Skip to main content
NCBI home page
The Cell Surface logoLink to The Cell Surface
. 2025 Aug 26;14:100151. doi: 10.1016/j.tcsw.2025.100151

Localization characteristics of cell wall synthesis protein Wag31 and penicillin binding protein C in Clavibacter michiganensis

Chengxuan Yu 1, Xiaoli Xu 1, Jia Shi 1, Wenqing Chu 1, Na Jiang 1, Jianqiang Li 1, Laixin Luo 1,⁎
PMCID: PMC12445707  PMID: 40978520

Abstract

CmWag31 is a member of the DivIVA family of proteins in Clavibacter michiganensis. The DivIVA family have been demonstrated to play a key role in the synthesis of cell wall peptidoglycan and cell division in most bacterial species. It has been previously confirmed that the pbpC (penicillin-binding protein C) deletion mutants affect bacterial division and cell wall synthesis. Based on the confirmation of the interaction between CmWag31 and CmPBPC, the present study conducted a systemic analysis on their localization characteristics. The results indicated that CmWag31 exhibited the capacity to interact with the transglycosylase (TG) and transpeptidase (TP) domain of CmPBPC, while CmPBPC only interacted with the NTD region of CmWag31. Co-localization analysis showed that CmWag31 co-localized with CmPBPC at the bacterial growth tips of Clavibacter michiganensis and Escherichia coli. The mutation of R19A, R19C, A99T, and A102T of CmWag31 resulted in abnormal localization in Escherichia coli. In the case of C. michiganensis, the CmWag31A102T protein exhibited a diffuse localization, which is a departure from the polar localization of its wild type. The co-localization of the CmWag31A102T mutation with CmPBPC exhibited discrepancies between C. michiganensis and E. coli. The diffused localization of CmWag31A102T can be restored by overexpression of CmPBPC in C. michiganensis, yet this restoration is not observed in E. coli. This result indicates that CmPBPC from C. michiganensis may not fully excute their function in E. coli due to species-specific differences.

Keywords: Wag31, Clavibacter michiganensis, PBPC, Co-localization

Highlights

  • •

    The proteins of CmWag31 and CmPBPC from Clavibacter michiganensis, can co-localize at the cell pole in C. michiganensis and E. coli.

  • •

    A102 is a key amino acid site for CmWag31 localization in both C. michiganensis and E. coli.

1. Introduction

Clavibacter michiganensis (Cm) is a Gram-positive plant pathogenic bacterium that causes tomato canker and wilt disease, significantly impacting both fresh tomato and seed production (Nandi et al., 2018). As a seedborne pathogen, C. michiganensis can be transmitted over long distances and is capable of causing severe epidemics among tomato-growing areas (Blank et al., 2016; Tsiantos, 1987;). Presently, there are no commercially resistant tomato varieties to Cm, and the effectiveness of existing bactericides is limited (Peritore-Galve et al., 2021). The primary strategies for management of tomato bacterial canker and wilt disease include the sowing or transplanting pathogen-free seeds or seedlings, the implementation of phytosanitary measures during seed transportation, and the spraying of copper-contained chemicals in the early stage of field disease in production (Jiang et al., 2016; Jahr et al., 1999).

The cell wall of Gram-positive (G+) bacteria is primarily composed of a solid and thick peptidoglycan layer and lipoteichoic acid, which is crucial for bacterial morphogenesis and pathogenicity. This structure maintains cell shape, collaborates with the cell membrane in material exchange and osmotic pressure stabilization, and serves as a protective barrier against damage (Jankute et al., 2015; Liu and Yang, 2011). Peptidoglycan peptide chains are usually cross-linked by five amino acids, forming a dense mesh structure, which results in the creation of a compact peptidoglycan layer (Mistou et al., 2016).

The process of bacterial division and cell wall synthesis is exquisitely regulated, involving the systematic assembly of multiple proteins at the division septum (Adams and Errington, 2009). The bacterial cell division is initiated by the formation of a contractile ring at the midpoint of the cell. In Escherichia coli, the central component of the divisome is the eukaryotic microtubule protein analog EcFtsZ (den Blaauwen et al., 2017; Löwe and Amos, 1998; Mukherjee et al., 1993), which assembles into a ring-like structure (Szwedziak et al., 2014; Mukherjee and Lutkenhaus, 1999). The FtsZ ring (Z-ring) is attached to the membrane through FtsA in Escherichia coli or SepF in Bacillus subtilis, both of which contain an amphipathic helix bound to the membrane (Duman et al., 2013; Pichoff and Lutkenhaus, 2005). Subsequently, the Z-ring interacts with other proteins (BsZapA, BsEzrA) that regulate BsFtsZ polymerization (Cleverley et al., 2014; Gueiros-Filho and Losick, 2002; Levin et al., 1999). In E. coli and B. subtilis, the core structure formed by these “early” cytokinesis proteins subsequently recruits “late” cytokinesis proteins, including enzymes necessary for septum cell wall biosynthesis (FtsW, PBP1, PBP2B) and scaffolding proteins (DivIB, DivIC, DivIVA, and FtsL) that link the cytoplasmic Z-ring to the extracellular cell wall (Taguchi et al., 2019; Gamba et al., 2009; Adams and Errington, 2009; Errington et al., 2003). FtsW has been reported to complex PbpB that synthesizes peptidoglycan in Enterococcus faecalis (Nelson et al., 2024). PBP1 is a transglycosylase/transpeptidase, and PBP2B is a transpeptidase that introduces cross-links into septal peptidoglycan (Nguyen-Distèche et al., 1998; Daniel et al., 2000). FtsL, DivIB and DivIC all have a single transmembrane span and a substantial extracellular domain (Errington et al., 2003), in Staphylococcus aureus, DiviB/Divic/FtsL subcomplexes are required to recruit MurJ, the flippase of peptidoglycan precursor lipid II to the splitting site, thereby doping peptidoglycan into the cell (Schäper et al., 2024), and DivIVA could ensure uniform septal cleavage and peripheral peptidoglycan synthesis around the division site and also affects the contribution of elongosomes and class A penicillin-binding proteins to cell elongation in Streptococcus pneumoniae (Trouve et al., 2024).

As a class of protein playing essential roles in cell wall synthesis, penicillin-binding proteins (PBPs), named for their covalent binding to penicillin and as the targets for β-lactam antibiotics, are widely present in most species of bacteria (Claessen et al., 2008; Suginaka et al., 1972). The PBPs have been shown to contain transglycosylase, transpeptidase, and D-alanine-D-alanine carboxypeptidase (D, D-carboxypeptidase) structural domains, which are crucial for synthesis and cross-linking of cell wall (Egan et al., 2020). However, how these structural domains interact with other proteins involved in cell wall synthesis remains unknown. The bacterial PBPs were divided into three groups based on their molecular size and function: class A high-molecular-weight (HMW) PBPs, class B HMW PBPs, and low-molecular-weight (LMW) PBPs (Valbuena et al., 2007). Previous studies from our research group have indicated that seven pbp genes are associated with cell wall peptidoglycan synthesis in C. michiganensis. Among them, CmPBPC is a Class A HMW PBP predicted to have both glycosyltransferase and transpeptidase structural domains. In the absence of the pbpC gene, the bacterial cell exhibited aberrant morphology, characterized by an enlargement at one end that deviated from the typical rod-like structure (Chen et al., 2021).

Wag31, a conserved class of coiled-coil proteins with the DivIVA structural domain of the cell division protein family, is ubiquitously present in Gram-positive bacteria, playing a crucial role in new cell wall synthesis and bacterial division. In Listeria monocytogenes, the DivIVA proteins have been demonstrated to possess the capacity to bind to lipid membranes, assembling in membrane-curved regions such as cell poles and septa, facilitating the recruitment of interacting proteins (Halbedel et al., 2012). Given the variations in the mechanisms of cell division among different bacterial species, the function and significance of DivIVA proteins are subject to variation as well. In Mycobacterium tuberculosis, M. smegmatis and Corynebacterium glutamicum, DivIVA is localized at cell poles and the cell division septum, playing a crucial role in polar growth, cell elongation and morphology maintenance (Melzer et al., 2018; Eskandarian et al., 2017; Cameron et al., 2015; Santi et al., 2013; Kang et al., 2008; Letek et al., 2008; Ramos et al., 2003). In spherical bacteria Enterococcus faecalis and Streptococcus pneumoniae, DivIVA also plays a vital function in cell division, cell growth, and nucleoid segregation (Fadda et al., 2007; Ramirez-Arcos et al., 2005). In the case of actinomyces Streptomyces coelicolor, DivIVA is located at the growing tips and lateral branches of hyphae and is essential for peptidoglycan synthesis (Flärdh, 2003). It is noteworthy that DivIVA is also localized at the cell pole and division septum in Bacillus subtilis and Staphylococcus aureus, however it is not indispensable for cell growth and division (Pinho and Errington, 2004; Cha and Stewart, 1997; Edwards and Errington, 1997). BsDivIVAA78T has been demonstrated to impact its localization in Bacillus subtilis (Edwards and Errington, 1997; Cha and Stewart, 1997; Thomaides et al., 2001). This alanine residue is situated in the hydrophobic core of the first predicted coiled structure (Edwards et al., 2000; Perry and Edwards, 2004). Mutations in amino acids at position 18 or 19 have been shown to influence the localization of BsDivIVA at the cell pole, but retains the ability to support nutrient growth and 50 % spore formation efficiency (Perry and Edwards, 2004). Also, in Streptococcus pneumoniae, the A78T mutant causes cells to be chain-like, and the localization of DivIVA is altered, where the bright diffuse fluorescence around the cell outline is clear, although it is visible at both the septum and the poles (Fadda et al., 2007).

In plant pathogenic bacteria, there is no report on the function of Wag31, the homologue of DivIVA. Previous studies predicted that the potential interacting protein of Wag31 in C. michiganensis (CmWag31) was CmPBPC. In current study, the interaction relationship between CmWag31 and CmPBPC was confirmed. Through the research on the synergistic function and localization of these two proteins, the key sites affecting the localization of CmWag31 were identified. Understanding the function of homologous proteins in the DivIVA family of Gram-positive plant pathogenic bacteria is paramount importance for the analysis of the cell wall synthesis mechanism in C. michiganensis.

2. Results

2.1. The N-terminal domain of CmWag31 interacts with CmPBPC

In the preceding screening study of interacting proteins by IP-MS, the results indicated that the Cell Division Protein CmWag31, which contains a DivIVA domain, could interact with CmPBPC (Chen, 2022). This interaction was confirmed by an in vitro pull-down assay (Fig. 1A). The CmPBPC is predicted to contain both transglycosylase and transpeptidase domains. In the present study, the CmPBPC was divided into three regions, the TG domain, the TP domain, and other regions. The GST tags were attached to each region for protein expression and purification (Fig. S1). The results of the Western blot analysis demonstrated that CmWag31 can interact with the TG or TP domains but not with the other regions of CmPBPC (Fig. 1B), thereby indicating the synergistic function between CmWag31 and CmPBPC. It has been reported that DivIVA proteins consist of an N-terminal lipid-binding domain (NTD) responsible for membrane association and a C-terminal domain (CTD) that supports oligomerization. Furthermore, DivIVA proteins have been demonstrated to interact with membrane proteins via the NTD and with cytoplasmic proteins via the CTD (Halbedel and Lewis, 2019). To elucidate the interaction relationship between different regions of CmWag31 and CmPBPC, in this study, the CmWag31 was divided into two regions, NTD and CTD, and the His tags were respectively attached for protein expression and purification (Fig. S1). The results indicated that the NTD region of CmWag31 exhibited a specific interaction with CmPBPC, while the CTD region did not (Fig. 1C).

Fig. 1.

Fig. 1

Open in a new tab

Interaction analysis of CmWag31 with CmPBPC by pull down (n = 2).

A. Interaction validation of CmPBPC with CmWag31 by GST pull down. (GST-PBPC: GST-tagged fusion protein with CmPBPC; GST-K: GST-tagged empty control protein; His-Wag31: His-tagged fusion protein with CmWag31.)

B. Interaction validation of different domains of CmPBPC with CmWag31 by His pull down. (GST-TG: GST-tagged fusion protein with TG domain of CmPBPC; GST-TP: GST-tagged fusion protein with TP domain of CmPBPC; GST-Other regions: GST-tagged fusion protein with other regions of CmPBPC.)

C. Interaction validation of different regions of CmWag31 with CmPBPC by GST pull down. (His-NTD: His-tagged fusion protein with NTD region of CmWag31; His-CTD: His-tagged fusion protein with CTD region of CmWag31.)

2.2. Wag31 and PBPC co-localized at the tip of the growing cell pole in C. michiganensis

The interaction of proteins and their subcellular localization are critical factors affecting their functions in biological processes. The localization of CmWag31 and CmPBPC was studied in E. coli and C. michiganensis, respectively. Four vectors, including the CmWag31 localization vector, the CmWag31 and CmPBPC co-localization vector, and their respective control vectors, were constructed and used for CmWag31 and CmPBPC localization analysis (Fig. S2). Among these vectors, CmWag31 was expressed in fusion with mCherry and CmPBPC was expressed in fusion with GFP.

The localization vector containing wag31 was introduced into Escherichia coli DH5α strain and the result showed that CmWag31 successfully localized at the pole in E. coli (Fig. 2A), consistant with its localization pattern in C. michiganensis (Fig. 2B). When CmPBPC and CmWag31 were co-expressed in the same vector, it was revealed that CmPBPC co-localized with CmWag31 at the cell pole in E. coli, while the fluorescence signal (mCherry for CmWag31 and GFP for CmPBPC) of their empty vector (EV) strains dispersed throughout the whole bacterial cell (Fig. 2C). The co-localization results in C. michiganensis cells were consistent with those of in E. coli (Fig. 2D). These findings suggested that CmWag31 and CmPBPC could be located at the same position within the bacteria cell, thereby facilitating the regulation of cell growth and division.

Fig. 2.

Fig. 2

Fig. 2

Open in a new tab

Subcellular localization of CmWag31 and CmPBPC in E. coli and C. michiganensis (n = 3).

A. E. coli strain DH-5α carried the plasmid vector pHN216 expressing mCherry protein (Up) or Wag31-mCherry fusion proteins (Down). Merge images of bacteria cells are shown as bright-field with overlaid fluorescence in red (Scale bar = 2 μm).

B. C. michiganensis strain BT-0505 carried the plasmid vector pHN216 expressing mCherry protein (Up) or Wag31-mCherry fusion proteins (Down) (Scale bar = 2 μm).

C. E. coli strain DH-5α carried the plasmid vector pHN216 expressing mCherry and GFP proteins (Up), a co-localization plasmid producing both GFP-PBPC (green) and mCherry-Wag31 (red) transferred to an E. coli strain DH-5α demonstrated the co-localization of PBPC and Wag31 (Down). Merge images of bacteria cells are shown as phase-contrast image with overlaid fluorescence both in green and red (Scale bar = 2 μm).

D. C. michiganensis strain BT-0505 carrying empty vector pHN216 with mCherry and GFP (Up), a co-localization plasmid producing both GFP-PBPC (green) and mCherry-Wag31 (red) transferred to a C. michiganensis strain BT-0505 demonstrated the co-localization of PBPC and Wag31 (Down) (Scale bar = 2 μm).

2.3. A102 is the key amino acid site that influences the localization of CmWag31

It was confirmed that CmWag31 and CmPBPC play crucial roles in cell wall synthesis and cell division in C. michiganensis. It has been reported that mutations in amino acids at position 18, 19 or 78 have the potential to influence the polar localization of BsDivIVA in Bacillus subtilis (Thomaides et al., 2001; Perry and Edwards, 2004), and the A78T mutant of DivIVA in Streptococcus pneumoniae has been shown to affect the localization (Fadda et al., 2007). To determine which amino acid site influence the localization function of CmWag31 and its interaction with CmPBPC, a multiple amino acid sequence alignment of CmWag31 with its homologous protein DivIVA from Bacillus subtilis and Streptococcus pneumoniae was conducted. The result showed that five amino acid residues, including R19, G21, I65, A99, and A102, may serve as the key sites that influence the function of CmWag31 (Fig. S3). Consequently, we mutated each of these five amino acid sites of CmWag31 and found that the mutants of CmWag31R19A, CmWag31R19C, CmWag31A99T, and CmWag31A102T affected its normal localization in E. coli, and the mCherry signals were not concentrated at cell poles, but instead exhibited diffuse localization. It is noteworthy that these point mutations affecting localization of CmWag31 also influence bacterial division, leading to the generation of more elongated cells in E. coli (Fig. 3A). However, the mutants of CmWag31G21A and CmWag31I65A do not affect the localization of CmWag31, nor do they affect the bacterial division in E. coli (Fig. S4). Consequently, the four amino acid sites with altered localization were selected for further investigation in C. michiganensis. Interestingly, only the Wag31A102T mutant affected its localization in C. michiganensis, while the other three sites did not (Fig. 3B). These results suggest that A102 of CmWag31 is the key amino acid responsible for its localization at the cell pole in C. michiganensis.

Fig. 3.

Fig. 3

Fig. 3

Open in a new tab

The effect of mutations at key amino acid sites on the localization of CmWag31 in E. coli and C. michiganensis, respectively (n = 3).

A. Mutants of R19A, R19C, A99T, A102T exhibited aberrant localization of CmWag31 in E. coli strain DH-5α (Scale bar = 2 μm).

B. Mutant of CmWag31A102T leads to a diffuse localization in C. michiganensis strain BT-0505 (Scale bar = 2 μm).

2.4. Co-expression with CmPBPC repaired the localization changes of CmWag31A102T mutant in C. michiganensis

Subsequent to determining that the CmWag31A102T mutation impacts its own localization in E. coli and C. michiganensis, we proceeded to investigate the effect of the CmWag31A102T mutation on the localization of its interacting protein CmPBPC. The results showed that co-expression of mutant CmWag31A102T and CmPBPC disrupted the co-localization of CmWag31 and CmPBPC in E. coli. The CmWag31A102T aggregates at one end of the cell, forming a non-polar localization, and the green fluorescent protein (GFP) signal fills the entire bacterial cell (Fig. 4A), suggesting that the mutation of CmWag31A102T impacts its co-localization with CmPBPC.

Fig. 4.

Fig. 4

Open in a new tab

The co-expression and localization of mCherry-CmWag31A102T and GFP-CmPBPC in E. coli and C. michiganensis (n = 3).

A. The localization of mCherry-CmWag31A102T and GFP-CmPBPC in E. coli is distinct. Specifically, mCherry-CmWag31A102T aggregates at the end of cells, rather than at the cell pole, while GFP-CmPBPC displays diffuse localization within the bacterial cells (Scale bar = 2 μm).

B. Co-expression with GFP-CmPBPC resulted in the re-localization of CmWag31A102T at the cell pole in C. michiganensis (Scale bar = 2 μm).

In contrast, the mutant CmWag31A102T and CmPBPC were observed to co-localize at the cell pole in C. michiganensis (Fig. 4B). Both the GFP and mCherry fluorescence were distributed on the tips of bacterial cells. These results suggested that through co-expression with CmPBPC, the diffuse localization of CmWag31A102T reverts to the pole distribution of wild type CmWag31. Recovery of the localization of CmWag31A102T indicates that co-expressed CmPBPC direct CmWag31A102T to its correct position.

2.5. Repair of the localization of CmWag31A102T in C. michiganensis is independent of the native expression of CmPBPC

To determine the effect of CmPBPC expression in wild-type strains on the localization of CmWag31A102T in C. michiganensis, the CmWag31 localization vector, the CmWag31A102T localization vector, and the CmWag31A102T-CmPBPC co-localization vector were introduced into the pbpC gene deletion mutant (ΔpbpC), respectively. The results revealed that CmWag31 exhibited a consistent localization at the cell pole in ΔpbpC spherical cells, which was located at one point on the edge of the sphere cells or at both ends of the diameter (Fig. 5A). However, CmWag31A102T exhibited distribution along the edge of the spherical cells, rather than at the ends of the diameter (Fig. 5B). This finding indicated that the native expression of CmPBPC does not impact the subcellular localization of CmWag31 that is transferred exogenously. Furthermore, CmWag31A102T demonstrated the capacity to co-localize with CmPBPC at the cell pole in the ΔpbpC mutant. It indicates that the abnormal localization of CmWag31A102T can be rectified by co-expression of CmPBPC. This specific repair mechanism is independent of the native expression of CmPBPC in wild-type C. michiganensis.

Fig. 5.

Fig. 5

Open in a new tab

The impact of native PBPC expression on the localization of CmWag31 and its mutant CmWag31A102T in C. michiganensis (n = 3).

A. The mCherry-CmWag31 localized at the cell poles while mCherry-CmWag31A102T showed abnormal localization in ΔpbpC mutants. The pole of the ΔpbpC spherical cells were defined as the ends of the diameter at the center of the sphere (Scale bar = 2 μm).

B. The mCherry-CmWag31A102T co-localized with GFP-CmPBPC at the cell poles of ΔpbpC mutants, and not affected by the expression of native PBPC. The empty vector pHN216 carried mCherry or GFP (Up) introduced into ΔpbpC mutant was used as the control for localization (Scale bar = 2 μm).

3. Materials and methods

3.1. Strains and culture conditions

The commercial Escherichia coli DH5α and the wild-type Clavibacter michiganensis BT_0505 which was isolated in Inner Mongolia, China (Luo, 2008), were used as the major bacterial strains in this study. E. coli cells were cultured on Luria-Bertani (LB) agar at 37 °C for 12 h, while C. michiganensis cells were cultured at 28 °C for 72 h. A single colony was selected and added to the LB broth medium, the E. coli cells were grown at 37 °C with shaking (200 rpm) for 12 h, whereas the C. michiganensis cells were grown at 28 °C with shaking (150 rpm) for 20 h. Then the bacterial suspension was used for subsequent experiments.

3.2. Protein purification by affinity chromatography

All fusion proteins were expressed in E. coli BL21DE3 cells (TransGen Biotech), which harbored the plasmid pGEX-4T, pGEX-4T-PBPC, pGEX-4T-TG, pGEX-4T-TP, pGEX-4T-Other regions, pETSUMO-Wag31, pETSUMO-NTD and pETSUMO-CTD respectively (Table 1). The cells were incubated at 37 °C in LB broth supplemented with ampicillin (50 μg/ml) or kanamycin (50 μg/ml). When the OD600 of the cell cultures reaches 0.6–0.8, expression of plasmid-encoded genes is induced by the addition of 0.1 mM IPTG. Subsequently, the cell cultures were incubated at 28 °C for 4 h, and then collected the cells by centrifugation at 4 °C, 8000 ×g, for 10 min. The harvested cells were washed three times by resuspension in phosphate buffered saline (PBS) and then resuspended with 10 ml of Lysis buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl). The bacterial suspension was incubated for 30 min at 4 °C, following the addition of 1 ml of TieChui E. coli Lysis buffer (ACE Biotech). The cell lysate was separated from cell debris by centrifugation at 4 °C, 10 min, 8000 ×g. Thereafter, the proteins were incubated with GST Resin (for CmPBPC proteins) or Ni-NTA Resin (for CmWag31 proteins) and washed with glutathione reduced (0.03 g/ml) (Coolaber) or imidazole concentrations ranging from 10 to 400 mM (Coolaber). The concentration of the eluted proteins was determined by measuring the optical density (OD) value at 280 nm.

Table 1.

Plasmids used in the study.

Plasmids Important features References
pGEX-4 T Cloning vector, AmpR
pGEX-4 T-PBPC pGEX-4T, pbpC+BT_0505 cloned via BamHI and XmaI This study
pGEX-4 T-TG pGEX-4T, TG domain of PBPC This study
pGEX-4 T-TP pGEX-4T, TP domain of PBPC This study
pGEX-4 T-Other regions pGEX-4T, Other regions of CmPBPC This study
pSESUMO Cloning vector, KmR
pSESUMO-Wag31 pSESUMO, wag31+BT_0505 cloned via NcoI and XhoI This study
pSESUMO-NTD pSESUMO, NTD domain of CmWag31 This study
pSESUMO-CTD pSESUMO, CTD domain of CmWag31 This study
pHN216 shuttle vector Laine et al., 1996
pHN216-Cherry pHN216, Cherry fluorescent label This study
pHN216-Cherry-GFP pHN216, Cherry and GFP fluorescent labels This study
pHN216-Cherry-Wag31 pHN216, Cherry fluorescent label fusion with CmWag31 This study
pHN216-Cherry-Wag31 (R19A) pHN216-Cherry-Wag31 but Arg 19 exchanged into Ala This study
pHN216-Cherry-Wag31 (R19C) pHN216-Cherry-Wag31 but Arg 19 exchanged into Cys This study
pHN216-Cherry-Wag31 (A99T) pHN216-Cherry-Wag31 but Ala 99 exchanged into Thr This study
pHN216-Cherry-Wag31 (A102T) pHN216-Cherry-Wag31 but Ala 102 exchanged into Thr This study
pHN216-Cherry-Wag31-PBPC-GFP pHN216, Cherry fluorescent label fusion with CmWag31, GFP fluorescent label fusion with CmPBPC This study
pHN216-Cherry-Wag31 (A102T)-PBPC-GFP pHN216-Cherry-Wag31-PBPC-GFP but Ala 102 of CmWag31 exchanged into Thr This study
Open in a new tab

3.3. Western blot analysis

The protein samples were treated with SDS loading buffer and separated by SDS-PAGE, then transferred to a PVDF membrane (Millipore). Purified proteins were incubated with HRP-conjugated anti-GST and anti-His antibodies (CWBIO). Subsequently, blots were incubated with Goat-Anti-Mouse IgG HRP Conjugate (CWBIO). The analysis of the blots was conducted by using the Immobilon Western Chemiluminescent HRP Substrate (Millipore).

3.4. Construction of localization vectors

As shown in Table 1, the plasmids used in this study were constructed by means of the ClonExpress Ultra One Step Cloning Kit (Vazyme Biotech, China), employing the shuttle vector pHN216 (Laine et al., 1996). The gfp gene was amplified by PCR using the forward primer (5’-ATGAGTAAAGGAGAAGAACTTTTC-3′) and the reverse primer (5’-GGCATGGATGAACTATACAAA-3′), mcherry was amplified with the forward primer (5’-GTGAGCAAGGGCGAGGAGG-3′) and the reverse primer (5’-GCATGGACGAGCTGTACAAG-3′). The sequence encoding CmWag31 and CmPBPC was amplified from C. michiganensis chromosomal DNA (Omega Biotech, America) with the forward primers (wag31 5’-ATGGCTCTCACTCCTGAAGA-3’ pbpC 5’-ATGAGGAAAACTGACCTGACCATCTC-3′) and the reverse primers (wag31 5’-CACCGCCTCGTTCGGCAACTAG-3’ pbpC 5’-CGCCCCTCCCGCGGAGGGGTGA-3′). The expression of all these genes is initiated by the promotor J23119, which belongs to a category of constitutive promoters (BBa_J23119 registered in standard biological parts, http://www.partsregistry.org). To facilitate the construction of the co-localization vectors, the HpaI restriction site was added between cherry and wag31, meanwhile, an AflII restriction site was inserted between gfp and pbpC, and the AscI site was inserted between wag31 and pbpC. All these localization vectors were transferred into targeted strains by electroporation as described in previous study (Kirchner et al., 2001).

3.5. Sequence analysis by Jalview

To identify the key sites that affect the localization of CmWag31, a sequence analysis of Wag31 from various Gram-positive bacteria was performed using Jalview. The Clustal color mode was selected to enhance the clarity and accessibility of the sites. To ensure a comprehensive analysis of the key sites of CmWag31, a variety of alignment methods were employed, including “Muscle with Defaults” and “Mafft with Defaults”.

3.6. Observation by confocal fluorescence microscopy

In order to observe the localization of CmWag31 and CmPBPC in C. michiganensis and E. coli, the bacterial cells were cultured at LB broth medium, adjust OD600 to 0.4, then the bacterial cells were harvested by centrifugation at 12, 000 rpm for 3 min, followed by three times washes with 0.85 % NaCl to remove the medium background. Ten microliters of the bacterial suspensions were deposited on a slide, which was then covered by a slip. The samples were observed using Confocol laser scanning microscopy (Zeiss LSM 800) with proper filter sets (GFP at 488 nm, mCherry at 610 nm).

3.7. Penicillin sensitivity assay

To determine whether the Wag31 protein with mutations at the key amino acid sites affected structure of the peptidoglycan and the sensitivity of the C. michiganensis cells to penicillin through interaction with PBPC, the minimum inhibitory concentration (MIC) of penicillin against various mutants was measured. The C. michiganensis cells were cultured in LB broth medium, and adjust to OD600 = 0.4. One hundred and thirty-five microliters of penicillin solution with series concentrations were added to each column of wells in a 96-well plate, with the final concentration of penicillin in the column is 8 μg/ml, then 15 μl of bacterial suspension of C. michiganensis was added to each well. The 96-well plates were incubated at 28 °C, 150 rpm for 40 h, and the MIC of penicillin against C. michiganensis was determined by observing the bacterial growth at specific concentrations of penicillin.

4. Discussion

The function of DivIVA and its homologous protein, Wag31, varies among Gram-positive bacteria. DivIVA is crucial for cell growth in Streptomyces coelicolor and plays a vital role in cell division in Streptococcus pneumoniae (Fadda et al., 2007; Flärdh, 2003). Notably, there is a high homology between the DivIVA protein in these two bacterial species and the Wag31 of C. michiganensis, with percentages of 48.39 % and 43.86 %, respectively. In contrast, divIVA was identified as a nonessential gene in Bacillus subtilis and Staphylococcus aureus, where its deletion did not result in cell death (Pinho and Errington, 2004; Cha and Stewart, 1997). Consequently, the DivIVA protein in these two bacterial species exhibits a comparatively low similarity to CmWag31, with percentages of 24.02 % and 17.67 %, respectively. This study attempted to knock out wag31 in C. michiganensis, but failed to obtain a gene deletion mutant, suggesting that wag31 may be essential for cell division and growth (data not shown).

In this study, the localization of proteins in the typical Gram-positive bacterium C. michiganensis was observed by confocal microscopy. The cells of C. michiganensis are diminutive in size and possess a thick cell wall, which poses a significant challenge in obtaining clearer images, even under optimal magnification conditions during microscopic observation. Subsequent attempts will be made to immobilize the organisms to prevent the quality of the photographs from being affected by the surface tension of the liquid. The cell wall is crucial for determining cell shape in bacteria. Peptidoglycan (PG) is a major component of the cell wall, and its synthesis depends on numerous proteins belonging to the murein (Mur) or Penicillin-binding protein (PBP) families (Typas et al., 2011). Previous studies have identified seven genes associated with peptidoglycan synthesis in C. michiganensis, including 2 Class A high-molecular-weight (HMW) PBPs (pbpC and pbpD), 2 Class B HMW PBPs (pbpA and pbpB1), and 3 low-molecular-weight (LMW) PBPs (pbpB, pbpX, and dacB) (Chen et al., 2021). These PBPs exhibit functional redundancy, with Class A HMW PBPs demonstrated to be essential in most bacteria (Pazos and Vollmer, 2021). Given the importance of CmPBPC, the present study focused on the synergistic effect between CmWag31 and CmPBPC. The results showed that CmWag31 interacted with the TG and TP domain of CmPBPC, while CmPBPC only interacted with the NTD region of CmWag31. The co-localization of these two proteins at the cell poles suggests that CmWag31 and CmPBPC can jointly regulate cell wall synthesis and cell division in C. michiganensis. However, further study is necessary to elucidate the synergistic function of these two proteins and the impact of CmWag31 on stress response and pathogenicity.

This study has for the first time confirmed the co-localization effect of CmWag31 and CmPBPC at the cell poles in C. michiganensis. In the courses of our observations of the localization of the point mutants CmWag31 protein in E. coli, it was determined that CmWag31R19A, CmWag31R19C, CmWag31A99T and CmWag31A102T exert an effect on cell division while concomitantly interfering with polar localization, resulting in the formation of elongated cells. However, point mutations that did not affect localization of Wag31 also did not cause cells with abnormal morphology in E. coli. It is noteworthy that CmWag31A102T exhibited no discernible effect on cell morphology, despite its aberrant localization within C. michiganensis. These findings suggest that CmWag31 functions inconsistently in E. coli and C. michiganensis, which may be due to the diversity of Wag31 between species and to the different modes of regulation of CmWag31 by other proteins in these two species of bacteria.

Notably, CmWag31A102T mutation and CmPBPC manifest distinct co-localization in E. coli and C. michiganensis. In C. michiganensis, the two proteins co-localized at the cell poles, thereby restoring the aberrant localization of the CmWag31A102T mutation. It can be hypothesized that the restoration is facilitated by the introduction and overexpression of CmPBPC in conjunction with CmWag31A102T, which results in the repositioning of the aberrantly localized CmWag31A102T to the cell poles-a conjecture that is confirmed by the co-localization results in ΔpbpC mutants. However, in E. coli, CmWag31A102T is diffusely localized and aggregates at the anterior end of the cell, while CmPBPC is distributed throughout the bacterial cell. It suggests that exogenously supplemented CmPBPC is incapable of restoring the abnormal localization of CmWag31A102T in E. coli. This differential result indicates that CmWag31 and CmPBPC from C. michiganensis have different functions in E. coli. It has been reported that DivIVA plays an essential role in recruiting the peptidoglycan biosynthetic machinery to the cell poles in actinomycetes such as Streptomyces coelicolor and Corynebacterium glutamicum (Hempel et al., 2008; Kang et al., 2008). In Bacillus subtilis, DivIVA has been observed to recruit the transmembrane protein MinJ, which has been demonstrated to mediate contact with the division inhibitors MinCD (Bramkamp et al., 2008; Patrick and Kearns, 2008). In addition, Gamba et al. have demonstrated that FtsZ ring assembles with FtsA, ZapA and EzrA. Subsequently, the complex can recruit DivIVA and PBP2B as a kind of “late” cytokinesis proteins to midcell (Gamba et al., 2009). In the present study, it was revealed that CmPBPC can restore the error localization of CmWag31A102T mutation in C. michiganensis. This funding suggests that CmPBPC localizes at cell poles earlier than CmWag31 and may act as a type of “early” cytokinesis protein.

Cell poles have been demonstrated to play a crucial role in regulating the biosynthetic activities, polar growth, and cell cycle development of bacteria. Therefore, it is imperative to comprehend the mechanisms underlying the localization of functional proteins at the poles. It has been reported that the “diffusion-capture” process is a general mechanism for protein recruitment at poles. This mechanism requires a “hub” protein to capture other diffusible proteins, and the DivIVA protein is regarded as a pivotal polar “hub” (Laloux and Jacobs-Wagner, 2014; Rudner and Losick, 2010). DivIVA has been observed to localize at cell poles, regulating the localization of related cytoplasmic cell wall synthesis and interacting with other enzymes (Meniche et al., 2014). The aberrant localization of CmWag31A102T mutation and CmPBPC in E. coli suggests that CmWag31 can function as a DivIVA homologue in E. coli, responsible for “recruiting” other cell division-related proteins to cell poles. When the localization of CmWag31A102T is disrupted, the “recruited” proteins are unable to localize at the poles, instead becoming diffuse within the bacterial cell. The hypothesis explaining the normal co-localization of CmWag31A102T mutation and CmPBPC in C. michiganensis may be attributed to CmWag31 not acting as a “hub” protein in C. michiganensis for “recruiting” other proteins to cell poles. Conversely, CmWag31 may be “recruited” by CmPBPC to the cell poles when its localization is disturbed. Therefore, further investigation is required to ascertain the effect of the key functional sites of CmPBPC on the localization of interacted CmWag31 and CmPBPC proteins. This investigation can determine whether CmPBPC can act as a “recruiting protein”. According to our results, A102 is located in the CTD region of CmWag31, which is not a region that interacts with CmPBPC (Fig. 1C), so the non-polar localization of Wag31A102T corrected by CmPBPC is not due to an interaction between the two proteins. The mechanism of repair of this mislocalization needs to be further investigated.

DivIVA, rich with coiled-coil structure domains, senses concave surfaces and self-assembles on the inner cytoplasmic side of the membrane at the cell poles (Surovtsev and Jacobs-Wagner, 2018; Wagstaff and Löwe, 2018; Lenarcic et al., 2009;). The prediction of the CmWag31 protein structure reveals its coiled-coil structures (analyzed by SPOMA), and the A102 is located at the beginning of this coiled-coil structure. The present study suggested that the A102T mutation of CmWag31 disrupts its coiled-coil structure, resulting in alterations in its localization and function. However, in contrast to the wild type, the CmWag31A102T mutation did not affect the sensitivity of C. michiganensis to penicillin, and overexpression of CmPBPC in conjunction with CmWag31A102T did not elicit any change in penicillin sensitivity (Fig. S5). This finding implies that although CmWag31 interacts with CmPBPC, CmWag31 does not affect the ability of C. michiganensis to against penicillin, which inhibits bacteria through preventing new cell wall formation. The synergistic function of CmWag31 and CmPBPC in influencing the structure of the bacterial cell wall requires further investigated.

CRediT authorship contribution statement

Chengxuan Yu: Writing – original draft, Visualization, Validation, Data curation. Xiaoli Xu: Formal analysis. Jia Shi: Formal analysis. Wenqing Chu: Formal analysis. Na Jiang: Visualization, Software, Formal analysis. Jianqiang Li: Supervision, Resources. Laixin Luo: Writing – review & editing, Supervision, Software, Resources, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.tcsw.2025.100151.

Appendix A. Supplementary data

Supplementary material 1

mmc1.docx (993.2KB, docx)

Supplementary material 2

mmc2.docx (2.6MB, docx)

Data availability

Data will be made available on request.

References

  1. Adams D.W., Errington J. Bacterial cell division: assembly, maintenance and disassembly of the Z ring. Nat. Rev. Microbiol. 2009;7:642–653. doi: 10.1038/nrmicro2198. [DOI] [PubMed] [Google Scholar]
  2. Blank L., Cohen Y., Borenstein M., Shulhani R., Lofthouse M., Sofer M., Shtienberg D. Variables associated with severity of bacterial canker and wilt caused by Clavibacter michiganensis subsp. michiganensis in tomato greenhouses. Phytopathology. 2016;106:254–261. doi: 10.1094/PHYTO-07-15-0159-R. [DOI] [PubMed] [Google Scholar]
  3. Bramkamp M., Emmins R., Weston L., Donovan C., Daniel R.A., Errington J. A novel component of the division-site selection system of Bacillus subtilis and a new mode of action for the division inhibitor MinCD. Mol. Microbiol. 2008;70:1556–1569. doi: 10.1111/j.1365-2958.2008.06501.x. [DOI] [PubMed] [Google Scholar]
  4. Cameron T.A., Zupan J.R., Zambryski P.C. The essential features and modes of bacterial polar growth. Trends Microbiol. 2015;23(6):347–353. doi: 10.1016/j.tim.2015.01.003. [DOI] [PubMed] [Google Scholar]
  5. Cha J.H., Stewart G.C. The divIVA minicell locus of Bacillus subtilis. J. Bacteriol. 1997;179:1671–1683. doi: 10.1128/jb.179.5.1671-1683.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen X. China Agricultural University; Beijing: 2022. Function of Penicillin Binding Proteins on Stress Resistance in Clavibacter michiganensis. PhD thesis. [Google Scholar]
  7. Chen X., Bai K.H., Lyu Q.Y., Jiang N., Li J.Q., Luo L.X. Role of penicillin-binding proteins in the viability, morphology, stress tolerance, and pathogenicity of Clavibacter michiganensis. Phytopathology. 2021;111(8):1301–1312. doi: 10.1094/PHYTO-08-20-0326-R. [DOI] [PubMed] [Google Scholar]
  8. Claessen D., Emmins R., Hamoen L.W., Daniel R.A., Errington J., Edwards D.H. Control of the cell elongation-division cycle by shuttling of PBP1 protein in Bacillus subtilis. Mol. Microbiol. 2008;68:1029–1046. doi: 10.1111/j.1365-2958.2008.06210.x. [DOI] [PubMed] [Google Scholar]
  9. Cleverley R.M., Barrett J.R., Basle A., Bui N.K., Hewitt L., Solovyova A., Xu Z.Q., Daniel R.A., Dixon N.E., Harry E.J. Structure and function of a spectrin-like regulator of bacterial cytokinesis. Nat. Commun. 2014;5:5421. doi: 10.1038/ncomms6421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Daniel R.A., Harry E.J., Errington J. Role of penicillin-binding protein PBP2B in assembly and functioning of the division machinery of Bacillus subtilis. Mol. Microbiol. 2000;35:299–311. doi: 10.1046/j.1365-2958.2000.01724.x. [DOI] [PubMed] [Google Scholar]
  11. den Blaauwen T., Hamoen L.W., Levin P.A. The divisome at 25: the road ahead. Curr. Opin. Microbiol. 2017;36:85–94. doi: 10.1016/j.mib.2017.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duman R., Ishikawa S., Celik I., Strahl H., Ogasawara N., Troc P., Lowe J., Hamoen L.W. Structural and genetic analyses reveal the protein SepF as a new membrane anchor for the Z ring. Proc. Natl. Acad. Sci. USA. 2013;110:E4601–E4610. doi: 10.1073/pnas.1313978110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edwards D.H., Errington J. The Bacillus subtilis DivIVA protein targets to the division septum and controls the site specificity of cell division. Mol. Microbiol. 1997;24:905–915. doi: 10.1046/j.1365-2958.1997.3811764.x. [DOI] [PubMed] [Google Scholar]
  14. Egan A.J.F., Errington J., Vollmer W. Regulation of peptidoglycan synthesis and remodeling. Nat. Rev. Microbiol. 2020;18(8):446–460. doi: 10.1038/s41579-020-0366-3. [DOI] [PubMed] [Google Scholar]
  15. Errington J., Daniel R.A., Scheffers D.J. Cytokinesis in bacteria. Microbiol. Mol. Biol. Rev. 2003;67:52–65. doi: 10.1128/MMBR.67.1.52-65.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eskandarian H.A., Odermatt P.D., Ven J.X.Y., Hannebelle M.T.M., Nievergelt A.P., Dhar N., McKinney J.D., Fantner G.E. Division site selection linked to inherited cell surface wave troughs in mycobacteria. Nat. Microbiol. 2017;2(9) doi: 10.1038/nmicrobiol.2017.94. [DOI] [PubMed] [Google Scholar]
  17. Fadda D., Santona A., D’Ulisse V., Ghelardini P., Ennas M.G., Whalen M.B., Massidda O. Streptococcus pneumoniae DivIVA: localization and interactions in a MinCD-free context. J. Bacteriol. 2007;189(4):1288–1298. doi: 10.1128/JB.01168-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Flärdh K. Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2) Mol. Microbiol. 2003;49:1523–1536. doi: 10.1046/j.1365-2958.2003.03660.x. [DOI] [PubMed] [Google Scholar]
  19. Gamba P., Veening J.W., Saunders N.J., Hamoen L.W., Daniel R.A. Two-step assembly dynamics of the Bacillus subtilis divisome. J. Bacteriol. 2009;191:4186–4194. doi: 10.1128/JB.01758-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gueiros-Filho F.J., Losick R. A widely conserved bacterial cell division protein that promotes assembly of the tubulin-like protein FtsZ. Genes Dev. 2002;16:2544–2556. doi: 10.1101/gad.1014102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Halbedel S., Lewis R.J. Structural basis for interaction of DivIVA/GpsB proteins with their ligands. Mol. Microbiol. 2019;111(6):1404–1415. doi: 10.1111/mmi.14244. [DOI] [PubMed] [Google Scholar]
  22. Halbedel S., Hahn B., Daniel R.A., Flieger A. DivIVA affects secretion of virulence-related autolysins in Listeria monocytogenes. Mol. Microbiol. 2012;83(4):821–839. doi: 10.1111/j.1365-2958.2012.07969.x. [DOI] [PubMed] [Google Scholar]
  23. Hempel A.M., Wang S.B., Letek M., Gil J.A., Flärdh K. Assemblies of DivIVA mark sites for hyphal branching and can establish new zones of cell wall growth in Streptomyces coelicolor. J. Bacteriol. 2008;190:7579–7583. doi: 10.1128/JB.00839-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jahr H., Bahro R., Burger A., Ahlemeyer J., Eichenlaub R. Interactions between Clavibacter michiganensis subsp. michiganensis and its host plants. Environ. Microbiol. 1999;1:113–118. doi: 10.1046/j.1462-2920.1999.00011.x. [DOI] [PubMed] [Google Scholar]
  25. Jankute M., Cox J.A., Harrison J., Besra G.S. Assembly of the mycobacterial cell wall. Ann. Rev. Microbiol. 2015;69:405–423. doi: 10.1146/annurev-micro-091014-104121. [DOI] [PubMed] [Google Scholar]
  26. Jiang N., Lyu Q.Y., Xu X., Cao Y.S., Walcott R.R., Li J.Q., Luo L.X. Induction of the viable but non-culturable state in Clavibacter michiganensis subsp. michiganensis and in planta resuscitation of the cells on tomato seedlings. Plant Pathol. 2016;65(5):826–836. [Google Scholar]
  27. Kang C.M., Nyayapathy S., Lee J.Y., Suh J.W., Husson R.N. Wag31, a homologue of the cell division protein DivIVA, regulates growth, morphology and polar cell wall synthesis in mycobacteria. Microbiology. 2008;154:725–735. doi: 10.1099/mic.0.2007/014076-0. [DOI] [PubMed] [Google Scholar]
  28. Kirchner O., Gartemann K.H., Zellermann E.M., Eichenlaub R., Burger A. A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis. Mol. Plant-Microbe Interact. 2001;14:1312–1318. doi: 10.1094/MPMI.2001.14.11.1312. [DOI] [PubMed] [Google Scholar]
  29. Laine M.J., Nakhei H., Dreier J., Lehtilä K., Meletzus D., Eichenlaub R., Metzler M.C. Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors. Appl. Environ. Microbiol. 1996;62:1500–1506. doi: 10.1128/aem.62.5.1500-1506.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Laloux G., Jacobs-Wagner C. How do bacteria localize proteins to the cell pole? J. Cell Sci. 2014;127:11–19. doi: 10.1242/jcs.138628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lenarcic R., Halbedel S., Visser L., Shaw M., Wu L.J., Errington J., Marenduzzo D., Hamoen L.W. Localisation of DivIVA by targeting to negatively curved membranes. EMBO J. 2009;28(15):2272–2282. doi: 10.1038/emboj.2009.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Letek M., Ordóñez E., Vaquera J., Margolin W., Flärdh K., Mateos L.M., Gil J.A. DivIVA is required for polar growth in the MreB-lacking rod-shaped actinomycete Corynebacterium glutamicum. J. Bacteriol. 2008;190:3283–3292. doi: 10.1128/JB.01934-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Levin P.A., Kurtser I.G., Grossman A.D. Identification and characterization of a negative regulator of FtsZ ring formation in Bacillus subtilis. Proc. Natl. Acad. Sci. USA. 1999;96:9642–9647. doi: 10.1073/pnas.96.17.9642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu F., Yang Y.H. Studies on bacterial cell wall peptidoglycan. J. Sichuan Univ. Sci. Eng. 2011;24(6):628–631. [Google Scholar]
  35. Löwe J., Amos L.A. Crystal structure of the bacterial cell-division protein FtsZ. Nature. 1998;391:203–206. doi: 10.1038/34472. [DOI] [PubMed] [Google Scholar]
  36. Luo L.X. China Agricultural University; Beijing: 2008. The Molecular Detection and Phylogenetic Analysis of Clavibacter michiganensis Subsp. Michiganensis. PhD thesis. [Google Scholar]
  37. Melzer E.S., Sein C.E., Chambers J.J., Siegrist M.S. DivIVA concentrates mycobacterial cell envelope assembly for initiation and stabilization of polar growth. Cytoskeleton. 2018;75(12):498–507. doi: 10.1002/cm.21490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Meniche X., Otten R., Siegrist M.S., Baer C.E., Murphy K.C., Bertozzi C.R., Sassetti C.M. Subpolar addition of new cell wall is directed by DivIVA in mycobacteria. Proc. Natl. Acad. Sci. USA. 2014;111(31):E3243–E3251. doi: 10.1073/pnas.1402158111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mistou M.Y., Sutcliffe I.C., van Sorge N.M. Bacterial glycobiology: Rhamnose-containing cell wall polysaccharides in gram-positive bacteria. FEMS Microbiol. Rev. 2016;40(4):464–479. doi: 10.1093/femsre/fuw006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mukherjee A., Lutkenhaus J. Analysis of FtsZ assembly by light scattering and determination of the role of divalent metal cations. J. Bacteriol. 1999;181:823–832. doi: 10.1128/jb.181.3.823-832.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mukherjee A., Dai K., Lutkenhaus J. Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein. Proc. Natl. Acad. Sci. USA. 1993;90:1053–1057. doi: 10.1073/pnas.90.3.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nandi M., Macdonald J., Liu P., Weselowski B., Yuan Z.C. Clavibacter michiganensis subsp. michiganensis: bacterial canker of tomato, molecular interactions and disease management. Mol. Plant Pathol. 2018;19(8):2036–2050. doi: 10.1111/mpp.12678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nelson M.E., Little J.L., Kristich C.J. Pbp4 provides transpeptidase activity to the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in Enterococcus faecalis. Antimicrob Agents Ch. 2024;68:9. doi: 10.1128/aac.00555-24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nguyen-Distèche M., Fraipont C., Buddelmeijer N., Nanninga N. The structure and function of Escherichia coli penicillin-binding protein 3. Cell. Mol. Life Sci. 1998;54:309–316. doi: 10.1007/s000180050157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Patrick J.E., Kearns D.B. MinJ (YvjD) is a topological determinant of cell division in Bacillus subtilis. Mol. Microbiol. 2008;70:1166–1179. doi: 10.1111/j.1365-2958.2008.06469.x. [DOI] [PubMed] [Google Scholar]
  46. Pazos M., Vollmer W. Regulation and function of class a penicillin-binding proteins. Curr. Opin. Microbiol. 2021;60:80–87. doi: 10.1016/j.mib.2021.01.008. [DOI] [PubMed] [Google Scholar]
  47. Peritore-Galve F.C., Tancos M.A., Smart C.D. Bacterial canker of tomato: revisiting a global and economically damaging seedborne pathogen. Plant Dis. 2021;105(6):1581–1595. doi: 10.1094/PDIS-08-20-1732-FE. [DOI] [PubMed] [Google Scholar]
  48. Perry S.E., Edwards D.H. Identification of a polar targeting determinant for Bacillus subtilis DivIVA. Mol. Microbiol. 2004;54:1237–1249. doi: 10.1111/j.1365-2958.2004.04363.x. [DOI] [PubMed] [Google Scholar]
  49. Pichoff S., Lutkenhaus J. Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA. Mol. Microbiol. 2005;55:1722–1734. doi: 10.1111/j.1365-2958.2005.04522.x. [DOI] [PubMed] [Google Scholar]
  50. Pinho M.G., Errington J. A divIVA null mutant of Staphylococcus aureus undergoes normal cell division. FEMS Microbiol. Lett. 2004;240(2):145–149. doi: 10.1016/j.femsle.2004.09.038. [DOI] [PubMed] [Google Scholar]
  51. Ramirez-Arcos S., Liao M., Marthaler S., Rigden M., Dillon J.A.R. Enterococcus faecalis divIVA: an essential gene involved in cell division, cell growth and chromosome segregation. Microbiology. 2005;151(5):1381–1393. doi: 10.1099/mic.0.27718-0. [DOI] [PubMed] [Google Scholar]
  52. Ramos A., Honrubia M.P., Valbuena N., Vaquera J., Mateos L.M., Gil J.A. Involvement of DivIVA in the morphology of the rod-shaped actinomycete Brevibacterium lactofermentum. Microbiology. 2003;149(12):3531–3542. doi: 10.1099/mic.0.26653-0. [DOI] [PubMed] [Google Scholar]
  53. Rudner D.Z., Losick R. Protein subcellular localization in bacteria. Cold Spring Harb. Perspect. Biol. 2010;2(4) doi: 10.1101/cshperspect.a000307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Santi I., Dhar N., Bousbaine D., Wakamoto Y., McKinney J.D. Single-cell dynamics of the chromosome replication and cell division cycles in mycobacteria. Nat. Commun. 2013;4:2470. doi: 10.1038/ncomms3470. [DOI] [PubMed] [Google Scholar]
  55. Schäper S., Brito A.D., Saraiva B.M., Squyres G.R., Holmes M.J., Garner E.C., Hensel Z., Henriques R., Pinho M.G. Cell constriction requires processive septal peptidoglycan synthase movement independent of FtsZ treadmilling in Staphylococcus aureus. Nat. Microbiol. 2024;9:4. doi: 10.1038/s41564-024-01629-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Suginaka H., Strominger J.L., Blumberg P.M. Multiple penicillin-binding components in Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli. J. Biol. Chem. 1972;247:5279. [PubMed] [Google Scholar]
  57. Surovtsev I.V., Jacobs-Wagner C. Subcellular organization: a critical feature of bacterial cell replication. Cell. 2018;172:1271–1293. doi: 10.1016/j.cell.2018.01.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Szwedziak P., Wang Q., Bharat T.A., Tsim M., Löwe J. Architecture of the ring formed by the tubulin homologue FtsZ in bacterial cell division. eLife. 2014;3 doi: 10.7554/eLife.04601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Taguchi A., Welsh M.A., Marmont L.S., Lee W., Sjodt M., Kruse A.C., Kahne D., Bernhardt T.G., Walker S. FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein. Nat. Microbiol. 2019;4(4):587–594. doi: 10.1038/s41564-018-0345-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Thomaides H.B., Freeman M., Karoui M.E., Errington J. Division site selection protein DivIVA of Bacillus subtilis has a second distinct function in chromosome segregation during sporulation. Genes Dev. 2001;15:1662–1673. doi: 10.1101/gad.197501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Trouve J., Zapun A., Bellard L., Juillot D., Pelletier A., Freton C., Baudoin M., Carballido-Lopez R., Campo N., Wong Y.S., Grangeasse C., Morlot C. DivIVA controls the dynamics of septum splitting and cell elongation in Streptococcus pneumoniae. MBIO. 2024;15:10. doi: 10.1128/mbio.01311-24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tsiantos J. Transmission of bacterium Corynebacterium michiganense pv. Michiganense by seeds. J. Phytopathol. 1987;119:142–146. [Google Scholar]
  63. Typas A., Banzhaf M., Gross C.A., Vollmer W. From the regulation of peptidoglycan synthesis to bacterial growth and morphology. Nat. Rev. Microbiol. 2011;10:123–136. doi: 10.1038/nrmicro2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Valbuena N., Letek M., Ordoñez E., Ayala J., Daniel R.A., Gil J.A., Mateos L.M. Characterization of HMW-PBPs from the rod-shaped actinomycete Corynebacterium glutamicum: peptidoglycan synthesis in cells lacking actin-like cytoskeletal structures. Mol. Microbiol. 2007;66(3):643–657. doi: 10.1111/j.1365-2958.2007.05943.x. [DOI] [PubMed] [Google Scholar]
  65. Wagstaff J., Löwe J. Prokaryotic cytoskeletons: protein filaments organizing small cells. Nat. Rev. Microbiol. 2018;16:187–201. doi: 10.1038/nrmicro.2017.153. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1

mmc1.docx (993.2KB, docx)

Supplementary material 2

mmc2.docx (2.6MB, docx)

Data Availability Statement

Data will be made available on request.

Articles from The Cell Surface are provided here courtesy of Elsevier

RESOURCES