Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada1
Department of Microbiology, University of Guelph, Guelph, ON, N1G 2W1, Canada2
Author for correspondence: Jo-Anne R. Dillon. Tel: +1 613 562 5459. Fax: +1 613 562 5452. e-mail: jdillon{at}uottawa.ca
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ABSTRACT |
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Keywords: Neisseria gonorrhoeae, minC, cell-division inhibitor, cell lysis, cocci
Abbreviations: Ap, ampicillin; Cm, chloramphenicol; GFP; green fluorescent protein; Kan, kanamycin; RT, reverse transcriptase; Str, streptomycin
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INTRODUCTION |
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In E. coli (Ec), proteins involved in correct division site selection are encoded by the minB operon and include MinCEc, MinDEc and MinEEc (de Boer et al., 1988 ; de Boer & Crossley, 1989
). MinCEc inhibits septum formation, acts as a cell-division inhibitor and is activated by the ATPase MinDEc (de Boer et al., 1988
, 1991
). If MinCEc and/or MinDEc are overexpressed, E. coli cells form long filaments, an indication that cell division has been blocked (de Boer & Crossley, 1989
). Together, MinCEc and MinDEc prevent division at all potential division sites by blocking assembly of FtsZEc (Bi & Lutkenhaus, 1993
; de Boer et al., 1992
; Huang et al., 1996
). However, a recent study showed that MinCEc did not prevent the oligomerization of FtsZ although it did prevent the formation of FtsAEc rings (Justice et al., 2000
). The N-terminal domain of MinCEc is responsible for inhibiting FtsZEc assembly whilst the C-terminal domain of MinCEc interacts with MinDEc (Hu & Lutkenhaus, 2000
). The Min proteins can be visualized inside cells by tagging them with green fluorescent protein (GFP). GFP-tagged MinCEc and MinDEc were found to oscillate from one pole to the other (Raskin & de Boer, 1999a
, b
; Hu & Lutkenhaus, 1999
) and this oscillation is dependent upon the presence of other Min proteins. MinEEc provides topological specificity to division site selection by forming a ring and counteracting the activity of MinCDEc at mid-cell (King et al., 1999
; Pichoff et al., 1995
; Raskin & de Boer, 1997
; Zhang et al., 1998
; Zhao et al., 1995
). MinEEc ring formation is independent of FtsZEc ring formation and the ring disassembles before, or shortly after, cell constriction begins (Raskin & de Boer, 1997
). When MinEEc is overexpressed, it prevents MinCDEc from acting at all potential division sites in E. coli. These cells divide asymmetrically to form round, anucleate cells, called minicells (Adler et al., 1967
; de Boer et al., 1988
).
The division site selection process in Bacillus subtilis (Bs) differs in several respects from E. coli. B. subtilis contains both minCEc and minDEc homologues but lacks a minEEc homologue (Varley & Stewart, 1992 ). MinC and MinD apparently do not oscillate in this organism. The topological specificity of the MinCDBs inhibitor complex in B. subtilis is controlled by the protein DivIVA which bears neither structural nor functional similarity to MinEEc (Edwards & Errington, 1997
). Inactivation of DivIVA results in minicell formation (Cha & Stewart, 1997
; Edwards & Errington, 1997
; Reeve et al., 1973
), and overexpression results in a filamentous phenotype, not minicell formation, as is observed with MinEEc (de Boer & Crossley, 1989
). DivIVAGFP localizes to both the mid-cell and polar regions and its main role may be to retain MinCDBs at the cell poles after division (Edwards & Errington, 1997
; Marston et al., 1998
; Marston & Errington, 1999
). Localization of GFPMinDBs to the poles is dependent on DivIVA. However, the targeting of GFPMinCBs to the mid-cell and its retention at the mature cell poles is dependent on FtsZBs, PbpBBs and MinDBs (Marston et al., 1998
; Marston & Errington, 1999
).
Proteins involved in division site selection in cocci have not been investigated. A key difference in cell division between cocci and rods is that division, in some cocci such as Neisseria gonorrhoeae (Ng), occurs sequentially, in alternating, perpendicular planes (Westling-Häggström et al., 1977 ). Division in N. gonorrhoeae comprises an incomplete asymmetric constriction, accompanied by septum formation dividing the cell into equal-sized daughter cells (Fitz-James, 1964
).
In the present study, which was initiated in order to determine how cocci select their division sites, we report on the identification of Min homologues in Neisseria species and have compared their genomic organization to min clusters in other bacteria. We show that the gonococcal and meningococcal min genes belong to a 17 kb cluster comprising 27 genes which are transcribed in the same direction. The transcription of min genes is linked to the transcription of upstream genes as determined by RT-PCR. Additionally, we have focused on the role of MinC from N. gonorrhoeae as a model system. By constructing a minC gene knockout in N. gonorrhoeae CH811, and by cloning and expressing minCNg in E. coli backgrounds, we show that the gonococcal MinC protein acts as a division inhibitor. The homologous and heterologous functionality of the gonococcal MinC protein in N. gonorrhoeae and E. coli, respectively, has been confirmed by complementation experiments and by Western blot analysis using anti-gonococcal MinC antibodies. Surprisingly, the deletion of minCNg causes gross enlargement of the round cells, leading them to lyse.
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METHODS |
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Reverse transcriptase polymerase chain reaction (RT-PCR).
Total RNA was obtained from overnight cultures of N. gonorrhoeae CH811 using the Qiagen RNeasy Total RNA kit. RT-PCR was used to determine whether the genes located between rpoA (upstream of minCNg) and minENg were co-transcribed. Primer CVME38 (Table 3) was used to produce cDNA using 1 µg total RNA from N. gonorrhoeae CH811 as a template in a reverse transcriptase reaction using Expand Reverse Transcriptase (Boehringer Mannheim) according to the instructions of the manufacturer. Primer pair CVRA41/CVRA42 (Table 3
) was used to amplify a 657 bp fragment corresponding to rpoA from the RT reaction.
Cloning, expression and complementation of min genes in E. coli and microscopy.
Based on the sequence analysis of the gonococcal minCDENg gene cluster, primer pair min10/min11 incorporating PstI and KpnI restriction sites, respectively (Table 3), was used to PCR amplify a 2810 bp fragment from N. gonorrhoeae CH811 cell suspensions. The PCR product consisted of minCDE, oxyR and the duplicated gonococcal uptake sequences at the 3' end. The amplicon was ligated into pBluescript KS II (+) (Stratagene) under the control of the T7 promoter, opposite to the direction of the lac promoter, to form pSR1 (Table 2
). This plasmid was obtained after transformation into E. coli DH5
by the CaCl2 method as described by Sambrook et al. (1989)
. A fragment of 720 bp corresponding to minCNg was PCR amplified from cell suspensions using primer pair min12/min29 into which EcoRI and BamHI restriction sites, respectively, had been incorporated (Table 3
). This amplicon was ligated into pUC18 (Pharmacia) under the control of the lac promoter and transformed into E. coli DH5
as mentioned above. The plasmid obtained was named pSR2 (Table 2
). Plasmid pSR1 was transformed into E. coli C43(DE3) (wild-type) and pSR2 was transformed into E. coli PB103 (wild-type) and DR105 (minC) for expression and complementation studies. Transformants were selected on LB agar supplemented with Ap. E. coli DR105 cells carrying pSR2 were transformed with pREP4 (Qiagen) to provide LacIq in trans in order to control the expression of the gonococcal minC. E. coli DR105 cells carrying both pSR2 and pREP4, were induced with 1·0 mM IPTG.
Samples from all E. coli transformants were collected from exponential-phase cells. These cells were fixed with 0·2% glutaraldehyde and 6% formaldehyde, prior to adhesion to coverslips pre-coated with 0·01% polylysine. Coverslips were then placed onto a slide containing a drop of 50% (v/v) glycerol. The morphology of all E. coli transformants was analysed by phase-contrast microscopy using a Zeiss Axioskop microscope (ZeissX100 oil immersion objective, 100W HBO lamp).
Construction and complementation of a N. gonorrhoeae minC knockout mutant.
To create an insertional mutant of minC in N. gonorrhoeae, the chloramphenicol acetyltransferase (cat) cassette from pACYC184 (New England Biolabs) was PCR amplified using primer pair min21/min22 (Table 3) and inserted into the unique SalI restriction site 35 bp from the start site of minCNg in pSR1 to generate pSR1cat3 (Table 2
). To facilitate homologous recombination with the gonococcal chromosome, a 504 bp BamHIPstI fragment corresponding to the region upstream of the translational start site of minCNg was PCR amplified using primer pair min28/min38 (Table 3
) and cloned into pSR1cat3 to generate pSR1cat4 (Table 2
) which would act as a suicide vector in N. gonorrhoeae. N. gonorrhoeae CH811 cells were transformed with pSR1cat4 as described by Janik et al. (1976)
. A transformant, selected on GCMBK supplemented with Str and Cm, was designated N. gonorrhoeae CSRC1. The insertion of cat into the chromosomal minCNg was confirmed by PCR analysis.
To complement N. gonorrhoeae CSRC1, plasmid pSR18 was constructed in several steps. These steps were required because the plasmid pLES94 (Silver & Clark, 1995 ), which is a gonococcal suicide vector, has a restricted number of cloning sites. A 1390 bp amplicon corresponding to the kan cassette of pET30a (Novagen) was amplified using the primer pair SRKan1/SRKan2 (Table 3
) incorporating KpnI and NcoI restriction sites, respectively. Kan selection was required since the host strain, CSRC1, was resistant to Cm because minCNg had been insertionally inactivated with a cat cassette. This amplicon was cloned into pEGFP (Clontech) to produce pEGFPKan (Table 2
). A 1300 bp amplicon containing all of minCNg and 550 bp upstream was generated using the primer pair SRpet1/HLC (Table 3
) incorporating NdeI and HindIII restriction sites, respectively, and was cloned into pET30a in fusion with 6xHis at the C-terminus of MinCNg to generate pSR16 (Table 2
). Using the primer pair min28/SRpet2 (Table 3
), which incorporated BamHI and KpnI restriction sites, respectively, a 1375 bp amplicon containing minCNg6xHis and the 550 bp region upstream of minCNg was PCR amplified from pSR16 and cloned into pEGFPKan to generate pSR17 (Table 2
). A fragment containing minCNg6xHis, a 550 bp upstream region, and the kan cassette was released from pSR17 using BamHI and NcoI endonuclease digestion. This 2765 bp fragment was cloned into pLES94 (Silver & Clark, 1995
), replacing a 3200 bp BamHINcoI fragment which contained lacZ and part of cat, to create pSR18 (Table 2
). The new insert in pSR18 was flanked by 2400 bp and 800 bp, at the 5' and 3' ends, respectively, with gonococcal proAB gene sequences to allow for homologous recombination into the gonococcal chromosome. N. gonorrhoeae CSRC1 cells were transformed with pSR18 and transformants were selected on GCMBK plates supplemented with Cm and Kan. A positive transformant strain, named N. gonorrhoeae CSRC2, was used for further studies. Confirmation of the insertion of the 2765 bp amplicon from pSR18 into the chromosome of N. gonorrhoeae CSRC2 was done by PCR and DNA sequence analysis (University of Ottawa Biotechnology Research Institute).
Microscopic analysis of N. gonorrhoeae.
Transmission electron microscopy was performed on N. gonorrhoeae strains CH811, CSRC1 and CSRC2 as described by Beveridge et al. (1994) . Phase-contrast microscopy analysis was also performed in these strains using a Zeiss Axioskop microscope (Zeiss x100 oil immersion objective, 100W HBO lamp).
Viability studies.
N. gonorrhoeae CH811, CSRC1 and CSRC2 cells were diluted 25-fold from overnight cultures into GCMBK liquid media to obtain an initial standard inoculum at OD550 of 0·05 for the three strains. Cultures were incubated at 35 °C with 5% CO2 with agitation (250 r.p.m.). One millilitre of culture was removed at fixed times to measure the OD550 and was serially diluted in liquid GCMBK. Then, 0·1 ml from selected dilutions was plated, in triplicate, on GCMBK agar medium supplemented with Str for N. gonorrhoeae CH811, with Str and Cm for N. gonorrhoeae CSRC1, and with Cm and Kan for N. gonorrhoeae CSRC2. The plates were initially incubated for 24 h prior to the first colony count and then recounted after incubation for an additional 48 h, if required. Viability of the colonies was confirmed by subculturing them on GCMBK plates and incubating for a further 2448 h.
Production of gonococcal anti-MinC and anti-MinD antibodies.
His-tagged MinCNg and MinDNg were purified by nickel affinity chromatography. Using the primer pair JSC1/JSC2 (Table 3) incorporating BamHI and EcoRI restriction sites, respectively, minCNg was PCR amplified and cloned into pQE30 (Qiagen), which encodes an N-terminal 6xHis tag, to produce pJSHC (Table 2
). Similarly, using the primer pair minD7pET30/minD2 (Table 3
), incorporating NcoI and BamHI restriction sites, respectively, gonococcal minD was amplified and cloned into pET30a, generating an N-terminal fusion to 6xHis in pJSHD2 (Table 2
). N. gonorrhoeae MinC was purified from E. coli M15 expressing minC from pJSHC and gonococcal MinD was purified from E. coli BL21(DE3) expressing minD from pJSHD2 after 1 mM IPTG induction. Inclusion bodies of MinCNg were purified with 8 M urea lysis buffer and eluted from the nickel affinity column with a pH 4·5 elution buffer as recommended by Qiagen. Purified 6xHisMinCNg was then used to immunize female New Zealand White rabbits for the production of polyclonal anti-MinCNg antibodies. Soluble MinDNg was eluted using a 60 mM imidazole buffer (Novagen). 6xHisMinDNg was then cut from SDS-PAGE gels and electroeluted prior to immunization. Each immunization consisted of 20 µg 6xHisMinCNg or 80 µg 6xHisMinDNg mixed with Gerbu adjuvant, according to the manufacturers instructions (GERBU Biotechnik). Three boosters of MinCNg and two of MinDNg using the same protein concentrations as above were administered. Anti-MinCNg sera were fractionated with 70% NH4SO4 and concentrated 10 times to obtain an antiserum rich in IgG (Hebert et al., 1973
).
Protein analysis and Western blots.
Protein samples from E. coli PB103 and DR105, and N. gonorrhoeae CH811, CSRC1 and CSRC2 were prepared by resuspending cells recovered from exponential phase in 1 ml PBS (pH 7·0). Cell suspensions were lysed by sonication using a SONIFIER cell disruptor 350 and then centrifuged at 4 °C for 30 min at 10000 g in a Sorvall MC 12V microcentrifuge or a Sorvall RC 5C Plus centrifuge (E. I. DuPont de Nemours and Company). Supernatants were removed and kept as soluble fractions. All protein samples were separated by SDS-PAGE as described by Sambrook et al. (1989) . The volume of protein loaded onto gels was standardized for each individual sample in order to have similar concentrations of total protein between samples as determined by densitometry.
Western blots of protein gels were performed following the instructions provided in the Mini Trans-Blot Electrophoretic Transfer Cell Instruction Manual (Bio-Rad) using Immobilon-P transfer membranes (Millipore) with the following modifications: 3% skimmed milk was used instead of gelatin to prepare the blocking solution and was eliminated from the antibody buffer. The concentrated anti-gonococcal MinC antisera was diluted 1:1000. Similarly, the anti-MinDNg antiserum was diluted 1:1000. Alkaline phosphatase conjugated goat anti-rabbit secondary antibody (Bio-Rad) was diluted 1:3000 and detected using Atto Phos Plus kit (JBL Scientific). Densitometric analysis to compare relative protein concentration was performed using the Alpha Imager 1220 v5.04 (AlphaEase version 5.00; Alpha Innotech).
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RESULTS |
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Genomic analysis of the min genes from N. meningitidis Z2491/serotype A using data provided at http://wit.mcs.anl.gov/WIT2/ showed that this bacterium had an identical gene organization to the gonococcal min cluster. Furthermore, the DNA sequences of the minNg genes were 97% identical to the meningococcal min genes. By contrast, min genes in E. coli are associated within a 1·7 kb gene cluster, whereas min genes in B. subtilis are contained in a 5 kb gene cluster (Fig. 1a).
The gonococcal and meningococcal minC, minD and minE genes encode proteins with the predicted sizes and molecular masses of 237 aa (26·3 kDa), 271 aa (29·6 kDa) and 87 aa (10·0 kDa), respectively. In both the minCDE gene clusters minC and minD are separated by 28 bp whereas minD and minE are separated by 3 bp. MinCNg shows 98%, 36% and 22% identity to MinC from N. meningitidis, E. coli and B. subtilis, respectively, whereas MinDNg is 98%, 73% and 39% identical to the N. meningitidis, E. coli and B. subtilis MinD proteins, respectively. Gonococcal MinE shares 97% and 42% identity to the N. meningitidis and E. coli MinE proteins, respectively. Comparison between MinC proteins from different Gram-positive and Gram-negative bacteria indicates that there are only five amino acids conserved in all sequences, corresponding to residues R136, G138, G157, G164 and G174 of gonococcal MinC.
Analyses of other genomes indicated that all three min genes are not ubiquitously present in different organisms (Table 4). Some bacteria such as Chlamydia trachomatis have only one min gene (minD), whilst others, such as Thermotoga maritima, have two (minCD). Interestingly, some species have multiple copies of a particular min gene. The archaeon Methanococcus jannaschii contains two copies of minD, whilst Borrelia burgdorferi has three minD-related genes (Table 4
). Thus far, the entire minCDE cluster appears to be present only in Gram-negative bacteria. A few species of bacteria (e.g. Mycoplasma spp.) do not contain any min homologues (Table 4
).
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Heterologous expression of MinNg proteins in E. coli
To determine whether Min proteins from cocci would disrupt cell division in a rod, the gonococcal minCDE cluster, encoded on pSR1, was overexpressed in E. coli C43(DE3). A large number of short filaments and minicells was produced (Fig. 4b). This phenotype was easily differentiated from the wild-type morphology of the same E. coli strain transformed with pBluescript KS II (+) as a control (Fig. 4a
). The appearance of the minicell phenotype suggested that gonococcal Min proteins disrupt cell-division site selection in E. coli, producing a phenotype similar to that observed from overexpression of all three E. coli Min proteins (de Boer & Crossley, 1989
).
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Heterologous complementation of an E. coli minC mutant with minCNg
The previous experiments suggested that gonococcal MinC had a similar function to E. coli MinC, possibly acting synergistically with the E. coli homologues to produce characteristic phenotypes when overexpressed. Therefore, one would expect that the gonococcal minC should complement E. coli minC mutants. E. coli DR105 (minC) was co-transformed with pSR2 (minCNg) and pREP4 in order to control the expression of the gonococcal minC by providing LacIq in trans. Most of the transformants exhibited a wild-type phenotype after 2 h of 1 mM IPTG induction (Fig. 5b) compared to the minicell phenotype characteristic of the E. coli minC mutant cells (Fig. 5a
). MinCNg expression in the complemented E. coli strain was confirmed by Western blot analysis. E. coli DR105 transformed only with pSR2, which displayed filamentation (data not shown), expressed three times MinCNg levels (Fig. 4d
, lane 5) in comparison to the same strain co-transformed with pSR2 and pREP4 (Fig. 4d
, lane 4). These results demonstrated that, under strict expression control, MinCNg can restore the wild-type phenotype and division pattern in the E. coli minC mutant, DR105.
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DISCUSSION |
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Neisserial min genes belong to a 17 kb gene cluster
The min homologues in N. gonorrhoeae and N. meningitidis are part of a large (17 kb) cluster of genes, which have the same transcriptional polarity and are flanked at the 5' and 3' ends by paired neisserial uptake sequences that presumably act as transcriptional terminators. This large gene cluster is somewhat surprising since neisserial gene clusters are normally shorter, reflecting the fact that the gonococcus has a series of mechanisms for frequent recombination and horizontal gene transfer, which lead to high genetic variability (Goodman & Scocca, 1988 ; Koomey, 1998
). Even the N. gonorrhoeae dcw cluster is distributed into five transcriptional units, whilst E. coli dcw genes belong to one long transcriptional unit (Ayala et al., 1994
; Francis et al., 2000
). However, there were no apparent transcriptional terminators in the neisserial min gene clusters, including the Correia element which has recently been found to act in this manner (Francis et al., 2000
). In addition, experimental evidence of the co-transcription of the minNg genes with at least two upstream genes, rpoA and rplQ, suggests that the minNg genes are part of a longer transcript. This indicates that the neisserial min gene cluster must be stable, and that it is conserved in pathogenic Neisseria species. Although a homologue to the E. coli Rho termination factor gene has been detected in N. gonorrhoeae (Miloso et al., 1999
), it remains to be determined whether its gene product could act to terminate transcription in N. gonorrhoeae as it does in E. coli (Platt & Richardson, 1992
). In addition, it is interesting to note that the gene content of min gene clusters is not conserved between bacteria, perhaps indicative of differing transcriptional regulation amongst genera.
MinC is required for correct cell division in N. gonorrhoeae
In this study, we have shown that MinC from N. gonorrhoeae acts as a cell-division inhibitor. We have also shown that in its absence, gonococci can lyse, a phenotype not observed before. The absence of MinCNg in N. gonorrhoeae CSRC1 generated uncontrolled cell division, which occurred along many planes rather than along perpendicular planes characteristic of the wild-type cells. This uncontrolled division resulted in the formation of abnormal cell clusters possessing cells of varying size and shape, with some cells having multiple, and sometimes incomplete, septa. We also frequently observed large ghost cells characterized by cell-wall structures lacking any obvious internal components. The lower viability of the minCNg mutant is probably due to the lysis observed in these cells or to improper nucleoid segregation within the numerous daughter cells generated.
The cell lysis observed in enlarged gonococci when MinCNg is depleted may be an indirect result of an increase of the surface tension of these cells. Based on the surface stress theory of Koch (1985) , where T=Pr/2 (T, surface tension; P, hydrostatic pressure; and r, radius) for cocci, any increase in internal pressure is accompanied by an increase in cell-wall tension that ultimately leads to cell-wall enlargement during normal growth of bacteria. However, a significant increase in cell size may upset this balance as cell-wall tension mounts and increases the stress of the peptidoglycan bonds, making them more susceptible to splitting. The tension may be sufficiently large such that the peptidoglycan may be hydrolysed without any enzymic aid. Alternatively, peptidoglycan bonds under stress are more susceptible to hydrolysis since autolytic enzymes increase their rate of action under stress conditions (Koch, 1985
). Since cocci need only half the hydrostatic pressure required in rods to increase their surface tension by the same amount (Koch, 1985
), round cells may be more susceptible to the effects of any change in normal cell growth or division, such as the one caused by the depletion of MinC.
The gonococcal minC mutant was complemented when a wild-type copy of minCNg, tagged to 6xHis at its C terminus, was inserted into the chromosomal proAB locus. A minority of cells showed an aberrant phenotype, possibly because the complemented strain expressed higher levels of MinCNg than the wild-type strain CH811. Since the C-terminal region of MinCEc has been shown to be responsible for the stability of the protein (Sen & Rothfield, 1998 ), it may be possible that the 6xHis tag at the C terminus of MinCNg renders the protein more resistant to proteases, thereby maintaining a higher amount of MinCNg in complemented cells. Alternatively, the activity of MinCNg in the complemented strain may be affected due to the 6xHis residues tagged to its C terminus, since altered biological function has been observed in other His-tagged proteins (Wu & Filutowics, 1999
).
MinC from N. gonorrhoeae acts as a cell division inhibitor in E. coli
Transformation of E. coli C43 with a plasmid containing all three minNg genes resulted in the formation of minicells and filaments. This phenotype might be explained, as has been done in E. coli (de Boer & Crossley, 1989 ), by assuming that the overexpression of MinENg would be dominant over the effects of increased MinCNg and MinDNg. The appearance of filamentous E. coli PB103 cells overexpressing MinCNg and the successful complementation of an E. coli minC mutant with minCNg showed that gonococcal MinC is able to retain its function as a cell-division inhibitor across species. Interestingly, ours is the first report of a heterologous complementation of a min mutant. However, other cell-division mutants, such as E. coli thermosensitive ftsZ mutants, have been heterologously complemented (Gaikwad et al., 2000
).
Comparison of MinC proteins from different bacteria revealed a single homologous region, containing one arginine and four glycines that are completely conserved. Arginine and glycine domains have been previously implicated in essential protein functions (Lee et al., 1999 ; Li & Rosen, 2000
; Thoden et al., 1999
; Saitoh et al., 1999
). The conserved residues in MinC may be involved in its activity as a division inhibitor, perhaps in proteinprotein interactions with MinD or FtsZ, which have been described for E. coli MinC (Huang et al., 1996
; Hu et al., 1999
). Recently, Hu & Lutkenhaus (2000)
showed that the C-terminal domain of MinCEc is responsible for its interaction with MinDEc. It is also possible that the conserved region in MinC proteins is responsible for its stability, as has been reported for MinC from E. coli, where mutations in the C-terminal region of the protein cause an increase in protease susceptibility (Sen & Rothfield, 1998
).
Concluding remarks
Most of our current knowledge of bacterial cell division has been assembled from studies based on rod-shaped organisms. However, even in rods that have similar Min proteins, the mechanism by which they act can be very different. For example, whilst MinC and MinD from both E. coli and B. subtilis are proposed to act as cell-division inhibitor complexes, their localization within cells, using GFP fusions, show important differences. Observations on cell-division site selection can now be extended into those cocci containing min genes. Coccal cells can lyse when MinC is not expressed, whereas in rods, deletion of MinC leads to a minicell phenotype. The present report is the first study to explore division site selection in Gram-negative cocci. We have shown that MinCNg is a cell-division inhibitor and that its role is essential to maintain proper site selection in dividing coccal cells.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ayala, J. A., Garrido, T., de Pedro, M. A. & Vicente, M.(1994). Molecular biology of bacterial septation. In Bacterial Cell Wall , pp. 73-102. Edited by J. M. Ghuysen & R. Hakenbeck. Amsterdam:Elsevier.
Beveridge, T. J., Popkin, T. J. & Cole, R. M.(1994). Electron microscopy. In Methods for General and Molecular Microbiology , pp. 44-70. Edited by P. Gerhardt, R. G. E. Murray, W. A. Woods & N. R. Krieg. Washington, DC:American Society for Microbiology.
Bi, E. & Lutkenhaus, J.(1993). Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. J Bacteriol 175, 1118-1125.[Abstract]
de Boer, P. & Crossley, R.(1989). A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in Escherichia coli. Cell 56, 641-649.[Medline]
de Boer, P., Crossley, R. & Rothfield, L.(1988). Isolation and properties of minB, a complex genetic locus involved in correct placement of the division site in Escherichia coli. J Bacteriol 170, 2106-2112.[Medline]
de Boer, P., Cook, W. & Rothfield, L.(1990). Bacterial cell division. Annu Rev Genet 24, 249-274.[Medline]
de Boer, P., Crossley, R. E., Hand, A. R. & Rothfield, L.(1991). The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site. EMBO J 10, 4371-4380.[Abstract]
de Boer, P., Crossley, R. & Rothfield, L.(1992). Roles of MinC and MinD in the site-specific septation block mediated by the MinCDE system of Escherichia coli. J Bacteriol 174, 63-70.[Abstract]
Cha, J. & Stewart, G.(1997). The divIVA minicell locus of B. subtilis. J Bacteriol 179, 1671-1683.[Abstract]
Edwards, D. & Errington, J.(1997). The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division. Mol Microbiol 25, 905-915.
Fitz-James, P.(1964). Thin sections of dividing Neisseria gonorrhoeae. J Bacteriol 87, 1477-1482.[Medline]
Francis, F., Ramirez-Arcos, S., Salimnia, H., Victor, C. & Dillon, J. R.(2000). Organization and transcription of the division cell wall (dcw) cluster in Neisseria gonorrhoeae. Gene 251, 141-151.[Medline]
Gaikwad, A., Babbarwal, V., Pant, V. & Mukherjee, S. K.(2000). Pea chloroplast FtsZ can form multimers and correct the thermosensitive defect of an Escherichia coli ftsZ mutant. Mol Gen Genet 263, 213-221.[Medline]
Goodman, S. D. & Scocca, J. J.(1988). Identification and arrangement of the DNA sequences recognized in specific transformation of Neisseria gonorrhoeae. Proc Natl Acad Sci U S A 85, 6982-6986.[Abstract]
Hebert, G. A., Pelham, P. L. & Pittman, B.(1973). Determination of the optimal ammonium sulfate concentration for the fractionation of rabbit, sheep, horse and goat antisera. Appl Microbiol 25, 26-36.[Medline]
Hu, Z. & Lutkenhaus, J.(1999). Topological regulation of cell division in Escherichia coli involved rapid pole to pole oscillation of the cell division inhibitor MinC under the control of MinD and MinE. Mol Microbiol 34, 82-90.[Medline]
Hu, Z. & Lutkenhaus, J.(2000). Analysis of MinC reveals two independent domains involved in interaction with MinD and FtsZ. J Bacteriol 182, 3965-3971.
Hu, Z., Mukherjee, A., Pichoff, S. & Lutkenhaus, J.(1999). The MinC component of the division site selection system in Escherichia coli interacts with FtsZ to prevent polymerization. Proc Natl Acad Sci U S A 96, 14819-14824.
Huang, J., Cao, C. & Lutkenhaus, J.(1996). Interaction between FtsZ and inhibitors of cell division. J Bacteriol 178, 5080-5085.[Abstract]
Janik, A., Juni, E. & Heym, G. A.(1976). Genetic transformation as a tool for detection of Neisseria gonorrhoeae. J Clin Microbiol 4, 71-81.[Medline]
Justice, S. S., Garcia-Lara, J. & Rothfield, L. I.(2000). Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery. Mol Microbiol 37, 410-423.[Medline]
King, G. F., Rowland, S. L., Pan, B., Mackay, J. P., Mullen, G. P. & Rothfield, L. I.(1999). The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain. Mol Microbiol 31, 1161-1169.[Medline]
Koch, A.(1985). How bacteria grow and divide in spite of internal hydrostatic pressure. Can J Microbiol 31, 1071-1084.[Medline]
Koomey, M.(1998). Competence for natural transformation in Neisseria gonorrhoeae: a model system for studies of horizontal gene transfer. APMIS Suppl 84, 56-61.[Medline]
Lee, S. J., Gray, M. C., Guo, L., Sebo, P. & Hewlett, E. L.(1999). Epitope mapping of monoclonal antibodies against Bordetella pertussis adenylate cyclase toxin. Infect Immun 67, 2090-2095.
Li, J. & Rosen, B. P.(2000). The linker peptide of the ArsA ATPase. Mol Microbiol 35, 361-367.[Medline]
Lutkenhaus, J. & Addinall, S.(1997). Bacterial cell division and the Z ring. Annu Rev Biochem 66, 93-116.[Medline]
Margolin, W.(2000). Self-assembling GTPases caught in the middle. Curr Biol 10, R328-R330.[Medline]
Marston, A. L. & Errington, J.(1999). Selection of the midcell division site in Bacillus subtilis through MinD-dependent polar localization and activation of MinC. Mol Microbiol 33, 84-96.[Medline]
Marston, A. L., Thomaides, H. B., Edwards, D. H., Sharpe, M. E. & Errington, J.(1998). Polar localization of the MinD protein of Bacillus subtilis and its role in selection of the mid-cell division site. Genes Dev 12, 3419-3430.
Miloso, M., Limauro, D., Alifano, P., Rivellini, F., Lavitola, A., Gulleta, E. & Bruni, C. B.(1999). Characterization of the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium. J Bacteriol 175, 8030-8037.[Abstract]
Miroux, B. & Walker, J. E.(1996). Overproduction of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260, 289-298.[Medline]
Nanninga, N.(1998). Morphogenesis of Escherichia coli. Microbiol Mol Biol Rev 62, 110-129.
Pagotto, F., Aman, A. T., Ng, L.-K., Yeung, K.-H., Brett, M. & Dillon, J. R.(2000). Sequence analysis of the family of penicillinase-producing plasmids of Neisseria gonorrhoeae. Plasmid 43, 24-34.[Medline]
Pichoff, S., Vollrath, B., Touriol, C. & Bouché, J.(1995). Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli. Mol Microbiol 18, 321-329.[Medline]
Platt, T. & Richardson, J. P.(1992). Escherichia coli Rho factor: protein and enzyme of transcription termination. In Transcriptional Regulation , pp. 365-388. Edited by S. L. McKnight & K. R. Yamamoto. Cold Spring Harbor, NY:Cold Spring Harbor Laboratory.
Raskin, D. M. & de Boer, P. A. J.(1997). The MinE ring: an FtsZ-independent cell structure required for selection of the correct division site in E. coli. Cell 91, 685-694.[Medline]
Raskin, D. M. & de Boer, P. A. J.(1999a). Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli. Proc Natl Acad Sci U S A 96, 4971-4976.
Raskin, D. M. & de Boer, P. A. J.(1999b). MinDE-dependent pole-to-pole oscillation of division inhibitor MinC in Escherichia coli. J Bacteriol 181, 6419-6424.
Reeve, J. N., Mendelson, N. H., Coyne, S. I., Hallock, L. L. & Cole, R. M.(1973). Minicells of Bacillus subtilis. J Bacteriol 114, 860-873.[Medline]
Roe, B. A., Lin, S. P., Song, L., Yuan, X., Clifton, S., Ducey, T., Lewis, L. & Dyer, D. W. (2000). Gonococcal Genome Sequencing Project (Funded by USPHS/ NIH grant AI 38399). University of Oklahoma.
Rothfield, L., Justice, S. & García-Lara, J. (1999). Bacterial cell division. Annu Rev Genet 33, 423-448.[Medline]
Rowland, S. L., Fu, X., Sayed, M. A., Zhang, Y., Cook, W. R. & Rothfield, L. I.(2000). Membrane redistribution of the Escherichia coli MinD protein induced by MinE. J Bacteriol 182, 613-619.
Saitoh, F., Kawamura, S., Yamasaki, N., Tanaka, I. & Kimura, M.(1999). Arginine-55 in the beta-arm is essential for the activity of DNA-binding protein HU from Bacillus stearothermophilus. Biosci Biotechnol Biochem 63, 2232-2235.[Medline]
Salimnia, H., Radia, A., Bernatchez, S. & Dillon, J. R.(2000). Characterization of the ftsZ cell division gene of N. gonorrhoeae: expression and localization in E. coli and N. gonorrhoeae. Arch Microbiol 173, 10-20.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sen, M. & Rothfield, L. I.(1998). Stability of the Escherichia coli division inhibitor protein MinC requires determinants in the carboxy-terminal region of the protein. J Bacteriol 180, 175-177.
Silver, L. E. & Clark, V. L.(1995). Construction of a translational lacZ fusion system to study gene regulation in Neisseria gonorrhoeae. Gene 166, 101-104.[Medline]
Thoden, J. B., Kappock, T. J., Stubbe, J. & Holden, H. M.(1999). Three-dimensional structure of N5-carboxyaminoimidazole ribonucleotide synthetase: a member of the ATP grasp protein superfamily. Biochemistry 38, 15480-15492.[Medline]
Varley, A. & Stewart, G.(1992). The divIVB region of the Bacillus subtilis chromosome encoded homologs of E. coli septum placement (MinCD) and cell shape (MreBCD) determinants. J Bacteriol 174, 6729-6742.[Abstract]
Westling-Häggström, B., Elmros, T., Normark, S. & Winblad, B.(1977). Growth pattern and cell division in Neisseria gonorrhoeae. J Bacteriol 129, 333-342.[Medline]
Wu, J. & Filutowics, M.(1999). Hexahistidine (His6)-tag dependent protein dimerization: a cautionary tale. Acta Biochim Pol 46, 591-599.[Medline]
Yu, X. & Margolin, W.(1999). FtsZ ring clusters in min and partition mutants: role of both the Min system and the nucleoid in regulating FtsZ ring localization. Mol Microbiol 32, 315-326.[Medline]
Zhang, Y., Rowland, S., King, G., Braswell, E. & Rothfield, L.(1998). The relationship between hetero-oligomer formation and function of the topological specificity domain of the Escherichia coli MinE protein. Mol Microbiol 30, 265-273.[Medline]
Zhao, C., de Boer, P. & Rothfield, L.(1995). Proper placement of the Escherichia coli division site requires two functions that are associated with different domains of the MinE protein. Proc Natl Acad Sci U S A 92, 4313-4317.[Abstract]
Received 26 June 2000;
accepted 11 October 2000.