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we used m. tuberculosis cdc1551 , a clinical isolate , as the host strain to generate a transposon insertion mutant in mt2594 ( ldtmt2::tn ) as described 14 .
this mutant carries a himar1 transposon insertion at + 872 base from the putative translation start site of the gene .
this is a single copy integrating plasmid based on pmh94 backbone 25 , which we modified into a gateway compatible destination vector ( invitrogen ) .
we cloned a wild - type copy of mt2594 along with its promoter into this destination vector pgs202 to generate pgs202_2594 .
we used middlebrook 7h9 liquid medium supplemented with 0.2% glycerol , 0.05% tween80 , 10% vol / vol oleic acid  albumin  dextrose  catalase
( oadc ) and 50 ug ml cycloheximide for in vitro growth , and middlebrook 7h11 solid medium ( becton - dickinson ) for enumerating colony forming units ( cfu ) in in vitro and in vivo growth studies .
we amplified a portion of ltdmt2 with primers 5-ttttcatgatcgccgatctgctggtgc-3 and 5-ttggatcccgccttggcgttaccggc-3 , digested with bsphi  bamhi ( underlined ) and cloned into pet2818 15 .
the resulting plasmid , encodes a fusion protein consisting of a methionine specified by the atg initiation codon of pet2818 , residues 55 to 408 of ldtmt2 , and a c - terminal polyhistidine tag with the sequence gsh6 .
we grew e. coli bl21(de3 ) harboring prep4groesl 26 and pet2818ldtmt2 at 37 c in 3
l of brain heart infusion broth containing 150 g ml ampicillin , induced expression using isopropyl 
we purified ldtmt2 from a clarified lysate by affinity chromatography on ni - nitrilotriacetate - agarose resin ( qiagen gmbh , germany ) followed by anion exchange chromatography ( monoq hr5/5 , amersham pharmacia ) with a nacl gradient in 50 mm tris 
we performed an additional size exclusion chromatography on a superdex hr10/30 column equilibrated with 50 mm tris 
finally , we concentrated the protein by ultrafiltration ( amicon ultra4 centrifugal filter devices , millipore ) and stored at 20 c in the same buffer supplemented with 20% glycerol .
we purified disaccharide - tetrapeptide containing amidated meso - diaminopimelic acid ( glcnac  murnac 
mesodapnh2d  ala ) from c. jeikeium strain cip103337 and determined the concentration after acid hydrolysis 27,28 .
next , we tested in vitro formation of muropeptide dimers in 10 l of 50 mm tris  hcl ( ph 7.5 ) containing 300 mm nacl , 5 m ldtmt2 , and 280 m disaccharide  tetrapeptide .
we incubated he reaction mixture for two hours at 37 c and analyzed the resulting muropeptides by nanoelectrospray tandem mass spectrometry using n2 as the collision gas 28 .
we used 45 week old , female balb / c mice ( charles river laboratories ) to study in vivo virulence of the strains and their susceptibility to drugs .
we infected mice with a log phase culture of wild  type m. tuberculosis , or ldtmt2::tn or the complemented strain in an aerosol chamber . for assessing in vivo growth of each strain
, we sacrificed four mice per group at days 1 , 7 , 14 , 28 , 56 and 98 following infection , obtained lungs and spleen , homogenized and cultured appropriate dilutions on middlebrook 7h11 medium to determine cfu .
we allocated 12 mice for each infection group to assess virulence of each strain , for which we determined median - survival - time that mice from each group survived following infection .
protocols for experiments involving mice were approved by johns hopkins university animal care and use committee .
we determined minimum inhibitory concentrations for amoxicillin  clavulanate , imipenem ( merck ) , isoniazid ( sigma ) and cycloserine ( sigma ) using the broth dilution method 29 .
we used augmentin ( glaxosmithkline ) , a preparation containing amoxicillin and calvulanate as m. tuberculosis contains lactamases 17 .
for this we inoculated 10 
 m. tuberculosis bacilli in 2.5 ml of 7h9 broth and added drugs at different concentrations .
we incubated these cultures at 37 c and evaluated for growth by visual inspection at 7 and 14 days .
for those samples with diminished growth compared to no - drug control , we determined cfu .
we used four - five week old , female balb / c mice for in vivo assessment of susceptibility of m. tuberculosis lacking ldtmt2 to amoxicillin .
we infected two groups of mice , 36 per group , with aerosolized cultures of either wild - type m. tuberculosis or ldtmt2::tn .
we allocated 12 mice from each group into 3 sub  groups and initiated daily treatment at 2 weeks following infection .
we provided each sub  group either 25 mg kg isoniazid , or 200 mg kg amoxicillin and 50 mg kg clavulanate , or no drug at all by oral gavage . for analysis
, we sacrificed four mice from each treatment sub - group at 1 , 2 and 4 weeks following initiation of therapy , obtained lungs , homogenized in 1 ml of pbs and determined cfu in each organ by plating appropriate dilutions of the homogenates on middlebrook 7h11 selective plates .
we used m. tuberculosis cdc1551 , a clinical isolate , as the host strain to generate a transposon insertion mutant in mt2594 ( ldtmt2::tn ) as described 14 .
this mutant carries a himar1 transposon insertion at + 872 base from the putative translation start site of the gene .
this is a single copy integrating plasmid based on pmh94 backbone 25 , which we modified into a gateway compatible destination vector ( invitrogen ) .
we cloned a wild - type copy of mt2594 along with its promoter into this destination vector pgs202 to generate pgs202_2594 .
we used middlebrook 7h9 liquid medium supplemented with 0.2% glycerol , 0.05% tween80 , 10% vol / vol oleic acid  albumin  dextrose  catalase
( oadc ) and 50 ug ml cycloheximide for in vitro growth , and middlebrook 7h11 solid medium ( becton - dickinson ) for enumerating colony forming units ( cfu ) in in vitro and in vivo growth studies .
we amplified a portion of ltdmt2 with primers 5-ttttcatgatcgccgatctgctggtgc-3 and 5-ttggatcccgccttggcgttaccggc-3 , digested with bsphi  bamhi ( underlined ) and cloned into pet2818 15 . the resulting plasmid , encodes a fusion protein consisting of a methionine specified by the atg initiation codon of pet2818 , residues 55 to 408 of ldtmt2 , and a c - terminal polyhistidine tag with the sequence gsh6 .
we grew e. coli bl21(de3 ) harboring prep4groesl 26 and pet2818ldtmt2 at 37 c in 3
l of brain heart infusion broth containing 150 g ml ampicillin , induced expression using isopropyl 
we purified ldtmt2 from a clarified lysate by affinity chromatography on ni - nitrilotriacetate - agarose resin ( qiagen gmbh , germany ) followed by anion exchange chromatography ( monoq hr5/5 , amersham pharmacia ) with a nacl gradient in 50 mm tris 
we performed an additional size exclusion chromatography on a superdex hr10/30 column equilibrated with 50 mm tris 
finally , we concentrated the protein by ultrafiltration ( amicon ultra4 centrifugal filter devices , millipore ) and stored at 20 c in the same buffer supplemented with 20% glycerol .
we purified disaccharide - tetrapeptide containing amidated meso - diaminopimelic acid ( glcnac  murnac 
mesodapnh2d  ala ) from c. jeikeium strain cip103337 and determined the concentration after acid hydrolysis 27,28 .
next , we tested in vitro formation of muropeptide dimers in 10 l of 50 mm tris  hcl ( ph 7.5 ) containing 300 mm nacl , 5 m ldtmt2 , and 280 m disaccharide  tetrapeptide .
we incubated he reaction mixture for two hours at 37 c and analyzed the resulting muropeptides by nanoelectrospray tandem mass spectrometry using n2 as the collision gas 28 .
we used 45 week old , female balb / c mice ( charles river laboratories ) to study in vivo virulence of the strains and their susceptibility to drugs .
we infected mice with a log phase culture of wild  type m. tuberculosis , or ldtmt2::tn or the complemented strain in an aerosol chamber . for assessing in vivo growth of each strain
, we sacrificed four mice per group at days 1 , 7 , 14 , 28 , 56 and 98 following infection , obtained lungs and spleen , homogenized and cultured appropriate dilutions on middlebrook 7h11 medium to determine cfu .
we allocated 12 mice for each infection group to assess virulence of each strain , for which we determined median - survival - time that mice from each group survived following infection .
protocols for experiments involving mice were approved by johns hopkins university animal care and use committee .
we determined minimum inhibitory concentrations for amoxicillin  clavulanate , imipenem ( merck ) , isoniazid ( sigma ) and cycloserine ( sigma ) using the broth dilution method 29 .
we used augmentin ( glaxosmithkline ) , a preparation containing amoxicillin and calvulanate as m. tuberculosis contains lactamases 17 .
for this we inoculated 10 
 m. tuberculosis bacilli in 2.5 ml of 7h9 broth and added drugs at different concentrations .
we incubated these cultures at 37 c and evaluated for growth by visual inspection at 7 and 14 days .
for those samples with diminished growth compared to no - drug control , we determined cfu .
we used four - five week old , female balb / c mice for in vivo assessment of susceptibility of m. tuberculosis lacking ldtmt2 to amoxicillin .
we infected two groups of mice , 36 per group , with aerosolized cultures of either wild - type m. tuberculosis or ldtmt2::tn .
we allocated 12 mice from each group into 3 sub  groups and initiated daily treatment at 2 weeks following infection .
we provided each sub  group either 25 mg kg isoniazid , or 200 mg kg amoxicillin and 50 mg kg clavulanate , or no drug at all by oral gavage . for analysis
, we sacrificed four mice from each treatment sub - group at 1 , 2 and 4 weeks following initiation of therapy , obtained lungs , homogenized in 1 ml of pbs and determined cfu in each organ by plating appropriate dilutions of the homogenates on middlebrook 7h11 selective plates .
the peptidoglycan layer is a vital component of the bacterial cell wall . 
 the existing paradigm describes the peptidoglycan network as a static structure that is cross - linked predominantly by 43 transpeptide linkages . 
 however , the non - classical 33 linkages predominate the transpeptide networking of the peptidoglycan layer of non - replicating m. tuberculosis 
 1,2 . 
 the molecular basis of these linkages , their significance to the physiology of the peptidoglycan layer , virulence and susceptibility of m. tuberculosis to drugs remain undefined . here , we identify mt2594 ( rv2518c ) as an l , d - transpeptidase that generates 33 linkages in m. tuberculosis . we show that the loss of this gene leads to altered colony morphology , loss of virulence and increased susceptibility to amoxicillin  
 clavulanate during the chronic phase of infection . 
 this suggests that 33 cross - linking is vital to the physiology of the peptidoglycan layer . 
 although a functional homolog exists , expression of mt2594 is dominant throughout the growth phases of m. tuberculosis . 
 the 43 transpeptide linkages are targeted by one of the most widely used classes of antibacterial drugs in human clinical use today , namely -lactams . 
 recently , meropenem  
 clavulanate was shown to be effective against drug resistant m. tuberculosis 
 3 . 
 our study suggests that a combination of an l , d - transpeptidase and a -lactamase inhibitors could effectively target persisters during chronic phase of tb .