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Journal of Bacteriology, June 2000, p. 3289-3291, Vol. 182, No. 11
Department of Microbiology and Immunology,
Laboratory of Molecular Biology, University of South Alabama
College of Medicine, Mobile, Alabama 36688
Received 12 January 2000/Accepted 10 March 2000
Rickettsia prowazekii, the etiologic agent of epidemic
typhus, is an obligate, intracytoplasmic, parasitic bacterium.
Recently, the transformation of this bacterium via electroporation has
been reported. However, in these studies identification of
transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin
resistance or expression of the green fluorescent protein and flow
cytometric analysis. In this paper we describe the expression in
R. prowazekii of the Escherichia coli ereB
gene. This gene codes for an erythromycin esterase that cleaves
erythromycin. To the best of our knowledge, this is the first report of
the expression of a nonrickettsial, antibiotic-selectable gene in
R. prowazekii. The availability of a positive selection for
rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.
Rickettsia prowazekii,
the etiologic agent of epidemic typhus, is an obligate, intracellular,
parasitic bacterium. Unlike the obligate, intracellular bacteria
of the genera Chlamydia, Coxiella, and
Ehrlichia, R. prowazekii grows only within the
cytoplasm of the eucaryotic host cell rather than within an
intracytoplasmic vesicle (9, 20, 22). Rickettsiae are
capable of entering a wide range of eucaryotic cells by a process of
induced phagocytosis and are well adapted to exploit the cytoplasmic
environment (22). After reaching the cytoplasm they
transport high-energy compounds, such as ATP, using specialized
transport systems (21), but they also retain the ability to
generate ATP via an intact tricarboxylic acid cycle and oxidative phosphorylation.
Recently, the genome sequence of R. prowazekii was
published, providing a complete rickettsial genotype for analysis
(1). In addition, progress in the development of techniques
for the genetic manipulation of rickettsiae has been made. Rachek et
al. (13) described the transformation of R. prowazekii to rifampin resistance via electroporation of a
rickettsial rpoB gene containing a rifampin resistance
mutation. Similarly, Troyer et al. (18) described the
successful transformation of the closely related Rickettsia
typhi, using green fluorescent protein to screen for transformants. Unfortunately, a method for the positive selection of
R. prowazekii transformants, using an antibiotic resistance gene encoding a product that would inactivate or destroy the
antibiotic, has not been described.
Erythromycin is a macrolide antibiotic that binds the procaryotic
ribosome, inhibiting protein synthesis. R. prowazekii is sensitive to this drug in in vitro assays, and erythromycin can be used
in the laboratory since it is not recommended for the treatment of
R. prowazekii infections (14, 15). In clinical isolates ribosomal modification is the predominant mechanism of resistance to erythromycin, although active efflux of the drug and
antibiotic inactivation mechanisms have been identified (10, 11,
19). One example of resistance resulting from the latter mechanism is the production of esterases that hydrolyze the lactone ring of the erythromycin molecule. Two genes (ereA and
ereB) have been identified in Escherichia coli
that code for such esterases (2, 3, 12). Interestingly,
ereB exhibits a usage of A+T-rich codons, unusual for
E. coli but similar to that of rickettsial genes
(2). In this report we describe the use of ereB
as a selectable marker in R. prowazekii transformation
and demonstrate its insertion into a selected site of the
R. prowazekii genome.
Bacterial strains, plasmids, and oligonucleotides used in this
study are listed in Table 1.
E. coli strains were grown on Luria-Bertani medium
(4). When required for selection of E. coli transformants, the antibiotic ampicillin or erythromycin was
added to a final concentration of 50 or 200 µg/ml, respectively. R. prowazekii Madrid E strain seed pool passage 282 was used
for infecting mouse L929 fibroblasts. Rickettsia-infected L929 cells were grown in an atmosphere of 5% CO2 at 34°C in
modified Eagle medium supplemented with 10% newborn calf serum (Sigma,
St. Louis, Mo.) and 1 mM L-glutamine (Sigma). For
selection, erythromycin (Fisher Scientific, Pittsburgh, Pa.) was added
to supplemented modified Eagle medium at a final concentration of 200 ng/ml, and the erythromycin-containing medium was changed every 2 to 3 days. Rickettsial growth was monitored by microscopic examination of Gimenez-stained (8) infected cells growing on glass
coverslips. All DNA manipulations were performed as described
previously (13). PCR amplifications for detection of
R. prowazekii and integration of the transforming plasmid
into the rickettsial chromosome were performed with the oligonucleotide
primers listed in Table 1. For DNA sequencing, the PCR products
obtained were purified using a GeneClean II kit (Bio 101, La Jolla,
Calif.) and sequenced directly using a ThermoSequenase cycle
sequencing kit from Amersham Life Science, Inc. (Cleveland,
Ohio). Probes used in Southern hybridizations (16)
were 32P labeled using the Multiprime DNA labeling system
(Amersham) and [
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Transformation of Rickettsia prowazekii to
Erythromycin Resistance Encoded by the Escherichia coli
ereB Gene
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-32P]dATP (ICN, Irvine, Calif.).
TABLE 1.
Strains, plasmids, and oligonucleotides
The transforming plasmid used in these studies was constructed with the
plasmid pMOB (17) as the base replicon. A 735-bp portion of
the ampicillin resistance gene of pMOB was removed by SspI
and BsaI digestion and replaced with a 1,918-bp
HincII-SmaI fragment containing the E. coli
ereB gene from pAT72 (2). The resulting plasmid,
pMW1041, conferred an erythromycin resistance phenotype when introduced
into E. coli strains. To provide a target for homologous
recombination into the R. prowazekii genome, a 1,881-bp
R. prowazekii EcoRV fragment from plasmid pMW264
(23), containing the gltA gene with its
constitutive promoter (6), was cloned into the
EcoRV site of pMW1041. This generated the plasmid,
designated pMW1047 (Fig. 1), used in the
transformation experiments. The entire coding sequence of
gltA was included in this construct to ensure that a single
crossover event within this region would not destroy the gene but would
instead produce a gltA duplication.
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Following electroporation with pMW1047, the rickettsiae were allowed to infect L929 cells, which were then incubated for 24 h. At 24 h after infection, nearly 100% of the L929 cells were infected, with each host cell containing approximately 10 to 15 rickettsiae/cell. Erythromycin (200 ng/ml) was added, and incubation was continued. Rickettsiae were slowly cleared from the host cells following erythromycin addition. At 7 days postinfection, rickettsiae could not be visually detected on stained coverslips or were detected at very low levels. However, at 21 days rickettsiae could once again easily be detected visually, with approximately 1% of the host cells containing 200 to 300 rickettsiae per cell. The cell cultures were incubated until approximately 20% of the host cells were infected before the L929 cells were harvested. Rickettsiae were isolated for analysis of their chromosomal DNA. PCR assays using primers DW318 (located upstream from the gltA gene) and DW331 (located within the ereB gene), which would generate a PCR product only if pMW1047 was inserted into the chromosome at the gltA gene, yielded the predicted fragment when DNA from the erythromycin-resistant rickettsiae was used as a template (data not shown). Direct sequencing of this PCR fragment revealed that the fragment consisted of gltA and ereB sequences, confirming that ereB-containing rickettsial transformants were present in the Emr population.
Since genetic analysis requires the isolation of clones derived from a
single transformed bacterium, clonal isolates of rickettsial Emr transformants were obtained by limiting dilution.
Rickettsia-positive microtiter dish wells were identified by PCR using
primers targeted to a rickettsial chromosomal gene. Wells positive for
rickettsiae were then analyzed for the ereB gene using
ereB-specific primers DW337 and DW338 (Table 1). A single
clone, designated RPMOB.001, was selected for additional
characterization. The replication time of 10 h for this clone did
not differ from that of the Madrid E parent strain. However, the MIC of
erythromycin for RPMOB.001 was determined to be 2 µg/ml, in contrast
to 0.2 µg/ml for the Madrid E strain. Southern hybridization analysis
(16) revealed the changes in mobility of
gltA-hybridizing sequences predicted from the genome
sequence and restriction map of pMW1047 (Fig. 1). For Madrid E
chromosomal DNA, the expected HpaI, XbaI, and PstI fragments of 4,767, 8,971, and 7,742 bp,
respectively (Fig. 2, lanes 1, 3, and 5),
were observed. For RPMOB.001 DNA, the predicted fragment of 9,738 bp for HpaI, two fragments of 2,720 and 11,222 bp for
XbaI, and two fragments of 4,880 and 6,259 bp for
PstI were observed (Fig. 2, lanes 2, 4, and 6), confirming
the presence of a pMW1047 insertion at the gltA locus of
R. prowazekii. DNA sequencing of PCR products spanning this
region confirmed the insertion of the ereB gene into the
gltA locus. All of the clones obtained from transformations
using pMW1047 were found to have the gene inserted at the same site as
in RPMOB.001. No Emr clones were obtained in which pMW1047
was inserted at another chromosomal site or in which the plasmid
replicated autonomously.
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Initially, the efficiency of R. prowazekii erythromycin resistance transformation (successful entrance of DNA into the cell, successful recombination, and ereB expression) was low. We achieved only 1 successful experiment in 17 electroporations attempted. This is in contrast to the two of three successful rifampin resistance transformations obtained previously (13). This prompted us to evaluate our transformation conditions and generate a revised protocol, which yielded a success rate of two out of five electroporations. The revised protocol differs from that used in the rifampin selection transformations in several critical parameters. First, electroporation conditions were changed by increasing the field strength from 17 to 24 kV/cm. Rickettsial viability was reduced approximately 50% at the higher field strength. Second, five to seven times more L929 cells (1 × 108 to 1.5 × 108) were used to ensure that every electroporated rickettsia had the opportunity to infect a host cell. Finally, the number of rickettsiae per cell had to be evaluated to ensure selection. We discovered that erythromycin selection is dependent upon the number of rickettsiae per cell at the time of initial selection. If the number per cell is greater than 20 at the time of initial selection (24 h after selection), erythromycin at 200 ng/ml is unable to stop the growth of these rickettsiae at a rate sufficient to prevent them from destroying the host cell. Since reinfection by the rickettsiae is not efficient, lysis at this early stage, when transformants are few, results in the loss of potential transformants. At lower numbers per cell, the sensitive rickettsiae stop growing before the host cells lyse. Thus, the goal is to have one or two electroporated rickettsiae infect each host cell, followed by erythromycin selection at 24 h, when the rickettsial numbers will be less than 20 per host cell.
With the isolation of RPMOB.001, we have demonstrated that the E. coli ereB gene can be inserted at a selected target site into the R. prowazekii genome via homologous recombination. The identification of a selectable marker that can be targeted to any rickettsial gene of interest is a significant accomplishment in efforts to establish a genetic system in this organism and, to our knowledge, is the first demonstration of the expression of a nonrickettsial antibiotic resistance gene in R. prowazekii.
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ACKNOWLEDGMENTS |
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We thank Patrice Courvalin for providing pAT72, containing the ereB gene used in this study, and Priscilla Wyrick for helpful discussions on the use of erythromycin as a selective agent for rickettsiae.
This work was supported by NIH grant AI20384 to D.O.W.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Laboratory of Molecular Biology, University of South Alabama College of Medicine, Mobile, AL 36688. Phone: (334) 460-6324. Fax: (334) 460-7269. E-mail: wood{at}sungcg.usouthal.edu.
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