Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan1
Author for correspondence: Hideyuki Takahashi. Tel: +81 3 5285 1111. Fax: +81 3 5285 1163. e-mail: hideyuki{at}nih.go.jp
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ABSTRACT |
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Keywords: IncQ plasmid, triparental conjugation, restriction/modification systems, complementation
Abbreviations: Ampr/Amps, ampicillin resistant/sensitive; Cmlr, chloramphenicol resistant; Ermr, erythromycin resistant; Kanr/Kans, kanamycin resistant/sensitive; Tetr/Tets, tetracycline resistant/sensitive; Strr/Strs, streptomycin resistant/sensitive
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INTRODUCTION |
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Some R-plasmids in Neisseria gonorrhoeae have been used for the construction of vector plasmids (Stein et al., 1983 ; Piffaretti et al., 1988
; Stein, 1989
; Kupsch et al., 1996
). Although it is well known that N. gonorrhoeae is genetically very closely related to N. meningitidis and a few plasmids were speculated to transfer from N. gonorrhoeae to N. meningitidis (Dillon et al., 1983
; Ison et al., 1986
; Knapp et al., 1988
), most of the plasmids in N. gonorrhoeae were not applicable to N. meningitidis (Genco et al., 1984
; Roberts et al., 1989
). As far as we know, only two plasmids, a 4·5 Mb R-plasmid of N. gonorrhoeae and pMGC10, a derivative of shuttle vector pLES2, could be transferred to N. meningitidis (Ikeda et al., 1986
; Nassif et al., 1991
). On the other hand, some R-plasmids have been also found in N. meningitidis (Bhatti et al., 1981
; Pintado et al., 1985
; Rotger et al., 1986
; Facinelli & Varaldo, 1987
; Roberts et al., 1989
). One of them was supposed to belong to the family of RSF1010, which is a broad-host-range vector of incompatibility group Q (IncQ) (Pintado et al., 1985
).
IncQ plasmids such as RSF1010 and R300B (Sharpe, 1984 ) are found among Gram-negative bacteria (Barth et al., 1981
). IncQ plasmids are not self-transmissible but mobile with the help of IncP plasmids. Since R300B is relatively small (8·68 kb; Meyer et al., 1982
) and low in copy number (about 10 copies per E. coli genome; Barth & Grinter, 1974
), it has been utilized for the construction of vectors (Sharpe, 1984
). pGSS33 is one of the derivatives of R300B and has been used as a cloning vector.
In this paper, we report that pHT128, a derivative of pGSS33, can be successfully introduced into N. meningitidis.
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METHODS |
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Strains.
Strains used in this study are listed in Table 1. HT1037 was constructed as follows. A 1·3 kb fragment containing the opc gene with BamHI and EcoRI linkers was amplified from H44/76 chromosomal DNA by PCR. All the PCR reactions done in this study were performed in a Gene Amp PCR System 2400 (Applied Biosystems) using ExTaq DNA polymerase (Takara Shuzo). The resulting DNA fragment was digested with BamHI and EcoRI, then cloned in the BamHI and EcoRI sites of pUC118 to construct pHT3 (see also Table 2
). A blunted 1·2 kb HindIIIHindIII fragment containing the ermC gene isolated from pHT24 was inserted into the blunted EcoT14I sites, located at 196 bp and 526 bp downstream from the start codon of the opc gene, to generate pHT148. pHT148 DNA (500 ng) linearized by digestion with SalI, the site of which is located on the vector, was transformed into H44/76 and Ermr clones were isolated, resulting in an opc deletion mutant of H44/76.
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Plasmids.
Plasmids used in this study are listed in Table 2. A Kans Tets derivative of RP4 (Pansegrau & Lanka, 1987
), pHT13, was constructed as follows. First, RP4 digested with HindIII and HpaI was blunted with a DNA blunting kit (Takara Shuzo) and self-ligated by DNA ligation kit version 2 (Takara Shuzo) to generate pHT12 (Kans). In the next step, pHT12 digested with BglII and partially digested with StuI was blunted, self-ligated and transformed to DH5
to select Ampr Kans Tets clones. The resultant plasmid was renamed pHT13 and used as a helper for conjugation of a derivative of pGSS33 (Fig. 1a
) from E. coli to N. meningitidis. pHT128 (Fig. 1b
), a Strs and Amps derivative of pGSS33, was constructed by removing the PstISacI region after blunting. pHT160 (see Fig. 4c) was constructed as follows. A 0·9 kb fragment containing the entire opc coding region with EcoRI and BamHI linkers was amplified by PCR and cloned in the BamHI and EcoRI sites of pTTQ18 (Stark, 1987
) to construct pHT157. A 4·3 kb AlwNIScaI fragment of pHT157 containing both the tac promoter fused to the opc gene and the lacIq gene was blunted and cloned in the PstI and SacI sites of pGSS33 after blunting. The resultant plasmid was pHT160. pHT161 (Fig. 4a) was constructed as follows. A 0·6 kb fragment containing the entire pilE1 coding region was amplified by PCR with the corresponding primers, which contained the EcoRI linker tagged by six CAC tandem repeats (His6) and a SmaI linker. The amplified fragment was cloned in the EcoRI and SmaI sites of pTTQ18 to construct pHT68. A 4 kb AlwNIScaI fragment of pHT68 containing both the tac promoter fused to the His6-pilE1 gene and the lacIq gene was blunted and cloned in the PstI and SacI sites of pGSS33 after blunting. The resultant plasmid was pHT161.
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DNA transfer.
For transformation by natural competence, N. meningitidis was transformed as previously described (Ryll et al., 1997 ). Plasmids were linearized prior to transformation unless otherwise stated.
Electroporation for Neisseria was performed as follows. N. meningitidis grown on GC agar plates at 37 °C in 5% CO2 was scraped and suspended in 30 ml TSB supplemented with 1% IsoVitaleX enrichment to adjust the supension to OD600 0·1. The bacteria were cultured at 37 °C with shaking to OD600 of 0·6 and then the cells were chilled and harvested by centrifugation at 4 °C. The pellets were rinsed with 1 ml ice-cold 10% (w/v) glycerol three times and the cells were resuspended to a final volume of 60 µl in ice-cold 10% glycerol. A 20 µl sample of the cell suspension was mixed with up to 1 µg of plasmid DNA and electroporation was performed under the following optimized conditions: electric field strength, capacitance and resistance were 1·25 kV, 25 µF and 400 , respectively, when using a 0·1 cm electrode gap cuvette (Bio-Rad) with the Gene Pulser (Bio-Rad). After being electroporated, cells were incubated in 1 ml TSB supplemented with 1% IsoVitaleX enrichment, 10 mM MgCl2 and 0·4% glucose at 37 °C for 1 h. The cells harvested by centrifugation were spread on GC agar plates with 5 µg chloramphenicol ml-1 and incubated at 37 °C in 5% CO2 for 18 h.
For conjugation, N. meningitidis grown on GC agar plates at 37 °C in 5% CO2 was scraped and suspended in TSB to adjust the supension to OD600 0·25. In order to transfer a cloned vector plasmid from E. coli to N. meningitidis, we used triparental conjugation. Overnight cultures of LE392 harbouring vector plasmid pGSS33 or pHT128, LE392 harbouring a helper plasmid pHT13 and N. meningitidis suspension prepared as described above were mixed at a volume ratio of 1:1:2 and then filtered through 0·45 µm membrane filters (Millipore) by use of a syringe. The filters were washed with 2 ml TSB, then incubated on GC agar plates at 37 °C in 5% CO2 for 8 h. Bacteria on the filters were washed out with 1 ml TSB. The bacteria, harvested by centrifugation, were then spread on GC agar plates containing 5 µg chloramphenicol ml-1 and 1% VCN inhibitor and incubated at 37 °C in 5% CO2 for 24 h. Transconjugants of N. meningitidis were picked up and streaked on the same kind of plates to isolate a single clone.
Checking the maintenance of pHT128 in N. meningitidis.
NIID57 harbouring pHT128 grown on a GC agar plate or E. coli strain LE392 harbouring pHT128 on an L agar plate was scraped and suspended in 10 ml TSB supplemented with 1% IsoVitaleX enrichment at an OD600 of 0·2 and cultured at 37 °C with shaking for 24 h. One millilitre of the bacterial culture was added to 9 ml fresh medium and continuously cultured under the same conditions as described above. This step was repeated a total of four times. Each time, appropriate dilutions of the bacterial culture were spread on GC agar plates with or without 5 µg chloramphenicol ml-1 for NIID57 and L agar plates with or without 10 µg chloramphenicol ml-1 for LE392 to count the number of bacteria harbouring pHT128 and the total number of bacteria, respectively. The number of bacterial generations was determined graphically from the values of OD600.
Isolation of DNA.
Mini-preparation of plasmid from E. coli was carried out by Wizard Plus SV Minipreps DNA Purification systems (Promega). Isolation of plasmids from N. meningitidis transconjugants and E. coli transformants cultured in more than 1 ml of medium was essentially as described by Kado & Liu (1981) .
For the isolation of total DNA, N. meningitidis was cultured in the same conditions as described in the preceding section except that chloramphenicol was added during the culturing. Total DNA from N. meningitidis transconjugants was isolated as described by Suker et al. (1994) .
Southern blotting.
One microgram of the total DNA of N. meningitidis was digested with ClaI and analysed on a 0·8% agarose gel. The gel was treated with 0·25 M HCl followed with 0·5 M NaOH and then transferred to Hybond-N+ membrane (Amersham). The resultant membrane was hybridized with the cat gene of pHT128 as a probe and signals were detected by ECL direct nucleic acid labelling and detection systems (Amersham) according to the manufacturers protocol.
Preparation of antibody for pili.
A peptide encompassing residues 4155 (EGQKSAVTEYYLNHG) of pilin, a region recognized by the monoclonal antibody SM1 made by Virji and co-workers (Virji & Heckels, 1983 ; Virji et al., 1989
), was synthesized. An antiserum specific for the oligopeptide was obtained by immunization of rabbits with the peptide coupled to keyhole limpet haemocyanin using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS).
Western blotting.
The N. meningitidis cells were suspended in 1xSDS buffer (2% SDS, 5% 2-mercaptoethanol, 62·5 mM Tris/HCl pH 8·0, 10% glycerol, 0·1% Bromophenol blue) and adjusted to OD600 10. After boiling for 5 min, 2·5 µl samples were subjected to 12 or 15% SDS-polyacrylamide gel electrophoresis. Western blotting was performed with Sequi-Blot PVDF membrane (Bio-Rad) and Horizeblot (ATTO). The resulting membrane was soaked in PBS containing 0·1% Tween 20 (T-PBS) and 5% skim milk at room temperature for 1 h with gentle shaking. The membrane was incubated with 103-fold diluted anti-pili serum or 104-fold diluted monoclonal antibody against Opc (B306; Achtman et al., 1988 ) at room temperature for 1 h. After washing the membrane in T-PBS for 10 min three times, it was incubated with 2000-fold diluted horseradish-peroxidase-conjugated anti-rabbit or anti-mouse antibody (Boehringer). After washing the membrane in T-PBS for 10 min three times, signals were detected by ECL detection kit (Boehringer) and ECL film (Boehringer).
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RESULTS |
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We tried to introduce the IncQ plasmid pHT128 into N. meningitidis strain NIID57 by three methods: transformation by natural competence, electroporation and conjugation. Although no Cmlr colonies were obtained with transformation by natural competence or electroporation, some Cmlr colonies appeared when triparental conjugation was used to transfer pHT128 from E. coli to NIID57. To optimize the conditions of triparental conjugation, we changed the concentration of the recipient strain NIID57 and found that the efficiency of conjugation seemed to be highest at a density of OD600 0·20·3, although the number of Cmlr transconjugants seemed to be almost equal (about 103 clones) independent of the concentration of the recipient strain (Fig. 2a). The efficiency of transconjugation was calculated to be about 10-5 (transconjugants/recipients) under the best conditions (Fig. 2b
). As a similar result was obtained with N. meningitidis strain H47/76 as recipient (data not shown), pHT128 seems to be introduced into N. meningitidis serogroup B regardless of strain specificity.
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Isolation of pHT128 from N. meningitidis
To confirm the existence of pHT128 in N. meningitidis, we isolated the plasmids from Cmlr N. meningitidis transconjugants. Plasmids the same size as pHT128 (12 kb) were isolated from all the Cmlr N. meningitidis colonies examined and the amount of plasmid DNA from N. meningitidis was as much as that in E. coli DH5 when examined by the density on agarose gel (data not shown). These results indicated that pHT128 could be conjugationally transferred from E. coli to N. meningitidis and existed at about 10 copies in N. meningitidis, as much as in E. coli.
Stability of pHT128 in N. meningitidis
To check the stability of pHT128 in N. meningitidis, we successively subcultured N. meningitidis harbouring pHT128 in liquid medium and counted total c.f.u. on GC agar plates with or without 5 µg chloramphenicol ml-1 as described in Methods. Although pHT128 was maintained at a frequency of 100% in E. coli strain LE392 even in drug-free liquid medium, the plasmid was gradually lost from N. meningitidis at a rate of about 79% loss per generation under the same conditions (Fig. 3a). This result indicated that maintenance of pHT128 in N. meningitidis was relatively unstable in the absence of selection by chloramphenicol.
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The extraneous genes on pHT128 can be expressed in N. meningitidis
Next, we examined whether pHT128 vector is available for the complementation of mutations in N. meningitidis. A derivative of pHT128 carrying the His6-tagged pilE1 or the opc gene under the control of tac promoter, pHT161 or pHT160, respectively (Fig. 4a, c
), was introduced into N. meningitidis strain HT1033 (pilE1) or HT1037 (
opc). Cell lysates of Cmlr transconjugants cultured in the presence and absence of IPTG were examined for the expression of pili and Opc protein by Western blotting with each antibody. As shown in Fig. 4(b
, d
), pili or Opc protein were expressed in N. meningitidis under the control of IPTG induction. These results confirmed that the foreign genes on pHT128 could be expressed in N. meningitidis.
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DISCUSSION |
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The difficulties of introducing R-plasmids into N. meningitidis may be partially attributed to some restriction/modification systems in N. meningitidis because naked DNA isolated from N. meningitidis transconjugants, but not from E. coli, could be easily transformed into N. meningitidis by electroporation (unpublished data). The influence of the restriction/modification systems on the efficiency of conjugation is still controversial (Stein et al., 1988 ; Butler & Gotschlich, 1991
). However, mobilization of plasmid by conjugation from E. coli to N. meningitidis may circumvent the barrier of modification/restriction systems by some unknown mechanism(s) because pHT128 or pMGC10 (Nassif et al., 1991
) was transferred at a considerable frequency.
The efficiency of plasmid DNA transfer in triparental conjugation was variable (Fig. 2). This might have been due to the difference of living state of N. meningitidis used for the preparation of recipient cells because neisseriae have rapid autolytic properties, even if cultured under steady conditions. In order to increase the transfer frequency, it would be critical to improve the recipiency of N. meningitidis.
To date, genetical analyses such as complementation tests in N. meningitidis have never been done because there were few useful plasmid vectors for N. meningitidis. We have shown here that pHT128 is applicable for complementation tests in N. meningitidis. We believe that our findings increase the possibilities for the molecular biological analysis of N. meningitidis.
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ACKNOWLEDGEMENTS |
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Received 8 June 2001;
revised 27 August 2001;
accepted 4 September 2001.