Borrelia burgdorferi ftsZ Plays a Role in Cell Division
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《细菌学杂志》
Departments of Microbiology and Immunology,Pathology, New York Medical College, Valhalla, New York 10595
ABSTRACT
ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZBbu). Its gene product (FtsZBbu) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZBbu might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZBbu antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZBbu could interfere with the function of E. coli FtsZ, ftsZBbu was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZBbu generated a filamentous phenotype. This suggested interference of ftsZBbu with E. coli FtsZ function and confirmed the role of ftsZBbu in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.
INTRODUCTION
Knowledge regarding cell division in bacteria has been primarily obtained using gram-positive and gram-negative bacteria as experimental models (8, 12, 41). Most bacterial cells grow in length with little change in cell diameter, until they reach a critical size that is generally twice their original length (14, 57). Cell division is then initiated in the middle diameter of the cell with the formation of a contractile ring comprised largely of FtsZ (4, 6, 14, 51, 57). The role of cell division genes has not been directly explored in spirochetes (41). However, null mutations in the recA gene in Leptospira spp. and the cfpA gene in Treponema spp. apparently interfered with cell division as judged by morphological alterations (25, 52). The Borrelia burgdorferi genome contains a homologue of ftsZ (ftsZBbu) (18) and the B. burgdorferi protein (FtsZBbu) encoded by ftsZBbu is highly homologous to FtsZ proteins of other bacteria (4, 6, 8, 16, 22, 39, 57, 59), but the physiologic roles of ftsZBbu and FtsZBbu in cell division is unknown.
FtsZ is essential for cell division and acts early in this process in Escherichia coli (12, 14, 33). High-temperature treatment of ftsZ temperature-sensitive mutant cells in E. coli results in complete and immediate cessation of division and formation of filamentous cells that lack visible constriction points and the contractile ring (8, 14). FtsZ is also rate limiting for septum initiation. Moderate increases in its level results in a minicell phenotype because there is an increase in division frequency at the cell poles and the average cell resulting from these divisions are smaller, suggesting that septation mediated by FtsZ is occurring earlier in the cell cycle (56). High levels of FtsZ, on the other hand, completely inhibit division (34, 36). The ftsZ gene is also essential for cell division in gram-positive organisms. In Bacillus subtilis, ftsZ is required for both cell division and the formation of the sporulation septum (4), while in Streptomyces coelicolor, ftsZ is required for septation but not for viability (47).
The lack of information regarding mechanisms of cell division and morphogenesis in B. burgdorferi and other spirochetes (41) is due at least in part to a lack of molecular genetic tools relative to species such as E. coli and B. subtilis. Despite the recent development of a number of genetic methods for manipulating B. burgdorferi (11, 17, 45), cell division mutants of B. burgdorferi have not yet been isolated, nor is it known whether they are lethal (which would preclude their isolation) (15, 23, 40, 55). Isolation of conditionally lethal mutants in other bacteria has permitted isolation of mutants in genes that play an essential role in bacterial metabolism or are essential for bacterial survival (23, 24, 27, 50, 58). Isolation of conditional lethal mutants as a genetic tool in B. burgdorferi has been limited by the lack of knowledge regarding the nutritional requirements of this bacterium (10, 45). Furthermore, its slow growth and the inability to use solid replica plating makes rapid identification of mutant bacteria unable to grow in limiting media difficult (10).
A regulatory system based on the tet operon of the E. coli Tn10 transposon (5, 19-21, 54) has been widely used for tight regulation of eukaryotic and prokaryotic gene expression (3, 5, 32, 50, 58) and permits analysis of conditionally lethal mutants (15, 26, 27, 29). In the absence of tetracycline or its nonantibiotic analogues such as anhydrotetracycline (ATc), the TetR repressor binds to the tetO operator that has been fused to the promoter of a target gene. Binding of TetR inhibits binding of RNA polymerase and transcription of the target gene. In the presence of tetracycline or its analogues, TetR conformation is altered so that it cannot bind to tetO, RNA polymerase can bind to the target gene promoter, and transcription occurs (5, 19-21). Gene expression in this system is tetracycline dose dependent, so that changes in its concentration permit variations in target gene expression with titration of the biological effects of a regulated phenotype (5, 54). Because the affinity of tetracycline and its derivatives is 1,000- to 100,000-fold greater for TetR than for the ribosome, induction of TetR-controlled genes occurs well before antibiotic-induced ribosomal inhibition (5).
The Tet system has been used to regulate expression of antisense RNA (asRNA) in bacteria and indirectly modulate gene expression so as to permit titration of gene expression and generate a functional equivalent of conditionally lethal mutants (26, 27, 29, 31, 35). This has permitted study of the in vitro and in vivo expression of multiple genes (including virulence genes) in S. aureus, Clostridium difficile, and mycobacteria (15, 26, 27, 58). Antisense RNA molecules also play a role in the natural regulation of gene expression of many bacteria, including Borrelia spp. (42). To date, neither Tet-regulated systems nor asRNA has been used to manipulate gene expression in Borrelia.
We have now applied the regulated control of gene expression by ATc, an approach used by others to obtain tightly regulated control of gene expression in bacteria (5, 15), to isolate the physiological equivalent of conditional lethal mutants. This has been accomplished by blocking FtsZBbu synthesis by regulated gene expression of asRNA to ftsZBbu (26, 27, 58). These experiments suggest that FtsZBbu plays a role in cell division in B. burgdorferi and that ftsZBbu expression in particular and B. burgdorferi gene expression in general can be negatively controlled by the use of tetracycline-regulated expression of asRNA.
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. E. coli DH5Z was obtained from H. Bujard (32). E. coli TOP10 and pCR2.1-TOPO were purchased (TOPO TA cloning kit, Invitrogen, Carlsbad, Calif.). Low-passage infectious B. burgdorferi strain 297 was provided by M. V. Norgard (1). pKFSS1 was provided by S. Samuels (17). E. coli cells were grown in Luria-Bertani (LB) broth (Gibco-BRL, Gaithersburg, MD). B. burgdorferi was grown in BSK-H medium (Sigma, St. Louis, Mo.) with 6% rabbit serum (Sigma).
DNA manipulations. DNA manipulations were performed by standard methods (46). All enzymes used in plasmid constructions were obtained from New England Biolabs, Beverly, MA. Total DNA was purified from cultures using the High Pure PCR Template Preparation kit (Roche, Mannheim, Germany), DNA restriction fragment isolation and PCR product purification was done using QIAquick gel extraction kit (QIAGEN, Valencia, Calif.); all methods were done according to the manufacturers' instructions. PCR amplification parameters were: denaturation for 2 min at 94°C for 1 cycle, followed by 38 cycles of 94°C for 10 s, 53°C for 10 s, and 72°C for 2 min, with a final extension at 68°C for 5 min. Oligonucleotide primers used in the present study were purchased (GenoSys Biotechnology, The Woodlands, Tex.). The structure of all constructs was confirmed by restriction enzyme analysis and by PCR amplifications with appropriate primers and DNA sequence analysis of amplicons. All primers used in the present study are listed in Table 1.
Construction of plasmid for creation of B. burgdorferi 297 containing chromosomal tetR. tetR was amplified by PCR from E. coli DH5Z chromosomal DNA using forward and reverse primers TetR1 and TetR2 and the B. burgdorferi B31 flaB promoter was amplified from total B. burgdorferi DNA with primers FlaB1 and FlaB2 (Table 1). These PCR products were digested with KpnI, ligated, and cloned into pCR2.1-TOPO (Invitrogen) to yield pCR2.1/tetR. This construct was electroporated into E. coli TOP10 (Invitrogen) and selected on ampicillin plates according to the manufacturer's instructions. The structure of tetR under the control of the flaB promoter was confirmed by DNA sequencing. tetR was inserted in the nonessential chromosomal luxS gene (BB0377) (7) as follows (Fig. 1A). A PCR fragment containing tetR under the flaB promoter was then amplified from pCR2.1/tetR with primers TetR3 and TetR4 (Table 1). TetR3 was designed to contain a sequence homologous to the aph(3')-IIIa kanamycin resistance gene from Enterococcus faecalis (53), TetR4 was designed to contain a sequence homologous to the B. burgdorferi luxS gene. A second PCR fragment containing aph(3')-IIIa under the control of its own promoter was amplified from pAT112 (53) by using primers KanR1 and KanR2 (Table 1). KanR1 was designed to contain a sequence homologous to B. burgdorferi luxS, KanR2 was designed to contain a sequence homologous to tetR. A third PCR fragment containing approximately 900 bp of the 3' end of B. burgdorferi metK (BB0376) and the 5' region of luxS was amplified from total DNA B. burgdorferi 297 with the primers LuxS1 and LuxS2; LuxS2 was designed to contain a sequence homologous to tetR. A fourth PCR fragment containing the 3' end of luxS and approximately 1,050 bp of 5' sequence of the BB0378 gene was amplified from B. burgdorferi strain 297 with primers LuxS3 and LuxS4 (Table 1); LuxS3 was designed to contain a sequence homologous to aph(3')-IIIa. All DNA fragments were purified with a QIAquick gel extraction kit, mixed together, and fused by long PCR (48) using the primers LuxS1 and LuxS4. To minimize polar effects, tetR and the kanamycin resistance marker aph(3')-IIIa were inserted in the opposite orientation to that of luxS. The resulting PCR product was cloned into pCR2.1-TOPO. The recombinant plasmid was electroporated into E. coli TOP10, and electroporants were selected on LB agar plates containing ampicillin and kanamycin according to the manufacturer's instructions. Plasmid DNA from ampicillin-kanamycin-resistant clones was purified. The presence of the insertion was detected by PCR with the primers LuxS5 and LuxS6, and its sequence was confirmed by restriction and sequence analysis.
Construction of plasmids for ATc-regulated expression. To create a hybrid promoter (Ptetl) that could interact with TetR and function in B. burgdorferi, nucleotides –190 to –35 bp upstream from the B. burgdorferi B31 bmpA starting codon were amplified from B. burgdorferi total DNA by PCR using forward and reverse primers Ptetl1 and Ptetl2 and purified (Table 1). A second amplicon containing the Tn10 tetO sequences fused to PbmpA on either side of the bmpA –35 promoter sequence was generated by PCR of B. burgdorferi total DNA using forward and reverse primers Ptetl3 and Ptetl4 and purified. Ptetl3 (Table 1) contained tetO DNA sequences (wavy underlines) on either side of the the –35 promoter. Ptetl4 (Table 1) contained ATG fused to nucleotides 120 to 139 of bmpA (double underline). These amplicons were fused by using long PCR (48) to generate the hybrid promoter Ptetl (Fig. 1B). This construct was purified, and its structure was confirmed by DNA sequencing. To create pLD6 (Fig. 1B), a plasmid containing DNA sequences coding for asftsZBbu RNA, primers Ptet1 and AftsZ were used in long PCR (48) to generate an amplicon with Ptetl fused to asftsZ (Fig. 1B). Primer AftsZ (Table 1) contained a KpnI site (underlined), a universal terminator sequence (boldface), asftsZBbu DNA sequences, and a sequence complementary to the Ptetl promoter (italicized). The resulting PCR product was cloned into pCR2.1-TOPO and subsequently electroporated into E. coli TOP10. Plasmid DNA from electroporants selected with ampicillin on LB agar plates was purified, and the DNA fragment containing Ptetl fused to asftsZBbu was excised and subcloned into the KpnI and PstI sites of pKFSS1 (Fig. 1B). To create pLD7 (Fig. 1C), DNA containing the complete ftsZBbu gene was amplified from B. burgdorferi genomic DNA and fused with a DNA segment containing Ptetl by long PCR (48) using primers FtsZ1 and FtsZ2 and purified. Primer FtsZ1 (Table 1) contained a DNA sequence complementary to Ptetl (italics). Primer FtsZ2 (Table 1) contained a KpnI site (underlined). This amplicon was fused to Ptetl using long PCR (48) and Tet1 and FtsZ2 as forward and reverse primers. The resulting amplicon was cloned into pCR2.1-TOPO, transformed into E. coli DH5Z and selected on LB agar plates with 50 μg of ampicillin/ml (Fig. 1C). Structures of all plasmid constructs were confirmed by PCR amplification and DNA sequence analysis.
Electroporation of B. burgdorferi. B. burgdorferi 297 was grown to mid-log phase (1 x 107 to 2 x 107 cells/ml) and electroporated with 10 to 30 μg of recombinant plasmid DNA. After overnight recovery, the electroporated cells were diluted and 1 x 106 to 2 x 106 cells in 200 μl of complete BSK-H medium containing 400 μg of kanamycin/ml for selection of clones of B. burgdorferi containing tetR and 400 μg of kanamycin/ml and 50 μg of streptomycin/ml for selection of clones containing 297/tetR and pLD6 were distributed in each well of 96 microwell plates (Corning, Inc., Corning, N.Y.). After 14 to 21 days, B. burgdorferi showing growth in microwells were cultured in 1 ml of complete BSK-H with appropriate antibiotics. DNA was extracted from these cultures by using High Pure PCR template preparation kit (Roche Diagnostics Corp., Indianapolis, Ind.). DNA of kanamycin-resistant colonies of B. burgdorferi 297/tetR was analyzed by PCR for the presence of the insertion of tetR-flaB promoter-kanamycin gene fusion using primers LuxS5 and LuxS6 to confirm it was the result of a double crossover. The fusion of asftsZBbu with the Ptetl promoter was confirmed by sequence analysis using Ptetl1.
Detection of TetR by immunoblotting. Total proteins of B. burgdorferi 297/tetR were extracted from 1 x 107 to 2 x 107 cells by lysing them in Laemmli buffer. Lysate proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting (49) with rabbit anti-E. coli TetR polyclonal antibody (a generous gift from Kai Schnig). Immunoblots were developed with alkaline phosphate-conjugated goat anti-rabbit immunoglobulin G (Jackson Immunoresearch, West Grove, Pa.) and enhanced chemifluorescence technology according to the manufacturer's instructions (ECF Western blotting kit; GE Healthcare Amersham Biosciences, Piscataway, N.J.), and read by using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).
Real-time quantitative reverse transcription-PCR (RT-PCR) analysis. Total RNA was prepared from B. burgdorferi 297 by using TRIzol (Invitrogen) according to the manufacturer's instructions. All RNA samples were treated with DNase I (Promega Corp., Madison, Wis.) to remove genomic DNA. DNA-free mRNA (50 to 100 ng in a volume of 20 μl) was reverse transcribed by using a reverse transcription system kit (Promega) according to the manufacturer's instructions. Reverse-transcribed samples were denatured for 5 min at 95°C and stored at –20°C until use. ftsZ mRNA and flaB mRNA were quantified by using an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, Calif.). TaqMan reactions were performed in a volume of 25 μl using the TaqMan Universal PCR Master Mix (Applied Biosystems). TaqMan probes and forward and reverse primers were designed with Primer Express 2 software (Applied Biosystems). For ftsZ quantitation, FtsZ3 and FtsZ4 were used as forward and reverse primers and TET/TAMRA was used as the probe (Table 1). For flaB quantitation, FlaB3 and FlaB4 were used as forward and reverse primers and 6FAM/TAMRA was used as the probe (Table 1). PCRs were performed under the following conditions: 50°C for 2 min, followed by 95°C for 10 min, and then 45 cycles of 95°C for 15 s and 60°C for 1 min. The threshold cycle (CT) for ftsZ and flaB mRNA was calculated by Sequence Detection software (Applied Biosystems); levels of ftsZ mRNA relative to flaB reference mRNA were calculated as described previously (43). PCR amplification was confirmed by measurement of amplicon sizes of ftsZ and flaB by agarose gel electrophoresis.
Growth of B. burgdorferi 297 and its derivatives. Cultures were inoculated at a final concentration of 105 cell/ml in 3 ml of complete BSK-H medium containing 6% rabbit serum, incubated at 34°C in 1% CO2 in the absence or presence of inducer to final concentrations of 108 cells/ml. Growth of B. burgdorferi cells was assessed by counting cells stained with acridine orange under a fluorescence microscope (49).
Microscopic analyses of cell length. Cultures of B. burgdorferi 297/tetR and derivatives containing pLD6 and pKFSS1 (106 cells/ml) were stained with acridine orange and examined by fluorescence microscopy without or with induction with 1.5 μg of ATc/ml. B. burgdorferi cells in three independent slides were examined (magnification, x1,250), and the cells in 10 fields (ca. 100 cells per field) in each slide were counted and assessed for the presence of the filamentous phenotype. Spirochete lengths on photomicrographs of similar fields were also measured, and a stage micrometer was used to convert these lengths to μm. A similar approach was used to analyze E. coli DH5Z and E. coli DH5Z(pLD7).
Statistical analysis. The effect of addition of ATc to B. burgdorferi 297 and derivatives in culture from three independent experiments was analyzed by a two-way analysis of variance with a repeated measures Bonferroni posttest. Significance levels were set at P < 0.05.
RESULTS
Comparison of ftsZBbu gene product and other bacterial FtsZ proteins. B. burgdorferi ftsZ open reading frame (ORF) codes for a protein with a molecular mass of 42,971 Da. Its deduced amino acid sequence shows strong similarity to other bacterial FtsZ amino acid sequences particularly over the first 312 codons and has 50, 50, 46, and 46% identity with FtsZ proteins of Treponema pallidum, B. subtilis, Leptospira interrogans, and E. coli, respectively (Fig. 2). Within the highly conserved N terminus region, FtsZBbu has a Gly-rich block, G118MGGGTGTG126, identical to the characterized GTP-binding site of E. coli FtsZ (37), and a N223IDFADV229 consensus sequence that is part of a putative GTP-hydrolyzing domain (13, 39). The C-terminal moiety of the borrelial FtsZ homologue also contains a D385DDIDVPTFLR395 conserved region that is thought to be important for interaction with FtsA and ZipA (33). The gene product of ftsZBbu is thus highly homologous to its counterparts in other bacteria.
Construction of a B. burgdorferi 297 strain expressing TetR from a chromosomally inserted tetR gene. Construction of the tetR insertion is shown in Fig. 1 and is described in detail in Materials and Methods. Electroporation of pCR2.1-TOPO containing the luxS flanking regions, tetR, and aph(3')-IIIa into B. burgdorferi 297 yielded four kanamycin-resistant clones; results from one of these clones are shown in Fig. 3A. The tetR insertion into the luxS gene was confirmed by PCR analysis, which showed that longer amplicons were generated from clones with the inserted gene than from wild-type B. burgdorferi 297. Immunoblotting demonstrated the presence of TetR (Fig. 3B). One of the clones was used for all subsequent studies.
Development of filamentous phenotype in B. burgdorferi in response to ATc-regulated production of asftsZ RNA. Construction of pLD6 to mediate regulated synthesis of asftsZ RNA in B. burgdorferi 297/tetR is shown in Fig. 1 and is described in detail in Materials and Methods. pLD6 contained an asDNA fragment complementary to the initial 62 nucleotides of the ftsZBbu coding strand under the control of the Ptetl promoter, a hybrid promoter containing two tetO sequences fused with bmpA promoter sequences (Fig. 1B). To prevent readthrough from the asDNA, a termination signal was placed at the end of the antisense sequence. After electroporation of pLD6 into B. burgdorferi 297/tetR, clones containing pLD6 were selected by using appropriate antibiotics. Twenty-four hours after expression of asftsZBbu RNA was induced in mid-log-phase cultures of B. burgdorferi 297/tetR(pLD6) by the addition of ATc, large numbers of abnormally elongated, filamentous spirochetes were visible under the microscope (Fig. 4C). We were unable to see any septations in these cells. No such cells were present in uninduced cultures of B. burgdorferi 297/tetR (pLD6) (Fig. 4B) or in control cultures of B. burgdorferi 297/tetR in the presence (Fig. 4A) or absence (not shown) of ATc.
ATc-induced B. burgdorferi 297/tetR (pLD6) (Fig. 4D) were significantly longer than uninduced B. burgdorferi 297/tetR (pLD6) or control B. burgdorferi 297/tetR in the absence or presence of ATc (P < 0.001). Although uninduced B. burgdorferi 297/tetR (pLD6) were slightly longer than uninduced or induced B. burgdorferi 297/tetR (compare Fig. 4C with Fig. 4A and B), this difference was not statistically significant (Fig. 4D). By 24hof induction, filamentous cells accounted for 78 ± 11% (mean ± standard error [SE]) of cells examined in cultures of B. burgdorferi 297/tetR (pLD6) and were significantly more frequent (P < 0.001) than in uninduced B. burgdorferi 297/tetR (pLD6) (25 ± 7%) or in control induced or uninduced B. burgdorferi 297/tetR cultures at this time (14 ± 3% and 19 ± 3%, respectively). At 48 h after ATc induction, the percentage of filamentous cells in ATc-induced cultures of B. burgdorferi 297/tetR (pLD6) had fallen to 48 ± 4% and remained at this level at 72 h. We were unable to detect formation of the division septum in any of these cells. These values were significantly less than at 24 h (P < 0.01) but still significantly higher than the percentages of filamentous spirochetes in uninduced cultures of B. burgdorferi 297/tetR (pLD6) (23 ± 2%) or in control induced or uninduced B. burgdorferi 297/tetR cultures (14 ± 1% and 13 ± 1%, respectively).
B. burgdorferi 297/tetR showed no significant differences in growth in the absence or presence of ATc (Fig. 4E). Growth of B. burgdorferi 297/tetR (pLD6) was significantly less (P < 0.001) than that of B. burgdorferi 297/tetR in the absence of 1.5 μg of ATc/ml (Fig. 4E). However, growth of this recombinant strain was even more inhibited in the presence of ATc (P < 0.001). This suggests that the growth and cell division of B. burgdorferi was slowed by the lack of FtsZ protein occasioned by induced asftsZBbu.
Quantitation of ftsZBbu mRNA after induction of production of asftsZBbu RNA. The filamentous borrelial phenotype was most probably a result of the inability of these cells to divide because of the lack of FtsZBbu generated by the induced asftsZBbu. As a first step toward understanding the mechanism of action of induced asftsZ RNA, ftsZBbu mRNA was examined by RT-PCR and real-time RT-PCR. RT-PCR indicated that induction of asftsZBbu RNA by 1.5 μg of ATc/ml for 24 h was associated with a sharp decrease in ftsZBbu mRNA compared to levels in uninduced B. burgdorferi. Culture of B. burgdorferi 297/tetR in the presence of ATc had no obvious effect on production of ftsZ mRNA (Fig. 5A). These results were quantitatively confirmed by real-time RT-PCR. Inhibition of ftsZ mRNA by induced production of asftsZ in B. burgdorferi 297/tetR (pLD6) was highly significant after 6 h of induction with ATc (Fig. 5B), indicating that the induction of asftsZBbu is associated with decreased ftsZBbu mRNA levels in these bacteria.
Expression of B. burgdorferi ftsZ in E. coli. Overexpression of autologous FtsZ in E. coli generates a minicell phenotype, whereas high levels of expression of heterologous FtsZ result in a filamentous phenotype (6, 34, 36, 55). To determine whether ftsZBbu could be ectopically expressed in E. coli and whether its gene product would interfere with E. coli FtsZ function, pLD7, containing ftsZBbu under the control of the Ptetl hybrid promoter (Fig. 1), was electroporated into E. coli DH5. No bacteria carrying pLD7 (Fig. 1C) were obtained after electroporation of pLD7 into E. coli lacking expression of TetR (data not shown), but electroporation of pLD7 into E. coli DH5Z expressing TetR (32) was successful. This could suggest that uncontrolled expression of FtsZBbu in E. coli was lethal. By 12 h after induction of synthesis of ectopic FtsZBbu in E. coli DH5Z (pLD7) with 1.5 μg of ATc/ml, numerous extremely long filamentous cells were visible (Fig. 6C). No septations were visible in these E. coli cells. No filamentous cells were detected in control cultures of E. coli DH5Z producing TetR in the absence (not shown) or presence of ATc (Fig. 6A). Small numbers of filamentous cells were also present in E. coli DH5Z (pLD7) in uninduced cultures (Fig. 6B), but they were much shorter than the filamentous cells seen in induced cultures (compare Fig. 6B and C), and their presence did not significantly increase mean bacterial length in these cultures (Fig. 6D). Both shorter and longer filamentous cells contained denser areas consistent with contractile rings (Fig. 6C and D). These experiments indicate that ectopically produced FtsZBbu can interfere with the function of the E. coli FtsZ protein in E. coli.
DISCUSSION
Although the deduced ftsZBbu gene product is highly homologous to other bacterial FtsZ proteins, the activity of ftsZBbu and FtsZBbu in cell division has not been previously examined. By combining tetracycline-regulated control of gene expression, a technique developed for eukaryotic cells but rarely used in bacteria (3), with antisense technology (29, 31, 35), it was possible to study the functionality of ftsZBbu and confirm its activity in cell division. Blocking of FtsZBbu production by ATc-mediated induction of asftsZBbu RNA resulted in decreased ftsZBbu mRNA, a filamentous phenotype, and slow growth. Furthermore, regulated ectopic expression of ftsZBbu in E. coli resulted in the appearance of filamentous E. coli as a result of functional protein interference of FtsZBbu with cell division proteins of E. coli, thus providing additional confirmation for the functionality of ftsZBbu in cell division.
It was not unexpected that the ATc-mediated downregulation of ftsZBbu expression resulted in a filamentous phenotype and slow growth in B. burgdorferi, since in the absence of FtsZ in other bacteria, the Z ring is not formed and there is a failure to assembly the complete divisome needed for cell division (34, 40, 55, 59). The observed low levels of ftsZ mRNA under the conditions of ATc-induced asRNA provided confirmation that the inhibition of ftsZBbu expression mediated by induced asftsZ was specific for production of FtsZBbu. Attempts to detect the presence of B. burgdorferi FtsZ protein in wild-type B. burgdorferi using cross-reacting anti-E. coli FtsZ rabbit antibodies in immunoblots of B. burgdorferi lysate proteins were unsuccessful (data not shown). This failure might be due to a lack of complete cross-reactivity of this antibody for the B. burgdorferi and E. coli FtsZ proteins or to the low levels of FtsZBbu present under the conditions of growth.
The functional role of the B. burgdorferi FtsZ homologue and its involvement in B. burgdorferi cell division was also indirectly confirmed by our inability to obtain transformants with pLD7 in E. coli not expressing TetR. (pLD7 contained FtsZBbu under the control of the TetR-susceptible Ptetl hybrid borrelial promoter). In contrast, transformants with pLD7 were easily obtained in the E. coli strain that expressed TetR. In this connection, it should be mentioned that many B. burgdorferi promoters are recognized by the E. coli protein synthesis machinery (11, 17, 49). These results suggested that constitutive expression of B. burgdorferi FtsZ was lethal for E. coli, as has been shown to be the case with other heterologously expressed bacterial FtsZ proteins (14). This hypothesis was confirmed by the appearance of filamentous E. coli after induction of expression of FtsZBbu in E. coli (pLD7), indicating inhibition of E. coli cell division by FtsZBbu (59). These findings indicate that FtsZBbu is a cell division protein with function similar to its homologues in other bacteria and thus able to block cell division when expressed in a heterologous background (30, 34, 36, 38, 47, 55, 59). This functional identity of the B. burgdorferi ftsZ gene and FtsZ protein with other bacterial homologues is fully consistent with the amino acid sequence and domain identity it shares with them (30, 36, 59).
B. burgdorferi is a genetically intractable organism. Its genome is unstable. There are no natural systems of horizontal gene transfer in spirochetes and, consequently, no simple and efficient method for introducing DNA into this bacterium. Its nutritional requirements are still incompletely known so that simple and defined media for its culture are lacking, and it grows slowly in the complex media that are available for its culture. Molecular genetic study of the B. burgdorferi genome has been hampered by the dearth of genetic tools to isolate and complement mutants and to manipulate gene expression although many strides have been made to remedy this in recent years (11, 45). It has therefore been difficult to isolate conditional lethal mutants in B. burgdorferi, a type of mutant that has been crucial for the development of genetic systems and physiological studies in other bacteria (5, 23, 26, 27, 32, 35, 45, 50, 58, 59).
Many advances to manipulate the genome of B. burgdorferi have been the result of adaptation of genetic systems developed in other bacteria to this pathogen (11, 45). The present study of the function of ftsZBbu was only possible because we were able to adapt two genetic tools developed for the study of other bacteria, namely, TetR-regulated control of gene expression and asRNA technology (3, 27, 31, 32, 35, 58). In adapting this system to B. burgdorferi, the tetR gene was placed under the control of the constitutive B. burgdorferi flaB promoter, and this DNA construct was inserted in the chromosome in luxS, a gene not required for B. burgdorferi growth and infectiousness (7); the DNA segment encoding antisense ftsZ RNA under the hybrid Ptetl TetR-responsive promoter was located extrachromosomally in a plasmid (17). The chromosomal location of the tetR gene under a constitutive promoter is preferable to a plasmid location. Variations in gene dosage as result of variations in copy number are minimized in the chromosomal location, thus ensuring that the synthesis of TetR will remain constant throughout the cell division cycle (32, 44). By permitting regulated expression of ftsZ asRNA, this system has for the first time generated the functional equivalent of a conditionally lethal mutant in B. burgdorferi.
B. burgdorferi cells containing both components of the Tet system regulated the expression of ftsZ asRNA and FtsZ in an ATc-dependent manner as measured by levels of ftsZ mRNA (Fig. 5). The reduced levels of FtsZ were associated with inhibition of cell division demonstrated by the emergence of B. burgdorferi cells with the filamentous phenotype and slow growth (Fig. 4). However, TetR repressor levels encoded from the B. burgdorferi chromosome were not totally able to suppress expression of ftsZ asRNA from the Ptetl hybrid promoter since uninduced cultures of B. burgdorferi 297/tetR (pLD6) contained cells that were slightly longer (Fig. 4B and D) and grew significantly more slowly (Fig. 4E) than the B. burgdorferi 297/tetR controls. We do not believe that filamentous cells divide after the blocking of cell division, and we have some preliminary evidence suggesting that they in fact are dead (data not shown). This escape of repression by TetR was evident in E. coli pLD7, where small numbers of filamentous cells were visible in the absence of ATc induction (Fig. 6B). It might be a result of titration of the TetR repressor by an increased number of the extrachromosomally located molecules of Ptetl generated by variations in the copy number of the pKFSS1 plasmid (2). However, we have previously demonstrated that pKFSS1 has a copy number of one relative to the B. burgdorferi chromosome (9), suggesting that other mechanisms may play a role in the imbalance generated between numbers of TetR molecules and Ptetl promoters (28). These might include changes in pKFSS1 supercoiling, which might decrease the access of the repressor to a region in the plasmid where Ptetl is located (2). Titration of the levels of TetR by excessive amounts of Ptetl molecules is a problem that could be readily corrected by placing an increased number of copies of the tetR gene in the chromosome and by putting its transcription under the control of a stronger B. burgdorferi constitutive promoter. Alternatively, this imbalance could be potentially corrected by placing both the tetR repressor gene and the putative gene controlled by the Ptetl promoter in the chromosome (15).
In summary, this is the first report to show the feasibility of the regulated control of gene expression in B. burgdorferi and the usefulness of asRNA for generating the physiologic equivalent of conditionally lethal mutants in this pathogen. These experiments suggest that FtsZBbu plays a role in cell division in B. burgdorferi and that ftsZBbu expression in particular, and gene expression in general, can be negatively controlled in this species by the use of tetracycline-regulated expression of asRNA.
ACKNOWLEDGMENTS
We thank H. Bujard for advice and for E. coli DH5Z and plasmids containing tetO and tetR; Kai Schnig for TetR antisera; Miguel Vincente, Pilar Palacios, and William Margolin for anti-E. coli FtsZ antisera; Dionysios Liveris for many useful discussions; Radha Iyer for help with real-time PCR; and Harriett Harrison for secretarial assistance.
This study was supported by grant R01 AI08856 (to F.C.C.) from the National Institutes of Health.
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ABSTRACT
ftsZ is essential for cell division in many microorganisms. In Escherichia coli and Bacillus subtilis, FtsZ plays a role in ring formation at the leading edge of the cell division septum. An ftsZ homologue is present in the Borrelia burgdorferi genome (ftsZBbu). Its gene product (FtsZBbu) is strongly homologous to other bacterial FtsZ proteins, but its function has not been established. Because loss-of-function mutants of ftsZBbu might be lethal, the tetR/tetO system was adapted for regulated control of this gene in B. burgdorferi. Sixty-two nucleotides of an ftsZBbu antisense DNA sequence under the control of a tetracycline-responsive modified hybrid borrelial promoter were cloned into pKFSS1. This construct was electroporated into a B. burgdorferi host strain carrying a chromosomally located tetR under the control of the B. burgdorferi flaB promoter. After induction by anhydrotetracycline, expression of antisense ftsZ RNA resulted in generation of filamentous B. burgdorferi that were unable to divide and grew more slowly than uninduced cells. To determine whether FtsZBbu could interfere with the function of E. coli FtsZ, ftsZBbu was amplified from chromosomal DNA and placed under the control of the tetracycline-regulated hybrid promoter. After introduction of the construct into E. coli and induction with anhydrotetracycline, overexpression of ftsZBbu generated a filamentous phenotype. This suggested interference of ftsZBbu with E. coli FtsZ function and confirmed the role of ftsZBbu in cell division. This is the first report of the generation of a B. burgdorferi conditional lethal mutant equivalent by tetracycline-controlled expression of antisense RNA.
INTRODUCTION
Knowledge regarding cell division in bacteria has been primarily obtained using gram-positive and gram-negative bacteria as experimental models (8, 12, 41). Most bacterial cells grow in length with little change in cell diameter, until they reach a critical size that is generally twice their original length (14, 57). Cell division is then initiated in the middle diameter of the cell with the formation of a contractile ring comprised largely of FtsZ (4, 6, 14, 51, 57). The role of cell division genes has not been directly explored in spirochetes (41). However, null mutations in the recA gene in Leptospira spp. and the cfpA gene in Treponema spp. apparently interfered with cell division as judged by morphological alterations (25, 52). The Borrelia burgdorferi genome contains a homologue of ftsZ (ftsZBbu) (18) and the B. burgdorferi protein (FtsZBbu) encoded by ftsZBbu is highly homologous to FtsZ proteins of other bacteria (4, 6, 8, 16, 22, 39, 57, 59), but the physiologic roles of ftsZBbu and FtsZBbu in cell division is unknown.
FtsZ is essential for cell division and acts early in this process in Escherichia coli (12, 14, 33). High-temperature treatment of ftsZ temperature-sensitive mutant cells in E. coli results in complete and immediate cessation of division and formation of filamentous cells that lack visible constriction points and the contractile ring (8, 14). FtsZ is also rate limiting for septum initiation. Moderate increases in its level results in a minicell phenotype because there is an increase in division frequency at the cell poles and the average cell resulting from these divisions are smaller, suggesting that septation mediated by FtsZ is occurring earlier in the cell cycle (56). High levels of FtsZ, on the other hand, completely inhibit division (34, 36). The ftsZ gene is also essential for cell division in gram-positive organisms. In Bacillus subtilis, ftsZ is required for both cell division and the formation of the sporulation septum (4), while in Streptomyces coelicolor, ftsZ is required for septation but not for viability (47).
The lack of information regarding mechanisms of cell division and morphogenesis in B. burgdorferi and other spirochetes (41) is due at least in part to a lack of molecular genetic tools relative to species such as E. coli and B. subtilis. Despite the recent development of a number of genetic methods for manipulating B. burgdorferi (11, 17, 45), cell division mutants of B. burgdorferi have not yet been isolated, nor is it known whether they are lethal (which would preclude their isolation) (15, 23, 40, 55). Isolation of conditionally lethal mutants in other bacteria has permitted isolation of mutants in genes that play an essential role in bacterial metabolism or are essential for bacterial survival (23, 24, 27, 50, 58). Isolation of conditional lethal mutants as a genetic tool in B. burgdorferi has been limited by the lack of knowledge regarding the nutritional requirements of this bacterium (10, 45). Furthermore, its slow growth and the inability to use solid replica plating makes rapid identification of mutant bacteria unable to grow in limiting media difficult (10).
A regulatory system based on the tet operon of the E. coli Tn10 transposon (5, 19-21, 54) has been widely used for tight regulation of eukaryotic and prokaryotic gene expression (3, 5, 32, 50, 58) and permits analysis of conditionally lethal mutants (15, 26, 27, 29). In the absence of tetracycline or its nonantibiotic analogues such as anhydrotetracycline (ATc), the TetR repressor binds to the tetO operator that has been fused to the promoter of a target gene. Binding of TetR inhibits binding of RNA polymerase and transcription of the target gene. In the presence of tetracycline or its analogues, TetR conformation is altered so that it cannot bind to tetO, RNA polymerase can bind to the target gene promoter, and transcription occurs (5, 19-21). Gene expression in this system is tetracycline dose dependent, so that changes in its concentration permit variations in target gene expression with titration of the biological effects of a regulated phenotype (5, 54). Because the affinity of tetracycline and its derivatives is 1,000- to 100,000-fold greater for TetR than for the ribosome, induction of TetR-controlled genes occurs well before antibiotic-induced ribosomal inhibition (5).
The Tet system has been used to regulate expression of antisense RNA (asRNA) in bacteria and indirectly modulate gene expression so as to permit titration of gene expression and generate a functional equivalent of conditionally lethal mutants (26, 27, 29, 31, 35). This has permitted study of the in vitro and in vivo expression of multiple genes (including virulence genes) in S. aureus, Clostridium difficile, and mycobacteria (15, 26, 27, 58). Antisense RNA molecules also play a role in the natural regulation of gene expression of many bacteria, including Borrelia spp. (42). To date, neither Tet-regulated systems nor asRNA has been used to manipulate gene expression in Borrelia.
We have now applied the regulated control of gene expression by ATc, an approach used by others to obtain tightly regulated control of gene expression in bacteria (5, 15), to isolate the physiological equivalent of conditional lethal mutants. This has been accomplished by blocking FtsZBbu synthesis by regulated gene expression of asRNA to ftsZBbu (26, 27, 58). These experiments suggest that FtsZBbu plays a role in cell division in B. burgdorferi and that ftsZBbu expression in particular and B. burgdorferi gene expression in general can be negatively controlled by the use of tetracycline-regulated expression of asRNA.
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. E. coli DH5Z was obtained from H. Bujard (32). E. coli TOP10 and pCR2.1-TOPO were purchased (TOPO TA cloning kit, Invitrogen, Carlsbad, Calif.). Low-passage infectious B. burgdorferi strain 297 was provided by M. V. Norgard (1). pKFSS1 was provided by S. Samuels (17). E. coli cells were grown in Luria-Bertani (LB) broth (Gibco-BRL, Gaithersburg, MD). B. burgdorferi was grown in BSK-H medium (Sigma, St. Louis, Mo.) with 6% rabbit serum (Sigma).
DNA manipulations. DNA manipulations were performed by standard methods (46). All enzymes used in plasmid constructions were obtained from New England Biolabs, Beverly, MA. Total DNA was purified from cultures using the High Pure PCR Template Preparation kit (Roche, Mannheim, Germany), DNA restriction fragment isolation and PCR product purification was done using QIAquick gel extraction kit (QIAGEN, Valencia, Calif.); all methods were done according to the manufacturers' instructions. PCR amplification parameters were: denaturation for 2 min at 94°C for 1 cycle, followed by 38 cycles of 94°C for 10 s, 53°C for 10 s, and 72°C for 2 min, with a final extension at 68°C for 5 min. Oligonucleotide primers used in the present study were purchased (GenoSys Biotechnology, The Woodlands, Tex.). The structure of all constructs was confirmed by restriction enzyme analysis and by PCR amplifications with appropriate primers and DNA sequence analysis of amplicons. All primers used in the present study are listed in Table 1.
Construction of plasmid for creation of B. burgdorferi 297 containing chromosomal tetR. tetR was amplified by PCR from E. coli DH5Z chromosomal DNA using forward and reverse primers TetR1 and TetR2 and the B. burgdorferi B31 flaB promoter was amplified from total B. burgdorferi DNA with primers FlaB1 and FlaB2 (Table 1). These PCR products were digested with KpnI, ligated, and cloned into pCR2.1-TOPO (Invitrogen) to yield pCR2.1/tetR. This construct was electroporated into E. coli TOP10 (Invitrogen) and selected on ampicillin plates according to the manufacturer's instructions. The structure of tetR under the control of the flaB promoter was confirmed by DNA sequencing. tetR was inserted in the nonessential chromosomal luxS gene (BB0377) (7) as follows (Fig. 1A). A PCR fragment containing tetR under the flaB promoter was then amplified from pCR2.1/tetR with primers TetR3 and TetR4 (Table 1). TetR3 was designed to contain a sequence homologous to the aph(3')-IIIa kanamycin resistance gene from Enterococcus faecalis (53), TetR4 was designed to contain a sequence homologous to the B. burgdorferi luxS gene. A second PCR fragment containing aph(3')-IIIa under the control of its own promoter was amplified from pAT112 (53) by using primers KanR1 and KanR2 (Table 1). KanR1 was designed to contain a sequence homologous to B. burgdorferi luxS, KanR2 was designed to contain a sequence homologous to tetR. A third PCR fragment containing approximately 900 bp of the 3' end of B. burgdorferi metK (BB0376) and the 5' region of luxS was amplified from total DNA B. burgdorferi 297 with the primers LuxS1 and LuxS2; LuxS2 was designed to contain a sequence homologous to tetR. A fourth PCR fragment containing the 3' end of luxS and approximately 1,050 bp of 5' sequence of the BB0378 gene was amplified from B. burgdorferi strain 297 with primers LuxS3 and LuxS4 (Table 1); LuxS3 was designed to contain a sequence homologous to aph(3')-IIIa. All DNA fragments were purified with a QIAquick gel extraction kit, mixed together, and fused by long PCR (48) using the primers LuxS1 and LuxS4. To minimize polar effects, tetR and the kanamycin resistance marker aph(3')-IIIa were inserted in the opposite orientation to that of luxS. The resulting PCR product was cloned into pCR2.1-TOPO. The recombinant plasmid was electroporated into E. coli TOP10, and electroporants were selected on LB agar plates containing ampicillin and kanamycin according to the manufacturer's instructions. Plasmid DNA from ampicillin-kanamycin-resistant clones was purified. The presence of the insertion was detected by PCR with the primers LuxS5 and LuxS6, and its sequence was confirmed by restriction and sequence analysis.
Construction of plasmids for ATc-regulated expression. To create a hybrid promoter (Ptetl) that could interact with TetR and function in B. burgdorferi, nucleotides –190 to –35 bp upstream from the B. burgdorferi B31 bmpA starting codon were amplified from B. burgdorferi total DNA by PCR using forward and reverse primers Ptetl1 and Ptetl2 and purified (Table 1). A second amplicon containing the Tn10 tetO sequences fused to PbmpA on either side of the bmpA –35 promoter sequence was generated by PCR of B. burgdorferi total DNA using forward and reverse primers Ptetl3 and Ptetl4 and purified. Ptetl3 (Table 1) contained tetO DNA sequences (wavy underlines) on either side of the the –35 promoter. Ptetl4 (Table 1) contained ATG fused to nucleotides 120 to 139 of bmpA (double underline). These amplicons were fused by using long PCR (48) to generate the hybrid promoter Ptetl (Fig. 1B). This construct was purified, and its structure was confirmed by DNA sequencing. To create pLD6 (Fig. 1B), a plasmid containing DNA sequences coding for asftsZBbu RNA, primers Ptet1 and AftsZ were used in long PCR (48) to generate an amplicon with Ptetl fused to asftsZ (Fig. 1B). Primer AftsZ (Table 1) contained a KpnI site (underlined), a universal terminator sequence (boldface), asftsZBbu DNA sequences, and a sequence complementary to the Ptetl promoter (italicized). The resulting PCR product was cloned into pCR2.1-TOPO and subsequently electroporated into E. coli TOP10. Plasmid DNA from electroporants selected with ampicillin on LB agar plates was purified, and the DNA fragment containing Ptetl fused to asftsZBbu was excised and subcloned into the KpnI and PstI sites of pKFSS1 (Fig. 1B). To create pLD7 (Fig. 1C), DNA containing the complete ftsZBbu gene was amplified from B. burgdorferi genomic DNA and fused with a DNA segment containing Ptetl by long PCR (48) using primers FtsZ1 and FtsZ2 and purified. Primer FtsZ1 (Table 1) contained a DNA sequence complementary to Ptetl (italics). Primer FtsZ2 (Table 1) contained a KpnI site (underlined). This amplicon was fused to Ptetl using long PCR (48) and Tet1 and FtsZ2 as forward and reverse primers. The resulting amplicon was cloned into pCR2.1-TOPO, transformed into E. coli DH5Z and selected on LB agar plates with 50 μg of ampicillin/ml (Fig. 1C). Structures of all plasmid constructs were confirmed by PCR amplification and DNA sequence analysis.
Electroporation of B. burgdorferi. B. burgdorferi 297 was grown to mid-log phase (1 x 107 to 2 x 107 cells/ml) and electroporated with 10 to 30 μg of recombinant plasmid DNA. After overnight recovery, the electroporated cells were diluted and 1 x 106 to 2 x 106 cells in 200 μl of complete BSK-H medium containing 400 μg of kanamycin/ml for selection of clones of B. burgdorferi containing tetR and 400 μg of kanamycin/ml and 50 μg of streptomycin/ml for selection of clones containing 297/tetR and pLD6 were distributed in each well of 96 microwell plates (Corning, Inc., Corning, N.Y.). After 14 to 21 days, B. burgdorferi showing growth in microwells were cultured in 1 ml of complete BSK-H with appropriate antibiotics. DNA was extracted from these cultures by using High Pure PCR template preparation kit (Roche Diagnostics Corp., Indianapolis, Ind.). DNA of kanamycin-resistant colonies of B. burgdorferi 297/tetR was analyzed by PCR for the presence of the insertion of tetR-flaB promoter-kanamycin gene fusion using primers LuxS5 and LuxS6 to confirm it was the result of a double crossover. The fusion of asftsZBbu with the Ptetl promoter was confirmed by sequence analysis using Ptetl1.
Detection of TetR by immunoblotting. Total proteins of B. burgdorferi 297/tetR were extracted from 1 x 107 to 2 x 107 cells by lysing them in Laemmli buffer. Lysate proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining or immunoblotting (49) with rabbit anti-E. coli TetR polyclonal antibody (a generous gift from Kai Schnig). Immunoblots were developed with alkaline phosphate-conjugated goat anti-rabbit immunoglobulin G (Jackson Immunoresearch, West Grove, Pa.) and enhanced chemifluorescence technology according to the manufacturer's instructions (ECF Western blotting kit; GE Healthcare Amersham Biosciences, Piscataway, N.J.), and read by using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).
Real-time quantitative reverse transcription-PCR (RT-PCR) analysis. Total RNA was prepared from B. burgdorferi 297 by using TRIzol (Invitrogen) according to the manufacturer's instructions. All RNA samples were treated with DNase I (Promega Corp., Madison, Wis.) to remove genomic DNA. DNA-free mRNA (50 to 100 ng in a volume of 20 μl) was reverse transcribed by using a reverse transcription system kit (Promega) according to the manufacturer's instructions. Reverse-transcribed samples were denatured for 5 min at 95°C and stored at –20°C until use. ftsZ mRNA and flaB mRNA were quantified by using an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, Calif.). TaqMan reactions were performed in a volume of 25 μl using the TaqMan Universal PCR Master Mix (Applied Biosystems). TaqMan probes and forward and reverse primers were designed with Primer Express 2 software (Applied Biosystems). For ftsZ quantitation, FtsZ3 and FtsZ4 were used as forward and reverse primers and TET/TAMRA was used as the probe (Table 1). For flaB quantitation, FlaB3 and FlaB4 were used as forward and reverse primers and 6FAM/TAMRA was used as the probe (Table 1). PCRs were performed under the following conditions: 50°C for 2 min, followed by 95°C for 10 min, and then 45 cycles of 95°C for 15 s and 60°C for 1 min. The threshold cycle (CT) for ftsZ and flaB mRNA was calculated by Sequence Detection software (Applied Biosystems); levels of ftsZ mRNA relative to flaB reference mRNA were calculated as described previously (43). PCR amplification was confirmed by measurement of amplicon sizes of ftsZ and flaB by agarose gel electrophoresis.
Growth of B. burgdorferi 297 and its derivatives. Cultures were inoculated at a final concentration of 105 cell/ml in 3 ml of complete BSK-H medium containing 6% rabbit serum, incubated at 34°C in 1% CO2 in the absence or presence of inducer to final concentrations of 108 cells/ml. Growth of B. burgdorferi cells was assessed by counting cells stained with acridine orange under a fluorescence microscope (49).
Microscopic analyses of cell length. Cultures of B. burgdorferi 297/tetR and derivatives containing pLD6 and pKFSS1 (106 cells/ml) were stained with acridine orange and examined by fluorescence microscopy without or with induction with 1.5 μg of ATc/ml. B. burgdorferi cells in three independent slides were examined (magnification, x1,250), and the cells in 10 fields (ca. 100 cells per field) in each slide were counted and assessed for the presence of the filamentous phenotype. Spirochete lengths on photomicrographs of similar fields were also measured, and a stage micrometer was used to convert these lengths to μm. A similar approach was used to analyze E. coli DH5Z and E. coli DH5Z(pLD7).
Statistical analysis. The effect of addition of ATc to B. burgdorferi 297 and derivatives in culture from three independent experiments was analyzed by a two-way analysis of variance with a repeated measures Bonferroni posttest. Significance levels were set at P < 0.05.
RESULTS
Comparison of ftsZBbu gene product and other bacterial FtsZ proteins. B. burgdorferi ftsZ open reading frame (ORF) codes for a protein with a molecular mass of 42,971 Da. Its deduced amino acid sequence shows strong similarity to other bacterial FtsZ amino acid sequences particularly over the first 312 codons and has 50, 50, 46, and 46% identity with FtsZ proteins of Treponema pallidum, B. subtilis, Leptospira interrogans, and E. coli, respectively (Fig. 2). Within the highly conserved N terminus region, FtsZBbu has a Gly-rich block, G118MGGGTGTG126, identical to the characterized GTP-binding site of E. coli FtsZ (37), and a N223IDFADV229 consensus sequence that is part of a putative GTP-hydrolyzing domain (13, 39). The C-terminal moiety of the borrelial FtsZ homologue also contains a D385DDIDVPTFLR395 conserved region that is thought to be important for interaction with FtsA and ZipA (33). The gene product of ftsZBbu is thus highly homologous to its counterparts in other bacteria.
Construction of a B. burgdorferi 297 strain expressing TetR from a chromosomally inserted tetR gene. Construction of the tetR insertion is shown in Fig. 1 and is described in detail in Materials and Methods. Electroporation of pCR2.1-TOPO containing the luxS flanking regions, tetR, and aph(3')-IIIa into B. burgdorferi 297 yielded four kanamycin-resistant clones; results from one of these clones are shown in Fig. 3A. The tetR insertion into the luxS gene was confirmed by PCR analysis, which showed that longer amplicons were generated from clones with the inserted gene than from wild-type B. burgdorferi 297. Immunoblotting demonstrated the presence of TetR (Fig. 3B). One of the clones was used for all subsequent studies.
Development of filamentous phenotype in B. burgdorferi in response to ATc-regulated production of asftsZ RNA. Construction of pLD6 to mediate regulated synthesis of asftsZ RNA in B. burgdorferi 297/tetR is shown in Fig. 1 and is described in detail in Materials and Methods. pLD6 contained an asDNA fragment complementary to the initial 62 nucleotides of the ftsZBbu coding strand under the control of the Ptetl promoter, a hybrid promoter containing two tetO sequences fused with bmpA promoter sequences (Fig. 1B). To prevent readthrough from the asDNA, a termination signal was placed at the end of the antisense sequence. After electroporation of pLD6 into B. burgdorferi 297/tetR, clones containing pLD6 were selected by using appropriate antibiotics. Twenty-four hours after expression of asftsZBbu RNA was induced in mid-log-phase cultures of B. burgdorferi 297/tetR(pLD6) by the addition of ATc, large numbers of abnormally elongated, filamentous spirochetes were visible under the microscope (Fig. 4C). We were unable to see any septations in these cells. No such cells were present in uninduced cultures of B. burgdorferi 297/tetR (pLD6) (Fig. 4B) or in control cultures of B. burgdorferi 297/tetR in the presence (Fig. 4A) or absence (not shown) of ATc.
ATc-induced B. burgdorferi 297/tetR (pLD6) (Fig. 4D) were significantly longer than uninduced B. burgdorferi 297/tetR (pLD6) or control B. burgdorferi 297/tetR in the absence or presence of ATc (P < 0.001). Although uninduced B. burgdorferi 297/tetR (pLD6) were slightly longer than uninduced or induced B. burgdorferi 297/tetR (compare Fig. 4C with Fig. 4A and B), this difference was not statistically significant (Fig. 4D). By 24hof induction, filamentous cells accounted for 78 ± 11% (mean ± standard error [SE]) of cells examined in cultures of B. burgdorferi 297/tetR (pLD6) and were significantly more frequent (P < 0.001) than in uninduced B. burgdorferi 297/tetR (pLD6) (25 ± 7%) or in control induced or uninduced B. burgdorferi 297/tetR cultures at this time (14 ± 3% and 19 ± 3%, respectively). At 48 h after ATc induction, the percentage of filamentous cells in ATc-induced cultures of B. burgdorferi 297/tetR (pLD6) had fallen to 48 ± 4% and remained at this level at 72 h. We were unable to detect formation of the division septum in any of these cells. These values were significantly less than at 24 h (P < 0.01) but still significantly higher than the percentages of filamentous spirochetes in uninduced cultures of B. burgdorferi 297/tetR (pLD6) (23 ± 2%) or in control induced or uninduced B. burgdorferi 297/tetR cultures (14 ± 1% and 13 ± 1%, respectively).
B. burgdorferi 297/tetR showed no significant differences in growth in the absence or presence of ATc (Fig. 4E). Growth of B. burgdorferi 297/tetR (pLD6) was significantly less (P < 0.001) than that of B. burgdorferi 297/tetR in the absence of 1.5 μg of ATc/ml (Fig. 4E). However, growth of this recombinant strain was even more inhibited in the presence of ATc (P < 0.001). This suggests that the growth and cell division of B. burgdorferi was slowed by the lack of FtsZ protein occasioned by induced asftsZBbu.
Quantitation of ftsZBbu mRNA after induction of production of asftsZBbu RNA. The filamentous borrelial phenotype was most probably a result of the inability of these cells to divide because of the lack of FtsZBbu generated by the induced asftsZBbu. As a first step toward understanding the mechanism of action of induced asftsZ RNA, ftsZBbu mRNA was examined by RT-PCR and real-time RT-PCR. RT-PCR indicated that induction of asftsZBbu RNA by 1.5 μg of ATc/ml for 24 h was associated with a sharp decrease in ftsZBbu mRNA compared to levels in uninduced B. burgdorferi. Culture of B. burgdorferi 297/tetR in the presence of ATc had no obvious effect on production of ftsZ mRNA (Fig. 5A). These results were quantitatively confirmed by real-time RT-PCR. Inhibition of ftsZ mRNA by induced production of asftsZ in B. burgdorferi 297/tetR (pLD6) was highly significant after 6 h of induction with ATc (Fig. 5B), indicating that the induction of asftsZBbu is associated with decreased ftsZBbu mRNA levels in these bacteria.
Expression of B. burgdorferi ftsZ in E. coli. Overexpression of autologous FtsZ in E. coli generates a minicell phenotype, whereas high levels of expression of heterologous FtsZ result in a filamentous phenotype (6, 34, 36, 55). To determine whether ftsZBbu could be ectopically expressed in E. coli and whether its gene product would interfere with E. coli FtsZ function, pLD7, containing ftsZBbu under the control of the Ptetl hybrid promoter (Fig. 1), was electroporated into E. coli DH5. No bacteria carrying pLD7 (Fig. 1C) were obtained after electroporation of pLD7 into E. coli lacking expression of TetR (data not shown), but electroporation of pLD7 into E. coli DH5Z expressing TetR (32) was successful. This could suggest that uncontrolled expression of FtsZBbu in E. coli was lethal. By 12 h after induction of synthesis of ectopic FtsZBbu in E. coli DH5Z (pLD7) with 1.5 μg of ATc/ml, numerous extremely long filamentous cells were visible (Fig. 6C). No septations were visible in these E. coli cells. No filamentous cells were detected in control cultures of E. coli DH5Z producing TetR in the absence (not shown) or presence of ATc (Fig. 6A). Small numbers of filamentous cells were also present in E. coli DH5Z (pLD7) in uninduced cultures (Fig. 6B), but they were much shorter than the filamentous cells seen in induced cultures (compare Fig. 6B and C), and their presence did not significantly increase mean bacterial length in these cultures (Fig. 6D). Both shorter and longer filamentous cells contained denser areas consistent with contractile rings (Fig. 6C and D). These experiments indicate that ectopically produced FtsZBbu can interfere with the function of the E. coli FtsZ protein in E. coli.
DISCUSSION
Although the deduced ftsZBbu gene product is highly homologous to other bacterial FtsZ proteins, the activity of ftsZBbu and FtsZBbu in cell division has not been previously examined. By combining tetracycline-regulated control of gene expression, a technique developed for eukaryotic cells but rarely used in bacteria (3), with antisense technology (29, 31, 35), it was possible to study the functionality of ftsZBbu and confirm its activity in cell division. Blocking of FtsZBbu production by ATc-mediated induction of asftsZBbu RNA resulted in decreased ftsZBbu mRNA, a filamentous phenotype, and slow growth. Furthermore, regulated ectopic expression of ftsZBbu in E. coli resulted in the appearance of filamentous E. coli as a result of functional protein interference of FtsZBbu with cell division proteins of E. coli, thus providing additional confirmation for the functionality of ftsZBbu in cell division.
It was not unexpected that the ATc-mediated downregulation of ftsZBbu expression resulted in a filamentous phenotype and slow growth in B. burgdorferi, since in the absence of FtsZ in other bacteria, the Z ring is not formed and there is a failure to assembly the complete divisome needed for cell division (34, 40, 55, 59). The observed low levels of ftsZ mRNA under the conditions of ATc-induced asRNA provided confirmation that the inhibition of ftsZBbu expression mediated by induced asftsZ was specific for production of FtsZBbu. Attempts to detect the presence of B. burgdorferi FtsZ protein in wild-type B. burgdorferi using cross-reacting anti-E. coli FtsZ rabbit antibodies in immunoblots of B. burgdorferi lysate proteins were unsuccessful (data not shown). This failure might be due to a lack of complete cross-reactivity of this antibody for the B. burgdorferi and E. coli FtsZ proteins or to the low levels of FtsZBbu present under the conditions of growth.
The functional role of the B. burgdorferi FtsZ homologue and its involvement in B. burgdorferi cell division was also indirectly confirmed by our inability to obtain transformants with pLD7 in E. coli not expressing TetR. (pLD7 contained FtsZBbu under the control of the TetR-susceptible Ptetl hybrid borrelial promoter). In contrast, transformants with pLD7 were easily obtained in the E. coli strain that expressed TetR. In this connection, it should be mentioned that many B. burgdorferi promoters are recognized by the E. coli protein synthesis machinery (11, 17, 49). These results suggested that constitutive expression of B. burgdorferi FtsZ was lethal for E. coli, as has been shown to be the case with other heterologously expressed bacterial FtsZ proteins (14). This hypothesis was confirmed by the appearance of filamentous E. coli after induction of expression of FtsZBbu in E. coli (pLD7), indicating inhibition of E. coli cell division by FtsZBbu (59). These findings indicate that FtsZBbu is a cell division protein with function similar to its homologues in other bacteria and thus able to block cell division when expressed in a heterologous background (30, 34, 36, 38, 47, 55, 59). This functional identity of the B. burgdorferi ftsZ gene and FtsZ protein with other bacterial homologues is fully consistent with the amino acid sequence and domain identity it shares with them (30, 36, 59).
B. burgdorferi is a genetically intractable organism. Its genome is unstable. There are no natural systems of horizontal gene transfer in spirochetes and, consequently, no simple and efficient method for introducing DNA into this bacterium. Its nutritional requirements are still incompletely known so that simple and defined media for its culture are lacking, and it grows slowly in the complex media that are available for its culture. Molecular genetic study of the B. burgdorferi genome has been hampered by the dearth of genetic tools to isolate and complement mutants and to manipulate gene expression although many strides have been made to remedy this in recent years (11, 45). It has therefore been difficult to isolate conditional lethal mutants in B. burgdorferi, a type of mutant that has been crucial for the development of genetic systems and physiological studies in other bacteria (5, 23, 26, 27, 32, 35, 45, 50, 58, 59).
Many advances to manipulate the genome of B. burgdorferi have been the result of adaptation of genetic systems developed in other bacteria to this pathogen (11, 45). The present study of the function of ftsZBbu was only possible because we were able to adapt two genetic tools developed for the study of other bacteria, namely, TetR-regulated control of gene expression and asRNA technology (3, 27, 31, 32, 35, 58). In adapting this system to B. burgdorferi, the tetR gene was placed under the control of the constitutive B. burgdorferi flaB promoter, and this DNA construct was inserted in the chromosome in luxS, a gene not required for B. burgdorferi growth and infectiousness (7); the DNA segment encoding antisense ftsZ RNA under the hybrid Ptetl TetR-responsive promoter was located extrachromosomally in a plasmid (17). The chromosomal location of the tetR gene under a constitutive promoter is preferable to a plasmid location. Variations in gene dosage as result of variations in copy number are minimized in the chromosomal location, thus ensuring that the synthesis of TetR will remain constant throughout the cell division cycle (32, 44). By permitting regulated expression of ftsZ asRNA, this system has for the first time generated the functional equivalent of a conditionally lethal mutant in B. burgdorferi.
B. burgdorferi cells containing both components of the Tet system regulated the expression of ftsZ asRNA and FtsZ in an ATc-dependent manner as measured by levels of ftsZ mRNA (Fig. 5). The reduced levels of FtsZ were associated with inhibition of cell division demonstrated by the emergence of B. burgdorferi cells with the filamentous phenotype and slow growth (Fig. 4). However, TetR repressor levels encoded from the B. burgdorferi chromosome were not totally able to suppress expression of ftsZ asRNA from the Ptetl hybrid promoter since uninduced cultures of B. burgdorferi 297/tetR (pLD6) contained cells that were slightly longer (Fig. 4B and D) and grew significantly more slowly (Fig. 4E) than the B. burgdorferi 297/tetR controls. We do not believe that filamentous cells divide after the blocking of cell division, and we have some preliminary evidence suggesting that they in fact are dead (data not shown). This escape of repression by TetR was evident in E. coli pLD7, where small numbers of filamentous cells were visible in the absence of ATc induction (Fig. 6B). It might be a result of titration of the TetR repressor by an increased number of the extrachromosomally located molecules of Ptetl generated by variations in the copy number of the pKFSS1 plasmid (2). However, we have previously demonstrated that pKFSS1 has a copy number of one relative to the B. burgdorferi chromosome (9), suggesting that other mechanisms may play a role in the imbalance generated between numbers of TetR molecules and Ptetl promoters (28). These might include changes in pKFSS1 supercoiling, which might decrease the access of the repressor to a region in the plasmid where Ptetl is located (2). Titration of the levels of TetR by excessive amounts of Ptetl molecules is a problem that could be readily corrected by placing an increased number of copies of the tetR gene in the chromosome and by putting its transcription under the control of a stronger B. burgdorferi constitutive promoter. Alternatively, this imbalance could be potentially corrected by placing both the tetR repressor gene and the putative gene controlled by the Ptetl promoter in the chromosome (15).
In summary, this is the first report to show the feasibility of the regulated control of gene expression in B. burgdorferi and the usefulness of asRNA for generating the physiologic equivalent of conditionally lethal mutants in this pathogen. These experiments suggest that FtsZBbu plays a role in cell division in B. burgdorferi and that ftsZBbu expression in particular, and gene expression in general, can be negatively controlled in this species by the use of tetracycline-regulated expression of asRNA.
ACKNOWLEDGMENTS
We thank H. Bujard for advice and for E. coli DH5Z and plasmids containing tetO and tetR; Kai Schnig for TetR antisera; Miguel Vincente, Pilar Palacios, and William Margolin for anti-E. coli FtsZ antisera; Dionysios Liveris for many useful discussions; Radha Iyer for help with real-time PCR; and Harriett Harrison for secretarial assistance.
This study was supported by grant R01 AI08856 (to F.C.C.) from the National Institutes of Health.
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