ORIGINAL RESEARCH published: 13 September 2021 doi: 10.3389/fmicb.2021.695572 Functional Characterization of the Cell Division Gene Cluster of the Wall-less Bacterium Mycoplasma genitalium Carlos Martínez-Torró 1†, Sergi Torres-Puig 1†, Marina Marcos-Silva 1, Marta Huguet-Ramón 1, Carmen Muñoz-Navarro 1, Maria Lluch-Senar 2, Luis Serrano 2, Enrique Querol 1, Jaume Piñol 1* and Oscar Q. Pich 1,3* 1 Departament de Bioquímica i Biologia Molecular, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain, 3 Laboratori de Recerca en Microbiologia i Malalties Infeccioses, Institut d’Investigació i Innovació Parc Taulí (I3PT), Hospital Universitari Parc Taulí, Universitat Autònoma de Edited by: Barcelona, Sabadell, Spain Joe Lutkenhaus, University of Kansas Medical Center Research Institute, United States It is well-established that FtsZ drives peptidoglycan synthesis at the division site in walled Reviewed by: bacteria. However, the function and conservation of FtsZ in wall-less prokaryotes such as William Margolin, mycoplasmas are less clear. In the genome-reduced bacterium Mycoplasma genitalium, the University of Texas Health Science Center at Houston, United States cell division gene cluster is limited to four genes: mraZ, mraW, MG_223, and ftsZ. In a previous Iain G. Duggin, study, we demonstrated that ftsZ was dispensable for growth of M. genitalium under University of Technology Sydney, laboratory culture conditions. Herein, we show that the entire cell division gene cluster of Australia M. genitalium is non-essential for growth in vitro. Our analyses indicate that loss of the mraZ *Correspondence: Jaume Piñol gene alone is more detrimental for growth of M. genitalium than deletion of ftsZ or the entire jaume.pinyol@uab.cat cell division gene cluster. Transcriptional analysis revealed a marked upregulation of ftsZ in Oscar Q. Pich oquijada@tauli.cat the mraZ mutant. Stable isotope labeling by amino acids in cell culture (SILAC)-based † These authors have contributed proteomics confirmed the overexpression of FtsZ in MraZ-deprived cells. Of note, we found equally to this work that ftsZ expression was upregulated in non-adherent cells of M. genitalium, which arise spontaneously at relatively high rates. Single cell analysis using fluorescent markers showed Specialty section: This article was submitted to that FtsZ localization varied throughout the cell cycle of M. genitalium in a coordinated manner Microbial Physiology and Metabolism, with the chromosome and the terminal organelle (TMO). In addition, our results indicate a a section of the journal possible role for the RNA methyltransferase MraW in the regulation of FtsZ expression at the Frontiers in Microbiology post-transcriptional level. Altogether, this study provides an extensive characterization of the Received: 15 April 2021 Accepted: 10 August 2021 cell division gene cluster of M. genitalium and demonstrates the existence of regulatory Published: 13 September 2021 elements controlling FtsZ expression at the temporal and spatial level in mycoplasmas. Citation: Martínez-Torró C, Torres-Puig S, Keywords: mycoplasmas, cell division, regulation, single cell analysis, FtsZ localization, cell cycle Marcos-Silva M, Huguet-Ramón M, Muñoz-Navarro C, Lluch-Senar M, Serrano L, Querol E, Piñol J and Pich OQ (2021) Functional INTRODUCTION Characterization of the Cell Division Gene Cluster of the Wall-less Cell division plays a central role in the life of all prokaryotic and eukaryotic organisms and Bacterium Mycoplasma genitalium. it requires the coordinated action of multiple proteins and regulatory circuits. In bacteria, Front. Microbiol. 12:695572. most genes necessary for cytokinesis and peptidoglycan wall biosynthesis are encoded in the doi: 10.3389/fmicb.2021.695572 division and cell wall (dcw) gene cluster. Organization and length of the dcw gene cluster are Frontiers in Microbiology | www.frontiersin.org 1 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium exquisitely conserved across phylogenetically distant species. of the cell division apparatus (Dai and Lutkenhaus, 1992; As an exception, several genes that lie within the dcw gene Dewar et al., 1992). In fact, it has been recently cluster of Gram-negative and Gram-positive rods, are located demonstrated that cell division in the Gram-positive bacterium elsewhere in the chromosome in Gram-positive cocci (Pucci Bacillus subtilis is mediated by filaments of FtsZ and FtsA, et al., 1997). Based on this observation, a relationship between which treadmill circumferentially around the division ring, the structure of the dcw gene cluster and cell morphology driving the motion of the peptidoglycan synthesizing enzymes was proposed. The underlying mechanism in this relationship, (Bisson-Filho et al., 2017). which involves the co-translational assembly of the protein Because of its pivotal role in cell division, FtsZ expression complexes involved in cell division, is referred to as genomic is controlled at multiple levels (Robin et al., 1990). Similarly, channeling (Mingorance et al., 2004). In mycoplasmas, the septal ring formation and localization are tightly regulated dcw gene cluster is usually limited to four genes: mraZ, mraW, (Vicente et al., 1998). The complex regulation observed within MG_223, and ftsZ (Figure 1A; Alarcón et al., 2007). Mycoplasmas the dcw gene cluster has a fundamental biological role and are phylogenetically related to Gram-positive bacteria, but they accordingly, dissociation of ftsZ expression from its natural have lost the peptidoglycan biosynthesis genes as a result of regulatory signals leads to important alterations in the physiology an extensive genome reduction. Although mycoplasma cells of cell division. However, so far, very little is known regarding are typically spherical, the presence in some species of a tip the factors controlling FtsZ expression, localization, and function structure instrumental for cytoadherence, results in an elongated, in mycoplasmas. The cell division gene cluster has an operonic flask-shaped morphology. structure in Mycoplasma genitalium and the close related species The function of mraZ and mraW, the first two genes of Mycoplasma pneumoniae (Benders et al., 2005). On the other the dcw gene cluster, has been elusive for many years. A hand, the N-terminal region of FtsZ from Mycoplasma pulmonis comprehensive study in Escherichia coli revealed that MraZ is could function for cell division in E. coli suppressor strains, a transcriptional repressor that controls its own expression provided that the C-terminal tail is replaced (Osawa and and that of other genes of the dcw gene cluster (Eraso et al., Erickson, 2006). In addition, in Mycoplasma hominis, FtsZ 2014). In the same study, the authors found that MraZ binds protofilaments can form spiral structures similar to Z-spirals to conserved sequences, designated as MraZ boxes, within the of B. subtilis and E. coli, suggesting also a functional role of upstream region of the dcw gene cluster. Perhaps surprisingly, FtsZ in cell division (Vishnyakov et al., 2009). Recent data loss of MraZ was not associated with any apparent phenotype also suggest that FtsZ stability is controlled by the Lon protease in this model bacterium. In spite of this, an antagonistic effect in M. pneumoniae (Burgos et al., 2020). between MraZ and MraW was disclosed. MraW is an RNA Remarkably, while the ftsZ gene is essential in most bacteria methyltransferase that targets the 16S ribosomal RNA (Kimura (Beall and Lutkenhaus, 1991; Dai and Lutkenhaus, 1991), it and Suzuki, 2010). Characterization of an mraW mutant in is dispensable in cell wall-deficient derivatives of some Gram- E. coli demonstrated an altered non-AUG initiation and a positive and Gram-negative bacteria known as L-forms (Mercier decreased translation fidelity, suggesting that MraW could play et al., 2014). Similarly, bacteria from the Planctomycetes and a role in start codon selection and recognition of classic STOP Chlamydiae superphylum lack the FtsZ protein (Pilhofer et al., codons. In Staphylococcus aureus, mraW mutants also exhibit 2008). In a previous study, our laboratory demonstrated that an anomalous translation fidelity, along with a reduced growth ftsZ was non-essential for in vitro growth of M. genitalium rate and an increased sensitivity to oxidative stress (Kyuma (Lluch-Senar et al., 2010), raising important questions as to et al., 2015). More recently, the MraW protein was found to the conservation of ftsZ in mycoplasmas. In keeping with this, also methylate DNA and alter gene expression in E. coli (Xu other species phylogenetically related to M. genitalium such et al., 2019). Of note, loss of MraW increases the expression as Mycoplasma mobile or Ureaplasma urealyticum, do not code of MraZ, which supports the antagonistic activity between these for a homolog of the ftsZ gene (Glass et al., 2000; Jaffe et al., two proteins described earlier (Eraso et al., 2014; Xu et al., 2019). 2004). Therefore, what is the role of the cell division genes By contrast, the function of the ancestral homologue of in mycoplasmas? We have addressed this question by constructing tubulin, FtsZ, is much better established. FtsZ polymerizes and and characterizing several cell division mutants in the genome- depolymerizes through GTP hydrolysis and forms a ring-like reduced bacterium M. genitalium. structure at the midcell known as the Z-ring that subsequently contracts during septation (Adams and Errington, 2009; Busiek and Margolin, 2015). The Z-ring recruits cell division proteins MATERIALS AND METHODS and septal peptidoglycan synthesizing enzymes to the division site, and constitutes the scaffold of the bacterial divisome Bacterial Strains and Culture Conditions (Errington et al., 2003). In E. coli, FtsA is the first protein All M. genitalium strains were grown in SP-4 broth at 37°C known to be recruited to the septal ring and it is important in a 5% CO2 atmosphere in tissue culture flasks. SP-4 for the stability of FtsZ (Vicente and Rico, 2006). Of note, plates were prepared supplementing the medium with 0.8% the ratio of FtsZ and FtsA is critical for the proper functioning agar (BD). Chloramphenicol (17 μg ml−1) or tetracycline (3 μg ml−1) were added for mutant selection. All M. genitalium Abbreviations: SEM, Scanning electron microscopy; TMO, Terminal organelle; strains used in this work are listed in the Supplementary Table S1. WGS, Whole genome sequencing. Escherichia coli strain XL-1 Blue was used for cloning and Frontiers in Microbiology | www.frontiersin.org 2 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium A B FIGURE 1 | Overview of the dcw gene cluster organization in selected bacteria. Scheme depicting the dcw gene cluster of three representative species of the Firmicutes phylum (A). Bacillus subtilis (17 genes, 18.9 kb), Staphylococcus aureus (nine genes, 10.7 kb), and Mycoplasma genitalium (four genes, 3.7 kb). (B) Scheme of the cell division gene cluster in the different M. genitalium mutants created in this study. The promoter region of the mraZ gene (PmraZ) was characterized in a previous report (Benders et al., 2005). The tetM or cat markers (in yellow) were introduced to select for the intended mutants. plasmid propagation. The strain was grown in Luria Bertani All primers used in this study are listed in the (LB) or LB agar plates containing 100 μg ml−1 ampicillin, 40 μg ml−1 Supplementary Table S2. Plasmids for M. genitalium X-Gal, and 24 μg ml−1 Isopropyl β-D-1-thiogalactopyranoside transformation were obtained using the GenElute HP Midiprep (IPTG) when needed. kit (Sigma). DNA Manipulation Mutant Construction and Screening Plasmid DNA was obtained using 5Prime kit. PCR products A detailed explanation of the methodology, including primers were purified from agarose gels using Nucleospin Gel and and plasmids, used to generate the different mutants created PCR Clean-up kit (Macherey-Nagel) and digested with the in this study is supplied in the Supplementary Material. corresponding restriction enzymes (Fermentas) when necessary. Integrity of the chromosome in the mutant strains, other than Frontiers in Microbiology | www.frontiersin.org 3 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium the intended deletions, was assessed and confirmed by whole RNA was extracted using the miRNeasy Mini Kit (Qiagen). genome sequencing (WGS; Supplementary Table S3). RNA libraries were prepared with TruSeq Stranded Total RNA Transformation of M. genitalium was carried out as previously Library Prep Kit (Illumina) and analyzed using a HiSeq 3000 described (Torres-Puig et al., 2015). Screening for mutants was System (Illumina) at the Genomics Unit from the Centre for performed using cell lysates as template for PCR or sequencing Genomic Regulation (CRG), Barcelona. cDNA clusters were reactions. Cell lysates were obtained by centrifugation of 1.5 ml immobilized in sequencing lanes of 2 × 50 reads. Reverse and cell cultures, disruption of pellets using 30 μl of Lysis Buffer complementary were computed for sequences coming from Read1 (Tris-HCl 0.1 M pH8.5, Tween-20 0.05%, and proteinase K primer. Data analysis and sequence alignment were performed 0.25 mg ml−1), and incubation for 1 h at 37°C followed by using Bowtie2 (Langmead and Salzberg, 2012) in the End-to-End inactivation at 95°C for 10 min. mode and Forward-Forward paired-ends. Sequences were piled up using SAMtools (Li et al., 2009) with no limited set to the DNA Sequencing number of sequences in the alignment. Counts in the different DNA sequencing reactions were performed using BigDye® v3.1 ORFs were performed with a standalone version of featureCounts Cycle Sequencing kit using 2.5 μl of genomic DNA or program (Liao et al., 2014) without counting the multi-mapping M. genitalium lysate, following manufacturer’s instructions. All reads and disabling multi-overlapping reads. reactions were analyzed in an ABI PRISM 3130xl Genetic Counted features were then submitted to the R/Bioconductor Analyzer at the Servei de Genòmica i Bioinformàtica (UAB). package DESeq2 (Love et al., 2014) for statistical analysis. DESeq2 analysis used a parametric fitType and a zero-mean normal prior on the non-intercept coefficients. Data were sorted Growth Rate Quantification by log2 fold change, and statistical significance was set at the Growth rates of M. genitalium cultures were determined using common threshold value of p < 0.05. Three independent biological an adaptation of the colorimetric protocol described by Karr repeats of each strain or condition were analyzed. et al. (2012). Strains were grown to mid-log phase in 25 cm2 flasks with 5 ml SP4. Attached cells were scraped off, recovered by centrifugation at 12,000 rpm, and resuspended in 3 ml of qRT-PCR fresh SP4. Then, 300 μl of the cellular suspension were inoculated RNA was extracted from mid-log phase cultures of M. genitalium in four different wells of a 96-well plate. Each of these wells using the RNAqueous Kit (Thermo Fisher Scientific) and constitutes the first sample of four technical repeats. About then treated with Turbo DNase (Thermo Fisher Scientific) 100 μl of the first well (sample 1) were inoculated into the following the manufacturer’s instructions. Reverse transcription next well (sample 2) already containing 200 μl of fresh SP4, was performed with iScript Reverse Transcriptase thus diluting 1/3 the initial concentration. This process was (Bio-Rad) and random primers as previously described (Torres- repeated four more times (samples 3, 4, 5, and 6), achieving Puig et al., 2015). Primers used for qPCR are listed in a final dilution of 1/243. The 96-well plate was sealed with Supplementary Table S2 and they were designed using Primer3 transparent tape, placed into a Tecan Sunrise Absorbance software. qPCR was performed with iTaq polymerase (Bio-Rad) Microplate Reader (Tecan), and incubated at 37°C for 7 days. and SYBR green in CFX96 or CFX384 PCR instruments (Bio- As M. genitalium grows, the bacterial metabolism acidifies the Rad). Relative gene expression was calculated using the Pfaffl culture medium changing the color of the phenol red indicator method (Pfaffl, 2001). Differential gene expression was judged from red (pH 7.8) to yellow (pH 6), which was detected by based on the common arbitrary 2-fold cutoff. Data presented measuring the absorbance at 550 nm. For each analysis, readings in the manuscript correspond to the analysis of RNAs isolated were taken every 30 min for up to 8 days (total of 400 reads from three independent biological repeats. per well). The readings were stored in an Excel datasheet and analyzed once the experiment was completed, and a curve Quantification of Protein Abundance was plotted for each dilution and the inflection point was Differences in relative protein abundance were assessed by stable inferred. Next, the inflection points were plotted in a graphic, isotope labeling by amino acids in cell culture (SILAC). To using the Napierian logarithm of the dilution as the x coordinate this end, M. genitalium cultures were grown to mid-log phase for each dilution. Once all the inflection points were plotted, in 25 cm2 flasks on Hayflick’s medium with 15 mM of light the slope (μ, growth rate constant) was inferred by linear (12C) or heavy (13C) lysine. Once grown, cells were split (1:10) regression, and the doubling time (g) was obtained and according and cultured in the same conditions. Then, cells were washed to the general equation for exponential growth of bacteria with PBS, scraped off, and pellets stored at −80°C. Protein [g = ln2/(1/μ)]. lysates were prepared as previously described (Sabidó et al., 2012). Protein quantification was performed using the Pierce RNA-Seq BCA Protein Assay Kit (Thermo Fisher Scientific), and protein RNA-Seq analyses were performed as previously described (Torres- extracts from heavy and light media cultures were mixed at a Puig et al., 2018; Martínez-Torró et al., 2020). Mid-log phase 1:1 ratio. Samples were then reduced with dithiothreitol (150 nmol, cultures of M. genitalium were scraped off in 1 ml of fresh SP-4 1 h, 37°C) and alkylated in the dark with iodoacetamide (300 nmol, and reinoculated in two new 25 cm2 tissue culture flasks with 30 min, 25°C). The resulting protein extract was diluted 1/3 fresh SP-4 medium for 6 h. Then, cells were lysed, and total with 200 mM NH4HCO3 and digested with 5 μg LysC (Wako) Frontiers in Microbiology | www.frontiersin.org 4 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium overnight at 37°C. Finally, the peptide mix was acidified with Phase Contrast and Fluorescence formic acid and desalted with a homemade Empore C18 column Microscopy (3 M, St. Paul, MN, United States; Rappsilber et al., 2007). Mycoplasma genitalium cells were grown in filtered SP-4 medium Samples were analyzed using an LTQ-Orbitrap Velos Pro mass (0.22 μm) on IBIDI chamber slides for 16 h, washed once with spectrometer (Thermo Fisher Scientific, San Jose, CA, 1 × PBS, and visualized on an inverted Nikon Eclipse TE 2000-E United States) coupled to an EasyLC [Thermo Fisher Scientific microscope. For the analysis of the mg191ftsZCh non-adherent (Proxeon), Odense, Denmark]. Peptides were loaded directly mutant, IBIDI chamber slides were previously treated with onto the analytical 25-cm column with an inner diameter of 0.2 mg/ml poly-l-lysine hydrobromide (Sigma-Aldrich) solution, 75 μm and packed with 5-μm C18 particles (Nikkyo Technos allowing surface adherence. In addition, cells were passed 10 Co. Ltd., Japan). Chromatographic gradients started at 97% times through a 25-gauge syringe to break the aggregates. buffer A and 3% buffer B, with a flow rate of 250 nl/min, and Hoechst 33342 0.01 mg ml−1 was added when necessary. All gradually increased to 65% buffer A/35% buffer B over 360 min. strains were grown and visualized under the same conditions. After each analysis, the column was washed for 10 min with Phase contrast, enhanced yellow fluorescent protein (eYFP), 10% buffer A/90% buffer B. Buffer A: 0.1% formic acid in 4',6-diamidino-2-phenylindole (DAPI), and TRITC water. Buffer B: 0.1% formic acid in acetonitrile. The mass epifluorescence images were captured with a Digital Sight spectrometer was operated in positive ionization mode with a DS-SMC Nikon camera controlled by NIS-Elements BR software. nanospray voltage set at 2.2 kV and source temperature at 250°C. Images were analyzed using ImageJ software and GDSC plug-in. Ultramark 1621 for the FT mass analyzer was used for external calibration prior to the analyses. Moreover, an internal calibration was also performed using the background polysiloxane ion RESULTS signal at m/z 445.1200. The instrument was operated in data- dependent acquisition (DDA) mode and full MS scans with Deletion of the Entire Cell Division Gene one micro scan at a resolution of 60,000 were used over a Cluster of Mycoplasma genitalium mass range of m/z 350–2,000 with detection in the Orbitrap. Previously, we demonstrated that the ftsZ gene was non-essential Auto gain control (AGC) was set to 106, and dynamic exclusion in M. genitalium (Lluch-Senar et al., 2010). Based on this (60 s) and charge state filtering disqualifying singly charged finding, we speculated whether the entire cell division gene peptides were carried out. In each cycle of DDA analysis, cluster could be dispensable in this wall-less bacterium. To following each survey scan, the top 10 most intense ions with ascertain this, we replaced the four genes of the cell division multiple charged ions above a threshold ion count of 5,000 gene cluster of M. genitalium by the chloramphenicol resistance were selected for fragmentation at normalized collision energy marker (Figure 1B). We recovered several chloramphenicol of 35%. Fragment ion spectra produced via collision-induced resistant transformants and confirmed the deletion of the entire dissociation (CID) were acquired in the ion trap, AGC was gene cluster (3.7 kb) by PCR and sequencing. We found that set to 5e4, with an isolation window of 2.0 m/z, activation the duplication time of the resulting dcw mutant was significantly time of 0.1 ms, and maximum injection time of 100 ms. All longer (10.13 ± 0.33 h) than that of the wild-type strain data were acquired with Xcalibur software v2.2. The MaxQuant (8.38 ± 0.28 h; Figure 2A). In addition, SEM analyses revealed software suite (v1.4.0.5) was used for peptide identification and an increased frequency of cells in division in the dcw mutant SILAC protein quantitation (Cox and Mann, 2008). The data (30.53%) as compared to the wild-type strain (13.35%; were searched against an in-house generated database containing Figure 2B). all M. genitalium proteins. Scanning Electron Microscopy Characterization of Cell Division Mutants Mycoplasma genitalium cultures were grown to mid-log phase of Mycoplasma genitalium over glass coverslips. Samples were fixed, dehydrated, and critical To dissect and better understand the phenotype of the dcw point dried as previously described (Pich et al., 2008). Coverslips mutant, we created strains defective for each gene of the cell were then sputter coated with gold and examined in a Merlin division gene cluster by allelic exchange. First, we obtained (Zeiss) scanning electron microscope (SEM). SEM micrographs mutants for the MG_223 and ftsZ genes using the were analyzed with the ImageJ software to determine the frequency chloramphenicol resistance marker for selection (Figure 1B; of cells in division. To this end, we determined the number of Supplementary Material). Our analyses revealed that the growth terminal organelles (TMOs) per cell. As mycoplasma cells duplicate rate of the ftsZ mutant (8.29 ± 0.09 h) was comparable to that the tip structure during cell division, cells with one TMO were of the wild-type strain (8.38 ± 0.28 h; Figure 2A). By contrast, classified as non-dividing, while cells with two or more TMOs the duplication time of the MG_223 mutant increased significantly were classified as dividing. On the other hand, we measured (9.80 ± 0.56 h). SEM analyses revealed an increased frequency the length of single cells from the tip of the TMO to the of cells in division in both mutant strains (ftsZ, 22.44%; MG_223, opposite pole. In addition, we determined the length of cytokinetic 29.15%; Figure 2B). cells by measuring the distance from the tip of one TMO to Next, we created mutants lacking either mraZ or mraW. In the tip of the opposite TMO. For this analysis, only cells with this case, to preserve the operonic structure of the gene cluster, one TMO at each cell pole were considered. the antibiotic resistance marker was placed at the 5' end of the Frontiers in Microbiology | www.frontiersin.org 5 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium A B C FIGURE 2 | Cell fitness analysis of the wild-type strain and several M. genitalium mutants. Growth rates (A) and percentage of cells in division (B) of different M. genitalium mutants of the cell division gene cluster. Statistical significance was assessed using the Student’s t-test and the value of p of significant values (p < 0.05) is indicated above each data point. An average of 900 cells for each strain were analyzed (C). Graph depicting the length of single or cytokinetic cells. An average of 320 cells for each strain were measured. mraZ UTR (Figure 1B; Supplementary Material). For control presence of small variants like SNPs or INDELS in different purposes, we also created a reference strain, designated mraREF, frequencies. Many of these variants were also identified in the carrying the selectable marker in the same chromosomal location, G37 strain. All new variants were found in recombinogenic, but with an intact cell division gene cluster (Figure 1B; variable repeated regions, being most of them in non-coding Supplementary Material). Cells from the mraREF strain grew genome locations, thus supporting the absence of additional at a similar rate (8.81 ± 0.29 h) than those of the wild-type strain mutations that could impact on the phenotype of the dcw strains (8.38 ± 0.28 h; Figure 2A). However, the duplication time increased (Supplementary Material). significantly upon the loss of MraZ (11.54 ± 0.38 h). Cells from Scanning electron microscopy analyses revealed a slight the mraW null mutant duplicated at a slower rate (9.96 ± 0.53 h), increase in the frequency of cells in division in the mraW indicating that the absence of MraW is also detrimental for mutant (19.0%) when compared to the wild-type (13.35%) or growth. All the mutant strains obtained were examined by the mraREF strains (12.99%; Figure 2B). Remarkably, the genome resequencing for the presence of mutations other than frequency of dividing cells found in the mraZ mutant was those introduced by genetic manipulation. No major genome markedly high (38.44%). Moreover, we observed that cytokinetic rearrangements like large deletions or translocations were detected cells from the mraZ mutant were markedly elongated in all the analyzed strains. However, all the strains showed the (Figures 3A,B). Quantitative analysis confirmed that cells in Frontiers in Microbiology | www.frontiersin.org 6 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium cytokinesis from the mraZ mutant were longer (1.651 ± 0.372 μm, transcription of the cell division gene cluster in a mutant n = 96) than those of the wild-type strain (1.118 ± 0.177 μm, strain lacking the mg191 gene, which codes for the major n = 24; Figure 2C). By contrast, analyses of the other cell cytoadhesin P140 of M. genitalium. Cells from the mg191 division mutants obtained in this study did not reveal mutant are pleomorphic and grow in suspension as large cell morphological changes in the cytokinetic cells (Figure 2C). aggregates (Burgos et al., 2006). This non-adherent mutant was analyzed to shed some light on the connection between Transcriptional Analysis of the Cell cell division and adherence in M. genitalium. In this sense, Division Gene Cluster in Different Mutant in a previous study (Lluch-Senar et al., 2010), we showed Backgrounds that, in contrast to WT cells, an ftsZ mutant could not generate Previous studies in E. coli indicated that MraZ is a transcriptional non-adherent variants. Based on this observation, repressor (Eraso et al., 2014). Therefore, we conducted a we hypothesized that non-adherent mutants rely exclusively genome-wide transcriptional analysis in M. genitalium by on the cell division cluster for division. Remarkably, RNA-Seq to identify transcriptional changes in the mraZ transcriptional analysis of the mg191 mutant revealed a mutant (Table 1). Our transcriptomic data revealed that mraW, significant upregulation of the cell division gene cluster ftsZ, and the MG_223 gene were significantly upregulated in (Figure 4). the absence of MraZ. By contrast, the transcription was largely unaffected in both the mraW mutant and the mraREF control Proteome Analysis of the mraZ and mraW strain. qRT-PCR analyses confirmed the increased expression Mutants of the cell division genes in the absence of MraZ (Figure 4). Next, we wondered whether the transcriptional changes observed To confirm that the altered gene expression observed in the in the mraZ null mutant were conserved at the protein level. mraZ mutant was due to the loss of the MraZ protein, To this end, we determined the proteome profile of the mraZ we reintroduced an ectopic copy of the mraZ gene under the mutant by SILAC (Supplementary Tables S4 and S5). Our control of its native promoter. Of note, several complementation analyses demonstrated a marked increase in the cellular levels attempts with a transposon encoded copy of the mraZ gene, of MraW (14.3-fold) and FtsZ (25-fold; Table 2). Along the alone or in combination with mraW, were unsuccessful. However, same lines, expression of the MG_223 protein could only insertion of the mraZ gene at the end of the cell division be detected when MraZ was absent. gene cluster in the mraZ mutant restored the transcriptional We also investigated the presence of proteomic changes in levels of mraW, ftsZ, and the MG_223 gene (Figure 4; the mraW mutant (Supplementary Tables S6 and S7). We found Supplementary Figure S1). On the other hand, we also assessed that several proteins, including a number of DNA and RNA methyltransferases, were differentially expressed in the absence of MraW. As these changes in protein abundance were not accompanied by changes in mRNA levels, we anticipate that MraW may modulate protein expression at the post- transcriptional level. Of note, we found that the levels of the FtsZ protein were slightly higher (by 2-fold) in the mraW mutant (Table 2). FtsZ Expression and Localization Dynamics Previous attempts in our laboratory to monitor FtsZ expression in M. genitalium using fluorescent markers were unsuccessful. In these studies, we characterized mutant strains carrying an TABLE 1 | Transcriptional changes in the mraZ and mraW mutants by RNA-Seq. mraZ mraW Gene Log2 fold change p Log2 fold change p mraZ −4.64 0 0.10 0.24 mraW 3.02 0 −3.84 0 MG_223 2.08 2 × 10−139 −0.21 0.02 ftsZ 2.34 9.3 × 10−218 −0.21 0.03 Transcriptional changes in the cell division gene cluster identified by RNA-Seq in the FIGURE 3 | Characterization of cell morphology of the mraZ mutant. mraZ and mraW mutants. Changes that are statistically significant (value of p < 0.05) Scanning electron micrographs of the wild-type strain (A) and the mraZ and biologically relevant (above or below the log2 ± 1 arbitrary cutoff) are highlighted in bold. Data were obtained from the analysis of three independent biological repeats of mutant (B). White arrows indicate some cells in cytokinesis. each mutant. Frontiers in Microbiology | www.frontiersin.org 7 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium A B FIGURE 4 | Transcriptional analysis of the cell division gene cluster of several M. genitalium mutants by qRT-PCR. Transcriptional changes of the cell division gene cluster in the mraREF mutant (A) or the mraZ, mraW, and mg191 mutants (B) compared to the wild-type strain. Bars represent the average log2 fold change of at least three independent biological repeats. Statistical significance was assessed using the Student’s t-test, and the value of p of biologically significant values (log2 ± 1) is stated above the corresponding bar if significant (value of p < 0.05). TABLE 2 | Comparative analysis of the abundance of cell division proteins in the wild-type strain and the mraZ and mraW mutants by stable isotope labeling by amino acids in cell culture (SILAC). G37 mraZ mraW Locus tag Gene Gene product AUC AUC FC AUC FC MG_221 mraZ Transcriptional regulator MraZ 4.12 × 108 ND – 4.65 × 108 – MG_222 mraW Ribosomal RNA small subunit 4.17 × 107 5.95 × 108 14.28 ND – methyltransferase H MG_223 – Uncharacterized protein ND 7.88 × 106 – ND – MG_224 ftsZ Cell division protein 4.40 × 106 1.10 × 108 24.96 8.50 × 106 1.93 Area under the curve (AUC) of the four proteins encoded in the cell division gene cluster in the wild-type (G37) and the mraZ and mraW mutants. Fold-change (FC) with respect to the G37 strain is indicated. Biologically significant fold changes (>2 and <0.5) are highlighted in bold. ND stands for not detected. ftsZ-mcherry fusion at its native locus, that is, at the 3' end we observed that some cells exhibited a conspicuous FtsZ- of the cell division gene cluster (Figure 5). Presumably, FtsZ associated fluorescence (Figure 5). expression was too low to allow detection of the fluorescent A detailed analysis of the mraZ mutant revealed that FtsZ fusion in single cells. However, we hypothesized that the foci displayed a polar location. Of note, M. genitalium cells increased levels of FtsZ expression observed in the mraZ mutant are polar because they exhibit a TMO instrumental for could facilitate the visualization of this protein in live cells. cytoadherence. For this reason, we wanted to test whether Therefore, we assessed FtsZ-mCherry expression in MraZ- FtsZ foci co-localized with the TMO of this bacterium. To deprived cells (Supplementary Material) and found a large this end, we tagged P65, a protein that localizes at the distal number (56.63%) of fluorescent cells, although at different end of the TMO (Burgos et al., 2008). Introduction of an extents (Figure 5). The presence of non-fluorescent cells suggests eYFP fluorescent marker at the P65 native locus (MG_217) that the expression and assembly of FtsZ are under the control allowed the identification of sharp P65-eYFP foci, providing of other factors in addition to MraZ (Burgos et al., 2020). a way to unambiguously identify the cell pole containing the Separately, we assessed FtsZ expression in the absence of MraW TMO (Figure 6). We found that FtsZ clustered at the cell and observed that a small subset of cells (1.58%) exhibited pole opposite to P65 in cells showing a single P65 focus. This mCherry fluorescence (Figure 5). This result is in keeping is important because as mycoplasma cells duplicate the TMO with the increased amount of FtsZ identified in the mraW prior to cytokinesis (Miyata and Seto, 1999), some cells displayed mutant by proteomics analysis. On the other hand, we tested two separate P65 foci. In addition, we observed that the intensity whether the FtsZ-mCherry fusion was visible in the P140 and localization of the FtsZ foci varied through the cell division adhesin mutant. In agreement with the transcriptional data, cycle. Based on the localization of the two tagged proteins Frontiers in Microbiology | www.frontiersin.org 8 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium FIGURE 5 | Single cell analysis of FtsZ expression in different M. genitalium mutants. Fluorescence microscopy images showing FtsZ-mCherry localization in live cells from the wild-type strain, mraZ, mraW, and mg191 mutants. Each row contains the phase contrast, TRITC channel, and the resulting overlay, respectively. The scale bar represents 2 μm. All images are shown at the same magnification. (FtsZ and P65) and the chromosome stained with Hoechst DISCUSSION 33258, we recognized eight different stages throughout the cell cycle (Figure 7). Non-dividing cells (stage 1) exhibit a single Mycoplasmas are wall-less bacteria that divide by binary fission. P65 focus and FtsZ signal is weak. In time, FtsZ is recruited So far, the factors mediating and coordinating cell division in at the cell pole opposite to the TMO (stage 2). Prior to these genome-reduced bacteria are poorly understood. Typically, cytokinesis, a new P65 focus forms close to the pre-existent the cell division gene cluster of mycoplasmas comprises four focus (stage 3). Then, one of these P65 foci migrates to the genes, including ftsZ. In walled bacteria, it is well-established opposite cell pole and localizes near the FtsZ focus (stage 4). that FtsZ drives peptidoglycan synthesis at the division site Next, likely coinciding with chromosome segregation, FtsZ (Bisson-Filho et al., 2017). However, the conservation in leaves the polar location and moves to the midcell to form mycoplasmas of FtsZ and other cell division related proteins, a diffuse band (stage 5). As cytokinesis proceeds, FtsZ signal such as MraZ or MraW, is puzzling. In this study, we constructed decreases (stage 6) and daughter cells display small FtsZ foci and characterized several cell division mutants of M. genitalium. with marginal fluorescence (stage 7). Finally, daughter cells We found that deletion of mraZ induces a strong activation of separate and FtsZ signal disappears (stage 8), but soon after, the other genes of the cell division gene cluster. Hence, our FtsZ accumulation starts within the cell body (stage 1). results indicate that MraZ functions as a transcriptional repressor. Frontiers in Microbiology | www.frontiersin.org 9 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium FIGURE 6 | Single cell analysis of FtsZ, P65, and chromosome localization in the mraZ mutant. Fluorescence microscopy analysis showing FtsZ-mCherry, P65-eYFP, and chromosome localization in live cells of M. genitalium. Each panel shows the TRITC, 4',6-diamidino-2-phenylindole (DAPI), and eYFP images or a combination of the three channels and the phase contrast. The scale bar represents 2 μm. All images are shown at the same magnification. This finding correlates well with proteomics data showing that occurrence of this motif in the chromosome of M. genitalium MraW, FtsZ, and MG_223 proteins are overexpressed in the lays within the promoter region of the mraZ gene. This finding absence of MraZ. Altogether, our results substantiate the repressor is consistent with the lack of transcriptional changes outside role of MraZ previously documented by Eraso et al. (2014). the cell division gene cluster in our mraZ mutant. Of note, Unlike in E. coli, MraZ depletion in M. genitalium results in a complementation of the mraZ mutant of M. genitalium could clear phenotype. The mraZ mutant exhibits a significant growth only be achieved in cis, that is, reintroducing a copy of the delay, important morphological alterations, and a high frequency mraZ gene at an ectopic site within the cell division gene cluster. of cells stuck in cytokinesis. These data indicate that expression This result reveals a complex interplay between the relative of the cell division proteins of M. genitalium at relative high location of the mraZ gene in the chromosome and MraZ activity. levels throughout the cell cycle is largely detrimental. In this sense, our results indicate that the repressor role of A previous study in Mycoplasma gallisepticum revealed that MraZ requires a transcriptional linkage between the mraZ gene, MraZ overexpression induces a characteristic cell filamentation the MraZ operator, and the cell division gene cluster. (Fisunov et al., 2016), which is reminiscent to the phenotype On the other hand, we found that cultures of the mraW described in the mraZ mutant of M. genitalium. Moreover, the mutant grew at a slower rate than those of the wild-type filamentation described in M. gallisepticum was also associated strain. More importantly, we could detect FtsZ-mCherry with increased levels of FtsZ. In the same study, the authors fluorescence in a small subset of cells from this mutant. This define a binding site for MraZ in Mollicutes, characterized by fact is relevant because we could not detect FtsZ-mCherry a series of direct repeats of the sequence AAAGTG[T/G]. The only expression in the wild-type background. Thus, MraW may Frontiers in Microbiology | www.frontiersin.org 10 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium FIGURE 7 | FtsZ localization dynamics throughout the cell cycle of M. genitalium. Scheme depicting different stages of the M. genitalium cell cycle based on the localization of FtsZ foci relative to the terminal organelle (TMO) and the chromosome. In this model, FtsZ accumulates at the cell body (1) and clusters at the cell pole opposite to the TMO (2). Then, the TMO duplicates (3) and one TMO moves to the opposite cell pole (4). Next, FtsZ foci move to the midcell (5). Finally, cytokinesis starts (6) and the FtsZ signal decreases (7) until the process is completed (8). play a role in regulating FtsZ expression in M. genitalium. role in DNA or rRNA methylation in this bacterium still This regulation must take place at the post-transcriptional needs to be fully addressed. level, as no transcriptional changes could be detected in the The increased levels of FtsZ expression in the mraZ mutant mraW mutant. In agreement with these data, our proteomics allowed us to visualize this protein in single cells and to determine analysis revealed a slight increase of FtsZ levels in the absence the FtsZ dynamics throughout the cell cycle of M. genitalium. of MraW. Of note, FtsZ expression in the mraW mutant is We found that FtsZ clusters at the cell pole opposite to the expected to be low because of the repressor activity of MraZ. TMO. When TMOs duplicate and migrate to the opposite cell The differential expression of several methyltransferases in the pole, FtsZ-associated foci move toward the midcell. Intensity of mraW mutant was also noticeable, which may represent a the FtsZ foci decreases when they arrive at the midcell and response of M. genitalium to the loss of the methyltransferase the two chromosomes start to segregate. This pattern strongly activity of MraW. The existence of species with orphan mraW suggests that FtsZ might play an active role in cell division and genes supports a role for MraW independent from MraZ facilitates cytokinesis in M. genitalium. In addition, the localization function in the regulation of cell division. It has been reported of FtsZ protein during the cell cycle seems to be tightly regulated. that MraW methylates the C1402 residue of the 16S rRNA Although the presence of a Min system (Lutkenhaus, 2007; (Kimura and Suzuki, 2010). This residue is involved in translation Rowlett and Margolin, 2015) has not been described in initiation and its mutation in E. coli leads to an increased mycoplasmas, it is presumed that unidentified regulatory factors doubling time (Jemiolo et al., 1985). Moreover, the loss of inhibit the formation of a Z-ring close to the TMO. Interestingly, MraW function has been directly related to an altered translation M. genitalium codes for a DivIVA domain-containing protein fidelity (Kyuma et al., 2015), DNA methylation, and regulation (MG211), a key component of the Min system of B. subtilis of gene expression (Xu et al., 2019). Our RNA-Seq data from (Edwards and Errington, 1997). In addition, we observed that the mraW mutant suggest that MraW does not regulate gene FtsZ foci were sometimes located between two DNA foci, expression in M. genitalium. However, whether MraW has a suggesting that nucleoid occlusion is also in place in M. genitalium. Frontiers in Microbiology | www.frontiersin.org 11 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium Altogether, the distinct localization of the FtsZ protein throughout Biotechnology Information (NCBI) BioProject database under the cell cycle suggests the existence of unknown factors regulating accession number PRJNA750275. cell division in this genome-reduced bacterium. These underlying regulatory mechanisms coordinate the cytokinesis process with chromosome segregation (Seto and Miyata, 1999). AUTHOR CONTRIBUTIONS The results of our study indicate that the cell division gene cluster of M. genitalium plays a minor role during in vitro CM-T, ST-P, ML-S, LS, EQ, JP, and OQP contributed to propagation of wild-type cells. This is consistent with previous conception and design of the study. CM-T, ST-P, MM-S, MH-R, reports showing that motility seems to be the primary force CM-N, ML-S, and OQP performed the experiments. CM-T facilitating cytokinesis in M. pneumoniae, a close relative species and ST-P performed the statistical analysis. CM-T and OQP (Hasselbring et al., 2006). A few years ago, we documented wrote the first draft of the manuscript. ST-P, EQ, and JP wrote that deletion of the ftsZ gene abrogated the occurrence of some parts of the manuscript. All authors contributed to the non-adherent phase variants in M. genitalium (Lluch-Senar article and approved the submitted version. et al., 2010). These non-adherent variants arise at relatively high frequencies and may play an important role to evade the immune response during infection (Burgos et al., 2018). FUNDING Based on this observation, we proposed that FtsZ could be important for cell division in non-adherent cells of M. This work was supported by the grant BIO2017-84166-R from genitalium. Of note, non-adherent derivatives of M. genitalium the Ministerio de Ciencia, Innovación y Universidades. are non-motile, largely pleomorphic and grow as large cell aggregates (Burgos et al., 2006). In the current study, we provide evidence that the cell division gene cluster is upregulated in ACKNOWLEDGMENTS a non-adherent mutant of M. genitalium. This finding is in agreement with our previous hypothesis that FtsZ and the We are grateful to the staff of the Servei de Genòmica formation of a rudimentary divisome may be required when i Bioinformàtica (UAB), the Genomics Unit (CRG) and the gliding motility is unfeasible. Therefore, the strict parasitic Proteomics Unit (CRG), for performing Sanger sequencing, lifestyle of M. genitalium and the intricate strategy to escape RNA-Seq and SILAC analyses, respectively. In addition, we immune surveillance, likely imposes the conservation of the are thankful to the thesis committee of CM-T and the two cell division gene cluster in this wall-less bacterium. Remarkably, reviewers of this manuscript for their helpful comments most Mycoplasma species are non-motile, which may also and constructive discussion. Finally, CM-T and MM-S want enforce the conservation of the cell division genes. Supporting to acknowledge a PIF fellowship from the UAB and a FI this notion, a recent report has demonstrated that ftsZ is predoctoral fellowship from the Generalitat de Catalunya, important to restore cell morphology and proficiency of cell respectively. division in synthetic minimal cells (Pelletier et al., 2021). SUPPLEMENTARY MATERIAL DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online The original contributions presented in the study are publicly at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.695572/ available. This data can be found here: National Center for full#supplementary-material REFERENCES Burgos, R., Pich, O. Q., Ferrer-Navarro, M., Baseman, J. B., Querol, E., and Piñol, J. (2006). Mycoplasma genitalium P140 and P110 cytadhesins are Adams, D., and Errington, J. (2009). Bacterial cell division: assembly, maintenance reciprocally stabilized and required for cell adhesion and terminal-organelle and disassembly of the Z ring. Nat. Rev. Microbiol. 7, 642–653. doi: 10.1038/ development. J. Bacteriol. 188, 8627–8637. doi: 10.1128/JB.00978-06 nrmicro2198 Burgos, R., Pich, O. Q., Querol, E., and Piñol, J. (2008). Deletion of the Alarcón, F., de Vasconcelos, A. T. R., Yim, L., and Zaha, A. (2007). Genes Mycoplasma genitalium MG_217 gene modifies cell gliding behaviour by involved in cell division in mycoplasmas. Genet. Mol. Biol. 30, 174–181. altering terminal organelle curvature. Mol. Microbiol. 69, 1029–1040. doi: doi: 10.1590/S1415-47572007000200003 10.1111/j.1365-2958.2008.06343.x Beall, B., and Lutkenhaus, J. (1991). FtsZ in Bacillus subtilis is required for Burgos, R., Weber, M., Martinez, S., Lluch-Senar, M., and Serrano, L. (2020). vegetative septation and for asymmetric septation during sporulation. Genes Protein quality control and regulated proteolysis in the genome-reduced organism Dev. 5, 447–455. doi: 10.1101/gad.5.3.447 Mycoplasma pneumoniae. Mol. Syst. Biol. 16:e9530. doi: 10.15252/msb.20209530 Benders, G. A., Powell, B. C., and Hutchison, C. A. (2005). Transcriptional Burgos, R., Wood, G. E., Iverson-Cabral, S. L., and Totten, P. A. (2018). analysis of the conserved ftsZ gene cluster in Mycoplasma genitalium and Mycoplasma genitalium nonadherent phase variants arise by multiple Mycoplasma pneumoniae. J. Bacteriol. 187, 4542–4551. doi: 10.1128/ mechanisms and escape antibody-dependent growth inhibition. Infect. Immun. JB.187.13.4542-4551.2005 86:e00866-17. doi: 10.1128/IAI.00866-17 Bisson-Filho, A. W., Hsu, Y. P., Squyres, G. R., Kuru, E., Wu, F., Jukes, C., Busiek, K. K., and Margolin, W. (2015). Bacterial actin and tubulin homologs et al. (2017). Treadmilling by FtsZ filaments drives peptidoglycan synthesis in cell growth and division. Curr. Biol. 25, R243–R254. doi: 10.1016/j. and bacterial cell division. Science 355, 739–743. doi: 10.1126/science.aak9973 cub.2015.01.030 Frontiers in Microbiology | www.frontiersin.org 12 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium Cox, J., and Mann, M. (2008). MaxQuant enables high peptide identification response to metal starvation in the emerging pathogen Mycoplasma rates, individualized p.p.b.-range mass accuracies and proteome-wide genitalium is mediated by fur-dependent and – independent protein quantification. Nat. Biotechnol. 26, 1367–1372. doi: 10.1038/ regulatory pathways. Emerg. Microbes Infect. 9, 5–19. doi: 10.1080/ nbt.1511 22221751.2019.1700762 Dai, K., and Lutkenhaus, J. (1991). ftsZ is an essential cell division gene Mercier, R., Kawai, Y., and Errington, J. (2014). General principles for the in Escherichia coli. J. Bacteriol. 173, 3500–3506. doi: 10.1128/ formation and proliferation of a wall-free (L-form) state in bacteria. eLife jb.173.11.3500-3506.1991 3:e04629. doi: 10.7554/eLife.04629 Dai, K., and Lutkenhaus, J. (1992). The proper ratio of FtsZ to FtsA is required Mingorance, J., Tamames, J., and Vicente, M. (2004). Genomic channeling in for cell division to occur in Escherichia coli. J. Bacteriol. 174, 6145–6151. bacterial cell division. J. Mol. Recognit. 17, 481–487. doi: 10.1002/jmr.718 doi: 10.1128/jb.174.19.6145-6151.1992 Miyata, M., and Seto, S. (1999). Cell reproduction cycle of Mycoplasma. Biochimie Dewar, S. J., Begg, K. J., and Donachie, W. D. (1992). Inhibition of cell division 81, 873–878. doi: 10.1016/S0300-9084(99)00209-6 initiation by an imbalance in the ratio of FtsA to FtsZ. J. Bacteriol. 174, Osawa, M., and Erickson, H. P. (2006). FtsZ from divergent foreign bacteria 6314–6316. doi: 10.1128/jb.174.19.6314-6316.1992 can function for cell division in Escherichia coli. J. Bacteriol. 188, 7132–7140. Edwards, D. H., and Errington, J. (1997). The Bacillus subtilis DivIVA doi: 10.1128/JB.00647-06 protein targets to the division septum and controls the site specificity Pelletier, J. F., Sun, L., Wise, K. S., Assad-Garcia, N., Karas, B. J., Deerinck, T. J., of cell division. Mol. Microbiol. 24, 905–915. doi: 10.1046/ et al. (2021). Genetic requirements for cell division in a genomically minimal j.1365-2958.1997.3811764.x cell. Cell 184, 2430.e16–2440.e16. doi: 10.1016/J.cell.2021.03.008 Eraso, J. M., Markillie, L. M., Mitchell, H. D., Taylor, R. C., Orr, G., and Pfaffl, M. W. (2001). A new mathematical model for relative quantification in Margolin, W. (2014). The highly conserved MraZ protein is a transcriptional real-time RT-PCR. Nucleic Acids Res. 29:e45. doi: 10.1093/nar/29.9.e45 regulator in Escherichia coli. J. Bacteriol. 196, 2053–2066. doi: 10.1128/ Pich, O. Q., Burgos, R., Ferrer-Navarro, M., Querol, E., and Piñol, J. (2008). JB.01370-13 Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins Errington, J., Daniel, R. A., and Scheffers, D.-J. (2003). Cytokinesis in in terminal organelle organization, gliding motility and cytadherence. bacteria. Microbiol. Mol. Biol. Rev. 67, 52–65. doi: 10.1128/ Microbiology 154, 3188–3198. doi: 10.1099/mic.0.2008/020636-0 MMBR.67.1.52-65.2003 Pilhofer, M., Rappl, K., Eckl, C., Bauer, A. P., Ludwig, W., Schleifer, K. H., Fisunov, G. Y., Evsyutina, D. V., Semashko, T. A., Arzamasov, A. A., et al. (2008). Characterization and evolution of cell division and cell wall Manuvera, V. A., Letarov, A. V., et al. (2016). Binding site of MraZ synthesis genes in the bacterial phyla Verrucomicrobia, Lentisphaerae, transcription factor in mollicutes. Biochimie 125, 59–65. doi: 10.1016/j. Chlamydiae, and Planctomycetes and phylogenetic comparison with rRNA biochi.2016.02.016 genes. J. Bacteriol. 190, 3192–3202. doi: 10.1128/JB.01797-07 Glass, J., Lefkowitz, E., Glass, J., Heiner, C. R., Chen, E. Y., and Cassell, G. H. Pucci, M. J., Thanassi, J. A., Discotto, L. F., Kessler, R. E., and Dougherty, T. J. (2000). The complete sequence of the mucosal pathogen Ureaplasma (1997). Identification and characterization of cell wall-cell division gene urealyticum. Nature 407, 757–762. doi: 10.1038/35037619 clusters in pathogenic gram-positive cocci. J. Bacteriol. 179, 5632–5635. doi: Hasselbring, B. M., Jordan, J. L., Krause, R. W., and Krause, D. C. (2006). 10.1128/jb.179.17.5632-5635.1997 Terminal organelle development in the cell wall-less bacterium Mycoplasma Rappsilber, J., Mann, M., and Ishihama, Y. (2007). Protocol for micro-purification, pneumoniae. Proc. Natl. Acad. Sci. U. S. A. 103, 16478–16483. doi: 10.1073/ enrichment, pre-fractionation and storage of peptides for proteomics using pnas.0608051103 StageTips. Nat. Protoc. 2, 1896–1906. doi: 10.1038/nprot.2007.261 Jaffe, J. D., Stange-Thomann, N., Smith, C., DeCaprio, D., Fisher, S., Butler, J., Robin, A., Joseleau-Petit, D., and D’Ari, R. (1990). Transcription of the ftsZ et al. (2004). The complete genome and proteome of Mycoplasma mobile. gene and cell division in Escherichia coli. J. Bacteriol. 172, 1392–1399. doi: Genome Res. 14, 1447–1461. doi: 10.1101/gr.2674004 10.1128/jb.172.3.1392-1399.1990 Jemiolo, D. K., Zwieb, C., and Dahlberg, A. E. (1985). Point mutations in the Rowlett, V. W., and Margolin, W. (2015). The min system and other nucleoid- 3' minor domain of 16S rRNA of E. coli. Nucleic Acids Res. 13, 8631–8643. independent regulators of Z ring positioning. Front. Microbiol. 6:478. doi: doi: 10.1093/nar/13.23.8631 10.3389/fmicb.2015.00478 Karr, J. R., Sanghvi, J. C., MacKlin, D. N., Gutschow, M. V., Jacobs, J. M., Sabidó, E., Quehenberger, O., Shen, Q., Chang, C. Y., Shah, I., Armando, A. M., Bolival, B., et al. (2012). A whole-cell computational model predicts phenotype et al. (2012). Targeted proteomics of the eicosanoid biosynthetic pathway from genotype. Cell 150, 389–401. doi: 10.1016/j.cell.2012.05.044 completes an integrated genomics-proteomics-metabolomics picture of cellular Kimura, S., and Suzuki, T. (2010). Fine-tuning of the ribosomal decoding metabolism. Mol. Cell. Proteomics 11:M111.014746. doi: 10.1074/mcp. center by conserved methyl-modifications in the Escherichia coli 16S rRNA. M111.014746 Nucleic Acids Res. 38, 1341–1352. doi: 10.1093/nar/gkp1073 Seto, S., and Miyata, M. (1999). Partitioning, movement, and positioning of Kyuma, T., Kimura, S., Hanada, Y., Suzuki, T., Sekimizu, K., and Kaito, C. nucleoids in Mycoplasma capricolum. J. Bacteriol. 181, 6073–6080. doi: 10.1128/ (2015). Ribosomal RNA methyltransferases contribute to Staphylococcus aureus JB.181.19.6073-6080.1999 virulence. FEBS J. 282, 2570–2584. doi: 10.1111/febs.13302 Torres-Puig, S., Broto, A., Querol, E., Piñol, J., and Pich, O. Q. (2015). A Langmead, B., and Salzberg, S. (2012). Fast gapped-read alignment with bowtie novel sigma factor reveals a unique regulon controlling cell-specific 2. Nat. Methods 9, 357–359. doi: 10.1038/nmeth.1923 recombination in Mycoplasma genitalium. Nucleic Acids Res. 43, 4923–4936. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., et al. doi: 10.1093/nar/gkv422 (2009). The sequence alignment/map format and SAMtools. Bioinformatics Torres-Puig, S., Martínez-Torró, C., Granero-Moya, I., Querol, E., Piñol, J., and 25, 2078–2079. doi: 10.1093/bioinformatics/btp352 Pich, O. Q. (2018). Activation of σ20-dependent recombination and horizontal Liao, Y., Smyth, G. K., and Shi, W. (2014). featureCounts: an efficient general gene transfer in Mycoplasma genitalium. DNA Res. 25, 383–393. doi: 10.1093/ purpose program for assigning sequence reads to genomic features. dnares/dsy011 Bioinformatics 30, 923–930. doi: 10.1093/bioinformatics/btt656 Vicente, M., Gomez, M. J., and Ayala, J. A. (1998). Regulation of transcription Lluch-Senar, M., Querol, E., and Piñol, J. (2010). Cell division in a minimal of cell division genes in the Eschericia coli dcw cluster. Cell. Mol. Life Sci. bacterium in the absence of ftsZ. Mol. Microbiol. 78, 278–289. doi: 10.1111/j. 54, 317–324. doi: 10.1007/s000180050158 1365-2958.2010.07306.x Vicente, M., and Rico, A. I. (2006). The order of the ring: assembly of Love, M. I., Huber, W., and Anders, S. (2014). Moderated estimation of fold Escherichia coli cell division components. Mol. Microbiol. 61, 5–8. doi: change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15:550. 10.1111/j.1365-2958.2006.05233.x doi: 10.1186/s13059-014-0550-8 Vishnyakov, I. E., Borchsenius, S. N., Basovskii, Y. I., Levitskiĭ, S. A., Lazarev, V. N., Lutkenhaus, J. (2007). Assembly dynamics of the bacterial MinCDE system Snigirevskaia, E. S., et al. (2009). Localization of division protein FtsZ and spatial regulation of the Z ring. Annu. Rev. Biochem. 76, 539–562. doi: in Mycoplasma hominis. Cell Tiss. Biol. 3, 254–262. doi: 10.1134/ 10.1146/annurev.biochem.75.103004.142652 S1990519X09030079 Martínez-Torró, C., Torres-Puig, S., Monge, M., Sánchez-Alba, L., Xu, X., Zhang, H., Huang, Y., Zhang, Y., Wu, C., Gao, P., et al. (2019). Beyond González-Martín, M., Marcos-Silva, M., et al. (2020). Transcriptional a ribosomal RNA methyltransferase, the wider role of MraW in DNA Frontiers in Microbiology | www.frontiersin.org 13 September 2021 | Volume 12 | Article 695572 Martínez-Torró et al. Cell Division in Mycoplasma genitalium methylation, motility and colonization in Escherichia coli O157:H7. 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