A D-alanine aminotransferase S180F substitution confers resistance to β-chloro-D-alanine in Staphylococcus aureus via antibiotic inactivation

A D-alanine aminotransferase S180F substitution confers resistance to β-chloro-D-alanine in Staphylococcus aureus via antibiotic inactivation | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A D-alanine aminotransferase S 180 F substitution confers resistance to β-chloro-D-alanine in Staphylococcus aureus via antibiotic inactivation Rakesh Roy , Yahani P. Jayasinghe , Sasmita Panda , View ORCID Profile Merve S. Zeden , Vinai C. Thomas , Donald R. Ronning , View ORCID Profile James P. O’Gara doi: https://doi.org/10.1101/2025.08.17.668425 Rakesh Roy 1 Microbiology, School of Biological and Chemical Sciences, University of Galway , Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yahani P. Jayasinghe 2 Department of Pharmaceutical Sciences, University of Nebraska Medical Center , Omaha, Nebraska, USA 3 Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center Omaha Nebraska 68198 USA. 4 UNMC Center for Drug Design and Innovation, University of Nebraska Medical Center Omaha Nebraska 68198 USA. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sasmita Panda 3 Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center Omaha Nebraska 68198 USA. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Merve S. Zeden 1 Microbiology, School of Biological and Chemical Sciences, University of Galway , Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Merve S. Zeden Vinai C. Thomas 5 Department of Pathology and Microbiology, University of Nebraska Medical Center , Omaha, Nebraska, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Donald R. Ronning 2 Department of Pharmaceutical Sciences, University of Nebraska Medical Center , Omaha, Nebraska, USA 3 Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center Omaha Nebraska 68198 USA. 4 UNMC Center for Drug Design and Innovation, University of Nebraska Medical Center Omaha Nebraska 68198 USA. Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: don.ronning{at}unmc.edu jamesp.ogara{at}universityofgalway.ie James P. O’Gara 1 Microbiology, School of Biological and Chemical Sciences, University of Galway , Ireland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for James P. O’Gara For correspondence: don.ronning{at}unmc.edu jamesp.ogara{at}universityofgalway.ie Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Alanine transport and metabolism impact MRSA pathophysiology by dictating the availability of D-alanine for cell wall synthesis, the target of β-lactam antibiotics. Furthermore cycA -dependent alanine transport controls MRSA β-lactam susceptibility in chemically defined medium (CDM) in a glucose-dependent manner. Here we report that S. aureus was auxotrophic for L-alanine in CDM, and that this growth defect was rescued by glucose (or compensatory mutations), but only when the alanine racemase ( alr1 ) and D-alanine aminotransferase ( dat ) genes were functional. No role was observed for the alanine dehydrogenase 1 ( ald1 ) and ald2 genes. As previously reported, alr1 and, to a lesser extent, cycA mutations increased susceptibility to D-cycloserine (DCS). In contrast, only alr1 mutation increased susceptibility to β-chloro-D-alanine (BCDA), suggesting distinct targets for these alanine analogue antibiotics, which act synergistically against MRSA. Genome sequencing of a BCDA-resistant mutant identified a C 539 T mutation in dat , predicted to result in a S 180 F substitution. Expression of the dat C539T operon in wild-type increased BCDA resistance. alr1/dat::Em and alr1/dat C539T double mutants were auxotrophic for D-alanine, indicating that Dat-S 180 F transaminase activity is impaired, a conclusion supported by in vitro enzyme assays. Structural modeling revealed an active-site loop shift in Dat-S 180 F that altered PLP co-factor binding. Molecular docking showed that the S 180 F substitution promotes BCDA-PLP adduct dissociation by releasing inactivated BCDA, thereby conferring resistance. These data reveal essential roles for Alr1 and Dat during growth under nutrient-limiting conditions and the potential of combination therapy separately targeting both enzymes with DCS and BCDA to extend the treatment options for MRSA infections. Introduction According to the Centers for Disease Control and Prevention, Staphylococcus aureus is a leading cause of healthcare-associated infections in the United States where in 2017, it caused more than 119,000 bloodstream infections and nearly 20,000 deaths ( 1 ). Methicillin-resistant S. aureus (MRSA) has been designated a high-priority pathogen by the World Health Organization in both its 2017 and 2024 Bacterial Priority Pathogens Lists, highlighting the ongoing and urgent need for novel, effective antimicrobial therapies targeting MRSA infections ( 2 ). The emergence of antibiotic resistance in S. aureus poses a critical threat to global public health, contributing significantly to the burden of AMR, with over 100,000 deaths attributable to MRSA in 2019 alone ( 3 , 4 ). S. aureus is well-known for its ability to acquire resistance to almost all currently available antibiotics. Among these, its resistance to β-lactam antibiotics, a cornerstone in the bacterial infection treatment, is particularly alarming. Resistance to methicillin and other β-lactams in S. aureus is conferred by the acquisition of the mecA gene ( 5 ). This gene encodes penicillin-binding protein 2a (PBP2a), a transpeptidase enzyme that exhibits reduced affinity for β-lactam antibiotics, allowing bacterial cell wall crosslinking in the presence of these drugs. However, β-lactam resistance in S. aureus is not solely driven by mecA , and additional genetic and metabolic factors also play a role in modulating this resistance phenotype ( 6 – 11 ). Understanding these complex resistance mechanisms is an important part of efforts to identify and target enzymes and pathways that may resensitise MRSA to β-lactam antibiotics. Bacterial cell wall biosynthesis is an important target of many antibiotics, and altering the physiology of the cell wall by targeting pathways associated with it can re-sensitize MRSA to β-lactam antibiotics ( 12 , 13 ). An important constituent of the cell wall is D-alanine, which is incorporated into the peptidoglycan stem peptide. Alanine transport via CycA and the pathway leading to the incorporation of the D-ala-D-ala residues into the peptidoglycan stem peptide plays an important role in the susceptibility of MRSA to β-lactam antibiotics ( 14 , 15 ). Our previous work showed that mutation of cycA or MRSA exposure to the alanine/serine analogue D-cycloserine (DCS), which blocks the activity of the D-alanine racemase (Alr) and D-alanine ligase (Ddl) enzymes, significantly potentiated oxacillin (OX) in vitro and in a mouse model of bacteremia ( 14 ). However, these phenotypes were only evident in complex media (Mueller Hinton Broth), or chemically defined media supplemented with glucose (CDMG). In CDM lacking glucose (CDM), alternative carbon sources such as amino acids become more important for growth ( 16 ), and the impact of the cycA mutation on DCS or OX susceptibility was significantly ameliorated ( 14 ), suggesting that an alternative alanine transporter(s) or altered alanine metabolism may compensate for CycA under these growth conditions. In particular, endogenous L- and D-alanine biosynthetic pathways may play a more significant role in the absence of CycA-mediated alanine transport in CDM. L-alanine can be synthesised from pyruvate by alanine dehydrogenase 1 and 2 (Ald1 and Ald2), while D-alanine can be synthesized from pyruvate and D-glutamate by D-alanine aminotransferase (Dat) ( Fig. 1 ). A S. epidermidis triple mutant lacking alr1 , alr2 and dat was previously shown to be auxotrophic for D-alanine ( 17 ). Notably exposure of MRSA to sub-inhibitory OX increased transcription of both ald1 and dat ( 18 ), which appears to be consistent with an increased requirement for alanine under β-lactam stress. Increased Ald1 or Dat activity may increase L-alanine and D-alanine biosynthesis, respectively, irrespective of reduced alanine transport in CycA or other putative alanine transporter mutants. The role of Alr2 which shares 29% homology with Alr1 remains unclear ( 19 ). Synergy between DCS and another alanine analogue antibiotic, β-chloro-D-alanine (BCDA) and β-lactams has previously been reported ( 20 ), but potential synergy between DCS and BCDA has not been investigated. BCDA has been reported to inhibit Alr1 activity in a number of Gram negative organisms ( 21 – 23 ), but not Mycobacterium tuberculosis ( 24 ) and the mechanism of action of BCDA in S. aureus remains to be determined. Download figure Open in new tab Fig. 1. Schematic depicting the major enzymes involved in production of the D-ala-D-ala dipeptide required for peptidoglycan (PG) biosynthesis in S. aureus . Our previous data have implicated CycA or an alternative transporter(s) in alanine uptake. Alanine racemase Alr1 converts L-alanine (L-ala) to D-alanine (D-ala), and D-ala-D-ala is synthesized by D-alanine ligase (Ddl). Within the cell L-alanine can be synthesized from pyruvate by alanine dehydrogenase 1 and 2 (Ald1 and Ald2), while D-alanine can be synthesized from pyruvate and D-glutamate by D-alanine aminotransferase (Dat). D-cycloserine blocks the activity of the Alr1 and Ddl enzymes. The antimicrobial mode of action of β-chloro-D-alanine in S. aureus remains largely unexplored. Created with Biorender.com. In this study, we examined the impact of mutations in the D-ala-D-ala and endogenous alanine biosynthetic pathways on MRSA susceptibility to OX, DCS and BCDA. An in vitro evolution experiment identified Dat as a target for BCDA and a C 539 T single nucleotide substitution resulting in a predicted S 180 F amino acid substitution was implicated in increased BCDA resistance. An alr1/dat C539T mutant was constructed and the enzymatic activity of purified recombinant Dat-S 180 F was measured. The impact of the S 180 F substitution on Dat activity was modelled after determining the X-ray crystal structure of Dat and its pyridoxal 5’-phosphate (PLP) co-factor. BCDA is expected to undergo β-elimination upon reacting with PLP to form a covalent complex with Dat residue Lys146 and irreversibly inactivating the enzyme ( 25 ). However, our data suggests that Dat-S 180 F may bind and react with BCDA but avoid covalent modification of the essential active site lysine residue. Combinations of β-lactams, BCDA (targeting Dat) and/or DCS (targeting Alr1 and Ddl) may have therapeutic potential to improve the treatment of MRSA infections. Results Reversal of MRSA L-alanine auxotrophy by glucose or compensatory mutations is dependent on alanine racemase (Alr1) and D-alanine aminotransferase (Dat) Alanine transport and the D-ala-D-ala pathway control MRSA susceptibility to β-lactams and early cell wall inhibitors such as DCS in a culture medium dependent manner ( 14 ). Building on our previous observation that growth in chemically defined medium (CDM) compared to CDM with glucose (CDMG) or Mueller Hinton broth (MHB) increased β-lactam and DCS resistance in the cycA alanine transport mutant ( 14 ), we compared growth of wild-type JE2 and the alanine metabolism mutants alr1 , alr2 , ald1 , ald2, cycA and dat in CDM ( Fig. 2A, B ) and CDMG ( Fig. 2C, D ). A ddl mutant is not available in the Nebraska Transposon Mutant Library (NTML). JE2 is auxotrophic for L-alanine in CDM ( Fig. 2B ) but not CDMG ( Fig. 2D ) consistent with an important role for carbon flux from central metabolism to the D-ala-D-ala pathway in the absence of exogenous alanine. However, it is notable that, apart from alr1 and dat , weak growth was detected for JE2, cycA , alr2 , ald1 and ald2 after a 16-18 h lag in CDM lacking L-alanine ( Fig. 2B ). Faster growth of these strains after subculture in fresh CDM lacking L-alanine (data not shown) suggested the acquisition of compensatory mutations that perhaps enhance the flux of alternative amino acids into central metabolism in the absence of L-alanine. The analysis of JE2 revertants to L-alanine prototrophy will be the subject of a separate study. Download figure Open in new tab Fig. 2. S. aureus is auxotrophic for L-alanine in CDM but not CDMG. A - D. Comparison of JE2, cycA ( cycA ::Em, NE810), ald1 ( ald1 ::Em, NE1136), ald2 ( ald2 ::Em, NE198), alr1 ( alr1 ::Em, NE1713, alr2 ( alr2 ::Em, NE799) and dat ( dat ::Em, NE1305) growth in CDM + L-alanine (A), CDM - L-alanine (B), CDMG + L-alanine (C) and CDMG - L-alanine (D). E. Comparison of JE2, alr1 and dat growth in CDMG - L-alanine + D-alanine. F. Comparison of JE2, ald1, ald2, dat, ald1 ::Spc /ald2 ::Em , ald1 ::Em/ dat:: Spc , ald2 ::Em/ dat ::Spc and ald1 ::Em/ ald2 ::Km/ dat:: Spc growth in CDM + L-alanine (G). G. Comparison of JE2, ald1 ::Spc /ald2 ::Em , ald1 ::Em/ dat:: Spc , ald2 ::Em/ dat ::Spc and ald1 ::Em/ ald2 ::Km/ dat:: Spc growth in CDM - L-alanine. Where indicated L- and D-alanine were added at a final concentration of 5mM. The data presented are the average of at least 3 biological replicates and standard deviations are shown. In CDMG, L-alanine was required for growth of the alr1 (but not alr2 ) and dat mutants ( Fig. 2C,D ). Presumably Dat is required for the synthesis of D-alanine from pyruvate in CDMG lacking L-alanine ( Fig. 1 ). However, it was less clear why Dat cannot compensate for alr1 under these growth conditions. One possibility is that Alr1 is required for conversion of D-alanine produced by Dat to L-alanine in CDMG lacking L-alanine. Supporting this idea, the growth defect of alr1 in CDMG lacking L-alanine ( Fig. 2D ) was not rescued by D-alanine alone ( Fig. 2E ). Compared to JE2, mutations in ald1 and ald2 did not impact growth under the conditions tested ( Fig. 2A-D ) including in CDMG lacking L-alanine ( Fig. 2D ), suggesting that these enzymes are not required for L-alanine biosynthesis or the D-ala-D-ala pathway when growing on glucose. Conversely, we recently demonstrated that L-alanine is fluxed to pyruvate during growth in CDM ( 19 ), indicating that Ald1 and/or Ald2 are involved in the conversion of L-alanine to pyruvate when this amino acid is available as a carbon source. However, as noted above, ald1 and ald2 mutants grew normally in CDM ( Fig. 2A ). Furthermore, to rule out the possibility that Ald1 and Ald2 can compensate for each other, an ald1 / ald2 double mutant was constructed and also shown to grow normally in CDM ( Fig 2F ). To investigate if Dat can play a role in compensating for the Ald enzymes, ald1 / dat, ald2 / dat and ald1 / ald2 / dat mutants were constructed. All three of these mutants grew normally in CDM ( Fig. 2F ) and all were growth impaired in CDM - L-alanine ( Fig. 2G ). Interestingly as observed for ald1 and ald2 single mutants ( Fig. 2B ), the ald1 / ald2 double mutant did start to grow after a 16 h lag in CDM - L-alanine ( Fig. 2G ) but all combination mutants lacking dat showed no growth even after 24 h ( Fig. 2G ). While the functions of Ald1 and Ald2 in the D-ala pathway remain unclear, these data do reveal that reversal of MRSA L-alanine auxotrophy in CDM by glucose or compensatory mutations is dependent on Dat-mediated conversion of pyruvate to D-alanine, and the subsequent conversion of D-alanine to L-alanine by Alr1. Mutation of alr1 but not cycA increases β-chloro-D-alanine (BCDA) susceptibility in CDM Consistent with our previous data ( 14 ), the alr1 mutant was dramatically more susceptible to OX and DCS in CDM ( Table 1 ). The cycA mutation also increased susceptibility to OX and DCS ( Table 1 ), albeit to a lesser extent to that previously observed in CDM supplemented with glucose ( 14 ). Interestingly, while the alr1 mutation significantly increased susceptibility to another alanine analogue antibiotic BCDA, the cycA mutation had no effect ( Table 1 ). The susceptibility of the ald1 and ald2 mutants to OX, BCDA and DCS was not significantly different to wild-type ( Table 1 ). Mutation of dat marginally reduced the BCDA MIC from 300 to 200 μg/ml and had no effect on OX or DCS susceptibility ( Table 1 ). The alr2 mutant had wild-type levels of OX, DCS and BCDA susceptibility ( Table 1 ). Taken together these data support important roles for Alr1 and Dat in the susceptibility of MRSA to DCS and BCDA. The differential effect of the cycA mutation on DCS and BCDA susceptibility suggests that these antibiotics have different targets or mechanisms of action in S. aureus . Furthermore, disk diffusion and checkerboard titration assays (ΣFIC = 0.17) revealed synergy between DCS and BCDA when used in combination against JE2 (Fig. S1A,B). View this table: View inline View popup Download powerpoint Table 1. Antibacterial activity (minimum inhibitory concentrations, MICs; μg/ml) of D-cycloserine (DCS), oxacillin (OX) and β-chloro-D-alanine (BCDA) against wild-type MRSA strain JE2 and alanine metabolism mutants grown in chemically defined media lacking glucose (CDM). A S 180 F substitution in D-alanine aminotransferase (Dat) confers resistance to β-chloro-D-alanine (BCDA) in S. aureus To investigate the mechanism of action of BCDA, a single-step, high selective pressure experiment was used to isolate a stable BCDA resistant mutant of JE2. This mutant, designated BCDA1, was isolated from a 96 well plate in which JE2 was grown in CDM lacking glucose supplemented with an inhibitory concentration (500 μg/ml) of BCDA. Whole-genome sequencing identified a single nucleotide mutation, C-to-T, at position 539 of the dat gene (SAUSA300_1696) predicted to result in a S 180 F amino acid substitution ( Fig. 3A ). No other genetic changes were identified in BCDA1 and the dat C539T mutation was confirmed by PCR amplification and Sanger sequencing. Download figure Open in new tab Fig. 3. The dat C539T mutation increases BCDA resistance in S. aureus . A. Genomic organisation of the dat locus in S. aureus. The 849 bp dat gene (SAUSA300_1696) is in a 2-gene operon with the dipeptidase gene pepV . A C 539 T mutation predicted to result in a S 180 F amino acid substitution was identified in the BCDA resistant strain BCDA1. The location of the Tn insertion in the NTML dat mutant NE1305 is also shown. B and C. Comparison of JE2, JE2 pLI50, JE2 p pepV , JE2 p pepV - dat , JE2 p pepV - dat C539T , BCDA1, BCDA1 p pepV - dat , NE1305 ( dat ::Em) and BCDA1 dat ::Em growth in CDM (A) and CDM supplemented with BCDA 500 μg/ml (B). The data presented are the average of at least 3 biological replicates and standard deviations are shown. The BCDA MIC for BCDA1 increased to 800 μg/ml compared to 300 μg/ml for wild-type JE2 and 200 μg/ml for NE1305 ( dat ::Em) ( Table 1 ). In liquid CDM cultures, growth of BCDA1 was the same as JE2 and NE1305 ( Fig. 3B ), whereas in CDM supplemented with 500 μg/ml BCDA, only the resistant mutant was able to grow ( Fig. 3C ). Phage 80α-mediated transduction of the dat ::Em allele from NE1305 into BCDA1 restored BCDA susceptibility ( Fig. 3C ) and reduced the BCDA MIC to wild-type levels (300 μg/ml) ( Table 1 ). Multicopy expression of the 2-gene pepV - dat C539T operon from BCDA1 increased BCDA resistance in CDM liquid cultures, albeit after a longer lag period ( Fig. 3C ) and increased the BCDA MIC to 600-700 μg/ml ( Table 1 ). In contrast multicopy expression of the wild-type pepV - dat operon in JE2 had no significant effect on growth in CDM supplemented with BCDA ( Fig. 3C ). Multicopy expression of the pepV dipeptidase gene alone in JE2 for control purposes also had no effect on BCDA resistance ( Fig. 3C ). Collectively these data support the conclusion that the dat C539T allele in BCDA1 is the critical determinant driving increased resistance to BCDA. BCDA1 is auxotrophic for D-alanine in the absence of alr1 Next the impact of the dat C539T mutation in BCDA1 on the D-ala-D-ala pathway and alanine auxotrophy was investigated. Phage 80α was used to transduce the alr1 ::Em allele from NE1713 into BCDA1. Consistent with recently published data ( 17 , 19 ), we hypothesized that in the absence of alanine racemase, synthesis of D-alanine will be entirely dependent on Dat activity ( Fig. 1 ). The alr1 / dat C539T double mutant was unable to grow in TSB, unless the growth media was supplemented with D-alanine ( Fig. 4A ). Supplementation of TSB, which already contains enough L-alanine to support the growth of alr1 ( Fig. 4A ), with additional L-alanine was unable to rescue growth of the alr1 / dat C539T mutant ( Fig. 4A ), further supporting the requirement for D-alanine. For control purposes an alr1 / dat double mutant carrying transposon insertions in both genes was also constructed. To facilitate this, the dat ::Em allele in NE1305 was first replaced with a Spc cassette and the dat ::Spc mutant then transduced with the alr1 ::Em allele. Growth of the alr1 ::Em/ dat ::Spc double mutant in TSB was also dependent on D-alanine ( Fig. 4A ). Consistent with growth data indicating that Alr2 is not involved in the D-ala-D-ala pathway ( Fig. 1 ), transduction of the alr2 ::Em allele from NE799 into BCDA1 had no effect on growth in TSB in the absence of D-alanine ( Fig. 4A ).The alr1 / dat C539T and alr1 ::Em/ dat ::Spc mutants were unable to grow in CDMG lacking L- and D-alanine ( Fig. 4B ) or supplemented with L-alanine ( Fig. 4C ) or D-alanine alone ( Fig. 4D ) but did grow when provided with both L- and D-alanine ( Fig. 4E ). Taken together, these data demonstrate that BCDA1 is auxotrophic for D-alanine in the absence of alr1 and suggest that the Dat-S 180 F mutation negatively impacts D-alanine aminotransferase activity. Download figure Open in new tab Fig. 4. BCDA1 is auxotrophic for D-alanine in the absence of alr1 . A. Growth of JE2, BCDA1 ( dat C539T ), NE1305 dat ::Em, NE1713 alr1 ::Em, NE799 alr2 ::Em, alr1 / dat C539T , alr2 / dat C539T , dat ::Spc and alr1 ::Em/ dat ::Spc for 18 h at 37°C in TSB supplemented as indicated with 5 mM L-alanine or D-alanine. Growth or no growth is indicated by the presence or absence of a cell pellet following microcentrifugation of 1 ml culture aliquots. This experiment was repeated 3 times, and the results of a representative experiment is shown. B, C, D and E. Comparison of JE2, BCDA1 ( dat C539T ), NE1305 dat ::Em, NE1713 alr1 ::Em, NE799 alr2 ::Em, alr1 / dat C539T , alr2 / dat C539T , dat ::Spc and alr1 ::Em/ dat ::Spc growth in CDMG with no L- or D-alanine (B) CDMG with 5mM L-alanine (C), CDMG with 5mM D-alanine (D) and CDMG with both L- and D-alanine (5mM) (E). The data presented are the average of at least 3 biological replicates and standard deviations are shown. The Dat-S 180 F variant has reduced activity and a lower IC 50 for BCDA The wild-type dat and dat C539T genes were cloned into pET28b and HIS-tagged recombinant proteins were purified from E. coli via nickel affinity chromatography. To compare the activity of Dat and Dat-S 180 F, we quantified pyruvate produced by both proteins using a lactate dehydrogenase coupled assay. The activity of Dat-S 180 F is 43% of the wild-type level ( Fig. 5A ). Inhibition of Dat-S 180 F by BCDA displayed a lower IC 50 (34.0 ± 4.7 µM; Fig. 5A ) as compared to wild-type Dat (IC 50 = 96.8 ± 12.0 µM). Similar to the alr1 / dat ::Spc mutant, the alr1 / dat C539T mutant was also auxotrophic for D-alanine ( Fig. 4A-E ) indicating that the reduced activity of the Dat-S 180 F variant is not sufficient to compensate for the absence of Alr1. Taken together, these data suggest that that the lower IC 50 observed for the S 180 F variant is likely a consequence of the lower intrinsic enzymatic activity and that the inhibition levels observed at BCDA concentrations higher than 400 μM are more reflective of inhibition levels at the determined MICs. Download figure Open in new tab Fig. 5. The S 180 F mutation reduces Dat enzymatic activity and increases the inhibitory activity of BCDA. A. Comparison of wild-type Dat (green) and Dat-S 180 F (violet) enzymatic production of pyruvate from D-alanine (which was coupled to that of lactate dehydrogenase) in increasing concentrations of BCDA (up to 500 μM). The enhanced inhibitory activity observed at BCDA concentrations below 400 μM is no longer evident at higher BCDA concentrations. B. Amino acid sequence alignment of the S. aureus (Sa) and Bacillus sp. Dat proteins. C. Superimposition of the S. aureus (salmon) and Bacillus sp. YM-1 (RCSB PDB: 1DAA) (green) Dat structures. The red arrow (bottom left) indicates the position of the structural differences. D. The interactions of PLP of aldimine with neighboring residues. The carbon atoms of PLP are in purple and the carbon atoms in the S. aureus Dat structure are in salmon color. The oxygen, nitrogen, and phosphate atoms are in red, blue, and orange, respectively. Structure of D-alanine aminotransferase in complex with its coenzyme pyridoxal phosphate (PLP) To gain mechanistic insights into how the S 180 F mutation impacts Dat activity, the X-ray crystal structure of Dat was solved at 2.9 Å resolution in a P4 1 2 1 2 space group with one molecule in the asymmetric unit. In its native biologically relevant form, Dat is a dimer. The crystallographic 2-fold axis relates the asymmetric unit contents to the second Dat subunit of the biological dimer. The crystal structure shows that PLP forms the expected covalent aldimine with Lys146 as indicated by continuous electron density. The comparison of S. aureus Dat with the published structure of Bacillus sp. YM-1 Dat (RCSB PDB: 1DAA) reveals only minor structural differences even though the sequence identity between the two proteins is 52% ( Fig. 5B ). Only helix α1 and the preceding loop deviate between the two orthologs ( Fig. 5C ). The active site residues of both structures exhibit similar conformations and interactions with PLP. The phosphate group of PLP interacts with the guanidium group nitrogen of Arg51, backbone nitrogen of Ile205, backbone nitrogen and side chain of Thr206 and backbone nitrogen of Thr242 by forming hydrogen bonds. The nitrogen of the pyridine forms an interaction with Glu178. The methyl group of PLP resides at 4.0 Å from the backbone carbonyl group of Ser180 forming a modest van der Waals interaction ( Fig. 5D ). Comparison of the Dat and Dat-S 180 F structures As Dat possesses a shared active site with residues from both subunits contributing to PLP and substrate binding, this necessitates the use of a biological dimer for docking experiments. The biological dimer of Dat was made using crystallographic symmetry, and energy minimization was performed in Glide to have all the residues in the lowest energy conformation. The corresponding Dat-S 180 F model was made from the wild-type crystal structure with residue 180 in both subunits being mutated to Phe. This was again followed by the energy minimization in Glide. Compared to the 4 Å in the crystal structure, docking with covalent adduct produces a slight shift of the PLP that reduces the distance between the methyl group of PLP and the Ser180 to 3.5 Å. This is unlikely to reflect use of the slightly larger PLP adducts in the docking, because the same shift is observed when PLP is attached to catalytic residue Lys146, but illustrates the possible movement in the 180-182 loop that may impact PLP binding or affinity during various steps of the catalytic reaction. Notably, the side chains in this loop have already been identified as important residues for interactions with D-amino acid substrates or products in other homologous proteins ( 26 ). In contrast, comparison of the Dat and Dat-S 180 F dimer structures revealed no significant differences apart from the deviation of the loop harboring Phe180 ( Fig. 6A ). Download figure Open in new tab Fig. 6. A. Conformational differences between amino acid residues in wild-type S. aureus Dat dimer structure (carbons in salmon) and Dat-S 180 F (carbons in gray). The nitrogen and oxygen atoms are in blue and respectively. The PLP is forming an internal aldimine with Lys146. B. The docking results of PLP-BCDA covalent adduct (external aldimine) to the wild-type Dat (carbons in salmon) and Dat-S 180 F (carbons in gray) variant indicating similar binding modes. C. Docking of the 2AA external aldimine. The carbons of the 2AA external aldimine produced by β-elimination of the PLP-BCDA adduct are in pink and grey in the wild-type Dat and Dat-S 180 F structures, respectively. The differing orientations of 2AA maybe be one way that the variant avoids covalent modification of Lys146. This otherwise minor structural change has multiple effects that may impact PLP-BCDA affinity following formation of that adduct. The alteration in the loop harboring residue 180 increases the distance between the methyl group of PLP with the backbone of Phe180 versus Ser180, resulting in a shift from 3.5 to 4.5 Å. As the aromatic ring of PLP also forms 7f-7f interactions with the peptide bond between residues 181 and 182, this minor structural shift due to the S 180 F mutation could result in a larger than expected affinity change. Also contributing to a possible affinity decrease, the B. subtilis Dat structure (RSCB PDB:3DAA) in a complex with a PLP-D-alanine adduct indicates that following formation of PLP-D-alanine, the hydroxyl moiety of PLP forms a through-water hydrogen bond with the backbone carbonyl oxygen of Ser179 ( 26 ). Residue Ser179 in the B. subtilis Dat structure corresponds to Ser180 in S. aureus . If similar interactions mediate PLP binding with S. aureus Dat, an approximately 1 Å deviation of the position of the backbone oxygen caused by the S 180 F substitution may create steric hindrance between PLP and the peptide bond connecting residues 180 and 181. It is reasonable to suggest that this shift in backbone position affects the binding affinity of PLP to Dat when PLP is not covalently attached to Lys146. Additionally, the water mediated hydrogen bond between the residue 180 side chain and PLP is lost due to the S 180 F mutation. There are additional minor conformational changes in the side chains of several other residues accommodating the added bulk of the Phe side chain in the S 180 F variant. The only prominent change to side chain structure is a shift of Lys157, which is pushed away from the active site due to steric hindrance with the Phe180 side chain. However, Lys157 does not appear to be important for substrate binding or catalysis. Other residues known to be important for Dat activity such as Tyr32, Arg99, and His101, which form the “carboxylate trap” in PLP-dependent enzymes catalyzing reactions on D-amino acids, position the carboxylate moiety of substrates to ensure correct binding orientation of substrate ( 26 ). The side chain conformations of the carboxylate trap residues are essentially unchanged in Dat-S 180 F when compared to wild-type Dat ( Fig. 5D ). Taken together, the likely structural differences in the Dat-S 180 F variant are small but disproportionately relate to interactions between the Dat polypeptide and the PLP co-factor. This suggests that the S 180 F mutant has an altered affinity for the PLP-BCDA adduct, which would enable release of that adduct before the active site Lys146 can react with the BCDA side chain. While this would sacrifice a PLP molecule, Dat would remain able to bind another PLP molecule, form the covalent linkage between PLP and the Lys146 side chain, and inactivate additional BCDA. The underpinning chemistry of this possibility is somewhat similar to the known mechanism of BCDA inactivation in M. tuberculosis ( 24 ). Docking of β-chloro-D-alanine covalent adducts The proposed mechanism of Dat inactivation by BCDA involves the initial formation of BCDA-PLP adduct followed by β-elimination of chloride via a 2-amino acrylate (2AA) external aldimine (2AA-PLP) intermediate ( 24 , 27 ). Here molecular docking experiments were performed to assess possible affinity changes in the Dat-S 180 F variant using the BCDA-PLP adduct and the 2AA-PLP intermediate. Predictably, the BCDA-PLP docking results revealed similar binding modes for both proteins but with minor differences. BCDA-PLP docked in a similar orientation as pyridoxal D-alanine in complex with B. subtilis Dat ( 26 ). The docking results suggest that the carboxylate moiety of the BCDA-PLP adduct forms interactions with the carboxylate trap but lacks the bidentate interaction with Arg99 ( Fig. 6B ) ( 24 , 25 ). The 2AA-PLP external aldimine also docked with a similar binding mode to that observed for the wild-type Dat crystal structure, but maintained a bidentate interaction between Arg99 and the carboxylate moiety of the 2AA-PLP also forming a hydrogen bond with the side chain of Tyr32 ( 26 ). Again, both residues are part of the carboxylate trap (RSCB PDB:3DAA). Additionally, Lys146 is positioned to afford a reaction with the 2AA-PLP, which would either simply reverse the initial reaction on PLP or, through multiple chemical steps, irreversibly inactivate the enzyme ( 24 , 25 ). In contrast, the Dat-S 180 F docked models vary the position of the 2AA-PLP carboxylate moiety placing it near the carboxylate trap residues in some models but exhibiting a 180° rotation around the substrate amino acid φ bond that places the β-carbon in the region near the carboxylate trap, possibly preventing irreversible covalent modification of Lys146. Although unlikely due to the multiple interactions between the residues of the carboxylate trap and the α-carboxylate of the amino acid substrate, this dihedral rotation may become feasible as a result of β-elimination of chloride from the BCDA-PLP adduct and formation of the 2-AA external aldimine ( Fig. 6C ). The structural and docking results suggest that differences in the PLP binding site caused by the S 180 F mutation likely modulate PLP-adduct affinity during the multi-step chemistry necessary for reaction with Lys146. In doing so, these structural differences may afford release of catalytic intermediates representing inactivated BCDA. Consistent with this the IC 50 of Dat-S 180 F is lower than wild-type. Contribution of D-ala-D-ala pathway enzymes to BCDA resistance Unlike the Dat-S 180 F allele in BCDA1, which as described above appears to retain enough catalytic activity to act on BCDA and chemically alter it, the dat ::Em mutant is presumably incapable of inactivating BCDA and its MIC is only slightly reduced ( Table 1 ). In contrast the BCDA MIC of alr1 is reduced to <50 μg/ml ( Table 1 ). These data suggest that either Alr1 activity can generate enough D-alanine to support growth in the presence of low BCDA concentrations even the absence of Dat and/or that BCDA may also be targeting the D-alanine ligase enzyme Ddl in the D-ala-D-ala pathway. To investigate this, the impact of exogenous D-alanine on the susceptibility of alr1 and dat ::Em to BCDA was measured. Consistent with its reduced MIC, growth of alr1 was completely inhibited in CDM supplemented with BCDA 100 μg/ml, whereas the dat ::Em mutant was capable of growing, albeit at reduced levels compared to JE2 (Fig. S2A). Exogenous D-alanine reversed the BCDA-mediated inhibition of growth of both alr1 and dat ::Em in a concentration dependent manner (Fig. S2B, C). Taken together these data reveal that in the absence of Alr1-mediated D-alanine synthesis, inhibition of Dat and Ddl by BCDA has a significant negative effect on growth. In contrast, in the absence of Dat activity, Alr1-mediated D-alanine synthesis enables growth at higher BCDA concentrations. The Dat-S 180 F mutation confers increased BCDA resistance not by altering D-alanine synthesis but via inactivation of the antibiotic. Discussion Alanine metabolism plays a crucial role in cellular processes, including the regulation of cell wall biosynthesis, amino acid homeostasis, and energy production. Despite its importance, the enzymes involved in alanine metabolism remain relatively underexplored in S. aureus , particularly in the context of metabolic adaptation to antibiotic exposure and expression of resistance. Here we sought to advance our understanding of these pathways and their contribution to growth under physiologically relevant, nutrient-limited conditions to generate new insights into antibiotic resistance in MRSA. Interconnected central metabolism and amino acid biosynthetic pathways are regulated in a manner dependent on the available carbon source(s). In the absence of its preferred carbon source, glucose, growth of S. aureus is supported by amino acids such as alanine and aspartate ( 28 , 29 ). Our data showing the reversal of L-alanine auxotrophy by glucose illustrates the metabolic flexibility of S. aureus and the interplay between central metabolism and amino acid biosynthesis. The Alr1 and Dat enzymes, and not Ald1 or Ald2, are essential for growth in chemically defined medium (CDM) lacking alanine. Furthermore, suppressor mutants enabling growth in CDM lacking L-alanine were readily isolated but only if alr1 and dat were intact. In CDM with glucose (CDMG), the dat mutant required either L- or D-alanine for growth, while the alr1 mutant could only grow when exogenous L-alanine was available. This underscores the critical role of Alr1 not only for the synthesis of D-alanine, but also for the conversion of D-alanine to L-alanine under alanine-limited growth conditions. Interestingly, the Alr2 homologue cannot compensate for the absence of Alr1 and indeed no growth defect or change in DCS/β-lactam susceptibility was observed for the alr2 mutant in CDM or CDMG. Beyond Alr2, it remains unclear why alanine dehydrogenase 1 and 2 (Ald1 and Ald2), which are predicted to catalyze the conversion of pyruvate to L-alanine ( Fig. 1 ), cannot compensate for the absence of Alr1 for the synthesis of L-alanine. Indeed our data revealed no growth defect for an ald1 / ald2 double mutant under any growth condition tested, which are largely consistent with a previous study in S. aureus which reported no detectable phenotype for ald2 and an extended lag phase for ald1 in CDM but no change in final cell density ( 16 ). These data raise questions about the functions of Ald1 and Ald2 in S. aureus compared to other microorganisms. Possibly Ald1 and Ald2 do not undertake this function in S. aureus or the enzymes are not expressed under these conditions. In S. aureus ald1 forms an operon with ilvA , encoding threonine dehydratase, while ald2 is located elsewhere in the genome. In Paeniclostridium sabinae , which also has two ald genes, ald1 , but not ald2 , facilitates alanine synthesis from ammonia and pyruvate ( 30 ). Mycobacterium smegmatis has a single ald gene implicated in alanine utilization and anaerobic growth ( 31 ). Bacillus subtilis also has a single alanine dehydrogenase gene, which is reported to support growth when alanine is the sole carbon source ( 32 ). Future characterization of the suppressor mutations that reverse alanine auxotrophy in CDM may provide insights on the pathways required for L-alanine biosynthesis under alanine-limited growth conditions, including the roles of ald1 and ald2 . Our previous paper ( 14 ) and the data presented in this study underscore the importance of alanine transport and biosynthesis in antibiotic resistance. Consistent with earlier findings, mutation of alr1 significantly increases susceptibility to oxacillin, DCS and BCDA in CDM. In contrast the dat mutation had no impact on oxacillin and DCS susceptibility, and only marginally reduced the BCDA MIC. Mutation of the alanine transporter cycA increased susceptibility to oxacillin and DCS, but not BCDA. These data indicate that DCS and BCDA, which are both alanine analogue antibiotics, have different mechanisms of action. One explanation for the observation that the alr1 mutant was significantly more susceptible to BCDA than the dat mutant (MIC <50 μg/ml versus 200 μg/ml) is that BCDA targets the Dat enzyme. Because D-alanine biosynthesis in the alr1 mutant is completely reliant on Dat activity, inhibition of Dat activity by BCDA significantly compromises D-alanine availability thereby exacerbating vulnerability to this antibiotic. We previously reported that alternative alanine transporter(s) can compensate for CycA in CDM ( 14 ), providing a possible explanation for why the D-alanine pathway is less perturbed in this mutant and why it is less vulnerable to BCDA-mediated inhibition of Dat. It is also notable that mutation of dat did not affect DCS susceptibility, indicating that Dat does not significantly contribute to production of D-alanine when Alr1 is active. This conclusion supports a previous report that Dat is more active in the conversion of D-alanine to pyruvate, rather than vice versa ( 19 ). Finally, aligned to their lack of impact on growth, alr2 , ald1 and ald2 mutations did not affect susceptibility to OX, DCS and BCDA, further excluding these enzymes from a significant role in the D-alanine pathway and peptidoglycan biosynthesis. Supporting the possibility that Dat is a target for BCDA, a novel C 539 T mutation in the dat gene was identified as the only genetic change in a BCDA resistant mutant of JE2. Swapping the dat C539T allele with dat ::Em reversed the increase in BCDA resistance, while multi-copy expression of the pepV - dat C539T operon in wild-type MRSA significantly increased BCDA resistance. In Bacillus sphaericus BCDA binds to and inactivates D-amino acid transaminase (D-AAT) ( 33 ). D-AAT has also been shown to bind to β-cyano-D-alanine, another D-alanine derivative, leading to inactivation of the enzyme ( 34 ). These studies suggest that BCDA may act as a competitive substrate and/or inhibitor of Dat in S. aureus . Dat functions as a bidirectional enzyme, catalyzing the reversible conversion of D-alanine to pyruvate. In vitro enzyme assays revealed that production of pyruvate by Dat-S 180 F was reduced to 43% of wild-type levels. Consistent with this, a R 179 G substitution in the active site of the Haliscomenobacter hydrossis DAAT also decreased enzyme function ( 35 ). Furthermore the strict D-alanine auxotrophy of both the alr1 / dat ::Em and alr1 / dat C539T double mutants support the conclusion that the Dat-S 180 F enzyme cannot compensate for the loss of Alr1 to meet the requirement for D-alanine synthesis. While the acquisition of resistance to BCDA through a mutation in dat seems logical, the more intriguing question is why or how this resistance is accompanied by altered Dat transamination activity? Given the hypothesis that BCDA targets Dat, the identification of a mutation in the dat gene leading to BCDA resistance was not unexpected. However the data showing that this mutation reduced the normal ability of the enzyme to catalyze the conversion of D-alanine to pyruvate and that the IC 50 of Dat-S 180 F for BCDA was significantly lower than wild-type Dat raises the intriguing question of how the S 180 F substitution in Dat leads to BCDA resistance. Dat is a pyridoxal phosphate (PLP)-dependent enzyme and we determined the X-ray crystal structure of Dat in complex with PLP to gain insight into its catalytic mechanism and cofactor interactions. Structural comparisons with Bacillus sp. YM-1 Dat showed that the active site residues, including those interacting with PLP, are conserved, indicating the functional importance of this region in catalysis. Further, the structural comparison between wild-type Dat and the S 180 F variant highlighted a subtle but functionally important shift in the active site loop, likely affecting the affinity of PLP binding. This finding aligns with a previous study on D-AAT, in which a Y 31 Q substitution was associated with significantly reduced enzyme activity and enhanced susceptibility to BCDA ( 36 ). A E 177 K mutation in D-AAT also significantly impacted coenzyme anchoring and catalytic efficiency ( 37 ) further emphasizing the significance of coenzyme binding site residues in enzyme stability and activity. Furthermore, these D-AAT mutations can alter the enzyme’s stereochemical fidelity, increasing its ability to convert L-alanine to D-alanine. This has not been examined in our study and may be important. Molecular docking analysis identified a possible mechanism through which the S 180 F substitution in Dat may change how the enzyme interacts with the BCDA-PLP adduct, leading to BCDA resistance. Thus, while the Dat_ Dat-S 180 F variant exhibits a reduced capacity to catalyze the reversible conversion of D-alanine to pyruvate, the IC 50 of the mutant enzyme for BCDA is decreased, which is consistent with the molecular docking analysis suggesting that Dat-S 180 F can bind BCDA, form the BCDA-PLP adduct, and release an inactivated form of BCDA, such as 2AA-PLP. If 2AA-PLP is released by Dat-S 180 F, this product would likely react with water to form pyruvate-PLP. Further validation of this predicted mechanism will be needed. Given the crucial role of PLP-dependent enzymes in bacterial physiology and antibiotic resistance, it would be interesting to investigate if similar resistance mechanisms exist in other bacterial species and if targeting the PLP interactions using modified BCDA could offer new strategies for overcoming BCDA resistance in S. aureus . Taken together our data reveal essential roles for Alr1 and Dat in alanine metabolism, growth and antibiotic resistance in MRSA. The ability of exogenous D-alanine to reverse growth inhibition of the alr1 and dat ::Em mutants by BCDA suggests that BCDA may competitively inhibit the Ddl D-alanine ligase enzyme as well as Dat. In contrast DCS targets Alr1 and Ddl revealing that these antibiotics together inhibit the three major enzymes in the D-ala-D-ala pathway. Consistent with this we report synergy between DCS and BCDA against MRSA and propose that their combined use may limit the emergence of resistance and have significant therapeutic potential, particularly if used in conjunction with β-lactams. Experimental procedures Bacterial strains, reagents and growth conditions The bacterial strains and plasmids used in this study are listed in Table S1. Staphylococcus aureus strains were grown in Tryptic Soy broth (TSB), Tryptic Soy agar (TSA), Mueller Hinton agar (MHA), chemically defined media (CDM) and CDM supplemented with glucose (5 g/L) (CDMG) ( 16 ). CDM and CDMG were further manipulated to remove L-alanine (CDM/CDMG - L-ala), to replace L-alanine with 5mM D-alanine (CDM/CDMG - L-ala + D-ala) or to add 5mM D-alanine (CDM/CDMG + L-ala + D-ala). Escherichia coli strains IMO8B, XL-1 Blue, and BL21 (DE3) were cultured in Luria Bertani broth (LB) or Agar (LBA). S. aureus JE2 mutants from the Nebraska Transposon Mutant library (NTML) used in this study were NE810 cycA ::Em, NE16 ald1 ::Em, NE198 ald2 ::Em, NE1713 alr1 ::Em, NE799 alr2 ::Em and NE1305 dat ::Em. E. coli - S. aureus shuttle plasmid pLI50 ( 38 ) was used for complementation analysis and plasmid pET28b was used for overexpression and purification of recombinant proteins. Growth media were supplemented with appropriate antibiotics, including β-chloro-D-alanine (BCDA, 500 µg/ml), erythromycin (Em, 10 µg/ml), chloramphenicol (Cm, 10 µg/ml), spectinomycin (Spc, 1000 µg/ml), kanamycin (Km, 50 µg/ml), and ampicillin (Amp, 100 µg/ml), oxacillin (OX) and D-cycloserine (DCS) as required. Mutant strain construction The ald1 ::Em, ald2 ::Em, and dat ::Em strains obtained from NTML were used to construct ald1 ::Em/ dat ::Spc and ald2 ::Em/ dat ::Spc double mutants, as well as the ald1 ::Em/ ald2 ::Km/ dat ::Spc triple mutant. To generate double mutants, the Em antibiotic cassette in the NE1305 dat ::Em transposon mutant was first replaced with Spc via allelic exchange using the plasmid pSPC, which harbours a temperature-sensitive origin as described previously ( 39 ). This was followed by transduction using a lysate prepared from the ald1 ::Em and ald2 ::Em alleles were then transduced into the dat ::Spc mutant using phage 80α. To construct the ald1 ::Em/ ald2 ::Km/ dat ::Spc triple mutant, the pKAN plasmid was used to generate the ald2 ::Km mutant. The ald2 ::Km was transduced into dat ::Spc, to generate the ald2 ::Km/ dat ::Spc double mutant, into which the ald1 ::Em allele was transduced. The dat ::Spc was transduced with a lysate prepared from the alr1 ::Em mutant to generate an alr1 / dat double mutant. Lysates from the alr1 ::Em and alr2 ::Em mutants were also transduced into BCDA1 to generate alr1 / dat C539T and alr2 / dat C539T mutants, respectively. All double and triple mutants were confirmed by PCR amplification of the target loci using primers listed in Table S2. Growth assays in CDM and CDMG The growth of S. aureus strains was measured using a Tecan Sunrise microplate reader, with data recorded and analyzed via Magellan software. In brief, overnight cultures of each strain grown in TSB supplemented with appropriate antibiotics were harvested by centrifugation at 10,000 x g for 5 min, washed twice in phosphate-buffered saline (PBS), and resuspended in PBS to an OD 600 = 1. A 10 µl of this suspension was inoculated into 190 µl of the respective medium in 96-well hydrophobic polystyrene plates, resulting in a final volume of 200 µl with a starting OD 600 = 0.05. Plates were then incubated with shaking at 35-37 □ for 18-24 hours in a pre-heated Tecan microplate reader, with the OD 600 recorded every 15-minutes. Growth assays were performed using at least three independent biological replicates, and the resulting data was plotted using GraphPad Prism software. Generation of a BCDA-resistant mutant To generate a BCDA-resistant mutant, a TECAN growth assay was performed using wild-type JE2. Briefly, overnight cultures were washed and resuspended in PBS at an OD 600 = 1, from which 10 µl was used to inoculate a 96-well plate containing 190 µl of CDM supplemented with BCDA at concentrations of 1, 50 and 500 µg/ml. The MIC of BCDA for JE2 is 300 µg/ml. Growth was monitored for 24 hours as described above. Growth was observed at 1 and 50 µg/ml BCDA, whereas 500 µg/ml BCDA inhibited growth in most wells. However, one of the wells inoculated with JE2 at 500 µg/ml showed visible growth after 24 h, from which single colonies were isolated on TSA. The BCDA resistant phenotype of one isolated colony was confirmed by TECAN growth assays, and designated BCDA1. Following whole genome sequencing analysis (described below), the dat C539T allele of BCDA1 was replaced with dat ::Em from NE1305 by phage 80α transduction as described above, and verified using FP_ dat and RP_ dat primers (Table S2). Genomic DNA (gDNA) extraction and whole genome sequencing (WGS) Cell pellets from 3 ml of overnight S. aureus cultures were pre-treated with lysostaphin (10 µg/ml; Ambi Products LLC) at 37 □ for 30 minutes prior to gDNA extraction using the Wizard Genomic DNA Purification Kit (Promega) ( 40 ), and sequencing was conducted by MicrobesNG using Illumina sequencing platform with 2×250 bp paired end reads. Genome sequence analysis was performed as described previously ( 41 ) using CLC Genomics Workbench software (Qiagen, Version 22.0.1). The S. aureus JE2 genome sequence was used as a reference, with a contig produced by mapping Illumina reads onto the closely related USA300_FPR3757 genome sequence (RefSeq accession number NC_007793.1 ). The Illumina short-read sequence of the BCDA1 strain was then compared to the assembled JE2 sequence to identify single nucleotide polymorphisms (SNPs), insertions, or deletions. Antibiotic minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) measurements MIC measurements by broth microdilutions were performed in accordance with CLSI methods for dilution susceptibility testing of staphylococci ( 42 ) with modifications. Briefly, strains were first grown at 37°C on MHA for 24 h and 5 - 10 colonies were resuspended in 0.85% saline before being adjusted to 0.5 McFarland standard ( A 600 = 0.1). The cell suspension was then diluted 1:20 in PBS and 10 ml used to inoculate 100 ml culture media (MHB or CDM) containing serially diluted antibiotics in 96-well plates. To investigate synergy between DCS and BCDA the FIC index (ΣFIC) for MRSA strain JE2 was calculated. The ΣFIC = FIC A + FIC B, where FIC A is the MIC of DCS in combination with BCDA / the MIC of DCS alone, and FIC B is the MIC of BCDA in combination with DCS / the MIC of BCDA alone. Antibiotic combinations are considered synergistic when the ΣFIC is ≤0.5, indifferent when the ΣFIC is >0.5 to 2. Complementation of the BCDA1 mutant To complement the BCDA1 mutant, the pepV - dat operon and upstream regulatory sequences from JE2 and BCDA1 were amplified and cloned into plasmid pLI50 using EcoRI and SalI restriction sites, generating recombinant plasmids p pepV - dat , and p pepV - dat C539T . For control purposes the pepV gene alone from JE2 was also amplified and cloned into pLI50 ( 38 ) to generate p pepV . These plasmids were first transformed into electrocompetent E. coli IMO8B cells, verified by Sanger sequencing (Eurofins Genomics) and subsequently transformed into JE2 and BCDA1 by electroporation. Expression and purification of recombinant S. aureus Dat in E. coli The dat and dat C539T genes were amplified from gDNA isolated from JE2 and BCDA1, respectively, using FP_Dat and RP_Dat primers (Table S2). The PCR products were digested with EcoR I and Sal I and ligated into the pET28b plasmid. The resulting recombinant plasmids, pET28b_Dat and pET28b_Dat C539T , were verified by Sanger sequencing (Eurofins Genomics) and transformed into E. coli BL21 (DE3). The BL21 cells containing a pET plasmid harboring the wild-type Dat enzyme were grown at 37°C in Luria broth media until OD 600 reached 0.6. 1 mM IPTG was added to induce protein production at 16 °C for 20 hours. The cells were harvested through centrifugation at 3756 x g . They were resuspended in 20 mM Hepes pH 7.5, 500 mM NaCl, 5 mM β-mercaptoethanol, and 5 mM Imidazole buffer. Cells were lysed and DNA degraded by adding lysozyme (Hampton Research) and DNase I (Roche) and incubated on ice for 30 minutes. Cells were further lysed by sonication (Sonicator 3000, Misonix) and crude cell lysate was separated by centrifugation at 16,000 × g (Fixed angle rotor, 5810-R centrifuge, Eppendorf). The supernatant was applied to a 5 mL HisTrap TALON crude cobalt column (Cytiva). After washing with running buffer (20 mM Hepes pH 7.5, 500 mM NaCl, 5 mM β-mercaptoethanol, and 5 mM imidazole) to remove unbound protein, the recombinant Dat was eluted using a similar buffer as running buffer with 150 mM imidazole. The protein was further purified by size exclusion chromatography using 20 mM Hepes pH 7.5, 150 mM NaCl and 0.5 mM Tris (2-carboxyethyl) phosphine (TCEP) buffer as the mobile phase. Fractions containing purified Dat were pool and 1 mM PLP was added and excess PLP was removed by ultrafiltration with a buffer composed of 20 mM Hepes pH 7.5, 150 mM NaCl and 0.5 mM TCEP buffer. BCDA inhibition of Dat enzyme kinetics Wild-type Dat and the Dat-S 180 F variant were purified as indicated above. To assess Dat enzymatic production of pyruvate from D-alanine, Dat enzymatic activity was coupled to that of Lactate Dehydrogenase (LDH). The reactions were monitored by continuously measuring absorbance at 340 nm using the Synergy H4 Hybrid Reader from BioTek. The assay component final concentrations were 50 nM DAT (WT or S 180 F), 100 mM Hepes pH 7.5, 10 mM α-ketoglutarate, 400 µM NADH, 25 mM D-alanine, 1.2 U LDH and 10 µM PLP. The inhibition of Dat by BCDA was determined using various concentrations of BCDA and the respective IC 50 values of BCDA against WT and S 180 F Dat were fit using GraphPad Prism software. Crystallization, X-ray diffraction and data processing The vapor diffusion hanging drop method was used for crystallization experiments. The Dat protein was in 20 mM Hepes pH 7.5, 150 mM NaCl and 0.5 mM TCEP buffer at 7 mg/mL concentration. The protein and well solution were mixed at a 1:1 volumetric ratio and equilibrated against the well solution containing 0.1 M BIS-TRIS pH 6.5, 45% v/v Polypropylene glycol P 400. The harvested crystals were flash cooled in liquid nitrogen. The X-ray diffraction experiments were performed at the University of Nebraska Medical Center, Structural Biology Core Facility. The diffraction data were processed in CCP4 using DIALS. The crystal structure of D-amino acid aminotransferase/PLP of Bacillus sp. YM-1 (RCSB PDB code: 1DAA) was used for molecular replacement in PHENIX ( 43 ). The PLP was modeled using eLBOW and the covalent adduct was made using PHENIX ( 44 ). PHENIX was used for the structure refinement and iterated with manual model building using Coo ( 45 , 46 ). The structure was validated using Molprobity ( 47 ). Molecular docking experiments Using a biological dimer derived from the S. aureus Dat X-ray crystal structure both wild-type Dat and the Dat-S 180 F variant were modeled in Glide (Schrödinger) using energy minimization. Schrödinger LigPrep was used to prepare the covalent adducts formed by the reaction of PLP and BCDA. Molecular docking experiments employed the PLP-BCDA adducts following receptor grid preparation for both the wild-type and S 180 F variant. The results were exported to PyMOL and visually evaluated. Data Availability The Full Worldwide Protein Data Bank (wwPDB) X-ray Structure Validation Report for Staphylococcus aureus D-alanine aminotransferase in complex with pyridoxal 5’-phosphate is available under accession ID: 9PXS / pdb_00009pxs. Fig. S1. DCS and BCDA are synergistic against MRSA. A. Disk diffusion assays with DCS (30 μg) and BCDA (1000 μg) against JE2 grown on Mueller-Hinton agar for 24 h at 37°C. B. Checkerboard titration assays conducted using DCS and BCDA with JE2 grown for 24 h at 37 °C in Mueller-Hinton broth in 96-well plates. The data shown are the OD 600 values for each well. The experiments were repeated at least three times and the data from a representative 96-well plate is shown. Red shaded boxes indicated wells in which growth was measured. Fig. S2. Exogenous D-alanine restores growth of alr1 and dat ::Em mutants inhibited by BCDA. A. Comparison of JE2, BCDA1, NE1305 ( dat ::Em) and alr1 growth in CDM supplemented with BCDA 200 μg/ml. B. Comparison of alr1 growth in CDM supplemented with BCDA 100 μg/ml alone or with exogenous D-alanine concentrations from 0.005 to 5 mM. C. Comparison of dat ::Em growth in CDM supplemented with BCDA 500 μg/ml alone or with exogenous D-alanine concentrations from 0.005 to 5 mM. The data presented are the average of at least 3 biological replicates and standard deviations are shown. ACKNOWLEDGEMENTS This work was supported by Research Ireland grant 19/FFP/6441 to J.P.O’G, Research Ireland Pathway Award 22/PATH-S/10804 to M.S.Z. and NIH/NIAID R01AI125588 to V.C.T. The funders had no role in the study design, data collection, interpretation, writing of the manuscript, or the decision to publish. Conceptualization: R.R., Y.J., M.S.Z., D.R.R. and J.P.O’G. Methodology: R.R., S.P., Y.J., and S.P. Formal analysis: R.R., J.Y., M.S.Z, V.C.T., D.R.R. and J.P.O’G. Writing-original draft: R.R., Y.J., D.R.R. and J.P.O’G. Writing-review, editing and approval: all authors. Funding acquisition: J.P.O’G, M.S.Z. and V.C.T. Project administration: J.P.O’G. Funder Information Declared Science Foundation Ireland, https://ror.org/0271asj38 , 19/FFP/6441 , 22/PATH-S/10804 NIH/NIAID , R01AI125588 References 1. ↵ Kavanagh , K. T. 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