Dpaa Detaches Braun's Lipoprotein from Peptidoglycan

bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 available under aCC-BY 4.0 International license.

1 DpaA detaches Braun's lipoprotein from peptidoglycan

3 Matthias Winkle1, Víctor M. Hernández-Rocamora1, Karthik Pullela2, Emily C. A. Goodall2,

4 Alessandra M. Martorana3, Joe Gray4, Ian R. Henderson2, Alessandra Polissi3*, Waldemar

5 Vollmer1*

7 1 Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle

8 upon Tyne, NE3 2AX, United Kingdom.

10 2 Institute for Molecular Bioscience, The University of Queensland, Brisbane Qld 4072,

11 Australia.

13 3 Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano,

14 Milan, Italy.

16 4 Biosciences Institute, Newcastle University, Newcastle upon Tyne, NE3 2AX, United

17 Kingdom.

21 * Corresponding authors: [email protected]; [email protected].

24 Keywords

25 Escherichia coli; cell envelope; peptidoglycan; Braun's lipoprotein (Lpp); periplasm; amidase;

28 ABSTRACT

29 Gram-negative bacteria have a unique cell envelope with a lipopolysaccharide-containing

30 outer membrane that is tightly connected to a thin layer of peptidoglycan. The tight connection

31 between the outer membrane and peptidoglycan is needed to maintain the outer membrane

32 as an impermeable barrier for many toxic molecules and antibiotics. Enterobacteriaceae such

33 as Escherichia coli covalently attach the abundant outer membrane-anchored lipoprotein Lpp

34 (Braun's lipoprotein) to tripeptides in peptidoglycan, mediated by the transpeptidases LdtA,

35 LdtB and LdtC. LdtD and LdtE are members of the same family of LD-transpeptidases but

36 they catalyse a different reaction, the formation of 3-3 cross-links in the peptidoglycan. The

37 function of the sixth homologue in E. coli, LdtF remains unclear, although it has been shown

38 to become essential in cells with inhibited LPS export to the outer membrane. We now show

39 that LdtF hydrolyses the Lpp-peptidoglycan linkage, detaching Lpp from peptidoglycan, and

40 have renamed LdtF to peptidoglycan meso-diaminopimelic acid protein amidase A (DpaA).

41 We show that the detachment of Lpp from peptidoglycan is beneficial for the cell under certain

42 stress conditions and that the deletion of dpaA allows frequent transposon inactivation in the

43 lapB (yciM) gene, whose product down-regulates lipopolysaccharide biosynthesis. DpaA-like

44 proteins have characteristic sequence motifs and are present in many Gram-negative bacteria

45 of which some have no Lpp, raising the possibility that DpaA has other substrates in these

46 species. Overall, our data show that the Lpp-peptidoglycan linkage in E. coli is more dynamic

47 than previously appreciated.

49 IMPORTANCE

50 Gram-negative bacteria have a complex cell envelope with two membranes and a periplasm

51 containing the peptidoglycan layer. The outer membrane is firmly connected to the

52 peptidoglycan by highly abundant proteins. The outer membrane-anchored Braun's lipoprotein

53 (Lpp) is the most abundant protein in E. coli and about one third of the Lpp molecules become

54 covalently attached to tripeptides in peptidoglycan. The attachment of Lpp to peptidoglycan

55 stabilizes the cell envelope and is crucial for the outer membrane to function as a permeability

56 barrier for a range of toxic molecules and antibiotics. So far the attachment of Lpp to

57 peptidoglycan has been considered to be irreversible. We have now identified an amidase,

58 DpaA, which is capable of detaching Lpp from PG and we show that the detachment of Lpp is

59 important under certain stress conditions. DpaA-like proteins are present in many Gram-

60 negative bacteria and may have different substrates in these species.

61 INTRODUCTION

62 Bacteria have to maintain the integrity of their complex cell envelope at all times when

63 propagating in diverse and often adverse environments (1). The peptidoglycan (PG) layer

64 surrounds the cytoplasmic membrane (CM) and protects the cell from rupture due to osmotic

65 challenges (2, 3). Diderm (Gram-negative) bacteria protect themselves from antimicrobial

66 compounds by surrounding their PG layer by a semi-permeable outer membrane (OM). In

67 Escherichia coli and related species, the PG and OM are tightly connected by multiple, highly

68 abundant proteins such as Lpp (Braun's lipoprotein), OmpA and Pal (4-7). The cell must

69 maintain these tight connections at all times and coordinate the expansion of the PG and OM

70 during growth and cell division to avoid leaks in the OM, the loss of OM vesicles or lysis due

71 to an instable cell envelope (6, 8, 9).

72 Lpp is the most abundant protein in E. coli with about 1 million copies per cell (5). In

73 pioneering work in the 1970s, Volkmar Braun and co-workers identified Lpp not only as the

74 first protein that is covalently attached to PG, but also as the first protein with a lipid

75 modification (4). The Lpp pre-protein uses the Sec system for translocation through the CM

76 where it then matures by (i) the addition of a diacylglycerol to a cysteine residue near the N-

77 terminus, (ii) the removal of the leader peptide and (iii) the addition of a third acyl chain to the

78 N-terminus (10). Mature Lpp is then transported through the periplasm and anchored to the

79 inner leaflet of the OM by the localisation of lipoprotein (Lol) transport system (11). About one

80 third of the protein molecules are covalently attached to PG via the -amino group of the C-

81 terminal lysine, which is linked to the -carboxylic group of meso-diaminopilemic acid (mDAP)

82 at position 3 of a PG stem peptide, by the LD-transpeptidases (LDTs) LdtA, LdtB and LdtC (4,

83 12, 13). About 5% of all PG subunits are attached to Lpp in exponentially growing cells and

84 15% in stationary phase (14).

85 In addition to stabilizing the cell envelope, Lpp helps to maintain a constant distance

86 between the PG and OM (15, 16). Mutants lacking Lpp or LdtA, LdtB and LdtC suffer from

87 hyper-vesiculation, losing OM-derived vesicles filled with periplasmic content into the

88 environment (9, 17, 18). These mutants become sensitive to sodium dodecyl sulphate (SDS)

89 due to abnormally high OM permeability (6). Pathogenic E. coli and Salmonella mutants

90 lacking Lpp are less virulent and able to invade their host, and an Lpp-deficient uropathogenic

91 E. coli strain is more susceptible to serum killing (19-21). In this strain, the attachment of Lpp

92 to PG was crucial for the expression of capsular polysaccharide, illustrating another key role

93 of PG-attached Lpp (21).

94 Given the important role of Lpp for cell envelope stability in E. coli, it was surprising

95 that Lpp is only present in closely related -proteobacteria (22). While another PG-attached

96 lipoprotein has been described in Pseudomonas spp., the covalent attachment of PG to OM

97 localized proteins has not been studied in depth in other diderm bacteria PG (23). This

98 changed recently when two publications reported the covalent attachment of certain OM β-

99 barrel proteins (OMPs) in a wide range of -proteobacteria (24, 25). Additionally to OMPs, one

100 of the studies identified the OM-anchored lipoprotein LimB attached to PG in Coxiella burnetti

101 (25). Similar to Lpp in E. coli, these proteins are attached to the mDAP residue in PG stem

102 peptides by LDTs and are required for OM stability. Different to Lpp, OMPs were found to be

103 attached to PG via N-terminal glycine or alanine residues. Many Firmicutes (Gram-positives)

104 attach multiple cell wall proteins to PG using the enzyme sortase (26). These are often

105 involved in interactions with host factors during bacterial infections and include, for example,

106 protein A in S. aureus or pilus subunits in S. pneumoniae (27, 28).

107 Although previous work identified several PG-attached proteins and two classes of

108 enzymes for protein-PG attachment (LDTs and sortase), to date no enzyme for the opposite

109 reaction, the detachment of proteins from PG, has been identified. Here, we show that an LDT

110 homologue of previously unknown function (DpaA, formerly named LdtF or YafK), has an

111 amidase activity hydrolysing the bond between Lpp and PG in E. coli. We also report that the

112 DpaA-mediated detachment of Lpp from PG helps the cell to cope with certain stress

113 conditions and that DpaA-like enzymes are present in many diderm bacteria.

114

115 RESULTS

116 DpaA is genetically linked to PG and OM biogenesis

117 In E. coli, all six LD-transpeptidases (LDTs) that contain the YkuD conserved motif

118 (Fig. 1) become important under cell envelope stress conditions. LdtA, LdtB and LdtC facilitate

119 cell envelope stability by covalently attaching the OM-anchored lipoprotein Lpp to PG (13).

120 LdtD forms 3-3 cross-links in PG to repair the PG layer upon defective OM assembly (29).

121 LdtE appears to form 3-3 cross-links when cells enter the stationary phase of growth, although

122 its activity has not been demonstrated with the purified enzyme. The function of the last LDT,

123 LdtF (now DpaA), remained unknown. Genetic depletion of the essential LPS export gene lptC

124 causes lysis in cells lacking dpaA (29), but these show morphological defects even in the

125 presence of inducer (arabinose), and the lysis of lptC-depleted ∆dpaA cells can be prevented

126 by the additional deletion of a novel amidase activator gene, actS (Gurnani et al., under

127 review). None of these phenotypes are observed for the other ldt mutants, suggesting that

128 DpaA has different (or additional) roles than LdtA-E.

129 To better understand the role of DpaA we first performed genome-wide transposon

130 insertion analysis (TraDIS) of the dpaA mutant. We constructed two libraries of transposon

131 mutants by electroporation of a mini-Tn5 transposon into a ∆dpaA strain, and the BW25113

132 parent strain as a control. To identify the transposon insertion sites, we sequenced the

133 transposon-genomic DNA junction and mapped the data to the BW25113 reference genome

134 as previously described (62). We identified 670,433 and 360,972 unique insertion sites for

135 dpaA and control library, respectively, and these are distributed around the chromosome (Fig.

136 S1, Table S1). There were no insertions within dpaA in the ∆dpaA library, confirming the library

137 genotype (Fig. 2B). To identify mutants with a fitness defect, or advantage, in the dpaA gene-

138 deletion background, we used the BioTraDIS EdgeR analysis tool to compare the frequency

139 of reads per gene between each library, using thresholds of >2-fold change and Q-value >

140 0.01. This analysis revealed relatively few genes with significantly more or less reads in the

141 dpaA mutant compared to wild-type. The most obvious differences we observed in the dpaA

142 library compared to the control were a 4.25 fold enrichment (Q-value = 1.84E-25) of reads

143 within lapB (yciM) and a 2.1 fold enrichment of reads with the neighbouring lapA (yciS) gene

144 (Q-value = 2.59E-5) (Fig. 2A and 2C). LapA and LapB are essential regulators of LPS

145 biosynthesis, regulating the degradation of LpxC by the membrane-bound protease FtsH (30,

146 31) and LapA physically interacts with the elongasome (31b). Hence, dpaA inactivation is

147 linked to phenotypes related to both LPS synthesis (lapB inactivation), peptidoglycan

148 synthesis (lapA inactivation) and LPS export (lptC depletion). To begin to understand these

149 genetic interactions, we wanted to determine the enzymatic activity of DpaA.

150

151 DpaA is not an LD-TPase

152 We purified LdtD, LdtE and DpaA and first tested for their ability to produce 3-3 cross-

153 links in PG isolated from an E. coli strain lacking all 6 LDTs (BW25113∆6LDT) at neutral (pH

154 7.5) or acidic (pH 5.0) conditions. The PG was digested with the muramidase cellosyl and the

155 resulting muropeptides were separated by high performance liquid chromatography (HPLC)

156 (Fig. S2A and S2B). LdtD produced muropeptides with 3-3 cross-links or tripeptides

157 [TetraTri(3-3), TriTri(3-3), TetraTri, Tri; Fig. S2C)], confirming LD-TPase and LD-CPase

158 activities (29). LdtE generated TetraTri(3-3) consistent with the presence of this TPase

159 products in the PG of BW25113∆6LDT cells overexpressing ldtE and dpaA (32). LdtE was

160 nearly inactive at pH 7.5 and did not show LD-CPase acitvity. DpaA did not show any LD-

161 TPase or LD-CPase activity under any condition tested, despite long incubation times of 12

162 hours. We noticed that DpaA lacked an active-site arginine residue conserved in other LDTs

163 (Fig. 1), suggesting that DpaA is an inactive enzyme of the LDT family or has a different

164 activity than the other LDTs.

165

166 DpaA releases Lpp from PG

167 We reasoned that rather than forming LD-bonds, DpaA might hydrolyse bonds

168 generated by other LDTs, however, in several experiments we could not detect any LD-

169 endopeptidase or LD-carboxypeptidase activity (not shown). Hence, we next tested if DpaA

170 hydrolyses the bond between the terminal L-lysine residue of Lpp and PG, which is formed by

171 LdtA-C (13). We first used purified muropeptides containing the terminal two amino acids

172 (lysine and arginine) from Lpp, as artificial substrates. The protease pronase E used in PG

173 purification removes contaminating proteins and most part of the covalently attached Lpp, with

174 the exception of the C-terminal Lys-Arg dipeptide; the residual dipeptide is used in PG analysis

175 to quantify the amount of PG-attached Lpp in strains (14).

176 We incubated Tri-LysArg and TetraTri-LysArg with DpaA or the catalytically inactive

177 DpaA(C143A) (with Ala replacing the active site Cys residue) and analysed the products by

178 HPLC. DpaA but not DpaA(C143A) converted both substrates to muropeptides lacking the

179 Lys-Arg dipeptide (Tri or TetraTri) (Fig. 3A and S2D). We confirmed the mass of the substrate

180 and product muropeptides by MS/MS analysis (Fig. S3). To test if this activity was specific for

181 DpaA we incubated Tri-LysArg with purified LdtD or LdtE and observed no activity with these

182 LDTs (Fig. S2E).

183 LDTs are known to be inhibited by copper ions (33). To test if this was true for DpaA,

184 we incubated PG from BW25113∆dpaA cells with DpaA in the presence of CuCl2, followed by

185 the quantification of Lys-Arg containing muropeptides by HPLC. In the absence of copper,

186 DpaA removed Tri-LysArg from the muropeptide profile, forming Tri whilst the presence of 0.2

187 mM CuCl2 inhibited the reaction (Fig. S4A). We observed half-inhibition at ~0.1 mM CuCl2

188 (Fig. S4B), indicating an approx. 6-fold lower IC50 than that for LdtD (33).

189 In a different assay for DpaA activity, we incubated the protein with PG sacculi from

190 BW25113∆dpaA cells that were not treated with pronase E and, hence contained covalently

191 attached full-length Lpp (rather than only the Lys-Arg dipeptide). Samples were centrifuged to

192 separate soluble proteins from insoluble PG, and proteins present in the supernatant were

193 analysed by SDS-PAGE (Fig. 3C) and Western Blot developed with a specific antibody

194 against Lpp (Fig. 3D). DpaA, but not inactive DpaA(C143A) was capable of releasing Lpp from

195 PG, confirming the results obtained with the soluble muropeptides with Lys-Arg dipeptides.

196 Altogether, our data prove that DpaA hydrolyses the bond between mDAP in PG and the C-

197 terminal Lys residue of Lpp, releasing Lpp from the PG.

198

199 DpaA has also CPase activity against glycine-containing muropeptides

200 When we further investigated the activity of DpaA. We found that it released LysArg

201 dipeptides from PG under acidic or neutral conditions (Fig. 4A). In the course of these

202 experiments we noticed that DpaA also acted on rare peptides in PG with glycine (instead of

203 D-Ala) at position 4, for example TetraGly4 (Peak 2) or its dimer version TetraTetraGly4 (Peak

204 5) (14). These structures were hydrolysed by DpaA particularly at acidic conditions via a

205 mDAP-Gly carboxypeptidase (CPase) activity.

206 We next tested if DpaA could remove other unusual amino acids present at the

207 terminus of a tetrapeptide. For this, we used LdtD to exchange the terminal D-alanine residue

208 by D-lysine, D-glutamine, D-valine or glycine in PG from BW25113∆6LDT and tested if these

209 can be removed by DpaA (Fig. S4C). As before, DpaA was active against muropeptides with

210 glycine but not against those with D-lysine, D-glutamine or D-valine. Together, these data

211 show that DpaA specifically removes Lys or Gly residues from position 4 of tetrapeptides, but

212 not D-alanine or any of the other D-amino acids tested.

213

214 DpaA is active in cells

215 Our biochemical experiments provided strong evidence that DpaA is able to release

216 Lpp from PG. To test if this reaction can happen in cells, we ectopically expressed plasmid-

217 borne dpaA or dpaA(C143A) in strains with deletions of the dpaA, lpp or ldtABC genes, and

218 analysed the PG composition of exponentially growing cells (Fig. 5A and 5B). Expression of

219 DpaA in BW25113∆dpaA resulted in a two-fold reduction of Tri-LysArg from 3.7 ± 0.1% to 1.6

220 ± 0.0%, compared to BW25113 expressing the empty plasmid (pGS100) (Fig. 5A and 5C).

221 BW25113∆dpaA expressing catalytically inactive DpaA(C143A) had an increased level of Tri-

222 LysArg compared to BW25113 pGS100, possibly due to the lack of DpaA activity. Tripeptides

223 were increased in all samples with decreased LysArg-muropeptides due to the expression of

224 active DpaA, for example, BW25113∆dpaA pDpaA contained 11.0 ± 0.3% tripeptides

225 compared to 4.9 ± 0.4% in BW25113 pGS100. This result suggests that cells counteract the

226 high DpaA activity by increasing the activities of LdtA-C to maintain a sufficient amount of PG-

227 attached Lpp.

228 The expression of DpaA had no significant effect on the PG composition in cells lacking

229 Lpp (BW25113∆lpp) or the Lpp-attaching enzymes (BW25113∆ldtA∆ldtB∆ldtC) which, as

230 expected, did not show detectable level of Tri-LysArg and therefore lack the substrate of DpaA

231 (Fig. 5A). To test if LdtD or LdtE affect the activity of DpaA, we also analysed the PG

232 composition of BW25113∆ldtD∆ldtE∆dpaA cells containing pDpaA, pDpaA(C143A) or

233 pGS100 (Fig. 5B). As expected, the PG of these cells did not contain 3-3 cross-links. The

234 level of Tri-LysArg was higher in strains lacking ldtD and ldtE, presumably because LdtA-C

235 compete with LdtD-E for tetrapeptide donors in PG. However, the expression of active DpaA

236 correlated with a reduction in Tri-LysArg and an increase in tripeptides in PG from BW25113

237 and BW25113∆ldtD∆ldtE∆dpaA, showing that the absence of LdtD and LdtE does not affect

238 DpaA. Altogether these data verify that DpaA detaches Lpp from PG in cells.

239

240 DpaA contributes to mecillinam resistance

241 Our previous work showed that DpaA is important in cells that experience severe OM

242 assembly stress. Here we wanted to further explore conditions at which DpaA becomes

243 important and searched a chemical genomics database for conditions that decreased the

244 fitness of the dpaA mutant strain (34). The dpaA mutant had negative fitness scores with the

245 inhibitor of LPS synthesis, CHIR-090, and the elongasome inhibitors A22 and mecillinam

246 which both cause cells to become spherical (35, 36). We hypothesised that the tight

247 connection between the OM and PG via PG-attached Lpp could complicate changes in cell

248 shape such as the transition of rod-shaped to spherical shape. To test this hypothesis, we

249 measured the sensitivity of strains towards mecillinam. When comparing the growth of

250 BW25113, BW25113∆dpaA, BW25113∆lpp, and BW25113∆dpaA∆lpp on LB agar plates

251 supplemented with mecillinam we observed a higher susceptibility of the ∆dpaA mutant (Fig.

252 6A). We also grew these strains in liquid LB medium in the presence or absence of 1 µg/ml

253 mecillinam. Cells of BW25113∆lpp still grew 8.5 h post addition of mecillinam, while

254 BW25113∆dpaA∆lpp cells arrested growth, and the optical density of BW25113 and

255 BW25113∆dpaA cultures declined (Fig. 6B). Expressing ftsQAZ from pTB63 decreased the

256 susceptibility to mecillinam as described (37), but the difference between the strains persisted.

257 BW25113∆dpaA pTB63 was more susceptible to mecillinam than the other strains and

258 expressing pDpaA form plasmid restored mecillinam resistance (Fig. S6B). This effect was

259 specific to mecillinam and the ∆dpaA strain showed similar susceptibility to tetracycline and

260 ampicillin, which do not cause cells to become spherical (Fig. S6A). Hence, detachment of

261 Lpp from PG appears to be beneficial for cells that undergo radical cell shape changes.

262

263 DpaA is present in many bacteria that lack Lpp

264 We next asked if DpaA co-occurs with Lpp in diderm bacteria. We analysed the

265 presence of proteins of the YkuD family (PF03734 in PFAM), Lpp-like proteins (PF04728) and

266 DpaA-like proteins within the AnnoTree database, which contains a set of representative, fully-

267 sequenced and consistently-annotated bacterial genomes (38). YkuD-like proteins are present

268 in 72% of the genomes, often with several copies (61,365 sequences in 16,986 genomes).

269 Lpp-like proteins are only present within a subset of -proteobacteria (~3% of all genomes)

270 (Fig. 7A). To identify DpaA-like proteins, we searched within the 61,365 YkuD-family

271 sequences in AnnoTree using BLAST and the E. coli DpaA sequence as query, resulting in

272 2451 hits from 2310 different genomes (Fig. 7A). The identified sequences are present in a

273 wide range of diderm bacteria lacking Lpp, including species within -, - and -proteobacteria,

274 Acidobacteria and Bacteriodetes (Fig. 7A). About a third of the genomes encoding Lpp did

275 not contain a DpaA-like protein, suggesting that those organisms are not capable of detaching

276 Lpp from PG or other proteins have this function (Fig. 7A). Alignment of the sequences of

277 DpaA-like proteins identified within AnnoTree revealed the catalytic cysteine and several

278 conserved positions common to all YkuD-like proteins (Fig. 7B). As in E. coli DpaA (Fig. 1),

279 most DpaA-like proteins have alanine or asparagine 2 positions after the catalytic cysteine

280 instead of an Arg that is present in LDTs with TPase activity.

281 Overall, our sequence analysis suggests that species without Lpp use DpaA for a

282 different purpose. Some of the DpaA homologues might function as LD-carboxypeptidase,

283 consistent with their classification in the MEROPS peptidase database (39) within the C82.A01

284 peptidase subfamily along with the LD-carboxypeptidases Csd6 from Helicobacter pylori and

285 Pgp2 from Campylobacter jejuni (40, 41). Alternatively, or in addition, DpaA could detach

286 different substrates from PG, for example OMPs in - and -proteobacteria (24, 25). Indeed,

287 a search within the genomes of - and -proteobacteria with PG-attached OMPs identified

288 genes encoding DpaA-like proteins in all of them (Fig. 7C).

289

290 DISCUSSION

291 DpaA is the first member of the YkuD family that does not act on LD-peptide bonds

292 and is not a transpeptidase. Our work demonstrated that DpaA hydrolyses the amide bond

293 between the L-centre of mDAP in PG and the -amino group of Lpp (Fig. 8). Hence, the name

294 LdtF (LD-transpeptidase F) was not appropriate anymore and we propose to name the

295 enzyme as Peptidoglycan mDap-protein amidase A (DpaA).

296 Lpp is the most abundant protein in E. coli and its attachment to PG substantially

297 contributes to the stability of the cell envelope and prevents excessive OM vesiculation (5, 6,

298 9, 17). Why does the cell then have an enzyme that detaches Lpp from PG? We do not yet

299 fully understand the importance of Lpp-detachment at different conditions. Our initial data

300 indicate that DpaA might be required under certain stress conditions. The dpaA mutant has

301 reduced fitness when cells grow in the presence of mecillinam, an antibiotic that induces a

302 stark change in cell shape (from rod-shape to spherical shape) in growing cells (35). We

303 hypothesize that under these conditions the detachment of Lpp from PG provides flexibility to

304 the cell envelope to better coordinate the remodelling of the PG and OM during the shape

305 change. The detachment of Lpp from PG could also be a means of controlling the amount of

306 OM-derived vesicles during an infection, affecting the interaction of a diderm bacterium with

307 the host organisms (42). In addition, the amount of PG-bound Lpp could affect bacterial cells

308 within a biofilm and the outcome of bacterial competition (42b). However, it remains to be

309 studied in more detail if DpaA affects virulence and bacterial fitness under environmental

310 conditions or cell envelope stress.

311 DpaA becomes essential when the export of LPS to the outer membrane is

312 compromised (29), presumably due to the spurious activation of peptidoglycan amidases via

313 ActS (Gurnani et al., under review). Our TraDIS data revealed another connection between

314 LPS and DpaA. The lapAB genes were interrupted by the transposon at much higher

315 frequency in the dpaA mutant than the wild-type. Deleting lapB stabilizes LpxC and

316 deregulates (enhances) LPS biosynthesis (43, 44), which causes lethality in wild-type cells

317 but not in a hypervesiculating lpp mutant, which presumably releases the excess of LPS into

318 outer membrane derived vesicles (30). The fact that transposon insertions in lapAB are

319 recovered at a higher frequency in the dpaA mutant when compared to the parent strain is

320 consistent with the lower fitness of the dpaA mutant in the presence of CHIR-090 (34), an

321 inhibitor of LpxC, and suggests that enhanced PG-bound Lpp (due to the absence of DpaA)

322 has a negative effect on LPS synthesis and/or export. We hypothesize that the higher amount

323 of PG-bound Lpp in the dpaA mutant hinders (but not completely abolishes) LPS export, which

324 alleviates problems caused by excessive LPS biosynthesis. This effect of the increased

325 amount of PG-attached Lpp points to a yet unrecognized regulatory function of Lpp in LPS

326 export.

327 Structure-based sequence alignment shows that LD-carboxypeptidases and DpaA-like

328 proteins are closely related and that both lack the conserved arginine residue present in LD-

329 TPases (Fig. S7). However, some residues conserved in DpaA-like proteins at the start of the

330 catalytic domain are absent in the LD-carboxypeptidases (Fig. S7), and a BLAST search with

331 E. coli DpaA failed to identify Csd6 while Pgp2 was identified with a low expect value (1e-4)

332 (40, 41). Consistent with this low similarity, E. coli DpaA was not able to remove terminal D-

333 alanine residues from PG stem peptides, however, more experiments will be necessary to

334 determine whether some DpaA-like proteins show LD-carboxypeptidase activity.

335 Depending on the growth medium, E. coli naturally contains a small proportion of stem

336 peptides with glycine residues at position 4 (14). Other bacteria use LDTs to incorporate non-

337 canonical D-amino acids produced by themselves or present in the environment into the same

338 position (45, 46). Of the 4 amino acids tested, DpaA was only capable of removing glycine

339 residues from position 4, but not the three D-amino acids tested. However, at present we

340 cannot exclude the possibility that some bacteria employ DpaA-like enzymes to remove non-

341 canonical D-amino acids, which are toxic when incorporated at a high amount (47). The recent

342 discovery of PG-attached OMPs in certain -proteobacteria suggests that the DpaA-like

343 enzymes in these species function to detach OMPs from PG (24, 25). In support of this

344 hypothesis, the attachment of OMPs to PG occurs via N-terminal glycine or alanine, and the

345 attachment of OM-lipoprotein LimB via an internal lysine residue, producing the very same

346 amide bonds as in TetraGly4 or in PG-bound Lpp in E. coli. Moreover, DpaA homologues are

347 present in many bacteria, including many species that do not contain Lpp, suggesting different

348 substrates. Hence, it remains to be tested if DpaA-like enzymes are capable of removing

349 OMPs from PG.

350 In summary, our work discovered that DpaA performs a yet unknown reaction in the

351 bacterial cell envelope, the hydrolytic removal of a protein from PG. Presumably, DpaA

352 provides flexibility to the bacterial cell to remodel the cell envelope when coping with certain

353 stress situations. The discovery of this reaction also illustrates that cell envelope remodelling

354 contributes to the robustness of bacterial cells and their lifestyles.

355

356 MATERIALS AND METHODS

357 Bacterial strains and growth conditions

358 Strains used in this study are listed in Table S2. Bacteria were grown aerobically at

359 30°C or 37°C on LB-plates or in liquid LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L

360 NaCl; 15 g/L agar for plates). Antibiotics were used at the following concentrations:

361 chloramphenicol (Cam), 25 µg/mL; kanamycin (Kan), 50 µg/mL; tetracycline (Tet), 5 µg/mL.

362 For monitoring cell cultures treated with mecillinam, 30°C pre-warmed LB medium was

363 inoculated with overnight cultures starting with a normalised OD600nm of 0.05. The cultures

364 were split in half at an OD600nm of 0.1. One-half was supplemented with 1 µg/mL mecillinam,

365 the other half remained untreated. The cultures were diluted 20-fold in fresh 30°C pre-warmed

366 LB supplemented with and without mecillinam at an OD600nm of 0.5.

367

368 Construction of E. coli deletion strains

369 Deletion strains were obtained by transducing kan-marked alleles from the Keio E. coli

370 single-gene knockout library (48) by P1 phage (49). The kan cassette was removed by pCP20-

371 encoded Flp recombinase to generate deletions with a FRT-site scar sequence (50). The

372 removal of the kan gene was verified by polymerase chain reaction. Strains with multiple

373 deletions were generated by sequential P1 transduction and kan cassette removal.

374

375 Plasmid construction

376 Plasmids and oligonucleotides used in this study are shown in Tables S2 and S3,

377 respectively. Genes were amplified by PCR using DNA from MC1061 as template. pET28a-

378 ldtE was constructed by ligase-independent cloning (51). Site directed mutagenesis of

379 plasmids were performed using Q5® Site-Directed Mutagenesis Kit (New England BioLabs)

380 following manufacturer’s instructions.

381 For pET29b-DpaA-his construction the signal sequence of dpaA was substituted with

382 the one of pelB by a three-step PCR method (52) using pET27b as template for pelBss. The

383 chimeric pelBss-dpaA gene was cloned in pET29b between the NdeI and NcoI restriction sites.

384 The thrombin cleavable sequence was inserted in the linker region between the 3’ end of the

385 gene and the sequence codifying the His-tag using the DpaA_pET29b_R primer.

386

387 Spot plate assay

388 The spot plate assays were performed as described (33). Serial dilutions of bacterial

389 cultures were spotted on LB plates supplemented with the appropriate antibiotics (Tet, 5

390 µg/mL; Cam, 25 µg/mL). The plates were incubated at 30°C for 33 h and images were taken

391 with an InGenius Syngene Bio Imaging system.

392

393 Mecillinam susceptibility testing

394 Minimal inhibitory concentration (MIC) of mecillinam towards BW25113 and mutants

395 with single or multiple gene deletions were determined with MIC stripes (mecillinam) from

396 Liofilchem (Roseto degli Abruzzi, Italy). Growing cells from cultures with an OD600nm of 0.5

397 were harvested by centrifugation (13,000 × g, 5 min), washed three times with 0.9% NaCl,

398 normalised to an OD600nm of 0.125 and distributed onto LB-plates with cotton swabs. Plates

399 contained tetracyclin (5 µg/mL) when used for strains with pTB63. An MIC-mecillinam test

400 stripe was applied and the plates were incubated at 30°C or 37°C. Images were taken 24 h

401 post incubation with an InGenius Syngene Bio Imaging system.

402

403 PG isolation and analysis

404 PG was isolated from E. coli cells and analysed by reversed-phase HPLC as described

405 (14). PG for DpaA activity assays containing bound Lpp was isolated as described (14) with

406 the omission of treatments with α-amylase and pronase E.

407

408 Purification of LdtE

409 E. coli LOBSTR-BL21(DE3) cells (Kerafast) harbouring the pET28a-LdtE-his plasmid

410 were grown in 2 L of LB-autoinduction medium (LB supplemented with 0.5% glycerol, 0.05%

411 glucose, 0.2% Lactose, for 20 h at 30°C (53). Cells were harvested by centrifugation for 15

412 min at 5,000 × g at 4°C. The cell pellet was resuspended in 100 mL of buffer I (20 mM

413 HEPES/NaOH pH 7.5, 1 M NaCl, 1 mM DTT, 10% glycerol) supplemented with 1 mM

414 phenylmethyl sulfonyl fluoride (Sigma Aldrich), 1 x protease inhibitor cocktail (Sigma Aldrich)

415 and desoxyribonuclease I (Sigma Aldrich). Cells were broken by sonication and insoluble cell

416 debris was removed by ultracentrifugation for 1 h at 130,000 × g at 4°C. The supernatant was

417 applied to a 5 mL HisTrap HP column equilibrated with buffer I on an ÄKTA PrimePlus. The

418 column was washed with 20 mL of buffer I containing 40 mM imidazole and bound protein was

419 eluted with buffer II (20 mM HEPES/NaOH pH 7.5, 500 mM NaCl, 1 mM DTT, 10% glycerol,

420 400 mM imidazole). Fractions containing LdtE were combined and dialysed against 3 L of

421 buffer III (20 mM HEPES/NaOH pH 7.5, 300 mM NaCl, 10% glycerol, 0.1 mM TCEP). The

422 sample was concentrated to 5 mL with a Vivaspin 6 filter. The protein was then further purified

423 by size exclusion chromatography on a HiLoad 16/60 Superdex 200 (GE Healthcare) column

424 using buffer III and a flowrate of 1 mL/min. LdtE-containing fractions were combined and

425 stored at -80°C.

426

427 Purification of DpaA and DpaA(C143A)

428 E. coli LOBSTR-BL21(DE3) (Kerafast) containing pET29b-DpaA-his (encoding PelB-

429 DpaA(20-246)-His6) or pET29b-DpaA(C143A)-his (PelB-DpaA(20-246,C143A)-His6; cysteine-

430 143 replaced by alanine) were grown at 25°C overnight in 4 L of TB-autoinduction medium

431 (54). Cells were harvested by centrifugation for 15 min, 5,000 x g at 14°C. The cell pellet was

432 resuspended in 150 mL of buffer I (20 mM HEPES/NaOH pH 7.5, 500 mM NaCl, 2 mM MgCl2,

433 10% glycerol) supplemented with 1 mM phenylmethyl sulfonyl fluoride (Sigma Aldrich), 1 x

434 protease inhibitor cocktail (Sigma Aldrich) and desoxyribonuclease I (Sigma Aldrich). Cells

435 were broken by sonication and the insoluble fraction was removed by ultracentrifugation for 1

436 h at 130,000 × g at 4°C. The supernatant was applied to a 5 mL HisTrap HP column pre-

437 equilibrated with buffer I using an ÄKTA PrimePlus. The column was washed with 20 mL buffer

438 I with 40 mM imidazole and protein was eluted in a 12-mL gradient of from buffer I with 40 mM

439 imidazole to buffer I with 400 mM imidazole. Fractions containing DpaA were combined and

440 split into two pools. Pool 1 was dialysed against 3 L of buffer III (20 mM HEPES/NaOH pH 7.5,

441 300 mM NaCl, 10% glycerol, 10 mM EDTA), pool 2 was incubated with 10 units of thrombin

442 (restriction grade, Novagen) to remove the His6-tag, during dialysis against 3 L buffer III.

443 Proteins were further purified by size exclusion chromatography on a HiLoad 16/60 Superdex

444 200 (GE Healthcare) column using buffer III and a flowrate of 1 mL/min. The protein-containing

445 fractions were aliquoted and stored at -80°C.

446

447 Purification of LdtD

448 LdtD was expressed in E. coli LOBSTR-BL21(DE3) cells harbouring the

449 overexpression plasmid pETMM82 (55) and purified as previously described (32).

450

451 HPLC activity assay

452 DpaA activity assays were carried out in a total volume of 50 µL in 20 mM

453 HEPES/NaOH pH 7.5 or 20 mM Na-acetate (pH 5.0), with 100 mM NaCl, 0.05% Triton X-100

454 (reduced), 0.1 mM TCEP, 10 µL substrate (muropeptides or PG), and 2 µM of protein. A

455 control sample contained no protein. Purified non-reduced and desalted muropeptides (Tri-

456 LysArg and TetraTri-LysArg) were a gift from J.-V. Höltje, MPI Tübingen, Germany). The

457 reaction mixture was incubated overnight in a thermoshaker at 37°C and 900 rpm. The

458 reaction was stopped by boiling the samples for 10 min at 100°C. Samples were reduced with

459 sodium borohydride and muropeptides were separated by reversed-phase HPLC (14), except

460 that the gradient was 90 min instead of 135 min for samples with purified muropeptides. For

461 activity assays on PG, the reaction products were treated overnight with the muramidase

462 cellosyl (0.5 µg/mL) at 37°C and 900 rpm in 80 mM sodium phosphate pH 4.8, and the

463 released muropeptides were reduced and separated by HPLC as described above. Tri-

464 LysArg, TetraTri-LysArg and the products generated by DpaA from these muropeptides were

465 collected during the HPLC run and analysed by MS/MS as described (56).

466

467 Amino acid exchange followed by DpaA reaction

468 The amino acid exchange reaction with glycine, D-valine, D-glutamine or D-lysine was

469 performed in a final volume of 50 µL in 20 mM HEPES/NaOH pH 7.5; 100 mM NaCl; 0.1 mM

470 TCEP; 10 mM MgCl2; 0.05% Triton X-100. Ten µL of muropeptides obtained from PG of

471 BW25113∆6LDT (~50 µg) were incubated with glycine, D-valine, D-glutamine or D-lysine (10

472 mM) and LdtD (5 µM) for 6 h at 37°C. The reaction was terminated by boiling the sample for

473 10 min. The sample was centrifuged and the supernatant containing the soluble muropeptides

474 were split into two equal aliquots. In one half, the pH was adjusted to 5.0 with Na-acetate,

475 DpaA (5 µM) was added and the sample was incubated overnight at 37°C. The other half was

476 treated the same way without the addition of DpaA. Samples were boiled for 10 min at 100°C,

477 treated with sodium borohydride and muropeptides were separated by HPLC as described

478 above.

479

480 DpaA activity assay against PG-Lpp

481 DpaA or DpaA(C143A) (2 µM) was mixed with 10 µL of PG with attached Lpp from

482 BW25113∆dpaA in 20 mM HEPES/NaOH pH 7.5, 100 mM NaCl, 0.05% Triton X-100

483 (reduced) and 0.1 mM TCEP. Samples were incubated overnight in a thermoshaker with 900

484 rpm at 37°C. Soluble proteins were separated from PG by centrifugation (13,000 × g, 10 min,

485 4°C). The supernatant was collected, proteins were separated by SDS-PAGE and stained with

486 Coomassie Brilliant Blue, or transferred to nitrocellulose for Western blotting and detection

487 with an anti-Lpp antibody (kind gift from Jean-François Collet, UC Louvain, Brussels, Belgium).

488

489 Analysis of DpaA conservation

490 We used the AnnoTree database to study the conservation of DpaA-like proteins within

491 bacteria (38). This database contains a set of archaeal and bacterial genomes which are

492 completely sequenced and consistently annotated. The sequences of proteins identified as

493 belonging to PFAM families PF03734 (LD-transpepetidase catalytic domain, 61,365

494 sequences in 16,986 genomes) and PF04728 (Lipoprotein leucine-zipper or Lpp, 717

495 sequences in 694 genomes) were downloaded from the AnnoTree website. We searched with

496 the PF03734 sequences for DpaA-like proteins using BLAST (57) and E. coli DpaA as query

497 with an e-value cut-off of 1e-8. This search resulted in 2451 hits from 2310 genomes. The

498 number of proteins of each type per genome were counted using custom python scripts and

499 the generated datasets were represented along a phylogenetic tree of all genomes in

500 AnnoTree using iTOL (58). The sequences for all identified DpaA-like proteins were aligned

501 using Clustal Omega (59). The resulting alignment was uploaded to the Skyling website to

502 generate a logogram by converting to a hidden Markov model after removing mostly empty

503 columns in the alignment (60). Next, the matrix with position frequencies was downloaded and

504 the final logogram was generated using the logomaker python package (61). The search for

505 DpaA-like genes in organisms attaching β-barrel outer membrane proteins to PG was

506 conducted using BLAST and the genomes analysed were: Agrobacterium fabrum str. C58

507 (ASM9202v1), Brucella abortus 2308 (ASM74219v1), Coxiella burnetii RSA 493 (ASM776v2),

508 and Legionella pneumophila subsp. pneumophila str. Philadelphia 1 (ASM848v1).

509

510 TraDIS method

511 Transposon mutant libraries were constructed by electrotransformation of the mini-Tn5

512 transposon carrying a kanamycin resistance cassette (Epibio). Two technical replicates of

513 each library were prepared for sequencing as described previously (62). Samples were

514 sequenced using an Illumina MiSeq with 150 cycle v3 cartridges. Raw data are available at

515 the European Nucleotide Archive (ENA) accession XXXX. Data were demultiplexed using the

516 Fastx barcode splitter, to remove the barcode unique to each sample (63). The transposon

517 was matched and trimmed allowing for 4 bp mismatch, and surviving reads were mapped to

518 the BW25113 reference genome using bwa mem (accession CP009273.1). 2,877,112 and

519 4,664,621 reads were mapped for BW25113 and BW25113ΔdpaA, respectively. Data are

520 available for viewing at the TraDIS-vault browser: https://tradis-vault.qfab.org/. The BioTraDIS

521 analysis package (version 1.4.5) was used to calculate the log-fold-change in read depth

522 between each gene in the control and dpaA transposon libraries, using a threshold of >2-fold

523 change and a Q-value < 0.01 (64).

524

525 ACKNOWLEDGEMENTS

526 We thank Jean-François Collet (Université catholique de Louvain) for the gift of anti-Lpp

527 antibodies.

528

529 FUNDING

530 A.P., W.V. and I.R.H received support from the European Commission via the International

531 Training Network Train2Target (721484). WV was also supported by BBSRC (BB/R017409/1).

532 533 FIGURE LEGENDS 534 Figure 1. Overview of the different LDT family members. E. coli has six proteins with a

535 YkuD (LDT) domain (top left). Protein regions: S, signal peptide (S); LysM and PGB,

536 peptidoglycan-binding domains. The numbers indicate amino acid positions. Sequence

537 alignment within the YkuD domain (bottom left) shows the active site cysteine (C, in red) and

538 other conserved amino acid residues in grey. DpaA (LdtF) misses a conserved arginine (R)

539 and its function was unknown. Right side: LdtA, LdtB and LdtC attach Lpp to mDAP in PG

540 stem peptides. LdtD and presumably LdtE form 3-3 cross-links in PG.

541

542 Figure 2. Comparison of the read depth between genes in each transposon library. (A)

543 Volcano plot showing a comparison of the relative mutant abundance between libraries.

544 Genes with a >2-fold change in read abundance and with a Q-value > 0.01 are coloured; these

545 thresholds are represented as solid red lines. (B,C) The insertion profile of dpaA (B) and the

546 lapAB operon (C) for both libraries, with transposon insertion sites identified in the control

547 library displayed above the gene track (black) and transposon insertion events in the dpaA

548 library shown below the gene track (blue). The insertion plot is capped at a read frequency of

549 1, scale bar = 100 bp.

550

551 Figure 3. DpaA detaches Lpp from PG. (A) Activity against the muropeptides Tri-LysArg

552 and TetraTri-LysArg. Muropeptides were incubated with DpaA and the reaction products

553 reduced with sodium borohydride and separated by HPLC. DpaA hydrolysed both substrates,

554 releasing the muropeptides lacking L-Arg-L-Lys. The released dipeptide is missing in the

555 chromatogram because it presumably co-elutes early with the salts at ~8 min. Fig. S3 shows

556 the MS data proving the identities of the muropeptide substrates used here, and of the

557 products. (B) Scheme of the reactions observed in A. Data in Fig. S2E show that LdtD and

558 LdtE are not active against Tri-LysArg. Fig. S4A,B shows the inhibition of DpaA by copper. (C,

559 D) Lpp release assay. DpaA was incubated with PG sacculi from BW25113∆dpaA that were

560 not treated with pronase E and hence contained PG-attached Lpp. Reaction mixtures were

561 boiled and proteins were separated by SDS-PAGE, following by staining with Coomassie-blue

562 (C) or Western Blotting and detection of Lpp with specific antiserum (D). DpaA, but not the

563 catalytically inactive DpaA(C143A), released Lpp from PG. M, protein size marker.

564

565 Figure 4. DpaA is active against TetraGly4 and TetraTetraGly4. (A) PG from strain

566 araBplptC ∆dpaA (+arabinose) was incubated with DpaA at pH 5.0 or 7.5, followed by

567 digestion with cellosyl and analysis of the muropeptide composition by HPLC. In addition to

568 its expected decrease of Tri-LysArg, DpaA was also active against muropeptides containing

569 glycine at position 4 (muropeptides 2 and 5). DpaA showed higher activity at acidic pH.

570 Chromatographs were cropped at 250 mAU to better observe the minor muropeptides. (B)

571 Structures of the relevant muropeptide labelled in A. Fig. S4C shows that DpaA is not active

572 against muropeptides with non-canonical D-amino acids at position 4 of the PG stem peptide.

573

574 Figure 5. DpaA is active in cells. (A) Wild-type (BW25113) or mutants with single or multiple

575 gene deletions (BW25113∆dpaA, BW25113∆lpp, BW25113∆ldtA∆ldtB∆ldtC) expressing

576 DpaA or DpaA(C143A) from a plasmid, or carrying the empty plasmid, were grown in LB

577 medium, harvested, and the muropeptide composition was determined. Only expression of

578 active DpaA, but not the inactive DpaA(C143A), reduced the Tri-LysArg peak when present.

579 (B) Muropeptide profiles of BW25113∆ldtD∆ldtE∆dpaA (lacking LDTs involved with 3-3 cross-

580 link formation and DpaA) expressing DpaA, DpaA(C143A) or no DpaA version.

581 Chromatographs were cropped above 250 mAU, the uncropped HPLC chromatographs of A

582 and B are shown in Fig. S5. (C) Quantification of the total tripeptides and Tri-LysArg in the

583 chromatograms shown in A and B. The values are mean ± variation in from two independent

584 biological replicates, except BW25113∆ldtA∆ldtB∆ldtC pDpaA, which was prepared once.

585 Significance was measured by a two-tailed, homoscedastic T-test. ns (not significant), p >

586 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

587

588 Figure 6. The dpaA mutant is more susceptible to mecillinam. (A) Serial dilutions of

589 BW25113 and mutants (∆dpaA, ∆lpp or ∆dpaA∆lpp) were spotted on LB plates with or without

590 mecillinam. Images were taken after incubation at 30°C for 33 h. (B) Growth of the same

591 strains in liquid medium in the absence of mecillinam or upon addition of 1 µg/ml mecillinam

592 (arrow) followed by measuring the optical density at 600 nm. Cultures were diluted 20-fold

593 when the OD600nm reached 0.5. Figure S6 shows that the effect is specific to mecillinam.

594

595 Figure 7. Conservation and distribution of DpaA. (A) Distribution of genes encoding

596 proteins of the YkuD family (PFAM PF03734, red), Lpp (PF04728, yellow) and DpaA-like

597 proteins (identified by BLAST within PF03734 proteins, blue) within the AnnoTree bacterial

598 genome database. The intensity of the colour corresponds to the number of genes identified

599 in each genome. The pie charts in the right bottom corner show the percentage of genomes

600 in the database encoding proteins in the PF03734 family, DpaA-like proteins or none of these

601 (left), and the percentage of PF04728-containing genomes also containing DpaA-like proteins.

602 (B) Logogram obtained from the multiple sequence alignment of the DpaA-like proteins

603 identified within the AnnoTree database. Top: full alignment; bottom: amino acid positions 65

604 to 140, highlighting same positions as in the alignment in Fig. 1A. (C) Table showing genes

605 encoding DpaA-like proteins identified in bacteria lacking lpp and known to attach OM β-barrel

606 proteins to PG. Proteins, NCBI protein annotation corresponding to the indicated gene; E-

607 Value, BLAST expect value indicating the similarity to the query sequence, E. coli DpaA.

608

609 Figure 8. Specificity of DpaA. DpaA hydrolyses the amide bond between the L-centre of

610 mDAP and Gly (middle) or the L-centre of mDAP and the -amino group of the C-terminal L-

611 lysine of Lpp. Both substrates have a CH2 group adjacent to the cleavage site (labelled with

612 *).

613 Supplemental material legends

614 Figure S1. Construction of transposon libraries. Genome maps representing the

615 transposon mutant libraries constructed in BW25113 (A) and BW25113∆dpaA (B). The sense

616 and anti-sense coding sequences are displayed in the two outermost tracks, respectively, in

617 pale blue. The position and frequency of transposon insertion events around the BW25113

618 reference genome are represented by the peaks in the innermost track; plotted using

619 DNAPlotter with a window size of 1 and a step size of 1.

620

621 Figure S2. Distinct activities of LdtD/LdtE and DpaA. PG from BW25113∆6LDT was

622 incubated with LdtD, LdtE or DpaA at pH 5.0 (A) or pH 7.5 (B). The PG was then digested

623 with cellosyl and the resulting muropeptides were separated by HPLC. The LD-TPase product

624 TetraTri(3-3) was present in reactions with LdtD or LdtE, but not DpaA or the control without

625 enzyme. (C) Structures of the relevant muropeptides. (D) Tri-LysArg incubated with DpaA,

626 DpaA(C143A) and (E) LdtD or LdtE. Muropeptides were reduced with sodium borohydride and

627 separated by HPLC. G, N-acetylglucosamine; M(r), N-acetylmuramitol; L-Ala, L-alanine; D-Glu,

628 D-glutamic acid; D-Ala, D-alanine; m-DAP, meso-diaminopimelic acid.

629

630 Figure S3. Mass spectrometry spectra of the substrates and products in DpaA

631 reactions. Muropeptides (Tri-LysArg, Tri, TetraTri-LysArg, TetraTri) were manually collected

632 during the HPLC runs (Fig. 3) and analysed by mass spectrometry (Bui et al., 2009). Spectra

633 on the top show the unfragmented ions, the bottom MS/MS spectra were obtained after

634 fragmentation. Middle spectra show zoomed regions of the relevant [M+nH]n+ ions to

635 determine the charges. MS spectra confirmed the identities of the muropeptides analysed.

636 Reference:

637 Bui NK, Gray J, Schwarz H, Schumann P, Blanot D, Vollmer W. 2009. The peptidoglycan

638 sacculus of Myxococcus xanthus has unusual structural features and is degraded

639 during glycerol-induced myxospore development. J Bacteriol 191:494-505.

640

641 Figure S4. DpaA is inhibited by copper and does not release D-Lys, D-Val or D-Gln from

642 PG. (A) PG from BW25113∆dpaA was incubated with DpaA in the presence of increasing

643 concentration of CuCl2. Muropeptides were generated, reduced and separated by HPLC.

644 DpaA activity is seen as the reduction in the Tri-LysArg peak which decreased by copper in a

645 concentration-dependent manner. Chromatographs were cropped above 250 mAU to better

646 observe Tri-LysArg. (B) Quantification of the relative amount of Tri-LysArg and Tri plotted

647 against the concentration of CuCl2. The data are mean ± variation of two independent

648 experiments. (C) PG from BW25113∆6LDT was incubated with LdtD and different D-amino

649 acids, which were incorporated into position 4 of the stem peptides. A star (*) labels

650 muropeptides with non-canonical D-amino acids. The PG was incubated with DpaA, followed

651 by generation of muropeptides, reduction and HPLC analysis. DpaA released glycine from

652 PG, but not the D-amino acids tested.

653

654 Figure S5. DpaA is active in cells. Full HPLC chromatograms of the cropped versions shown

655 in Figure 5.

656

657 Figure S6. Growth defect of the dpaA mutant is specific to mecillinam. (A) Spot-plate

658 assays with tetracycline (TET) or ampicillin (AMP) showed no altered sensitivity of the dpaA

659 mutant. (B) Spot-plate assay of strains carrying the pTB63 plasmid, expressing ftsQAZ alone

660 or in combination with empty plasmid (pGS100, ep) or plasmids expressing DpaA, in the

661 presence or absence of mecillinam. Cells overexpressing ftsQAZ were more susceptible to

662 mecillinan when they lacked dpaA.

663

664 Figure S7. Sequence alignment of DpaA-like proteins and members of the YkuD family.

665 Structure-based sequence alignment of DpaA-like proteins, LD-carboxypeptidases and LD-

666 transpeptidases obtained with PROMALS3D (Pei et al., 2008), using the structures of E. coli

667 LdtD (PDB accession: 6NTW), Bacillus subtilis YkuD (1Y7M) and Helicobacter pylori Csd6

668 (4XZZ) as guides. The residues corresponding to the 4 structural loops surrounding the

669 substrate-pocket in Csd6 are highlighted in different colors. The Red dots indicate the catalytic

670 triad in Csd6, purple dots mark positions conserved in DpaA-like proteins but different in LD-

671 carboxypeptidases and the blue dot indicates the conserved Arg in LD-TPases but not in

672 hydrolytic YkuD-family proteins. The sequences aligned include E. coli DpaA (Uniprot

673 P0AA99, residues 45-159), Agrobacterium fabrum ATU3332 (Q7CS68, 43-168) and ATU3631

674 (A0A3G2CVN4, 58-176), Brucella abortus BAB1_2034 (C4ITM3, 50-169), Coxiella burnetii

675 CBU_1157 (Q83CG1, 67-183), Legionella pneumophila LPG1514 (Q83XL0, 72-186),

676 Pseudomonas aeruginosa PA3756 (Q9HXN9, 10-166), Helicobacter pylori Csd6 (O25255, 73-

677 181) and Campylobacter jejuni Pgp2 (A7H3E2, 66-196), E. coli LdtD (P22525,377-543) and

678 Bacillus subtilis YkuD (O34816, 48-164).

679 Reference:

680 Pei J, Kim B-H, Grishin NV. 2008. PROMALS3D: a tool for multiple protein sequence and

681 structure alignments. Nucleic Acids Research 36:2295-2300.

682

683 Table S1. TraDIS raw data.

684 Table S2. Strains and plasmids used in this work.

685 Table S3. Oligonucleotides used in this study.

686

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897

Figure 1

1 21 96 230 310 LdtA S YkuD 1 24 101 231 306 LdtB S YkuD Lpp 1 23 47 91 103 240 320 D-Ala LdtC S LysM YkuD R 1 30 268 312 377 543 615 R K LdtD S PGB YkuD K NH2 1 23 40 84 97 232 334 LdtE S LysM YkuD 1 19 45 159 246 DpaA (LdtF) PGB S YkuD Lpp attachment by LdtA, LdtB, LdtC LdtA(189-216) AIHGTNAN.F GIGLR.VSQG CIRLRNDDIK D-Ala LdtB(192-219) AIHGTNAN.F GIGLR.VSHG CVRLRNEDIK LdtC(198-226) LLHGTNAD.F GIGMR.VSSG CIRLRDDDIK NH2 m-DAP LdtD(508-537) LHDTPNHNLF KRDTRALSSG CVRVNKASDL D-Glu LdtE(201-218) LIHGTSAP.D SVGLR.VSSG CIRMNAPDIK L-Ala DpaA(123-152) RAHGYEGKYL MIHGDCVSIG CYAMTNQGID Cons l.hg.n...f .i..r.vSsG C.r.....i. LD-(3-3) cross linkage by LdtD and LdtE bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this 1000 preprint (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 available under aCC-BY 4.0 International license.

750 Figure 2

A 1000 B •ColourdpaA enriched value a dpaA − dpaA enriched 500 • dpaA sick • anulldpaA sick a null Log2 Q 750

− 100 bp dpaA C

Colour 250 value - dpaA enrichedlapA lapB 500 dpaA sick null Log2 Q

- lapB dpaA 100 bp

0 −10 −5 250 0 5 10 Log2 Fold−change, dpaA − WT lapB

0 -10 -5 0 5 10 Log2 Fold-change, dpaA - WT bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure 3

A B C

Tri-LysArg dpaA Tri-LysArg

R Tri kDa M - K DpaA R + 180 K 130 70 50 Trii 55 40 DpaA TetraTri-LysArg 35 25 DpaA R TetraTri TetraTri-LysArgi K 15 DpaA R + 10 K Lpp

TetraTrii Coomassie-blue stain D

Absorbance at 205 nm (A.U.) DpaA R GM(r) L-Arg-L-Lys 10 K L-Ala Lpp 0 20 40 60 D-Ala D-Glu m-DAP Time (min) anti-Lpp Figure 4 bioRxiv preprint preprint (whichwasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplayin A Absorbance at 205 nm (A.U.) 200 0 doi: https://doi.org/10.1101/2021.02.21.432140 20 1 perpetuity. Itismadeavailableundera 2 Time (min) Time 3 4 40 5 6 pH 7.5 pH 5.0 pH PG ; this versionpostedFebruary21,2021. + DpaA B TetraTetraGly4 TetraTetraGly4 CC-BY 4.0Internationallicense Tri 1 5 TetraGly4 2 TetraTri 6 Tri - LysArg 3 K R . TetraTri(3 K R The copyrightholderforthis GM(r) L-Ala Gly D-Glu m D-Ala L-Arg-L-Lys -DAP 4 - 3) 3) bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure 5

A C B TetraTri TetraTrii ldtDldtEdpaA TetraTri i Trii (3-3)i Trii + DpaA

dpaA + DpaA Tri- ldtDldtEdpaA LysArgi + DpaA(C143A)

Tri- LysArgi dpaA ldtDldtEdpaA + DpaA(C143A) + empty plasmid Absorbance at 205 nm (A.U.)

BW25113 0 20 40 60 80 100 120 + empty plasmid C Time (min) C ** ns ** *** 12 ** ** 10 8 all lpp 6 (%) 4 tripeptides Absorbance at 205 nm (A.U.) + DpaA 2 0 12 BW pGS100* ns 10 8 ** ** ** ** ldtAldtBldtC Muropeptides 6 Tri-LysArg + DpaA 4 140.00min2 0 BW pGS100 pLdtF* pLdtF pLdtF 0 20 40 60 80 100 120 Time (min) bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure 6

A B LB BW MEC (1 µg/mL) 6 ∆dpaA LB ∆lpp 1 ∆dpaA∆lpp MEC (1 µg/mL) 600nm BW OD 0. 1 + MEC ∆dpaA wt ∆dpaA∆lpp ∆lpp • • ∆dpaA ∆lpp ∆dpaA∆lpp 0. 01 • • 0 150 300 450 600 750 100 7 Serial dilutions 10 Time (min) bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure 7

A YkuD DpaA- Lpp (PF03734) like (PF04728) 1 1 1

Number of hits

23 4 3

All genomes

YkuD

No YkuD DpaA

Genomes with Lpp

DpaA

no DpaA

2.5

0 25 50 75 100 125 150 175 200 4

2 Information content (bits) 0 70 80 90 100 110 120 130 140 position C Species Gene Protein -Value

Agrobacterium fabrum atu3332 AAK90059.2 3e-57

atu3631 AAK89766.2 2e-59

Brucella abortus BAB1_2034 WP_002965097.1 7e-63 Coxiella burnetii cbu_1157 NP_820154.1 8e-15

Legionella pneumophila lpg1514 AAU27596.1 2e-16 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure 8

OM Lpp

D-AA Gly L-Lys R NH OH * OH * OH mDAP HN D mDAP HN mDAP HN L H2N O H2N O H2N O D L O D L O D L O NH NH NH HO O HO O HO O D-Glu D-Glu D-Glu L-Ala L-Ala L-Ala PG GM GM GM bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure S1 A B 0 Mb 0 Mb

4 Mb 4 Mb

1 Mb 1 Mb BW25113 BW25113 dpaA

3 Mb 3 Mb

2 Mb 2 Mb bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure S2

A B C pH 5.0 pH 7.5 TetraTri(3-3) TetraTetra TetraTetra TetraTri TetraTri(3-3) TriTri(3-3) Tri Tetra Tetra D-Ala TriTri(3-3) m-DAP D-Glu Tetra Tri Tri LdtD L-Ala

200 LdtD

200 GM(r)

TriTri(3-3) TetraTri(3-3)

LdtE

DpaA TetraTri TetraTetra Absorbance at 205 nm (A.U.) Absorbance at 205 nm (A.U.)

DpaA Control

0 20 40 60 0 20 40 60 Time (min) Time (min)

D E

Tri-LysArg 50 40 Tri-LysArg Control

Control

Tri DpaA

LdtD

DpaA(C143A) LdtE Absorbance at 205 nm (A.U.) Absorbance at 205 nm (A.U.)

0 20 40 60 80 0 20 40 60 Time (min) Time (min) bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure S3.1

Tri-LysArg

Tri bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure S3.2

TetraTri-LysArg

TetraTri bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure S4

A C Tri-LysArg TetraTri(3-3) TetraTetra CuCl2 conc.

Control Tetra - 200 LdtD 0.2 mM DpaA

0.15 mM * * - 200 LdtD 0.1 mM D-Lys

DpaA 0.05 mM

Absorbance at 205 nm (A.U.) * Tri * 0 mM - LdtD

0 20 40 60 D-Gln Time (min) B DpaA 100 Absorbance at 205 nm (A.U.) * * * - 80 LdtD (%)

D-Val 60 Tri-LysArg IC50 = 0.1 mM DpaA

40 Tri TetraGly4 * - LdtD Relative muropeptide 20 Gly Tri DpaA 0 0 0.05 0.1 0.15 0.2 0 10 20 30 40

Concentration of CuCl2 (mM) Time (min) bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure S5

A B

dpaA + DpaA ldtDldtEdpaA

+ DpaA

dpaA 200 + DpaA(C143A) ldtDldtEdpaA + DpaA(C143A) 200

BW25113 + empty plasmid

ldtDldtEdpaA

Absorbance at 205 nm (A.U.) + empty plasmid

Absorbance at 205 nm (A.U.) lpp 0 20 40 60 80 100 120 140 + DpaA Time (min)

ldtAldtBldtC + DpaA

0 20 40 60 80 100 120 140 Time (min) bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Figure S6

A TET (2 µg/mL) AMP (32 µg/mL) BW BW dpaA dpaA lpp lpp dpaAlpp dpaAlpp 0 10 Serial dilutions 107 100 Serial dilutions 107 B MEC (5 µg/mL), ftsQAZ MEC (5 µg/mL), IPTG (1 mM), ftsQAZ BW BW ep dpaA dpaA ep lpp dpaA pDpaA dpaAlpp lpp ep 100 Serial dilutions 107 lppdpaA ep

100 Serial dilutions 107 IPTG (1 mM), ftsQAZ ftsQAZ BW ep BW ep dpaA ep dpaA ep dpaA pDpaA dpaA pDpaA lpp ep lpp ep lppdpaA ep lppdpaA ep 100 Serial dilutions 107 100 Serial dilutions 107 DpaA E. coli The copyright holder for this holder The copyright ATU3332 A. fabrum . ATU3631 A. fabrum BAB1_2034 B. abortus CBU_1157 C. burnetii LPG1514 L. pneumophila PA3756 P. aeruginosa Csd6 H. pylori Pgp2 C. jejuni LdtD E. coli YkuD B. subtilis

DpaA E. coli

CC-BY 4.0 International license 4.0 International CC-BY ATU3332 A. fabrum ATU3631 A. fabrum this version posted February 21, 2021. 21, 2021. February posted this version

; BAB1_2034 B. abortus CBU_1157 C. burnetii LPG1514 L. pneumophila PA3756 P. aeruginosa Csd6 H. pylori Pgp2 C. jejuni LdtD E. coli YkuD B. subtilis

DpaA E. coli ATU3332 A. fabrum ATU3631 A. fabrum perpetuity. It is made available under a available It is made perpetuity. BAB1_2034 B. abortus CBU_1157 C. burnetii LPG1514 L. pneumophila PA3756 P. aeruginosa https://doi.org/10.1101/2021.02.21.432140 Csd6 H. pylori doi: Pgp2 C. jejuni

LdtD E. coli YkuD B. subtilis preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in preprint to display the a license bioRxiv has granted who the author/funder, review) is by peer not certified (which was preprint bioRxiv preprint bioRxiv Figure S7 Figure bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

Table S2. Strains and plasmids used in this work.

Strain / plasmid Relevant features Reference / source Strain BL21(DE3) Overexpression strain F- ompT, hsdSB(rB-mB-) Novagen

(placUV5::T7gene1), λ(DE3) LOBSTR BL21(DE3) F– ompT hsdSB(rB– mB–) gal dcm (DE3), Kerafast carries genomically modified copies of arnA and slyD

BW25113 lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 (1)

ΔrhaBADLD78 BW25113∆lpp BW25113∆lpp::frt This work (AMM14) BW25113∆ldtA::frt ∆ldtB::frt ∆ldtC::frt (2) BW25113∆ldtABC (AMM28) BW25113∆ldtD::frt ∆ldtE::frt ∆dpaA::frt (2) BW25113∆ldtDE∆dpaA

BW25113∆6LDT lacIq rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 (3)

ΔrhaBADLD78ΔldtA ΔldtB ΔldtC ΔldtD ΔldtE ∆dpaA (AMM24) BW25113∆dpaA::frt (4) BW25113∆dpaA BW25113∆dpaA∆lpp BW25113∆dpaA::frt ∆lpp::frt This work (AMM30) BW25113 Φ(kan araC araBplptC)1 ∆dpaA::frt (4) araBplptC ΔdpaA Plasmid

pETMM82 pETMM82 derivative; expresses LdtD with an N- (5) terminal DsbC and a 6×His tag

1-334 R pET28a-LdtE-his LdtE -His6, native signal peptide, Kan This work

20-246 R pET29b-DpaA-his PelBss- DpaA -His6, Kan This work

20-246 R pET29b-DpaA(C143A)- PelBss- DpaA -His6 C143A, Kan This work his pGS100 pGZ119EH derivative, contains TIR sequence (6) downstream of ptac, CamR pGS124 (pDpaA) pGZ119H derivative; expresses DpaA under the (4) tac promoter; CamR pGS124-DpaA(C143A) pGZ119H derivative; expresses DpaA This work pDpaA(C143A) DpaA(C143A), tac promoter; CamR pTB63 tetA PftsQAZ::ftsQAZ (7) pCP20 FLP expression, temperature sensitive (1) replication; CamR and AmpR. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.21.432140; this version posted February 21, 2021. The copyright holder for this preprint (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 available under aCC-BY 4.0 International license.

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Table S3. Oligonucleotides used in this study.

Primer Sequence 5’3’ Construct(s)

DpaA_C143A_F TTC CAT CGG CGC CTA CGC AAT GAC pET29b-DpaA(C143A)-his, pGS124-DpaA(C143A) DpaA_C143A_R ACA CAA TCG CCG TGG ATC pET29b-DpaA(C143A)-his, pGS124-DpaA(C143A) LdtE_pET28a_F TTA GTT CCT CGT GGT TCT CTC GAG pET28a-LdtE-his CAC CAC CAC CAC LdtE_pET28a_R CCA TGG TAT ATC TCC TTC TTA AAG pET28a-LdtE-his TTA AAC AAA ATT LdtE_Insert_F GAA GAA GAT ATA CCA TGG ATG AAA pET28a-LdtE-his CGC GCG TCT TTG LdtE_Insert_R AGA ACC ACG AGG AAC TAA CTG CGT pET28a-LdtE-his CAC GCG TAA CAT

PelBss- GCGCC CAG CCG GCG ATG GCc ggt ttg pET29b-DpaA-his DpaA_pET29b_F ctg ggc agc ag DpaA_pET29b_R GGA TCC GAT ATC GCC ATG GAA CCG pET29b-DpaA-his CGT GGC ACC AGg gta cct ttt gcc tcg ggg agc gtg PelBss_F GGC CAT CGC CGG CTG GGC pET29b-DpaA-his PelBss_R ACT ATA GGG GAA TTG TGA GCG G pET29b-DpaA-his