Mutation of lipoprotein processing pathway gene lspA or inhibition of LspA activity by globomycin increases MRSA resistance to β-lactam antibiotics

The Staphylococcus aureus cell envelope comprises numerous components, including peptidoglycan (PG), wall teichoic acids (WTA), lipoteichoic acids (LTA), targeted by antimicrobial drugs. MRSA resistance to methicillin is mediated by the mecA-encoded β-lactam-resistant transpeptidase, penicillin binding protein 2a (PBP2a). However, PBP2a-dependent β-lactam resistance is also modulated by the activity of pathways involved in the regulation or biosynthesis of PG, WTA or LTA. Here, we report that mutation of the lipoprotein signal peptidase II gene, lspA, from the lipoprotein processing pathway, significantly increased β-lactam resistance in MRSA. Mutation of lgt, which encodes diacylglycerol transferase (Lgt) responsible for synthesis of the LspA substrate did not impact β-lactam susceptibility. Consistent with previous reports, lgt and lspA mutations impaired growth in chemically defined media, but not in complex broth. MRSA exposure to the LspA inhibitor globomycin also increased β-lactam resistance. Mutation of lgt in an lspA background restored β-lactam resistance to wild type. The lspA mutation had no effect on PBP2a expression, PG composition or autolytic activity indicating a potential role for WTA or LTA. The lspA and lgt mutants exhibited marginally increased resistance to the D-alanine pathway inhibitor D-cycloserine. In addition, mutation of lgt and multicopy lspA expression, but not mutation of lspA, significantly increased susceptibility to the lipoteichoic acid synthase inhibitor Congo red revealing complex interplay between lipoprotein processing mutations and the expression/stability of cell surface glycopolymers. These findings indicate that accumulation of the LspA substrate, diacylglyceryl lipoprotein, increases MRSA resistance to β-lactam antibiotics through impacts on cell envelope components other than PG.


Introduction 44
The cell envelope of Staphylococcus aureus comprises a cytoplasmic membrane surrounded 45 by a thick peptidoglycan layer, cell wall-anchored proteins, lipoteichoic acids (LTA), wall 46 teichoic acids (WTA) and cell surface proteins. Accurate biosynthesis, assembly and stability 47 of these cell envelope components is essential for the growth and pathogenesis of S. aureus, 48 and is the target of numerous antimicrobial agents (1). The peptidoglycan layer determines 49 cell shape and protects the cell from osmotic lysis, cell surface proteins have important roles 50 in adhesion, biofilm formation, and immune evasion, and teichoic acids are involved in 51 protecting the cell from the activity of cationic antimicrobial peptides. 52 Methicillin resistance in MRSA is mediated by the mecA-encoded, low-affinity penicillin-53 binding protein 2a (PBP2a) carried on the mobile staphylococcal cassette chromosome mec 54 resistance (HeR) under laboratory growth conditions. HeR strains can become highly or 55 homogeneously resistant (HoR) after selection on elevated β-lactam concentrations via 56 poorly understood mechanisms, which require accessory mutations at other chromosomal 57 loci frequently associated with activation of the stringent response, cyclic-di-adenosine 58 monophosphate (c-di-AMP) signalling pathway (2-5), the activity of RNA polymerase (6) and 59 the ClpXP chaperone-protease complex (7,8). In addition, methicillin susceptible S. aureus 60 strains (MSSA) lacking mecA can also acquire low-level resistance through adaptive mutations 61 impacting the c-di-AMP signalling pathway and ClpXP activity (9). 62 Bacterial lipoproteins are a class of lipid-modified membrane proteins, involved in a range of 63 diverse functions such as; nutrient acquisition (10), signal transduction (11), respiration (12), 64 protein folding (13), virulence (14), antibiotic resistance (15) and host invasion (16). Mature cleavage of the signal peptide between the amino acid at position -1 and the +1 cysteine (22). 90 The first enzyme in the lipoprotein processing pathway is diacylglycerol transferase (Lgt) 91 which covalently attaches a diacylglycerol molecule from phosphatidyl glycerol onto the 92 sulfhydryl group of the invariant cysteine, resulting in a prolipoprotein (24). This diacylglycerol 93 serves as a membrane anchor. Next, the type II lipoprotein signal peptidase (Lsp) cleaves the 94 signal peptide between the amino acid at position -1 and +1, leaving the invariant cysteine 95 residue as the new terminal amino acid (25). Lgt and Lsp are conserved in all bacterial species. 96 In Gram-negative bacteria, a third step is catalysed by the enzyme N-acyl transferase (Lnt), 97 which transfers an N-acyl group onto the invariant cysteine residue at the N-terminal of the 98 protein (26). Lnt homologs have been identified in high-GC Gram-positive bacteria (27) but 99 not in low-GC Firmicutes. Despite the lack of an apparent Lnt homolog, N-acylated 100 lipoproteins have been identified in S. aureus (28) and recent work has identified two novel 101 non-contiguous genes lnsA and lnsB which catalyse the N-terminal acylation of lipoproteins 102 in S. aureus (29). The membrane metalloprotease Eep and the EcsAB transporter were shown 103 to be involved in the processing and export of linear peptides, including the signal peptide 104 cleaved by LspA in the lipoprotein processing pathway (30)(31)(32). Lgt and Lsp are essential for 105 the viability of Gram-negative bacteria (33). In contrast, lgt and lspA mutations do not impact 106 viability in Gram-positive bacteria (34), but are associated with changes in growth, 107 immunogenicity (35) and virulence (14) phenotypes. 108 In this study, we characterised the impact of lspA and lgt mutations, alone and in 109 combination, on susceptibility to b-lactams, D-cycloserine and Congo red, growth, PBP2a 110 expression, peptidoglycan structure, and autolytic activity in MRSA. The impact of 111 globomycin, which is known to inhibit LspA activity, on b-lactam susceptibility was also 112 characterised. Our data suggest that accumulation of the LspA substrate, diacylglyceryl-113 prolipoprotein, modulates resistance to b-lactam antibiotics in MRSA. 114

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Mutation of lspA in MRSA increases resistance to β-lactam antibiotics. The Nebraska 117 Transposon Mutant Library (NTML) (36) was screened to identify mutants exhibiting altered 118 susceptibility to cefoxitin, which is recommended as a surrogate for measuring mecA-119 mediated oxacillin resistance in clinical laboratories, in accordance with Clinical and 120 Laboratory Standards Institute (CLSI) guidelines for disk diffusion susceptibility assays. 121 Mutants identified by this screen included NE869 (yjbH) (37), NE1909 (sagA) (38) and NE810 122 (cycA)(39), all of which have previously been implicated in β-lactam resistance. A new mutant 123 identified in this screen was NE1757 (lspA::Tn), which exhibited increased resistance to 124 cefoxitin ( Fig 1A, B). PCR was used to confirm the presence of lspA::Tn allele in NE1757 (data 125 not shown). Using E-test strips, as described in the methods, oxacillin MIC of the lspA mutant 126 NE1757 was found to be 128 -256 μg/ml, compared to 32 -64 μg/ml for JE2 (Fig. 1C). Two 127 JE2 transductants carrying the lspA::Tn allele from NE1757 also exhibited increased resistance 128 to oxacillin (Fig. 1C). 129 The increased oxacillin resistance phenotype of NE1757 was also complemented by the 130 introduction of a plasmid (pLI50)-borne copy of the wild type lspA gene (plspA) into the 131 mutant. Growth of JE2, NE1757, NE1757 pLI50 and NE1757 plspA on MHA 2% NaCl 132 supplemented with oxacillin 32 μg/ml visually demonstrated that carriage of the 133 complementation plasmid reversed the increased oxacillin resistance phenotype of NE1757 134 ( Fig. S1). Measurement of oxacillin MICs by agar dilution showed that NE1757 and NE1757 135 pLI50 had MICs of 256 µg/ml, while JE2 and the complemented strain NE1757 plspA had MICs 136 of 64 µg/ml (Table 1). 137 Comparison of JE2 and NE1757 growth in MHB, MHB 2% NaCl, TSB and TSB 0.5 mg/ml oxacillin 138 revealed no significant differences (Fig. S2). Similarly, population analysis profiling revealed 139 that the heterogeneous pattern of oxacillin resistance expressed by JE2 was unchanged in 140 NE1757 (Fig. S3). These observations indicate that the increased β-lactam resistance 141 phenotype of NE1757 was not attributable to any growth advantage or change in the 142 heterogeneous/homogeneous oxacillin resistance profile. 143 Comparative WGS analysis confirmed that the only change in the NE1757 genome was the 144 insertion of the Bursa aurealis transposon in the lspA gene at position 1192002 (Table S1) and 145 there were no SNPs present. The NE1757 genome was also checked manually for zero 146 coverage regions to confirm the absence of any large deletions and insertions. Taken together 147 these data indicate that mutation of lspA, which encodes lipoprotein signal peptidase II 148 involved in the lipoprotein processing pathway (Fig. 2) Quantitative peptidoglycan compositional analysis was performed using UPLC analysis of 161 muramidase-digested muropeptide fragments extracted from exponential or stationary 162 phase cultures of JE2 and NE1757 grown in MHB or MHB supplemented with oxacillin 3 µg/ml 163 or 32 µg/ml. The PG profile of JE2 and the lspA mutant NE1757 were similar under all growth 164 conditions tested (Fig. 3B). Thus, supplementation of MHB with oxacillin was associated with 165 significant changes in muropeptide oligomerization and reduced crosslinking, but these 166 effects were the same in both JE2 and NE1757 (Fig. S4). The total PG concentrations extracted 167 from JE2 and NE1757 cell pellets were also the same (data not shown). Comparison of Triton 168 X-100-induced autolysis in JE2 and NE1757 also revealed identical autolytic profiles (Fig. S5). 169 Finally, the NaCl tolerance phenotypes of JE2 and NE1757 were also similar ( Fig. S6), indicating 170 that c-di-AMP signalling, which has previously been implicated in the control of β-lactam 171 resistance, autolytic activity and NaCl tolerance (4,5) was unaffected by the lspA mutation. 172 These data indicate that increased b-lactam resistance in the lspA mutant was not associated 173 with significant changes in PG abundance, structure, crosslinking, c-di-AMP signalling or 174 autolytic activity. 175

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Exposure to the LspA inhibitor globomycin also increases β-lactam resistance. Globomycin 177 is a natural peptide antibiotic, first discovered in 1978, produced by 4 different strains of 178 actinomycetes (41,42). It is an inhibitor of LspA and works by sterically blocking the active site 179 of the enzyme (43). Globomycin has moderate to strong antibacterial activity against many 180 Gram-negative species and has been proposed to cause disruption of cell surface integrity 181 (44). However, despite its ability to inhibit LspA, globomycin does not have significant 182 antimicrobial activity against Gram-positive bacteria including S. aureus, with MICs >100 183 µg/ml (41,42,45). 184 Because disruption of lspA increased resistance to β-lactams, we hypothesized that chemical 185 inhibition of LspA by globomycin may also be associated with increased β-lactam resistance. 186 To test this hypothesis, the susceptibility of JE2 and NE1757 to oxacillin was determined in 187 the presence or absence of globomycin. A series of JE2 and NE1757 cultures grown in MHB 188 2% NaCl were used to determine that oxacillin 40 µg/ml inhibited growth of JE2 but not 189 NE1757 ( Fig. 4A) as predicted. Next, JE2 MHB 2% NaCl 40 µg/ml oxacillin cultures were further 190 supplemented with 10, 20, 30, 40 or 50 µg/ml globomycin (Fig. 4B). Oxacillin-induced 191 inhibition of JE2 growth was rescued globomycin, optimally at 10, 20 and 30 µg/ml (Fig. 4B). In contrast to the observation that globomycin increased b-lactam resistance in wild type JE2, 204 USA300 and ATCC43300, the growth of NE1757 in a range of globomycin concentrations from 205 10 -50 μg/ml had a dose-dependent and negative effect on growth in the presence of 206 oxacillin 40µg/ml (Fig. S7). Taken together, these data suggest that globomycin antagonises 207 b-lactam antibiotics, increasing MRSA resistance to oxacillin and cefotaxime in a LspA-208 dependent manner. However, in the absence of lspA, the combination of globomycin and 209 oxacillin interferes with growth of JE2, particularly at higher concentrations of globomycin, 210 perhaps due to off-target effects. 211 To determine if globomycin could also increase resistance to other classes of β-lactam 212 antibiotics, its effect on cefotaxime resistance in JE2, USA300 and ATCC43300 was evaluated. 213 Cefotaxime was chosen because it is a 3 rd generation cephalosporin with broad spectrum 214 activity against Gram-positive and Gram-negative bacteria commonly used in the clinic, 215 whereas oxacillin is a narrow-spectrum penicillin antibiotic. Cefotaxime 40 μg/ml inhibited 216 growth of JE2 and USA300 ( Fig. 5A and B), while 30 μg/ml inhibited growth of ATCC43300 (  and lspA/lgt mutant cultures were spotted onto TSA 0.125% Congo Red. Our data showed 226 that mutation of lgt dramatically increased susceptibility to the selective LtaS inhibitor Congo 227 red ( Fig. 6) suggesting that impaired lipoprotein processing affects the expression or stability 228 of LTA. However, while the lgt/lspA double mutant was even more susceptible to Congo red 229 than the single lgt mutant, the single lspA mutations in NE1757 and NE1757 MM did not 230 significantly alter Congo red susceptibility and complementation of NE1757 was also 231 associated with increased susceptibility (Fig. 6). These data show that mutation of lgt or 232 multicopy expression of lspA both increase susceptibility to this LTA inhibitor indicating that 233 the lipoprotein processing pathway impacts LTA synthesis/stability in a complex manner. The 234 mutations in the lgt and lspA genes alone, and in particular when combined, significantly 235 increased resistance to the alanylation pathway inhibitor D-cycloserine (DCS)( Table 1). 236 However lack of correlation between the effects of lgt and lspA mutations on susceptibility to 237 Congo red, DCS and b-lactams indicates that further analysis is needed to better understand 238 the interactions between lipoprotein processing pathway intermediates and the 239 expression/stability of LTA and WTA. 240 241 Mutation of lgt in the lspA background restores wild type levels of b-lactam resistance. LspA 242 catalyses the second major step in the lipoprotein processing pathway (Fig. 2). To probe the 243 contribution of lipoprotein processing to LspA-controlled oxacillin resistance, we compared 244 the impact of lgt, lspA and lgt/lspA mutants on growth and resistance to oxacillin, as well as 245 cefotaxime, nafcillin and vancomycin. Lgt catalyses the addition of a diacylglycerol moiety 246 onto preprolipoproteins, from which the signal peptide is then cleaved by LspA (Fig. 2). To 247 construct a lspA/lgt double mutant the erythromycin resistance marker of the lspA::Tn allele 248 in NE1757 was first exchanged for a markerless transposon to generate a strain designated 249 NE1757 MM into which the erythromycin-marked lgt::Tn allele from NE1905 was transduced. The lgt mutant NE1905 exhibited no changes in susceptibility to oxacillin, cefotaxime, nafcillin 257 or vancomycin (Table 1). As observed for oxacillin, the lspA mutant NE1757 was more 258 resistant to cefotaxime and nafcillin and these phenotypes were complemented by the plspA 259 plasmid (Table 1). Neither the lspA nor lgt mutations increased resistance to vancomycin 260 (Table 1). Oxacillin and nafcillin MICs were restored to wild type levels in the lspA/lgt double 261 mutant, and the cefotaxime MIC was significantly reduced (Table 1). 262 Taken together, these data indicate that while mutation of lspA or lgt or both impact growth nafcillin MICs of the ecsB mutant from the NTML were the same as wild type (Table 1) 268 indicating that downstream processing of the LspA-cleaved signal peptide is not associated 269 with altered b-lactam resistance. 270

Discussion 271
Advances in our understanding of the accessory factors that control levels of mecA/PBP2a-272 dependent resistance to methicillin has the potential to reveal new therapeutic targets and 273 drugs that may facilitate the reintroduction of other b-lactam antibiotics for the treatment of 274 MRSA infections. In this study, we demonstrated that mutation of the lipoprotein processing 275 pathway gene lspA, or inhibition of LspA with globomycin increased resistance to b-lactam 276 antibiotics. Although numerous mutations impacting the stringent response (ppGpp) and c-277 di-AMP signalling are associated with the transition from a heterogeneous to homogeneous 278 pattern of resistance and elevated PBP2a expression (2,3), our data show that the lspA 279 mutation was not associated with a HoR phenotype, increased PBP2a expression, or altered 280 NaCl tolerance (which is controlled by c-di-AMP) (4,5). On the other hand, changes in b-lactam 281 resistance independent of altered PBP2a regulation has long been known (47-49), and several 282 auxiliary factors known to influence b-lactam resistance in MRSA have been described (49-283 54). In addition to unchanged PBP2a expression, no evidence of peptidoglycan remodelling 284 was observed in NE1757 after growth in the presence or absence of oxacillin, potentially 285 implicating wall teichoic acid (WTA) or lipoteichoic acid (LTA) synthesis or stability in the lspA 286 mutant phenotype. Inhibition of WTA synthesis was previously shown to decrease b-lactam 287 resistance in a PBP2a-independent manner (50). Reduced LTA stability, as evidenced by 288 Western blotting and increased susceptibility to the selective lipoteichoic acid synthase 289 inhibitor Congo red, was recently correlated with a PBP2a-independent reduction in b-lactam 290 resistance in auxiliary factor auxA and auxB mutants (49). Interestingly AuxA is structurally 291 similar to SecDF (49) and may interact with Sec pathway and lipoprotein processing (Fig. 8). 292 The lgt mutant was significantly more susceptible to Congo red than the lspA mutant and 293 multicopy expression of lspA also increased Congo red susceptibility suggesting that although 294 the lipoprotein processing pathway modulates LTA synthesis/stability, the relationship 295 between these two pathways appears to be complex. 296 Consistent with previous studies of lipoprotein pathway processing mutants in S. aureus (35), 297 Listeria monocytogenes (55) and Streptococcus agalactiae (56), our analysis also showed that 298 the lgt, lspA and lgt/lspA double mutants all exhibited impaired growth in CDM but not in TSB 299 or MHB indicating that the impact of lipoprotein processing pathway mutations on nutrient 300 acquisition and growth under nutrient limiting conditions can be compensated in rich media. 301 Mutation of the lgt gene from the lipoprotein processing pathway (Figs. 2, 7) did not affect b-302 lactam resistance and introduction of the lgt mutation into a lspA mutant restored wild type 303 levels of resistance. These data implicate accumulation of diacylglycerol lipoprotein in 304 elevated b-lactam resistance. Consistent with this possibility, lipoproteins were retained in 305 the membrane of a S. agalactiae lspA mutant, but were released into the supernatant in large 306 concentrations by lgt and lgt/lspA mutants (56). Lipoproteins synthesised by the S. agalactiae 307 lspA mutant retained their signal peptide, which was absent in a lgt mutant with LspA activity. 308 Importantly, signal peptide processing also occurred in the lgt/lspA double mutant, albeit 309 with cleavage occurring between different amino acids, implicating the involvement of an 310 alternative signal peptidase. Our analysis revealed no change in oxacillin susceptibility in the 311 MRSA lgt/lspA double mutant, indicating that even if an alternative peptidase can cleave the 312 signal peptide, this may not impact b-lactam resistance. Furthermore, mutation of ecsB, 313 which has recently been implicated in export of linear peptides from the lipoprotein 314 processing pathway in S. aureus (32), did not change the oxacillin MIC in JE2 (Table 1) also 315 indicating that downstream processing of signal peptides cleaved from lipoproteins by LspA 316 is not associated with altered b-lactam resistance. In L. monocytogenes, deletion of lgt also 317 led to significant release of lipoproteins into the supernatant. However, treatment of the L. 318 monocytogenes lgt mutant with globomycin (inhibiting LspA activity) resulted in enhanced 319 lipoprotein retention in the membrane (55), suggesting that the impact of globomycin and 320 lspA mutation on lipoprotein processing is not necessarily the same. In a S. aureus lgt mutant, 321 the Götz group reported that the majority of lipoprotein (lacking signal peptide) was released 322 into the supernatant (35). Taken together, the data suggest that accumulation of membrane-323 anchored diacylglycerol lipoprotein with uncleaved signal peptide, or lipoprotein that is mis 324 localised or released due to aberrant signal peptide processing by an alternative peptidase, is 325

Experimental procedures 328
Bacterial strains and culture conditions. Bacterial strains and plasmids used in this study are 329 listed in Table S2. Escherichia coli strains were grown in Luria-Bertani (LB) broth or agar (LBA). supplemented with 2% NaCl where indicated, were used for antimicrobial susceptibility 333 testing (AST). Antibiotic concentrations used were 10 µg/ml erythromycin, 10 µg/ml 334 chloramphenicol, 75 µg/ml kanamycin, 100 µg/ml ampicillin. 335 Two hundred and fifty ml flasks were filled with 25 ml growth media, and overnight cultures 336 were used to inoculate the media at a starting OD600 of 0.05. Overnight cultures were grown 337 in TSB, and washed once in 5 ml PBS before being used to inoculate the CDM cultures. Flasks 338 were incubated at 37°C shaking at 200 rpm. OD600 readings were measured at 1 -2 h intervals. Genetic manipulation of S. aureus. Phage 80α transduction was used to verify the association 349 between antibiotic resistance phenotypes and transposon insertion-marked mutations from 350 the NTML as described previously (39). Transductants were verified by PCR amplification of 351 the target locus using primers listed in Table S3. The plasmid pTnT, which contains a 352 truncated, markerless transposon was used to construct a markerless lspA mutant designated 353 NE1757 MM, as described previously (58 for 24 h and 5 -6 colonies were resuspended in 0.85% saline to OD600 of 0.08 -0.1 (0. before 10 µl (approximately 5 × 10 5 CFU/ml ) was added to each well and the plates were 411 incubated at 35°C with shaking on a Tecan Sunrise microplate instrument for 20 h 412 (cefotaxime) or 24 h (oxacillin). Globomycin ranging from 10 -100 µg/ml was added to the 413 cefotaxime or oxacillin cultures to measure potential synergism or antagonism. Three 414 independent biological replicates were performed for each strain and antibiotic combination. 415

416
PBP2a western blot analysis. Overnight MHB cultures were used to inoculate 25 ml of MHB 417 2% NaCl, with or without 0.5 µg/ml oxacillin to a starting OD600 of 0.05, incubated at 35°C (200 418 rpm shaking) until an OD600 of 0.8 was reached before the cells were pelleted and 419 resuspended in PBS to an OD600 of 10. Six µl of lysostaphin (10 µg/ml) and 1 µl of DNase (10 420 µg/ml) was added to 500 µl of this concentrated cell suspension before being incubated at 421 37°C for 40 min. Next, 50 µl of 10% SDS was added and the incubation continued for a further 422 20 min. The lysed cells were then pelleted in a microcentrifuge for 15 min, following which 423 the protein-containing supernatant was collected and total protein concentration 424 determined using the Pierce® BCA Protein Assay Kit. Samples containing 8 µg total protein 425 were mixed 1:1 with protein loading buffer (2x) (National Diagnostics) and incubated at 95°C 426 for 5 min and loaded onto a 7.5% Tris-Glycine gel and separated at 120 V for 60 mins. 427 Electrophoretic transfer to a PVDF membrane was carried out at 30 V for 30 min on the Trans-428 Blot Turbo Transfer System (Biorad). The PVDF membrane was blocked overnight in 5% skim 429 milk powder in PBS at 4°C. The following day, the membrane was washed in fresh PBS. Anti- Antibiotic tolerance assay. Tolerance assays were performed as described previously (61). 447 Briefly overnight TSB cultures were sub-cultured into 25 ml of fresh TSB in 250 ml flasks at a 448 starting OD600 of 0.05 and grown to an OD600 of 0.5 at 37°C with 200 rpm shaking. At this time 449 (T0) an aliquot was removed for CFU enumeration and 12.5 µg/ml oxacillin promptly added 450 before the cultures were re-incubated. Antibiotic tolerance was expressed as the % CFU/ml 451 after 2, 4, 6, 8 and     and growth curves were plotted in Prism software (GraphPad). The data presented are the 586 average of 3 independent biological replicates, and error bars represent standard deviations.   peptidoglycan extracted from JE2 and NE1757. Cells were collected from cultures grown to 677 exponential phase in MHB or MHB supplemented with oxacillin 3 µg/ml or 32 µg/ml. Each 678 profile shown is a representative of 3 biological replicates. Significant differences determined 679 using Students t-test (**P < .01; ***P < .001; ****P<.0001). 680 681 682 683 Supplementary Fig. S5. Autolytic activity is unaffected by the lspA mutation. Triton X-100-684 induced autolysis of JE2, NE1757 (lspA::Tn) and NE406 (atl::Tn, negative control). The strains 685 were grown to OD600 = 0.5 in MHB medium at 37°C, before being washed in cold PBS and 686 resuspended in 0.1% Triton X-100. The OD600 was monitored, and autolysis was expressed as 687 a percentage of the initial OD600. The experiments were repeated 3 independent times, 688 plotted using Prism software (GraphPad) and standard deviations are shown. Overnight cultures of JE2, NE1757, NE1757 pLI50, NE1757 plspA, JE2 lspA::Tn #2 transductant 695 and NE788 (JE2 ktr::Tn, NaCl-sensitive control) were grown in TSB and cell density was 696 standardised to OD600 of 1. Four µl aliquots from 10-fold serial dilutions were spotted onto 697 TSA supplemented with 2.2 M NaCl and the plates incubated overnight at 37°C. Three 698 independent experiments were carried out and a representative image of a plate is shown 699 700 701 702 Supplementary Fig. S7. Globomycin does not increase oxacillin resistance in the lspA mutant 703 NE1757. NE1757 was grown in MHB 2% NaCl supplemented with a sub-inhibitory 704 concentration of oxacillin (40 µg/ml) and a range (10 -50 µg/ml) of globomycin 705 concentrations. The solvent for globomycin, 0.6% ethanol, was included as a control. The 706 cultures were grown in a Tecan Sunrise incubated microplate reader for 24 h at 35 o C. OD600 707 was recorded at 15 min intervals and growth curves were plotted in Prism software 708 (GraphPad). The data presented are the average of 3 independent biological replicates, and 709 error bars represent standard deviations.