Deletion of the cell-division inhibitor MinC results in lysis of Neisseria gonorrhoeae

Sandra Ramirez-Arcos1, Jason Szeto1, Terry J. Beveridge2, Charles Victor1, Finola Francis1 and Jo-Anne R. Dillon1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The minCDE genes involved in division site selection in Neisseria gonorrhoeae were identified using raw data from the N. gonorrhoeae genome project and are part of a cluster of 27 genes. When gonococcal min genes were heterologously expressed as a cluster in Escherichia coli, minicells and filaments were produced, indicating that gonococcal min genes disrupted cell division in other genera. The insertional inactivation of the minC gene of N. gonorrhoeae CH811 resulted in a strain (CSRC1) with decreased viability and grossly abnormal cell division as observed by phase-contrast and electron microscopy analysis. Western blot analysis of N. gonorrhoeae CSRC1 confirmed that MinCNg was not produced. Complementation of CSRC1 by integrating a minC–6xHis tag fusion at the proAB locus by homologous recombination restored viability and 1·9 times wild-type levels of MinCNg expression. This slight increase of expression caused a small percentage of the complemented cells to divide aberrantly. This suggested that the 6xHis tag has partially affected the stability of MinC, or that the chromosomal position of minC is critical to its regulation. Comparison of MinC proteins from different bacteria showed a homologous region corresponding to residues 135–230 with five conserved amino acids. Overexpression of MinCNg in wild-type E. coli cells induced filamentation and an E. coli minC mutant was successfully complemented with minCNg. Therefore, the evidence indicates that MinC from N. gonorrhoeae acts as a cell-division inhibitor and that its role is essential in maintaining proper division in cocci.

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


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial rods usually divide symmetrically at mid-cell, in single parallel planes with the formation of the division septum occurring in DNA-free spaces (Nanninga, 1998 ). The key protein that drives the septation process is considered to be FtsZ (de Boer et al., 1990 ; Lutkenhaus & Addinall, 1997 ; Salimnia et al., 2000 ), a highly conserved protein in bacteria which is encoded in the division cell wall (dcw) cluster (Ayala et al., 1994 ; Francis et al., 2000 ). The poles of bacilli are also zones where cell division could occur; however, septa do not form there under normal circumstances. Thus, rods are generally considered to have three potential division sites where FtsZ ring formation causing cellular constriction could occur, one site at mid-cell and one at each pole (de Boer et al., 1990 ). Recently, it has been shown that FtsZ rings can form throughout the length of Escherichia coli cells and that correct placement of the FtsZ ring at the mid-cell is dependent on the inhibitory effect of the nucleoid as well as proteins involved in mid-cell site selection (Yu & Margolin, 1999 ).

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 ). DivIVA–GFP 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 GFP–MinDBs to the poles is dependent on DivIVA. However, the targeting of GFP–MinCBs 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and growth conditions.
The strains used in this study are listed in Table 1. E. coli DH5{alpha} was used as a host to clone minNg genes. E. coli C43(DE3) (Miroux & Walker, 1996 ) was used to express the cloned minCDENg cluster present on plasmid pSR1 (Table 2). This strain is a {lambda}DE3 lysogen encoding T7 RNA polymerase which can initiate minCDENg gene expression under the control of the T7 promoter of pSR1. E. coli PB103 was used for minCNg expression. E. coli DR105 (minC) was used for the complementation studies with minCNg. E. coli M15 and BL21(DE3) were used for the expression and purification of His-tagged MinCNg and MinDNg proteins, respectively. All E. coli cells were grown at 37 °C on Luria–Bertani (LB) medium (Difco). N. gonorrhoeae CH811 streptomycin-resistant strain (Strr) was used to generate the minCNg insertional knockout mutant, N. gonorrhoeae CSRC1. The latter strain was complemented by inserting a minCNg–6xHis fusion using the suicide vector pLES94 (Silver & Clark, 1995 ) inserted at the proAB locus by homologous recombination generating N. gonorrhoeae CSRC2. Gonococcal strains were grown on GC medium base (GCMB) (Difco), supplemented with Kellogg’s defined supplement (GCMBK; Pagotto et al., 2000 ), for 18–24 h at 35 °C in a humid, 5% CO2 environment. GCMBK broth cultures of gonococci containing 0·04% NaHCO3 were incubated with shaking at 200–250 r.p.m., at 35 °C and 5% CO2.When required, antibiotics were added to media in the following concentrations: 100 µg ampicillin (Ap) ml-1, 25 µg kanamycin (Kan) ml-1 and 25 µg chloramphenicol (Cm) ml-1 for E. coli; and 1 mg Str ml-1, 18 µg Kan ml-1 and 5 µg Cm ml-1 for N. gonorrhoeae.


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Table 1. Bacterial strains used in this study

 

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Table 2. Plasmids used in this study

 
Oligonucleotide primers and PCR.
The oligonucleotide primers used for PCR amplification of the min genes were designed using Primer Designer (Scientific and Education Software) and were synthesized by the University of Ottawa Biotechnology Research Institute (Table 3). All PCR reactions were carried out in a Gene Amp PCR System 9600 Thermocycler (Perkin Elmer) using the following program: 3 min at 94 °C; 30 cycles of denaturation for 15 s at 94 °C, annealing for 15 s at temperatures varying from 47 °C to 51 °C (depending on the primer pair used) and extension at 72 °C for 0·5–3 min (depending on the expected product size); and a final 5 min extension at 72 °C. Reactions were carried out in a final volume of 100 µl containing the following reagents: 1x PCR buffer containing 1·5 mM MgCl2, 0·2 mM dNTPs, 0·2 µg each primer and 2·5 U Taq DNA polymerase (Boehringer Mannheim). To detect insertions in the chromosome of N. gonorrhoeae, whole cell suspensions of putative transformants were used to provide chromosomal DNA templates for PCR. Cell suspensions were prepared by diluting cells from overnight cultures on GCMBK plates in double-distilled water. Cell concentrations were adjusted using the McFarland Equivalence Turbidity Standard 0·5 (Remel).


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Table 3. Primer sequences used for PCR and RT-PCR

 
Genomic analysis of min gene clusters and identification of minCDENg genes.
BLAST analysis of raw sequence data from the N. gonorrhoeae FA1090 genome project (Roe et al., 2000 ) was used to identify and align the minCDENg cluster. Annotations of the min genes and the order of the min cluster from N. meningitidis Z2491/serotype A were analysed using information provided at the WIT web site (http://wit.mcs.anl.gov/WIT2/). BLAST searches for min homologues in other organisms were carried out in their respective sequence databases provided by the Institute for Genomic Research (http://www.tigr.org/tdb/mdb/mdb.html). Protein translations were made using PCGene (IntelliGenetics).

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{alpha} 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{alpha} 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 BamHI–PstI 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 minCNg–6xHis 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 minCNg–6xHis, 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 BamHI–NcoI 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 24–48 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 6xHis–MinCNg 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). 6xHis–MinDNg was then cut from SDS-PAGE gels and electroeluted prior to immunization. Each immunization consisted of 20 µg 6xHis–MinCNg or 80 µg 6xHis–MinDNg mixed with Gerbu adjuvant, according to the manufacturer’s 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).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
min genes are present in Neisseria species
We identified homologues of minC, D and E from the raw data of the N. gonorrhoeae genome project (Roe et al., 2000 ), which is now complete and with annotation under way (September, 2000). We determined that the minNg genes appear to be part of a large 17 kb gene cluster, with rpoA ({alpha} subunit of RNA polymerase), secY (secretion protein Y), IF1 (initiation factor 1) and 20 structural ribosomal genes upstream, and with oxyR (oxidative stress transcriptional regulator) downstream. All genes are transcribed in the same direction. This 17 kb cluster is flanked by two stem–loop structures containing neisserial uptake sequences (T1; Fig. 1a). Previous studies have shown that gonococcal gene clusters are generally separated by stem–loop structures containing neisserial uptake sequences which act as transcriptional terminators (Goodman & Scocca, 1988 ; Francis et al., 2000 ). Interestingly, there are no stem–loop transcriptional terminator structures between the structural ribosomal gene L3, at the 5' end of the large cluster, and oxyR located at the 3' end of the gene cluster. Recently, we have described the Correia element as another functional transcriptional terminator (Francis et al., 2000 ); nevertheless, this terminator is also absent in the 17 kb gene cluster containing the minNg genes.



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Fig. 1. The min gene cluster of N. gonorrhoeae. (a) Comparison of the genomic organization of min clusters in different bacteria. Grey arrows represent the direction of transcription. White arrows show the orientation of the primers used in RT-PCR. T1, transcriptional terminator consisting of a stem–loop structure containing neisserial uptake sequences (US); T2, rho-independent transcription terminator sequences. (b) Co-transcription of rpoANg–minENg by RT-PCR. Lanes: 1, rpoA PCR amplicon from cDNA amplified with primer CVME38; 2, PCR control, amplicon from chromosomal DNA of N. gonorrhoeae CH811; M, 1 kb DNA ladder.

 
Since the genes upstream of the minNg cluster are not apparently related to cell-division processes and there is no obvious transcriptional terminator in the 149 bp silent region upstream of minCNg, we used RT-PCR to investigate whether the minNg genes could be expressed from promoters further upstream of the minNg cluster. Primer CVME38, which annealed at the 3' end of minENg (Fig. 1a, Table 3), was used to generate cDNA from total RNA isolated from N. gonorrhoeae CH811. This cDNA was used as a template for PCR using primers CVRA41 and CVRA42 (Fig. 1a, Table 3) which hybridized internally to the rpoA gene upstream of minCNg. An amplicon of the expected size of 657 bp was produced from this RT reaction which would only occur if an mRNA transcript containing at least rpoA to minE was synthesized (Fig. 1b); therefore, the genes rpoA, rplQ, minC, minD and minE are co-transcribed.

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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Table 4. minCDE homologues in completed prokaryotic genome projects

 
A minC knockout in N. gonorrhoeae CH811 causes cell aberrations
An insertional knockout of minC in N. gonorrhoeae CH811 was created (N. gonorrhoeae CSRC1). If MinCNg acts as a division inhibitor, gonococcal cells lacking this protein should appear as having uncontrolled cell division, not the sequential cell division along perpendicular planes, found in wild-type cells. We show by phase-contrast and electron microscopy (Fig. 2) that, in stark contrast to the wild-type CH811 cells (Fig. 2a, c), the phenotype of the minCNg mutant (CSRC1) cells included clusters of cells of differing sizes and a large number of lysed cells (Fig. 2b, d, e). From 50 randomly counted cells of wild-type N. gonorrhoeae CH811, 48 had a normal cell-division phenotype and two cells showed asymmetric cell division (i.e. one cell larger than the other). By contrast, of 50 cells in N. gonorrhoeae CRSC1, 6 were normal, 16 were aberrant (i.e. multi-lobed cells) and 28 were ghost cells (i.e. empty cells). Under electron microscopy analysis, these abnormal morphologies in N. gonorrhoeae CSRC1 appeared as multi-lobed cells having bulges at the septa, incomplete divisions and ghost cells (Fig. 2d, e). These observations are consistent with cell division occurring along several planes other than the characteristic perpendicular planes described for the wild-type strain (Fig. 2c).



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Fig. 2. Effects of minC knockout on morphology of N. gonorrhoeae CSRC1: (a, b) phase-contrast micrographs; (c–e) electron microscopy micrographs. (a, c) Control N. gonorrhoeae CH811 exhibit wild-type morphology. Cell division results in equal-sized daughter cells. (b) N. gonorrhoeae CSRC1, minC knockout mutant, displays lysed (ghost) cells. (d) N. gonorrhoeae CSRC1, minC knockout mutant, showing asymmetric division patterns resulting in different-sized daughter cells (arrows). (e) Ghost cells of N. gonorrhoeae CSRC1. The scale bar in (a) is 10 µm and (b) is the same magnification. The scale bar in (c) is 500 nm and (d) and (e) are the same magnification.

 
In addition to its gross phenotypic alterations, the N. gonorrhoeae minC mutant CSRC1 grew slowly and formed colonies of quite different sizes (0·5–4·0 mm diameter). The number of c.f.u. ml-1 of the minCNg mutant was significantly lower than the wild-type strain (Fig. 3a), despite similar initial inoculum sizes. The initial viable counts of N. gonorrhoeae CSRC1, which required 72 h incubation before colonies were observed, averaged 105 c.f.u. ml-1. The initial viable count for N. gonorrhoeae CH811, in which colonies were observed within the normal incubation time of 18–24 h, was 107 c.f.u. ml-1 (Fig. 3a). This 100-fold difference between the two strains was maintained over 9 h. Western blot analysis using anti-MinCNg antisera on cell extracts (Fig. 3b, lane 2) indicated that MinCNg was not expressed in the mutant N. gonorrhoeae strain (CSRC1) compared to the wild-type strain CH811 (Fig. 3b, lane 1).



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Fig. 3. Comparison of the viability of the wild-type, minCNg mutant and complemented N. gonorrhoeae strains. (a) Colony counts of wild-type N. gonorrhoeae CH811 (squares), minC mutant N. gonorrhoeae CSRC1 (triangles) and minC-complemented strain N. gonorrhoeae CSRC2 (circles). Colonies could be counted after 24 h incubation for CH811 and CSRC2 and only after 72 h incubation for CSRC1. (b) Western blot using anti-MinCNg. Soluble fractions of: lanes 1, N. gonorrhoeae CH811; 2, N. gonorrhoeae minC mutant, CSRC1; 3, complemented minC mutant, N. gonorrhoeae CSRC2. (c) Western blot using anti-MinDNg. Lanes: 1, purified 6xHis–MinDNg as a control; 2, N. gonorrhoeae CH811; 3, N. gonorrhoeae CSRC1.

 
Reversal of mutant minC phenotype in N. gonorrhoeae CSRC1 by complementation
N. gonorrhoeae CSRC1 was complemented by the insertion of one copy of a C-terminal 6xHis-tagged minCNg into the gonococcal chromosome at the proAB locus using the suicide plasmid pSR18 (see Methods). Expression of MinCNg in the complemented strain, N. gonorrhoeae CSRC2, was confirmed by Western blot analysis (Fig. 3b, lane 3). Densitometry analysis indicated that the expression of MinCNg in CSRC2 was ~1·9 times greater than in the wild-type strain N. gonorrhoeae CH811 (Fig. 3b, lane 1). However, the viability of N. gonorrhoeae CSRC2 was similar to that of CH811 (Fig. 3a) and different-sized colonies were not observed in this strain, unlike the minCNg knockout mutant CSRC1. In addition, colonies from N. gonorrhoeae CSRC2 were obtained after 24 h incubation and not after 72 h as observed within CSRC1. Phase-contrast examination of CSRC2 indicated that the majority of the cells had a wild-type phenotype, as exemplified by the appearance of diplococci and cell tetrads (data not shown). From 50 randomly counted cells, 41 appeared to be dividing normally whereas 6 were dividing abnormally (i.e. multilobed) and 3 burst/ghost cells were seen. Since the wild-type phenotype was not completely recovered on complementation, we investigated whether the minCNg mutation exerted a polar effect on MinDNg expression in N. gonorrhoeae CSRC1. Western blots using anti-MinDNg antibodies showed that MinDNg is expressed in N. gonorrhoeae CSRC1 (Fig. 3c, lane 3) at similar levels as the wild-type CH811 (Fig. 3c, lane 2).

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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Fig. 4. Expression of minNg in E. coli. (a) Control. E. coli C43(DE3) transformed with pBluescript KS II (+) exhibiting wild-type morphology. (b) E. coli C43(DE3) cells transformed with pSR1 (minCDENg) exhibit a minicell phenotype: minicells and short filaments. (c) E. coli PB103 cells transformed with pSR2 (minCNg) exhibit long filaments. The scale bar in (a) is 10 µm and all other figures are the same magnification. (d) Western blot using anti-MinCNg. Lanes: 1, purified 6xHis–MinCNg; 2, wild-type E. coli PB103 transformed with pUC18; 3, E. coli PB103 overexpressing MinCNg from pSR2; 4, E. coli DR105 (minC) expressing MinCNg from pSR2 under in trans control of LacIq from pREP4; 5, E. coli DR105 (minC) overexpressing MinCNg from pSR2.

 
Since overexpression of E. coli MinC produces filamentation (de Boer & Crossley, 1989 ), which is indicative of cell-division inhibition, we investigated whether gonococcal MinC alone produced a similar phenotype when overexpressed in E. coli. Transformation of pSR2 (minCNg) into E. coli PB103 produced a high percentage of filamentous cells (Fig. 4c). Western blot experiments indicated that E. coli PB103 transformed with pSR2 overexpressed MinCNg (Fig. 4d, lane 3) compared to the same strain transformed with pUC18 as a control, which only showed the background signal which would correspond to the cross-reacting resident E. coli MinC (Fig. 4d, lane 2).

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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Fig. 5. Complementation of E. coli DR105 (minC) with minCNg. (a) E. coli DR105 (minC) transformed with pUC18, as a control, exhibit a minicell phenotype. The generation time is 50 min. (b) E. coli DR105 (minC) co-expressing pSR2 (minCNg) and pREP4 (lacIq) after 2 h 1 mM IPTG induction. The generation time is 2 h. Most cells present wild-type phenotype and normal mid-cell division (arrow).The scale bar in (a) is 10 µm and both figures are the same magnification.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have shown that a minCDE gene cluster is present in a pathogenic Neisseria species and that the genes are involved in cell-division site selection. This is the first study that addresses the function of min genes from cocci. The division pattern in some cocci, such as N. gonorrhoeae, alternates in perpendicular planes, differing substantially from rods, which divide along parallel planes (Westling-Häggström et al., 1977 ; Nanninga, 1998 ). Models for division site selection in rods may not adequately explain division site selection in cocci because: (a) there is no obvious mid-cell location in cocci and therefore the manner in which a spherical cell defines its middle for division must be determined; (b) it is not intuitively clear how the concept of ‘middle’ would be maintained in cells that divide in perpendicularly alternating, and not parallel, planes. In addition, due to the differences in the presence and the combination of min genes in various genera (Table 4), other mechanisms or proteins for selecting division sites must exist. Based on our genomic analysis, it became evident that MinD is the most ubiquitously distributed Min protein amongst bacteria. This may be due to its ATPase activity and its other possible functions in the cell, aside from its activation of MinC (de Boer et al., 1991 ). In addition, it is interesting that of all bacteria sequenced to date, only Gram-negative bacteria have complete minCDE clusters. The absence of MinE in Gram-positive organisms may indicate that fundamental differences in wall structure, which contribute to septation, may require different proteins for controlling cell-division site selection. It is also suspected that none of the {alpha}-Proteobacteria have Min systems (Margolin, 2000 ).

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 protein–protein 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.


   ACKNOWLEDGEMENTS
 
This project was partially funded by the Canadian Bacterial Diseases Network (Centers of Excellence) through grants to J. R. Dillon and to T. J. Beveridge and through an operating grant from the Medical Research Council to J. R. Dillon. Electron microscopy was performed in the NSERC Guelph Regional SDM Facility, which is partially funded by a Major Facilities Access Grant from NSERC to T. J. Beveridge. We thank Dr W. Margolin for helpful comments and suggestions regarding this manuscript. We also thank Dr P. de Boer and Dr J. E. Walker for providing the E. coli strains used in expression studies, and Dr V. Clark for supplying the cloning vector pLES94. We are grateful to D. Moyles (University of Guelph) for her technical expertise in the EM studies and to Dr A. Krantis (University of Ottawa) for providing the phase-contrast microscope.


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Received 26 June 2000; accepted 11 October 2000.