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Journal of Bacteriology, December 2008, p. 8204-8214, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00752-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Deletion of cgR_1596 and cgR_2070, Encoding NlpC/P60 Proteins, Causes a Defect in Cell Separation in Corynebacterium glutamicum R ^,

Yota Tsuge,^1,2,, Hidetaka Ogino,^1, Haruhiko Teramoto,¹ Masayuki Inui,¹ and Hideaki Yukawa^1,2*

Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizugawa, Kyoto 619-0292, Japan,¹ Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Nara 630-0101, Japan²

Received 27 May 2008/ Accepted 1 October 2008

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In previous work, random genome deletion mutants of Corynebacterium glutamicum R were generated using the insertion sequence (IS) element IS31831 and the Cre/loxP excision system. One of these mutants, C. glutamicum strain RD41, resulting from the deletion of a 10.1-kb genomic region (cgR_1595 through cgR_1604) from the WT strain, showed cell elongation, and several lines appeared on the cell surface (bamboo shape). The morphological changes were suppressed by overexpression of cgR_1596. Single disruption of cgR_1596 in WT C. glutamicum R resulted in morphological changes similar to those observed in the RD41 strain. CgR_1596 has a predicted secretion signal peptide in the amino-terminal region and a predicted NlpC/P60 domain, which is conserved in cell wall hydrolases, in the carboxyl-terminal region. In C. glutamicum R, CgR_0802, CgR_1596, CgR_2069, and CgR_2070 have the NlpC/P60 domain; however, only simultaneous disruption of cgR_1596 and cgR_2070, and not cgR_2070 single disruption, resulted in cell growth delay and more severe morphological changes than disruption of cgR_1596. Transmission electron microscopy revealed multiple septa within individual cells of cgR_1596 single and cgR_1596-cgR_2070 double disruptants. Taken together, these results suggest that cgR_1596 and cgR_2070 are involved in cell separation and cell growth in C. glutamicum.

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The gram-positive, high-GC-content bacterium Corynebacterium glutamicum has been one of the most important bacteria in industry for several decades due to its high production of amino acids such as glutamate (16). The bacterium often shows a V-shaped cell form under the microscope, and its coryneform rod-shaped cells have one side of their poles slightly wider than the other. Since its unique characteristics enable it to produce such large amounts of amino acids, most C. glutamicum research has remained focused on the creation of highly productive strains on the one hand and analysis of metabolic flow regulation on the other (14, 15, 32). Consequently, the unusual behavior of the bacterium to produce V-shaped cells has elicited less research attention. The cell division system resulting in V-shaped cells is known as snapping division (37). Besides C. glutamicum, other Actinomycetales such as Arthorobacter, Nocardia, and Mycobacteria are known to produce V-shaped cells (9, 18, 28, 38). Although several genes involved in cell division (13, 17, 20, 30, 34, 47) and cell morphology (21, 35, 44, 45) in C. glutamicum have been characterized, the molecular mechanism of the snapping division is still largely unknown.

Cell division is achieved by the consecutive actions of cell extension, chromosome replication and segregation, and cell separation. In most prokaryotic and eukaryotic species, cell separation starts by constriction and the subsequent formation of two equivalent daughter cells. After completion of chromosome replication and segregation of the daughter chromosomes to the two halves of the cell, constriction occurs at a predetermined site and two progeny cells are produced. In most bacteria, cell separation is achieved by the simultaneous constriction of the cytoplasmic membrane, the peptidoglycan layer, and any other cell envelope layers, such as the outer membrane of the gram-negative bacteria (26). In Escherichia coli, separation of the daughter cells by cleavage of the central part of the septal cell wall occurs together with septum formation and constriction (27). In Bacillus subtilis, cell separation is delayed compared to septum formation, resulting in formation of a cell septum. Although there is a time lag between septum formation and cell separation in B. subtilis, the fundamental mechanism of cell separation that requires constriction of the cell wall is conserved between the two model microorganisms (12, 22). In contrast, in Arthrobacter crystallopoietes, a member of Actinomycetales, a two-layer cell wall is observed and two daughter cells are sealed off by the outer layer of the cell wall, even after septum formation is completed (18). It is unknown whether or not C. glutamicum has the same cell septum formation mechanism as Arthrobacter; however, at least different cell separation systems in C. glutamicum and model microorganisms are likely to exist even within the group of rod-shaped bacterial cells.

Cell separation is initiated by hydrolysis of peptidoglycan by cell wall hydrolases. In E. coli, endopeptidase EnvC and amidases AmiA, -B, and -C are implicated in cell separation. EnvC and AmiC are part of the configuration factors of the FtsZ ring, a constriction ring responsible for cell division (3, 4, 12). Disruption of these genes results in a chained-cell phenotype. In B. subtilis, several cell wall hydrolases, including endopeptidases LytC, LytE, LytF, and YojL, have been implicated in cell separation (11, 48). In both bacteria, the cell length of a mutant increases in proportion to the number of genes inactivated, even though disruption of some cell wall hydrolases imparts no phenotypic change. In other bacteria, various other cell lytic enzymes have been identified, e.g., Aml from Streptococcus mutans, Cse from Streptococcus thermophilus, and AcmA from Lactococcus lactis (7, 39, 49).

In this study, we show that deletion of cgR_1596 and cgR_2070, encoding a catalytic domain of cell wall hydrolase in the carboxyl-terminal region, causes defect of cell separation and delay of cell growth in Corynebacterium glutamicum R. Single disruption of cgR_1596 and simultaneous disruption of cgR_1596 and cgR_2070 resulted in cell elongation and a defect in cell separation. The mutants with cgR_1596 and/or cgR_2070 disrupted show more sensitivity to β-lactam antibody than the wild type (WT). The mechanism of the snapping division is also discussed, based on transmission electron microscopy (TEM) observation of the mutants defective in cell separation.

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Bacterial strains, plasmids, and culture conditions. The bacterial strains, plasmids, and primers used in this paper are listed in Tables 1 and 2. C. glutamicum strains were grown in a minimal medium or in A medium, as a rich medium, at 33°C with aeration. Minimal medium contained 2 g liter^–1 urea, 7 g liter^–1 (NH₄)₂SO₄, 0.5 g liter^–1 K₂HPO₄, 0.5 g liter^–1 KH₂PO₄, 0.5 g liter^–1 MgSO₄·7H₂O, 6 mg liter^–1 FeSO₄·7H₂O, 6 mg liter^–1 MnSO₄·7H₂O, 200 µg liter^–1 biotin, and 200 µg liter^–1 thiamine-HCl. For A medium, 2 g liter^–1 yeast extract, and 7 g liter^–1 Casamino Acids were added to the minimal medium. Glucose (4%, wt/vol) was added to both media as the sole carbon source. E. coli strains were grown in Luria-Bertani (LB) medium at 37 or 30°C with aeration. When necessary, spectinomycin was added to a final concentration of 200 µg ml^–1, ampicillin to 100 µg ml^–1, and kanamycin to 50 µg ml^–1. Chloramphenicol was used at 50 µg ml^–1 for E. coli strains and at 5 µg ml^–1 for C. glutamicum strains.

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TABLE 1. Strains and plasmids used in this study

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TABLE 2. Oligonucleotides used in this study

DNA techniques and PCR methodology. E. coli plasmid DNA was extracted using a QIAprep spin miniprep kit (Qiagen, Hilden, Germany), and C. glutamicum genomic DNA was extracted using a GenomicPrep cells and tissue DNA isolation kit (GE Healthcare, Little Chalfont, United Kingdom), according to the manufacturer's instructions. PCRs were performed using Takara LA Taq DNA polymerase (Takara, Kyoto, Japan) or Pyrobest DNA polymerase (Takara) in a GeneAmp PCR System 9700 (Applied Biosystems, CA). PCR products were electrophoresed on 1% agarose gels and recovered by using a QIAquick gel extraction kit (Qiagen). DNA ligation was performed using ligation kit version 2.1 (Takara).

Transformation of C. glutamicum. All plasmid DNA used for the transformation of C. glutamicum was extracted from E. coli JM110 (dam dcm). Plasmid DNA extracted from a dam⁺ dcm⁺ E. coli strain cannot transform C. glutamicum efficiently due to the presence of a methyl-specific restriction system in C. glutamicum (46). One microgram of unmethylated plasmid was used to transform C. glutamicum cells using a GenePulser II (Bio-Rad). Electroporated cells were added to 1 ml of A medium supplemented with glucose and incubated for 2 h at 33°C. An appropriate volume of culture was plated on medium containing the appropriate antibiotic to select for transformants.

Construction of recombinant plasmids for complementation experiments. For cloning of cgR_1596, including the full length of cgR_1596 up to 200 bp upstream from the start codon, 1596F-KpnI and 1596R-KpnI were used to generate a DNA fragment with KpnI cohesive ends. The PCR amplicon was subsequently ligated to KpnI-digested pCRB1 plasmid DNA, yielding plasmid pCRB608 (Table 1). For cloning of cgR_2070, including the full length of cgR_2070 up to 200 bp upstream from start codon, 2070F-EcoRI and 2070R-KpnI were used to generate a DNA fragment with EcoRI and KpnI cohesive ends. The PCR amplicon was subsequently ligated to EcoRI- and KpnI-digested pCRB1 plasmid DNA, yielding plasmid pCRB609 (Table 1). For cloning of cg1735, including the full length of cg1735 up to 470 bp upstream from start codon, 1735F-SphI and 1735R-SphI were used to generate a DNA fragment with SphI cohesive ends. The PCR amplicon was subsequently ligated to SphI-digested pCRB1 plasmid DNA, yielding plasmid pCRB610 (Table 1).

Construction of a mutant with multiple disruptions of putative cell wall hydrolases. A mutant with multiple disruptions of putative cell wall hydrolases was constructed using the mutant lox system as described previously (42). cgR_2070, cgR_0802, and cgR_2069 were successfully disrupted based on cgR_1596::Tn5. Each disruptant was screened by spectinomycin resistance. For disruption of cgR_0802 and cgR_2070, homologous regions of the amino and carboxyl termini of each gene were cloned into pHSG398. For cloning cgR_0802 homologous regions, primers 802FF-SmaI and 802FR-XbaI and primers 802RF-SalI and 802RR-SphI were used. For cloning cgR_2070 homologous regions, primers 2070FF-SmaI and 2070FR-XbaI and primers 2070RF-SalI and 2070RR-SphI were used. A spectinomycin cassette was inserted between both regions amplified with primers SpF-Lelox-XbaI and SpR-Relox-XbaI. The primers contained mutant lox sequences lox66 and lox71, respectively, at the 5' end. For cloning cgR_2069, the whole region of cgR_2069 was cloned by first using primers 20692070F-EcoRI and 20692070R-XbaI. After digestion with BamHI, which has two restriction sites in cgR_2069, a spectinomycin resistance cassette amplified using primers SpF-Lelox-BamHI and SpR-Relox-BamHI was inserted into the BamHI-digested plasmid. The primers have lox66 and lox77 sequences, respectively, at the 5' end. The constructed plasmids were integrated into the chromosome by double crossover and screened for spectinomycin resistance and chloramphenicol sensitivity. Finally, the spectinomycin cassette was deleted by Cre expression using the plasmid pCRA406 to allow successive deletion of putative hydrolases (1).

Light and fluorescence microscopy. Microscopy was performed on an Olympus AX70 microscope equipped with a 100x differential interference contrast (DIC) objective and appropriate filter sets (Chroma Technology, VT) and a Photometric cool snap HQ camera (Nikon, Tokyo, Japan). Images were processed with Metamorph 5.0 (Universal Imaging) and Adobe Photoshop 5.0. Time-lapse analysis was performed essentially as descried elsewhere (24). C. glutamicum cells were placed on a 0.5% agar-padded slide containing A medium with 4% glucose. 4',6-Diamino-2-phenylindole (DAPI) (Wako, Japan) was used to stain nucleoids. FM4-64 (Molecular Probes, CA) and Nile Red (Invitrogen) were used to stain cytoplasmic membranes. fDHPE (Invitrogen) was used to stain mycolate layers. The cells were then fixed for examination at a later time. For fixation, cell samples from the culture were washed in phosphate-buffered saline (PBS) prior to suspension in 1.6% (wt/vol) formaldehyde in PBS and left on ice for 1 hour. The fixed cells were washed with PBS three times and then placed on slides.

-Amylase assay on agar plates. The secretion of -amylase from Geobacillus stearothermophilus fused signal peptides was detected by the starch iodine reaction. Signal sequences of both the cgR_1596 and cgR_2070 genes were amplified by PCR using primers 1596SPF-EcoRV and 1596SPR-EcoRV primers and 2070SPF-EcoRV and 2070SPR-EcoRV, respectively. PCR fragments were cloned into plasmid pCRC900 (J. Watanabe, unpublished data), which contains the -amylase gene lacking the start codon under control of the tac promoter. The resulting plasmids, named pCRB600 and pCRB601, were transformed into C. glutamicum R, and transformants were used to inoculate a solid A medium plate containing 4% starch for 2 days at 33°C. After 3 ml of iodine solution (1.3 mM iodine and 40 mM potassium iodide) was dropped onto the plate, the presence of a white halo around transformants with a purple background indicated extracellular -amylase activity. pCRC900 lacking a signal sequence was used as a negative control.

Preparation of fusion proteins. The cgR_1596 and cgR_2070 genes excluding the start codons were amplified by PCR using primers 1596SP+NdeI and 1596R-KpnI and primers 2070SP+NdeI and 2070R-KpnI, respectively. The cgR_1596 gene, excluding signal peptides, was amplified with PCR using 1596SP-SacI and 1596R-KpnI. The resulting PCR products were digested with appropriate restriction enzymes and cloned into pCold-I vector which was digested with appropriate restriction enzymes (Takara). The resulting plasmids (pCRB606, pCRB607, and pCRC611) were sequenced to ensure that no mutations had been introduced during cloning and transformed into E. coli strain BL21. Transformants were used to inoculate into 100 ml of LB medium containing 100 µg ml^–1 ampicillin, incubated at 37°C to an optical density at 610 nm (OD₆₁₀) of 0.5, and transferred to 15°C. After 30 min of incubation at 15°C, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the medium. Twenty-four hours after induction, cells were harvested by centrifugation (10,000 x g, 10 min, 4°C), and the pellet was frozen at –80°C. The pellet was resuspended in PBS and sonicated, and the lysate was centrifuged at 10,000 x g for 10 min at 4°C. His-tagged proteins were recovered using His-trap HP (GE Healthcare) according to the manufacturer's instructions.

Localization of CgR_1596 and CgR_2070 proteins. To visualize the locations of the CgR_1596 and CgR_2070 proteins, anti-CgR_1596 antibody was produced and a CgR_2070-Myc fusion was constructed. The anti-CgR_1596 antibody was produced against a His₆-CgR_1596 recombinant protein as an antigen (Scrum, Tokyo, Japan). cgR_2070 was amplified with PCR using primers 2070F-EcoRI and 2070R-myc-SalI. The 2070R-myc-SalI primer contains the whole Myc sequence and the carboxyl terminus of cgR_2070 except for the stop codon. The PCR product was cloned into pCRB1, and the resulting vector, pCRB605, was transformed into WT cells. The transformant was cultured in A medium containing 4% glucose and chloramphenicol to exponential phase (OD₆₁₀ of ca. 1.0). WT and WT/pCRB605 cells were fixed by incubation with PBS (pH 7.4) containing 4% paraformaldehyde for 15 min at room temperature. Fixed cells were washed with PBS for 5 min twice. The cells were permeabilized by incubation in 0.25% (vol/vol) Triton X-100 in PBS for 10 min and then washed with PBS for 5 min twice. Blocking was performed by incubation with 10% bovine serum albumin (BSA) in PBS for 30 min at 37°C. The cells were suspended in 400 µl of BSA-PBS containing a 1:100 dilution of mouse anti-CgR_1596 polyclonal antibody or a 1:500 dilution of mouse anti-Myc M2 monoclonal antibody (Sigma, Taufkirchen, Germany) and then incubated at 37°C for 2 h (primary antigen-antibody reaction). After washing of the cells with PBS for 5 min twice, they were suspended in 400 µl of BSA-PBS containing a 1:10,000 dilution of Alexa Fluor 488 goat anti-mouse immunoglobulin G (2 mg ml^–1) (Molecular probes) and then incubated at 37°C for 45 min (secondary antigen-antibody reaction). After washing the cells with PBS for 5 min for two times, the cells were placed on slides and examined.

Electron microscopy. For scanning electron microscopy analysis, WT and cgR_1596 mutant cultures at exponential phase (OD₆₁₀ of ca. 1.0) were centrifuged, fixed with 2.5% (wt/vol) glutaraldehyde, and washed with 50 mM PBS three times, and then samples were dehydrated with a graded series of ethanol solutions (60, 80, and 99%) at –30°C for 30 min, 30 min, and overnight, respectively. Samples were then immersed in thiobarbituric acid and were dehydrated with a Hitachi ES-203 instrument. Vapor deposition of platinum was performed with a Hitachi E-1010 instrument. Samples were examined in a Hitachi S-4700 scanning electron microscope at an acceleration voltage of 1.5 kV. For TEM analysis, 10 ml of WT and cgR_1596 mutant cells were grown to exponential phase (OD₆₁₀ of 1.0) and were fixed with 2.5% (wt/vol) glutaraldehyde diluted with 50 mM PBS, followed by 2 hours of incubation at 4°C. After cells were washed with distilled water for 10 min at 4°C five times, cells were fixed with 1% (wt/vol) osmium tetroxide diluted with distilled water at 4°C overnight. Cells were then washed with distilled water for 10 min at 4°C three times and dehydrated with a graded series of ethanol solutions (25, 60, 80, 99, and 100%), using 2 hours for the first four steps at 4°C and 20 min for the last step at room temperature three times. Subsequently, cells were incubated in mixtures of ethanol and propylene oxide at 2:1 and 1:2 at room temperature for 20 min each; cells were then incubated in mixtures of propylene oxide and Spurr resin at 3:1, 1:1, and 1:3 at room temperature for 1 hour each, followed by incubation in Spurr resin for 2 hours twice and then overnight. Polymerization was performed at 60°C for 2 days. Ultrathin sections were cut with a diamond knife, collected onto copper grids, counterstained with 4% (wt/vol) uranyl acetate for 5 min, washed with distilled water, and air dried. Samples were examined in a Hitachi H-7100 TEM at an acceleration voltage of 75 kV.

Determination of MIC. MICs were determined by using A medium plates containing 4% glucose and 1.5% agarose. Inocula were prepared in A medium containing 4% glucose and standardized to yield ca. 10⁶ CFU ml^–1. The MIC was determined as the first dilution at which no colony formation could be detected visually.

Purification of C. glutamicum cell walls. C. glutamicum cell walls were prepared essentially as described elsewhere (5). C. glutamicum R was grown in 1 liter of liquid A medium containing 4% glucose until the stationary phase. Cells were harvested by centrifugation (5,000 x g, 10 min, 4°C), and then 0.5 g of wet cells was resuspended in breaking buffer (2% [wt/vol] Triton X-100 in PBS) and sonicated for 5 min at 4°C (using 30 5-s cycles of sonication and cooling). After sonication, the suspension was centrifuged (10,000 x g, 10 min, 4°C), and the pellet was resuspended in breaking buffer again. After incubation at room temperature overnight, cells were centrifuged (10,000 x g, 10 min, 4°C). The pellet was resuspended in 2% sodium dodecyl sulfate (SDS) and incubated at 95°C for 1 hour twice. The suspension was centrifuged (10,000 x g, 10 min, 4°C), and the pellet was washed with distilled water, 80% acetone, and 100% acetone. The pellet was dried for 30 min in a Speed-Vac and pounded in a mortar. The cell wall was resuspended in distilled water. The suspension was stored at 4°C.

Zymogram assay. Purified His₆-CgR_1596 and His₆-CgR_2070 were subjected to SDS-polyacrylamide gel electrophoresis analysis with gels containing 0.1% (wt/vol) purified C. glutamicum cell walls as a substrate. SDS-polyacrylamide gels were run at 15 mA on ice. Following electrophoresis, gels were rinsed in distilled water, transferred to 300 ml of renaturation solution (25 mM Tris-HCl [pH 7.2], 1% [vol/vol] Triton X-100), and incubated at 37°C for 16 h with gentle shaking. The gel were rinsed with distilled water, stained with 0.01% (wt/vol) methylene blue in 0.01% (wt/vol) KOH for 3 h, and destained with distilled water. Lysozyme and BSA were used as positive and negative controls, respectively. For native assay to detect cell wall hydrolase, fusion proteins were spotted onto an agar plate containing 0.1% (wt/vol) purified C. glutamicum cell walls, and the plate was incubated at 37°C for 3 h. Staining and destaining procedures were the same as described above.

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Disruption of cgR_1596 causes cell elongation and "bamboo" shape. We obtained random genome deletion mutant in previous work (43). In this study, one of these mutants, RD41, with the 10 genes cgR_1595 through cgR_1604 deleted, was examined. RD41 showed cell elongation, and several lines appeared on the cell surface (bamboo shape) (Fig. 1). Of the 10 genes, only cgR_1596 was implicated in the morphological changes, as only the complementation of cgR_1596 in RD41 suppressed the morphological changes (Fig. 1). Additionally, a cgR_1596 single disruptant created by transposon mutagenesis of the WT strain showed morphological changes similar to those of RD41, with cells elongated about threefold compared to WT cells and several lines, thought to be cell septa, observed on the cell surface under the microscope (Fig. 1).

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FIG. 1. cgR_1596 is responsible for the morphological change of the random genome deletion strain RD41. DIC images of WT, RD41, RD41/pcgR_1596, cgR_1596 (cgR_1596::Tn5) and cgR_1596/pcg1735 cells are shown. The cells were cultivated to mid-log phase in A medium (containing 4% glucose). Bars, 1 µm.

CgR_1596 has 611 amino acid residues in total, with a 49-amino-acid signal peptide at the amino terminus as predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/) (29). There is also an NlpC/P60 domain at the carboxyl terminus (Fig. 2A). This domain is the catalytic domain of cell wall hydrolases such as the LytE, LytF, and YojL in B. subtilis and P60 in Listeria monocytogenes (11, 23, 48) (Fig. 2B). The NlpC/P60 domain of CgR_1596 shows sequence similarity to the corresponding domains of B. subtilis LytE (26% amino acid identity), LytF (33%), YojL (34%), and YvcE (33%) and L. monocytogenes P60 (42%), with conserved cysteine and histidine residues. On the other hand, the amino terminus of the CgR_1596 protein shows 24% amino acid identity to that of Streptococcus agalactiae PscB, which has been reported as an essential protein for cell separation and proper growth (36). An orthologue of CgR_1596, Cg1735 (98% amino acid identity), exists in the genome of C. glutamicum ATCC 13032. Overexpression of cg1735 suppressed the morphological defect of the cgR_1596 disruptant (Fig. 1).

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FIG. 2. (A) Schematic representation of all four predicted NlpC/P60 proteins in C. glutamicum R. The total number of amino acid residues encoded by each gene is shown under the gene name. Numbers under the pictures represent the amino acid numbers of each motif. aa, amino acid; SP, predicted signal peptide region; NlpC/P60, predicted NlpC/P60 domain. (B) Multiple-sequence alignment of the NlpC/P60 proteins in C. glutamicum and B. subtilis. Multiple-sequence alignments of the NlpC/P60 proteins were constructed using ClustalW at the DDBJ website. The catalytic and conserved residues are highlighted in black and gray, respectively. The numbers within the alignment are nonconserved inserts that are not shown. The rightmost numbers show the lengths of proteins. Accession numbers of proteins are as follows: CgR_1596, BAF54588; CgR_2070, BAF55069; CgR_0802, BAF53774; CgR_2069, BAF55068; LytE, BG11406; LytF, BG13010; YojL, BG13564; YvcE, BG11023; P60, CAC98661.

To investigate how cgR_1596 disruptant cells separate, a time-lapse analysis of the cells on an agarose pad containing rich medium was carried out under a DIC microscope and was compared with that for the WT. The WT cell snapped and divided within 1 second from the first to the second picture in Fig. 3, resulting in V-shaped cells (Fig. 3; see Movie S1 in the supplemental material) (20). The cgR_1596 disruptant, despite having the morphological changes, still could separate into progeny cells, resulting in V-shaped cells like those of the WT (Fig. 3; see Movie S2 in the supplemental material).

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FIG. 3. Time-lapse analysis of WT and cgR_1596 single disruptant showing snapping division. Predivisional cells rapidly divided into two daughter cells. cgR_1596, cgR_1596::Tn5.

Simultaneous disruption of cgR_1596 and cgR_2070 delays cell growth and causes more cell elongation than is caused by single cgR_1596 disruption. In addition to cgR_1596, C. glutamicum R has three other genes, cgR_0802, cgR_2069, and cgR_2070, which have an NlpC/P60 domain at the carboxyl terminus. The products of all the genes except CgR_0802 have predicted signal peptides at their amino termini in addition to the catalytic residues of the NlpC/P60 domain (Fig. 2A). Four genes encoding NlpC/P60 proteins are also found in the genome of C. glutamicum ATCC 13032, i.e., Cg1735 (corresponding to CgR_1596), Cg0784 (99% amino acid identity to CgR_0802), Cg2401 (99% amino acid identity to CgR_2069), and Cg2402 (98% amino acid identity to CgR_2070).

The amino-terminal regions of these proteins are not conserved and vary in length. The NlpC/P60 domain of CgR_1596 has amino acid identities of 26%, 25%, and 27%, respectively, to those of CgR_0802, CgR_2069, and CgR_2070. However, individual disruption of any of these genes did not result in any of the morphological changes seen in the cgR_1596 disruptant (data not shown). In B. subtilis, multiple-gene disruption mutants of cell wall hydrolases exhibited longer cells than single-gene disruptants (31). Therefore, it was expected that multiple disruption of C. glutamicum putative cell wall hydrolases in concert with cgR_1596 would result in more elongated cells than for the cgR_1596 single disruptant. Indeed, a cgR_2070-cgR_1596 double disruptant produced longer cells than the cgR_1596 single disruptant (see Fig. S1 in the supplemental material), and cell membrane staining with FM4-64 revealed that multiple septa were formed in both cgR_1596 single and cgR_1596-cgR_2070 double disruption mutant cells (Fig. 4). Double staining of the chromosome and cell membrane with DAPI and FM4-64 showed that the mutants did not create anucleate cells (Fig. 4). To check how many septa were contained in an individual mutant cell, cells at an OD₆₁₀ of 2.0 were stained with FM4-64, and the number of septa in 500 cells each of the WT, cgR_1596 single disruptant (cgR_1596), cgR_2070 single disruptant (cgR_2070), cgR_1596-cgR_2070 double disruptant (cgR_1596-cgR_2070), cgR_1596-cgR_2070 double disruptant with plasmid pCRB608 containing cgR_1596 (cgR_1596-cgR_2070/pcgR_1596), and cgR_1596-cgR_2070 double disruptant with plasmid pCRB609 containing cgR_2070 (cgR_1596-cgR_2070/pcgR_2070) were counted. In the WT and cgR_2070, about 90% of the cells had 0 or 1 septum per cell, while over 50% of cgR_1596-cgR_2070 cells and over 30% of cgR_1596 cells had multiple septa per cell (Fig. 5A). The multiple-septum formation of cgR_1596-cgR_2070 was suppressed by introduction of plasmid pCRB608 carrying cgR_1596 but was not suppressed by pCRB609 carrying cgR_2070. The multiple septa observed per cell for cgR_1596 and the cgR_1596-cgR_2070 implied that cell separation was delayed compared to cell elongation and septum formation.

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FIG. 4. FM4-64 and DAPI staining of WT, cgR_1596 single-disruptant, cgR_2070 single-disruptant, and cgR_1596-cgR_2070 double-disruptant cells. DIC and corresponding fluorescence images are shown. The cells were cultivated to mid-log phase in A medium (containing 4% glucose). Bars, 1 µm. cgR_1596, cgR_1596::Tn5; cgR_2070, cgR_2070::Tn5; cgR_1596-cgR_2070, cgR_1596::Tn5 cgR_2070.

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FIG. 5. cgR_1596 single and cgR_1596-cgR_2070 double disruptants show multiseptum formation and growth delay. (A) Number of septum per individual WT or mutant cell. A total of 500 cells in exponential growth, stained with FM4-64, were counted. The average numbers of septa per cell were 0.39 for the WT, 1.39 for cgR_1596, 0.36 for cgR_2070, 2.42 for cgR_1596-cgR_2070, 0.26 for cgR_1596-cgR_2070/pcgR_1596, and 1.74 for cgR_1596-cgR_2070/pcgR_2070. (B) Growth of WT and mutant cells. OD610s for WT, cgR_1596, cgR_2070, cgR_1596-cgR_2070, cgR_1596-cgR_2070/pcgR_1596, and cgR_1596-cgR_2070/pcgR_2070 cells are shown. The doubling times are 80 min for WT, 91 min for cgR_1596, 85 min for cgR_2070, 164 min for cgR_1596-cgR_2070, 93 min for cgR_1596-cgR_2070/pcgR_1596, and 118 min for cgR_1596-cgR_2070/pcgR_2070. The graph shows the mean values with standard deviations obtained from three independent experiments. cgR_1596, cgR_1596::Tn5; cgR_2070, cgR_2070::Tn5; cgR_1596-cgR_2070, cgR_1596::Tn5 cgR_2070; cgR_1596-cgR_2070/pcgR_1596, cgR_1596::Tn5 cgR_2070/pCRB608; cgR_1596-cgR_2070/pcgR_2070, cgR_1596::Tn5 cgR_2070/pCRB609.

The effect of cgR_1596 and/or cgR_2070 disruption on cell growth was examined (Fig. 5B). The growth rate of the single-gene disruptants, cgR_1596 and cgR_2070, was slightly lower (about 1.1-fold) than that of the WT. On the other hand, the growth rate of the cgR_1596-cgR_2070 double disruptant was twofold lower than that of the WT. This growth delay was suppressed by introduction of plasmid pCRB608 carrying cgR_1596 and was partially suppressed by pCRB609 carrying cgR_2070. These results show that cgR_1596 and cgR_2070 are involved not only in cell separation but also in cell growth. Complementation of only cgR_1596 eliminated the multiple-septum formation and cell growth delay of cgR_1596-cgR_2070 (Fig. 5A and B), while introducing cgR_2070 into cgR_1596-cgR_2070 led to the cgR_1596 single-disruptant phenotype. These results suggest that CgR_1596 could complement the cell separation and cell growth activity of CgR_2070, though CgR_2070 could not complement the activity of CgR_1596. Successive disruption of cgR_0802 and/or cgR_2069 along with cgR_1596 and cgR_2070 did not lead to any more pronounced morphological changes than those observed with the cgR_1596-cgR_2070 double disruptant (data not shown). These results indicate that both CgR_1596 alone and CgR_2070 in concerted action with CgR_1596 are involved in cell separation and cell growth.

Signal peptides of the CgR_1596 and CgR_2070 proteins are functional for extracellular secretion. According to the SignalP program (29), CgR_1596 and CgR_2070 contain putative signal peptides of 49 and 35 amino acids, respectively, suggesting that they are secretory proteins. To confirm this, each of the signal peptides was fused to an -amylase gene from Geobacillus stearothermophilus on plasmid pCRC900 (J. Watanabe et al., unpublished data) under the control of lac promoter. The resulting vectors, pCRB600 and pCRB601, were transformed into C. glutamicum R. Following growth, the transformants were transferred onto a starch-containing solid medium, allowing detection of extracellular amylase activity by visualization of a clear halo after addition of iodine solution, indicating that the signal peptides were functional for secretion (Fig. 6A).

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FIG. 6. (A) The signal peptides of CgR_1596 and CgR_2070 are functional for secretion in C. glutamicum R. Promoterless -amylase from Geobacillus stearothermophilus was used to check secretion activity of the signal peptides (SPs). Amylase activity was detected by the iodo-starch reaction. The plate was incubated overnight. SP less, WT/pCRC900; CgR_1596SP, WT/pCRB600; CgR_2070SP, WT/pCRB601. (B) Localization of CgR_1596 protein in WT cells. The cells were cultivated to mid-log phase (OD610 = 2.0). The exposure times were 0.1 s for phase-contrast microscopy and 10 s for anti-CgR_1596 immunofluorescence microscopy. Bars, 1 µm.

CgR_1596 protein localizes at the center of the cell. In order to visualize the localization of CgR_1596 and CgR_2070, fusion proteins with green fluorescent protein at carboxyl termini of their respective genes were constructed. However, introduction of fusion genes into the cgR_1596 and cgR_1596-cgR_2070 mutants did not eliminate the mutants' morphological changes, indicating that the fusion proteins either were not functional or could not pass through the cytoplasmic membrane. To overcome this, anti-CgR_1596 antibody was obtained. Unfortunately, anti-CgR_2070 antibody could not be produced successfully in this study. In place of anti-CgR_2070 antibody, we used anti-Myc antibody for a Myc tag fusion protein (CgR_2070-Myc). CgR_2070-Myc eliminated the morphological changes, proving CgR_2070-Myc to be functional (data not shown). The localization of the proteins was determined by immunofluorescence microscopy with anti-CgR_1596 antibody and anti-Myc antibody. As a result, CgR_1596 was shown to be localized mainly at the cell septum and slightly at the cell poles in WT cells (Fig. 6B; see Fig. S2 in the supplemental material). The specific fluorescent signal was not observed in the cgR_1596 single disruptant (cgR_1596) cells but was observed in the disruptant with a plasmid carrying the cgR_1596 gene (cgR_1596/pcgR_1596) cells (see Fig. S3 in the supplemental material). In contrast, the specific fluorescent signal of CgR_2070-Myc could not be detected under the experimental conditions used (data not shown).

Separation is delayed in cells lacking cgR_1596 and cgR_2070. To confirm whether cgR_1596 and cgR_1596-cgR_2070 are impaired in septum formation or cell separation, fDHPE and Nile Red staining were tested. It was previously reported that fDHPE stains a mycolate layer and Nile Red stains cytoplasmic membrane in C. glutamicum (19). Mycolate layer formation is known to occur after cytoplasmic membrane and peptidoglycan layer formation (19). Both Nile Red and fDHPE stained the septum in both cgR_1596 and cgR_1596-cgR_2070 (Fig. 7). These results suggested that formation of the mycolate layer was completed at the multiple septa in a mutant cell, indicating that cell elongation was caused by a defect in cell separation but not in septum formation.

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FIG. 7. Nile Red and fDHPE staining of WT, cgR_1596 single-disruptant, cgR_2070 single-disruptant, and cgR_1596-cgR_2070 double-disruptant cells. DIC, Nile Red-stained, and fDHPE-stained images are shown. The cells were cultivated to mid-log phase in A medium (containing 4% glucose). Bars, 1 µm. cgR_1596, cgR_1596::Tn5; cgR_2070, cgR_2070::Tn5; cgR_1596-cgR_2070, cgR_1596::Tn5 cgR_2070.

To investigate the structure of the cell septa of the WT and the mutants in detail, TEM analysis was performed. Two daughter cells were still cross-linked with an outer layer of the cell wall even after septum formation was complete (Fig. 8A). After cell separation, one side of the junction point was still cross-linked, although the other side was separated and scars were visible (Fig. 8A). In cgR_1596 and cgR_1596-cgR_2070, multiple septa were observed within individual cells, clearly showing a cell separation defect in the mutant cells (Fig. 8B). Larger numbers of cell septa than in the cgR_1596 cells were observed in the cgR_1596-cgR_2070 cells by TEM analysis (Fig. 8B). In the cgR_1596 and the cgR_1596-cgR_2070 cells, two daughter cells were still cross-linked with an outer layer of the cell wall, even though the daughter cells had started cell extension and cell poles had become round (Fig. 8C). These results indicate that both cgR_1596 and cgR_2070, in concerted action with cgR_1596, were necessary for cell separation in C. glutamicum R.

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FIG. 8. (A) TEM analysis of cell separation in a WT strain. The black arrow shows the point of cross-linking with the cell wall. White arrows show scars caused by snapping cell division. (B) Multiple septa were observed in the cgR_1596 single disruptant and the cgR_1596-cgR_2070 double disruptant. (C) Curved septa of the cgR_1596 single disruptant and the cgR_1596-cgR_2070 double disruptant. cgR_1596, cgR_1596::Tn5; cgR_1596-cgR_2070, cgR_1596::Tn5 cgR_2070.

Antibiotic susceptibility upon disruption of cgR_1596 and/or cgR_2070. Cell wall hydrolases are postulated to have widespread roles in the dynamics of the bacterial cell wall. Defects in cell wall biosynthesis and turnover possibly alter susceptibility to antibiotics. We examined the antibiotic susceptibility of the C. glutamicum R WT, cgR_1596, cgR_2070, cgR_1596-cgR_2070, cgR_1596-cgR_2070/pcgR_1596, and cgR_1596-cgR_2070/pcgR_2070 strains. The antibiotics cephalexin, amdinocillin (mecillinam), cefsulodin, rifampin, and tetracycline were chosen for this analysis. cgR_1596-cgR_2070 showed about threefold higher sensitivity to all the β-lactam antibiotics tested in this study than the WT (Table 3). cgR_2070 showed greater sensitivity to cephalexin, an inhibitor of FtsI/PBP3, and to amdinocillin, an inhibitor of PBP2a/2b (44), than the WT. On the other hand, cgR_1596 showed greater sensitivity to cefsulodin, an inhibitor of PBP1a/PBP1b (44), than the WT. The sensitivity of cgR_1596-cgR_2070 to cephalexin was suppressed by pCRB609 containing cgR_2070 (Table 3). In the cases of mecillinam and cefsulodin, only partial suppression was observed in cgR_1596-cgR_2070/pcgR_1596 or cgR_1596-cgR_2070/pcgR_2070. cgR_1596-cgR_2070 and other strains showed no difference in sensitivity to rifampin, which is known to inhibit the bacterial RNA polymerase beta subunit, or to tetracycline, which is known to inhibit the bacterial 30S ribosomal subunit. These results show that the sensitivity alteration is specific to β-lactam antibiotics in these mutants.

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TABLE 3. MICs of various antibiotics for growth

Test of purified CgR_1596 and CgR_2070 proteins for cell wall hydrolase activity in vitro. To determine whether CgR_1596 and CgR_2070 represent cell wall hydrolases of C. glutamicum R in vitro, purified His₆-CgR_1596 with or without the putative signal peptides and His₆-CgR_2070 with the putative signal peptides were tested in a Zymogram assay using purified cell walls of the cgR_1596 and the cgR_1596-cgR_2070 mutants as substrates. The purified proteins showed no lytic activity (data not shown). To account for the denaturing conditions of SDS-polyacrylamide gel electrophoresis, the purified proteins were tested for autolytic activity using C. glutamicum cell walls in a native assay. Here again, the fusion proteins were not able to hydrolyze the C. glutamicum cell walls (data not shown). No in vitro cell wall hydrolase activity was detectable using the Zymogram assay in this study.

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In this study, of four genes encoding putative cell wall hydrolases containing the NlpC/P60 domain, cgR_1596 and cgR_2070 were shown to be putatively involved in cell separation in C. glutamicum R. NlpC/P60 proteins are widely represented in various bacteria and act in peptidoglycan degradation for proper cell separation and cell growth (2). cgR_1596 showed defects in cell separation, i.e., cell elongation with multiple septa, whereas cgR_2070 showed no obvious morphological changes. The cell separation defect of a cgR_1596-cgR_2070 double mutant was significantly more severe than that of cgR_1596. Full elimination of the defect of cgR_1596-cgR_2070 was observed upon complementation with only cgR_1596, while introducing cgR_2070 into the double disruptant resulted in the cgR_1596 phenotype. fDHPE staining and TEM analysis indicated that septum formation is completed in these mutants. These results suggest that CgR_1596 and CgR_2070 are responsible for cell separation. CgR_1596 plays a greater role in cell separation than CgR_2070. Localization of CgR_1596 at the cell septum supported the notion that the CgR_1596 is involved in septum separation for cell separation.

CgR_1596 and CgR_2070 were predicted to have an NlpC/P60 domain at the carboxyl terminus. The domain has been reported to be a catalytic domain of cell wall hydrolases, i.e., B. subtilis LytEF and L. monocytogenes P60, involved in cell separation (33, 48). The amino-terminal regions of CgR_1596 and CgR_2070 do not have any known peptidoglycan binding domain, such as LysM, which LytEF and P60 do have. Instead, the amino-terminal region of CgR_1596 has similarity to that of S. agalactiae PcsB, a putative cell wall hydrolase involved in cell separation. The carboxyl-terminal region of PcsB showed no similarity to that of CgR_1596. In this study, we were not able to detect hydrolase activity of the purified CgR_1596 protein using a Zymogram assay. It should be noted that hydrolase activity of PcsB also is not detected in vitro (36). Thus, the amino-terminal region may contain a unique domain for specific interaction with cell division proteins for cell wall degradation that is involved in their specific aspects of cell division and/or substrate recognition. The reason why we were unable to detect enzymatic activity of purified CgR_1596 may be the requirement for other factors such as coenzymes or scaffolding proteins for cell wall degradation, although it cannot be excluded that the protein was inactivated during purification or renaturing. Both CgR_1596 and CgR_2070 have signal peptides, which were confirmed to function in secretion in this study. However, only CgR_1596 localization was detected using immunofluorescence microscopy. CgR_2070 may not be able to interact with the cell wall rigidly, and consequently we were unable to detect its localization and enzymatic activity. Further study will be needed to elucidate the mechanism of action of putative cell wall hydrolases in cell separation.

Different types of cell wall hydrolases are coordinately involved in cell wall turnover and contribute to cell wall maintenance. In E. coli and B. subtilis, mutants with defective lytic enzymes, for instance, amiABC or lytC disruptants, show antibiotic resistance (6, 12). In contrast, an S. agalactiae pcsB mutant shows more sensitivity to β-lactam antibiotics than WT strains (36). It is possible that the NlpC/P60 proteins in C. glutamicum are involved not only in cell separation but also in other aspects of cell growth. Indeed, simultaneous disruption of C. glutamicum cgR_1596 and cgR_2070 caused greater sensitivity to β-lactam antibiotics than that of the WT strain. It is noteworthy that the contribution of CgR_2070 to cephalexin resistance is larger than that of CgR_1596, which is in contrast with the finding that CgR_2070 plays a minor role in cell separation. Sensitivity was altered in cgR_2070 and cgR_1596 and depended on the different types of β-lactam antibiotics, which inhibit different class of penicillin binding proteins (PBPs). Interestingly, it has been reported that C. glutamicum PBP deletion mutants show higher sensitivity to β-lactam antibiotics than their parent strain. Moreover, a pbp2 disruptant shows a phenotype similar to that of cgR_1596 (44). CgR_1596 and CgR_2070 may be involved in peptidoglycan maintenance with high-molecular-weight PBPs. These results suggest that both CgR_1596 and CgR_2070 are involved in cell wall maintenance, though their relative weights appear to be different. Although the mechanism of alteration of susceptibility to antibiotics is unclear, effects of putative cell wall hydrolase inactivation may be altered depending on the topological organization of cell wall biosynthesis, which is different in different bacterial species.

As observed in Fig. 8, C. glutamicum had a pair of daughter cells that were sealed off from each other by a cell wall but still connected at the septum by a layer of outer cell wall material after septum formation was completed (Fig. 8A). A member of the Actinomycetales, Arthrobacter crystallopoietes, also has a similar two-layer cell wall which joins two daughter cells after septum formation (18). C. glutamicum cell separation occurs instantly via the unusual process called snapping division, which results in formation of V-shaped cells (Fig. 3) (20). After cell separation, scars seemed to remain at the junction of two daughter cells caused by snapping division (Fig. 8A). On the other hand, cell separation of the daughter cells by cleavage of the central part of the cell wall can occur gradually at almost the same time as septum formation in E. coli and B. subtilis (3, 12, 22, 27). There is another interesting feature of cell division in C, glutamicum. The cells synthesize new peptidoglycan at the cell poles (polar growth), unlike E. coli and B. subtilis, which synthesize new peptidoglycan at the lateral cell wall (side wall growth) (8, 10). These features of cell division in C. glutamicum are likely to affect the phenotype of mutants with cell separation defects, which is different from the chained cells observed for mutants deficient in cell wall hydrolase genes in E. coli and B. subtilis. In cgR_1596 and cgR_1596-cgR_2070, the septum in single cells of individual compartments was frequently curved outward, indicating that the curved septum is a consequence of cell elongation (Fig. 8BC). The cell separation event is achieved within 1 second (see Movie S1 in the supplemental material). Before the dynamic cell separation, a precursor "earthquake slip," i.e., slight movement of the cell toward the opposite direction, was observed (see Movie S1 in the supplemental material). Considering these observations, it is speculated that the two daughter cells press against each other due to the cell elongation. Therefore, the snapping division is likely to be accomplished by hydrolysis of the outer layer cross-linked point, involved in CgR_1596 and CgR_2070, and by the pressure of the daughter cells.

 
We thank Ian Smith, Crispinus Omumasaba, and Roy H. Doi for critical reading of the manuscript. We are grateful to Megumi Iwano for her support with electron microscopy analysis.

This work was supported by grants from the New Energy and Industrial Technology Development Organization (NEDO). Y.T. is a JSPS research fellow.

 
* Corresponding author. Mailing address: Research Institute of Innovative Technology for the Earth, 9-2 Kizugawadai, Kizugawa, Kyoto 619-0292, Japan. Phone: (81) 774 75 2308. Fax: (81) 774 75 2321. E-mail: mmg-lab{at}rite.or.jp

Published ahead of print on 17 October 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520.

These authors contributed equally to this work.

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Journal of Bacteriology, December 2008, p. 8204-8214, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00752-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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