrossRef][Medline].
|
| 14.
|
Hotta, K.,
T. Ogata,
J. Ishikawa,
M. Okanishi,
S. Mizuno,
M. Morioka,
H. Naganawa, and Y. Okami.
1996.
Mechanism of multiple aminoglycoside resistance of kasugamycin-producing Streptomyces kasugaensis MB273: involvement of two types of acetyltransferases in resistance to astromicin group antibiotics.
J. Antibiot.
49:682-688[Medline].
|
| 15.
|
Ishikawa, J., and K. Hotta.
1991.
Nucleotide sequence and transcriptional start point of the kan gene encoding an aminoglycoside 3-N-acetyltransferase from Streptomyces griseus SS-1198PR.
Gene
108:127-132[CrossRef][Medline].
|
| 16.
|
Ishikawa, J.,
Y. Koyama,
S. Mizuno, and K. Hotta.
1988.
Mechanism of increased kanamycin-resistance generated by protoplast regeneration of Streptomyces griseus. II. Mutational alteration and gene amplification.
J. Antibiot.
41:104-112[Medline].
|
| 17.
|
Katz, E.,
C. J. Thompson, and D. A. Hopwood.
1983.
Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans.
J. Gen. Microbiol.
129:2703-2714[Abstract/Free Full Text].
|
| 18.
|
Lambert, T.,
G. Gerbaud, and P. Courvalin.
1994.
Characterization of the chromosomal aac(6')-Ij gene of Acinetobacter sp. 13 and the aac(6')-Ih plasmid gene of Acinetobacter baumannii.
Antimicrob. Agents Chemother.
38:1883-1889[Abstract/Free Full Text].
|
| 19.
|
Lambert, T.,
G. Gerbaud,
M. Galimand, and P. Courvalin.
1993.
Characterization of Acinetobacter haemolyticus aac(6')-Ig gene encoding an aminoglycoside 6'-N-acetyltransferase which modifies amikacin.
Antimicrob. Agents Chemother.
37:2093-2100[Abstract/Free Full Text].
|
| 20.
|
Payei, K. G.,
P. N. Rather, and A. J. Clarke.
1995.
Contribution of gentamicin 2'-N-acetyltransferase to the O acetylation of peptide glycan in Providencia stuartii.
J. Bacteriol.
177:4303-4310[Abstract/Free Full Text].
|
| 21.
|
Rather, P. N., and E. Orosz.
1994.
Characterization of aarA, a pleiotropic negative regulator of the 2'-N-acetyltransferase in Providencia stuartii.
J. Bacteriol.
176:5140-5144[Abstract/Free Full Text].
|
| 22.
|
Rather, P. N.,
E. Orosz,
K. J. Shaw,
R. Hare, and G. Miller.
1993.
Characterization and transcriptional regulation of the 2'-N-acetyltransferase gene from Providencia stuartii.
J. Bacteriol.
175:6492-6498[Abstract/Free Full Text].
|
| 23.
|
Rather, P. N.,
M. R. Paradise,
M. M. Parojcic, and S. Patel.
1998.
A regulatory cascade involving AarG, a putative sensor kinase, controls the expression of the 2'-N-acetyltransferase and an intrinsic multiple antibiotic resistance (Mar) response in Providencia stuartii.
Mol. Microbiol.
28:1345-1353[CrossRef][Medline].
|
| 24.
|
Rather, P. N.,
K. A. Solinsky,
M. R. Paradise, and M. M. Parojcic.
1997.
aarC, an essential gene involved in density-dependent regulation of the 2'-N-acetyltransferase in Providencia stuartii.
J. Bacteriol.
179:2267-2273[Abstract/Free Full Text].
|
| 25.
|
Shaw, K. J.,
P. N. Rather,
F. J. Sabatelli,
P. Mann,
H. Munayyer,
R. Mierzwa,
G. L. Petrikkos,
R. S. Hare,
G. H. Miller,
P. Bennett, and P. Downey.
1992.
Characterization of the chromosomal aac(6')-Ic gene from Serratia marcescens.
Antimicrob. Agents. Chemother.
36:1447-1455[Abstract/Free Full Text].
|
Antimicrobial Agents and Chemotherapy, February 2000, p. 437-440, Vol. 44, No. 2
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Top
Abstract
Text
