A symmetric molecule produced by mycobacteria generates cell

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Letter

A symmetric molecule produced by mycobacteria generates cell-length asymmetry during cell-division and thereby cell-length heterogeneity Nagaraja Mukkayyan, DEEPTI SHARAN, and Parthasarathi Ajitkumar ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00080 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 16, 2018

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ACS Chemical Biology

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Letters

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A symmetric molecule produced by mycobacteria generates cell-

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length asymmetry during cell-division and thereby cell-length

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heterogeneity

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Nagaraja Mukkayyan, Deepti Sharan and Parthasarathi Ajitkumar*

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Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru,

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Karnataka.

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*Corresponding author: [email protected]

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Keywords: Mycobacteria; Asymmetric constriction during division; Cell-length

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asymmetry; Diadenosine hexaphosphate; Ap6A; Cell-length heterogeneity

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1 ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT: Diadenosine polyphosphates, Ap(2-7)A, which contain two adenosines

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in a 5’,5’ linkage through phosphodiester bonds involving 2-7 phosphates, regulate

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diverse cellular functions in all the organisms, from bacteria to humans, under

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normal and stress conditions. We had earlier reported consistent occurrence of

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asymmetric constriction during division (ACD) in 20-30% of dividing mycobacterial

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cells in culture, irrespective of different growth media, implying exogenous action of

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some factor of mycobacterial origin. Consistent with this premise, concentrated

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culture supernatant (CCS), but not the equivalent volume-wise concentrated unused

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medium, dramatically enhanced the ACD proportion to 70-90%. Mass spectrometry

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and biochemical analyses of the bioactive fraction from CCS revealed the ACD-

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effecting factor to be Ap6A. Synthetic Ap6A showed mass spectrometry profile,

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biochemical characteristics and bioactivity identical to native Ap6A in the CCS. Thus,

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the present work reveals a novel role for Ap6A in generating cell-length asymmetry

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during mycobacterial cell-division and thereby cell-length heterogeneity in the

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population.

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All single cellular organisms and cells in multicellular organisms use a variety of

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small molecules for cell-to-cell communication to modulate diverse cellular functions.

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Among the small molecules, the conserved diadenosine polyphosphates, Ap(2-7)A,

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are involved in diverse cellular processes in all the organisms, from bacteria to

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humans1-14. Bacterial systems use ApnA molecules to modulate oxidative/heat stress

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response, competence, host invasion, bioluminescence, virulence, cell-division

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timing, and biofilm formation, although their mechanism of action remain unknown1-6.

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It is well known that bacteria use small molecules in quorum sensing to generate

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phenotypic heterogeneity among individual members in a subpopulation or among

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subpopulations for survival advantage15. Since bacteria generate and maintain

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phenotypic heterogeneity with genetic identity to survive under stress conditions,

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knowledge of the mechanisms and mediators used by bacteria to generate different

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types of heterogeneity will be useful for the development of antibacterial compounds.

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Bacterial populations harbour phenotypic heterogeneity in cell-length, cell-size,

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morphology, biomolecular contents, metabolic status and in several other

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parameters, for survival under diverse growth conditions16,17. Both pathogenic and

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non-pathogenic mycobacteria maintain phenotypic heterogeneity in in vitro cultures,

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mice, guinea pigs and tuberculosis patients18-25. Three research groups and our

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laboratory had earlier shown that 70-80% of Mycobacterium tuberculosis (Mtb),

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Mycobacterium smegmatis (Msm), Mycobacterium xenopi (Mxe), Mycobacterium

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bovis BCG (Mbo BCG) and Mycobacterium marinum (Mma) cells in culture divided

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with 5-10% deviation of the constriction position from the median, producing slightly

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unequal-sized sister-daughter cells19,20,26-28. Despite the slight deviation in the final

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constriction position, due to the accurate median placement of FtsZ/septum26, the



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division is still considered symmetric. Hence, we call it symmetric constriction during

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division (SCD).

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As a striking deviation from SCD, we recently showed that the remaining 20-

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30% of the septating mid-log phase (MLP; 0.6 OD600

nm,

herein after called OD)

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population of Mtb, Msm and Mxe cells, divided with the final division constriction

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position at 11-31% deviated from the median, irrespective of growth media19,20. This

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generated short-sized cells (SCs) and normal/long-sized cells (NCs) as sister-

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daughter cells, thereby creating cell-length heterogeneity in the population19,20. We

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call this asymmetric constriction during division (ACD). In the present study, we

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identified and structurally and functionally characterised a small molecule, Ap6A,

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produced by Mtb and Msm into the culture medium at specific proportions, which act

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on mycobacterial cells to effect ACD on specific proportions of dividing cells in a

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growth phase dependent manner.

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Since 5-10% deviation of division constriction position occurring in 70-80% of

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mycobacterial cells is still called symmetric26, we defined the extent of deviation of

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division constriction position in ACD for cell-length measurements in the present

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study. For this purpose, we determined the distribution frequency of percentage

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deviations of constriction position by staining the septum and cell membrane with

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FM-4-64 and measuring the lengths of sister-daughter cells of the septum-

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constricted V-snapped mother cells (n=1000), using co-localised images of FM-4-64

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stained septum fluorescence and DIC of constriction. These measurements showed

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that 20-30% of the cells divide with constriction position deviation of ≥11% (Figure

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S1). This cut-off for constriction position deviation for ACD was consistent with the

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11-31% deviation in the constriction position in 20-30% of the V-snapped septated

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cells, using only DIC images19,20. Hence, in the present study, the V-snapped


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division constrictions showing deviation of ≥11% in DIC images were taken as ACD

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for both Mtb and Msm cells.

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Since ACD occurred consistently in 20-30% of the dividing Mtb, Msm and Mxe

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cells in the MLP population19,20, we hypothesised that there might exist an ACD-

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effecting molecule (ACD-EM) synthesised and maintained at specific proportion in

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the culture medium by mycobacteria to effect ACD on a definite proportion of the

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dividing cells. Consequentially, exposure of MLP cells to the cell-free concentrated

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culture supernatant (CCS) should significantly increase the proportion of cells

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undergoing ACD (Figure 1A,B). Proving this premise true, exposure of Mtb and

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Msm MLP cultures to the CCS from the respective MLP (0.6 OD) and 0.8 OD

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cultures dramatically reversed the ACD:SCD ratio from ~20:80 to ~70:30 (Figure

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1C,D, respectively; Figure S2A,B, respectively). Neither the volume-wise

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equivalently concentrated unused growth medium (negative control) nor the

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unconcentrated fresh growth medium altered the 20:80 ACD:SCD ratio in the Mtb

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and Msm MLP cultures (Figure 1C,D; Figure S2A,B, respectively). These

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observations confirmed that the culture medium contained an ACD-EM that came

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from the mycobacterial cells. Live cell imaging further confirmed that significantly

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high number of Msm MLP cells undergo ACD in the presence of CCS from Msm

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MLP and 0.8 OD cultures (Supporting videos 1-3; Figures S3-S5).

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Since the biochemical nature of the ACD-EM needs to be known for its isolation

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and purification from CCS, the CCS of Mtb and Msm MLP and 0.8 OD cultures were

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exposed to DNase I, snake venom phosphodiesterase, RNase A, lipase, and

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proteinase K, and subsequently tested for the bioactivity. The expectation was that

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susceptibility of the ACD-EM to any one or more of these enzymes and

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consequential loss of bioactivity might reveal the biochemical nature of the molecule 5 ACS Paragon Plus Environment


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(Figure 1E,F). Exposure of CCS from Mtb and Msm MLP and 0.8 OD cultures to

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DNase I and snake venom phosphodiesterase (SVP) abolished the bioactivity of

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ACD-EM in the CCS (Figure 1G,H and I,J, respectively; Figure S6A,B,

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respectively). But RNase A, lipase or proteinase K (the latter two enzymes were

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filtered out from the CCS before adding to the MLP cells, to avoid damage to the

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cells) did not abolish the bioactivity of ACD-EM in the CCS of Msm MLP and 0.8 OD

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cultures (Figure S6C-G). In the control experiment, exposure of Msm MLP cells in

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culture directly to DNase I, SVP, RNase A, and lipase did not change the ~30:70

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ACD:SCD ratio (Figure S6 A-E). Thus, the abolition of the bioactivity of ACD-EM in

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the CCS by DNase I and SVP was not due to the direct action, if any, of the

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enzymes present in the enzyme-exposed CCS on the cells per se. However, Msm

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MLP CCS could not change the 20:80 ACD:SCD ratio of the Msm MLP culture pre-

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exposed directly to proteinase K (Figure S6E). Probably cell surface integrity is

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required for the action of ACD-EM on the cells. These biochemical characteristics of

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ACD-EM indicated that it contains phosphodiester bond(s) and might be DNA-like,

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but not RNA/lipid/peptide-like.

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Since ACD-EM was likely to be anionic due to the presence of phosphodiester

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bond(s), the cell-free CCS from Mtb and Msm 0.8 OD cultures were extracted with

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weak anion exchange (WAX) resin cartridges (see online methods). Strong anion

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exchange (SAX) column based HPLC purification of ACD-EM from the respective

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lyophilised Mtb WAX and Msm WAX eluates showed a distinct peak of 36.5 ± 0.5

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min retention time for the Mtb WAX sample (n=3; Agilent instrument) and of 32.5 ±

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0.5 min retention time for the Msm WAX sample (n=3; Thermo Finnigan Surveyor

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instrument) (Figure S7A,B). Both the Mtb SAX and Msm SAX peaks showed

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identical retention time of 36.0 ± 0.5 min on the Agilent instrument (n=3) (Figure


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S7C). The dramatic reversal of the ~20:80 ACD:SCD ratio to ~90:10 and ~75:25,

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effected by the Mtb SAX and Msm SAX peaks, respectively, was abolished by

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DNase I and SVP (Figure S8A,B). ESI-MS analyses of the bioactive Mtb and Msm

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HPLC peaks, which showed identical retention time of 5.6 ± 0.2 min (n=3) on the

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reverse phase column (Figure S9A,B), gave comparable MS1 profiles (Figure

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S10A-C). High-resolution ESI-MS (HRMS) analyses, which were performed for the

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CID (collision-induced dissociation) fragmentation patterns of the parent ion of m/z,

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994.986 and 994.898 (where z = 1) of Mtb and Msm bioactive peaks, respectively,

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showed identical profile of the daughter ions (Figure 2A,B). In-depth analyses of the

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MS2 spectra, with the set of prominent daughter ions from the respective parent ions

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(Figure 2A,B), revealed that the ACD-EM present in the Mtb and Msm bioactive

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peaks was diadenosine 5’, 5’’’’-P1, P6-hexaphosphate (Ap6A) – a symmetric

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molecule in which two adenosines are linked 5’, 5’ through five phosphodiester

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bonds involving six phosphates (Figure 2D; Table S1).

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Synthetic Ap6A also showed HPLC retention time of 36.0 ± 0.5 min (n=3; SAX

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column; Agilent instrument) and 5.6 ± 0.2 min (n=3; reverse phase column) (Figures

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S7C,S9C, respectively), identical to those of the native Ap6A of Mtb and Msm

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bioactive peaks on these columns (Figure S7C,S9A,B). Similarly, the MS and

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MS/MS profiles of synthetic Ap6A were identical to those of the native Ap6A in the

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Mtb and Msm bioactive peaks (Figure 2C, Figure S10D, respectively). Consistent

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with these identical characteristics, synthetic Ap6A effected significant reversal of the

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~20:80 ACD:SCD ratio of Mtb and Msm MLP cultures to ~88:12 and ~73:27 ratio,

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respectively (Figure 2E,F, respectively). Moreover, DNase I and SVP abolished the

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ACD-effecting activity of synthetic Ap6A on Msm cells (we did not check the same on

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Mtb cells) (Figure 2F). Live cell imaging showed that synthetic Ap6A effected ACD



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on ~95% of Msm MLP cells (Figure 2G,H; Supporting video 4). Identical

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bioactivities, biochemical properties, and MS/MS profiles of native Ap6A in the Mtb

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and Msm culture supernatants and of synthetic Ap6A unequivocally established the

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identity of ACD-EM as Ap6A.

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Quantitation of Ap6A using ESI-MS (see online methods) showed that the 0.8

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OD CCS contained highest concentrations of Mtb and Msm Ap6A, as compared to

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the CCS from other OD cultures (Figure 3A,B, respectively). Interestingly, the

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concentration of Ap6A at 0.8 OD was six times higher in Mtb CCS than in the Msm

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CCS prepared from cultures containing equivalent number of cells. The higher levels

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of Ap6A in the respective 0.8 OD CCS correlated with the higher proportions of Mtb

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and Msm cells dividing by ACD in the 0.8 OD culture than in the respective MLP (0.6

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OD) cultures (Figure 3C,D & E,F, respectively). Further, the relative proportions of

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SCs and NCs were found to be significantly high in all the Mtb and Msm cultures

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exposed to the CCS from MLP and 0.8 OD, and synthetic Ap6A (Figure S11A-H).

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However, in the 1 OD culture, although the proportion of cells undergoing ACD:SCD

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was ~90:10, the levels of Ap6A in the CCS of 1.0 OD culture was not as high as that

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in the 0.8 OD CCS. It is possible that the high concentration of Ap6A produced by 0.8

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OD cells would have already acted upon the target cells for an ACD:SCD ratios of

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~90:10 and ~84:16 for Mtb and Msm respectively, which was sustained till the

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culture reached 1.0 OD (Figure 3D,F). This response is reminiscent of the quorum

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sensing phenomenon where the levels of quorum sensing molecules build up to a

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threshold concentration when the cells in the population respond in a sustained

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manner29. Drawing parallels, the response of the 0.8 OD cells to the high levels of

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Ap6A by increasing ACD proportion to all-time high seemed to have been sustained

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till the culture reached 1.0 OD even though by which time the levels of the effector


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decreased. Thus, the bacilli seem to regulate the production of Ap6A such that only

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specific proportion of the population are acted upon by Ap6A to undergo ACD during

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a specific window during the growth phase. The 5-10% deviation in the constriction

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found in 70-80% of cells (SCD), may be occurring naturally without being affected by

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Ap6A.

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Since the ACD proportion at 0.8 OD was ~90% for Mtb cells and ~84% for Msm

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cells (see Figure 3D, F, respectively), as compared to other OD values of growth,

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and exposure of 0.6 OD cells to Ap6A could also increase the ACD proportion to

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similar levels, we examined whether the response-readiness of cells to Ap6A was

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only at 0.8 OD where nutrient stress may begin to set in. However, the Mtb and Msm

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cells from 0.3 OD cultures also responded like the cells from the respective 0.6 OD

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cultures, to increasing concentrations of Ap6A, with the ACD proportion increased to

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~80-90% (Figure S12). This ruled out the possibility of growth phase specific,

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nutrient stress dependent response of cells to Ap6A. However, a link between the

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increase in the levels of Ap6A at 0.8 OD and the nutrient stress associated with 0.8

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OD growth phase may be a possibility.

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Consistent with Mtb and Msm cells maintaining Ap6A in the culture medium to

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effect ACD, the CCS of the Msm MLP and 0.8 OD cultures and that of Mtb MLP and

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0.8 OD cultures could cross-effect significantly high proportion of ACD on the MLP

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cells of each other (Figure S13A,B). The 0.8 OD CCS of Escherichia coli K12 cells

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grown in different media did not alter the ~25:75 ACD:SCD ratio of Msm MLP cells

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(Figure S14), indicating that Ap6A is not present in the E. coli culture supernatant

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and may be conserved among mycobacterial species. Further, Msm genomic DNA

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sonicate did not alter the ~25:75 ACD:SCD ratio showing that sheared genomic

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DNA, if any, present in the CCS from lysed cells, was not the effector of ACD



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(Figure S15). Extended exposure of Mtb and Msm MLP cells to the respective MLP

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CCS did not further change the ~80:20 ACD:SCD ratio (Figure S16A,B,

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respectively). Finally, from a technical point of view, the measurement of the

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proportions of Msm cells undergoing ACD and SCD using para-formaldehyde fixed

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cells and unfixed adhered cells were comparable, indicating that cell fixation used for

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the length measurements did not influence the quantitation (Figure S16C).

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Thus, the present study reveals that mycobacteria maintain specific

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concentrations of Ap6A in the growth medium to effect ACD for the generation of cell-

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length asymmetry between sister-daughter cells at birth to produce SCs and NCs.

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Cell-length heterogeneity being a characteristic feature of mycobacterial populations,

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it is possible that the SCs and NCs formed with difference in length at birth from the

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ACD of mother cells of varying lengths can contribute to cell-length heterogeneity in

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the population. We verified this possibility by determining the distribution of cell-

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lengths of sister-daughter cells of V-snapped (just completed constriction) Mtb and

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Msm mother cells (n=1000) undergoing ACD at 0.3, 0.6, 0.8 and 1.0 OD values and

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of 0.6 OD mother cells exposed to Ap6A (positive control). A wide range of cell-

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lengths were found in the distribution of the sister-daughter cells from the V-snapped

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Mtb and Msm mother cells at these OD values where the bacilli produced different

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levels of Ap6A (Figure 4, Table S2, Figure 3A,B). Interestingly, cell-length variation

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at birth between the sister-daughter cells arising from ACD was found confined to the

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longer-sized sister-daughter cells. Thus, although ACD seems to be generating only

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a binary product, the variations in the length of the longer-sized sister-daughter cells

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contribute to cell-length heterogeneity. These observations show that the

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physiological role of Ap6A may be to generate cell-length heterogeneity in the

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population by effecting asymmetric constriction.


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However, the heterogeneity in cell-length may be what is phenotypically

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apparent while the SCs and NCs produced by ACD may have significantly different

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molecular and metabolic profiles. This possibility is alluded to by the low density of

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SCs and high density of NCs, found during their enrichment on Percoll density

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gradient, indicative of atleast difference in lipid content30. The possibility for

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significant metabolic differences between them was confirmed by the significantly

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higher tolerance of NCs than SCs to oxidative/nitrite stress and anti-tuberculosis

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antibiotics30. Thus, the fundamental physiological role of Ap6A and thereby of ACD in

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mycobacteria may be to generate metabolic heterogeneity among subpopulations.

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Among the roles of the conserved diadenosine polyphosphates, Ap(2-7)A, in diverse

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cellular processes in all the organisms from bacteria to higher eukaryotes1-14, the

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novel role of Ap6A in mycobacteria seems to be different from the role of cell division

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timing control proposed for diadenosine tetraphosphate (Ap4A) in E. coli9.

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Asymmetric constriction during cell division could be a consequence of

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differential polar (cell-tip) growth26,31 or asymmetric condensation of nucleoids and

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consequential asymmetric positioning of nuceloids19,20 or asymmetric segregation of

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chromosomes as in Xanthomonas citri ssp. citri32 or of asymmetric partitioning of

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cellular components between sister-daughter cells during cell division33,34. In view of

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these possibilities, it is tempting to speculate that Ap6A might be prompting any one

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or more of these processes resulting in asymmetric constriction during division.

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Although both pathogenic and non-pathogenic mycobacteria use Ap6A for ACD,

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Ap6A-prompted ACD in Mtb may have clinical relevance as alluded to by the

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presence of asymmetrically sized cells (SCs and NCs) in the sputum of pulmonary

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tuberculosis patients19 and differential antibiotic tolerance of SCs and NCs30.

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METHODS

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Experimental Methods are given in detail in the Supporting Information.

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ASSOCIATED CONTENT

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Supporting Information includes Methods, Supporting Figures (Figures S1-S16),

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Legends for Supporting Videos (Videos S1-S4), Supporting Tables (Tables S1, S2)

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and Supporting References.

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AUTHOR INFORMATION

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Corresponding Author

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E-mail: [email protected]

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*Address: Department of Microbiology and Cell Biology, Indian Institute of Science,

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Bengaluru, Karnataka.

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Author Contributions

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PA conceived the study. PA, NM and DS designed the experiments. NM and DS

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performed the experiments. PA, NM and DS analysed the data. PA and NM wrote

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the manuscript.

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Funding

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This work was supported by funds received by PA from the DBT-IISc Partnership

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Programme and Indian Institute of Science, and by the infrastructure support from

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DST-FIST, UGC Centre for Advanced Study, ICMR Centre for Advanced Study in

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Molecular Medical Microbiology and Indian Institute of Science. NM and DS received

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senior research fellowship from Indian Institute of Science and UGC, respectively.


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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS

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Authors acknowledge the DBT-supported LC-ESI-MS/MS facility at Biological

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Sciences Division and are grateful to A. Roy for technical help with mass

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spectrometry and M. Naik for HPLC analyses. Authors thank P. D. Deepalakshmi

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(CeNSE, IISc) for technical discussions on the mass spectrometry data & analyses

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and lab colleagues for critical suggestions on the work.

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REFERENCES

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Visual Abstract for the manuscript 7x6mm (600 x 600 DPI)

ACS Paragon Plus Environment

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Figure 1. The presence and biochemical nature of ACD-EM. Cartoon: ACD:SCD ratio of Mtb and Msm cells (A) at MLP, (B) exposed to CCS. Quantitation of (C) Mtb MLP cells and (D) Msm MLP cells, exposed to respective CCS from MLP and 0.8 OD cultures. Cartoon: ACD:SCD ratio of Mtb and Msm cells (E) if the enzymes affected ACD-EM in the CCS and (F) if the enzymes did not affect ACD-EM in the CCS. The CCS of Mtb (G) MLP, (H) 0.8 OD and Msm (I) MLP (J) 0.8 OD exposed to DNase I (2 U) and SVP (0.001 U). Bars represent the percentage of Mtb and Msm cells dividing by ACD (black) and SCD (grey) (n=300; biological triplicates with mean values ± s.d. ***P < 0.001 via two-tailed t-test). 18x23mm (600 x 600 DPI)



Figure 2. Structure of ACD-EM in the Mtb and Msm CCS. Negative ion mode tandem mass (MS2) spectra after CID fragmentation of ion with m/z = 994.986, 994.898, and 994.939 (z = 1) obtained for (A) Mtb, (B) Msm and (C) synthetic Ap6A, respectively. (D) Molecular structure and prominent ESI-CID fragmentation pattern generated for the monoisotopic mass of synthetic Ap6A (m/z 995.98). Exposure of MLP cells of (E) Mtb and (F) Msm to Ap6A (166 pM) and DNase I (2 U) and SVP (0.001 U) treated Ap6A (166 pM). Bars represent the percentage of Mtb and Msm cells dividing by ACD (black) and SCD (grey) (n=300; biological triplicates with mean values ± s.d. and ***P < 0.001 via two-tailed t-test. (G) Representative live cell timelapse micrograph panels of Msm MLP cells exposed to synthetic Ap6A recorded at the time points indicated (hr:min). The arrow head indicates the site of constriction. (H) The percentage of Msm cells dividing by ACD (black) and SCD (grey) (n = 54 cell divisions; biological replicates with mean values ± s.d. Micrograph corresponds to Supplementary video 4). 15x15mm (600 x 600 DPI)


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Figure 3. Concentration of Ap6A and ACD:SCD ratio in Mtb and Msm culture. The concentration of Ap6A ml-1 at 0.3, 0.6, 0.8 and 1.0 OD CCS obtained from (A) Mtb and (B) Msm cultures. Bar graph represents the concentration of Ap6A (nM) per ml of Mtb and Msm culture (n=3; biological triplicates with mean values ± s.d. ***P < 0.001 via two-tailed t-test). (C) Micrographs and (D) quantitation of Mtb ACD:SCD ratio at 0.3, 0.6, 0.8 and 1.0 OD. (E) Micrographs and (F) quantitation of Msm ACD:SCD ratio at 0.3, 0.6, 0.8 and 1.0 OD. In the micrographs, ACD (white star) and SCD (white diamond). Bars represent the percentage of Mtb and Msm cells dividing by ACD (black) and SCD (grey). (n=300; biological triplicates with mean values ± s.d. ***P < 0.001 via two-tailed t-test). 14x13mm (600 x 600 DPI)



Figure 4. Ap6A generates cell-length heterogeneity in mycobacteria. Mtb sister-pairs cell-length distribution at 0.3, 0.6, 0.8 and 1.0 OD during ACD (A,B), and SCD (C,D). Msm sister-pairs cell-length distribution at 0.3, 0.6, 0.8 and 1.0 OD during ACD (E,F), and SCD (G,H). Mtb MLP cells exposed to Ap6A (166 pM) and its sister-pairs cell-length distribution during ACD (I,J), and SCD (K,L). Msm MLP cells exposed to Ap6A (166 pM) and its sister-pairs cell-length distribution during ACD (M,N), and SCD (O,P). Lines represent the percentage of cells at 0.3 OD (blue), 0.6 OD (saffron), 0.8OD (grey) and 1.0 OD (yellow), and MLP cells of Mtb and Msm exposed to Ap6A (purple) (n=1000). See Table S2 for coefficient of variance. 11x9mm (600 x 600 DPI)


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A symmetric molecule produced by mycobacteria generates cell

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