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1
Plant peptidoglycan precursor biosynthesis: Conservation 2
between moss chloroplasts and Gram negative bacteria 3
Amanda J. Dowson,a2 Adrian J. Lloyd,a Andrew C. Cuming,b David I. Roper,a Lorenzo 4
Frigerioa and Christopher G. Dowsona3 5
aSchool of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom 6
bCentre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom 7
1This work was supported by a Pump Priming Initiative from the University of Warwick. 8
2Corresponding author 9
3Senior author 10
A.J.D generated the heterologous expression vectors, prepared and assayed proteins, purified peptidoglycan intermediates 11
from P. patens and wrote the manuscript; A.J.L. conceived use of TCA extraction, performed the mass 12
spectrophotometric analysis and analysed the data; A.C.C. assisted with P. patens handling; D.I.R, and L.F initiated the 13
enzymatic analysis and Pump Prime funding, C.G.D assisted with funding and A.J.L., D.I.R. and C.G.D. assisted with 14
experimental discussion and critiqued the manuscript. 15
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the 16
policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: 17
A.J.Dowson (a.j.dowson@warwick.ac.uk). 18
19
Short title: 20
Bacterial Peptidoglycan Precursors in Land Plants 21
22
Abstract 23
An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the 24
chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte 25
ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a 26
twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium 27
(Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with 28
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the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in 29
bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted 30
intermediates from UDP-GlcNAc through to the UDP-MurNAc-D,L-diaminopimelate (DAP)-pentapeptide. 31
Most Gram negative bacteria, including cyanobacteria, incorporate meso-diaminopimelate (D,L-DAP) into the 32
third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram positive bacteria. To 33
establish the specificity of D,L-DAP incorporation into the P.patens precursors, we analysed the recombinant protein, 34
UDP-MurNAc-tripeptide ligase (MurE), from both P.patens and the cyanobacterium Anabaena sp. strain PCC 7120. 35
Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric 36
data, with catalytic efficiencies similar to previously documented Gram negative bacterial MurE ligases. We discuss how 37
these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases 38
and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway. 39
40
Introduction 41
The endosymbiotic theory for the origin of photosynthetic eukaryotes proposes that an engulfed cyanobacterium evolved 42
into the first ancestors of chloroplasts (Dagan et al., 2013; Ponce-Toledo et al., 2017). As with bacteria, these organelles 43
(cyanelles) were surrounded by a peptidoglycan (or murein) wall (Scott et al., 1984). In bacteria, peptidoglycan covers 44
the organism in a mesh-like ‘sacculus’ confering resistance to osmotic stress, and a species-specific shape and size. 45
Although originally considered likely that peptidoglycan was lost from all photosynthetic organelles immediately after 46
the glaucophyte branch (Pfanzagl et al., 1996), there has been an accumulation of evidence including sensitivity of 47
chloroplast division to peptidoglycan-directed antibiotics, fluorescent labelling studies and gene knockout phenotypes to 48
indicate that many streptophytes, including the charophyte algae (Matsumoto et al., 2012; Takano et al., 2018) and some 49
bryophytes and pteridophytes (sister lineages to seed plants) (Takano and Takechi, 2010; Hirano et al., 2016), may have 50
chloroplasts that synthesize peptidoglycan. Furthermore, in gymnosperms (Lin et al., 2017) and also a diverse number of 51
eudicots (van Baren et al., 2016) all the critical genes for peptidoglycan synthesis have been identified, although a 52
potential penicillin binding protein (PBP) typically required for peptidoglycan cross-linking has not been confirmed in 53
eudicots. 54
The earliest evidence for peptidoglycan in embryophytes was uncovered when antibiotics affecting bacterial 55
peptidoglycan synthesis in the bryophyte moss P. patens (Kasten and Reski, 1997; Katayama et al., 2003) and lycophytes 56
and ferns (Izumi et al., 2008) were found to cause a decrease in chloroplast number with the formation of giant 57
(macro)chloroplasts. Subsequently, genomics and in silico analyses confirmed the presence of all essential bacterial genes 58
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for peptidoglycan biosynthesis (Rensing et al., 2008). These genes are nuclear-encoded, predominantly plastid-targeted 59
(Machida et al., 2006; Homi et al., 2009) and transcribed, as revealed by expressed sequence tags (ESTs). More recently, 60
a peptidoglycan layer surrounding P. patens chloroplasts has been visualized using a fluorescently labelled substrate 61
(Hirano et al., 2016) and electron microscopy (Sato et al., 2017). 62
Peptidoglycan in Gram negative bacteria has a repeating disaccharide backbone of β-(1,4) linked N-acetylglucosamine 63
(GlcNAc) and N-acetylmuramic acid (MurNAc) to which is appended a stem peptide comprising L-Ala, D-Glu, D,L-64
DAP, D-Ala--D-Ala. Variations in the amino acid residues have been identified and are consequent on either the 65
specificity of the Mur ligases (MurC-F) or later modifications in peptidoglycan biosynthesis. In Gram positive bacteria 66
MurE typically incorporates L-Lys as opposed to D,L-DAP, although Bacilli are a notable exception and several other 67
amino acids have been identified in this position (Schleifer and Kandler, 1972; Barreteau et al., 2008; Vollmer et al., 68
2008). The stem peptides of adjacent saccharide strands are crosslinked by transpeptidation to stabilize the mature 69
peptidoglycan (see biosynthetic pathway Figure 1). 70
Knock-out of P. patens homologs of bacterial peptidoglycan synthesis genes Ddl, MurA, MurE, MraY, MurJ or PBP1A, 71
results in a macrochloroplast phenotype, similar in appearance to antibiotic treatments that target their gene products, 72
while complementation with the intact genes restores the wild type number of about 50 typical chloroplasts per cell 73
(Machida et al., 2006; Homi et al., 2009; Hirano et al., 2016; Takahashi et al., 2016; Utsunomiya et al., 2020). Cross-74
species complementation using a P.patens MurE (PpMurE) knockout showed that Anabaena MurE (AnMurE) fused to 75
the plastid-targeting signal of PpMurE can also restore the wild type chloroplast phenotype (Garcia et al., 2008). In 76
contrast, the homologous Arabidopsis thaliana gene, AtMurE, failed to complement the PpMurE mutant (Garcia et al., 77
2008). Interestingly, MurE knockouts in both A. thaliana and Zea mays, appear bleached as opposed to having a 78
macrochloplast phenotype, are deficient in chloroplast thylakoids and lack many plastid RNA polymerase-regulated 79
chloroplast transcripts, indicating that angiosperm MurE has a primary function in plastid gene expression and biogenesis 80
rather than plastid division per se (Garcia et al., 2008; Williams-Carrier et al., 2014). 81
Although data suggestive of the formation of chloroplast peptidoglycan is available, no direct observation of the 82
peptidoglycan precursors or the operation of the chloroplast peptidoglycan synthetic pathway has yet been made. 83
Therefore, here, using pathway-inhibiting antibiotics to drive the accumulation of peptidoglycan intermediates, we 84
establish that in a basal land plant, P. patens, the six Mur genes and Ddl actively synthesize all the main precursors of the 85
peptide stem of peptidoglycan. Furthermore, we show that the pentapeptide building blocks are identical to those of most 86
typical Gram negative bacteria, including the cyanobacteria, plus the Chlamydiae, the ‘acid fast’ Mycobacterium spp. and 87
some Gram positive bacilli, where D,L-DAP is incorporated instead of L-Lys. Consistent with and supportive of this 88
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observation, we show that in vitro the moss MurE ligase, PpMurE, incorporates D,L-DAP in strict preference to L-Lys as 89
the third amino acid within the stem peptide, as would be consistent with the cyanobacterial ancestral origin of the 90
chloroplast, and the enzyme kinetics of PpMurE are similar to cyanobacterial and other Gram negative D,L-DAP-91
incorporating MurE ligases. 92
Materials and Methods 93
Plant Material 94
P. patens (Gransden strain, GrD13) was grown on modified KNOPS medium with 5 mM diammonium tartrate, to 95
promote chloronemata formation (Schween et al., 2003). The medium was solidified with 0.85 % (w/v) plant agar 96
(Sigma) and overlaid with 9 cm cellophane discs (AA Packaging). Plants were grown in 90 mm diameter x 20 mm vented 97
tissue culture dishes sealed with Micropore (3M) surgical tape in a plant growth room at 21°C under continuous light 98
from Sylvania white F100W tubes at 65-100 μmol.m−2.s−1. After being homogenised axenically in water in a 250 ml flask 99
using an IKA T18 digital Ultra Turrax homogeniser, for one to two 12 s bursts, P. patens protonemata were cultured as 2 100
ml aliquots per 25 ml solid KNOPS plus tartrate. 101
Confocal Microscopy of Antibiotic Treated P. patens Protonemata 102
Confocal single plane images and Z-series stacks were acquired on a Leica SP5 microscope, using a 63 x 1.4 Oil UV 103
immersion objective with the 405 nm and 496 nm laser lines and transmitted light, and photo multiplier tube spectral 104
detection adjusted for the chlorophyll emission (735-790 nm). Images were processed using the Fiji distribution 105
of ImageJ v2.0.0. 106
Trichloroacetic Acid (TCA) Extraction of Plant Metabolites 107
Antibiotics were added to KNOPS plus tartrate agar at 100 µg ml-1 carbenicillin, 100 µg ml-1 D-cycloserine or 200 µg ml-
108
1 phosphomycin. After 15 days tissue was harvested, weighed and ground in liquid nitrogen using a pestle and mortar 109
before being frozen at -80°C. To extract TCA-soluble plant metabolites the tissue was ground again in 5 ml g-1 of ice cold 110
10% (w/v) TCA (Fisons AR grade) before being mixed gently in 50 ml Falcon tubes on a rolling shaker for 30 min at 4°C 111
(Roten et al., 1991). Insoluble material was pelleted at 48,000 xg, 10 min, 2°C, the supernatant was retained and the pellet 112
reextracted twice more, first with 2.5 ml.g-1 and then with 1.25 ml.g-1 (of the original pellet weight) of ice cold 10% 113
(w/v) TCA. The pooled supernatants were extracted into an equal volume of diethyl ether, to remove TCA, by manually 114
shaking for 3 x 20 s in a separating funnel before recovering the lower, aqueous layer. The ether extraction of the aqueous 115
phase was repeated twice more. The pH of the combined lower phases was restored to pH 7-8 using 1 M NaOH and 116
residual ether was removed in vacuo at which point, the sample was lyophilised. 117
Purification of Muropeptide Precursors 118
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The nucleotide precursors in the TCA-soluble metabolite extracts were first partially purified by size exclusion using a 119
Superdex Peptide 10/300GL column. The freeze-dried pellets were resuspended in deionised water, applied to the column 120
as a 500 µl aliquot, eluted with deionised water and collected as 0.5 ml fractions at 0.5 ml min
-1. The likely elution 121
volume of molecules of interest was established by elution of 20 nmols UDP-MurNAc-DAP-pentapeptide and 20 nmols 122
UDP-N-acetyl-glucosamine (Sigma) standards. 123
The A260 of pooled Superdex Peptide fractions of 1-2 ml was used to determine the upper linit of the total concentration of 124
UDP species and an estimated 2, 10 or 20 nmols UDP species in 2 ml 10 mM ammonium acetate, pH 7.5, was loaded 125
onto a MonoQ 50/5 GL column equilibrated in the same buffer. Bound molecules were eluted with a 27 ml linear gradient 126
of 10 mM to 0.81M ammonium acetate (pH 7.5), at 0.7 ml.min-1 and collected as 1ml fractions using an Äkta Pure where 127
the eluate absorbance was recorded at A230, A254 and A280. Peaks with an absorbance ratio of usually 1:2 A280:A254 were 128
selected for freeze drying and mass spectrometry. 129
Mass Spectrometric Nano-spray Time of Flight Analysis of Peptidoglycan UDP-MurNAc Precursors 130
Identity of UDP-MurNAc precursors were confirmed by negative ion time of flight mass spectrometry using a Waters 131
Synapt G2Si quadrupole-time of flight instrument operating in resolution mode, equipped with a nanospray source 132
calibrated with an error of less than 1 ppm with sodium iodide over a 200-2500 m/z range (Catherwood et al., 133
2020). Samples, freeze dried three times to remove ammonium acetate, were diluted in LCMS grade 50% v/v 134
acetonitrile to between 1 µM and 5 µM. They were introduced into the instrument using Waters thin wall nanoflow 135
capillaries and up to 20 minutes of continuum data were collected at a capillary voltage of 2.0 kV, cone and source 136
offset voltages of 100 V and a source offset of 41 V, respectively. Source and desolvation temperatures were 80°C 137
and 150°C respectively, desolvation and purge gas flow rates were both 400 l.min-1. Scan time was 1 s with an 138
interscan time of 0.014 s. Scans were combined into centred mass spectra by Waters Mass Lynx software. Resolution 139
(m/z/half-height spectral peak width) was measured as 1 in 20,100. 140
Construction of Heterologous Expression Plasmids 141
PpMurE (derived from Pp3c23_15810V3.2) and AnMurE (derived from Anabaena sp. strain PCC 7120 MurE 142
WP_010995832.1 Q8YWF0|MURE_NOSS1) were inserted into the vector pPROEX HTa (Addgene) in order to be 143
expressed in frame with an amino terminal, TEV protease-cleavable, hexa-histidine (His6) tag. The MurE sequences were 144
PCR amplified from their respective cDNAs (Machida et al., 2006; Garcia et al., 2008) in pTFH22.4 using the primers 145
PpMurE_L63_Forward (TTTGCGACATGTTGAAAATGGGGTTTGGGGATTCGAAATTGACGGATCG) and 146
PpMurE_Reverse (AAACGCGCGGCCGCTTATTTTCTAAGTCGCAAAGCCTCCCGACATTCCTC) and 147
Anabaena_PCC7120_MurE_Forward 148
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(TTTGCGGGTCTCTCATGAAATTGCGGGAATTACTAGCGACAGTAGACAGTG) and 149
Anabaena_PCC7120_MurE_Reverse 150
(AAACGCGCGGCCGCTTATAATTTTTCTCTTTCTGTCAAAGCGGCGCGTGCG). The amplified region for 151
PpMurE started at leucine 63, effectively deleting the chloroplast transit peptide at the cleavage site predicted by the 152
ChloroP1.1 Prediction server (Emanuelsson, 1999) and introducing a unique Nco1-compatible Pci1 site around the novel 153
ATG and a Not1 site immediately 3’ to the stop codon. A TAA stop codon was substituted for the native TGA. The 154
AnMurE primers amplified the cDNA and novel Bsa1 and Not1 sites were introduced 5’and 3’ to the ATG start and TAA 155
stop codons, respectively. The former was sited to create a Nco1-compatible 5’ cohesive end. The vector pPROEX HTa 156
was restricted with Nco1 and Not1 and gel purified before being ligated to Pci1-Not1 restricted PpMurE_L63 or Bsa1-157
Not1 restricted AnMurE PCR fragments that had been cleaned up with a PCR clean up kit (Qiagen). Coding sequences 158
were confirmed by Sanger sequencing (Eurofins). 159
Expression of PpMurE_L63 and AnMurE and Protein Purification 160
For protein purification Escherichia coli strains were tested for optimal expression: BL21 DE3 (Thermofisher) was 161
selected for PpMurE_L63_pPROEX and BL21(DE3), with the chaperone plasmid pG-KJE8 (Takara Bio Inc.), was 162
selected for AnMurE_pPROEX. These were grown in L-Broth plus 0.2% v/v glucose, 100 µg ml-1 ampicillin and 35 µg 163
ml-1 chloramphenicol at 37°C to an A600 of 0.6 when PpMurE protein expression was induced with 0.5 mM IPTG and 164
AnMurE expression was induced by 0.5 mM IPTG with 1.5 mg.ml-1 arabinose and 8 ng.ml-1 tetracycline to induce pG-165
KJE8 chaperones. Over-expressing cells were then grown overnight at 19°C. Bacteria were harvested by centrifugation at 166
5600 x g, 15 min at 4°C and resuspended in Buffer A: 50 mM HEPES-NaOH, 0.5 M NaCl, 10 mM imidazole and 10% 167
v/v glycerol (pH 7.5) containing EDTA-free protease inhibitor tablets, as recommended by the supplier (Pierce), and 2.5 168
mg ml-1 lysozyme, with gentle mixing for 30 min at 4°C. Lysis was by sonication on ice for 10 x 15 s bursts at 70%, 169
interspersed by 1-2 min cooling on ice. Insoluble material was pelleted at 50,000 xg for 30 min at 4°C and the supernatant 170
loaded directly onto a 5 ml His Trap HP (GE Healthcare) at 2 ml.min-1 and washed with 50 ml Buffer A at 4 m.min-1 at 171
4°C. Bound material was eluted with an 100 ml linear gradient to 100% Buffer B: 50 mM HEPES-NaOH, 0.5 M NaCl, 172
5% w/w glycerol and 0.5 M imidazole (pH 7.5) at 4 ml.min-1. Selected peak fractions were pooled and concentrated in 173
either 30 or 50 kDa MWCO Vivaspin concentrators (GE Healthcare), for AnMurE or PpMurE_L63, respectively, at 174
2,800 xg at 4°C. Proteins were further purified by size exclusion chromatography on Superdex G200 XK26 (GE Life 175
Sciences) pre-equilibrated and eluted with 50 mM HEPES-NaOH, 150 mM NaCl (pH 7.5) and purity of the eluted MurE 176
proteins was established by SDS-PAGE (Supplemental Figure S3). Pooled peak fractions were dialysed against DB2: 30 177
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mM HEPES-NaOH, 1 mM MgCl2, 50 mM NaCl, 50% v/v glycerol with 0.2 mM PMSF, 1 µM leupeptin, 1 µM pepstatin, 178
3 mM dithiothreitol (pH 7.6) overnight at 4°C, before storage at -20°C and -80°C. 179
TEV Protease-Cleaved Protein Preparation 180
Bacteria were expressed as above, harvested and lysed using a cell disruptor and the proteins first purified on 5ml His 181
Trap HP columns, using Buffer A and B (pH 8.0) as above, except that 100 mM Tris replaced 50 mM HEPES and Buffer 182
A included 2% v/v glycerol, 10 mg.l-1 DNase1 (DN25) and 1 mM DTT. Pooled fractions were exchanged into a buffer of 183
50 mM PIPES, 100 mM NH4SO4, 200 mM KCl, 20 mM MgCl2, 1 mM DTT, 30 mM imidazole, 2% v/v glycerol (pH 184
7.7). using a stack of four 5ml HiTrap Desalting columns (Pharmacia). Peak fractions were incubated for 48h at 4°C in 185
the ratio 1 mg TEV protease: 50 mg protein before reverse His-tag purification, collecting the column flow through. 186
Samples were concentrated using 50 kDa concentrators as above. 187
Streptococcus pneumoniae MurE and Pseudomonas aeruginosa MurF were over-expressed and purified exactly as 188
described (Blewett et al., 2004; Majce et al., 2013). 189
Mur Ligase Activity Assays 190
The assays employed a continuous spectrophotometric method following ATP consumption at 37oC in a Cary 100 191
UV/Vis double beam spectrophotometer. Mur ligase catalysed ADP release, coupled to NADH oxidation by pyruvate 192
kinase and lactate dehydrogenase, led to stoichiometric consumption of NADH measured by a fall in the A340. Assay 193
volumes were 0.2 ml and contained 50 mM PIPES, 10 mM MgCl2 adjusted to pH 6.7 for AnMurE or 50 mM Tricine, 10 194
mM MgCl2 adjusted to pH 8.7 for PpMurE_L63, 1 mM dithiothreitol, 0.2 mM NADH, 2 mM phosphoenol pyruvate, 195
1mM ATP, 50 mM.min-1 pyruvate kinase and 50 mM.min-1 lactate dehydrogenase (as assayed by the manufacturer, 196
Sigma). Ligases were diluted prior to assay as required in 50 mM HEPES pH 7.7, 50 mM KCl, 1 mM MgCl2, 3 mM 197
DTT, 50% v/v glycerol, 0.2 mM PMSF. Concentrations of UDP-MurNAc dipeptide, Mur ligase and amino acid 198
substrates were as described in the text or table legends. Control rates were collected usually in the absence of the amino 199
acid, or UDP-MurNAc-dipeptide as specified, and the activity of the enzyme was initiated by addition of the missing 200
component. Mur ligase initial rates were recorded as mols ADP.mol Mur ligase-1.s-1 (ADP/s) within the linear range of 201
the time course of the assay. 202
203
204
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Results 205
Identification of antibiotics with the most profound effect on P. patens chloroplast division 206
P.patens was grown on a variety of antibiotics that impact peptidoglycan biosynthesis in bacteria in order to select those 207
best able to cause an accumulation of peptidoglycan intermediates in the moss, so that they might be more readily 208
detected. Of the antibiotics tested the three that appeared most specific at inhibiting peptidoglycan synthesis, as measured 209
by a widespread macrochloroplast phenotype with least effect on chlorophyll intensity, were phosphomycin (500 µg.ml-
210
1), a PEP analog that inhibits MurA by alkylating an active site cysteine residue (Figure 11), the ß-lactam ampicillin (100 211
µg.ml-1), which binds covalently to the active site serine of PBPs (Figure 17), and D-cycloserine (20 µg.ml-1), with at least 212
two enzyme targets in peptidoglycan biosynthesis, DDL (Figure 12) and alanine racemase (Figure 2 B, D and G) . Even at 213
higher concentrations, where growth rate was impaired, the protonemata were green indicating chlorophyll synthesis and 214
therefore chloroplast function was not significantly impaired. The impact of antibiotics that had either a more profound 215
and pleiotropic effect or that had little impact on phenotype are described in Supplemental Text S1 and include 216
vancomycin, bacitracin, murgocil and A22 (Figure 2 C, E, F and H). 217
The TCA-extracted metabolome contains peptidoglycan precursors in P. patens 218
P. patens was grown separately on the three most specific and effective antibiotics, phosphomycin (400 µg.ml-1), D-219
cycloserine (100 µg.ml-1), and carbenicillin (100 µg.ml-1) to facilitate accumulation of different peptidoglycan precursor 220
molecules (Figure 11, 2 and 7
). After size exclusion and anion exchange chromatography to purify UDP-linked 221
intermediates from the TCA-extracted metabolome, mass spectrophotometric analysis identified precursors common to 222
most Gram negative bacterial cell wall syntheses (Table 1 and Figure 3 C, identified precursors numbered 1-5). Precursor 223
molecules were detected only in the earlier fractions from the Superdex Peptide column (Figure 3 B), as expected from 224
the elution profiles of UDP-GlcNAc and UDPMurNAc-pentapeptide standards (not shown). 225
The identification of UDP-MurNAc-Ala-Glu-D,L-DAP in three of the samples as well as the D,L-DAP pentapeptide 226
(Table 1 and Figure 3 C, numbers 4 and 5), together with the inability to identify UDP-MurNAc-Ala-Glu-Lys or UDP-227
MurNAc-Lys-pentapeptide suggested that in vivo, PpMurE specifically incorporated DL-DAP in the third position of 228
stem peptide. By comparison, when the plant was grown on phosphomycin (Figure 11), anticipated to block synthesis of 229
UDP-MurNAc, only the UDP-GlcNAc precursor was identified (Table 1 and Figure 3 C, number 1). Interestingly, this 230
metabolite was not detected in the samples treated with the other antibiotics. 231
Similarly, the MurC and D products, UDP-MurNAc-Ala and UDP-MurNAc-Ala-Glu were detected in the D-cycloserine-232
grown extract consistent with the accumulation of precursors up to the UDP-MurNAc-tripeptide MurF substrate (Figure 3 233
C, numbers 2 and 3). From the MonoQ anion exchange chromatograms (Figure 3, C) and the mass spectral data 234
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(Supplemental Figure S2) we can conclude that use of the different antibiotics proved to be an effective way to ensure 235
most of the intermediates were detected, confirming the utility of this method for the purpose. 236
P. patens MurE incorporates DL-DAP into the peptidoglycan stem peptide 237
To account for the composition of the P.patens peptidoglycan stem peptide, we analysed the activity and substrate 238
specificity of the MurE ligase protein product of its murE gene, with the predicted 62 residue chloroplast transit peptide 239
sequence deleted (PpMurE_L63). The enzyme was compared with the cyanobacterial Anabaena MurE ligase. Analysis of 240
the ability of both AnMurE and PpMurE_L63 to utilise D,L-DAP D,D-DAP L,L-DAP and L-Lys revealed that both 241
MurE enzymes were catalytically active in the aminoacylation of UDP-MurNAc-dipeptide, Removal of the His tag by 242
TEV protease cleavage did not enhance the efficiency of either enzyme (Figure 4, B and Supplemental Figure S4) and 243
significantly, both proteins favoured D,L-DAP as a substrate over the other DAP diastereoisomers (Figure 4, A). 244
Noticeable was the slow rate of turnover of D,D-DAP by PpMurE_L63, in particular, possibly indicative of a weak 245
stereo-selectivity for the L- over the D stereocentre of DAP utilised by the enzyme when at high concentrations. 246
Significantly, neither enzyme incorporated L-Lys. As a control, lysylation of UDP-MurNAc-Ala-Glu was also assayed 247
with the L-Lys specific Streptococcus pneumoniae Pn16 MurE (Blewett et al., 2004) and resulted in a rate (vo) of 1.94 248
ADP.s-1 at 150 µM L-Lys, with the same UDP-MurNAc-dipeptide and ATP concentrations as the other assays (data not 249
shown). 250
That the assay followed the aminoacylation of UDP-MurNAc-dipeptide by D,L-DAP to yield D,L-DAP tripeptide was 251
confirmed by the ability of the assay product to be utilised as a substrate by Pseudomonas aeruginosa MurF (PaMurF). 252
This was achieved in the same coupled assay by adding PaMurF at t=0, initiating the MurE ligase reaction with D,L-DAP 253
and then the MurF ligase with D-Ala-D-Ala as the second substrate once the MurE reaction had reached completion to 254
yield the UDP-MurNAc-pentapeptide (Supplemental Figure S5). 255
pH and Temperature Optima of P. patens and Anabaena MurE 256
Prior to kinetic investigation of the properties of PpMurE the pH optimum was determined, with that of the 257
cyanobacterial AnMurE, by comparing rate of ADP generated (vo) at pH 5.7-9.7 at approximately saturating 258
concentrations of its substrates (Supplemental Figure S6, A-D). Neither of the coupled enzymes in the MurE/ADP release 259
assay was a major factor affecting rate over the pH range studied as evidenced by the independence of the measured 260
MurE rate from coupling enzyme concentration. Additionally, the similarity of activities of the MurE proteins in 261
different buffers allowed us to discount the impact of buffers over the pH range under consideration (Supplemental Figure 262
S6, B and D). Assuming saturation with substrates and the only variable responsible for a change in enzyme activity was 263
pH range we fitted vo data vs pH to an equation that follows the relationship of activity versus pH. From these data it was 264
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concluded that the optimum for AnMurE is 7.5 and that for PpMurE_L63 is approximately pH 7.5-8.5. The data fit for for 265
PpMurE_L63 (R2 = 0.94 and 0.89, for 1 and 2 x coupling enzymes, respectively) is better than that for AnMurE (R2 = 266
0.78) indicating that the assumption that other variables (kinetic constants and substrate ionization) are not influenced by 267
pH may be less true for AnMurE. 268
P. patens MurE has similar kinetic properties to cyanobacterial MurE 269
The two enzymes AnMurE and PpMurE_L63 were assayed to calculate their kinetic efficiency for the preferred substrate 270
D,L-DAP. From the tabulated data PpMurE_L63 was more sensitive to substrate inhibition from D,L-DAP than AnMurE, 271
as indicated by its greater R2 value for fit of data to the kinetics of substrate inhibition compared to those for standard 272
Michaelis Menten kinetics (Figure 4, B and the two fitted curves in Supplemental Figure S4, C and D). However, the 273
KcatApp/KMApp ratio for the plant enzyme were similar to the cyanobacterial one, the most marked difference being the lower 274
D,L-DAP KMApp value, indicative that the plant enzyme may operate at lower substrate concentrations in vivo. These 275
figures were compared with reported data for other MurE activities (Supplemental Figure S7) and reveal that the plant 276
and cyanobacterial MurE are at least as catalytically active, as indicated by the KcatApp/KMApp ratio, as the bacterial 277
homologs. It was apparent that removal of the His tag by TEV protease cleavage did not enhance the efficiency of either 278
enzyme (Figure 4, B and Supplemental Figure S4, B and D). 279
Conservation of amino acid residues common to DL-DAP-incorporating MurE ligases 280
BLASTP searches and ClustalW (EMBL-EBI) alignment indicated that the closest bacterial homolog to PpMurE is the 281
MurE of Gemmatomonidates bacterium (50.0% homology), which is a photoheterotrophic Gram negative bacterium in a 282
phylum quite distal to the cyanobacteria (Zeng et al., 2014). The next closest is the Gram positive Bacillus fortis (43.0%), 283
which would be anticipated to incorporate D,L-DAP (Barreteau et al., 2008). Both are considerably more closely related 284
than the cyanobacterial AnMurE (37.8%), determined in this paper to be D,L-DAP incorporating, E.coli MurE
D,L-DAP
285
(34.9%) and Mycobacterium tuberculosis MurE
D,L-DAP (34.7%). The L-Lys incorporating enzymes, all from Gram 286
negative species, share still less homology: Thermatoga maritima MurEL-Lys (33.0%), Streptococcus pneumoniae MurE 287
(30.1%) and Staphylococcus aureus MurEL-Lys (26.6%). Likewise, the neighbour-joining phylogram computed in Jalview 288
(Supplemental Figure S8) placed AnMurE as more distantly related than Gemmatimonadetes to plant MurE, as 289
represented by PpMurE and the algal streptophytes Mesotaenium endlicherianum MurE and Coleochaete scutata MurE. 290
M. endlicherianum (66.2%) represents a late charophyte ancestor within the Zygnemophyceae which are predicted to be 291
on a branch point preceding embryophyte evolution (Donoghue and Paps, 2020), whereas MurE from Klebsormidium 292
nitens (41.8%) and C. scutata (51.3%) in the Klebsomidiophyceae and Coleochaetaceae, respectively, and also within 293
the charophyte algae, are on more divergent branches. 294
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To relate homology to functionality, PpMurE was aligned in Clustal Omega (EMBL-EBI) with homologs of both L-Lys- 295
and DL-DAP-incorporating MurE ligases (Supplemental Figure S9). Many amino acid residues are conserved not only 296
between MurE from bacterial and early plant species but also across the Mur ligase family (as indicated by asterisks on 297
Supplemental Figure S9). Mur ligases comprise three domains: an N-terminal Rossmann-fold domain responsible for 298
binding the UDP-MurNAc substrate; a central ATP-binding domain and a C-terminal domain associated with binding the 299
incoming amino acid. Most of the amino acids conserved between the different Mur ligases lie within the central ATP-300
binding domain, those in the N- and C-termini generally do not co-localise with the known substrate binding residues. 301
Amino acids of published importance for ATP binding (species abbreviation subscripted); the P-loop within 302
TGTXGKTSa, E220Mt, D356Sa, N347Mt, R377Mt and R392Mt are conserved in the plant enzymes M. endlicherianum MurE 303
and PpMurE, as well as a lysine, K219Sa, carbamylated in MurD for positioning the MgATP complex for the generation 304
of a transient UDP-MurNAc-phosphodi-peptide intermediate (Dementin et al., 2001). K360Sa and Y343Mt have undergone 305
conservative changes. Similarly, residues that bind UDP-MurNAc, S28Ec, HQA45Ec, NTT158Ec, E198Mt, S184Ec, 306
QXR192Ec and H248Mt are no less conserved in plants than they are between bacteria. 307
Although most of the UDP-MurNAc-tripeptide interactions are within the MurE central domain, those made in relation to 308
the appended amino acid, D,L-DAP or L-Lys, are within the C-terminal domain. All of the identified bacterial MurE 309
residues that interact with D,L-DAP are highly conserved in plant MurE proteins. More specifically, with reference to E. 310
coli MurE and M. tuberculosis MurE, it is possible to distinguish those that interact with either the D- or L-stereocentre 311
carboxylates of D,L-DAP : G464Ec, E468Ec, D413Ec and N414Ec, which bond to the D-stereocentre, R389Ec, which bonds 312
with the L-stereocentre, and especially R416Ec, which interacts with both the L- and D-centre carboxylates. Of these 313
R389Ec, N414Ec, R416Ec, G464Ec and E468Ec are less consistently present in MurE ligases from Gram positive bacteria 314
that incorporate L-Lys, a decarboxylated derivative of D,L-DAP, which has only been reported to interact with the 315
R383Sa, D406Sa and E460Sa residues (Ruane et al., 2013). Similarly, the pattern of charged residues in the C-terminal 316
domain of the basal streptophyte MurE (those highlighted red or purple in Supplemental Figure S9) would indicate a 317
binding cleft for the amino acid substrate that is more basic and resembles that of the Gram negative MurE ligases. 318
Together these data are in complete accord with our kinetic findings that D,L-DAP is the preferred substrate in plants and 319
AnMurE, rather than L-Lys. As would be anticipated from the phylogeny, the more closely related G. bacterium MurE 320
aligns strongly with the Gram negative DL-DAP incorporating enzymes, and includes the DNPR motif, which confers 321
specificity for the D-stereocentre carboxyl and amino groups of D,L-DAP, indicating that this phylum is most likely to 322
incorporate DL-DAP. 323
324
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Discussion 325
P. patens peptidoglycan is synthesized from a UDP-MurNAc-D,L-DAP-pentapeptide 326
Growth of P. patens on the antibiotics phosphomycin, D-cycloserine and ampicillin facilitated the detection, by mass 327
spectrophotometric analysis of the TCA-extracted metabolome, of peptidoglycan intermediates up to UDP-MurNAc-Ala-328
Glu-DAP-Ala-Ala in the moss. These data enable us to conclude that the identical basic building blocks for the Gram 329
negative bacterial cell wall are found in basal embryophytes. With evidence for knock-out phenotypes for P.patens 330
homologs of bacterial MraY, MurJ and PBP1A and the presence of mRNA for MurG (Machida et al., 2006; Homi et al., 331
2009; Utsunomiya et al., 2020) it would be expected that the D,L-DAP-containing pentapeptide within the stroma is lipid-332
linked then flipped across the chloroplast inner envelope membrane and polymerised into peptidoglycan to form a 333
‘sacculus’ bounding the organelle, as indicated from fluorescent-labelling using a D-Ala-D-Ala analogue (Hirano et al., 334
2016). By analogy with bacteria and from the predicted transit peptides of the peptidoglycan-maturing proteins it is 335
anticipated that the peptidoglycan will lie between the inner and outer membranes of the chloroplast, although this has yet 336
to be determined (Figure 1). 337
PpMurE appends D,L-DAP to UDPMurNAc-Ala-Glu 338
From our data, it is evident that the moss MurE ligase, with the transit peptide omitted, PpMurE_L63, can efficiently 339
append D,L-DAP to UDP-MurNAc-L-Ala-D-Glu in vitro, as can the cyanobacterial enzyme from Anabaena sp. strain 340
PCC 7120, AnMurE. This is in accordance with the D,L-DAP content of peptidoglycan in the cyanobacteria 341
Synechococcus sp. and Synechocystis sp. (Jurgens et al., 1983; Woitzik, 1988) and is inconsistent with the observation 342
that Anabaena cylindrica may incorporate L-Lys (Hoiczyk and Hansel, 2000). Our in vitro MurE enzymological data also 343
complement the mass spectrometric analysis of the antibiotic-grown P. patens which identified UDP-MurNAc-D,L-DAP 344
intermediates as being present in vivo in the TCA-extracted metabolome. 345
That UDP-MurNAc-L-Ala-D-Glu is an efficient substrate for PpMurE_L63 is significant in that there is no obvious 346
homolog in most green plants for glutamate racemase (MurI), exceptions include the glaucophyte alga Cyanophora 347
paradoxa (Contig25539), the charophyte alga K. nitens (GAQ85716.1) but not M. endlicherianum, a zygenematophycean 348
alga proposed to be closest to the embryophyte branch point. Here, this function may be replaced by a D-alanine amino 349
transferase (DAAA), of which there are two genes having weak homology to Bacillus subtilis DAAA in both P. patens 350
and M. endlicherianum (Phytozome v.13 P. patens: Pp3c6_5420 (15.7%), Pp3c16_17790 (14.7%) and OneKP M. 351
endlicherianum: WDCW scaffolds 2009723 (17.6%) and 2007189 (16.5%)). Alternatively P. patens diaminopimelate 352
epimerase (DapF), like Chlamydial DapF, may possess the dual specificity required to racemase L-Glu to D-Glu in 353
addition to its epimerization of L,L-DAP to D,L-DAP (De Benedetti et al., 2014). 354
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Substrate Preference of AnMurE and PpMurE 355
The high degree of specificity of both AnMurE and PpMurE_L63 for D,L-DAP, over the alternatives L,L-DAP, D,D-356
DAP and L-Lys, is consistent with other D,L-DAP-incorporating enzymes assayed in vitro, including E. coli MurE, M. 357
tuberculosis MurE and Chlamydia trachomatis MurE , for which L-Lys is either a very poor substrate or is not accepted 358
at all (Supplemental Table S7). Similarly, the L-Lys-incorporating S. aureus MurE does not incorporate D,L-DAP in 359
vitro. Not all MurE ligases are as selective, Thermotoga maritima MurE incorporates L-Lys and D-Lys in almost equal 360
amounts in vivo (Huber, 1986) and can efficiently incorporate D,L-DAP in vitro (Boniface et al., 2006). In this regard it is 361
notable that T. maritima MurE possesses a DDPR motif, which includes the arginine residue of the consensus DNPR of 362
D,L-DAP-incorporating enzymes which hydrogen bonds to and stabilises D,L-DAP, consequently the almost complete 363
absence of D,L-DAP in T. maritima peptidoglycan has been attributed to its low intracellular concentration. This almost 364
absolute specificity of most MurE ligases is indicative of a requirement that the stem peptide be composed of the correct 365
amino acids to facilitate optimal transpeptidation (Vollmer et al., 2008). 366
PpMurE is a slow but efficient MurE ligase 367
Kinetic analyses of PpMurE_L63 demonstrated an enzymatic efficiency similar to bacterial MurE homologs, as estimated 368
by comparison of KcatApp/KMApp (Supplemental Table S7). Further comparisons with other D,L-DAP-incorporating 369
enzymes, and in particular those of the obligate intracellular pathogens C. trachomatis and M. tuberculosus, revealed the 370
plant MurE to have a similarly low KM for the amino acid substrate relative to the L-Lys-incorporating enzymes. This 371
may reflect either (or both) a lower abundance of D,L-DAP or the potential toxicity of the D,L-diamino acid, particularly 372
in a eucaryotic cell (Kolukisaoglu and Suarez, 2017). A higher KM for L-Lys-incorporating MurE ligases has been 373
attributed to the much greater abundance of this amino acid in bacteria (Mengin-Lecreulx et al., 1982; Ruane et al., 2013). 374
The availability of the D,L-DAP substrate in plants, as in cyanobacteria and Chlamydiae, is not in question as the 375
biosynthesis of L-Lys is catalysed by DAP decarboxylase (LysA) from D,L-DAP which is ultimately derived from 376
aspartate (Hudson et al., 2006). 377
Comparison of the PpMurE_L63 KcatApp with the bacterial enzymes reveals the rate of turnover to be quite low, possibly 378
reflecting the apparent low density of peptidoglycan surrounding the chloroplast and a concomitant slower rate of 379
synthesis compared to rapidly dividing, free-living bacteria. Moreover, the plant enzyme has UDP-MurNAc-Ala-Glu 380
kinetics best fitted to a substrate inhibition model, possibly to ensure that peptidoglycan synthesis proceeds at a rate 381
insufficient to consume the majority of available prenyl phosphates that are otherwise required for other pathways. 382
It is important to mention that the P. patens genome encodes two MurE homologs (PpMurE1: Pp3c23_15810, studied in 383
this paper, and PpMurE2: Pp3c24_18820) which have 72.2% amino acid identity to each other over the conventional 384
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bacterial MurE ligase domains and 48.4% identity overall. PpMurE2 primarily differs from PpMurE1 in encoding a long, 385
relatively unstructured extension at the amino terminus and a short carboxy terminal extension (expanded description in 386
Supplemental Figure S10). Although the DNPR motif and other amino acids associated with D,L-DAP binding are 387
retained in PpMurE2, knock out mutations of PpMurE1 alone results in a comprehensive macrochloroplast phenotype 388
(Machida et al., 2006; Garcia et al., 2008), consistent with the hypothesis that this protein is sufficient for peptidoglycan 389
synthesis in the moss. Moreover, preliminary in vitro experiments indicate that intact PpMurE2 does not function as a 390
MurE ligase (data not shown) and we would suggest that both the amino and carboxy terminal extensions have been 391
acquired during streptophyte evolution to participate in novel interactions thereby facilitating an alternative function for 392
MurE within the chloroplast transcription and translation apparatus. 393
In contrast to P. patens (and the Polypodiidae ferns, Supplemental Figure S10), many in the same and closely related 394
phylla encode a single MurE homolog with both the amino and carboxy terminal extensions and aligning more closely to 395
PpMurE2, yet these proteins would be anticipated to function as MurE ligases. We propose the shorter MurE in P. patens 396
and the Polypodiidae ferns represents a de-evolution of streptophyte MurE to more closely resemble its bacterial 397
counterpart. It has yet to be determined at what point in streptophyte evolution the function of MurE changed and whether 398
in any plants it remains a bifunctional protein capable of both MurE ligase activity and interaction with chloroplast RNA 399
polymerase in chloroplast transcription. 400
That basal embryophyte MurE has evolved a new role essential to plastid photomorphogenesis in seed plants indicates an 401
exaptation from its original function in peptidoglycan biosynthesis and plastid division (Williams-Carrier et al., 2014). 402
This raises the intriguing question why important residues of the D,L-DAP-binding motif are retained, in similar 403
proximity to the ATPase domain, in these proteins. We would speculate that the novel function of the MurE-like proteins 404
in seed plants could have evolved consequent on the two whole gene duplication events which occurred in an ancestral 405
moss, as opposed to in the liverworts or hornworts (Lang et al., 2018). 406
Predicted streptophyte peptidoglycan structure from peptidoglycan gene homologies 407
The moss ‘sacculus’, like that of Chlamydiae, has been recalcitrant not only to visualisation by electron microscopy but 408
also to common extraction protocols, making analysis of the mature polymer a future goal. The moss chloroplast 409
envelope membranes were found to be closely appended with little dense intervening material (Takano and Takechi, 410
2010; Matsumoto et al., 2012; Sato et al., 2017), likewise in Chlamydiae the apparent deficit of a bounding sacculus lead 411
to the term the ‘chlamydial anomaly’ (Packiam et al., 2015). This is in marked contrast to most cyanobacteria where the 412
cell wall is highly cross-linked and forms a broad, electron dense layer (Hoiczyk and Hansel, 2000). Intermediate 413
between these extremes is the earliest side branch in plant evolution, the glaucophyte algae, where the cyanelles comprise 414
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a peptidoglycan layer that has been more tractable to visualisation and analysis (Pfanzagl et al., 1996; Higuchi et al., 415
2016). 416
It would appear that progressive transition of a bacterium from free-living to endosymbiont or pathogen and thence to an 417
integrated organelle is associated with a reduction in substance of the sacculus. Presumably there are not the same 418
osmotic constraints and risks of dehydration within the host cell and the vestigial peptidoglycan may function primarily 419
or exclusively for the purpose of assembly of the division apparatus. Additionally, it may be that for cyanobacterial 420
evolution into a cyanelle and subsequently a plastid that a finer, net-like cell wall would be a prerequisite if extensive 421
exchange of larger molecules, including lipids and proteins, were to occur. Supportive of this suggestion is the fact that 422
most of the bacterial PBPs which cross-link the lipid-linked GlcNAc-MurNAc-pentapeptide precursor, have been 423
identified as having no predicted product from RNA-seq data (data not shown). Currently the only reported exception is a 424
PBP1A homolog, the transpeptidase and transglycosylase functions of which have an almost complete knock out 425
phenotype (Machida et al., 2006; Takahashi et al., 2016). 426
We also propose that streptophyte peptidoglycan must differ in its mature form by being uniquely modified to distinguish 427
it from the peptidoglycan of potential plant pathogens. The P. patens genome encodes a battery of proteins that include 428
peptidoglycan-binding and LysM domains and which frequently but not invariably include cell export signals (data not 429
shown). Many of these proteins will be part of the defences of the plant cell which are activated on detection of fungal 430
and bacterial cell wall material. To evade the host cell defences it is anticipated that an endosymbiont, obligate pathogen 431
or evolving organelle must protect its peptidoglycan from the host defences, conceivably by modification of the peptide 432
stem (Wolfert et al., 2007) or the GlcNAc-MurNAc backbone (Davis and Weiser, 2011). Predictions as to what those 433
modifications might be in streptophytes are hampered by the fact that the ancestry of the modifying enzymes is not 434
necessarily cyanobacterial. We have reported here the closer homology of PpMurE to MurE in the Gemmatimonadetes 435
phylum and we can further include P. patens PBP1A, MurF, MurD, MurG and Ddl as most closely related to homologs 436
within the same Fibrobacteres-Chlorobi-Bacteroidetes group of Gram negative bacteria (data not shown). The diverse 437
origins of several peptidoglycan biosynthesis-related proteins have previously been reported (Sato and Takano, 2017). 438
Therefore, it appears highly probable that a horizontal gene transfer event of a distinct Gram negative peptidoglycan-439
related gene cluster must have occurred early in the plant lineage. Hence we conjecture a simultaneous transfer of 440
peptidoglycan-modifying genes could have occurred that would introduce novel modifications to the mature polymer, 441
distinct from any in cyanobacteria. This is not without precedent, as the divergent glaucophyte algae were found to 442
append N-acetyl-putrescine to the second residue in the stem peptide (Pfanzagl et al., 1996). 443
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Here we have determined that chloroplast peptidoglycan in the streptophyte, P. patens, is constructed from typical Gram 444
negative UDP-MurNAc-D,L-DAP-pentapeptide peptidoglycan precursor. However, we propose that the final 445
polymerised structure derived from this building block differs from its cyanobacterial progenitor by being both less 446
highly polymerised and, to distinguish it from plant pathogens and thereby evade the plant immune response, 447
significantly modified. 448
449
Supplemental Data 450
Supplemental Text S1. Effects of antibiotics on P. patens 451
Supplemental Figure S2. Negative ion nanospray TOF mass spectra of TCA-extracted peptidoglycan intermediates 452
Supplemental Figure S3. PAGE gel of AnMurE and PpMurE_L63 after gel filtration 453
Supplemental Figure S4. D,L-DAP substrate curves for AnMurE and PpMurE_L63 454
Supplemental Figure S5. Assay data demonstrating PaMurF utilises the product of AnMurE and PpMurE_L63 455
Supplemental Text S6. Activities of AnMurE and PpMurE_L63 with pH and buffer 456
Supplemental Table S7. Comparison of AnMurE and PpMurE_L63 kinetics with published data for other MurE ligases 457
Supplemental Figure S8. Neighbour joining phylogram of MurE of Gram-negative bacteri and early plant species 458
Supplemental Figure S9. Clustal Omega multiple sequence alignment of MurE homologs 459
Supplemental Figure S10. Phylogram of evolutionary relationship of both PpMurE proteins to selected MurE homologs 460
461
Acknowledgements 462
The authors thank Professor Hiroyoshi Takano (Kumamoto University, Japan) for kindly providing the pTFH22.4 vectors 463
with Anabaena (PCC7120 Q8YWF0|MURE_NOSS1) and P.patens (Pp3c23_15810V3.2) MurE cDNA and Dr Sven 464
Gould (Heinrich Heine University, Düsseldorf, Germany) for helpful discussion on chloroplast evolution. We are also 465
grateful to Julie Tod and Anita Catherwood (University of Warwick, UK) for synthesis of UPD-MurNAc-dipeptide, -466
tripeptide and -pentapeptide and providing Streptococcus pneumoniae MurE and Pseudomonas aeruginosa MurF and Ian 467
Hands-Portman for access to and training in the School of Life Sciences Imaging Suite, University of Warwick, UK). We 468
also gratefully acknowledge Prof. Rebecca Goss (St Andrews, UK) for provision of pacidamycin. 469
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P
Ac
L-A D-E
DAP
D-A D-A
PP
Ac
L-A D-E
DAP
D-A D-A
Ac
Ac
PP
PP
Ac
L-A D-E
DAP
D-A D-A
P
P
PP
Ac
L-A D-E
DAP
PP
Ac
L-A D-E
D-E
PP
Ac
L-A
PP
Ac
L-A
PP
Ac
EP
NADPH
NADP+
P
PEP
PP
Ac
D-A D-A
D-A D-A
DAP
PP
Ac
L-A D-E
DAP
D-A D-A
Ac
Ac
Ac
Ac
Ac
L-A
L-A
Ac
Ac
Ac
Ac
L-A D-E
DAP
D-A D-A
D-E
DAP
D-A D-A
D-E
DAP
D-A D-A
Ac
Ac
Ac
Ac
L-A
L-A
Ac
Ac
Ac
Ac
L-A D-E
DAP
D-A
D-E
DAP
D-A
D-E
DAP
D-A
MurA
1
MurB
MurC
MurD
MurE MurF
MurG
5
MurJ
MraY
3,4
PBP
7,8
PP
Ddl
2
PP
UDP
GlcNAc
Ac
P
Undecaprenyl phosphate
(C55-P)
MurNAc
Ac
PEP
L-A
D-E
DAP
EP
D-A
4-3 cross links
(Phospho)enolpyruvate
L-alanine
D-glutamate
DL-DAP or L-Lysine
D-alanine
+
UMP
PP
Undecaprenyl pyrophosphate
(C55-PP)
P
P
UDP
6
Figure 1 Schematic of the fundamental cytoplasmic and periplasmic enzyme steps in peptidoglycan (murein) biosynthesis. Enzymes: MurA-J,
murein synthases A-J; Ddl, D-Ala--D-Ala ligase; MraY, phospho-N-acetylmuramoyl-pentapeptide-transferase and PBP, transglycosylase and
transpeptidase activities of penicillin-binding proteins. Superscript numbers indicate targets for the following antibiotics: 1, phosphomycin, 2,
D-cycloserine, 3, pacidamycin, 4, tunicamycin, 5, murgocil, 6, bacitracin, 7, penicillins and 8, vancomycin.
The cytoplasmic Mur proteins MurA and MurB catalyze the formation of UDP-N-acetylmuramic acid (UDP-MurNAc), Mur ligases (MurC, D, E
and F) sequentially append amino acids to form UDP-MurNAc-pentapeptide. The transmembrane protein MraY attaches MurNAc-pentapeptide
to C55-P to yield C55-PP-MurNAc-pentapeptide (lipid I) and MurG GlcNAc transferase creates C55-PP-MurNAc-(pentapeptide)-GlcNAc (lipid
II). Finally, the disaccharide pentapeptide monomer is flipped into the periplasm, polymerized by the transglycosylase activities of
penicillin-binding-proteins (PBPs), or functionally related shape, elongation, division and sporulation (SEDS) proteins, and the peptides are 4-3
cross-linked to pre-existing peptidoglycan by the transpeptidase activities of PBPs or 3-3 cross-linked by L,D-transpeptidases. C55-PP is then
subject to pyrophosphatase activity and C55-P recycled.
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Figure 2 Confocal microscope images showing the effects
of antibiotics on P. patens chloronemata. Chlorophyll
autofl uorescence (red) reveals macrochloroplasts consequent
on growth on phosphomycin, D-cycloserine, vancomycin,
bacitracin, ampicillin and A22. A. untreated, B. phosphomycin
(500 µg.ml-1), C. vancomycin (25 µg.ml-1), D. D-cycloserine (20
µg.ml-1, two images), E. bacitracin (100 µg.ml-1), F. murgocil (10
µg.ml-1), G. ampicillin (100 µg.ml-1), H. A22 (2.5 µg.ml-1) and I.
A22 (10 µg.ml-1). Sequential fl uorescence and transmitted light
images, from a Leica SP5 with 63 x oil immersion lens, were
processed using LAS AF lite to optimise intensity and combined
as hyperstacks using Fiji on Image J. Scale bars 10µm.
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Peptidoglycan Intermediate
Number
on
MonoQ
Trace
(Fig. 3)
Con
ducti
vity
(mS
cm
-1
)
Growth
Medium
Superdex
Peptide
and
MonoQ
Fractions
Species of
Inter-
mediate
Detected
Expected
mZ
Nanospray
TOF value
consistent
with
expected
UDP-GlcNAc
1
18.3
KNOPS +
Phos400
C3-C4 F15
(m-1)/1
606.0738
606.0814
(m+Na+-1)/1
628.0557
628.0628
(m-2)/2
302.5330
302.5352
UDP-MurNAc-L-Ala
2
29.41
33.77
KNOPS +
D-cyclo100
C3-C4 F19
C3-C4 F20
(m-2)/2
374.0621
374.0696
374.0698
(m-1)/1
749.1320
749.1476
749.1488
(m+Na+-1)/1
771.1139
771.1294
771.1281
(m+2Na+-1)/1
793.0959
793.1107
793.1127
(m+3Na+-1)/1
815.0778
815.0909
815.0963
(m+Na+-2)/2
385.0531
385.0607
385.0611
UDP-MurNAc-L-Ala-D-
gg
Glu
3
44.78
50.97
KNOPS +
D-cyclo100
C3-C4 F23
C3-4 F24-
25
(m-2)/2
438.5833
438.5928
438.5935
(m+Na+-2)/2
449.5744
449.5839
449.5858
(m+2Na+-2)/2
460.5653
460.5750
460.5716
3
41.73
KNOPS
+Cb100
C7-C8 F23
(m-2)/2
438.583
438.5916
(m+Na+-2)/2
449.5744
449.5829
(m+2Na+-2)/2
460.5653
460.5743
(m-3)/3
292.0530
292.0575
UDP-MurNAc-L-Ala-D-
gg
Glu-meso-
DAP
4
29.6
32.74
KNOPS
alone
C7-C8 F18-
19
C7-C8 F20
(m-2)/2
524.6258
524.6289
524.6289
(m+Na+-2)/2
535.6168
-
535.6196
(m+2Na+-2)/2
546.6077
-
546.6108
(m-3)/3
349.4146
-
349.4155
4
33.77
KNOPS +
D-cyclo100
C3-C4 F20
(m-2)/2
524.6258
524.6377
(m+Na+-2)/2
535.6168
535.6290
(m+2Na+-2)/2
546.6077
546.6197
(m+3Na+-2)/2
557.5987
557.6108
4
29.68
32.83
KNOPS
+Cb100
C7-C8 F19
C7-C8 F20
(m-2)/2
524.6258
524.6317
524.6324
(m+Na+-2)/2
535.6168
535.6230
535.6234
(m+2Na+-2)/2
546.6077
546.6139
546.6144
(m+3Na+-2)/2
557.5987
-
557.6004
(m-3)/3
349.4146
349.4173
349.4178
UDP-MurNAc-L-Ala-D-
gg
Glu-meso-
DAP-D-Ala-D-Ala
5
29.6
KNOPS
alone
C7-C8 F18-
19
(m-2)/2
595.6629
595.6646
(m+Na+-2)/2
606.6539
606.6553
(m-3)/3
396.7726
396.7740
5
29.68
KNOPS
+Cb100
C7-C8 F19
(m-2)/2
595.6629
595.6693
(m+Na+-2)/2
606.6539
606.6602
(m+2Na+-2)/2
617.6446
617.6509
(m-3)/3
396.7726
396.7763
Table 1 UDP-linked intermediates in peptidoglycan biosynthesis as detected by mass spectrometry of the P. patens TCA-extracted
metabolome, with expected mass:charge (mZ) ratios and actual TOF nanospray values as listed. (Figures in italics represent where a
species was detected in more than one fraction). P. patens was grown on KNOPS medium with or without antibiotics, including Phos400
(phosphomycin 400 μg.ml-1), D-cyclo100 (D-cycloserine 100 μg.ml-1) and Cb100 (carbenicillin 100 μg.ml-1). Superdex Peptide (C) and MonoQ
fractions (F) where the different species were identified are listed with their peak conductivities on MonoQ, as detailed in Figure 3. The
negative ion nanospray TOF mass spectra from which the data are derived are in Supplemental Figure S2.
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Supplemental Text S1 Effects of antibiotics on P. patens
Antibiotics which only rarely effected macrochloroplast development included vancomycin (1.0, 5.0 and 25 µg.ml-1), a 1.45 kDa glycopeptide which
binds D-Ala-D-Ala, thereby inhibiting PBP transpeptidation, and the 1.42 kDa bacitracin (20 and 100 µg.ml-1), which complexes with C55-isoprenyl
pyrophosphate, inhibiting recycling (Figure 18,6 and Figure 2, C and E). Neither antibiotic typically traverse cytoplasmic membranes, and therefore
a strong phenotype was not expected as it is anticipated they would have to penetrate not only the cytoplasmic membrane but also, potentially,
the outer chloroplast membrane. It may be that any observed effect of these antibiotics was restricted to damaged or senescing cells. At high
concentrations (500 µg.ml-1) bacitracin did cause premature senescence.
A22 hydrochloride, a smaller molecule at 271.6 kDa, was tested at 2.5 and 10 µg.ml-1 and was likewise found to result in macrochloroplast
formation in some but not most cells, although at higher concentrations its impact was more pleiotropic and chloroplasts were considerably
bleached (Figure 2, H and I). A22 inhibits MreB, an actin homolog and cytoskeletal protein that controls bacterial width in rod-shaped bacteria by
spatiotemporal regulation of peptidoglycan synthesis. Since there is not an evident MreB homolog in the moss (Ozdemir et al., 2018), any effect of
A22 may be consequent on a less specic effect on chloroplast heat shock proteins having homology to MreB, especially HSP70 (Gao and Gao,
2011).
Another antibiotic clearly pleiotropic in its effect was tunicamycin (0.2, 1.0 and 5.0 µg.ml-1), a glycoprotein that inhibits the transfer of phospho-
MurNAc-pentapeptide to the lipid carrier undecaprenyl pyrophosphate by MraY (Figure 14). At concentrations equal to or above 1 µg.ml-1 it caused
chloroplast malformation, slow growth and apoptosis (data not shown). This could be attributed to its effect on the maturation of glycoproteins in the
endoplasmic reticulum since, in eucaryotes, tunicamycin also blocks the transfer of UDP-GlcNAc to dolichol phosphate.
Pacidamycins 1 and 5, cationic peptides with homology to the bacteriophage øX174 lysis protein Arg-Trp-x-x-Trp motif, believed to bind the
cytoplasmic surface of MraY and thereby inhibiting it (Figure 13) (Rodolis et al., 2014; Bugg and Kerr, 2019), had little effect on either growth rate or
chloroplast division (data not shown). Likewise, Murgocil, a 448Da steroid-like molecule, which inhibits peptidoglycan synthesis in Staphylococcus
aureus and is predicted to bind in the MurG active site blocking UDP-GlcNAc access (Figure 15), when tested at 1, 5 and 25 µg.ml-1 was found to
have little effect on protonemata phenotype (Figure 2, F).
The effect of the three antibiotics, phosphomycin, D-cycloserine and ampicillin (Figure 3 B,D and G), subsequently selected for investigating the
accumulation of peptidoglycan intermediates is detailed in the text of the paper.
Bugg TDH, Kerr RV (2019) Mechanism of action of nucleoside antibacterial natural product antibiotics. J Antibiot (Tokyo) 72: 865-876
Gao H, Gao F (2011) Evolution of the chloroplast division machinery. Frontiers in Biology 6: 398-413
Ozdemir B, Asgharzadeh P, Birkhold AI, Mueller SJ, Rohrle O, Reski R (2018) Cytological analysis and structural quantication of FtsZ1-2 and
FtsZ2-1 network characteristics in Physcomitrella patens. Sci Rep 8: 11165
Rodolis MT, Mihalyi A, Ducho C, Eitel K, Gust B, Goss RJ, Bugg TD (2014) Mechanism of action of the uridyl peptide antibiotics: an unexpected
link to a protein-protein interaction site in translocase MraY. Chem Commun (Camb) 50: 13023-13025
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Supplemental Figure S2 Negative ion nanospray TOF mass spectra of TCA-extracted peptidoglycan intermediates, with the expected
mass:charge (mz) values for the different species in red boxes. P. patens was grown on KNOPS medium with and without antibiotics, including
Phos400 (phosphomycin 400 μg.ml-1), D-cycloserine100 (D-cycloserine 100 μg.ml-1) and Cb100 (carbenicillin 100 μg.ml-1). UDP-linked intermediates
were puri ed by chromatography on Superdex Peptide and then MonoQ columns and their respective fractions (C and F) are indicated in
brackets in the headers.
UDP-GlcNAc
KNOPS + Phos400 (C3-4 F15)
Expected (m-1)/1
606.0738
Expected (m-1)/1
606.0738
Expected (m+Na+-1)/1
628.0557
Expected (m+Na+-1)/1
628.0557
Expected (m-2)/2
302.5330
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UDP-MurNAc-Ala
KNOPS + D-cycloserine100 (C3-4 F19)
KNOPS + D-cycloserine100 (C3-4 F20)
Expected (m-2)/2
374.0621
Expected (m-1)/1
749.1320
Expected (m-1)/1
749.1320
Expected (m+Na+-1)/1
771.1139
Expected (m+Na+-1)/1
793.0959
Expected (m+Na+-1)/1
815.0778
Expected (m-2)/2
374.0621
Expected (m+Na+-2)/2
385.0531
Expected (m-2)/2
374.0621
Expected (m+Na+-2)/2
385.0531
Expected (m-2)/2
374.0621
Expected (m-1)/1
749.1320
749.1488
Expected (m-1)/1
749.1320
Expected (m+Na+-1)/1
771.1139
Expected (m+Na+-1)/1
793.0959
Expected (m+Na+-1)/1
815.0778
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UDP-MurNAc-dipeptide (Ala:Glu)
KNOPS + D-cycloserine100 (C3-4 F23)
KNOPS + D-cycloserine100 (C3-4 F24-25)
KNOPS +Cb100 (C7-8 F23)
438.5928
Expected (m-2)/2
438.5833
Expected (m-2)/2
438.5833
Expected (m+Na+-2)/2
449.5744
Expected (m+2Na+-2)/2
460.5653
Expected (m-2)/2
438.5833
438.5935
Expected (m-2)/2
438.5833
Expected (m+Na+-2)/2
449.5744
Expected (m+2Na+-2)/2
460.5653
460.5716
Expected (m-2)/2
438.583
Expected (m-2)/2
438.583 Expected (m+Na+-2)/2
449.5744
Expected (m+2Na+-2)/2
460.5653
Expected (m-3)/3
292.0530
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UDP-MurNAc-tripeptide (Ala:Glu:DAP)
KNOPS alone (C7-C8 F20)
KNOPS + D-cycloserine100 (C3-4 F20)
KNOPS +Cb100 (C7-8 F19)
524.6317
Expected (m-2)/2
524.6258
Expected (m-2)/2
524.6258
Expected (m+Na+-2)/2
535.6168
Expected (m+2Na+-2)/2
546.6077
Expected (m-3)/3
349.4146
Expected (m-2)/2
524.6258
Expected (m-2)/2
524.6258
Expected (m+Na+-2)/2
535.6168 Expected (m+2Na+-2)/2
546.6077
Expected (m+3Na+-2)/2
557.5987
Expected (m-2)/2
524.6258
Expected (m-2)/2
524.6258
Expected (m+Na+-2)/2
535.6168
Expected (m+2Na+-2)/2
546.6077
Expected (m-3)/3
349.4146
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UDP-MurNAc-tripeptide (Ala:Glu:DAP)
KNOPS +Cb100 (C7-8 F20)
Expected (m-3)/3
349.4146
Expected (m-2)/2
524.6258
Expected (m+Na+-2)/2
535.6168
Expected (m+2Na+-2)/2
546.6077
Expected (m+3Na+-2)/2
557.5987
Expected (m-3)/3
349.4146
Expected (m-2)/2 =
524.6258
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UDP-MurNAc-pentapeptide (Ala:Glu:DAP:Ala:Ala)
KNOPS alone (C7-C8 F18-19)
KNOPS +Cb100 (C7-8 F19)
Expected (m-3)/3
396 .772 6
Expected (m-2)/2
595.6629
Expected (m+Na+-2)/2
606.6539
Expected (m-3)/3
396.7726
Expected (m-2)/2
595.6629
Expected (m-3)/3
396.7726
Expected (m-2)/2
595.6629
Expected (m-3)/3
396.7726
Expected (m-2)/2
595.6629
Expected (m+Na+-2)/2
606.6539
Expected (m+Na+-2)/2
617.6448
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15.60 Gb
10.97 Pp1
11.46 Me
0.47
12.32 Cs
5.19
4.23
18.31 An
0.50
18.95 Ec
10.63
10.63 Mt
Supplemental Figure S8 Neighbour joining phylogram of MurE of
Gram negative bacteria and early plant species, computed using
percentage identity in Jalview. Ec E. coli (strain K12), An Anabaena
nostoc PCC7120, Mt Mycobacterium tuberculosum, Pp1 P. patens
(Pp3c24_18820V3.2 v3.3 from Phytozome), Me Mesotaenium endliche-
rianum (WDCW from Onekp CNGBDB), Cs Coleochaete scutata (VQBJ
from Onekp), Gb Gemmatimonadetes bacterium. All sequences are from
the Uniprot or NCBI databases unless stated otherwise.
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Sp/1-481
Sa/1-494
Tm/1-490
Mt/1-535
Me/1-531
Pp1/1-606
Ec/1-495
An/1-496
Sp/1-481
Sa/1-494
Tm/1-490
Mt/1-535
Me/1-531
Pp1/1-606
Ec/1-495
An/1-496
Sp/1-481
Sa/1-494
Tm/1-490
Mt/1-535
Me/1-531
Pp1/1-606
Ec/1-495
An/1-496
Sp/1-481
Sa/1-494
Tm/1-490
Mt/1-535
Me/1-531
Pp1/1-606
Ec/1-495
An/1-496
Sp/1-481
Sa/1-494
Tm/1-490
Mt/1-535
Me/1-531
Pp1/1-606
Ec/1-495
An/1-496
Sp/1-481
Sa/1-494
Tm/1-490
Mt/1-535
Me/1-531
Pp1/1-606
Ec/1-495
An/1-496
Sp/1-481
Sa/1-494
Tm/1-490
Mt/1-535
Me/1-531
Pp1/1-606
Ec/1-495
An/1-496
---------------------------------------------------------------------------------MIKIET VLDIL---KKDGL
----------------------------------------------------------------------------------------------MDASTL
----------------------------------------------------------------------------------------------MNISTI
--------------------------------------------------MSSLARGISRRRT EVAT---QVEAAPT GLR----PNAV----VGV RLAAL
-----------------------------------------------------------------------------------------ERAY SM TLGEL
MA LQWIQKPLLAQ FSIHSSHA SAVRER LSSQ SGLANFSFSHKPC SIPLTPSGRLSGRV GR SR LKMGFGDSKLTDRSFSLKSSTHEEAVLDQTDRMTLRKL
-------------------------------------------------------------------------------------------MADRNLRDL
----------------------------------------------------------------------------------------------MKLREL
----FR EI IDQGHYHYNYSKVV FDSI SY DSRKVTEDTLFFAKGA--AFKKEYLLSAITQ GLAWYVAEKDY EV GI ---------PVI IVNDIKKAMSLIAM
----FK--KVKVKRVLGSLEQQ IDDI TTDSRTAREGSIFVA SV GYTVDSHKFCQNVADQGC KLVVVNKEQSLPANV-------TQVVVPDTLRV ASI LAH
----VSN LKDL ILEVRAPYDLEITGVSN HSSKVKKGDLFICRRGEK FDSH EI IPEVMEK GAVAVVV ER EIDLDFPY--------- IQVFDSRYFEAKVAS
ADQVGAALAEGPAQRAVTEDRT VTGV TLRAQDVSPGD LFAALTGSTTHGARHVGDAIARGAVAV LTDPAGVAEI---AGRAAVPV LVHPAPRGV LGGLAA
----LR IADVEP IAYEGD LDVEVTGIQQDSREVQ PGDLFVCC EGLKTDGHMYANEA LERGAIAIFSSK EVEI YEPV- R-----ALVLLEDT SA ALSA LAD
----LNEA RV SP LSTEGD LDVEITGIQQDSRLVAPGDLFVCVKGLK SDGHQFA IQAIEKGAVA IISLMEVSLT EGL- K-----AAV IVEDTSVI LSALAG
----LAP------WVPDAPSRALREMT LDSR VAAAGDLFVAVVGHQADGRRY IPQAIAQGVAA IIAEAKDEA TDGEIREMHGVPVIYLSQLNER LSALAG
----LATVDSVENLPPVLADA EVKG IKTNSHACGAGD LFIGMPGT RV DGGEFWPSAIASGA IAAIVSPQAV EK NPP---HDEAVV ISSNNM TKACAAIAA
EFYGNPQEK LK ILAFTGTKGKTTAAY FAYN ILSQ-RYPTALLSTMNTTLDGTTFFKSSFSTPENIDLFDMMAQAVKNGR SH LVMEVSSQAY LVHRVYGLT
TLYDYP SHQLVTFGVTGTNGKTSIATMIHLIQRK LQ KNSA YLGTNGFQ INETK- TKGA NTTPET VSLTKK IKEAVDAGAESM TLEV SSHGLVLGRLRGVE
LFFEDPWKDV LT FGVTGT NGKTTTTMM IYHM LT SLGERGSV LTTAVKR ILGNS- YYDDITTPDA ITILSAMKENR EGGGK FFALEV SSHALVQQRV EGVR
TVYGHPSERLTVIGITGT SGKTTTTYLVEA GLRAAGRVAGLIGT IGIRVGGA D- LPSA LTTP EAPT LQAM LAAMVERGVDTVVMEV SSHA LALGRVDGTR
AFYGHP SQ SLTVVG ITGTNGKTTTSY LVRSIYDAMGLKTGLLGT IAYSIGSKQ-QEATHTTPDA INVQKLMASM VHQRCDACIMEVSSHALALGRCTRVE
VIYGHPSKKLSVVG ITGTNGKTTT SY LLQSLYEAM GLQVGLLGT IQYYIGGK NK LEADHTTPEALNLQ NLMASM VQ NGTEVC IMEV SSHGLVLGRCED IE
RFYHEP SDNLRLVGVTGT NGKTTTTQLLAQWSQLLGEISAVMGTVGNGLLGKV- IPTENTTGSAVDVQ HELAGLVDQGATFCAMEVSSHGLVQHRVAA LK
AFYGYPGQKLKLVGVTGTNGKTTTTHLI EFFLTKAKLSTALMGT LYTRWP GFE- QTATHTTPFAVELQQQLAQAVNAGCEFGVMEV SSHALAQGRVLGCP
FDVGVFLNITPDHIGP IEHP SF EDYFYHKRLLMENSRA -------VIINSDMDHFSVLK------------ EQ VEDQDHDFY GSQFDNQ I- ENSK AFSFS
FDVA IFSN LTQDHLD-- FHGTMEAYGHAKSLLFSQ LGEDLSKEKYVVLNNDDSF SEYLRTV- TPYEV- -FSYGI DE-EAQFM AKN I- -QESLQGV SFDFV
FDVGIFTN ISRDH LD--FHGT FENYLKAK LHLFDLLKD--DGV --AVL--NESLADAFNRKS-----RKITFGT SK-NADYRLGNIEV S- -WEGTQ FV LE
FAVGAFTNLSRDHLD--FHPSMADY FEAKASLFDPD SA LRART--AVVCIDDDAGR AMAARAADAITVSAAD-----RPAHWRATDVAPTDAGGQQ FTAI
FDVAVFT NLTRDHMD--FHATPEEYRDAKAQLFQRMVDPARHR--KVVNLDDPAAD FFVDQGHPD-VPTVT YGLEREDADVYPLEVSLSLFE--TELVVR
FDVAVFT NLTRDHMD--FHKT EEEYRRAKGLLFAKMVDP ERQR--KVVNIDDPNV SY FV SQ GNQD- VPVVT FGMGDKSADVYP LAVK LSLVE- -SEV LVR
FAASVFTNLSRDH LD--YHGDM EHYEAAKWLLY SE-H--HCGQ--AIINADDEVGR RWLAKLPDAVAVSM EDHINP NCHGRWLKAT EV NYHD SGAT IRFS
FEVGVFSNLTQDHLD--YHSDM EDYFAAKALLFSPEY --LKGR --AIINADDT YGQRLIKALSP EKVWS--YSVNDSSADLWMS--DLSYEPNGVTGTIH
ATGK LAGDYD IQLIGN FNQENAVAAGLACLRLGASL ED IKKG IAAT-RVPGRMEV LTQ--KNGAKVFIDYAHNGDSLKKLINVVETHQ TGKIALVLGSTG
TPFG-TYPVKSPYVGKFNISN IMAAMI AVWSKGTSLETI IKAV EN LEPVEGRLEV LDP- -SLP IDLI IDYAHTADGMNKLIDAVQPFVKQKLIFLVGM AG
TPDG-LLKVFTRAIGD FNAYNAAAA IAALHQ LGYDPKDLASSL ET FTGV EGRFEVVRGAKK IGLNVVVDFAHSPDALEKLLKNVRKISQ GRVIVVFGAGG
DPAGVGHH IGIRLPGRYNVANCLVA LA ILDT VGVSPEQAVPGLR EI-RVPGRLEQ IDR--GQGF LALVDYAHKP EA LRSV LTTL-AHP DRRLAVV FGAGG
TPKG-PLEISSGLLGRHNV SN ILAA IAVG IAVGADLEDIQKGIEEVDAVPGRCEL IDE- -EQAFAV IVDYAHTPDALGRLLDTVRECGPTRI ITVVGCGG
TPQG- DVEI SSRLLGRHNVYNILTAVAVGIAVGAPLEDIVRGIEAVDAVPGRCEL IDE--GQTFAVLVDYAHTPDAVARLLDTVRECGPKR IITVLGCGG
SSWG -DGEIESHLM GA FNVSNLLLALATLLALGYPLADLLKTAARLQPVCGRMEVFTA--PGKPT VVVDYAHTPDALEKALQ AARLHCAGK LWCV FGCGG
TPEG-NVSFRSP LVGQ YN LENL LAAVGAVLHLGLNLQ LIANAIPEFPGVPGRM ERVQ INPDQDISV IVDYAHTPDSLENLLKAARPFIPGRMICVFGCGG
NKGESRRKDFGLLLNQHPEIQVFLTADDPNY ED PMAIADEI SSYINH---------------------PVEKIADRQEA IKAAMA ITNHELDAVI IAGKG
ERDLTKTPEMGR VAC-RADY- VIFT PDNPANDDPKM LT AELAKGAT HQ --------------------NYIEFDDRA EG IKHAIDIAE- PGDTVVLA SKG
NSDRGKRPMMSEVASKLADV- VI LTTDDPRGED PEQIMEDLIKG IDKR-------------------KPY LVLFDRREA IETALT IAN- RGDSVV IAGR G
DRDPGK RAPMGR IAAQLADL-VVVTDDNP RDEDPT AIRREI LAGA AEVG----------------GDAQVVEIADRRDA IRHAVAWAR- PGDVVL IAGKG
ERDRGKRPIMGKIATDK SD I- TILT SDNP RNED ACEI IDDM LAGVGWDM EQ YLAYGEQ GYYPPLKNGHR LFVHDCRDIAVRAAVAMGE- EGDA IVVAGKG
DRDKGK RP IMAKIAADKSDV -CIITSDNPRT EKPLDI IDDM LAGV GWSM EQ YCKWEEDSSYPLLPNGHRLFCQEI RSKAIRAAVAMA E- EGDAVV IAGKG
DRDKGK RP LMGA IAEEFADV- AVVTDDNPRT EEPRAI INDI LAGM LDA-------------------GHAKVMEGR AEAVTCAVMQAK- ENDVVLVAGKG
DRDRTKRPKMGKIVAELADL- AFVTSDNPRT ED PDRILDDI LAGIPDT-------------------VQPT VIGDRA IAIRTA ILQAQ -PGDGV LLAGKG
ADCYQIIQGKKESYPGDTAVAENYL-------------------
REPYQIMPGHIKVPHRDDLIGL EAAYKK FGGGPVDQ--------
HERYQIIDEEKKVPFQDR EV VEEI IRDKLKGRKYAQ--------
HETGQRGGGR-VRP FDDRV ELAAA LEALERRA------------
HETYQIDVKG-KRY FDDREECREA LQ NVAEI RQ HFDT SEIPWR L
HETYQIIGEI- KGHFDDREECREA LRLRK---------------
HEDYQIVGNQ-RLDYSDRVTVARLLGV IA---------------
HEDYQILGTE- KIHFDDREH ARAALT ER EK L-------------
1
1
1
1
1
1
1
1
16
6
6
39
11
100
9
6
17
7
7
40
12
101
10
7
101
93
93
136
101
190
99
99
102
94
94
137
102
191
100
100
200
192
192
235
200
290
198
198
201
193
193
236
201
291
199
199
280
284
276
326
293
383
291
288
281
285
277
327
294
384
292
289
377
381
375
422
390
480
388
387
378
382
376
423
391
481
389
388
456
458
454
504
488
578
467
466
457
459
455
505
489
579
468
467
481
494
490
535
531
606
495
496
**
Streptophyte Carboxy-Terminal Loop SCTL
▼
▼
▼
S28Ec HQA45Ec
TGTXGKT115Sa NTT158Ec
▼
▼S184Ec QXR192Ec
K224Ec
F300Sa D356SaK360Sa
R389Ec
R383Sa
DNXR416Ec
D406Sa
E460Sa
G464Ec
▼
▼ ▼
*
*
**
*
**
* *
**
*
*
*
*
***
** *
Supplemental Figure S9 Clustal Omega (EMBL-EBI)(Madeira et al., 2019) multiple sequence alignment of MurE homologs displayed using
Jalview (Waterhouse et al., 2009) with Clustalx designated colours: Sp Streptococcus pneumoniae, Sa Staphylococcus aureus, Mt Mycobacterium
tuberculosum, Pp1 P. patens (Pp3c24_18820V3.2 v3.3 from Phytozome), Tm Thermotoga maritima, Me Mesotaenium endlicherianum (WDCW
from Onekp CNGBDB), Ec E. coli (strain K12), An Anabaena nostoc PCC7120. All sequences are from the Uniprot or NCBI databases unless
stated otherwise. Green arrows indicate ChloroP predicted cleavage site for PpMurE and red arrows the domain hinge points (Smith, 2010). Black
arrows indicate residues with a reported role in MtMurE catalysis (Basavannacharya et al 2010). Letter labels indicate numbered residues with
published ligand interractions: Ec for EcMurE (Gordon et al., 2001), Mt for MtMurE (Basavannacharya et al., 2010; Maitra et al., 2019) and Sa for
SaMurE (Ruane et al., 2013) with colours indicating binding to UDP (blue), MurNAc sugar (green), ATP or ADP (mauve) and DL-DAP (orange)
ligands. Blue asterisks indicate residues common to the Mur ligase family, which includes folylpolyglutamate synthetase, cyanophycin synthetase
and the capB enzyme from Bacillales (Gordon et al., 2001; Smith, 2010) and pink asterisks indicate residues common to MurC, D, E and F ligases
(Basavvanacharya et al., 2010). Two streptophyte-specific features are identified by black boxes and the DNPR consensus by a red box.
E468Ec
E220Mt
R230Mt
S222Mt
LXAQ70Mt
GKT158Mt
H248Mt
R377Mt D392Mt
▼
▼
S456Sa
GS84Mt
K219Sa
*
*
**
YXXXN347Mt
TTXE198Mt
*
*
*
DDREECREAL motif
Basavannacharya C, Moody PR, Munshi T, Cronin N, Keep NH, Bhakta S (2010) Essential residues for the enzyme activity of ATP-dependent
MurE ligase from Mycobacterium tuberculosis. Protein Cell 1: 1011-1022
Gordon E, Flouret B, Chantalat L, van Heijenoort J, Mengin-Lecreulx D, Dideberg O (2001) Crystal structure of UDP-N-acetylmuramoyl-L-ala-
nyl-D-glutamate: meso-diaminopimelate ligase from Escherichia coli. J Biol Chem 276: 10999-11006
Maitra A, Munshi T, Healy J, Martin LT, Vollmer W, Keep NH, Bhakta S (2019) Cell wall peptidoglycan in Mycobacterium tuberculosis: An
Achilles' heel for the TB-causing pathogen. FEMS Microbiol Rev 43: 548-575
Ruane KM, Lloyd AJ, Fulop V, Dowson CG, Barreteau H, Boniface A, Dementin S, Blanot D, Mengin-Lecreulx D, Gobec S, Dessen A,
Roper DI (2013) Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE
enzyme ternary complex. J Biol Chem 288: 33439-33448
Smith CA (2006) Structure, function and dynamics in the mur family of bacterial cell wall ligases. J Mol Biol 362: 640-655
Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) Jalview Version 2--a multiple sequence alignment editor and analysis
workbench. Bioinformatics 25: 1189-1191
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted January 6, 2022. ; https://doi.org/10.1101/2022.01.05.475093doi: bioRxiv preprint
Eudicotyledon Thymus vulgaris
Eudicotyledon Micromeria fruticosa
Eudicotyledon Tabebuia umbellate
Eudicotyledons Mimulus guttatus
Eudicotyledon Solanum xanthocarpum
Eudicotyledons Arabidopsis thaliana
Eudicotyledons Carica papaya
Magnoliidi Magnolia grandiflora
Amborellales Amborella trichopoda
Poaceae Zea mays
Poaceae Neurachne lanigera
Poaceae Oryza sativa
Poaceae Brachypodium stacei
Brommeliaceae Ananas comosus
Eudicotyledons Curcuma olena
Asparagales Urginea maritima
Araceae Spirodela polyrhiza
Polypodiidae Argyrochosma nivea MurE2
Polypodiidae Adiantum capillus-veneris MurE2
Polypodiidae Notholaena montieliae MurE2
Polypodiidae Pityrogramma trifoliata MurE2
Polypodiidae Leucostegia immersa MurE2
Polypodiidae Blechnum spicant MurE2
Polypodiidae Anemia tomentosa MurE2
Gnetopsida Welwitschia mirabilis
ConifersII Lagarostrobos franklinii
ConifersII Saxegothaea conspicua
ConifersII Falcatifolium taxoides
ConifersII Cupressus dupreziana
ConifersII Sciadopitys verticillata
Cycadopsida Encephalartos barteri
Cycadopsida Stangeria eriopus
ConifersI Cedrus libani
ConifersI Pinus radiata
ConifersI Larix gmelinii
Ophioglossidae Sceptridium dissectum
Ophioglossidae Botrypus virginianus
Ophioglossidae Psilotum nudum
Equisetidae Equisetum hymale
Lycopodiopsida Diphasiastrum digitatum
Lycopodiopsida Selaginella moellendorfii
Lycopodiopsida Isoetes sp
Marchantiophyta Marchantia polymorpha
Marchantiophyta Riccia berychiana
Marchantiophyta Porella pinnata
Marchantiophyta Bazzania trilobata
Marchantiophyta Lejeuneaceae sp
Anthocerotopsida Megaceros vincentianus
Anthocerotopsida Paraphymatoceros hallii
Anthocerotopsida Buxbaumia aphylla
Bryophyta Sphagnum fallax
Bryophyta Physcomitrium patens MurE2
Bryophyta Leucobryum albidum
Bryophyta Funaria
Bryophyta Aulacomnium heterostichum
Bryophyta Ceratodon purpureus
Bryophyta Racomitrium varium
Coleochaetophyceae Coleochaete scutata
Coloechaetophyceae Coleochaete irregularis
Zygnemophyceae Pleurotaenium trabecule
Zygnemophyceae Cosmocladium cf. constrictum
Zygnemophyceae Staurodesmus convergens
Zygnemophyceae Phymatodocis nordstedtiana
Zygnemophyceae Roya obtusa
Zygnemophyceae Mesotaenium endlicherianum
Zygnemophyceae Zygnemopsis sp
Zygnemophyceae Cylindrocystis brebissonii
Zygnemophyceae Mougeotia sp
Charophyceae Chara braunii
Bryophta Physcomitrium patens MurE1
Polypodiidae Notholaena montieliae MurE1
Polypodiidae Argyrochosma nivea MurE1
Polypodiidae Adiantum capillus-veneris MurE1
Polypodiidae fern Pityrogramma trifoliata MurE1
Polypodiidae Leucostegia immersa MurE1
Polypodiidae Blechnum spicant MurE1
Polypodiaceae Anemia tomentosa MurE1
Klebsormidiophyceae Interfilum paradoxum
Klebsormidiophyceae Klebsormidium nitens
Zygnemophyceae Entransia fimbriat
Mesostigmatophyceaea Spirotaenia minuta
Chlorokybophceae Chlorokybus atmophyticus
Glaucocystophyceae Gloeochaete wittrockiana
Glaucocystophyceaea Glaucocystus nostochinearum
FCB Bacteria Gemmatimonadetes bacterium
Prasinodermophyceae Prasinoderma coloniale
Cyanobacteria Gloeomargarita lithophora
Cyanobacteria Anabaena PCC7120
Proteobacteria Escherichia coli
Actinobacteria Mycobacterium tuberculosa
Thermatogae Thermotoga maritima
Firmicute Staphylococcus aureus
Firmicute Streptococcus pneumoniae
0.00
0.20
0.40
0.60
Grasses
Bacteria
Charophyte algae
Mosses and liverworts
Polypodiidae ferns
Ophioglossidae ferns, club mosses and horsetails
Supplemental Figure S10 Evolutionary relationship of both PpMurE proteins to selected MurE homologs. P. patens, as well as many ferns in the
Polypodiidae, encodes two MurE homologs: PpMurE1 and PpMurE2, labelled in green. Different taxonomic groups are boxed to highlight the
relationship of the P. patens proteins to bacterial, algal and streptophyte phylla. Sequences, except P. patens, were sourced from the ONEKP
database and selected to represent each group (Leebens-Mack et al 2019, Carpenter et al 2019). The evolutionary history was inferred using the
Minimum Evolution method (Rzhetsky and Nei,1992) and computed using MegaX software (Kumar et al 2018). The evolutionary distances, are in
the units of the number of amino acid substitutions per site. PpMurE1 and the shorter Polypodiidae fern ‘MurE1’ homologs are evolutionarily closer
to charophyte algae than land plants. PpMurE2 is closer to most marchantiophytes and other bryophytes, which lack a second MurE homolog,
whereas the longer Polypodiidae ‘MurE2’ are closer to the Acrogymnsopermae (conifers).
PpMurE2 primarily differs from PpMurE1 in comprising a long, relatively unstructured extension at the amino terminus and a short carboxy terminal
extension. The former is considerably longer than a conventional transit peptide (290 residues longer than typical bacterial homologs, compared to
94 residues for PpMurE1) and is common to most seed plant MurE-like proteins, as well as some streptophyte algal and bryophyte MurE
homologs.The extended amino terminus is typically proline-rich in the amino terminal residues, being more glycine-rich in lower orders, and, in the
later residues, more conserved within different plant divisions. The carboxy terminal extension (24 residues in PpMurE2 beyond a consensus
streptophyte DDREECREAL motif in PpMurE1 (Supplemental Figure S9) is more highly conserved, with a consensus sequence
(DDREECREALQXVDXLHXAGIDTFESPWRXPESX) that is common to most streptophyte MurE homologs, although streptophyte algae lack the
terminal PESX. However, where there are two distinct MurE homologs, as there are for P. patens and some ferns, this carboxy terminal extension
is typically absent from the shorter MurE homologs and these proteins appear to have de-evolved to more closely resemble their bacterial
counterparts. The retention of a DNPR motif is common not only to the non-seed plants but also most seed plant MurE homologs, with the similarly
charged DNPK also being common, and the Poaceae and a few Pinaceae being notable exceptions (DNPA and DNSR, respectively). In contrast
to P. patens and the Polypodiidae ferns, many in the same and closely related phylla, including the Acrogymnospermae (Lin et al., 2017) do not
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