Asymmetric diversification of mating pheromones in fission yeast.

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4-8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation-perhaps as a first step toward prezygotic isolation in S. pombe.

Division-site localization of RodZ is required for efficient Z ring formation in Escherichia coli.

Bacteria such as Escherichia coli must coordinate cell elongation and cell division. Elongation is regulated by an elongasome complex containing MreB actin and the transmembrane protein RodZ, which regulates assembly of MreB, whereas division is regulated by a divisome complex containing FtsZ tubulin. These complexes were previously thought to function separately. However, MreB has been shown to directly interact with FtsZ to switch to cell division from cell elongation, indicating that these complexes collaborate to regulate both processes. Here, we investigated the role of RodZ in the regulation of cell division. RodZ localized to the division site in an FtsZ-dependent manner. We also found that division-site localization of MreB was dependent on RodZ. Formation of a Z ring was delayed by deletion of rodZ, suggesting that division-site localization of RodZ facilitated the formation or stabilization of the Z ring during early cell division. Thus, RodZ functions to regulate MreB assembly during cell elongation and facilitates the formation of the Z ring during cell division in E. coli.

Exonuclease III (XthA) Enforces In Vivo DNA Cloning of Escherichia coli To Create Cohesive Ends.

Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinases but is dependent on XthA, a 3' to 5' exonuclease. Here, in vivo cloning of E. coli by XthA is referred to as in vivoE. coli cloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3' ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5' overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.IMPORTANCE Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, an in vitro recombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly, E. coli potentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for this in vivo cloning have realized a high level of usability, comparable to that by in vitro recombination reactions. However, the mechanism of in vivo cloning is highly controversial. Here, we clarified the fundamental mechanism underlying in vivo cloning and also constructed a strain that was optimized for in vivo cloning. Additionally, we streamlined the procedure of in vivo cloning by using a single microcentrifuge tube.

The periplasmic disordered domain of RodZ promotes its self-interaction in Escherichia coli.

Rod shape of bacterial cells such as Escherichia coli is mainly regulated by a supramolecular complex called elongasome including MreB actin. Deletion of the mreB gene in rod-shaped bacterium E. coli results in round-shaped cells. RodZ was isolated as a determinant of rod shape in E. coli, Caulobacter crescentus and Bacillus subtilis and it has been shown to be an interaction partner and a regulator of assembly of MreB through its cytoplasmic domain. As opposed to functions of the N-terminal cytoplasmic domain of RodZ, functions of the C-terminal periplasmic domain including a disordered region are still unclear. To understand it, we adopted an in vivo photo-cross-linking assay to analyze interaction partners to identify proteins which interact with RodZ via its periplasmic domain, finding that the RodZ self-interacts in the periplasmic disordered domain. Self-interaction of RodZ was affected by MreB actin. Deletion of this region resulted in aberrant cell shape. Our results suggest that MreB binding to the cytoplasmic domain of RodZ causes structural changes in the disordered periplasmic domain of RodZ. We also found that the disordered domain of RodZ contributes to fine-tune rod shape in E. coli.

Exclusion of assembled MreB by anionic phospholipids at cell poles confers cell polarity for bidirectional growth.

Cell polarity determines the direction of cell growth in bacteria. MreB actin spatially regulates peptidoglycan synthesis to enable cells to elongate bidirectionally. MreB densely localizes in the cylindrical part of the rod cell and not in polar regions in Escherichia coli. When treated with A22, which inhibits MreB polymerization, rod-shaped cells became round and MreB was diffusely distributed throughout the cytoplasmic membrane. A22 removal resulted in restoration of the rod shape. Initially, diffuse MreB started to re-assemble, and MreB-free zones were subsequently observed in the cytoplasmic membrane. These MreB-free zones finally became cell poles, allowing the cells to elongate bidirectionally. When MreB was artificially located at the cell poles, an additional pole was created, indicating that artificial localization of MreB at the cell pole induced local peptidoglycan synthesis. It was found that the anionic phospholipids (aPLs), phosphatidylglycerol and cardiolipin, which were enriched in cell poles preferentially interact with monomeric MreB compared with assembled MreB in vitro. MreB tended to localize to cell poles in cells lacking both aPLs, resulting in production of Y-shaped cells. Their findings indicated that aPLs exclude assembled MreB from cell poles to establish cell polarity, thereby allowing cells to elongate in a particular direction.

The Ras1-Cdc42 pathway is involved in hyphal development of Schizosaccharomyces japonicus.

Dimorphic yeasts transform into filamentous cells or hyphae in response to environmental cues. The mechanisms for the hyphal transition of dimorphic yeasts have mainly been studied in Candida albicans, an opportunistic human fungal pathogen. The Ras1-MAPK pathway is a major signal transduction pathway for hyphal transition in C. albicans. Recently, the non-pathogenic dimorphic yeast Schizosaccharomyces japonicus has also been used for genetic analyses of hyphal induction. We confirmed that Ras1-MAPK and other MAPK pathways exist in Sz. japonicus. To examine how hyphal transition is induced by environmental stress-triggered signal transduction, we studied the hyphal transition of deletion mutants of MAPK pathways in Sz. japonicus. We found that the MAPK pathways are not involved in hyphal induction, although the mating response is dependent on these pathways. However, only Ras1 deletion caused a severe defect in hyphal development via both DNA damage and environmental stressors. In fact, genes on the Cdc42 branch of the Ras1 (Ras1-Cdc42) pathway, efc25Sj, scd1Sj and scd2Sj, are required for hyphal development. Cell morphology analysis indicated that the apical growth of hyphal cells was inhibited in Ras1-Cdc42-pathway deletion mutants. Thus, the control of cell polarity by the Ras1-Cdc42 pathway is crucial for hyphal development.

DDK phosphorylates checkpoint clamp component Rad9 and promotes its release from damaged chromatin.

When inappropriate DNA structures arise, they are sensed by DNA structure-dependent checkpoint pathways and subsequently repaired. Recruitment of checkpoint proteins to such structures precedes recruitment of proteins involved in DNA metabolism. Thus, checkpoints can regulate DNA metabolism. We show that fission yeast Rad9, a 9-1-1 heterotrimeric checkpoint-clamp component, is phosphorylated by Hsk1(Cdc7), the Schizosaccharomyces pombe Dbf4-dependent kinase (DDK) homolog, in response to replication-induced DNA damage. Phosphorylation of Rad9 disrupts its interaction with replication protein A (RPA) and is dependent on 9-1-1 chromatin loading, the Rad9-associated protein Rad4/Cut5(TopBP1), and prior phosphorylation by Rad3(ATR). rad9 mutants defective in DDK phosphorylation show wild-type checkpoint responses but abnormal DNA repair protein foci and decreased viability after replication stress. We propose that Rad9 phosphorylation by DDK releases Rad9 from DNA damage sites to facilitate DNA repair.

Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library.

BACKGROUND: The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features. RESULTS: Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics. CONCLUSIONS: Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD-PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan).

The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of ß-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked ß-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.

Mating response and construction of heterothallic strains of the fission yeast Schizosaccharomyces octosporus.

The fission yeast Schizosaccharomyces octosporus is one of four species in the genus Schizosaccharomyces. Recently released genome sequence data provide useful information for comparative studies. However, Sz. octosporus has not yet been genetically characterized because there have been no heterothallic strains of this species. Here we report the construction of stable heterothallic strains of Sz. octosporus for genetic crosses. First, we continuously observed the mating process of a homothallic strain, yFS286, and determined the mating frequency of Sz. octosporus on various sporulation media. It showed, on average, 30% zygote formation on mating, and a higher frequency of zygotes (43.8 ± 4.7%) on PMG medium. Regardless of sporulation, the number of spores within an ascus was variable. Schizosaccharomyces octosporus forms eight-spored asci, but preferentially produced four-spored asci on MEA or YMoA medium. To obtain heterothallic strains essential for genetic analyses, we isolated spontaneous mutants showing heterothallic-like phenotypes. We also constructed stable heterothallic strains by deleting the silent mat region. As a result, we established the following heterothallic strains, TS162 as h+ and TS150/TS161 as h-, which successfully mated with each other. These genetic tools will be useful for yeast genetics such as molecular cloning, gene complementation tests and tetrad (octad) analysis.

Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

Aurantimicrobium minutum gen. nov., sp. nov., a novel ultramicrobacterium of the family Microbacteriaceae, isolated from river water.

A Gram-stain-positive, aerobic, non-motile, curved (selenoid), rod-shaped actinobacterium, designated KNCT, was isolated from the 0.2 µm-filtrate of river water in western Japan. Cells of strain KNCT were ultramicrosized (0.04-0.05 µm3). The strain grew at 15-37 °C, with no observable growth at 10 °C or 40 °C. The pH range for growth was 7-9, with weaker growth at pH 10. Growth was impeded by the presence of NaCl at concentrations greater than 1 %. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KNCT showed relatively high sequence similarity (97.2 %) to Alpinimonas psychrophila Cr8-25T in the family Microbacteriaceae. However, strain KNCT formed an independent cluster with cultured, but as-yet-unidentified, species and environmental clones on the phylogenetic tree. The major cellular fatty acids were anteiso-C15 : 0 (41.0 %), iso-C16 : 0 (21.8 %), C16 : 0 (18.0 %) and anteiso-C17 : 0 (12.9 %), and the major menaquinones were MK-11 (71.3 %) and MK-12 (13.6 %). The major polar lipids were phosphatidylglycerol and two unknown glycolipids. The cell-wall muramic acid acyl type was acetyl. The peptidoglycan was B-type, and contained 3-hydroxyglutamic acid, glutamic acid, aspartic acid, glycine, alanine and lysine, with the latter being the diagnostic diamino acid. The G+C content of the genome was unusually low for actinobacteria (52.1 mol%), compared with other genera in the family Microbacteriaceae. Based on the phenotypic characteristics and phylogenetic evidence, strain KNCT represents a novel species of a new genus within the family Microbacteriaceae, for which the name Aurantimicrobium minutum gen. nov., sp. nov. is proposed. The type strain of the type species is KNCT ( = NBRC 105389T = NCIMB 14875T).

RecA protein recruits structural maintenance of chromosomes (SMC)-like RecN protein to DNA double-strand breaks.

Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recA(Q300R), is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recA(Q300R) fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecN(K35A) binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.

Mutations in cell elongation genes mreB, mrdA and mrdB suppress the shape defect of RodZ-deficient cells.

RodZ interacts with MreB and both factors are required to maintain the rod shape of Escherichia coli. The assembly of MreB into filaments regulates the subcellular arrangement of a group of enzymes that synthesizes the peptidoglycan (PG) layer. However, it is still unknown how polymerization of MreB determines the rod shape of bacterial cells. Regulatory factor(s) are likely to be involved in controlling the function and dynamics of MreB. We isolated suppressor mutations to partially recover the rod shape in rodZ deletion mutants and found that some of the suppressor mutations occurred in mreB. All of the mreB mutations were in or in the vicinity of domain IA of MreB. Those mreB mutations changed the property of MreB filaments in vivo. In addition, suppressor mutations were found in the periplasmic regions in PBP2 and RodA, encoded by mrdA and mrdB genes. Similar to MreB and RodZ, PBP2 and RodA are pivotal to the cell wall elongation process. Thus, we found that mutations in domain IA of MreB and in the periplasmic domain of PBP2 and RodA can restore growth and rod shape to ΔrodZ cells, possibly by changing the requirements of MreB in the process.

Regulation of nuclear envelope dynamics via APC/C is necessary for the progression of semi-open mitosis in Schizosaccharomyces japonicus.

Three types of mitosis, which are open, closed or semi-open mitosis, function in eukaryotic cells, respectively. The open mitosis involves breakage of the nuclear envelope before nuclear division, whereas the closed mitosis proceeds with an intact nuclear envelope. To understand the mechanism and significance of three types of mitotic division in eukaryotes, we investigated the process of semi-open mitosis, in which the nuclear envelope is only partially broken, in the fission yeast Schizosaccharomyces japonicus. In anaphase-promoting complex/cyclosome (APC/C) mutants of Sz. japonicus, the nuclear envelope remained relatively intact during anaphase, resulting in impaired semi-open mitosis. As a suppressor of apc2 mutant, a mutation of Oar2, which was a 3-oxoacyl-[acyl carrier protein] reductase, was obtained. The level of the Oar2, which had two destruction-box motifs recognized by APC/C, was increased in APC/C mutants. Furthermore, the defective semi-open mitosis observed in an apc2 mutant was restored by mutated oar2+. Based on these findings, we propose that APC/C regulates the dynamics of the nuclear envelope through degradation of Oar2 dependent on APC/C during the metaphase-to-anaphase transition of semi-open mitosis in Sz. japonicus.

Schizosaccharomyces japonicus: the fission yeast is a fusion of yeast and hyphae.

The clade of Schizosaccharomyces includes 4 species: S. pombe, S. octosporus, S. cryophilus, and S. japonicus. Although all 4 species exhibit unicellular growth with a binary fission mode of cell division, S. japonicus alone is dimorphic yeast, which can transit from unicellular yeast to long filamentous hyphae. Recently it was found that the hyphal cells response to light and then synchronously activate cytokinesis of hyphae. In addition to hyphal growth, S. japonicas has many properties that aren't shared with other fission yeast. Mitosis of S. japonicas is referred to as semi-open mitosis because dynamics of nuclear membrane is an intermediate mode between open mitosis and closed mitosis. Novel genetic tools and the whole genomic sequencing of S. japonicas now provide us with an opportunity for revealing unique characters of the dimorphic yeast.

Synchronous activation of cell division by light or temperature stimuli in the dimorphic yeast Schizosaccharomyces japonicus.

Many fungi respond to light and regulate fungal development and behavior. A blue light-activated complex has been identified in Neurospora crassa as the product of the wc-1 and wc-2 genes. Orthologs of WC-1 and WC-2 have hitherto been found only in filamentous fungi and not in yeast, with the exception of the basidiomycete pathogenic yeast Cryptococcus. Here, we report that the fission yeast Schizosaccharomyces japonicus responds to blue light depending on Wcs1 and Wcs2, orthologs of components of the WC complex. Surprisingly, those of ascomycete S. japonicus are more closely related to those of the basidiomycete. S. japonicus reversibly changes from yeast to hyphae in response to environmental stresses. After incubation at 30°C, a colony of yeast was formed, and then hyphal cells extended from the periphery of the colony. When light cycles were applied, distinct dark- and bright-colored hyphal cell stripes were formed because the growing hyphal cells had synchronously activated cytokinesis. In addition, temperature cycles of 30°C for 12 h and 35°C for 12 h or of 25°C for 12 h and 30°C for 12 h during incubation in the dark induced a response in the hyphal cells similar to that of light. The stripe formation of the temperature cycles was independent of the wcs genes. Both light and temperature, which are daily external cues, have the same effect on growing hyphal cells. A dual sensing mechanism of external cues allows organisms to adapt to daily changes of environmental alteration.

The Ty1 retrotransposon harbors a DNA region that performs dual functions as both a gene silencing and chromatin insulator.

In various eukaryotic kingdoms, long terminal repeat (LTR) retrotransposons repress transcription by infiltrating heterochromatin generated within their elements. In contrast, the budding yeast LTR retrotransposon Ty1 does not itself undergo transcriptional repression, although it is capable of repressing the transcription of the inserted genes within it. In this study, we identified a DNA region within Ty1 that exerts its silencing effect via sequence orientation. We identified a DNA region within the Ty1 group-specific antigen (GAG) gene that causes gene silencing, termed GAG silencing (GAGsi), in which the silent chromatin in the GAGsi region is created by euchromatin-specific histone modifications. A characteristic inverted repeat (IR) sequence is present at the 5' end of this region, forming a chromatin boundary between promoter-specific chromatin upstream of the IR sequence and silent chromatin downstream of the IR sequence. In addition, Esc2 and Rad57, which are involved in DNA repair, were required for GAGsi silencing. Finally, the chromatin boundary was required for the transcription of Ty1 itself. Thus, the GAGsi sequence contributes to the creation of a chromatin environment that promotes Ty1 transcription.

Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis.

The number of copies of rRNA (rrn) operons in a bacterial genome differs greatly among bacterial species. Here we examined the phenotypic effects of variations in the number of copies of rRNA genes in the genome of Bacillus subtilis by analysis of eight mutant strains constructed to carry from two to nine copies of the rrn operon. We found that a decrease in the number of copies from ten to one increased the doubling time, and decreased the sporulation frequency and motility. The maximum levels for transformation activity were similar among the strains, although the competence development was significantly delayed in the strain with a single rrn operon. Normal sporulation only occurred if more than four copies of the rrn operon were present, although ten copies were needed for vegetative growth after germination of the spores. This behaviour was seen even though the intracellular level of ribosomes was similar among strains with four to ten copies of the rrn operon. Furthermore, ten copies of the rrn operon were needed for the highest swarming activity. We also constructed 21 strains that carried all possible combinations of two copies of the rrn operons, and found that these showed a range of growth rates and sporulation frequencies that all fell between those recorded for strains with one or three copies of the rrn operon. The results suggested that the copy number of the rrn operon has a major influence on cellular processes such as growth rate and sporulation frequency.

Relationship between the Rod complex and peptidoglycan structure in Escherichia coli.

Peptidoglycan for elongation in Escherichia coli is synthesized by the Rod complex, which includes RodZ. Although various mutant strains of the Rod complex have been isolated, the relationship between the activity of the Rod complex and the overall physical and chemical structures of the peptidoglycan have not been reported. We constructed a RodZ mutant, termed RMR, and analyzed the growth rate, morphology, and other characteristics of cells producing the Rod complexes containing RMR. The growth and morphology of RMR cells were abnormal, and we isolated suppressor mutants from RMR cells. Most of the suppressor mutations were found in components of the Rod complex, suggesting that these suppressor mutations increase the integrity and/or the activity of the Rod complex. We purified peptidoglycan from wild-type, RMR, and suppressor mutant cells and observed their structures in detail. We found that the peptidoglycan purified from RMR cells had many large holes and different compositions of muropeptides from those of WT cells. The Rod complex may be a determinant not only for the whole shape of peptidoglycan but also for its highly dense structure to support the mechanical strength of the cell wall.