1 Departamento de Ecología, Genética y Microbiología, Área de Microbiología, Facultad de Biología, Universidad de León, 24071 León, Spain
2 Departamento de Biología Celular, Facultad de Biología, Universidad de León, 24071 León, Spain
Correspondence
José A. Gil
degjgs{at}unileon.es
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ABSTRACT |
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INTRODUCTION |
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Hermann et al. (1998) identified the presence of a typical mycobacterial antigen (Antigen 84, Ag84) in C. glutamicum by protein microsequencing after two-dimensional gel electrophoresis (Hermann et al., 1998
). The Mycobacterium tuberculosis gene encoding cytoplasmic Ag84 (wag31) (Cole et al., 1998
) has been cloned (Hermans et al., 1995
), and expressed as a 34 kDa protein in Escherichia coli; the recombinant protein corresponded to Ag84 in a crossed immunoelectrophoresis reference system. The homologous gene for Ag84 was also cloned from Mycobacterium leprae and its amino acid sequences showed 85 % identity to the M. tuberculosis sequence, which indicates that Ag84 constitutes a group of conserved highly immunogenic mycobacterial antigens. The antibodies of almost 60 % of lepromatous leprosy patients responded to Ag84 (Hermans et al., 1995
).
Ag84 from M. tuberculosis is related to DivIVA, a protein encoded in the dcw cluster of several Gram-positive micro-organisms (Massidda et al., 1998), including all Gram-positive bacteria sequenced to date. DivIVA has been extensively studied in Bacillus subtilis, in which inactivation of divIVA produces a minicell phenotype, whereas overproduction of DivIVA results in a filamentation phenotype (Cha & Stewart, 1997
). Previous work has shown that in vegetatively growing Bac. subtilis cells, DivIVA is involved in cell division, and its role appears to be the sequestration of 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 E. coli MinE, which sequesters MinCD at the cell poles using a completely different mechanism (Marston et al., 1998
). DivIVA has a second, quite separate role in sporulating cells of Bac. subtilis. Again, it acts at the cell pole but in this case it interacts with the chromosome segregation machinery to help position the oriC region of the chromosome at the cell pole, in preparation for the polar division event that initiates spore formation (Thomaides et al., 2001
). More recently Harry & Lewis (2003)
, using a synchronous model system in Bac. subtilis (spore germination and outgrowth), found that DivIVA localizes to poles of germinated and outgrowing cells without the prior assembly of the division apparatus at this site, suggesting that its localization does not occur via a direct interaction with components of the division apparatus as proposed previously (Edwards et al., 2000
). Later on DivIVA localizes, probably by interaction with a component of the cell division apparatus, to the division site before the final stages of Z-ring constriction (Harry & Lewis, 2003
). Once the Z-ring constricts, DivIVA is attracted to the cell poles by an unidentified protein or by a physical property unique to the cell poles (Edwards et al., 2000
; Harry & Lewis, 2003
).
Here we show that the Brev. lactofermentum homologue of the wag31/divIVA is important in cell shape and morphology.
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METHODS |
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Plasmids to be transferred by conjugation from E. coli to Brev. lactofermentum were introduced by transformation into the donor strain E. coli S17-1. Brev. lactofermentum R31 was used as the recipient. The protocol for conjugation was a slight modification of the method developed by Schäfer et al. (1990).
DNA fragments were purified using the Gene Clean Kit (Bio 101). Restriction enzymes were purchased from Promega and New England Biolabs.
A library of Brev. lactofermentum ATCC 13869 DNA constructed in lambda gt11 (Honrubia et al., 1998) was checked for DNADNA hybridization screening using a 675 bp HindIIIXhoI fragment containing the 3' end of ORF5, the intergenic region, and the ORF6 gene from Brev. lactofermentum present in plasmid pPHFZ8 (probe A), and with a 600 bp BstXIEcoRI fragment of the non-coding region (probe B) (Fig. 1
). Two clones giving a positive signal with probe B and no signal with probe A were found, and one of them, which contained a 1·5 kb NotI insert, was subcloned into NotI-digested pBSK- and pBKS-, giving plasmids pARX2A and pARX2B.
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Samples of total DNA from different Brev. lactofermentum transconjugants were digested with BamHI and hybridized with a 660 bp PvuII internal fragment (probe C, Fig. 1) of divIVABL labelled with digoxigenin according to the manufacturer's (Boehringer Mannheim) instructions.
RNA from Brev. lactofermentum strains containing plasmid pECM2 or plasmid pECAG1 was isolated at different culture times in TSB medium using the RNeasy commercial kit (Qiagen). For Northern experiments, 20 µg RNA was loaded onto a 1·5 % formaldehyde-agarose gel and transferred to nylon membranes. Filters were hybridized with an internal fragment of divIVABL (513 bp HincII fragment; probe D; Fig. 1) from Brev. lactofermentum labelled by nick-translation.
Plasmid constructions.
To disrupt divIVABL by single recombination, two different constructions were made by cloning two internal overlapping DNA fragments separately into the conjugative suicide plasmid pK18mob (Table 1). A 500 bp internal BglI fragment (Klenow-filled) of divIVABL (Fig. 1
) was cloned into SmaI-digested pK18mob and the resulting plasmid was named pKD8-1B (Table 1
). A single reciprocal crossover event would create two interrupted DivIVABL versions, one lacking 89 amino acids from its C-terminus, and the other lacking 100 amino acids from its N-terminus (see Fig. 2
). The second plasmid (pKD8-1P) contained a 660 bp internal PvuII fragment (Fig. 1
) cloned in SmaI-digested pK18mob. The resulting truncated DivIVABL proteins would lack 20 amino acids from the C-terminus or 150 amino acids from the N-terminus (Fig. 2
; Table 1
).
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To detect the presence of a functional promoter immediately upstream from divIVABL, a 160 bp DNA fragment was amplified by PCR using the following primers: upper primer, 5'-GGGGTCCTTGTGGCCTTGAAAGTGTGCAGG-3'; lower primer, 5'-GGGAATTCCATATGCGGATTCCCTTCGATTTAACGGTGG-3'. Owing to the presence of a NdeI (CATATG) site in the lower primer and a single EcoRI in the amplified fragment, the PCR product was digested with EcoRI and NdeI and cloned into the promoter-probe vectors pECMel-1 and pJMFA24, both digested with EcoRI+NdeI.
To overexpress divIVABL in Brev. lactofermentum, a 1·3 kb EcoRIBamHI fragment containing the entire divIVABL gene (and upstream sequences) (Fig. 1) was cloned in the high-copy-number conjugative bifunctional plasmid pECM2 (Jager et al., 1992
), giving pEAG1 (Table 1
). The plasmid was then transferred to Brev. lactofermentum by conjugation.
Two different divIVABLgfp translational fusions were made. In both cases, the 3' end of divIVABL was amplified by PCR using the following primers: upper primer, 5'-ACGCGTCGACACTTCCGAGGCTCGCTCCGAATCCAAGTCCATG-3'; lower primer, 5'-GGGAATTCCATATGCTCACCAGATGGCTTGTTGTTGGTTGG-3'. These primers were designed to replace the stop codon (TAA) of the divIVABL by CAT (His), which after NdeI digestion and ligation with gfp will immediately be followed by the ATG start of gfp. Owing to the presence of restriction sites in the primers [SalI (GTCGAC) and NdeI (CATATG)] and a single XhoI in the amplified fragment, the SalINdeI-amplified fragment (480 nt) was cloned together with gfp (as a NdeIXbaI fragment) in plasmid pET28a digested with SalI+XbaI. The gfp gene used was egfp2 from Clontech, and includes the mutations V163A and S175G introduced by Siemering et al. (1996). The in-frame-fused
divIVABLgfp gene was isolated as a SalIXbaI fragment, sequenced (see below), and cloned into plasmid pK18mob (digested with SalI and XbaI), to give pKAG1, which was introduced by conjugation into Brev. lactofermentum, and integrated into the chromosome by single recombination.
The divIVABLgfp gene was also isolated from the pET28a derivative as an XhoIXbaI fragment and cloned into plasmid pEAG1 (digested with XhoI and XbaI) to replace the 3' end of divIVABL (24 amino acids) by the 3' end of divIVABL fused to gfp. The resulting plasmid, pEAG2, was constructed in E. coli and transferred by conjugation to Brev. lactofermentum.
To express divIVABL in E. coli, a 1·3 kb EcoRIBamHI fragment containing the entire divIVABL gene (and upstream sequences) (Fig. 1) was cloned into plasmids pT7-5 and pT7-6 (both digested with EcoRI+BamHI), giving plasmids pTAG1 and pTAG2 respectively (Table 1
).
Sequencing and DNA analysis.
The 1·5 kb NotI fragments from plasmids pARX2A and pARX2B were trimmed down using the Erase a Base Kit (Promega). DNA sequencing was carried out by the dideoxy nucleotide chain termination method of Sanger. Computer analysis was performed with DNASTAR; database similarity searches were done at the BLAST and FASTA public servers (NCIB and EBI, Hinxton Hall, UK), and multiple alignments of sequences were accomplished using CLUSTAL W (EBI). The DNA sequence was deposited in the EMBL/GenBank database under accession number AJ242594. Plasmid constructions carrying divIVABLgfp were confirmed to be correct by sequencing.
To localize the gene located downstream from divIVABL, we PCR amplified Brev. lactofermentum DNA using an upstream primer (5'-CCGCATCCACCTCTCGT-3') designed from divIVA and a downstream primer (5'-TCTCGCCCTTGTCCTTGATGC-3') from the downstream gene (ileS) in the C. glutamicum genome. A DNA fragment of the expected size (1230 nt) was amplified and sequenced, and it was confirmed that ileS is the gene located downstream from divIVA in Brev. lactofermentum, as in Streptomyces coelicolor, Streptococcus pneumoniae and Corynebacterium diphtheriae.
Preparation of cell-free extracts, PAGE and Western blotting.
E. coli JM109(DE3) cells transformed with pT7-5, pT7-6, pTAG1 or pTAG2 were grown at 37 °C in LB broth with 100 µg ampicillin ml-1 until the OD600 reached 0·4. IPTG was added at a final concentration of 0·5 mM and the cultures were incubated for 3 h.
Brev. lactofermentum, M. smegmatis, S. coelicolor and Bac. subtilis cells were disrupted by sonication as follows. Cells (1 g wet weight) were suspended in 5 ml TES buffer (25 mM Tris/HCl, 25 mM EDTA, 10·3 % sucrose, pH 8). Sonication was carried out over periods of 30 s with 1 min intervals in an ice-cooled tube using a Branson sonifier (model B-12) at 75100 W, until the cells had been completely disrupted as observed microscopically. Cell debris was removed by centrifugation, and the supernatants were used as cell extracts. E. coli cells were washed, resuspended in loading buffer, and boiled for 5 min. SDS-PAGE of cell extracts from the different micro-organisms 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 with monoclonal antibodies (F126-2) raised against purified Ag84 from Mycobacterium kansasii provided by Professor A. H. J. Kolk (Royal Tropical Institute, Amsterdam, The Netherlands).
Microscopic techniques.
Brev. lactofermentum cells containing constructions carrying GFP were observed under a Nikon E400 fluorescence microscope. Pictures were taken with a DN100 Nikon digital camera and assembled using Corel Draw.
For scanning electron microscopy, Brev. lactofermentum cells were collected by centrifugation, fixed for 2 h at room temperature in 2·5 % glutaraldehyde in 100 mM cacodylate buffer (pH 7·4), rinsed three times in cacodylate buffer (pH 7·4), and postfixed for 2 h in 1 % osmium tetroxide. Cells were washed twice with cacodylate buffer and recovered by filtration in Millipore filters (0·20 µm diameter). After passage through 20 %, 50 %, 75 %, 95 %, and 100 % ethanol, filters containing the cells were dried using the critical-point method and finally coated with gold, giving a layer 40 nm thick. Cells were observed with a scanning electron microscope (JEOL JSM-6100) at an accelerating voltage of 20 kV.
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RESULTS |
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A 1505 bp downstream sequence (GenBank accession no. AJ242594) contained two possible ORFs (ORF7 and ORF8). ORF7 encodes a small protein of 95 amino acids and showed similarities with ORF Rv2146c of M. tuberculosis (FASTA E value 2x10-6), and with ORF SCO2078 from S. coelicolor A3(2) (FASTA E value 1x10-5). These proteins belong to the group of YlmG proteins, a group of small (approx. 10 kDa) conserved hypothetical proteins with a transmembrane domain, localized downstream from ftsZ in most of the dcw clusters from Gram-positive bacteria analysed (Massidda et al., 1998).
ORF8 is located 488 bp downstream from ORF7, and encodes a protein of 365 amino acids with a predicted molecular mass of 38·7 kDa. A stemloop-forming sequence (1231GCTCCCGGGTCCGGA1245------1261TCCGGATCCGGGAGC1275) is located 28 nt downstream from the ORF8 gene and may be a transcriptional terminator. When the amino acid sequence of ORF8 was aligned with protein databases using BLAST (NCBI) it showed significant scores with Ag84 from M. tuberculosis (E value 1x10-21), M. leprae (E value 7x10-20) and ORF SCO2077 from S. coelicolor A3(2) (E value 8x10-8), all of which are encoded by genes located in the same position with respect to the dcw cluster. A very low score with Bac. subtilis DivIVA was found (E value 1·8), and none with DivIVA from Streptococcus pneumoniae or Staphylococcus aureus. The gene was designated divIVABL like the S. coelicolor A3(2) paralogue (Flardh, 2003) to avoid misunderstanding. No similar gene was detected in the chromosome of Brev. lactofermentum on the basis of Southern analysis.
The N-terminal region of DivIVA homologues is highly conserved (Fig. 1B) and is predicted to adopt a coiled-coil conformation using the COILS algorithm (Lupas et al., 1991
). Another coiled-coil domain is predicted at the C-terminus in all DivIVA proteins analysed. In S. coelicolor DivIVAs, the predicted coiled-coil regions are interrupted by a low-complexity region (from amino acid 69 to 201), mainly composed of glycine, proline and glutamic acid. DivIVABL showed an alanine-rich region from amino acid 58 to 302 and two extra coiled-coil regions. It is also interesting to note that the DivIVA from Brev. lactofermentum has been included by InterPro within the Pollen allergen Poa pI signature group of proteins (InterPro Entry IPR001778) and it has three clear domains characteristic of these proteins (Fig. 1B
). These Poa signatures are absent in the Ag84 from M. tuberculosis, and in the DivIVA from S. coelicolor or Bac. subtilis (Fig. 1B
).
Once the complete genome of Corynebacterium glutamicum had become available (accession no. AX114121), we analysed the DNA sequence located downstream from divIVABL in Brev. lactofermentum. By PCR analysis we found that the next gene is oriented in the same direction, and encodes a putative tRNA synthetase (ileS), as in S. coelicolor, Streptococcus pneumoniae, C. diphtheriae and C. glutamicum.
Attempts to disrupt the divIVABL gene in Brev. lactofermentum by single and double recombination
In order to see whether divIVABL is necessary for the viability of Brev. lactofermentum, we attempted disruption experiments using internal fragments of divIVABL (single crossover event, single recombination) or an in vitro-disrupted divIVABL gene (double crossover event, double recombination).
The conjugative suicide plasmid pKD8-1B (containing an internal fragment of divIVABL; Fig. 2A) and pK18-thrB1 (used as control) were introduced separately into E. coli S17-1 and mated with Brev. lactofermentum R31 cells. Brev. lactofermentum kanamycin-resistant transconjugants were obtained only after mating with E. coli/pK18-thrB1, suggesting the lethality of the divIVABL gene disruption or the lethality of its polar effects. However, the comparatively small size of divIVABL (500 bp, compared with the 750 bp thrB1 fragment) might also have greatly reduced the frequency of integration events. Therefore, to further test whether divIVABL is essential for the growth and viability of Brev. lactofermentum we attempted to replace the chromosomal divIVABL by an in vitro-interrupted version of the gene. A knockout mutation (
divIVABL : : hyg) conferring hygromycin resistance was created in plasmid pK18mob (containing kan), giving rise to pKD8-2. Since pKD8-2 cannot replicate in Brev. lactofermentum, hygromycin-resistant transconjugants were expected to arise by single or double recombination between the Brev. lactofermentum insert in the plasmid and homologous sequences in the chromosome (see Fig. 2B
). However, all the transconjugants analysed contained the original divIVABL gene and the
divIVABL : : hyg, suggesting that only a single crossover event had taken place. No hygromycin-resistant kanamycin-sensitive strains were found, even after several transfers on TSA lacking kanamycin and checking more than 10 000 hygromycin-resistant colonies. These results strongly suggest that divIVABL is essential for the growth/viability of Brev. lactofermentum. Comparable results have been obtained in Bac. subtilis (Edwards & Errington, 1997
) and in S. coelicolor A3(2) (Flardh, 2003
).
Another plasmid (pKD8-1P) was constructed to interrupt divIVABL by single recombination using an internal 660 bp PvuII fragment overlapping the 500 bp BglI fragment used above. pKD8-1P was introduced into E. coli S17-1 and mated with Brev. lactofermentum R31 cells. In this case kanamycin-resistant transconjugants were obtained, and Southern blotting experiments revealed the expected pattern of two BamHI DNA fragments (1·2 and 4·5 kb) (Fig. 2A). Because the N-terminus of Ag84/DivIVA/DivIVABL is a highly conserved region, its C-terminus (final 20 amino acids) may perhaps be unnecessary for activity.
Expression of divIVABL in Brev. lactofermentum
Overexpression of DivIVA in Bac. subtilis is lethal and leads to filamentation due to inhibition of cell division (Cha & Stewart, 1997). To determine the phenotype when divIVABL is expressed in Brev. lactofermentum, the multicopy plasmid pEAG1 (Table 1
) was introduced into E. coli S17-1 and mated with Brev. lactofermentum R31 cells. As can be observed in Figs 3 and 4
, transconjugants from both solid and liquid media showed an altered morphology (rounder, large and swollen cells that tended to grow at the poles), indicating the possible participation of DivIVABL in the maintenance of cell morphology in Brev. lactofermentum. Brev. lactofermentum carrying the empty vector pECM2 never showed this kind of morphology (Fig. 3A and 4A
). This aberrant morphology is obviously different from that observed in M. tuberculosis when overexpressing ftsZ (Dziadek et al., 2002
), in Rhizobium (Sinorhizobium) meliloti overexpressing ftsZEC or ftsZRM (Latch & Margolin, 1997
), or in Brev. lactofermentum overexpressing ftsZBL (A. Ramos, unpublished data); it was concluded that in rod-shaped micro-organisms branching and swelling are default pathways for increasing mass when cell division is blocked after ftsZ overexpression (Latch & Margolin, 1997
).
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Construction of divIVABLgfp fusions
Previous results in Bac. subtilis had suggested that DivIVA is recruited to mid-cell once the FtsZ ring has matured, and after cell division DivIVA and MinCD is localized to the cell poles (Harry & Lewis, 2003; Marston et al., 1998
). To investigate a possible role of divIVABL in corynebacterial morphogenesis, plasmids pKAG1 and pEAG2 were introduced by conjugation into Brev. lactofermentum, to give strains bearing a single copy of divIVABLgfp in the chromosome or multiple copies of divIVABLgfp, respectively.
Microscopical observation of both strains revealed that DivIVAGFP was located mainly at the cell poles (Fig. 6), but in both cases there was a stronger accumulation at one pole than the other. This was especially noticeable in Brev. lactofermentum AR30 (multicopy fusion; Fig. 6B
), where swollen and aberrant cells were formed. These accumulations were not detected by phase-contrast microscopy, indicating that they were not insoluble inclusions, but real structures. In those cells, and to a lesser extent in Brev. lactofermentum AR20 (single-copy fusion; Fig. 6A
), after cell division the elongation of the daughter cells seemed to be asymmetrical, suggesting a nascent mycelial growth in this lower actinomycete. The size and intensity of the fluorescent region of DivIVAGFP at the pole of the cell, especially in Brev. lactofermentum AR30, indicated that a large number of molecules was concentrated there. These observations were made clearer by the negligible fluorescence background seen in Brev. lactofermentum control strains, compared with the significant autofluorescence observed with some other bacteria (e.g. S. coelicolor). We assumed that the GFPDivIVABL fusions are functional because Brev. lactofermentum/pEAG1 showed comparable cell morphology to Brev. lactofermentum/pEAG2, and the morphology of Brev. lactofermentum AR20 is similar to that of Brev. lactofermentum R31 and Brev. lactofermentum/pECM2.
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DISCUSSION |
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In Brev. lactofermentum, divIVABL seems to be essential for viability, since no disruptants were obtained using different strategies, and due to the fact that divIVABL is expressed as a monocistronic transcript of 1·5 kb during growth, no polar effects are expected on the downstream essential ileS gene. Owing to the lack of more effective systems to manipulate Brev. lactofermentum, it is difficult to introduce a second copy of divIVABL into the chromosome of Brev. lactofermentum and disrupt one of them. Nevertheless, the present data strongly suggest that the gene is essential for viability.
In Brev. lactofermentum overexpression of DivIVA is not lethal but it might partially affect cell division because cells containing pEAG1 are bigger, exhibit aberrant morphology and grew at a slower rate than cells carrying the empty vector pECM2. Therefore, the more visible effects of divIVABL overexpression were on cell size and cell morphology. Moreover, we assume that DivIVABL does not act through a MinCD system since no homologue of MinCD has been detected in the C. glutamicum genome (A. Ramos, unpublished results) nor in S. coelicolor A3(2) (Flardh, 2003). The above data therefore suggest that DivIVABL is mainly important in the determination of cell shape, but we cannot exclude the possible role of DivIVABL as a positive regulator of apical growth.
In Bac. subtilis DivIVAGFP is targeted to a committed division site, and once daughter cells have become separated it remains at the cell poles, attracting MinD, and preventing these potential division sites from being used again, which would generate minicells (Marston et al., 1998). In corynebacteria, DivIVAGFP is mainly localized to the cell poles, although to a lesser extent it can be found in the mid-cell area. The intensity of fluorescence, even when DivIVAGFP is integrated as a single copy into the Brev. lactofermentum chromosome, suggests that DivIVA could form an oligomeric (perhaps through the coiled-coil regions) structure involved in the apical growth of corynebacterial cells. Thus, overexpression leads to dramatic changes in morphology. The asymmetric terminal location of the DivIVA protein may be widespread among actinomycetes, with different levels of morphological complexity, since a homologous protein in S. coelicolor A3(2) is located at the tips of the hyphae, where it appears to determine growth polarity (Flardh, 2003
). On the other hand, staining of S. coelicolor mycelia with vancomycin labelled with fluorescein (Van-FL) gave bright staining at the ends of the hyphae and at intermediate sites corresponding to branching points (Daniel & Errington, 2003
).
Staining of Bac. subtilis and C. glutamicum with Van-FL has clearly shown that using different growth strategies both Gram-positive bacteria achieve a rod-shaped morphology (Daniel & Errington, 2003). In Bac. subtilis, staining with Van-FL was mainly concentrated in the mid-cell but not at the cell poles, whereas in C. glutamicum Van-FL staining was observed at the mid-cell but mainly at the poles. It was concluded that in C. glutamicum cell elongation occurs from the new cell poles (Daniel & Errington, 2003
) and therefore DivIVABL is localized close to the sites of peptidoglycan assembly in Brev. lactofermentum cells as well as in S. coelicolor A3(2) (Flardh, 2003
). The Van-FL staining of C. glutamicum is also in agreement with the cellular location of the inorganic pyrophosphatase from Brev. lactofermentum at the cell poles (Ramos et al., 2003
). Because pyrophosphate is a by-product in the biosynthesis of UDP-N-acetylglucosamine by the UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23), high levels of pyrophosphate should be formed during the biosynthesis of cell wall intermediates. Moreover, in the biosynthesis of the cell wall there is a strong requirement of ATP for the synthesis of UDP-N-acetylmuramic acid (UDP-MurNAc)-pentapeptide (Falk et al., 1996
). Therefore hydrolysis of pyrophosphate is important for driving the above anabolic pathway in the direction of biosynthesis and replenishes the orthophosphate required for ATP biosynthesis (Perez-Castineira et al., 2002
).
From all the data presented here it can be proposed that DivIVA is an essential protein with a possible structural function at Brev. lactofermentum growing cell poles, and probably accumulates there by an unknown affinity mechanism for curved surfaces (Harry & Lewis, 2003) or through interaction with proteins involved in peptidoglycan biosynthesis.
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ACKNOWLEDGEMENTS |
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Received 18 July 2003;
revised 4 September 2003;
accepted 16 September 2003.
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