1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada K1H 8M5
2 Centre for Research in Biopharmaceuticals and Biotechnology, University of Ottawa, Ottawa, ON, Canada K1H 8M5
Correspondence
Jo-Anne R. Dillon
j.dillon{at}usask.ca
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AF414352.
Present address: Infectious Diseases, Canadian Blood Services, 1800 Alta Vista Drive, Ottawa, Ontario K1G 4J5, Canada.
Present address: College of Arts and Science, University of Saskatchewan, Room 226, Arts Building, 9 Campus Drive, Saskatoon, Saskatchewan, Canada S7N 5A5.
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INTRODUCTION |
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In other micro-organisms, such as Gram-negative bacteria, the MinC, MinD and MinE proteins are involved in controlling cell division site selection (de Boer et al., 1989; Ramirez-Arcos et al., 2001
; Szeto et al., 2001
). However, Gram-positive organisms lack MinE and instead possess an unrelated protein, DivIVA. This protein has mostly been studied in the rod Bacillus subtilis (Bs), where it is involved in cell division and chromosome segregation. DivIVABs, together with MinCBs and MinDBs, defines the midcell. MinCBs and MinDBs interact to form an inhibitor complex which prevents division at the cell poles (Marston & Errington, 1999
). The topological specificity of the MinCDBs inhibitor complex is controlled by DivIVABs, which retains MinCDBs at the cell poles after division (Edwards & Errington, 1997
). DivIVABs-GFP localizes to both the midcell and polar regions of B. subtilis, further supporting its role at both old and nascent cell poles (Edwards & Errington, 1997
). In chromosome segregation during sporulation, DivIVABs helps to position the oriC region of the chromosome at the cell pole in preparation for polar division (Thomaides et al., 2001
).
In other Gram-positive organisms, such as the actinomycete Streptomyces coelicolor (Flardh, 2003) and Brevibacterium lactofermentum (Ramos et al., 2003
), DivIVA is involved in controlling apical growth and morphology. Although DivIVA is present mostly in Gram-positive bacteria, a DivIVA homologue, FruD, was shown to be involved in cell growth and development in the Gram-negative soil bacterium Myxococcus xanthus (Akiyama et al., 2003
).
Unlike the rod B. subtilis, cocci such as E. faecalis, Streptococcus pneumoniae and Staphylococcus aureus do not have Min protein homologues, although all contain DivIVA homologues. Thus, the mechanism of cell division site selection in these organisms is unknown, as is the function of DivIVA. Recently, Pinho & Errington (2004) reported that disruption of divIVA in Staphylococcus aureus did not have an effect on chromosome segregation or cell morphology. In contrast, inactivation of divIVA in S. pneumoniae resulted in severe growth inhibition, producing defects in cell shape, nucleoid segregation and cell division (Fadda et al., 2003
). It was also revealed that S. pneumoniae divIVA is co-transcribed with other genes likely to be involved in cell division (Fadda et al., 2003
). Similarly, our bioinformatic analysis of the genome project strain E. faecalis V583, presented in this study, suggests that divIVA is transcribed with other genes of the division cell wall (dcw) gene cluster.
In this study, we show that divIVA in E. faecalis (Ef) is essential and is involved in maintaining proper cell division, viability and chromosome segregation. In addition, our studies of DivIVAEf overexpression in round mutant Escherichia coli, a heterologous model host, confirmed the role of this protein in maintaining proper cell division in cocci. The inability of divIVAEf to complement B. subtilis and S. pneumoniae divIVA mutants and the differences between DivIVA sequences from the three species suggest that either the function of DivIVA differs in each micro-organism or that DivIVAEf may not interact with other proteins of the B. subtilis or S. pneumoniae cell division machineries.
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METHODS |
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PCR.
Oligonucleotide primers used for PCR amplification are listed in Table 2. Primers were designed using Primer Designer (Scientific and Education Software) and were synthesized by the Core DNA Synthesis and Sequencing Facility, Centre for Research in Biopharmaceuticals and Biotechnology, University of Ottawa (UOCDSSF). PCR reactions were carried out in a Perkin Elmer GenAmp PCR System 9600 Thermocycler (Perkin Elmer) as follows: 5 min at 94 °C; 30 cycles of denaturation for 15 s at 94 °C, annealing for 15 s at temperatures varying from 47 to 51 °C (depending on the primer pair used) and extension for 11·5 min at 72 °C (depending on the expected product size), 5 min at 72 °C and hold at 4 °C. PCR reactions were carried out in a final volume of 100 µl containing the following reagents: 75·5 µl double distilled H2O (ddH2O), 10 µl 10x PCR buffer containing 15 mM MgCl2 (Roche), 2 µl 10 mM dNTPs (Boehringer Mannheim), 1 µl each primer (0·2 µg ml1), 0·5 µl Taq DNA polymerase (Roche) and 10 µl template DNA. E. faecalis JH2-2, B. subtilis 1756 and S. pneumoniae Rx1 (divIVA) cell suspensions were prepared by diluting cells from overnight cultures in ddH2O. Cell concentrations were adjusted using a 0·5 McFarland Equivalence Turbidity Standard (Remel) to provide chromosomal DNA templates for PCR. Plasmid DNA templates were adjusted to 0·01 µg ml1 with ddH2O.
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The divIVA gene from E. faecalis JH2-2, along with its own promoter, was PCR-amplified using primers IVA-PRO and IVA-4, containing PstI and XbaI restriction sites, respectively (Table 2). This amplicon was cloned into pMSP3545 (Table 1
), a plasmid used for protein overexpression in enterococcal backgrounds (Bryan et al., 2000
), generating plasmid pMSPSRDiv-2 (Table 1
). Co-transformation experiments of E. faecalis JH2-2 with plasmids p3ERMdiv-kan and pMSPSRDiv-2 were carried out as described above. A transformant, selected on BHI with 125 µg Ery ml1 and 500 µg Kan ml1, was designated E. faecalis JHSR1. This strain was then successively subcultured (five times) on BHI agar supplemented only with 1000 µg Kan ml1. The insertion and orientation of kan into chromosomal divIVAEf was confirmed by PCR amplification followed by DNA sequence analyses. E. faecalis JH2-2 was also transformed with pMSPSRDiv-2 as a control. PCR amplification of wild-type and inactivated divIVAEf was performed to estimate whether the copy number of pMSPSRDiv-2 in E. faecalis JHSR1 differs in cells grown in BHI with or without Ery. Relative concentrations of the PCR amplicons were determined by densitometry. The DNA sequence of all plasmid inserts was obtained to confirm frame conservation and gene integrity at UOCDSSF.
Morphological studies of E. faecalis JHSR1 (divIVA).
To determine whether the insertional inactivation of divIVAEf affected the morphology and size of enterococcal mutant cells, phase-contrast microscopy using a Zeiss Axioskop microscope was performed on strains E. faecalis JH2-2 and JHSR1 as described previously (Ramirez-Arcos et al., 2004). E. faecalis JHSR1 was grown in BHI supplemented either with 1000 µg Kan ml1, or with 125 µg Ery ml1 and 500 µg Kan ml1. To investigate whether high levels of Kan contributed to the morphology of E. faecalis JHSR1, this strain was grown either in non-supplemented BHI or in Ery-supplemented BHI. DAPI staining was also applied to wild-type and mutant strains as described by Fadda et al. (2003)
. At least 10 microscopy fields were examined for each strain, grown in different conditions, each containing a minimum of 100 cells. Photographs were obtained using a Sony Power HAD 3CCD Colour Video Camera and Northern Eclipse software version 6.0. Transmission electron microscopy was performed on E. faecalis strains JH2-2 and JHSR1 as described previously (Ramirez-Arcos et al., 2001
).
Viability studies.
E. faecalis JH2-2 and JHSR1 cells were diluted from overnight cultures into BHI liquid medium to obtain an initial standard inoculum at OD600 of 0·05. To avoid the effect that Kan may have on these experiments, E. faecalis JHSR1 was grown either in non-supplemented BHI or in BHI containing 125 µg Ery ml1. Cultures were incubated at 37 °C without agitation. One millilitre of culture was removed every 2 h for a period of 10 h to measure the OD600, and was serially diluted in liquid BHI. Then, 0·1 ml from selected dilutions was plated, in duplicate, on BHI agar. The wild-type strain was plated on non-supplemented medium while E. faecalis JHSR1 was plated either on non-supplemented BHI or on BHI containing 125 µg Ery ml1. Plates were incubated for 24 h followed by determination of c.f.u. ml1.
Overexpression of DivIVAEf in round E. coli cells.
To determine whether DivIVAEf could disrupt cell division in Gram-negative cocci, round E. coli KJB24 (rodA) (Table 1) was used as a heterologous model system for DivIVAEf expression. E. faecalis divIVA was PCR-amplified using primer pair IVA-1 and IVA-2, containing EcoRI and BamHI restriction sites, respectively (Table 2
). This amplicon was cloned into pUC18 (Table 1
), which had been previously digested with the same restriction enzymes, generating plasmid pSM6 (Table 1
). The non-cell division gene prgX, which encodes a transcriptional regulator on the enterococcal conjugative plasmid pCF10 (Bae et al., 2000
), and which was expressed from plasmid pMSP3535X, was used as a control. The gene was PCR-amplified from E. faecalis OG1RF carrying pMSP3535X (Bryan et al., 2000
) using primer pair PRGX-1 and PRGX-2, containing EcoRI and BamHI restriction sites, respectively (Table 2
). The prgX gene was ligated into pUC18 (Table 1
) previously digested with the same restriction enzymes, generating plasmid pSR-X (Table 1
). Plasmids pSM6 and pSR-X were confirmed by restriction and DNA sequence analyses at UOCDSSF. These plasmids along with pUC18 were separately transformed in E. coli KJB24. Transformants were selected on LB medium supplemented with Amp and thymine. The morphology of the transformants and host strain was analysed by phase-contrast microscopy as described above.
Complementation of an S. pneumoniae divIVA mutant strain with DivIVAEf.
S. pneumoniae Rx1 (divIVA) was transformed with plasmid pMSPSRDiv-2, which expresses the divIVAEf gene (Table 1), following protocols outlined at http://www.btxonline.com/applications/protocols/pdfs/PR0133.pdf. To prepare competent cells, S. pneumoniae Rx1 (divIVA) (Table 1
) was grown overnight in 2 ml TS broth containing 4·5 µg Cm ml1. Samples (500 µl) of overnight cultures were inoculated into 20 ml TS broth supplemented with Cm and incubated until an OD600 of 0·3 was achieved. Cells were then washed twice and concentrated tenfold with electroporation buffer (0·5 M sucrose, 7 mM potassium phosphate, pH 7·5, and 1 mM MgCl2). Of these competent cells, 400 µl was electroporated with 10 µl pMSPSRDiv-2 (0·1 µg ml1). Electroporated cells (150 µl) were plated on TS agar plates and incubated for 2 h at 35 °C with 5 % CO2. A layer of 20 ml TS agar containing 4 µg Cm ml1 and 4 µg Ery ml1 was added to each plate before further incubation for 24 h under the same conditions. Colonies were then subcultured on TS agar plates containing Cm and Ery. The presence of E. faecalis divIVA was confirmed by PCR and Western blotting. The transformed strain was named S. pneumoniae (divIVA)EfDiv (Table 1
). Phase-contrast microscopy was performed on S. pneumoniae strains as described above.
Complementation of a B. subtilis divIVA mutant strain with DivIVAEf.
E. faecalis divIVA was PCR-amplified along with its own ribosome-binding site using primer pair IVA-XYL and IVA-2, containing EcoRI and BamHI restriction sites, respectively (Table 2). This amplicon was cloned into pJPR1 (Table 1
), which acts as a suicide plasmid in B. subtilis and which had been previously digested with the same restriction enzymes, generating plasmid pJPR1div (Table 1
). The divIVAEf gene in pJPR1div is under the control of the xylose promoter (Pxyl) and is flanked at its 5' and 3' ends by the B. subtilis amyE gene. pJPR1div was transformed into B. subtilis 1756 (divIVA) following the protocol of Marston & Errington (1999)
. B. subtilis 1756 has an inactivated divIVABs gene as well as a wild-type divIVABs gene inserted into the chromosome, which is expressed under the control of the PSpac promoter (inducible with IPTG) (Thomaides et al., 2001
). A transformant was selected on PAB plates supplemented with Cm and Ery and designated B. subtilis 1756Efdiv. The insertion and orientation of divIVAEf into the chromosomal amyEBs gene was confirmed by PCR and DNA sequence analyses, as described above. Insertion of divIVAEf at this chromosomal position was also verified by amylase activity of the B. subtilis 1756 and 1756EfDiv strains. To ascertain amylase activity, these strains were subcultured on starch agar (Difco) followed by the addition of Lugol's iodine solution to the cultures. Strains with amylase activity produced a clear zone surrounding the colonies where the starch was hydrolysed upon the addition of iodine, while negative amylase activity was observed by the unchanged purple colour of the starchiodine complex. DivIVAEf expression was induced with xylose while DivIVABs expression was induced with IPTG. B. subtilis 1756 and 1756EfDiv strains were diluted from overnight cultures into S liquid medium [0·2 % (w/v) (NH4)SO4, 1·4 % (w/v) K2HPO4, 0·6 % (w/v) KH2PO4, 0·1 % sodium citrate, 0·02 % (w/v) MgSO4, 0·056 % (w/v) MnSO4, 0·5 % (w/v) glucose] (Marston & Errington, 1999
) supplemented with Ery and Cm to obtain an initial inoculum at an OD600 of
0·07. Five millilitres of each culture was then distributed in duplicate into tubes containing different concentrations [none, 0·01, 0·02, 0·05, 0·1, 0·2, 0·5, 1 and 2 % (w/v)] of D-xylose. One series of tubes was also supplemented with 0·1 mM IPTG. Cultures were incubated at 37 °C with agitation (250 r.p.m.) until they reached an OD600 of
0·7 and they were then split into two aliquots followed by centrifugation (5 min at 5000 r.p.m.). One aliquot was kept for phase-contrast microscopy analyses, and the other aliquot was used to prepare protein extracts for Western blot analysis.
Purification of DivIVAEf-6xHis and production of anti-DivIVAEf antibodies.
To purify DivIVAEf, the coding region of divIVAEf was PCR-amplified from E. faecalis JH2-2 with primers IVApet-1 and IVApet-2, incorporating NdeI and XhoI restriction sites at its 5' and 3' ends, respectively (Table 2). The gene was fused in-frame to the C-terminal 6xHis tag of pET30a (Novagen), generating pSRDiv (Table 1
). The fusions were confirmed to be in-frame by DNA sequencing. Samples (25 ml) of exponential-phase cultures of E. coli C41(DE3) carrying this plasmid were induced with 0·4 mM IPTG for 4 h at 37 °C with shaking at 250 r.p.m. Protein purification was done as described previously (Ramirez-Arcos et al., 2004
) with the following modifications: the protein was washed with a buffer containing 60 mM imidazole, 0·5 M NaCl and 20 mM Tris/HCl (pH 7·9) and eluted with the same buffer containing 250 mM imidazole, as described by Novagen. Protein concentration was determined by the Bradford (1976)
method using the Bio-Rad protein assay dye reagent. Purified DivIVAEf-6xHis (100 ng) was mixed with Gerbu adjuvant according to the manufacturer's instructions (Gerbu Biotechnik) and were used to immunize female New Zealand white rabbits for the production of polyclonal anti-DivIVAEf antibodies. Three boosters were administered using the same protein concentration. The antiserum IgG was purified with Protein G Sepharose (Pharmacia) as described by the manufacturer.
Protein analysis and Western blotting of bacterial and yeast extracts.
Protein extracts from E. faecalis, B. subtilis and S. pneumoniae strains were prepared as described previously (Ramirez-Arcos et al., 2004). Protein extracts from Saccharomyces cerevisiae expressing DivIVAEf and DivIVABs fused to the GAL4 activation domain (AD) and GAL4 DNA binding domain (BD) were prepared according to the method described by Hoffman et al. (2002)
. Cells were grown to an OD600 of 0·5; then 1 ml cells were harvested, washed in sterile ddH2O and resuspended in 50 µl PBS with 10 µl glass beads (Sigma) followed by rigorous vortexing for 1 min. The suspension was mixed with 50 µl sample buffer [100 mM NaOH, 10 mM EDTA, pH 8·0, 10 % (v/v) glycerol, 10 % (w/v) SDS]. Samples were then boiled for 5 min, followed by centrifugation at 14 000 r.p.m. for 5 min, and supernatants were kept as total protein fractions. Bacterial and yeast protein fractions were separated by SDS-PAGE and protein concentrations were standardized prior to membrane transfer by densitometry as described previously (Ramirez-Arcos et al., 2004
). SDS-PAGE-resolved proteins were transferred to Immobilon-P membranes (Millipore) and Western blotting was conducted according to methods described previously (Ramirez-Arcos et al., 2004
). Membranes were incubated with 1 : 2000 (0·3 µg ml1) anti-DivIVAEf antiserum, overnight at 4 °C. Differences in protein expression levels were calculated by densitometry.
Yeast two-hybrid assays.
diviIVAEf was PCR-amplified from E. faecalis JH2-2 using primer pair IVA-1 and IVA-2, incorporating EcoRI and BamHI restriction sites at the 5' and 3' ends, respectively (Table 2). The gene was ligated in-frame with the GAL4 DNA binding domain (BD) and the GAL4 activation domain (AD) of pGBT9 and pGAD424 (Clontech), respectively. Similarly, divIVABs was PCR-amplified using primer pair BsDivF and BsDivR1, incorporating BamHI and BglII restriction sites at the 5' and 3' ends, respectively (Table 2
). This gene was ligated in-frame with the GAL4 AD of pGAD424. divIVABs was also PCR-amplified using primer pair BsDivF and BsDivR2, incorporating BamHI and PstI restriction sites at the 5' and 3' ends, respectively (Table 2
). This gene was ligated in-frame with the GAL4 DNA BD of pGBT9. The plasmids containing divIVAEf and divIVABs were pSRAD-Div (AD-DivIVAEf), pSRBD-Div (BD-DivIVAEf), pGBTDivBs (BD-DivIVABs) and pGADDivBs (AD-DivIVABs), respectively (Table 1
). All plasmids were transformed into Saccharomyces cerevisiae SFY526, singly as a negative control or in pairs to test for proteinprotein interactions, using the lithium acetate (LiAc) method (Clontech Yeast Two-Hybrid Manual). Yeast transformants were assayed for
-galactosidase activity using the colony-lift method (with X-Gal as a substrate) and liquid assays [using o-nitrophenyl-D-galactopyranoside (ONPG) as a substrate] as described in the Clontech Yeast Two-Hybrid Manual. Both colony-lift and liquid assays were performed in triplicate at least twice for each interaction. Standard deviations were determined on the means of
-galactosidase activities. Unpaired t-test analysis was done to calculate the differences between the strength of proteinprotein interactions.
Bioinformatic analyses.
The sequence of divIVA from E. faecalis JH2-2 was ascertained (GenBank accession no. AF414352) and proved to be identical to the divIVA sequence from the genome project strain E. faecalis V583 (http://www.tigr.org/). Analysis of the gene order and the presence of putative promoters and transcriptional terminators in the E. faecalis dcw gene cluster was completed using
http://sun1.softberry.com/berry.phtml?topic=annot&group=data&subgroup=annotation
. DivIVAEf, DivIVABs and S. pneumoniae DivIVA sequences were obtained from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). Percentage identity between proteins was calculated using the sequence manipulation software available at (http://www.bioinformatics.org/sms/index.html) and BLAST analysis at the NCBI website.
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RESULTS |
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To minimize the expression of wild-type DivIVAEf from pMSPSRDiv-2 in E. faecalis JHSR1, this strain was subcultured five times on medium containing 1000 µg Kan ml1 and no Ery, hence selecting against the expression of the wild-type protein from pMSPSRDiv-2. PCR amplification of wild-type divIVAEf from E. faecalis JHSR1 grown in Kan-supplemented medium indicated that the copy number of pMSPSRDiv-2 was decreased approximately fivefold (Fig. 2a, lane 2) as compared to the copy number of the plasmid from cells grown with both Kan and Ery (Fig. 2a
, lane 1). As expected because of its chromosomal location, the copy number of the inactivated gene, divIVAEf : : kan, was maintained in cells grown under either condition (Fig. 2a
, lanes 1 and 2). Correspondingly, Western blot analyses revealed that DivIVAEf protein levels in E. faecalis JHSR1 grown in BHI supplemented with both Ery and Kan were about fivefold higher (Fig. 2b
, lane 2) as compared to E. faecalis JHSR1 grown only with Kan (Fig. 2b
, lane 1). These results indicated that expression levels of wild-type DivIVAEf in E. faecalis JHSR1 were dependent on the copy number of pMSPSRDiv-2. Interestingly, expression of DivIVAEf from pMSPSRDiv-2 did not provide full complementation to the mutant strain since DivIVAEf protein levels in E. faecalis JHSR1 grown with Ery (Fig. 2b
, lane 2) were significantly decreased (
20-fold) as compared to wild-type E. faecalis JH2-2 (Fig. 2b
, lane 4). Protein levels of DivIVAEf from wild-type E. faecalis JH2-2 transformed with pMSPSRDiv-2 revealed only a slight increase (
0·2-fold) (Fig. 2b
, lane 3) as compared to wild-type DivIVAEf levels from non-transformed JH2-2 cells (Fig. 2b
, lane 4).
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DivIVAEf does not complement a B. subtilis divIVA mutant and inhibits homologous complementation
We investigated whether E. faecalis divIVA could complement B. subtilis 1756, a strain in which divIVA was inactivated. This strain also contains a wild-type copy of divIVABs inserted into the chromosome under the control of the Spac promoter (PSpac), which can be induced with IPTG (Thomaides et al., 2001). E. faecalis divIVA was inserted into the chromosomal amyE gene of B. subtilis 1756, generating B. subtilis 1756EfDiv. Enterococcal divIVA is expressed under the control of the xylose promoter (Pxyl) in B. subtilis 1756EfDiv. The insertion of divIVAEf into amyEBs of strain 1756EfDiv was confirmed by disruption of amylase activity (data not shown). Inactivation of divIVA in B. subtilis produces a minicell phenotype, characterized by the presence of short filaments and minicells (Cha & Stewart, 1997
). B. subtilis 1756EfDiv cells presented a minicell phenotype (Table 3
), which reverted to wild-type morphology upon induction of the homologous divIVABs gene with 0·1 mM IPTG, similar to the parental strain B. subtilis 1756 (Table 3
). When expression of divIVAEf was induced with 0·5, 1 or 2 % (w/v) D-xylose in B. subtilis 1756EfDiv (no IPTG added), the divIVABs mutation was not complemented and the minicell phenotype was maintained (Table 3
). Western blotting showed that DivIVAEf was expressed in these cells (data not shown), indicating that the failure of DivIVAEf to complement was not due to lack of protein expression. Comparison of DivIVA sequences from E. faecalis and B. subtilis revealed that these proteins are 34 % identical, with DivIVAEf being 40 aa longer at the C terminus. Furthermore, there is a region of 20 aa, corresponding to positions 95116 of DivIVAEf, which is missing in the Bacillus protein.
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DISCUSSION |
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Due to the essentiality of divIVA in E. faecalis, an enterococcal divIVA mutant strain was obtained only through rescue experiments by expressing wild-type divIVAEf in trans from pMSPSRDiv-2. However, the wild-type gene could not complement all deleterious effects caused by the mutation, such as cell clustering and decreased viability. The lack of full complementation was due to the fact that DivIVAEf levels from rescued cells were not the same as those from wild-type E. faecalis JH2-2 cells. One might hypothesize that in wild-type enterococcal cells, divIVAEf expression is controlled by two promoters, its own and one upstream of ftsAEf, as suggested by our bioinformatic analyses. In contrast, expression of divIVAEf from pMSPSRDiv-2 is only under the control of its own promoter. In E. coli, it has been demonstrated that genes of the dcw cluster have a complex transcriptional regulation and that full expression of some genes depends on more than one promoter (Flardh et al., 1998; de la Fuente et al., 2001
).
Overexpression of DivIVAEf in E. coli KJB24 resulted in enlarged cells with disrupted cell division, suggesting that this round E. coli mutant strain can be used as an indicator for functionality of cell division proteins from Gram-positive cocci. Similar morphological changes have been observed when cell division proteins from Gram-negative bacteria were expressed in round E. coli backgrounds (Ramirez-Arcos et al., 2002; Corbin et al., 2002
).
DivIVAEf did not complement the divIVA mutation in B. subtilis 1756EfDiv. We propose that this occurred either because DivIVA from E. faecalis did not recognize DivIVABs-specific target sites, or because it may not interact with other proteins of the B. subtilis cell division machinery. DivIVABs regulates the positioning of the MinCDBs inhibitor complex in B. subtilis during vegetative growth and E. faecalis does not possess Min protein homologues. In addition, DivIVAEf and DivIVABs differ in their amino acid composition, especially at the C-terminal regions, which may also account for differences in protein function. Furthermore, the interaction between DivIVAEf and DivIVABs is likely to be responsible for the failed homologous complementation of the B. subtilis divIVA mutation in cells co-expressing both proteins. This proteinprotein association would result in dilution of the DivIVABs levels necessary for complementation.
Similar to the role of DivIVA in chromosome segregation in E. faecalis, DivIVA from B. subtilis is involved in anchoring the chromosomal oriC region to the poles of sporulating cells. In B. subtilis, DivIVA forms part of the sporulation-specific Spo0J-RacA-DivIVA protein system (Wu & Errington, 2003). Recently, the coordination of cell division and chromosome segregation by nucleoid occlusion has been proposed in B. subtilis (Wu & Errington, 2004
). A non-specific DNA-binding protein, Noc, which is similar to Spo0J, was identified as a specific effector of nucleoid occlusion which prevents the division machinery from assembling in the vicinity of the nucleoid (Wu & Errington, 2004
). Since there is no evidence that B. subtilis Noc interacts with components of the cell division machinery, it remains to be elucidated which other effectors might be involved in the nucleoid occlusion process. Our bioinformatic analyses of the enterococcal genome project revealed that E. faecalis chromosome partitioning proteins ParA and ParB are homologues to B. subtilis Spo0A and Spo0B, respectively. Furthermore, we found that B. subtilis Noc is homologous to an enterococcal protein that belongs to the ParB family. Therefore, it is possible that the Par proteins act along with DivIVA during chromosome segregation in E. faecalis, which would need to be further investigated.
The diverse functionality of DivIVA is demonstrated by our studies and reports by others. In organisms such as Streptomyces coelicolor (Flardh, 2003) and Brevibacterium lactofermentum (Ramos et al., 2003
), DivIVA has been implicated in controlling polar growth and morphogenesis. Furthermore, the FruD protein, a DivIVA homologue, is involved in cell growth and development in Myxococcus xanthus (Akiyama et al., 2003
). Surprisingly, Staphylococcus aureus DivIVA is not involved in cell division or chromosome segregation, despite being localized at the division septum of staphylococcal cells (Pinho & Errington, 2004
). Interestingly, we found significant differences between the function of DivIVA in E. faecalis in comparison to the morphologically related S. pneumoniae. Although divIVA is essential for E. faecalis viability, the gene is not essential in S. pneumoniae (Fadda et al., 2003
). In addition, E. faecalis divIVA did not complement an S. pneumoniae divIVA mutation and sequence alignments of DivIVA from both micro-organisms revealed differences in protein length and amino acid composition. Although enterococci and streptococci have the same lancet-shaped cells and diplococcal morphology, there are significant phenotypic differences between these two genera based on growth in broth containing 6·5 % NaCl and at extreme temperatures, and in their ability to hydrolyse 40 % bile salts (Facklam et al., 2002
). Moreover, genetic evidence based on DNADNA reassociation experiments, protein profile and sequencing of 16S rRNA genes showed that the genus Enterococcus is phylogenetically distant to Streptococcus and is more related to the genera Vagococcus, Tetragenococcus and Carnobacterium, all catalase-negative Gram-positive cocci (Facklam et al., 2002
). Our current knowledge of DivIVA function shows that the mechanism by which it acts differs in each species. This function might not only depend on the differences in protein composition, but it may reflect species-specific protein interactions.
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ACKNOWLEDGEMENTS |
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Received 20 October 2004;
revised 11 February 2005;
accepted 11 February 2005.
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