Comparative Analysis of an Integral Component of Bacterial Cell Division from the Lactococcus and Bacillus Genera

FtsZ proteins have been well-characterized to play a crucial role in cell division. Unfortunately, data are scarce on FtsZ proteins in the Lactococcus and Bacillus genera. The objective of this study was to analyze the features of FtsZ proteins in the Bacillus and Lactococcus genus groups. By exploring the available genomes, we identified and characterized FtsZ proteins in 19 Bacillus and 22 Lactococcus species. The sizes and weights of the FtsZ proteins ranged from 376 to 410 aa residues and 39.53 to 44.15 kDa in the Bacillus genus, respectively, and from 387 to 430 aa residues and 41.14 to 45.11 kDa in the Lactococcus genus, respectively. All the FtsZ proteins in the Bacillus and Lactococcus species were acidic and globular, and localized in the cytoplasm. Next, 3D modeling and multiple alignments were performed. We realized that the FtsZ proteins in the Bacillus and Lactococcus species exhibited five specific regions. Taken together, our study could provide a general background for further functional characterization of the FtsZ proteins in Bacillus and Lactococcus species.


Introduction
Cell division is a crucial process for living organisms.Principally, cell division in bacteria is orchestrated by a divisome.In this step, FtsZ, a polymer-forming guanosine-5′-triphosphatease (GTPase), drives bacterial cell division (de Boer et al., 1992;Löwe and Amos, 1998).
Particularly, FtsZ proteins play the role as the pacemaker of the formation of the divisome and the cytokinesis process (Margolin, 2005) as it assembles into proto-filaments to construct a ring-like template (McQuillen and Xiao, 2020).The formation of this ring initiates and facilitates cellular division, enabling a single parent cell to give rise to two offspring cells, while the divisome plays a pivotal role in the constriction of the cell envelope and the generation of new cell wall segments at the division site.FtsZ also has an extended role beyond cellular division where it contributes to determining cell morphology and establishing polarity in certain bacterial species.In a nutshell, the FtsZ protein is indispensable in the orchestration of bacterial cell division, thereby significantly influencing the growth and structural dynamics of bacterial cells.The FtsZ proteins in bacterial species have been reported to share a similar manner with the tubulins in eukaryotic cells.Up until now, information about the FtsZ proteins in various prokaryotic cells, unfortunately, has been unclearly (Pal et al., 2019).
The Lactococcus and Bacillus genus groups are two very common types of bacteria.Firstly, Lactococcus constitutes some of the genera forming the lactic acid bacteria family.This genus has been reportedly used in the food industry (Song et al., 2017) such as in the production of dairy products (Li et al., 2020).On the other hand, Bacillus species signify Grampositive, spore-forming, rod-shaped, and aerobic bacteria (Miljaković et al., 2020).This genus can be isolated from various sources, like soil (mostly), air, water, animal guts, vegetables, and other food items (Elshaghabee et al., 2017).At the molecular scale, the cell division process, particularly the FtsZ proteins of these genus groups, has been not fully described (Pal et al., 2019).Recently, a comprehensive search of the FtsZ proteins from approximately 70 bacterial families (belonging to 40 orders) was performed in order to find out the core set of codons in the coding region of the gene sequence (Pal et al., 2019).
The aims of this study was to comprehensively describe the FtsZ proteins found in the Lactococcus and Bacillus genus groups.

In silico searches of FtsZ proteins in databases
The well-characterized ftsZ gene from Escherichia coli (strain ATCC 47076) (McQuillen and Xiao, 2020) was used as the seed sequence for comprehensively searching against the sequences of Lactococcus and Bacillus genera (Loman et al., 2012;Tatusova et al., 2014) available from the NCBI GenBank database (Wheeler et al., 2008).The coding DNA sequences (CDSs) of the FtsZ proteins were then BlastP-ed to obtain the full-length amino acid (aa) sequences.

Analysis of features of the FtsZ proteins
The full-length aa sequences of the FtsZ proteins in the Lactococcus and Bacillus genus groups were applied in the Expasy Protparam portal (Gasteiger et al., 2003;Gasteiger et al., 2005).Particularly, six typical features of the molecules, namely protein sizes (aa residues), protein weights (kilo Dalton, kDa), iso-electric points (pI-s), instability indexes (II-s), aliphatic indexes (AI-s), and grand average of hydropathicity (GRAVY), were identified (Gasteiger et al. 2003).Basically, pI scores of > 7 and < 7 indicated basic and acidic proteins, respectively, while II values of > 41 and < 41 suggested unstable and stable proteins, respectively.GRAVY scores below 0 were more likely for hydrophilic proteins, while scores above 0 were more likely for hydrophobic proteins.

Prediction of subcellular localization of the FtsZ proteins
The subcellular localizations of the FtsZ proteins in the Lactococcus and Bacillus genus groups were predicted by using the YLoc portal (Briesemeister et al., 2010a;2010b).Particularly, the full-length aa sequence of each FtsZ protein was searched against the YLoc (Briesemeister et al., 2010b) to suggest the putative localizations, like the nucleus, cytoplasm, mitochondrion, plasma membrane, extracellular space, endoplasmic reticulum, peroxisome, Golgi apparatus, and vacuole.The probability (%) and confidence scores were used to validate the predictions (Briesemeister et al., 2010b).

Construction of 3D models of the FtsZ proteins
The full-length aa sequences of the FtsZ proteins in the Lactococcus and Bacillus species were used for the model predictions as previously described (La et al., 2022).Particularly, the Phyre2 tool (Kelley et al., 2015) was applied to analyze the secondary structures of the proteins, including the rates of alpha and beta subunits.Then, the 3D models of the FtsZ proteins in the Lactococcus and Bacillus species were constructed based on the available structures (Kelley et al., 2015).

Analysis of conserved domains of the FtsZ proteins
The full-length aa sequences of the FtsZ proteins in the Lactococcus and Bacillus species were applied for alignment using the ClustalX software (Thompson et al., 1997;Thompson et al., 2002).The Pfam domain (Finn et al., 2014;Mistry et al., 2021) was then used to obtain the conserved domain of the bacterial FtsZ proteins (Löwe and Amos, 1998;Silber et al., 2020).The conserved regions found in the FtsZ proteins in the Lactococcus and Bacillus species were graphically viewed by the BioEDIT software (Hall, 1999).

Identification and annotation of the FtsZ proteins in the Lactococcus and Bacillus genera
In order to screen the FtsZ proteins from the Lactococcus and Bacillus genera, we selected the well-annotated FtsZ protein in E. coli from a previous study (McQuillen and Xiao, 2020) for a comprehensive search against the assemblies of Lactococcus and Bacillus genera (Loman et al., 2012;Tatusova et al., 2014).As shown in Tables 1 and 2, a total of 19 and 22 FtsZ proteins were found in these genus groups, respectively.Particularly, we reported the occurrences of FtsZ proteins in 13 Bacillus species, namely B. anthracis, B. benzoevorans, B. aquiflavi, B. amyloliquefaciens, B. massiliigabonensis, B. carboniphilus, B. vallismortis, B. dakarensis, B. solitudinis, B. paralicheniformis, B. infantis, B. mediterraneensis, and B. methanolicus (Table 1).
Next, we also found information of the FtsZ proteins in Lactococcus sp.For instance, FtsZ proteins were identified and annotated in a variety of Lactococcus species, namely L. allomyrinae, L. chungangensis, L. cremoris, L. formosensis, L. fujiensis, L. garvieae, L. hircilactis, L. hodotermopsidis, L. insecticola, L. lactis, L. nasutitermitis, L. petauri, L. piscium, L. plantarum, L. protaetiae, L. raffinolactis, L. reticulitermitis, L. taiwanensis, and L. termiticola.The detailed information of the FtsZ proteins in Lactococcus sp. has been provided in Table 2.In this study, the CDS and full-length aa sequences from all the FtsZ proteins in the Bacillus and Lactococcus genus groups were then collected for further in silico analyses.

Analysis of conserved domains of the FtsZ proteins
In this study, we analyzed the general characteristics of the FtsZ proteins in the two genus groups using a web-based tool (Gasteiger et al., 2003;Gasteiger et al., 2005).Six features of the proteins, namely molecular lengths and weights, pI, II and AI scores, and GRAVY from the Bacillus and Lactococcus species, are subsequently provided in Tables 3 and 4, respectively.
We found that the majority (18 out of 19) of the FtsZ proteins in the Bacillus genus exhibited sizes of less than 400 aa residues (Table 3).Particularly, the sizes of the FtsZ proteins varied from 376 (in B. carboniphilus) to 410 aa residues (in B. anthracis) (Table 3).The molecular masses of the FtsZ proteins in the Bacillus genus ranged from 39.53 (in B. carboniphilus) to 44.15 kDa (in B. anthracis) (Table 3).Next, the pI scores of the FtsZ proteins in the Bacillus genus were all less than 7.0 (Table 3), which suggested that these proteins were acidic.The II scores of all the FtsZ proteins in the Bacillus species were less than 40, ranging from 27.2 (in B. amyloliquefaciens) to 38.7 (in B. infantis) (Table 3).These findings predicted that the FtsZ proteins in the Bacillus genus were stable.Additionally, the AI values of these FtsZ proteins varied from 82.3 (in B. amyloliquefaciens) to 95.5 (in B. benzoevorans) (Table 3).Interestingly, the GRAVY values of the FtsZ proteins were less than 0, ranging from -0.35 (in B. anthracis) to -0.09 (Table 3).The hydrophobicity scores of the FtsZ proteins in the Bacillus species suggested that these proteins were more likely globular.
As compared to the FtsZ proteins in the Bacillus genus, the FtsZ proteins found in the Lactococcus species were also investigated and found to share similar phenomena (Tables 3 and  4).Briefly, the protein sizes and masses of the FtsZ proteins in the Lactococcus genus varied from 387 (in L. lactis subsp.cremoris TIFN1) to 430 aa residues (in L. hodotermopsidis), and 41.14 (in L. lactis subsp.cremoris TIFN1) to 45.11 kDa (in L. hodotermopsidis), respectively (Table 4).Next, all the identified FtsZ proteins in the 22 Lactococcus species were demonstrated to be acidic (pI scores were less than 7.0) and hydrophilic (GRAVY scores were negative) (Table 4).A variety (eight out of 22) of the FtsZ proteins in the Lactococcus genus were less than 40, which suggested that these proteins were stable, whereas the remaining (14 out of 22) proteins were unstable (Table 4).Furthermore, the AI scores of these proteins were found to range from 85.56 (in L. hircilactis) to 91.54 (in L. insecticola) (Table 4).
Previously, the typical characteristics of FtsZ proteins in other bacterial genus groups have also been reported.For example, the sizes and molecular weights of the FtsZ protein found in Bartonella bacilliformis (strain KC583) were recorded to be 593 aa residues and 63.61 kDa, respectively, while the FtsZ protein in Geobacter sulfurreducens (strain PCA) exhibited a length of 384 aa residues and a mass of 40.83 kDa (Pal et al., 2019).Based on a recent report, the protein size and weight of the FtsZ molecule in E. coli (strain ATCC 47076) were found to be 383 aa residues and 40.32 kDa, respectively (McQuillen and Xiao, 2020).In Alcaligenes faecalis subsp.faecalis NCIB 8687, the FtsZ protein exhibited a size of 387 aa residues and a weight of 40.60 kDa (Pal et al., 2019).Interestingly, all the FtsZ .proteins found in these organisms were realized to be acidic and hydrophilic because the pI values were less than 7.0 and the GRAVY values were negative, respectively.

Predictions of subcellular localization and construction of 3D models of the FtsZ proteins in the Lactococcus and Bacillus genus groups
The FtsZ proteins in Bacillus were predicted to be distributed in the cytoplasm (Table 3).The probability scores were more than 90%, except for the prediction of the FtsZ protein found in B. amyloliquefaciens (Table 3).The confidence was strong (0.91) to very strong (0.99) (Table 3).Similarly, we demonstrated that all the FtsZ proteins found in the Lactococcus genus were localized in the cytoplasm with high confidence (Table 4).The percentages of probability and confidence scores of the prediction algorithms varied from 96.25 to 98.99%, and from 0.81 (strong confidence) to 0.99 (very strong confidence), respectively (Table 4).
Previously, the subcellular localization of the FtsZ proteins in several bacterial species has been reported.Briefly, the FtsZ proteins found in E. coli (strain K12) (McQuillen and Xiao, 2020), Blautia sp.(strain MCC269), A. faecalis (strain NCIB 8687), B. bacilliformis (strain KC583), and Geobacter sulfurreducens (Pal et al., 2019) were predicted to be localized in the cytoplasm with high confidence levels.Taken together, these findings strongly suggest that the FtsZ proteins in the Bacillus and Lactococcus species, and perhaps in other bacterial genera as well, are distributed in the cytoplasm.
Next, we analyzed the secondary structures and simulated 3D models of the FtsZ proteins in the Lactococcus and Bacillus species by the Phyre2 web-based platform (Kelley et al., 2015) as previously reported (La et al., 2022).Here, two elements of the secondary structure, namely the alpha-helix and beta-pleated sheets, were the focus.The alpha-helices and beta-pleated sheets of the FtsZ proteins in the Bacillus species varied from 0.35 to 0.39, and from 0.18 to 0.20, respectively (Figure 1A).This phenomenon was also reported in the FtsZ proteins in the Lactococcus genus.Particularly, the alphahelices of the FtsZ proteins in the Lactococcus species ranged from 0.32 to 0.36, whereas the beta-pleated sheets varied from 0.17 to 0.18 (Figure 1B).We found two 3D models, namely 'c2vxyA' (Figure 2A) and 'c4dxdA' (Figure 2B), representative of the FtsZ proteins in the Bacillus species.Particularly, out of the 19 FtsZ proteins found in the Bacillus species, 17 had the 'c2vxyA' model and two had the 'c4dxdA' model.Meanwhile, the FtsZ proteins found in the 22 Lactococcus species were predicted to exhibit the 'c2vxyA' model (Figure 2A).Taken together, the construction of the 3D models of the FtsZ molecules could provide a solid foundation for further functional characterization of these proteins in the Bacillus and Lactococcus genus groups.

Investigation of the core set of conserved domains in the structure of the FtsZ proteins in the Bacillus and Lactococcus genus groups
We analyzed the conserved domains of the FtsZ proteins in the Lactococcus and Bacillus species by using various software tools (Thompson et al., 1997;Thompson et al., 2002;Finn et al., 2014;Mistry et al., 2021).The multiple alignments of the FtsZ proteins found in the 19 and 22 Bacillus and Lactococcus species, respectively, and six other bacterial strains were consequently well-described (Figures 3 and 4).As compared with the typical domains of FtsZ proteins (Löwe and Amos, 1998;Silber et al., 2020) found in the Pfam domain (Finn et al., 2014;Mistry et al., 2021), the FtsZ proteins in the Lactococcus and Bacillus species clearly exhibited the existence of five distinct functional regions, namely the N-terminal peptide (NTP) region, a GTP-binding pocket, a C-terminal linker (CTL), a C-terminal tail (CTT), and a Cterminal variable region (CTV) (Figures 3 and 4).
Particularly, the NTP regions identified in the FtsZ proteins from the Lactococcus and Bacillus genus groups were both reported to contain 25 aa residues (Figures 3 and 4).The NTP regions of these FtsZ proteins were recognized to end with the highly conserved isoleucine (I) residue (Figures 3 and 4).Of interest, the GTP-binding site was the conserved core domain of the FtsZ proteins.This region was specific with the appearance of the seven-aamotif GGGTGTG (G and T meaning glycine and threonine, respectively) (Figures 3 and 4).The CTT region of the FtsZ proteins in the Lactococcus and Bacillus genus groups were recognized to contain approximately 10 aa residues (Figures 3 and 4).Among them, proline (P) and phenylalanine (F) were two highlyconserved aa found in the CTT region of the FtsZ proteins (Figures 3 and 4).Finally, the CTV region was defined as highly variable and harbored several aa residues.We found that two aa residues, arginine-lysine (RK), were highly conserved in the CTV region from the Lactococcus species (Figure 3), while three typical aa residues, leucine-arginine-asparagine (LRN), were specific in the CTV region from the Bacillus species (Figure 4).
Previously, the functional regions of the FtsZ proteins in various microorganism species have also been summarized (Silber et al., 2020).For example, the NTP domain was poorly conserved among various bacterial genera and could contain a variety of dozens of aa residues (Silber et al., 2020).Recent studies also confirmed the ending point of the NTP region with an I residue (Rossmann et al., 1974;Silber et al., 2020).For example, the NTP regions of the FtsZ proteins found in Methanococcus jannaschii and B. subtilis harbored 39 and 13 aa residues, respectively (Löwe, 1998;Raymond et al., 2009).Up until now, the function of this part has been not assigned (Silber et al., 2020).Interestingly, the GTP-binding region of the FtsZ proteins was reported to provide the interface for a head-to-tail polymerization of FtsZ proteins into proto-filaments (Scheffers et al., 2002).Next, the CTV region of the FtsZ protein found in B. subtilis was reported to be highly positively charged with six conserved aa residues (NRNKRG) (Raymond et al., 2009).This region plays an important role in the lateral interaction between FtsZ proteins and proto-filaments.The CTL region is an unstructured domain localized between the GTP-binding site and the CTT/CTV regions.The size of this region has been reported to be highly variable, up to 330 aa residues (Vaughan et al., 2004).Recently, two substrate  binding sites, namely the nucleotide-binding domain and the inter-domain binding sites, on the FtsZ proteins were well-characterized in Staphylococcus epidermidis (Vemula et al., 2023).The presence of these domains in the FtsZ proteins significantly made them perfect candidates for the development of broadspectrum inhibitors (Battaje et al., 2023;Di Somma et al., 2023).Next, a comprehensive search revealed a total of eight and 113 FtsZ proteins in diverse archaea and bacteria, respectively (Makarova and Koonin 2010).Among them, the signature GTP-binding loop sequence, as well-characterized as GGGTGTG in the Bacillus and Lactococcus genera (Figures 3 and 4), were recognized to be the GTPase loop, which plays a crucial role in the hydrolysis of GTP and the subsequent disassembly of the protein (Makarova and Koonin, 2010).

Conclusions
In this study, a total of 19 and 22 FtsZ proteins were reported in the Bacillus and Lactococcus genus groups, respectively.Our analysis indicated that the FtsZ proteins in the Bacillus and Lactococcus species were slightly variable in size, mass, and II and AI scores, while these proteins were acidic and hydrophilic.All the FtsZ proteins in the Bacillus and Lactococcus genus groups were predicted to be localized in the cytoplasm.The multiple alignments obviously indicated that the conserved domain of the FtsZ proteins contained five distinct regions.

Figure 1 .
Figure 1.The ratios of alpha and beta subunits in the FtsZ proteins in (A) Bacillus and (B) Lactococcus species

Figure 4 .
Figure 4. Functional regions of the FtsZ proteins found in the Bacillus genus group

Table 1 .
Information of the FtsZ proteins found in the Bacillus genus Note: -: No information.

Table 2 .
Information of the FtsZ proteins found in the Lactococcus genus Note: -: No information.

Table 3 .
Characteristics of the FtsZ proteins in the Bacillus genus Note: Protein size (aa residues), protein weight (kDa), pI -Iso-electric point, II -Instability index, AI -Aliphatic index, GRAVY -Grand average of hydropathicity.

Table 4 .
Characteristics of the FtsZ proteins in the Lactococcus genus