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1 Departamento de Ecología, Genética y Microbiología, Área de Microbiología, Facultad de Biología, Universidad de León, 24071 León, Spain
2 Centro Biología Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Científicas, CSIC-UAM, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
3 Institut fur Genomforschung, Universitat Bielefeld, Universitatsstrasse 25, D-33615 Bielefeld, Germany
Correspondence
José A. Gil
degjgs{at}unileon.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present Address: Departamento de Biología Funcional, Área de Microbiología, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Nakayama et al., 1978).
In recent years, different approaches have been used to characterize cell division genes in corynebacteria, and the possible relationship between amino acid production and growth inhibition (Honrubia et al., 1998; Kobayashi et al., 1997; Ramos et al., 2003b; Wachi et al., 1999). Recent studies have shown that corynebacteria might follow an archaic pattern of mycelial growth that involves an asymmetric mechanism of division (Ramos et al., 2005), with cell elongation occurring at the tip of the daughter cells (Daniel & Errington, 2003). As a member of the order Actinomycetales, corynebacteria show apical growth, similar to that seen in Corynebacterium diphtheriae (Umeda & Amako, 1983). The availability of the complete genome sequence of C. glutamicum (GenBank accession nos NC_003450 and BX927154) has enabled us to study the expression profile/regulation of the cell division gene ftsI, and perform proteomic studies in the organism.
FtsI homologues have been described in different bacteria, such as Escherichia coli (Begg et al., 1992; Botta & Park, 1981) or Bacillus subtilis (Daniel et al., 1996; Marston et al., 1998). FtsI (also called penicillin-binding protein 3, PBP3) is a well-characterized protein that has been reported to be expressed in very low amounts in the cell (about 100 molecules) (Dougherty et al., 1996). The protein consists of a short cytoplasmic domain, a single membrane-spanning segment and a large periplasmic domain that encodes a transpeptidase activity that is involved in the biosynthesis of septal peptidoglycan. Immunofluorescence microscopy has shown that in E. coli during the later stages of cell growth FtsI localizes to the division site at the septum. The septal localization of FtsI, however, depends upon prior localization of the other cell division proteins, such as FtsZ, FtsA, FtsK, FtsQ, FtsL and FtsW (Mercer & Weiss, 2002; Weiss et al., 1999), and therefore, it appears that FtsI is a late recruit to the division site. In B. subtilis, the septal localization of PBP3/FtsI is also delayed, but is needed for the localization of the division inhibitor MinC (Marston & Errington, 1999).
DivIVA is another cell division protein that has been extensively studied in B. subtilis. It enables cell division in the organism by sequestering the cell division inhibitors MinC and MinD at the cell poles (Cha & Stewart, 1997; Edwards & Errington, 1997; Marston et al., 1998). In this respect, its role is similar to that of MinE of E. coli, which repels MinCD inhibitors at the cell poles (Marston et al., 1998). DivIVA also acts at the cell pole by interacting with the chromosome segregation machinery, and it is involved in the correct localization of the oriC region at the cell pole, a step that precedes asymmetric division during sporulation (Thomaides et al., 2001). More recently, Harry & Lewis (2003) found that DivIVA localizes at the poles of germinated and outgrown cells in B. subtilis without prior assembly of the division apparatus at this site, suggesting that its localization does not occur by direct interaction with components of the division machinery, as proposed by Edwards et al. (2000).
In C. glutamicum, as in many other bacteria, divIVA is located downstream from the dcw cluster. Its encoded product (DivIVA) appears to be an essential protein playing an important role at the cell poles in the organism (Ramos et al., 2003b). Overexpression of DivIVA-GFP translational fusion in C. glutamicum leads to an altered morphology showing rounder larger and swollen cells, with the DivIVA-GFP product being preferentially localized at the cell poles. It has been previously suggested that DivIVA participates in the maintenance of cell morphology in C. glutamicum (Ramos et al., 2003b).
In the present study we show that ftsI in C. glutamicum, as in E. coli, is an essential gene that is required for the maintenance of cell shape and morphology. We further show that a partial depletion of FtsI induces an increased concentration of DivIVA, opening new questions about the regulation of cell division and polar growth in this micro-organism.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. E. coli strains were grown at 37 °C in Luria–Bertani broth (Hanahan, 1983), supplemented with agar where appropriate. When necessary, the antibiotics kanamycin, apramycin and ampicillin were used at a final concentration of 50 µg ml–1. C. glutamicum cells were grown in trypticase soy broth (TSB; Difco) or trypticase soy agar (TSA) (TSB containing 2 % agar) at 30 °C.
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. E. coli cells (DH5
and S17-1) were transformed by the method of Hanahan (1983).
All the mobilizable plasmids (integrative and bifunctional) were introduced into the donor strain (E. coli S17-1) and then transferred to C. glutamicum RES167 (or additional recombinant strains) following the method described previously (Mateos et al., 1996).
Purification of DNA fragments was carried out using a GENECLEAN kit (Bio 101). Restriction enzymes were purchased from Promega and New England Biolabs.
Total DNA from C. glutamicum was isolated using the Kirby method described for Streptomyces (Kieser et al., 2000), except that the cells were treated with 5 mg lysozyme ml–1 for 4 h at 30 °C.
DNA probes for Southern blots were labelled with DIG-High Prime, according to the manufacturer's (Roche) instructions.
RNA isolation, RT-PCR analysis, Q-PCR and RACE-PCR.
For total RNA isolation from C. glutamicum, cells were grown in TSB medium to OD600 1.5. RNA was isolated using the RNeasy kit (Qiagen).
RT-PCR analysis of the total RNA preparation was carried out in order to detect the presence of a polycistronic transcript originating from the upstream mraZ that includes ftsI; 1 µg total RNA was used as the template to generate single strand cDNA using a first strand cDNA synthesis kit (Roche), essentially according to the manufacturer's recommendations. Primers F6, P2, P4, P6, P8 and P10 (Table 2), which were used to generate cDNA corresponding to the upstream regions of the dcw genes (Fig. 1b), were designed using Primer Express, v2.0 (Applied Biosystems). The generated cDNAs were used as templates for subsequent PCR amplification using the primer pairs P0/F6 (for the upstream mraZ), P1/P2 (for the intergenic mraZ–mraW region), P3/P4 (for the intergenic mraW–ftsL region), P5/P6 (for the intergenic ftsL–ftsI region), P7/P8 (for the intragenic ftsI region) and P9/P10 (for the intergenic ftsI–murE region) (Table 2, Fig. 1b). The PCR amplified products were analysed by electrophoresis on 2 % agarose gels. The absence of DNA contamination of the RNA samples was ascertained by PCR using appropriate primers as negative and positive controls.
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). A 1/20 volume of the generated cDNA sample was then used as a template in the second step of the PCR in which both the forward and the reverse primers (P11 and P12, respectively; Table 2) were used in the reaction mixture, in a total volume of 25 µl. Reactions were performed using an ABI Prism 7000 sequence detection system (Applied Biosystems). Results are indicated relative to the Ct (cycle threshold) value. Ct is defined as the cycle at which fluorescence is determined to be statistically significant compared to the background, being inversely proportional to the log of the initial copy number; this value was calculated automatically by the ABI Prism 7000 SDS software.
RACE-PCR experiments were performed according to the 5'/3' RACE kit, 2nd generation (Roche), protocol. In order to identify promoters that were located upstream from mraZ and ftsI, 2 µg total RNA preparation was used as a template to generate single strand cDNA using primers F5 and P11, respectively (Table 2). A homopolymeric A tail was added to the 3' end of the purified cDNA preparation using terminal transferase, the dA-tailed cDNA that was obtained was used in two further PCR amplifications steps; the first one using the primer pair dT-primer/F5 (for mraZ) and dT-primer/F3 (for ftsI) (Table 2). The amplified DNA product was used again in a second round PCR amplification using the primer pair dT-primer/F6 (for mraZ) and dT-primer/F4 (for ftsI) (Table 2). The amplified fragments were cloned into pGEM-T Easy vector (Table 1), utilizing a T–A cloning technique, and used to transform E. coli DH5; five plasmids isolated from different clones (per assay) were sequenced.
Plasmid constructions.
In order to subclone the complete ftsI gene from C. glutamicum using PCR-amplified DNA, primers F1 and F2 were designed (Table 2). These primers amplify ftsI including the second GTG (position 2 293 165) but not any upstream elements that are likely to contain the RBS and promoter. The 1.9 kb Taq-amplified PCR product was subcloned into pGEM-T Easy vector, creating plasmid pFtsI (Table 1). This plasmid was digested with NdeI and XhoI (target sites of which are present in F1 and F2 primers, respectively, Table 2), and the 1.9 kb fragment (corresponding to the ftsI gene) was used to replace the xysA gene from pXHis1-Npro, yielding plasmid pKFtsI (Table 1).
In order to detect the presence of the promoter of ftsI, a 150 bp DNA fragment immediately upstream of the gene was PCR amplified using the primer pair F7/F8 (Table 2); the PCR product was digested with EcoRI and NdeI, the sites of which were included in the forward and reverse primers, respectively, and subcloned in the promoter probe vectors pEMel-1 and pEGFP, creating pEMel-FtsI and pEGFP-FtsI plasmids, respectively (Table 1).
To construct a GFP-FtsI translational fusion, we used a variant of GFP (Clontech) that includes the V163A and S175G mutations introduced by Siemering et al. (1996); this variant was found to be efficiently expressed in ‘Brevibacterium lactofermentum’/C. glutamicum (Ramos et al., 2003a). The whole gfp gene was amplified from plasmid pXEGFP2 (Table 1) using primers F9 and F10 (Table 2). These primers were designed to replace the stop codon (TAA) of the gfp gene with CAT (His), which after NdeI digestion and ligation with the aforementioned ftsI gene will be immediately followed by the ATG start of ftsI. Because of the presence of two NdeI sites in the gfp gene, one at the primer F10 region and the other one at the start of the gene, the amplified fragment was digested with NdeI and cloned into NdeI-digested pKFtsI (see above) yielding plasmid pNV3 (Table 1), which contained the gfp-ftsI gene fusion flanked by BglII sites.
Plasmid pNV4A was obtained by cloning the BglII cassette from pNV3 (BglII-Pkan-gfp-ftsI-BglII) into plasmid pOJ260 (BamHI digested); pNV4A was then transferred by conjugation to C. glutamicum RES167, giving rise to the merodiploid strain MAPF (Table 1, Fig. 1), which carries the normal ftsI gene copy and an additional copy of the gfp-ftsI gene fusion under Pkan on the chromosome.
Plasmid pNV4A was digested with StuI and EcoRV (to remove the 3' end of ftsI) and autoligated, affording plasmid pNV5A, which carries gfp-
ftsI under the control of Pkan (Table 1). When pNV5A was transferred by conjugation to C. glutamicum RES167, the resulting transconjugant strain C. glutamicum APF contained an incomplete copy of ftsI and the fused gfp-ftsI under Pkan on the chromosome (Table 1, Fig. 2b).
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, Fig. 1a) was subcloned at the SmaI and BamHI sites of pK18mob (Table 1). A single crossover integration of pKInt1 in the chromosome at the ftsI locus would create two deleted FtsI versions: one lacking 385 amino acids from its C-terminus and the other lacking 154 amino acids from its N-terminus.
pOJPB vector was designed to disrupt the chromosomal copy of ftsI and place a second functional copy of ftsI under the control of the Plac promoter. This was achieved by subcloning the 462 bp EcoRI–BamHI 5' region of ftsI (which encodes the first 154 amino acids) from plasmid pFtsI into the EcoRI and BamHI sites of pOJ260, downstream of the Plac promoter (Table 1).
Vector pALacI was constructed as follows: the E. coli lacIq gene present in plasmid pECXK99E (Table 1) was PCR amplified using the primer pair L1/L2 (Table 2), digested with BamHI and subcloned at the same site in pABK (Table 1).
All of the aforementioned plasmid constructs were confirmed by DNA sequencing.
DNA sequencing.
DNA sequencing was carried out by the dideoxy nucleotide chain termination method of Sanger et al. (1977). Computer analysis was performed with DNASTAR (dnastar); database similarity searches were carried out using the BLAST and FASTA public servers (National Center for Biotechnology Information, NCBI, and European Bioinformatics Institute, EBI), and multiple alignments of sequences were achieved using CLUSTAL W (EBI).
Preparation of cell-free extracts, SDS-PAGE, Western blotting and 2D electrophoresis.
Cell-free extracts of C. glutamicum cells were disrupted by sonication as follows. One gram wet weight cells was suspended in 5 ml ice-cold TES buffer (25 mM Tris/HCl, 25 mM EDTA, 10.3 % sucrose, pH 8). Sonication was carried out for periods of 30 s with 1 min intervals in an ice-cooled tube using a Branson sonicator (model B-12) at 75–100 W, until the cells were completely disrupted, as observed microscopically. Cell debris was removed by centrifugation (8000 g), and supernatants were used as cell extracts.
SDS-PAGE was carried out essentially as described by Laemmli (1970). Electrophoresis was performed at room temperature in a vertical slab gel (170x130x1.5 mm), using 10 % (w/v) polyacrylamide at 100 V and 60 mA. After electrophoresis, proteins were stained with Coomassie blue or electroblotted to PVDF membranes (Millipore), and immunostained using the following antibodies: mouse monoclonal antibodies (F126-2) raised against purified DivIVA/Ag84 from Mycobacterium kansasii (provided by Professor A. H. J. Kolk, Royal Tropical Institute, Amsterdam, The Netherlands) and goat anti-mouse IgG alkaline phosphatase-conjugated antibodies (Santa Cruz Biotechnology), or rabbit polyclonal anti-GFP and goat anti-rabbit IgG alkaline phosphatase-conjugated antibodies (both from Santa Cruz Biotechnology).
2D gel electrophoresis was performed as described by Vohradsky et al. (1997). The IEF of proteins in the first dimension was carried out using 24 cm Immobiline DryStrips, pH 4–7 (Amersham Pharmacia) followed by electrophoretic separation on 10 % SDS-PAGE gels in the second dimension. Preparative gels were loaded with 400 µg total protein and stained with Coomassie blue. Protein sizes and isoelectric point ranges of the 2D gels were determined using 2D-gel marker proteins (Bio-Rad). The 2D gels were matched and quantified by image analysis using the Z3 2D-gel analysis system (Compugen). Protein spots were identified by peptide mass fingerprinting after they were excised from Coomassie blue-stained preparative gels, destained, gel purified and digested with trypsin. The mass spectra of the peptides after proteolytic digestion were determined with a MALDI-TOF mass spectrometer (Bruker Biflex III).
Fluorescence microscopy.
C. glutamicum cells expressing GFP-FtsI or DivIVA-GFP were observed under a Nikon E400 fluorescence microscope. Images were captured with a DN100 Nikon digital camera and assembled using Corel Draw, Adobe Photoshop and Metamorph.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and BX927154 (Kalinowski et al., 2003), and the region of the dcw cluster is shown in Fig. 1(a). The structural similarity in the organization of the genes that are 5' to the dcw cluster around the ftsI region in both strains suggests that mraZ, mraW, ftsL, ftsI and murE might be cotranscribed. In order to test this possibility, total RNA was extracted from C. glutamicum cells that were grown in TSB to exponential phase, and used in RT-PCR using specific primers for the intergenic mraZ–mraW, mraW–ftsL, ftsL–ftsI, ftsI–murE regions, as well as for an internal region of ftsI (see Fig. 1b). RT-PCR products of the expected sizes were obtained in all cases, strongly suggesting that there is a transcriptional relationship between these five genes (Fig. 1c). No amplification band was seen when primer pair P0/F6 was used, indicating a lack of cotranscription with mraZ and upstream genes (Fig. 1c, lanes 11 and 12).
The main promoter of the dcw cluster was located by RACE PCR to be at the G residue, at position 2 295 904, which is 7 nucleotides downstream from the putative –10 promoter region TGCAGC (Fig. 1b) (Patek et al., 2003) and 99 nt upstream from the C. glutamicum mraZ gene (at the beginning of the mra/dcw cluster of cell division and cell envelope biosynthesis genes) as seen in E. coli (Hara et al., 1997; Mengin-Lecreulx et al., 1998).
Two different initiation codons for ftsI were suggested for C. glutamicum; the first being a GTG codon at position 2 293 318, which would encode a 704 amino acid protein with a calculated molecular mass of 75.7 kDa (Wijayarathna et al., 2001). The second being the other GTG at position 2 293 165, which would encode a 651 amino acid protein with a calculated molecular mass of 69.7 kDa (Kalinowski et al., 2003). In order to determine which of the two protein products might be correct, and also to find a possible transcriptional start point, additional RACE-PCR experiments, using different primers upstream from ftsI, were performed. The results showed the presence of an mRNA starting at C (at position 2 293 257), which is located 13 nucleotides downstream from a putative –10 promoter region (GAAGAT) in the intergenic ftsL–ftsI region (Fig. 1a). A 150 bp fragment containing the above-mentioned region was PCR amplified using primers F7 and F8 (Table 2), and subcloned into the promoter probe vectors pEMel-1 and pEGFP (Table 1), creating pEMel-FtsI and pEGFP-FtsI, respectively. These vectors were mobilized into C. glutamicum, and the detection of melanin production and GFP activity in the corynebacteria confirmed the presence of a promoter activity in that region. Therefore, it can be concluded that ftsI is mainly cotranscribed along with the mraZ, mraW, ftsL and murE genes of the operon probably from the Pmra promoter, as described in E. coli and B. subtilis (Hara et al., 1997), and probably also from a minor promoter (PftsI) as in B. subtilis (Daniel et al., 1996). This result also suggests that the second GTG (at position 2 293 165) most probably is the start codon of ftsI.
Taking into account the above results, a 1.9 kb fragment containing the gene encoding FtsI (Kalinowski et al., 2003) was PCR amplified using specific primers, and ligated into the pGEM-T Easy E. coli vector using the A–T strategy, to create pFtsI (Table 1). However, all of the E. coli transformants that were tested for the recombinant plasmid were found to have the ftsI insert in the opposite orientation to the Plac promoter. This observation strongly suggests that overexpression of FtsI might be toxic in E. coli. Furthermore, we also observed that expression of ftsI was unable to complement the temperature-sensitive ftsIts E. coli AX655 strain, as has been described for similar complementation assays (Wijayarathna et al., 2001).
Visualization of FtsICG-GFP fusions
In order to analyse the role of FtsI in cell division in C. glutamicum, various strains of the organism were constructed in which expression of the gfp-ftsI fusion was studied. The Pkan promoter of the kan gene from Tn5 is efficiently expressed in corynebacteria (Cadenas et al., 1991). C. glutamicum transformed with pNV4A vector (MAPF strain) contains a wild-type copy of ftsI in addition to the gfp-ftsI cassette, which is under the control of the Pkan promoter and inserted as a single copy in the chromosome (Fig. 2c); in C. glutamicum transformed with pNV5A vector (APF strain) gfp-ftsI expressed under the control of the Pkan promoter is the only functional copy of the gene since this strain carries a truncated form of ftsI (Fig. 2b). Phase-contrast microscopy analysis of C. glutamicum APF (Fig. 2b) revealed typical corynebacterial cell morphology similar to the parental strain. Despite the chromosomal integration, no polar effects on the downstream genes were perceptible. The GFP-FtsI fluorescence signal was mainly located as foci in the mid-cell region (Fig. 2b, indicated by arrowheads). However, although the GFP signal in the merodiploid strain MAPF (Fig. 2c) clearly shows a reduced level of fluorescence, this is probably due to the competition between the original FtsI and GFP-FtsI. Nevertheless, the signal is well above the background autofluorescence that is seen in the parent C. glutamicum RES167 strain, which has no GFP fusion (not shown). Furthermore, the localization of GFP-FtsI at the mid-cell is strongly indicative of the involvement of FtsI in C. glutamicum cell division, most likely in the biosynthesis of septum peptidoglycan as described for other bacteria.
ftsI seems to be an essential gene in C. glutamicum
ftsI has been shown to be an essential gene in E. coli (Begg et al., 1992) and in B. subtilis (Daniel et al., 1996). Therefore, in order to determine whether ftsI was also necessary for the viability of C. glutamicum, we performed gene disruption experiments using the suicide plasmid pKInt1 (Table 1); all attempts to inactivate the ftsI gene using internal fragments were unsuccessful, similar to our earlier studies involving murC, another essential gene in C. glutamicum (Ramos et al., 2004).
Disruption of ftsI was only possible in the merodiploid strain C. glutamicum MAPF using plasmid pKInt1. Cell morphology and growth rate of the transconjugants expressing the GFP-FtsI fusion (Fig. 2d) were found to be similar to the host MAPF strain. Disruption of the original chromosomal copy of ftsI was confirmed by Southern blotting using DNA isolated from ten fluorescent transconjugants (not shown). These results unambiguously substantiate that ftsI is essential in C. glutamicum, as it is in E. coli and B. subtilis.
Decreased expression of ftsI causes severe defects in cell morphology
It has been reported previously that the promoter of the lactose operon of E. coli (Plac) is not well recognized by the C. glutamicum RNA polymerase (Ramos et al., 2005). As ftsI appears to be an essential gene in C. glutamicum, and since no ftsI null mutants could be obtained, the function of the FtsI was investigated by partial depletion of FtsI levels in the organism. To do this, C. glutamicum transformed with the plasmid pOJPB (Table 1) was used. Southern blotting of the transconjugant strain (C. glutamicum RESF1) revealed the pattern expected of Campbell integration of pOJPB at the ftsI locus (Fig. 3b). The strain carries a disrupted non-functional copy of ftsI and a functional copy under the control of Plac, and has a distinctive phenotype (see Fig. 3b). This may be due to a reduced expression of ftsI (2.9 times less than the wild-type strain as quantified by Q-PCR analysis); furthermore, cells were found to be irregularly shaped, swollen and larger than the parent C. glutamicum RES167 strain (Fig. 3a). However, the possibility exists that the aberrant phenotype may be due to the expression of the truncated ftsI or to polar effects on expression of other genes in the dcw cluster located downstream from ftsI (see Fig. 1a).
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). To reduce the levels of FtsI, we attempted to introduce the lacIq gene of E. coli, which is present in the plasmid pALacI (Table 1), into C. glutamicum RESF1. All of our attempts to obtain viable kanamycin- and apramycin-resistant transconjugants using the vector pALacI were unsuccessful, while control plasmid pABK readily yielded transconjugants under the same conditions. These results seem to indicate that a more reduced ftsI expression due to the possible effect of the lacZ repressor (lacIq) is lethal for C. glutamicum.
Decreased expression of ftsI induces the expression of several genes in C. glutamicum
Total cytoplasmic proteins synthesized by C. glutamicum RES167 (parent strain) and C. glutamicum RESF1 (partially depleted FtsI strain) were characterized by 2D gel electrophoresis (IEF SDS-PAGE). Representative gels depicting a consistent pattern of the protein profiles are shown in Fig. 4. Twenty-two proteins of various molecular sizes seem to be clearly overexpressed in C. glutamicum RESF1 compared to the parent RES167 strain. An attempt was made to identify these 22 proteins by the peptide mass mapping technique. Only eight proteins, namely the ribosomal protein L10, 
70 sigma factor, pyruvate carboxylase, enolase, arginine succinate synthase, m-diaminopimelate dehydrogenase, DivIVA and 6-phosphofructokinase, were positively identified. Except for DivIVA, which is a part of the cell division machinery, the identified proteins are known to be involved in central metabolic pathways. Therefore, these results indicate that partial depletion of FtsI in C. glutamicum induces the expression of several genes including divIVA, whose protein product is involved in the apical growth of corynebacterial cells (Ramos et al., 2003b). The use of monoclonal antibodies raised against DivIVA/Ag84 of M. kansasii enabled us to measure the levels of DivIVA in C. glutamicum RES167 and C. glutamicum RESF1 strains, and we found 3–4 times more DivIVA protein in RESF1 than in RES167 (Fig. 5).
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divIVA-gfp transcriptional fusion (Table 1), was introduced into C. glutamicum RESF1 strain and transconjugants were selected in TSA medium containing apramycin and kanamycin. Depending upon the region of integration two types of transconjugants were expected: (i) those that would arise by a single recombination event between plasmid pOJPB, which was previously integrated into the chromosome of C. glutamicum RESF1, and homologous sequences of the incorporated plasmid pKAG1, and (ii) those integrated at the chromosomal 3' end of divIVA. Forty kanamycin- and apramycin-resistant transconjugants were observed under the fluorescence microscope for the expression of DivIVA-GFP. Five clones among the forty obtained showed fluorescence, and these were then tested by Southern blot hybridization to confirm the integration of pKAG1 vector at the 3' end of divIVA (data not shown). One among these transconjugants was named C. glutamicum RESF12 and was used in our studies. Its genetic structure is shown in Fig. 3(d).
The expression product of divIVA-gfp in C. glutamicum RESF12 strain was found to accumulate not only at the cell poles (Fig. 3d) but also at the mid-cell as previously shown (Ramos et al., 2003b). The level of expression was comparable to that observed in a strain of C. glutamicum/pEAG2, which contains the divIVA-gfp fusion being expressed from a multicopy plasmid (Ramos et al., 2003b), and higher than C. glutamicum AR200 strain in which divIVA-gfp was expressed as a single copy on the chromosome (Fig. 3c). Cell-free extracts from C. glutamicum AR200 and RESF12 were electrophoresed by SDS-PAGE, transferred and analysed by Western blotting, using anti-DivIVA (Fig. 5a) and anti-GFP antibodies (data not shown). In both cases, the level of DivIVA-GFP was 3–4 times higher in C. glutamicum RESF12 than in AR200 (Fig. 5a).
The 
-lactam antibiotic cephalexin is a specific inhibitor for FtsI that blocks cell division but does not affect the level of FtsZ or FtsI in E. coli (Pogliano et al., 1997). C. glutamicum cell division was blocked by growing cells in TSA media containing a subinhibitory concentration of cephalexin (0.6 µg ml–1) (Fig. 5c) to see if the inactivation of FtsI by this drug would increase the level of DivIVA. As shown in Fig. 5(a), inhibition of FtsI by cephalexin did not increase the level of DivIVA.
It was recently described that C. glutamicum cell division was partially blocked when the chromosomal copy of ftsZ was expressed under the control of Plac (C. glutamicum AR2), and even more when the lacIq repressor was introduced in AR2 (C. glutamicum AR20) (Fig. 5b); the resulting strains showed aberrant cells but no filaments (Ramos et al., 2005). The level of DivIVA in C. glutamicum AR2 and AR20 is similar to that in the wild-type strain (Fig. 5a).
Therefore, it may be concluded that divIVA is being overexpressed when FtsI is partially depleted in C. glutamicum and not when cell division is blocked either by inactivation of FtsI by cephalexin treatment or by partial depletion of FtsZ.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). FtsZ is also an important component of the internal cytoskeleton and its polymerization at the septum facilitates FtsI in redirecting peptidoglycan synthesis in E. coli or B. subtilis (Nanninga, 1998). Peptidoglycan synthesis in those model micro-organisms proceeds by diffuse intercalation of new material being synthesized along the length of each cell. Peptidoglycan at the cell poles is inert, not being recycled or only being recycled at an extremely low rate (de Pedro et al., 1997).
The situation is different in C. glutamicum, where peptidoglycan synthesis takes place at the septum and also at the cell poles (Daniel & Errington, 2003). It has been suggested that cell elongation occurs from the new cell poles (Daniel & Errington, 2003) as was also described for C. diphtheriae (Umeda & Amako, 1983). Corynebacteria probably have an apical growth reminiscent of the characteristic apical growth of actinomycetes. The localization of GFP-FtsI in this organism appears to be similar to that seen in E. coli (Weiss et al., 1999) and B. subtilis (Daniel et al., 2000), and this suggests the participation of FtsI in the biosynthesis of peptidoglycan for septum formation. Sometimes, and in a non-repetitive way, it was also possible to see accumulation of GFP-FtsI at the cell poles, which is reminiscent of previous septa or artefacts due to non-specific accumulation of GFP-FtsI as described in E. coli (Weiss et al., 1999).
Our results suggest that ftsI is transcribed both from a minor promoter (PftsI), as in B. subtilis (Daniel et al., 1996), and also as a part of the polycistronic mraZ, mraW, ftsL and murE transcript from an upstream promoter (Pmra), as described for E. coli and B. subtilis (Hara et al., 1997; Mengin-Lecreulx et al., 1998). The ftsI gene seems to be essential for the viability of C. glutamicum since gene disruption was possible only in the merodiploid strain C. glutamicum MAPF. No transformants were recovered when we attempted to elicit a stronger reduction in the expression of ftsI under the control of Plac in C. glutamicum RESF1 transformed with a plasmid carrying lacIq.
When ftsI was expressed as a single copy in the chromosome under the control of Plac (strain C. glutamicum RESF1) morphologically abnormal cells (filamentous or branched filaments) were obtained. The shape of RESF1 cells contrasts with the bulky and elongated cells obtained when cell division is inhibited by cephalexin treatment or by partial FtsZ depletion (Fig. 5). These abnormal cells were considered as a general strategy by the bacteria for increasing cell mass when division is blocked in rod-shaped micro-organisms that lack actin homologues as was suggested by Latch & Margolin (1997) and Ramos et al. (2005). The cephalexin treatment has no effect on the levels of ftsI or ftsZ (Pogliano et al., 1997), suggesting that the characteristic shape of C. glutamicum RESF1, as a consequence of a severe reduction (2.9-fold) in the expression of ftsI (Fig. 3b), is not only due to a block in cell division. This possibility prompted us to compare the proteome of C. glutamicum RESF1 with that of the wild-type. It was noticeable from our results that the level of DivIVA, among other proteins, increases in the strains that are partially depleted for FtsI.
Several hypotheses can be proposed to explain why a reduction in the levels of FtsI could account for an increase in the levels of DivIVA, a cell division-associated protein with a possible structural function at C. glutamicum growing cell poles (Ramos et al., 2003b). The first possibility is that the filamentous phenotype may be due to the expression of the truncated ftsI in C. glutamicum RESF1 (Fig. 3b), although no filaments were observed in C. glutamicum APF (having a truncated form of ftsI and a unique copy of gfp-ftsI under the control of Pkan) (Fig. 2b). The observed effect of DivIVA overexpression could also be due to polar effects on the expression of other genes in the dcw cluster located downstream from ftsI, such as murE (encoding the tripeptide synthetase MurE) or murF (encoding pentapeptide synthase MurF) (Fig. 1a). It has been described for E. coli that the balance between pentapeptide and tripeptide precursors determines whether the cells will divide or elongate (Begg et al., 1990). We cannot rule out this option, but no effect on the morphology was observed when different C. glutamicum strains were obtained by simple recombination in the chromosome (Fig. 2). A third possibility is that depletion of FtsI, and not the inhibition of FtsI by cephalexin, alters the turnover or stability of DivIVA, or even the expression of the divIVA gene. Because the amount of FtsI protein, and not the activity of FtsI, could be the start point of a mechanism leading to DivIVA overexpression, it is possible that a balance between FtsI and DivIVA would be needed for cell division in C. glutamicum. The requirement of a specific ratio between two proteins of the dcw cluster has been described by Dewar et al. (1992) and Flardh et al. (1998).
The filamentous phenotype observed when ftsI is under the control of Plac seems to be the result of a lack of FtsI to synthesize peptidoglycan at the septum and an increase in the concentration of enzymes involved in central metabolism and DivIVA, which might form a scaffold structure that guides cell wall biosynthesis and maintains the cell diameter in MreB-lacking rod-shaped corynebacteria.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 15 December 2005;
revised 10 March 2006;
accepted 30 March 2006.
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, Lambda DNA digested with PstI.
