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Center for Environmental Genomics, Department of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada
Correspondence
Turlough M. Finan
finan{at}mcmaster.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The first known step in the formation of the prokaryotic division machinery is the polymerization of the cytosolic tubulin homologue FtsZ into a ring-like structure (Z-ring) encircling the dividing cell at the position of a potential septum (Bi & Lutkenhaus, 1991). Proper localization of the Z-ring is determined by at least two proposed mechanisms: nucleoid occlusion and the Min system (Errington et al., 2003). The nucleoid occlusion model states that all sites along the length of the cell are potential division sites competent for cell division; however, division is inhibited at positions occupied by nucleoids. Therefore, upon nucleoid segregation the midpoint becomes available for the placement of an FtsZ ring and the central division site.
In Escherichia coli the MinCDE proteins act together as a negative regulatory element, restricting the formation of septa at polar division sites, ensuring that the FtsZ ring is placed correctly at the midcell (Errington et al., 2003). MinC acts as an inhibitor of septation and is given topological specificity by the action of MinE, whereas MinD is required for the recruitment of MinC and MinE to the cytoplasmic membrane (Hale et al., 2001). The MinCD proteins form a MinE-dependent spiral complex that oscillates from pole to pole and inhibits the formation of the Z-ring at polar locations while allowing proper localization of the Z-ring at the midcell (Hale et al., 2001; Shih et al., 2003).
The Min system is found in a diverse range of bacteria (Errington et al., 2003). The Gram-positive organism Bacillus subtilis contains homologues to both MinC and MinD, but lacks a MinE counterpart (Cha & Stewart, 1997; Edwards & Errington, 1997). The MinC and MinD proteins do not oscillate from pole to pole and midcell selection is controlled by a protein, DivIVA, which lacks significant amino acid sequence similarity to MinE. The DivIVA protein stably associates with the B. subtilis cell poles and retains MinCD at the poles, thereby preventing division at those sites (Cha & Stewart, 1997; Edwards & Errington, 1997; Marston & Errington, 1999). Additionally, MinCDE homologues have been characterized in coccal Neisseria gonorrhoeae (Ramirez-Arcos et al., 2001; Szeto et al., 2001) and the spherical-celled cyanobacterium Synechocystis (Mazouni et al., 2004).
Sinorhizobium meliloti is a Gram-negative, rod-shaped alphaproteobacterium that induces N2-fixing root nodules on the legume alfalfa (Medicago sativa). The genome of this micro-organism consists of three replicons: a circular chromosome (3.7 Mb) and two megaplasmids pSymA (1.3 Mb) and pSymB (1.7 Mb) (Galibert et al., 2001). The S. meliloti genome sequence reveals a single copy of minCDE gene homologues on the pSymB megaplasmid (Finan et al., 2001; Galibert et al., 2001). During nodule formation, S. meliloti cells invade the host plant through root hairs and enter the plant cells where they differentiate from 1–2 µm long small rods into enlarged filaments and branched cells 5–10 µm in length (Mergaert et al., 2006; Paau et al., 1977). In a recent study Mergaert et al. (2006) examined the viability of S. meliloti differentiated bacteroids and concluded that they are non-dividing. They also reported that the genome content of the bacteroids is 24C (C being the haploid DNA content), while the content in free-living bacteria was 1–2C. The genes and factors involved in and controlling the morphological and genome content changes during bacteroid differentiation have not been well characterized. Here we report on experiments designed to investigate the requirement of min genes for cell division and symbiotic N2 fixation in S. meliloti. We have constructed various min mutants and min reporter gene fusions, and overexpressed the min genes in S. meliloti and E. coli. Our findings demonstrate that while minDE genes are expressed in free-living cells and within nodules, they are not absolutely required for cell division and symbiotic N2 fixation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The E. coli cultures were grown at 37 °C in Luria broth (LB), and S. meliloti strains were grown at 30 °C in LB containing 2.5 mM CaCl2 and 2.5 mM MgSO4 (LBmc). Defined M9 medium contained M9 salts (Miller, 1972) supplemented with 0.25 mM CaC12, 1 mM MgSO4, 0.3 µg biotin ml–1, 5 ng CoCl2 ml–1 (Watson et al., 2001) and 15 mM succinate or glucose. When necessary, antibiotics were used as described previously (Finan et al., 1986). For Min protein overexpression and electron microscopy, E. coli strains were grown to an OD600 of 0.1 and the growth continued for 4 h after IPTG was added to a final concentration of 0.1 mM. S. meliloti cultures were grown to an OD600 of 0.1 and then induced with IPTG (1 mM) for 16 h.
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). Synthesis of oligonucleotides (Table 2) and sequencing of PCR products were performed at MOBIXLab (McMaster University, Hamilton, Ontario, Canada). Bacterial matings were done as described previously (Finan et al., 1986).
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Sp cassette from pHP45 (Prentki & Krisch, 1984), the Sp cassette was cloned between the 844 and 911 bp PCR-amplified DNA fragments (primer pairs AB32663/AB32664 & AB32661/AB32662, respectively) flanking the outside of minC and minE, serially cloned into pJQ200-mp18 (Quandt & Hynes, 1993). The resultant plasmid, pTH1426 (Fig. 1a), was transferred into S. meliloti wild-type strain Rm1021 and a single crossover recombinant was selected on an LB plate containing (µg ml–1) streptomycin, (200), gentamicin (60) and spectionmycin (200). The double crossover integrant SmK850 was obtained by plating the transconjugants on LB agar with 5 % sucrose and screening the colonies for gentamicin sensitivity and spectinomycin resistance.
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), to obtain plasmid pTH1218 (Fig. 1a
). Integration of pTH1218 by homologous recombination into the S. meliloti genome resulted in a minC and gusA gene fusion, and promoterless minC'DE genes in SmK690 (Fig. 1b).
To make a polar minD mutation (i.e. minC+D–E–) a 773 bp DNA fragment carrying minC (without its translational start codon) was obtained by PCR using primers AB31700/AB31697 and cloned upstream of gusA and downstream of the trp terminator in pVO155 to yield pTH1219 (Fig. 1a). Homologous recombination of pTH1219 into S. meliloti genomic DNA produced a functional copy of minC and disrupted the minCDE operon in SmK691 (Fig. 1b). To disrupt the minE gene, a 1657 bp DNA fragment carrying the minCD gene region was PCR-amplified using primers AB31700/AB31699 and cloned downstream of the trp terminator and upstream of gusA in pVO155 to obtain pTH1220 (Fig. 1a). Strain SmK692, resulting from integration of pTH1220 into the S. meliloti genome, contained functional minC and minD genes with a gusA gene fusion at the 3' end of minD (Fig. 1b).
To overexpress S. meliloti min genes in both E. coli and S. meliloti, a broad-host-range expression vector, pTH1227, was constructed. A 1510 bp DNA fragment carrying the lacIq-Ptac cassette from pMAL-c2x (New England Biolabs) was PCR-amplified using primers AB32193/AB32194 and inserted upstream of gusA in a broad-host-range plasmid, pFUS1 (Reeve et al., 1999), to obtain pTH1227. Overexpression of the gusA gene after induction with IPTG was determined by measuring GusA activities in E. coli M65 and S. meliloti SmK700, both of which contained the vector pTH1227. DNA fragments carrying min genes with a modified ribosome-biding site were PCR-amplified using primers AB31697/AB32186 (807 bp) for minC, AB31699/AB32187 (850 bp) for minD, AB32188/AB31979 (336 bp) for minE, AB31699/AB32186 (1694 bp) for minCD, AB32186/AB31979 (1995 bp) for minCDE and AB32187/AB31979 (1151 bp) for minDE. The resulting PCR products were cloned into pTH1227 to obtain pTH1369 (Ptac-minC), pTH1228 (Ptac-minD), pTH1229 (Ptac-minE), pTH1230 (Ptac-minCD), pTH1231 (Ptac-minCDE) and pTH1587 (Ptac-minDE). Transfer of these plasmids to S. meliloti Rm1021 resulted in strains SmK701 (pTH1228), SmK702 (pTH1229), SmK703 (pTH1230), SmK704 (pTH1231), SmK705 (pTH1369) and RmP321 (pTH1587), respectively.
To measure min gene expression and identify promoter(s) in the minCDE region, an 844 bp DNA fragment from 837 bp upstream of minC was PCR-amplified using primers AB32663/AB32664 and cloned into pUCP30T (Schweizer et al., 1996). The SphI–KpnI fragment from the resulting plasmid (the promoter region) was then cloned into the reporter plasmid pFUS1 to give pTH1261 (Fig. 1a). Another 839 bp DNA fragment carrying the minC ORF and the minC and minD intergenic region was obtained by PCR using primers AB32919/AB32920 and then cloned upstream of gusA in pFUS1 to obtain pTH1273 (Fig. 1a). Following transfer of pTH1261 and pTH1273 into Rm1021, strains SmK712 and SmK710 were obtained, respectively. Plasmid pTH1445 was constructed for amplifying minC mRNA for primer extension. A DNA fragment carrying minC and the 1 kb region upstream of minC was PCR-amplified using primer AB32664/AB31697 and cloned into the ApaI/XbaI sites in pBluescript SK (Stratagene) to obtain pTH1423. The S. meliloti DNA was then subcloned into the KpnI/XbaI sites in pFUS1 to obtain pTH1445. Strain SmK857 was obtained after transfer of pTH1445 into S. meliloti Rm1021.
RNA isolation and primer extension.
S. meliloti SmK857 containing pTH1445 was grown in 20 ml LBmc and M9 glucose to an OD600 between 0.4 and 0.6. After addition of 2 ml 5 % (v/v) phenol in ethanol (Bernstein et al., 2002), cells were collected by centrifugation at 2500 g for 10 min at 4 °C. The cell pellet was resuspended in 325 µl 10 mM sodium acetate (pH4.2) buffer containing 0.3 M sucrose and 50 mM EDTA. Following addition of 375 µl lysis buffer (2 %, w/v, SDS in 10 mM sodium acetate, pH 4.2), the mixture was incubated at 65 °C for 5 min. Pre-warmed acidic phenol (700 µl, 65 °C, pH 4.2) was added to the lysate and incubation was continued for a further 5 min with vortexing every 1 min. Total RNA was extracted with the acidic phenol twice, phenol/chloroform (50 : 50) twice and then chloroform, and the RNA was precipitated with ethanol. The RNA was treated with RNase-free DNase I at 37 °C for 30 min and then purified using a Qiagen RNeasy mini column, according to the manufacturer's instructions. The purity and quality of the RNA were checked by electrophoresis through a 1.2 % agarose TBE gel.
To identify the transcriptional start site through primer extension, primers ML2050 and ML9882 were end-labelled with 
-32P-ATP (Amersham) by using T4 polynucleotide kinase (New England Biolabs) at 37 °C for 45 min. Unincorporated label was removed by passing the labelling mixture through Qiagen oligopurification columns. The primer extension reaction was carried out as described previously (Yuan et al., 2006). The primer extension product was loaded onto a 6 % acrylamide/7 M urea sequencing gel and run alongside a sequencing ladder generated by using the same primer with plasmid template pTH1423 and the Sequenase Version 2 DNA Sequencing kit (USB).
-Glucuronidase activity assay.
To measure expression of the gusA gene encoding -glucuronidase (GusA), S. meliloti cultures were grown up in 96 deep-well plates (ABgene) in 0.5 ml LBmc medium with antibiotics, and then subcultured into 150 µl LBmc or M9 media in 96-well plates (Costar). After 16 h growth in LBmc or 36 h growth in M9 media, 5 µl cultures were transferred to 95 µl assay buffer (50 mM sodium phosphate, pH 7.0, 1 mM EDTA, 0.0125 %, w/v, SDS and 0.438 mg p-nitrophenyl--glucuronide ml–1) in 96-well plates. The reaction was stopped with 100 µl 1 M Na2CO3 after 1 h. A405 was measured using a Safire microplate reader (Tecan). GusA specific activities were calculated using the equation: specific activity=A405x1000/60x0.005xOD600.
For nodule extracts, GusA specific activity was calculated using the equation: A405x1000x(time, min)–1x(volume, ml)–1x(mg protein)–1.
Plant tests.
Symbiotic N2 fixation was assessed using alfalfa (Medicago sativa) grown under nitrogen-deficient conditions in Leonard assemblies as described previously (Finan et al., 1986). Plants were harvested 28 days after inoculation and shoot dry weights were determined following drying in an oven. Measurement of GusA activity in nodules and histochemical staining for GusA were performed as described by Yuan et al. (2006). To determine the viable cell numbers in nodules, fresh nodules were surface-sterilized for 5 min in 2.5 % sodium hypochlorite, rinsed three times in 0.85 % (w/v) NaCl and then put into ice-cold MMS buffer (20 µl mg–1) containing 40 mM MOPS (pH 7.0), 20 mM KOH, 2 mM MgSO4 and 0.3 M sucrose. The nodules were crushed and then centrifuged for 2 min at 400 g. The supernatant was diluted serially in 0.85 % NaCl and then plated onto LB agar plates.
Environmental scanning electron microscopy (ESEM).
ESEM was performed as described previously (Sibley et al., 2006). Briefly, primary fixation of mid-exponential-phase LB-grown cells was done at room temperature for 30 min in 2 ml 0.2 M sodium cacodylate buffer (pH 7.4) containing 2.5 % (v/v) glutaraldehyde. Bacteroids were fixed in MMS buffer containing 2.5 % glutaraldehyde for 1 h. Fixed cells were washed five times in ddH2O and then 1 µl was spotted onto a gold-coated cover glass (5 nm thick) that was mounted on an ESEM stub with conductive glue. Wet samples were viewed with an Electronscan 2020 Environmental Scanning Electron Microscope at 2.4–4.0 Torr with an accelerating voltage of 20–30 KeV.
Transmission electron microscopy.
Primary fixed samples were washed twice in acetate/veronal buffer (59 mM sodium acetate, 35 mM sodium barbital, 145 mM NaCl and 12 mM CaCl2, pH 6.1), resuspended in the same buffer containing 1 % (w/v) osmium tetroxide and then incubated at room temperature for 4 h. The cells were then washed twice in the acetate/veronal buffer and resuspended in 2 % (w/v) Nobel Agar (45 °C) and cooled on a glass slide. Cells embedded in the agar cubes (1 mm3) were then stained with 0.5 % (w/v) uranyl acetate in the acetate/veronal buffer at 4 °C for 12 h. Samples were dehydrated at room temperature with sequential washes of increasing ethanol concentrations and finally with propylene oxide. Infiltrated samples were embedded in Spurr's resin (Spurr, 1969) and 60–90 nm sections were cut with an Ultracut Microtome. Thin sections were post-stained in a 1 % (w/v) aqueous uranyl acetate solution for 20 min on carbon-coated copper EM grids (100 mesh) that had been previously covered with 0.3 % (w/v) Formvar. Sections were then post-stained with Reynold's lead citrate for 6 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and there is a single nucleotide overlap between minD and minE (schematically outlined in Fig. 1). To identify promoters associated with these genes, an 844 bp DNA fragment encompassing the region upstream from the minC translational start codon and a 847 bp DNA fragment containing the minC ORF and the minC–minD intergenic region were cloned into the replicating gusA reporter plasmid pFUS1 to generate plasmids pTH1261 and pTH1273, respectively (Fig. 1). Analysis of GusA activity from S. meliloti strains carrying these plasmids (Fig. 2a) demonstrated strong promoter activity (20-fold above background) from upstream of minC (SmK712). The promoter activities from cells grown in both rich medium (LB) and minimal medium (M9-glucose) were comparable although the growth rate for S. meliloti was about twofold slower in M9 minimal medium compared to LBmc. Consistent with an absence of promoter activity, only background GusA activity was observed from constructs carrying the minC–minD intergenic region (SmK710). These data demonstrated that, under the conditions examined, transcription emanated from upstream of minC. To map the transcriptional start site(s) upstream of minC, we used primer extension and two distinct primers (ML9882, Fig. 2b; ML2050, data not shown). Using mRNA extracted from both LBmc- and M9-glucose-grown cells, a transcript initiating from 174 nt upstream from the minC start codon was identified in both extension reactions. The deduced –35 and –10 hexanucleotide promoter sequences, 5'-GCCTGC-N17-CCATGC-3', showed little similarity to the recently derived S. meliloti promoter consensus sequences 5'-CTTGAC-N17-CTATAT-3', particularly within the more conserved –35 region (MacLellan et al., 2006). We note that the 174 nt untranslated region is relatively large for S. meliloti genes. This could reflect the presence of regulatory sequences and, in this respect, it is interesting that two possible CtrA protein-binding sites (Barnett et al., 2001) are positioned in this region (Fig. 2c), implying that the CtrA protein might be involved in regulation of minCDE expression in S. meliloti.
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Sp cassette. We confirmed that the min genes were absent from the resulting strain SmK850 (Rm1021 
minCDE : : Sp) by PCR, while a fragment from upstream of minC to the Sp gene was readily amplified in four separate mutants. Additional min mutant derivatives were made via a single cross-over recombination between plasmids carrying fragments from the min gene region and the min genes in the genome. These co-integrates readily generated single cross-over polar mutations in the minC gene in strain SmK690 (minC– minD– minE–), in the minD gene in strain SmK691 (minD– minE–) and in the minE gene in strain SmK692 (minE–) (see Fig. 1b).
The growth of the resulting strains in LBmc and M9 media was examined and the viability of these cultures was determined by plating dilutions onto LB agar (data not shown). In addition, the effects of the mutations on cell structure were determined by examining the cells by phase-contrast microscopy and by both scanning and transmission electron microscopy (Fig. 3).
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minCDE : : Sp) (Fig. 3b), SmK690 (minC– minD– minE–) (data not shown) and SmK691 (minD– minE–) (Fig. 3c) were indistinguishable from the rod-shaped, 1–2 µm long wild-type cells (Fig. 3a). This differs from the minicell phenotype observed with E. coli minCDE and minDE mutants (de Boer et al., 1989). Strikingly the cells from the minE– mutant strain SmK692 were swollen and branched with single or multiple asymmetric septa (Fig. 3d). This phenotype also contrasts with the cell filamentation phenotype seen with an E. coli minE mutant (de Boer et al., 1989). Transmission electron micrographs showed that wild-type S. meliloti cells had normal mid-cell invaginations and an intact membrane surrounding the entire cell (Fig. 3e). The minE cells were very different from wild-type (Fig. 3f), as seen by the membrane connectivity of the cytoplasm with shapes never seen in the wild-type sections. Thus, the branched cells appeared to represent individual bacteria with atypical morphology.
Symbiotic phenotype of S. meliloti min mutants
The symbiotic phenotype of the S. meliloti wild-type and min mutant strains was examined following their inoculation onto alfalfa seedlings. The min mutants formed wild-type-like pink nodules. With the exception of the minE– mutant SmK692 (discussed below), the dry weights of the alfalfa plants inoculated with the mutants were similar to those inoculated with the wild-type strains (Table 3). The dry weights of alfalfa inoculated with minE– mutant SmK692 were 30 % of the parent Rm5000, while the uninoculated plants were less than 5 % of the wild-type dry weight (Table 3). Thus the minE mutation resulted in a large decrease in symbiotic N2 fixation.
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minCDE) and SmK691 (minD–E–) nodules were similar to that of the wild-type [9x107 (g nodule)–1], while the number of viable cells in SmK692 (minE–) nodules was slightly reduced [3x107 (g nodule)–1] relative to the wild-type. The morphology of the bacteroids was examined using ESEM. Wild-type strain Rm1021 bacteroids showed a heterogeneous morphology as described by Vasse et al. (1990) with enlarged filaments and Y shapes of 3–8 times the length of vegetative cells, and the various min mutant strains, SmK690, SmK850, SmK691 or SmK692, were indistinguishable from the wild-type (data not shown). We suggest that the reduced N2-fixation phenotype of the minE– mutation is related to the aberrant cell division phenotype that results when minC and minD are expressed in the absence of minE. Our results demonstrate that the min genes are not essential for the development of N2-fixing nodules.
Expression of min genes in nodules
The lack of a symbiotic phenotype for the minCDE deletion mutant coupled with the reduced fix phenotype of the minE– mutant prompted us to investigate the expression of these genes in nodules. Expression of the minCDE genes in nodules was measured following the inoculation of alfalfa seedlings with the minD : : gusA reporter gene fusion in strain SmFL5810. The single cross-over that generated this strain also reconstituted a wild-type minCDE gene region (see Fig. 1b). As a control we employed strain RmP319 carrying a nifH : : gusA gene fusion since nifH is highly expressed in bacteroids (Barnett et al., 2004). Examination of nodules 4 weeks after inoculation revealed that the nifH gene was highly expressed with a GusA specific activity of 7739±311 while the GusA specific activity from SmFL5810 nodule extracts was 1675±26 or about 22 % that of nifH. These data demonstrate that the minCDE genes are expressed in nodules.
To investigate whether min gene expression was uniform throughout the nodule, longitudinal sections of nodules were examined following histochemical staining for GusA. The staining revealed that expression of the nifH gene occurred in the previously described interzone II to III (Vasse et al., 1990) (data not shown), as reported by Labes et al. (1993) and Soupene et al. (1995). In contrast, the minD gene was expressed in all regions of the nodule containing the bacteria (SmFL5810, minD : : gusA), including infection zone II (data not shown).
Overexpression of min genes in S. meliloti generated different phenotypes
With the exception of the aberrant cell division phenotype that resulted from the minE– mutation, the expression data and min mutant phenotypes were not informative as to a direct role for min genes in cell septation in S. meliloti. We therefore examined whether expressing the min genes from a strong plasmid-borne promoter would affect the cell morphology and viability of S. meliloti. Individual or combinations of min genes with ribosome-binding sites were cloned downstream of the Ptac promoter in the expression vector pTH1227 which also carries the lacIq gene upstream of the promoter. These cell structures of IPTG-induced S. meliloti cells carrying these plasmids were examined by ESEM, and cell viability was evaluated by diluting cultures of known OD600 and plating onto LB agar. In the absence of added IPTG, alterations to the wild-type cell structures were not observed. As the gusA gene is located downstream of the Ptac promoter in pTH1227, we first examined S. meliloti cells overexpressing gusA alone. These cells resembled the short rods of wild-type Rm1021 (SmK700, Fig. 4a, b) and their viability was the same as wild-type cells (data not shown). When the minC gene was expressed in pTH1227, several cell phenotypes were observed, including wild-type-like short rods, filaments, and swollen and branched cells (SmK705, Fig. 4c). The viability of these cells was similar to the wild-type. Expression of the minD gene from pTH1227 generated multi-branched cells (Fig. 4d, e) and the cell viability of these cultures was over 10 000-fold lower than the wild-type. Hence overproduction of MinD had a much more inhibitory effect on cell growth and division than overproduction of MinC. A similar inhibitory effect and a filamentation phenotype was observed in E. coli upon increased synthesis of MinD (de Boer et al., 1989). Neither cell division nor cell viability appeared compromised in cells overexpressing minE (SmK702, Fig. 4a, f). When minE was overexpressed with minD in RmP321, a mixture of wild-type and branching cells was generated (Fig. 4k, l) and the viability of these cells was about tenfold higher than the cells overexpressing minD alone. These data indicated that MinE could relieve the inhibitory effect of MinD on cell division and growth, although it is also possible that minD expression levels were reduced in these cells.
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) generated a branching cell morphology with the reduced cell viability observed for cells in which minD alone was overexpressed. The overexpression of minCDE (SmK704, Fig. 4i, j) from pTH1227 also generated the cell morphology and cell viability phenotypes observed from when minD alone was overexpressed (Fig. 4g, h). We note that for these constructs the cell growth, cell viability and cell morphology were not affected under non-inducing conditions.
Overexpression of S. meliloti Min proteins in E. coli
The morphology of E. coli DH5
cells carrying the various pTH1227 min derivatives was examined after IPTG-induced expression of min gene expression. Without IPTG induction all cells appeared like wild-type. IPTG-induced expression of the gusA gene from pTH1227 did not alter the rod-shaped morphology of E. coli (Fig. 5a). IPTG-induced overexpression of the entire minCDE operon resulted in a large number of short filaments and minicells (Fig. 5f), a classic minicell phenotype of E. coli (de Boer et al., 1989). When viewed by ESEM, the same minicell phenotype was also observed when the S. meliloti minE gene was expressed in E. coli (Fig. 5d). In addition, expression of the minD or minCD genes in E. coli caused formation of long, non-septate filaments (Fig. 5c, e), whereas S. meliloti MinC produced various sizes of filaments in E. coli (Fig. 5b). Filamentation resulting from overexpression of S. meliloti MinC and MinCD proteins was the same as that resulting from E. coli Min proteins (de Boer et al., 1989). Taken together, the morphological changes in E. coli cells resulting from overexpression of the S. meliloti Min proteins imply that the S. meliloti and E. coli MinCDE proteins act similarly with respect to cell division.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The parAB genes are essential in Caulobacter crescentus (Figge et al., 2003; Mohl et al., 2001). Our data clearly show that the min genes are expressed in free-living cells, and both the cell phenotype of the minE mutant and that of cells expressing minD from the Ptac promoter are consistent with these genes playing a role in cell septa location/formation. In the absence of MinE, it appears that the MinC and MinD proteins interfere with septum formation. This is consistent with the E. coli model where MinE prevents the MinCD division inhibitor from accumulating at the midcell, while permitting it to block division at other unwanted sites along the length of the cell (Shih et al., 2003). In E. coli, the Min system prevents septation at potential division sites near cell poles without blocking septum formation at the desired midcell location. MinC interacts with the division protein FtsZ to prevent formation of stable FtsZ rings, a vital first step in assembly of the division machinery. MinE is responsible for giving site specificity to MinC, limiting its activity to the sites away from the desired midcell. MinD is responsible for the membrane association of MinC and MinE (Errington et al., 2003). According to the E. coli Min model, deletion of S. meliloti minCDE genes, or a polar minC mutation or a polar minD mutation, should result in a minicell phenotype which was not observed in this study. Instead, the mutant cells were indistinguishable from wild-type. Alternatively, expression of the genomic copy of the minC and minD genes in the absence of the minE gene in S. meliloti would be predicated to produce long filaments, but cells actually showed a large, swollen and branched morphology (Fig. 4g, h).
Deletion of the min genes in E. coli and B. subtilis generates a minicell phenotype in which a large fraction of cells divide at polar positions of the cell instead of the middle (de Boer et al., 1989; Cha & Stewart, 1997). In contrast, the Synechocystis minCDE mutant displays spiralled, heart-shaped cells and minicells (Mazouni et al., 2004). The minicells of Synechocystis are able to further divide, but those of E. coli and B. subtilis are anucleate. The minC and minD mutants of Neisseria gonorrhoeae do not show a minicell phenotype, but rather a gross enlargement of their round shape, causing cell lysis (Ramirez-Arcos et al., 2001; Szeto et al., 2001). Thus while the Min system is required for proper septum location in a wide range of bacteria, the phenotypes resulting from min gene mutations can be very different.
Both the free-living and nodule phenotypes of the minCDE mutant demonstrate that in the absence of the Min proteins, other systems in S. meliloti may direct appropriate cell septation. Mutations in such systems may be lethal in the min mutant background, as in the case of the slmA mutants of E. coli (Bernhardt & de Boer, 2005). Many bacterial species lack Min homologues (Caulobacter crescentus, Haemophilus influenzae and Staphylococcus aureus) and thus must have other mechanisms to replace the role of the MinCDE proteins in determining Z-ring assembly (Margolin, 2003; Figge et al., 2003). In this respect, we note that the megaplasmid location of the min genes in S. meliloti is consistent with a dispensable function. Moreover, the minCDE genes are located on the p42e plasmid of Rhizobium etli (Gonzalez et al., 2006) and the pRL11 plasmid in Rhizobium leguminosarum (Young et al., 2006).
Six of the nine genes required for cytokinesis in E. coli [ftsA, ftsI, ftsK (2), ftsQ, ftsW and ftsZ (2)] have been annotated in the S. meliloti genome (Galibert et al., 2001). Recent transcriptome analysis of S. meliloti shows that the expression of these genes is down-regulated in bacteroids (Barnett et al., 2004). Our mutant data show that while minCDE genes are not absolutely required for the bacteroid differentiation process, the min genes are expressed in nodules. Both the free-living and nodule phenotypes of the minCDE mutant demonstrate that there is redundancy in the pathway(s) determining septa formation in S. meliloti.
The cellular concentrations of MinCDE proteins are maintained within certain levels to avoid disrupting cell division, as overexpression of S. meliloti min genes in wild-type or disruption of the minE gene resulted in aberrant morphology and growth. We identified a promoter upstream of the minC gene and identified two putative CtrA-binding sites (Fig. 2c). S. meliloti CtrA is essential and binds to the five CtrA-binding sites in the ctrA promoter region (Barnett et al., 2001). CtrA is a DNA-binding response regulator and regulates expression of about 100 genes involved in cell cycle progression and morphogenesis in Caulobacter crescentus (Laub et al., 2000). The presence of CtrA-binding sites upstream of the minC gene indicates that CtrA may be involved in regulating minCDE expression, as implied for Brucella abortus minC expression (Bellefontaine et al., 2002; Hallez et al., 2004).
The complex cell morphological changes resulting from a minE mutation or overproduction of Min proteins are similar to those in S. meliloti overexpressing CcrM (Wright et al., 1997), DnaA (Sibley et al., 2006) and FtsZ (Latch & Margolin, 1997), or treatment with DNA-damaging agents (Latch & Margolin, 1997). This phenotype is correlated with blocked cell division (Latch & Margolin, 1997; Wright et al., 1997; Sibley et al., 2006) and loss of normal control of DNA replication initiation (Wright et al., 1997). It remains to be established whether a blockage in cell division always leads to branching in S. meliloti; however, this possibility is of considerable interest given the cellular morphology of N2-fixing bacteroids. We do not exclude the possibility that the MinCDE proteins may be involved in other cell process such as genome partitioning and segregation, as in E. coli (Åkerlund et al., 2002).
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ACKNOWLEDGEMENTS |
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Edited by: C. W. Ronson
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 16 August 2006;
revised 11 October 2006;
accepted 12 October 2006.
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