The effects of mycobacterial RmlA perturbation on cellular dNTP pool, cell morphology, and replication stress in Mycobacterium smegmatis.

The concerted action of DNA replication and cell division has been extensively investigated in eukaryotes. Well demarcated checkpoints have been identified in the cell cycle, which provides the correct DNA stoichiometry and appropriate growth in the progeny. In bacteria, which grow faster and less concerted than eukaryotes, the linkages between cell elongation and DNA synthesis are unclear. dTTP, one of the canonical nucleotide-building blocks of DNA, is also used for cell wall biosynthesis in mycobacteria. We hypothesize that the interconnection between DNA and cell wall biosynthesis through dTTP may require synchronization of these processes by regulating dTTP availability. We investigated growth, morphology, cellular dNTP pool, and possible signs of stress in Mycobacterium smegmatis upon perturbation of rhamnose biosynthesis by the overexpression of RmlA. RmlA is a cell wall synthetic enzyme that uses dTTP as the precursor for cross-linking the peptidoglycan with the arabinogalactan layers by a phosphodiester bond in the mycobacterial cell wall. We found that RmlA overexpression results in changes in cell morphology, causing cell elongation and disruption of the cylindrical cell shape. We also found that the cellular dTTP pool is reduced by half in RmlA overexpressing cells and that this reduced dTTP availability does not restrict cell growth. We observed 2-6-fold increases in the gene expression of replication and cell wall biosynthesis stress factors upon RmlA overexpression. Using super-resolution microscopy, we found that RmlA, acting to crosslink the nascent layers of the cell wall, localizes throughout the whole cell length in a helical pattern in addition to the cellular pole.

Introduction

The unique intricate cell wall impermeable for most antibiotics is a specific hallmark of mycobacteria. This cell wall makes infections caused by pathogenic mycobacteria extremely difficult to treat [1, 2]. Mycobacterium tuberculosis (M. tuberculosis) has been a leading cause of mortality worldwide, accounting for 1.4 million deaths in 2020, commensurable only with the death toll caused by SARS-CoV-2 [3]. Although effective combination chemotherapy exists to combat M. tuberculosis, drug-resistant tuberculosis accounts for 450,000 new cases annually. Almost a third of all tuberculosis-related deaths are due to antimicrobial resistance [4]. The thick cell wall constitutes of mycolic acid, peptidoglycan, and arabinogalactan (Fig 1). The galactan region of the arabinogalactan layer is bound to the peptidoglycan layer via a phosphodiester linkage of the α-L-rhamnopyranose-(1→3)-α-D-GlcNAc-1-phosphate disaccharide [5]. This linker, composed of a rhamnosyl residue, a sugar not found in humans, is critical to the structural integrity of the mycobacterial cell wall (Fig 1), as well as for the viability and pathogenicity of pathogenic mycobacteria [6, 7]. Rhamnose has also been reported to mediate virulence, adhesion, and pathogenesis in several other bacteria [8-11]. L-Rhamnosyl residues are synthesized in the rhamnose pathway by four enzymes: RmlA, B, C, and D from α-D-glucose-1-phosphate (G1P) and dTTP as the main precursors [7]. No salvage pathway is known for the synthesis of dTDP-L-rhamnose. Consistent with this, the RmlA enzyme (D-glucose-1-phosphate thymidylyltransferase), which catalyzes the first step of rhamnose biosynthesis, is essential for bacterial growth [12]. The importance of this enzyme for mycobacterial viability, together with the absence of the rhamnose pathway in humans, make this enzyme a potentially valid drug target for the development of RmlA-related anti-mycobacterial drugs for tuberculosis treatment [13].

Fig 1

Hypothesis: A dual role of thymidylate biosynthesis in mycobacterium.

In mycobacteria, dUMP, the precursor for dTTP biosynthesis, is synthesized exclusively by dCTP deamination and coupled dUTP hydrolysis by Dcd:dut. dTTP is used not only for DNA replication by DNA polymerases but also by RmlA that uses this nucleotide for the first step of L-rhamnosyl biosynthesis, a critical step in cell wall biosynthesis. (Abbreviations: linker–L-rhamnosyl linker; Dcd:dut—bifunctional dCTP deaminase:dUTPase; TS–thymidylate synthase; RmlA—D-glucose-1-phosphate thymidylyltransferase; Pol–DNA polymerase).

RmlA catalyzes the condensation of G1P with dTTP to produce dTDP-D-glucose, which is processed further by the RmlB-D enzymes [7]. RmlA is homologous to other bacterial sugar nucleotide transferases, although the tetrameric arrangement of RmlA is distinct from these [14, 15]. The competitive and non-competitive product inhibition of RmlA by dTDP-L-rhamnose has long been known [16]. In addition, the M. tuberculosis rhamnose pathway is likely regulated by c-di-GMP and the serine/threonine protein kinases PknG and PknB [13, 17]. PknG is required for the intrinsic multidrug resistance [18] and virulence [19] of mycobacteria; moreover, it interacts with and phosphorylates two essential components of the rhamnose pathway, RmlA and Wbbl2 [17], thereby inhibiting the enzymatic activity of RmlA [17]. The inhibition of cell wall assembly has proven to be effective for inhibiting mycobacterial growth. Drugs such as ethambutol (EMB), isoniazid (INH), and D-cycloserine, which target the synthesis of various cell wall components, are successfully used in tuberculosis therapy. As a result, the biosynthesis of the mycobacterial cell wall has been a significant research objective over the last decade [1, 5, 20].

As dTTP, one of the canonical nucleotide building blocks of DNA, is utilized not only for DNA replication but also for cell wall biosynthesis and assembly, we hypothesized that this connection might serve as a checkpoint allowing mycobacteria to synchronize these processes by monitoring and regulating dTTP availability (Fig 1). To this end, we investigated growth, morphology, cellular dNTP pool, and possible signs of stress in Mycobacterium smegmatis (M. smegmatis) upon perturbation of the cellular RmlA level. Since rmlA is essential [12] and attempts to inhibit M. smegmatis RmlA using a previously reported inhibitor [21] were unsuccessful, we chose to overexpress RmlA to influence and study its cellular function. In addition, by overexpressing RmlA, cellular dTTP used for replication could be restricted. We found that RmlA overexpression caused morphological changes, including cell elongation and the appearance of large polar bulbs at the tip of the cell. RmlA overexpression induced 2-6-fold changes in the mRNA levels of various stress factors, including LexA, WhmD, and IniA. We also found that RmlA overexpression resulted in significant dTTP depletion in a M. smegmatis PknG knock-out strain (PknG(-)). Finally, live-cell imaging of M. smegmatis cells expressing fluorescently tagged RmlA revealed an intriguing helix-like distribution of RmlA along the cylindrical portion of the cell.

Materials and methods

Bacterial strains, media, and growth conditions

M. smegmatis mc2155 [22] strains were grown in Lemco broth. The Lemco broth was supplemented with 15 g L−1 Bacto agar for a solid medium. Streptomycin was added at 20 μg/ml, hygromycin B at 50 μg/ml, and tetracycline (TC) at the final concentration of 0.05–0.2 μg/ml.

Construction of RmlA expressing M. smegmatis strains

The RmlA coding region was PCR amplified and cloned into the vectors pKW08-Lx [23] (Addgene #25012), pTEC16-mOrange [24] (Addgene plasmid # 30175), and pLL192 [25] using the primers and restriction sites indicated in S1 Table in S1 File to construct the overexpressing and the fluorescently tagged constructs, respectively. Successful cloning was verified with the sequencing of the appropriate region of the plasmid. 0.5–0.5 μg of the appropriate plasmids were electroporated into electrocompetent wild type (WT) [26] or PknG(-) [18] M. smegmatis strain according to Table 1. Three parallels of each strain were used in the experiments.

Table 1

Strains and plasmids used in the study.

	resistance	relevant characteristics	usage	source or reference
strains
mc2 155	-	WT		Snapper et al. 1990
RmlAi	strep, hyg	wt harboring pKW08-RmlA	inducible (TC) overexpression of RmlA	this study
RmlAc	hyg	wt harboring pTEC-RmlAover	constant overexpression of RmlA	this study
Lxi	hyg	wt harboring pKW08-Lx	inducible (TC) overexpression of luciferase, control strain	Hirmondo et al. 2015
RmlA-gfp	strep	wt harboring pLL192-RmlA	expression of RmlA-gfp	this study
RmlA-mOrange	hyg	wt harboring pTEC16-RmlA	expression of RmlA-mOrange-2	this study
mOrange	hyg	wt harboring pTEC16	expression of mOrange-2; control strain	this study
PknG-	hyg	pknG-	control strain	Wolff et al. 2009
RmlAi, pknG-	strep, hyg	pknG- harboring pKW08-RmlA	inducible (TC) overexpression of RmlA in PknG- background	this study
plasmids
pKW08-Lx	hyg	expression from TetRO promoter	inducible (TC) overexpression of luciferase	Williams et al. 2010; Addgene #25012
pKW08-RmlA	hyg, strep	expression from TetRO promoter	inducible (TC) overexpression of RmlA	this study
pTEC-RmlAover	hyg	expression from hsp60 promoter	constant overexpression of RmlA	this study
pLL192-RmlA	strep	GFP tag, expression from own promoter	localization of RmlA	this study
pTEC16	hyg	mOrange-2 expression from MSP promoter	localization control	Takaki et al. 2013; Addgene #30175
pTEC16-RmlA	hyg	mOrange-2 tag, expression from own promoter	localization of RmlA; super-resolution imaging	this study

Verification of RmlA expression by qPCR

Overexpression was verified by measuring mRNA levels of RmlA in WT and inducible or constitutive overexpressing strains. Cells were grown in 50 ml liquid culture until saturation; then, in the RmlAi strain, overexpression was induced by adding 0.05 μg/ml TC at OD(600) = 0.2–0.4. Cells were harvested by centrifugation at 4000 g for 20 min. Cells were washed in ice-cold PBS before resuspension in 2 volumes of RNA Protect Reagent (Qiagen). Samples were stored at -80°C. RNA was purified with the Rneasy RNA Clean-up kit (Qiagen) according to the manufacturer’s instructions. Mycobacterial RNA yield was assayed using the Nano-Drop ND-2000 Spectrophotometer (NanoDrop Technologies). cDNA samples were amplified from 0.1 μg total RNA by random hexamer primers using High-Capacity Reverse Transcription Kit (Applied Bioscience). The resulting cDNA was quantified by qPCR using EvaGreen (Bioline) and MyTaq PCR master mix (Bioline) in a BioRad CFX96 qPCR instrument. Non-reverse transcribed, and no template controls were used as controls for DNA contamination. We prepared three technical and three biological replicates for all measurements. sigA (MSMEG_2758) and Ffh (MSMEG_2430) [27] were used as endogenous reference genes to normalize input cDNA concentration. The relative expression ratios of the examined genes were calculated using the comparative Ct method (ΔΔCt) by the BioRad CFX Maestro software. Primers, primer efficiency, and all measured data of the qPCR analysis are compiled in the supplementary archive.

Growth inhibition assay

The control WT and PknG(-) M. smegmatis strains, the WT and PknG(-) M. smegmatis strains overexpressing RmlA (RmlAi), and the control M. smegmatis strain overexpressing luciferase (Lxi) were grown in M9 minimal media containing 0.05% Tween-80 in liquid culture overnight. The precultures were then diluted to OD (600) = 0.1 and grown in the presence of various concentrations of TC (0, 0.05, or 0.1 μg/ml TC) in a plate reader (Biotek Synergy MX) at 37°C with constant shaking. OD (600) was measured every 10 min.

Microscopy

RmlA was overexpressed in WT and PknG(-) strains for morphological studies. Overexpression was induced by 0.1 or 0.2 μg/ml TC. The strains were grown in agar plates containing TC in the indicated concentration, then streaked onto microscopy slides covered with 0.1% low melting agarose (Sigma). Cell membranes were stained with 1 μg/ml Bodipy (522/529) dye (Thermo Fischer). Samples were investigated in phase-contrast, and fluorescence modes under a Leica DM IL LED microscope. Microscopic images were analyzed using Fiji [28]. Cell length was measured using the software Bacstalk [29].

RmlA was expressed using its own promoter for localization studies, tagged with GFP or mOrange-2. If appropriate, the strains were grown in agar plates containing specific drugs, then streaked onto microscopy slides covered with 0.1% low melting agarose (Sigma). RmlA-GFP strains were stained with 10 μg/ml Propidium-iodide. Samples were investigated using a Leica TCS SP8 STED microscope. In the case of the GFP tagged constructs, the confocal mode was used, while the mOrange-2 signal was suitable for the STED mode to analyze localization patterns. Deconvolution was performed using the Huygens Professional software. Microscopic images were refined and analyzed using Fiji [28].

Gene expression analysis

RmlA was overexpressed in WT and PknG(-) strains for gene expression studies. Overexpression was induced by 0.1 or 0.2 μg/ml TC. For CIP and EMB treatments, drugs were added to the cultures at OD (600) = 0.1. CIP was used at 0.3 μg/ml, EMB at 100 μg/ml final concentration, where cell mortality was 20–80%. Cells were grown in 50 ml liquid culture until saturation, washed in ice-cold PBS, and harvested by centrifugation (4000 g, 20 min). Bacterial pellets were resuspended in 2 volumes of RNA Protect Reagent (Qiagen) and stored at -80°C. RNA was purified with the Rneasy RNA Clean-up kit (Qiagen) according to the manufacturer’s instructions. Mycobacterial RNA yields were assayed using the Nano-Drop ND-2000 Spectrophotometer (NanoDrop Technologies). 0.1 μg of total RNA was reverse transcribed to cDNA using random hexamer primers and the High-Capacity Reverse Transcription Kit (Applied Bioscience). The resulting cDNA was quantified by qPCR using EvaGreen (Bioline) and MyTaq PCR master mix (Bioline) in a BioRad CFX96 qPCR instrument. Non-reverse transcribed, and no-template controls were used for checking genomic and exogenous DNA contamination, respectively. For all measurements, three technical and three biological replicates were used. sigA (MSMEG_2758) and Ffh (MSMEG_2430) were used as endogenous reference genes to normalize input cDNA concentrations. The relative expression ratios of the examined genes were calculated using the comparative Ct method (ΔΔCt) by the BioRad CFX Maestro software. Primers, primer efficiency, and all measured data of the qPCR analysis are compiled in S1 Table in S1 File.

dNTP extraction and determination of the dNTP pool size

dNTP extraction and quantification were performed according to Szabo et al. [30]. Cells were grown until the culture reached the mid-exponential phase OD (600) ~ 0.7. The total CFUs were determined for each culture. The cultures were centrifuged (20 min, 4000 g, 4°C), and the cell pellets were extracted in precooled 0.5 ml 60% methanol overnight at −20°C. After 5 minutes of boiling at 95°C, cell debris was removed by centrifugation (20 min, 13 400 g, 4°C). The methanolic supernatant containing the soluble dNTP fraction was vacuum-dried (Eppendorf) at 45°C. The dNTP pellet was dissolved in 50 μl nuclease-free water.

Determination of the dNTP pool size in each extract was as follows: 10 pmol template oligo (Sigma), 10 pmol probe (IDT), and 10 pmol NDP1 primers (Sigma) were present per 25 μl reaction. The concentration of each non-specific dNTP was kept at 100 μM. VWR® TEMPase Hot Start DNA Polymerase (VWR) was used at 0.9 unit/reaction in the presence of 2.5 mM MgCl2. To record calibration curves, the reaction was supplied with 0–12 pmol or 0–25 pmol specific dNTP depending on the applied dT1 or dT2 template, respectively. Sequences of used primers and probes are presented in the supplementary archive. Fluorescence was recorded every 13 seconds in a CFX96 Touch™ Real-Time PCR Detection System. The thermal profile was 95°C 15 min, (60°C 13 s) × 260 cycle for dATP and dTTP measurement. In the case of dCTP and dGTP measurements, the polymerization temperature was 55°C. The results were analyzed using the nucleoTIDY software (http://nucleotidy.enzim.ttk.mta.hu/) [30].

Results

RmlA overexpression produces elongated cells and altered cell morphology

To decipher how dTTP metabolism and cell wall biosynthesis affect each other, we investigated the cellular function of RmlA, the cell wall biosynthetic enzyme that potentially links these two processes together using dTTP in the first step of the rhamnose biosynthetic pathway. As RmlA is essential in Mycobacteria [12], we could not knock out its gene. Alphey and his colleagues described a RmlA inhibitor [21] effective against Pseudomonas aeruginosa and M. tuberculosis. However, we could not detect any growth inhibition using this compound in M. smegmatis, even at the highest possible concentration limited by water solubility (200 μg/ml; 8-fold the MIC determined in M. tuberculosis [21]). Therefore, we chose to overexpress RmlA and investigate its effects in WT and PknG(-) M. smegmatis cells. RmlA activity has been described to be regulated by serine/threonine protein kinases PknB and PknG mediated phosphorylation. In PknG(-) cells, RmlA regulation by PknG is supposed to be switched off [17], i.e., RmlA is likely to be active in this strain. To overexpress the protein, we constructed a constitutive (RmlAc) and an inducible expression system (RmlAi). Protein expression in the latter case could be induced by adding TC to the growth medium. The degree of RmlA expression was verified by mRNA quantification using qPCR. sigA [31] and Ffh [27] were used as reference genes. We detected a 25-40-fold increase (p < 0.008) in the RmlA mRNA levels in the RmlAc and RmlAi strains upon TC induction, respectively (Fig 2A). We chose the inducible system for subsequent experiments. RmlAi M. smegmatis cultures grew at a WT rate (Fig 2B). Intriguingly, RmlA overexpression in the PknG(-) strain fully restored the growth arrest of the parental strain in a minimal medium (Fig 2B).

Fig 2

RmlA overexpression in M. smegmatis and its effect on cell growth.

A) Overexpression was evaluated by measuring mRNA levels of RmlA in WT and overexpressing strains (RmlAi–induced overexpression by the addition of 0.05 μg /ml TC, or RmlAc—constitutive overexpression). RmlA levels in RmlAc and RmlAi strains were 25 and 40 fold higher (p < 0.008) than in the WT M. smegmatis, respectively. B) WT, RmlAi, PknG(-), and PknG(-); RmlAi M. smegmatis strains were grown in liquid culture without any treatment or with 0.1 μg/ml TC. OD (600) was measured every 10 min in a plate reader.

We investigated the possible phenotypes resulting from RmlA overexpression under the microscope. The distribution of cells on microscopy slides prepared from exponentially growing cultures indicates that under the same growth conditions (the same amount of detergent applied in the medium), RmlAi cells are more prone to form aggregates than WT cells (Fig 3). This observation is consistent with the noisiness of the RmlAi growth curves (Fig 2B).

Fig 3

RmlAi cells are more prone to aggregation.

The indicated strains were grown in liquid culture and streaked onto microscopy slides as described in Methods. RmlA overexpression was induced by the addition of 0.2 μg/ml TC. The control strains do not carry the RmlAi plasmid.

The effects of RmlA overexpression on M. smegmatis morphology were investigated using phase contrast and epifluorescence microscopy (Fig 4). WT, PknG(-), and luciferase-expressing (Lxi) strains were used as controls to exclude artifacts potentially caused by TC and protein overexpression, respectively. We observed two distinct morphological changes upon the induction of RmlA overexpression in both WT and PknG(-) backgrounds. The cells became more variable in size and shape than the WT; on average, they became longer (Fig 4A). In addition, the normal rod shape of M. smegmatis was disrupted at the cell poles, especially at the 0.2 μg/ml TC concentration. Fig 4B shows that the cylindrical shape is changed into spherical at the tip of the cells. To statistically analyze the observations, we defined cells with a diameter larger than 0.6 μm deformed. In the RmlAi strain, 4.6% of the cells were deformed, 0 spherical (bubble-like) structure was observed (n = 119) at 0 μg/ml TC; 11% of the cells were deformed, spherical structures were observed in 9% of the cells (n = 86) at 0.1 μg/ml TC, and 52% of the cells were deformed, spherical structures were observed in 40% of the cells (n = 89) at 0.2 μg/ml TC. In the PknG(-); RmlAi strain, 3% of the cells were deformed, 0 spherical (bubble-like) structure was observed (n = 390) at 0 μg/ml TC; 11% of the cells were deformed, spherical structures were observed in 9% of the cells (n = 277) at 0.1 μg/ml TC, and 34% of the cells were deformed, spherical structures were observed in 34% of the cells (n = 259) at 0.2 μg/ml TC. In the control strains, 1–5% of the cells were deformed, with no spherical structure observed (n = 75–549 / strain).

Fig 4

Effects of RmlA overexpression on the morphology of M. smegmatis.

A) RmlA overexpression in both WT and PknG(-) backgrounds resulted in cell elongation and the appearance of spherical, bubble-like structures breaking off the normal rod shape. B) High-resolution visualization of the morphological changes in the RmlAi; PknG(-) strain upon TC induction. The cell membrane was stained by Bodipy 522/529. PC denotes phase-contrast images.

We quantified the observed elongation of the RmlAi cells and subjected the results to statistical analysis (Fig 5). We calculated 2.9 and 1.6 fold increase in the mean cell length in the RmlAi and RmlAi; PknG(-) strains compared to the WT, respectively.

Fig 5

Cell elongation upon RmlA overexpression.

The normalized distribution of cell length is shown as bars. Gaussian curve fitting to the data is shown as smooth lines. The number of cells counted in each sample (n) is shown in the legend. RmlA overexpression was induced by 0.1 or 0.2 μg/ml TC (TC 0.1 and TC 0.2 respectively). TC 0 stands for non-induced controls. The highest probability cell length yielded by the Gaussian curves in each case is the following in the order of 0.2 μg/ml TC; 0.1 μg/ml TC and no TC: 2.02±0.03 μm; 2.10±0.02 μm; 2.06±0.08 μm for the WT; 1.98±0.01 μm; 2.22±0.01 μm; 2.20±0.01 μm for the PknG(-); 5.48±0.53 μm; 1.94±0.16 μm; 1.91±0.02 μm for the RmlAi; 3.17±0.20 μm; 1.99±0.01 μm; 2.01±0.02 μm for the PknG(-); RmlAi and 2.32±0.01 μm; 2.10±0.02 μm; 2.01±0.02 μm for the Lxi samples.

Effects of RmlA overexpression on the cellular dNTP pool

As RmlA uses dTTP to synthesize dTDP-rhamnose, we expected that the overexpression of RmlA could deplete dTTP from the cellular nucleotide pool, which, in turn, could cause replication defects. Therefore, we measured the cellular dNTP concentrations in all investigated strains using our improved fluorescence-based dNTP quantification assay (Fig 6) [30]. The principle of the assay is similar to that of the TaqMan assay. All four dNTPs are determined separately using dNTP-specific templates. The fluorescence output signal upon DNA elongation is directly proportional to the dNTP to be quantified. Interestingly, we could not detect any significant decrease in dTTP concentration caused by RmlA overexpression in the WT strain (Fig 6). However, RmlA overexpression in the PknG(-) background resulted in ~ 2 fold decrease in the dTTP concentration (p < 0.016) (Fig 6). Changes in the cellular dGTP, dATP, and dCTP concentrations were statistically insignificant (Fig 6).

Fig 6

The effect of RmlA overexpression on the cellular dNTP pool.

Concentrations of dNTPs in the extracts were measured according to [30]. The dTTP level in the RmlA overexpressing PknG(-) strain was significantly lower than that in the PknG(-) strain (p < 0.016), although it is unchanged when RmlA was overexpressed in the WT background. The cellular concentration of other dNTPs was not significantly changed in the RmlA overexpressing strains.

Effects of RmlA overexpression on replication and cell wall biosynthesis stress factors

RadA and LexA were chosen to indicate stress for DNA replication, while IniA and WhmD served as cell wall biosynthesis stress indicators. The upregulation of iniA and whmD upon treatment with the cell wall biosynthesis inhibitors EMB and INH [31, 32] was shown earlier and used as a positive control for cell wall biosynthesis stress. The upregulation of the SOS-response protein LexA and the DNA repair protein RadA in response to CIP treatment was used as a positive control for replication stress [33, 34]. The concentrations of the applied drugs were chosen to inhibit cell growth while allowing downstream analysis of the remaining cells (Fig 7). To investigate the response of genes in the thymidylate biosynthesis pathway to RmlA overexpression, we measured the expression levels of Dcd:dut, ThyA, and ThyX enzymes. We also quantified the mRNA of the members of the rhamnose biosynthesis pathway (RmlA-D) and PknG known to regulate rhamnose biosynthesis [17]. The transcription level of these genes was measured by qPCR following different treatments or RmlA overexpression.

Fig 7

Effects of CIP and EMB treatments on cell viability.

Treatments were performed on exponentially growing WT bacteria in liquid cultures for 8 h using 100 μg/ml EMB and 0.3 μg/ml CIP. CFU was counted on antibiotic-free agar plates. Concentrations were chosen so that growth inhibition was in the range of 20–80%.

mRNA expression levels were normalized to that of the nontreated WT strain. RmlA overexpression had only a mild effect on the expression of the investigated genes in the WT background (Fig 8). However, in the PknG(-) background, all investigated stress factors were upregulated 2-6-fold (IniA 3.3-fold; WhmD 5.6-fold; LexA 2.15-fold and RadA 6-fold upregulation, respectively). We also detected a mild upregulation for RmlB and RmlC (~2-fold overexpression). However, RmlD was downregulated (-1.5-fold in the PknG-, and -2.5-fold in the WT background). The expression level of the thymidylate synthesis pathway enzymes was not changed significantly. The CIP treatment increased the expression of LexA (~13.5-fold), RadA (~12-fold), and IniA (~1.8-fold), while the EMB treatment resulted in RadA (~3.5-fold), WhmD (~5.5-fold), and IniA (~ 100-fold) overexpression. Interestingly, the expression level of RmlA was significantly decreased in the PknG(-) strain (-5.5-fold) and also as a result of the CIP treatment (-14-fold). In summary, the 2-6-fold increase in the expression of cell wall biosynthetic and replication stress factors indicates that RmlA overexpression induces quantifiable stress in the cellular processes studied.

Fig 8

Effects of RmlA overexpression on the expression of different genes.

RadA and LexA were used as indicators for replication stress, while IniA and WhmD indicated cell wall biosynthesis stress. Dcd:dut, ThyA, and ThyX represent the thymidylate biosynthesis pathway, while RmlA-D belongs to the rhamnose biosynthetic pathway. PknG, known to regulate rhamnose biosynthesis, was also measured. The following treatments were used as positive controls: CIP for replication stress and EMB for cell wall biosynthesis stress. Expression levels were normalized to those of the WT strain. Changes in expression levels are shown as a heat map. Blue depicts downregulation, while red is for upregulation. Black star indicates p < 0.05 for the change in expression.

RmlA localizes nearby the cell membrane in a helix-like pattern

To investigate the cellular localization of RmlA, strains expressing the fluorescently tagged proteins RmlA-mOrange-2 or RmlA-GFP were constructed. To avoid artifacts from overexpression, the expression of these fluorescent constructs was driven by the RmlA promoter. We found that RmlA is accumulated at the cell perimeter in a helix-like pattern (Fig 9). The signal is most abundant at the tip of cells (Fig 9), where growth is thought to occur in mycobacteria.

Fig 9

The cellular localization pattern of fluorescently tagged RmlA.

To investigate the cellular localization of RmlA, we constructed M. smegmatis strains expressing GFP (A) and mOrange-2 (B) tagged RmlA under the control of the endogenous RmlA promoter. A) The localization pattern of the GFP tagged protein was observed using confocal microscopy. Of the 57 cells analyzed, all exhibited a helical fluorescence pattern here. DNA was stained with propidium iodide. B) The localization of mOrange-2 tagged protein was investigated using STED super-resolution imaging. We found that in all 68 cells analyzed, RmlA localized to the cell perimeter in a helix-like pattern, while no helicity was observed in the mOrange controls (mOrange panel in Fig 9B). Cell boundaries are indicated with a dashed line.

Discussion

To reveal possible interconnections between dTTP metabolism and cell wall biosynthesis, we set out to investigate the cellular function of RmlA. This enzyme uses dTTP as a precursor in the rhamnose biosynthetic pathway. Qu and his colleagues showed [12] that RmlA is essential in mycobacteria and constructed a M. smegmatis rmlA null strain that conditionally expressed RmlA from M. tuberculosis. At a permissive temperature, cells exhibited the normal rod shape. In contrast, cells showed irregular surface wrinkles, and subsequent lysis at a non-permissive temperature at which the plasmid expressing M. tuberculosis RmlA could not replicate [12]. The morphological changes we observed upon RmlA overexpression are in line with those resulting from rmlA depletion [12]. Our RmlA overexpressing cells also appeared significantly longer than the WT, indicating a role of RmlA in cell growth. Furthermore, 40% of the RmlA overexpressing cells developed spheroidal structures at the cell poles. These results indicate that both the absence and the excess of RmlA activity cause morphological abnormalities in M. smegmatis.

We found that RmlA localizes in a helical pattern at the cylindrical part of the cell in addition to its enrichment at the poles. This pattern indicates that crosslinking of peptidoglycan and arabinogalactan layers is not restricted to the cell poles despite the observation that newly synthesized peptidoglycan in mycobacteria is limited to the poles [35]. The helical localization pattern of RmlA is reminiscent of the helical cables formed by cytoskeletal proteins (FtsZ, MreB, and Mbl) in rod-shaped bacteria that grow via cylindrical extension [36-38]. In the absence of MreB homologs in mycobacteria [39], the rod shape must be achieved by yet unknown mechanisms. The observed disrupted rod morphology at the poles in the RmlAi strains and the localization pattern of RmlA-GFP using epifluorescence microscopy are also reminiscent of what Plocinski et al. detected when studying the cell wall synthesis protein cwsA at a similar resolution [40, 41].

Since RmlA uses dTTP to synthesize dTDP-D-glucose, it could potentially deplete the cellular dTTP pool causing replication defects and growth inhibition if not appropriately regulated, as shown with other dTTP deficient conditions [42]. However, we could not detect any significant growth arrest in RmlA overexpressing strains. On the contrary, the RmlA-overexpressing PknG(-) strain grew better than its parental strain in a minimal medium. The reduced growth of the PknG‐deficient M. tuberculosis strain in the minimal medium was shown in an earlier report [43], in which Cowley et al. proposed that growth arrest is caused by a decrease in the de novo glutamine synthesis [43]. Interestingly, in our experiments, RmlA overexpression fully rescued the growth arrest observed in PknG(-) M. smegmatis in a minimal medium. We also found that the RmlA overexpressing strains (both RmlAi and PknG(-); RmlAi) are more prone to aggregation than parental strains. Hsu et al. and Hardy et al. showed that RmlA influences biofilm formation in Listeria monocytogenes [10] and Caulobacter crescentus [11], respectively. A similar influence of RmlA in M. smegmatis would be consistent with the observed increase in aggregation. The importance of RmlA regulation by phosphorylation is also discernible from the results on cellular dTTP concentrations in the RmlA overexpressing strains. While we could not detect any significant cellular dTTP concentration change in PknG proficient cells, the dTTP level decreased to its half upon RmlA overexpression in the PknG(-) background, devoid of negative regulation by PknG phosphorylation. Similarly, RmlA overexpression only resulted in significant expression changes in the PknG(-) background. In addition to its PknG regulation, the feedback inhibition of RmlA also seems to be efficient in avoiding dTTP depletion. In a 40-fold excess of RmlA in the RmlAi;PknG(-) cells, dTTP should have been entirely depleted without feedback inhibition. Moretti et al. showed that Salmonella enterica typhimurium RmlA could use all dNTPs and NTPs in vitro [44], although with a dTTP bias. We found that M. smegmatis RmlA perturbed the cellular concentration of only dTTP without affecting other dNTPs, which suggests that it is specific for dTTP in the cellular environment.

RmlA overexpression resulted in both cell wall biosynthetic and replication stress indicated by the 2-6-fold moderate but solid increase in the mRNA levels of the selected stress factors (Fig 8). Although severe dNTP depletion was not observed in cell extracts, transient dTTP depletion in the cells may have led to thymine starvation which can induce replication stress.

Due to the efficient feedback inhibition mechanism of RmlA, we could not establish at what point dTTP availability may serve as a link between replication and cell wall biosynthesis. We observed, however, that both cell wall biosynthesis and replication stresses can be moderately induced by RmlA overexpression, and there might be some interconnection between the two processes. On the other hand, our investigations unveiled that the cylindrical part of the cell may not be as inert as previously thought. The fact that RmlA, acting to crosslink the peptidoglycan and arabinogalactan layers, localizes throughout the whole cell length in a helical pattern strongly suggests that cell wall synthesis also occurs in the cylindrical part of the cell, not only at the poles. Consistent with this, the morphological changes upon RmlA overexpression indicate that RmlA plays a role in determining cell shape driven by a yet unknown mechanism in mycobacteria.

The “raw data” archive contains the sequences of oligos used in this study, raw data for qPCR, dNTP quantitation, growth measurements, microscopy images, and data for the determination of cell length distribution.

(ZIP)

Click here for additional data file.

(DOCX)

Click here for additional data file.

13 Oct 2021

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Reviewer #1: This paper describes the construction and characterization of an overexpression strain of RmlA in M. smegmatis. Overall, the experiments were conducted well with appropriate controls. However the conclusions are not supported by the data. The manuscript should either be strengthened with additional data or the overstated claims removed.

Major Points

L43. Essentiality of a gene is not a “strong basis” for the development of anti-tubercular agents. This is a gross over-statement.

L62 There is no evidence that RmlA is a checkpoint, this is a stretch from the published data. It is a hypothesis.

L307 No measurements of cell length were presented to support the statement that they were significantly longer. the link to cell growth division is weak.

The authors refer to extensive clumping in their cultures. What was the rationale for culturing M. smegmatis without Tween 80, as is the norm in the field to reduce aggregation? If clumping was an issue, how was OD determined reliably?

The changes in gene expression for the stress response are marginal. Although they achieved statistical significance, the magnitude of the change was very small and much lower than the response to the control agents. Therefore, the claim that RmlA overexpression induces these stresses is not supported by the data. The gene expression studies did not include any no RT controls to account for the presence of genomic DNA.

For the experiments with CIP and EMB, how was the concentration used determined/selected?

Some more explanation of the methods to measure dNTPs is needed. It was not clear. In the results how this was done.

The discussion was very lengthy. It should be restricted to a discussion of the results in context of the published literature instead of a literature review. For example, Lines 311 to 361 read like a literature review.

L388 The conclusion that RmlA overexpression leads to cell wall and replication stress is not supported by the data in the paper. In fact, it's more likely that RmlA overexpression does not lead to cell wall or replication stress based on the very small changes in gene expression. the discussion should be revised to remove the overstated claims and limit discussion to what is shown in the data.

The supporting information should be included in the main body of the paper as it is required to follow the methods.

Minor points

There was a lot of unscientific language and claims which were not supported by refernces e.g.

L23 what is “outstanding genetic adaptability”?

L24 “most dreaded member”

L144 what does “2-2 stress”mean?

References are missing to support the statements in L24, L38.

L193 what does the “X’ stand for? What was the maximum concentration tested against M. smegmatis?

L206 “somewhat disturbed by the intensive clumping”

L240 what does “quasi unchanged” mean?

L198 and others “constitutive” not “constant”

L245 Reference in wrong format

L330 is “bulgy” a real word?

L87-90 The methods section should describe the methods, not the rationale or interpretation of data. Why did the authors use TC and not anhydrotetracycline?

L101. Table legend should just be the legend.

Abbreviations are inconsistent. For example, why is M. smegmatis used and not M. tuberculosis? Why is Cip lower case, but EMB upper case. Why is there an abbreviation for EMB but no isoniazid?

Abbreviations should be defined at first use.

Figure 1 is not required as the data do not support this conclusion. It was also not written in English.

Reviewer #2: In this manuscript entitled “Perturbation of rhamnose biosynthesis reveals crosstalk between mycobacterial cell wall synthesis and DNA replication” Hirmondo et al. have attempted to elucidate the crosstalk between cell wall synthesis and DNA replication in M. smegmatis, by overexpressing RmlA. RmlA, is an essential gene synthesizing L-Rhamnosyl residues using D-glucose-1-phosphate (G1P) and dTTP as the precursors. The rhamnosyl residues synthesised are critical to the structural integrity of the mycobacterial cell wall. They have investigated the role of RmlA in the biosynthesis and assembly of cell wall in M.smegmatis by RmlA overexpression which caused morphological changes including cell elongation and the appearance of polar bulbs at the tip of the cell. Although the over expression of RmlA in PknG deficientM. smegmatis results in significant dTTP depletion, intracellular dTTP concentration was not affected which lead them to suggest a role for PknG in the regulation of dTTP uptake. However, the manuscript and the experiments presented(including grammatical and spelling errors) do not adequately justify the title and the abstract.

1. In the conditional knockdown of RmlA (Qu et al) the effect on cellular morphology has been investigated. The overexpression levels of rmlA transcript (40 fold in constitutive and 80 fold in inducible expression) is way too high to see physiological effect. At protein level (not estimated) it could be much more. The overexpression levels should be between 5 to 10 fold to be physiologically relevant. Either they could use the knock down strain of Qu et al or generate a conditional knockdown of rmlA using a CRISPR-Cas or another approach to address how rmlA affects cell wall bio-synthesis pathway and cellular dNTP pool.

2.What is the effect of over expression of RmlA on the expression levels of Rml B-D? The connection between replication and cell wall biosynthesis needs to be clearly demonstrated. Increase in transcript level of replication stress factors like whmD and iniA upon RmlA overexpression does not establish the connection between cell wall synthesis and replication. Increase in lexA would imply SOS connection?

3. Increase in RmlA would mean higher utilization of dTTP. Although they claim that there is no depletion of dTTP, transient depletion may lead to thymine starvation which can induce stress.

4. In Figure 3, the cells depicting bulging can should be quantitated and a zoomed clear figure of the bulged cells would be appropriate. The phase contrast images are not in focus and should be improved. Similarly in both confocal and super resolution microscopy (Fig. 6), the number of cells analyzed should be provided with statistical analyses. Two cells in RmlA m-orange panel are looking like cells in m-orange panel, with no helical pattern. Statistical data for helicaly patterned RmlA containing cells should be provided. How does localization of RmlA affects the conclusion of the authors linking cell wall biosynthesis and replication is not clear.

5. Line 204 – 208 : Tween 80 concentration can be increased (upto 0.5%) to ensure clumping is reduced, to obtain better growth patterns. Line 206 -208: vaguely written.

I am listing a few more of corrections. There are more.

1. Line 71: ‘that’ should be ‘this’.

2. Line 74: ‘overexpression’ is a better word

3. Line 80 should be: an M.smegmatis PknG knock out strain.

4. In Materials and methods: write the methods only and not why a method was used. You can provide the rationale for the experiment in Results section.

5. Line 140-141: Rephrase

6. Line 144: rephrase ‘2-2 stress factors’

7. Line 153,154: ‘Quigene’ should be ‘Qiagen’.

8. Lines 207-209: vaguely written; Rephrase

9. Line 213: constant should be ‘constitutive’.

10. Fig 4 legend: No need to explain in the method in figure legend.

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Reviewer #2: No

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7 Jan 2022

Comments to the Author

Reviewer #1: This paper describes the construction and characterization of an overexpression strain of RmlA in M. smegmatis. Overall, the experiments were conducted well with appropriate controls. However the conclusions are not supported by the data. The manuscript should either be strengthened with additional data or the overstated claims removed.

Thank you for your constructive comments. In the revised version, we provided additional data and removed claims that are not fully supported by the data.

Major Points

L43. Essentiality of a gene is not a “strong basis” for the development of anti-tubercular agents. This is a gross over-statement.

We agree that essentiality alone is not a “strong basis” for drug development. We originally wrote that the essentiality of the gene together with the absence of a similar gene product in the host provides a strong basis.

Following your comment, we rephrased the message to “potentially valid drug target” in order to avoid any overstatement.

We also searched for the definition of a good drug target. According to a recent paper in Nat Rev Drug Discovery:

“Relevant characteristics of microbial drug targets include: essentiality for microbial growth and survival; pharmacological tractability and accessibility; similarity to related mammalian molecules; presence in important pathogens; potential for the development of resistance; and lack of target-based cross-resistance.” (Emmerich et al.: Improving target assessment in biomedical research: the GOT-IT recommendations. 20, 64–81 (2021). https://doi.org/10.1038/s41573-020-0087-3)

Considering these relevant characteristics, RmlA may qualify as a valid target. It was mentioned so recently here, as well: Qu D, Zhao X, Sun Y, Wu F-L and Tao S-C (2021) Mycobacterium tuberculosis Thymidylyltransferase RmlA Is Negatively Regulated by Ser/Thr Protein Kinase PknB. Front. Microbiol. 12:643951. doi: 10.3389/fmicb.2021.643951

L62 There is no evidence that RmlA is a checkpoint, this is a stretch from the published data. It is a hypothesis.

We corrected this and stated that it is a hypothesis.

L307 No measurements of cell length were presented to support the statement that they were significantly longer. the link to cell growth division is weak.

We quantified the length of ~ 3700 cells and now provide thorough statistics for cell length distribution in the various strains in Fig 5.

Reference to division regulation is removed.

The authors refer to extensive clumping in their cultures. What was the rationale for culturing M. smegmatis without Tween 80, as is the norm in the field to reduce aggregation? If clumping was an issue, how was OD determined reliably?

We did cultivate smegmatis cells in Tween 80 as described in Materials and methods. Extensive clumping happened only upon RmlA overexpression in spite of the presence of Tween in the medium. We added this comment to the text.

Although the OD measurements were noisier than in the WT cell cultures, the aggregation did not compromise the experiments. We consider the increased tendency to aggregate a phenotype upon RmlA overexpression, consistent with the fact that cell wall biosynthesis is perturbed.

The changes in gene expression for the stress response are marginal. Although they achieved statistical significance, the magnitude of the change was very small and much lower than the response to the control agents. Therefore, the claim that RmlA overexpression induces these stresses is not supported by the data.

We agree that changes upon RmlA overexpression in the WT strain are small. As we observed dTTP decrease only in the PknG-;RmlAi strain and not in the WT strain, we expected that the effect of RmlA overexpression in the PknG-;RmlAi strain could be larger. We, therefore, repeated the measurement and obtained, indeed, a larger change in the expression levels of all investigated stress factors (Fig 8). In the case of the established stress response genes whmD and radA, the measured overexpression in the PknG(-)/RmlAi strain is commensurable with that of the positive controls. The M. tuberculosis homologue of WhmD, Whib2 is overexpressed 2-3-fold in response to EMB and INH as shown below (Geiman et al., (2006) Differential Gene Expression in Response to Exposure to Antimycobacterial Agents and Other Stress Conditions among Seven Mycobacterium tuberculosis whiB-Like Genes, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2006, p. 2836–2841, doi:10.1128/AAC.00295-06):

The 3.3-fold overexpression of iniA in the PknG(-)/RmlAi strain is commensurable with the effect of induction by ampicillin, and by the combination B-lactam–B-lactamase inhibitor Unasyn (ampicillin/sulbactam), which was consistently three- to fivefold greater than in control cultures (Alland et al., (2000) Characterization of the Mycobacterium tuberculosis iniBAC Promoter, a Promoter That Responds to Cell Wall Biosynthesis Inhibition, JOURNAL OF BACTERIOLOGY, doi: 10.1128/JB.182.7.1802-1811.2000.). Following gamma irradiation (!), 2.5-fold increase in the mRNA level of radA was found in Pyrococcus furiosus (Williams et al., (2007) Microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus exposed to gamma irradiation, Extremophiles, doi: 10.1007/s00792-006-0002-9.) to be compared to our 6-fold increase in the PknG(-)/RmlAi strain. In view of these data, we hope that the Reviewer agrees that the measured stress response in our experiments is relevant in addition to being statistically significant.

The gene expression studies did not include any no RT controls to account for the presence of genomic DNA.

We now included the no RT and no template controls in the excel table submitted in the supplementary zip file as part of the data availability requirements.

For the experiments with CIP and EMB, how was the concentration used determined/selected?

We scanned a range of drug concentrations with the objective to arrive at a level of growth inhibition between 20-80 % in order to have enough cells left to analyse. We included Fig 7 in the manuscript showing that the selected concentrations were in this regime.

Some more explanation of the methods to measure dNTPs is needed. It was not clear. In the results how this was done.

We now included a brief description of the principle of the method used in addition to the reference for the published method:

“The principle of the assay is similar to that of the TaqMan assay. All four dNTPs are determined separately using dNTP-specific templates. The fluorescence output signal upon DNA elongation is directly proportional to the dNTP to be quantified.”

The discussion was very lengthy. It should be restricted to a discussion of the results in context of the published literature instead of a literature review. For example, Lines 311 to 361 read like a literature review.

We agree and now restrict the discussion to subjects immediately pertinent to the results.

L388 The conclusion that RmlA overexpression leads to cell wall and replication stress is not supported by the data in the paper. In fact, it's more likely that RmlA overexpression does not lead to cell wall or replication stress based on the very small changes in gene expression. the discussion should be revised to remove the overstated claims and limit discussion to what is shown in the data.

As RmlA overexpression in the WT background did not result in solid replication and cell wall stress, we repeated the measurement in the PknG-;RmlAi strain, as well. In the case of the established stress response genes whmD and radA, the measured overexpression in the PknG(-)/RmlAi strain is commensurable with that of the positive controls (Fig 8). Please see above a longer version of this response with references.

Nevertheless, we revised the text and removed overstated claims.

The supporting information should be included in the main body of the paper as it is required to follow the methods.

Following your suggestion, we included the supporting figures in the main body of the paper (Fig 2B and Fig 3 in the revised version).

Minor points. There was a lot of unscientific language and claims which were not supported by refernces e.g.

Thank you for pointing these out. We reworked the text extensively.

L23 what is “outstanding genetic adaptability”?

This expression was not necessary to support the claim and was removed.

L24 “most dreaded member”

Removed.

L144 what does “2-2 stress”mean?

The “2-2 stress factors” referred to RadA and LexA, and IniA and WhmD. The sentence is rephrased for clarity.

References are missing to support the statements in L24, L38.

References are now added.

L193 what does the “X’ stand for? What was the maximum concentration tested against M. smegmatis?

X remained in the manuscript by error. We used the maximum concentration limited by water solubility: 200 µg/ml. This is 8-fold the MIC determined in M. tuberculosis.

L206 “somewhat disturbed by the intensive clumping”

Expression replaced using scientific language.

L240 what does “quasi unchanged” mean?

Expression replaced using clear statements.

L198 and others “constitutive” not “constant”

Corrected.

L245 Reference in wrong format

Reference corrected.

L330 is “bulgy” a real word?

Replaced using scientific language.

L87-90 The methods section should describe the methods, not the rationale or interpretation of data.

We removed rationales and interpretations.

Why did the authors use TC and not anhydrotetracycline?

In Williams et al: Improved Mycobacterial Tetracycline Inducible Vectors (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3495547/#!po=73.5294) (Fig 3), the induction of protein expression was investigated using TC, ATC, and doxycycline. TC proved to be the most efficient of the three, and therefore, we also used TC.

L101. Table legend should just be the legend.

Corrected.

Abbreviations are inconsistent. For example, why is M. smegmatis used and not M. tuberculosis? Why is Cip lower case, but EMB upper case. Why is there an abbreviation for EMB but no isoniazid?

Corrected.

Abbreviations should be defined at first use.

Corrected.

Figure 1 is not required as the data do not support this conclusion.

This figure presents in a transparent way the hypothesis we examined. It does not contain any suggestions to the conclusions. We highlighted in the figure legend that this is a hypothesis.

Reviewer #2: In this manuscript entitled “Perturbation of rhamnose biosynthesis reveals crosstalk between mycobacterial cell wall synthesis and DNA replication” Hirmondo et al. have attempted to elucidate the crosstalk between cell wall synthesis and DNA replication in M. smegmatis, by overexpressing RmlA. RmlA, is an essential gene synthesizing L-Rhamnosyl residues using D-glucose-1-phosphate (G1P) and dTTP as the precursors. The rhamnosyl residues synthesised are critical to the structural integrity of the mycobacterial cell wall. They have investigated the role of RmlA in the biosynthesis and assembly of cell wall in M.smegmatis by RmlA overexpression which caused morphological changes including cell elongation and the appearance of polar bulbs at the tip of the cell. Although the over expression of RmlA in PknG deficientM. smegmatis results in significant dTTP depletion, intracellular dTTP concentration was not affected which lead them to suggest a role for PknG in the regulation of dTTP uptake. However, the manuscript and the experiments presented(including grammatical and spelling errors) do not adequately justify the title and the abstract.

Thank you for your insightful comments. In the revised version, we removed overstated claims and changed the title and abstract accordingly.

1. In the conditional knockdown of RmlA (Qu et al) the effect on cellular morphology has been investigated. The overexpression levels of rmlA transcript (40 fold in constitutive and 80 fold in inducible expression) is way too high to see physiological effect. At protein level (not estimated) it could be much more. The overexpression levels should be between 5 to 10 fold to be physiologically relevant.

It is not clear to us what the Reviewer means by physiological in this case. When we perturb a system to investigate the role of a protein, we intentionally move away from physiologically relevant cellular concentrations. Using KO strains represents the extreme of this logic and is yet entirely accepted as a mode of perturbation.

Either they could use the knock down strain of Qu et al

This was our original intention. We contacted Prof Ma and his colleagues several times with no response (see the request for the strain in the screenshot below). That is when we changed our strategy.

or generate a conditional knockdown of rmlA using a CRISPR-Cas or another approach to address how rmlA affects cell wall bio-synthesis pathway and cellular dNTP pool.

This is a very good approach. The reason why we chose another one after several trials to ask for the knock-down already generated by Qu et al is that we were primarily interested in the dNTP pool and in the potentially resulting replication stress. Therefore, we thought that overexpression and in consequence, the expected depletion of dTTP could be a good tool to investigate the formulated hypothesis. Looking at the morphological changes resulting from RmlA overexpression, it still seems like a valid tool for the perturbation of the relevant physiological pathways.

2.What is the effect of over expression of RmlA on the expression levels of Rml B-D?

We completed the experiment with the inclusion of the RmlB-D transcripts, as suggested. RmlA overexpression did not significantly change the level of RmlB-D mRNAs (Fig 8).

In the repeated experiment, we changed the 16S RNA reference gene to Ffh as we found the latter to be more stable in this experimental system. The change of one of the reference genes naturally modified fold-change values but not tendencies and conclusions.

The connection between replication and cell wall biosynthesis needs to be clearly demonstrated. Increase in transcript level of replication stress factors like whmD and iniA upon RmlA overexpression does not establish the connection between cell wall synthesis and replication.

In the revised version, we removed overstatements.

Increase in lexA would imply SOS connection?

Yes. Replication stress generally induces SOS response.

3. Increase in RmlA would mean higher utilization of dTTP. Although they claim that there is no depletion of dTTP, transient depletion may lead to thymine starvation which can induce stress.

Thank you for this comment; we included it in the text.

4. In Figure 3, the cells depicting bulging can should be quantitated and a zoomed clear figure of the bulged cells would be appropriate.

We conducted further microscopy experiments to better show this change in cell morphology (currently Fig 4). We also included fluorescence imaging to show morphology (Fig 4B) and quantitated all our cell biology experiments.

The phase contrast images are not in focus and should be improved.

We tried our best to achieve high-quality images using a microscopy objective HCX PL FLUOTAR 100x; NA.=1.30 OIL. The average length of M. smegmatis in our cultures is 2 microns, the average width is 0.5 microns. The width of the cells is therefore only 2-3x larger than the resolution limit of our system. In our view, this technical limitation does not hinder the clear presentation of the phenotype upon RmlA overexpression.

Similarly in both confocal and super resolution microscopy (Fig. 6), the number of cells analyzed should be provided with statistical analyses.

We agree and now provide the requested statistical analyses.

Two cells in RmlA m-orange panel are looking like cells in m-orange panel, with no helical pattern.

The referred cells are overexposed in the settings presented. Setting a lower gain to visualize these cells, we obtain the same helical pattern as in the other cells chosen for demonstration. Both the low and high gain pictures are available in the row data zip file. The presented statistical analysis now shows that the helical pattern is present in 100% of the cells visualized.

Statistical data for helicaly patterned RmlA containing cells should be provided.

Data are now provided.

How does localization of RmlA affects the conclusion of the authors linking cell wall biosynthesis and replication is not clear.

The currently available data do not establish any linkage between the subcellular localization of RmlA and its potential role in linking cell wall biosynthesis and replication. We rephrased the text so that our position on this issue is clear.

5. Line 204 – 208 : Tween 80 concentration can be increased (upto 0.5%) to ensure clumping is reduced, to obtain better growthpatterns.

Although the OD measurements were noisier than in the WT cell cultures, the aggregation did not compromise the experiments. We consider the increased tendency to aggregate a phenotype upon RmlA overexpression consistent with the fact that cell wall biosynthesis is perturbed.

Line 206 -208: vaguely written.

Replaced using scientific language.

I am listing a few more of corrections. There are more.

1. Line 71: ‘that’ should be ‘this’.

Corrected.

2. Line 74: ‘overexpression’ is a better word

Corrected.

3. Line 80 should be: an M.smegmatis PknG knock out strain.

Corrected.

4. In Materials and methods: write the methods only and not why a method was used. You can provide the rationale for the experiment in Results section.

Corrected.

5. Line 140-141: Rephrase

Rephrased.

6. Line 144: rephrase ‘2-2 stress factors’

Rephrased.

7. Line 153,154: ‘Quigene’ should be ‘Qiagen’.

Corrected.

8. Lines 207-209: vaguely written; Rephrase

Rephrased.

9. Line 213: constant should be ‘constitutive’.

Corrected.

10. Fig 4 legend: No need to explain in the method in figure legend.

Explanation removed.

Submitted filename: 220107 Point by point answers to referees.pdf

Click here for additional data file.

2 Feb 2022

The effects of mycobacterial RmlA perturbation on cellular dNTP pool, cell morphology, and replication stress in Mycobacterium smegmatis

PONE-D-21-26930R1

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Reviewer #2: The authors have addressed most of my concerns an explained if they could not carry out other suggested experiments. Although they have interesting observations, it is not clear how Rml impacts cellular functions especially replication stress and cell architecture.

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11 Feb 2022

PONE-D-21-26930R1

The effects of mycobacterial RmlA perturbation on cellular dNTP pool, cell morphology, and replication stress in Mycobacterium smegmatis

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