ribosomally synthesized 
 and post - translationally modified peptides 
 ( ripps ) with attached adenine residues form a family of trojan - horse 
 antibiotics encoded by gene clusters present in diverse bacteria . here , we show that one such ripp cluster actually 
 directs biosynthesis of an unprecedented cytidylated peptide , which 
 is biologically active and has modifications that allow it to overcome 
 a resistance mechanism that effectively detoxifies related adenylated 
 peptide antibiotics . the data underscore how limited is our knowledge 
 about diversity of ripp modifications performed by evolutionarily 
 related enzymes . this information can be used for creation of new 
 antibiotics while avoiding or limiting bacterial resistance . trojan - horse antibiotic microcin c ( mcc ) is a peptide - adenylate 
 produced by some strains of escherichia coli . mcc - producing 
 cells carry a plasmid - borne six - gene mcc biosynthesis / self - immunity 
 cluster ( figure 1a ) . the ribosomally synthesized mcc precursor heptapeptide mrtgnan , the 
 product of mcca gene , 
 is adenylated by mccb , a thif - type nad / fad adenylyltransferase 
 that attaches amp to the c - terminal asn residue of mcca via a phosphoramidate bond . the reaction 
 proceeds through formation of a succinimide intermediate and requires 
 two atp molecules . as a result of modification 
 the terminal asparagine residue of the peptide is converted to an 
 aspartate - adenylate with phosphoramidate linkage . the products of 
 the mccd and mcce genes jointly are responsible 
 for an additional decoration of the phosphate of adenylated mcca with aminopropyl group , increasing 
 the bioactivity . the peptide 
 part of mcc is responsible for the import into sensitive e.  coli cells through the yejabef transporter . inside the host cells mcc undergoes proteolysis 
 by cellular aminopeptidases , which causes the release of processed 
 mcc  a nonhydrolyzable aspartyl - adenylate that binds aspartyl - trna 
 synthetase ( asp - rs ) and inhibits it , leading to cessation of protein 
 synthesis . the products of mcce and mccf contribute to self - immunity by acetylating processed 
 mcc and cleaving the amidate bond connecting the terminal aspartate 
 and amp , respectively . stand - alone cellular homologues 
 of mcce and mccf that are not part of mcc operon 
 also contribute to basal levels of mcc resistance . bioinformatics searches revealed mcc - like 
 operons 
 in diverse gram - negative and gram - positive bacteria . some of predicted mccb - mcca pairs were subsequently validated in 
 vitro by demonstrating that recombinant mccb proteins adenylate cognate 
 precursor peptides and the resulting compounds inhibit bacterial growth 
 by the same mechanism as e.  coli mcc . such minimal arrangement 
 is apparently sufficient to produce a toxic adenylated peptide without 
 additional decorations and export it outside the producing cells . the mccd , mccf , and mcce homologues are detected in only a few of non - e . some mcc operons contain diverse additional genes that are absent from the 
 prototypic e.  coli operon . these genes may contribute 
 to self - immunity or be responsible for additional modifications of 
 the basic peptide - adenylate scaffold . a group of microcin c - like 
 ripp clusters from bacillus 
 amyloliquefaciens dsm7 , streptococcus bovis jb1 , and various strains of yersinia sp . and serratia sp . is characterized by an extended mccb gene and the presence of an additional gene mccs . the simplest of these gene clusters mcc , from b.  amyloliquefaciens dsm7 , consists of four genes , mcca , mccb ( bamf_rs40475 ) , mccs ( bamf_rs29790 ) , and mccc ( bamf_rs29795 ) ( figure 1a ) . here we show that mccb performs an unprecedented 
 reaction of cytidylation of the terminal residue of cognate mcca . we further show that mccs cooperates with 
 the c - terminal methyltransferase domain of mccb to attach 
 carboxymethyl to the base of the cytidine moiety . we finally show 
 that peptide - cytidylates inhibit aspartyl - trna synthetase , leading 
 to cessation of cell growth . the presence of carboxymethyl modifications 
 inactivates the self - immunity / resistance mechanism mediated by mcce . our results underscore the diversity of modifications carried out 
 by ripp biosynthesis machinery and may provide new tools for rational 
 design of novel antibacterial compounds . the 
 mccb homologue from b.  amyloliquefaciens dsm7 was identified earlier . unlike 
 most other mccb proteins , mccb contains , in addition 
 to the n - terminal adenylyltransferase part , a 30 kda c - terminal 
 extension ( mccbctd ) homologous to sam - dependent 
 methyltransferases . the mccb gene is followed by mccs , whose product is homologous to carboxy - s - adenosyl - l - methionine ( cxsam ) synthetase cmoa of e.  coli . a gene encoding the export pump of the major 
 facilitator superfamily ( mfs ) mccc is located downstream of mccs . in most validated mcc clusters 
 the precursor peptide - encoding mcca genes are located 
 upstream of mccb to ensure an optimal ratio of production 
 of the peptide and its cognate adenylyltranferase ( figure 1a ) . visual inspection 
 of the extended mccs - mccc intergenic region identified 
 a short open reading frame encoding a 19-amino acid peptide mlkirkvkivraqnghytn . ( a ) the structure 
 of mcc clusters from e.  coli and b.  amyloliquefaciens is schematically 
 presented . arrows represent genes and are colored 
 according to validated ( e.  coli ) or predicted 
 ( b.  amyloliquefaciens ) functions of their products . upper panel shows the background signals from b.  subtilis cells bearing empty pht01 plasmid . lower panel shows the additional 
 difference mass peaks detected in b.  subtilis cells carrying plasmid - borne mcc cluster . series of peaks derived from n - terminally formylated mcca are marked in red ; peaks , corresponding to deformylated mcca peptide and its post - translationally modified forms are 
 marked in blue . m / z 2251.3 and 2279.3 
 correspond to peptide - succinimides , m / z 2269.3 and 2297.3 to unmodified mcca , m / z 2574.4 and 2602.4 correspond to cytidylated peptide , m / z 2632.4 and 2660.5 correspond to mcca modified with an unidentified nucleotide . ( c ) fragments 
 of ms / ms spectra of parent mass - ions 2574.4 and 2632.4 shown in ( b ) 
 are presented . both spectra contain m / z 2269 peaks corresponding to the peptide part of the compounds and 
 additional peaks indicating nucleotide monophosphate modification 
 on its c - end terminus . mass shift of 111 da on the upper pannel ( m / z 2574 to m / z 2463 ) matches cytosine while mass shift of 169 da on the 
 lower panel ( m / z 2632 to m / z 2463 ) does not correspond to any known 
 nucleobase . if any mcc - like compounds are produced by b.  amyloliquefaciens dsm7 we performed mass - spectrometric 
 analysis of cell lysates and 
 conditioned media after growth under various conditions . a mass - ion 
 of predicted mcca peptide ( m / z 2269.3 ) or its adenylated form ( m / z 2598.4 ) along with mass - ions corresponding to species 
 with n - formylated peptide and , possibly , forms carrying additional 
 modifications were expected . however , no such peaks were observed . 
 we therefore next cloned the entire mcc cluster into multicopy pht01 plasmid and introduced 
 it into a surrogate host b.  subtilis 168 . mass - spectrometric 
 analysis of transformed cells revealed a series of peaks some of which 
 could have been the products of the b.  amyloliquefaciens 
 mcc cluster ( figure 1b ) . there were [ mh ] signals corresponding to predicted 
 mcca peptide in n - formylated ( 2297.3 ) and deformylated 
 ( 2269.3 ) forms . there were also peaks of corresponding succinimide 
 intermediates ( 2251.3 and 2279.3 ) that should be produced at the first 
 step of mccb - catalyzed reaction . however , 
 peaks corresponding to adenylated mcca ( 329 da shift 
 from unmodified peptide peaks ) were not observed . instead , peaks with 
 305 and 363 da shifts were present . ms / ms analysis of these compounds 
 unambiguously showed that they contain terminally modified mcca ( figure 1c ) . fragmentation of the m / z 2574.4 
 compound resulted in major daughter ions corresponding to unmodified 
 mcca ( m / z 2269 ) and 
 its dehydrated form ( m / z 2251 ) . signals matching mcca - phosphate ( m / z 2331 ) and mcca - phosphate - ribose ( m / z 2463 ) were also clearly seen . therefore , 
 the putative nucleobase of the m / z 2574.4 compound has a molecular weight of 111 da . analysis of fragmentation 
 spectrum of the m / z 2602.4 compound , 
 which produces a 2279 daughter ion matching n - formylated mcca , led to the same conclusion . in contrast , fragmentation 
 of the m / z 2632.4 compound suggested 
 a 169 da putative nucleobase mass . while neither calculated mass difference 
 matched adenine ( 135 da ) , the 111 da mass shift matches cytosine . the 169 da mass change might correspond to an unknown or modified 
 known nucleobase . to check if mccb indeed modifies 
 the precursor peptide 
 with cmp , recombinant mccb protein was prepared and 
 combined with chemically synthesized mcca peptide in 
 the presence of atp , ctp , utp , or gtp . mass - spectrometric analysis 
 revealed the presence of the peptide ( m / z 2269.3 ) and succinimide intermediate ( m / z 2251.3 ) peaks in all four reactions ( figure 2a ) . the apparent efficiency 
 of in vitro modification decreased in the order ctp  atp  
 utp  gtp . if the reaction was carried out in the presence 
 of all four nucleotide triphosphates , only the mcca - cmp 
 product was detected ( figure 2a , lower panel ) . the observed preference for ctp is consistent 
 with detection of mcca - cmp in the b.  subtilis surrogate production host . nucleotide specificity of e.  coli and b.  amyloliquefaciens mccb nucleotidyltransferases 
 in 
 in vitro reactions . ( a ) mccb was combined with mcca ( m / z 2269.3 ) in the presence 
 of indicated individual ntps or an equimolar mixture of four ntps 
 (  + ntp  ) . on mass spectra shown , the differences between the parental peptide 
 mass peak ( m / z 2269.3 ) and peptidyl - nucleotide 
 products are indicated . ( b ) . as 
 in ( a ) but mccbntd , an mccb derivative lacking the c - terminal domain was used instead 
 of mccb . ( c ) reaction products formed in the presence 
 of mccb and mcca ( m / z 763.4 ) . ( d ) as in ( c ) but 
 using formylated mcca ( m / z 791.5 ) . the peaks of sodium salt of mcca and its peptydyl - adenilates 
 are marked with asterisks . the unexpected nucleotide preference of mccb could 
 have been caused by the presence of an additional domain mccbctd that is absent from the e.  coli and most other homologues . the n - terminal fragment of mccb lacking the methyltransferase domain was prepared and its ability 
 to modify cognate mcca peptide was investigated . the mutant , mccbntd , variant exhibited the same promiscuity toward 
 ntp substrates ( figure 2b ) , indicating that the c - terminal domain is not involved in substrate 
 specificity modulation . intrigued by substrate promiscuity of 
 the mccb we 
 tested if mccb also can modify its cognate peptide mcca mrtgnan with various nucleotides . under our reaction conditions , yet , when we tested formylated mcca , which should be considered as a bona fide ribosomally 
 synthesized mccb substrate , modification products by 
 each of the four individual nucleotides were observed ( figure 2d ) . however , the preference 
 to atp was retained when the enzyme was combined with an equimolar 
 mixture of all four nucleotides ( figure 2d , lower spectrum ) . cmoa uses sam and prephenate to produce cxsam , which is then used by the second enzyme , cmob , as a donor of carboxymethyl 
 group in the reaction of carboxymethylation of 5-hydroxyuridine in 
 wobble positions of various trnas . we hypothesized 
 that mccs and mccbctd can also 
 act as a synthase - transferase pair and modify the peptide cytidylated 
 by mccbntd . indeed , the observed mass difference 
 of 58 da between cytidylated mcca peptide ( m / z 2574.4 ) and another peptide nucleotide 
 ( m / z 2632.4 ) detected in b.  subtilis ( figure 1b ) matches carboxymethylation . to test this hypothesis , 
 we determined whether ( i ) mccs can produce cxsam from 
 sam and prephenate and ( ii ) mccbctd can use 
 cxsam to carboxymethylate mcca - cmp . upon incubation 
 of mccs or e.  coli cmoa with sam 
 and prephenate , a new peak appeared during hplc separation of reaction 
 products that was absent from the mixture of sam and prephenate incubated 
 without added proteins ( compare the trace shown in figure 3a with those shown in figures 3bc ) . the same peak 
 was observed among the multiple products of a reference reaction of 
 where cxsam was chemcially synthesized from s - adenosylhomocysteine 
 and iodoacetic acid ( figure 3d ) . in all cases , these hplc peaks contained 
 a substance with [ mh+ ] of 443.2 , which matches cxsam . the main signals 
 in fragmentation spectra of these mass - ions were those of adenine 
 ( m / z 136 ) and its complementary 
 part ( m / z 308 ) ; adenosine ( m / z 250 ) and its complementary part ( m / z 192 ) ; and the 3-amino-3-carboxypropyl 
 tail of methionine ( m / z 102 ) and 
 its complementary part ( m / z 342 ) 
 ( figure 3e ) . since 
 the adenosine moiety of the [ mh+ ] 443.2 compound was not modified , 
 the methionine part must have been carboxylated . since 3-amino-3-carboxypropyl 
 tail was not modified , the carboxylic group must be attached to the 
 s - methyl group of the methionine moiety . we therefore conclude that 
 mccs indeed synthesizes carboxy - s - adenosyl - l - methionine from sam and prephenate , and is therefore a functional 
 analog of e.  coli cmoa . mccs synthesizes 
 cxsam which is used as a donor of 
 carboxymethyl group during modification of mcca - cmp 
 by mccbctd . left sides of panels ( a  d ) 
 show hplc traces of cxsam synthesis reactions products . collected hplc fractions used 
 for in vitro modification of mcca - cmp with mccb are marked with dashed lines . on the right of each panel 
 maldi ms spectra of reactions products obtained after incubation of 
 mcca - cmp ( m / z 2574.4 ) 
 with recombinant mccb and material from the indicated 
 hplc fractions are shown . the newly appearing m / z 2632.4 mass peak matches that obtained in b.  subtilis cells carrying plasmid - borne mcc cluster 
 ( figure 1b ) . ( e ) a 
 representative fragmentation spectrum of m / z 443.2 parental ion from hplc fractions indicated in ( b  d ) . m / z peaks of complementing cxsam fragments 
 are shown in same colors . for details of peaks interpretation ( f ) maldi ms analysis of mcca - cmp carboxymethylation 
 reaction by recombinant mccbctd in the presence 
 of cxsam . we next tested if mccb can use cxsam to modify the 
 cytidine moiety of mcca - cmp . when purified peptidyl - cytidylate 
 and full - sized mccb were combined with enzymatically 
 prepared or chemically synthesized cxsam , an m / z = 2632.4 mass - ion appeared indicating that conversion 
 of the mcca - cmp to mcca - carboxymetylated 
 cmp ( -cxcmp ) had taken place ( figure 3b  d , right panels ) . fragmentation patterns of 
 the newly synthesized compounds matched that of m / z 2632.4 compound produced in b.  subtilis ( figure 1c , lower 
 panel ) , suggesting that carboxymethylation of peptidyl - cytidylate 
 takes place both in vivo and in vitro . we tested if mccbctd alone is sufficient 
 for transferring the carboxymethyl moiety from cxsam to peptidyl - cytidylate . as can be seen from figure 3f , mccbctd successfully caboxymethylated 
 the purified mcca - cmp in the presence of cxsam . when 
 mcca - amp , mcca - ump , and mcca - gmp were tested in reactions with mccbctd and cxsam , no modification was observed ( data not shown ) , indicating 
 that this modification is specific for cytidine . the entire 
 biosynthesis pathway of mcca - cxcmp was 
 reconstituted in vitro in the presence of mcca , mccb , mccs , ctp , sam , and prephenic acid ( figure s1 ) . omission of either component led 
 to the disappearance of the mcca - cxcmp product peak . the mcca - cxcmp was , however , present , when e.  coli cxsam synthase cmoa was used instead of mccs in the 
 coupled mcca modification reaction ( figure s1 ) . the peak corresponding to mcca - cxcmp 
 was absent when mccbntd was used instead 
 of full - sized mccb . overall , the data clearly show that 
 mccbctd is carboxymethyl transferase that 
 uses cxsam produced by mccs to modify mcca cytidylated 
 by mccbntd . attempts to detect biological activity 
 of in vitro synthesized mcc , with or without the carboxymethyl 
 modification , were unsuccessful . neither b.  subtilis 168 nor several e.  coli strains appeared to be sensitive to these compounds . at least in the case of e.  coli , the result 
 could be due to poor intracellular transport , lack of processing , 
 or the absence of appropriate target . the serendipitous observation 
 about decreased nucleotide specificity of mccb when 
 presented with formylated cognate peptide substrate allowed us to 
 obtain , in addition to fmcca - amp , the fmcca - cmp , fmcca - ump , and fmcca - gmp compounds . 
 an mcca - cxcmp variant was also prepared by in vitro 
 carboxymethylation of fmcca - cmp with the mccbctd - mccs pair ( figure s2 ) . the resulting compounds contained the e.  coli fmcca transport peptide that in the context of amp modification 
 is recognized by the yejabef transporter and is efficiently processed by intracellular aminopeptidases . each of the compounds was tested against a wild - type e.  coli strain and isogenic yej mutant 
 in the spot bioactivity test . an sbma mutant lacking 
 a transporter that is not involved in mcc transport 
 was included as a control . as can be seen in figure 4a , mcca - amp , mcca - cmp , and mcca - cxcmp inhibited the growth of wild - type 
 and sbma strains . the mic values determined using 
 spot bioactivity test were 1 , 40 , and 20 m for mcca - amp , mcca - cmp , and mcca - cxcmp , respectively . ( a ) five microliters 
 of 50 m solutions of hplc - purified peptidyl - nucleotides were 
 deposited onto lawns of e.  coli b cells . panels are arranged in rows showing 
 results obtained with different tester strains : ( 1 ) wild type e.  coli b ; ( 2 ) e.  coli b yejb ; ( 3 ) e.  coli b sbma ; ( 4 ) e.  coli b overexpressing 
 mcce ; ( 5 ) e.  coli b overexpressing mccf ; ( 6 ) e.  coli b overexpressing asp - rs ; and ( 7 ) e.  coli b overexpressing pro - rs . growth inhibition zones are seen as clear 
 circles on the turbid surface of cell lawn . ( b ) in vitro inhibition 
 of aminoacylation of trna by peptidyl - nucleotides . s30 
 extracts prepared from wild - type e.  coli cells 
 were supplied with hplc - purified compounds and incorporation of radioactive 
 aspartate was measured . e.  coli cells 
 that express mcce become resistant 
 to mcc due to acetylation of processed mcc ; cells that express mccf become resistant due to cleavage of intact 
 or processed mcc . cells expressing mccf 
 were resistant to both mcca - cmp and mcca - cxcmp . in contrast cells expressing mcce were resistant to mcca - cmp but remained sensitive to mcca - cxcmp 
 ( figure 4a ) . thus , 
 carboxymethylation of cytidine moiety allows to overcome one of the 
 two resistance mechanisms to mcc - like peptide  nucleotide antibiotics . cells expressing asp - rs but not pro - rs became resistant to mcca - amp , mcca - cmp , and mcca - cxcmp 
 ( figure 4a ) . to directly 
 demonstrate that peptidyl - cytidylates target asp - rs , in vitro trna aminoacylation reactions were carried out . nucleotides to preprocess the peptide part 
 of antibiotics and trna aminoacylation reaction was 
 next carried out . as can be seen from figure 4b , mcca - amp , mcca - cmp , and mcca - cxcmp inhibited incorporation of radioactive 
 asp in acid - insoluble fraction , while mcca - ump and mcca - gmp were inactive . a mimic of processed mcca - amp aspartyl sulfamoyl adenosine ( dsa ) and aspartyl sulfamoyl cytidine 
 ( dsc ) , a mimic of processed mcca - cmp , were also tested 
 ( figure s3 ) . aminoacylation reactions were 
 carried out immediately after dsa or dsc addition , since no processing 
 is required . in both cases , inhibition of aminoacylation was observed . both with peptidyl - nucleotides and with aminoacyl sulfamoyl nucleotides , 
 adenylated compounds were better inhibitors than cytidylate ones . there are roughly a dozen of known natural cytosine - containing 
 antibiotics , all of them being the products 
 of nonribosomal synthesis . in this work we describe the first bioactive 
 ripp modified with cytidine . during the synthesis of e. coli microcin c the attachment of the nucleotide moiety to the peptide 
 part is carried out by adenylyltransferase mccb via 
 a two - step mechanism : first , carboxyl group of the 
 c - terminal asn residue of mcca is activated with atp forming a liable 
 peptidyl - amp intermediate , which undergoes intramolecular cyclization 
 resulting in the formation of peptidyl - succinimide ; second , a new 
 molecule of atp is used for generation of stable peptidyl - nucleotide 
 with nonhydrolyzable n our work reveals , surprisingly , 
 that the mccb enzymes have relaxed specificity toward the nucleobase 
 moiety of ntp substrate at both stages of the reaction . thus , the 
 nucleotide - binding site of mccb enzymes should be able to accommodate 
 different nucleoside triphosphates substrates . mccb has a clear preference for ctp at both stages of the reaction . in 
 contrast interestingly , the specificity 
 of mccb is strongly modulated by the presence of the 
 formyl group located at the other end of the peptide substrate . the 
 mechanism of this modulation remains unknown but can be caused by 
 more avid binding of formylated mcca to the enzyme , allowing the presumably 
 slower reaction of modification with less preferred nucleotide to 
 occur . mccb proteins are part of a very large family of thif - type 
 nad / fad adenylyltransferases . while many enzymes from the family were 
 shown to function as adenylyltransferases , most have not been studied . our work suggests that many enzymes of the family may be transferring 
 nucleotides other than amp , raising a possibility that there remains 
 a very large class of post - translational modifications that remain 
 to be discovered . at the last stage of mcc maturation , the c - terminal 
 domain of mccb transfers the carboxymethyl group to 
 the nucleobase of peptidyl - cytidylate using the cxsam as a donor ( scheme 1 ) . the cxsam cofactor 
 was discovered during analysis of the e.  coli cmoa - cmob enzyme pair . cmoa participates in modification 
 of uridine to 5-oxyacetyl uridine ( cmo5u ) at the wobble position of 
 anticodon loop of trnas by generating cxsam used by the cmob enzyme . cmoa - like enzyme mccs encoded 
 in the mcc cluster also synthesizes 
 cxsam . n - terminal residues of mcca 
 ( mlkirkvkivraqnghyt ) are denoted as r for clarity . to the best of our knowledge , our study reveals only 
 the second 
 instance of the use of cxsam in living cells . linked genes encoding 
 cmoa - cmob homologues are characteristic for proteobacteria and some cyanobacteria , where they likely perform a function similar to that in e.  coli , but are extremely rare in gram - positive bacteria . the c - terminal domain of mccb does not show any significant 
 similarity to cmob from e.  coli . since it uses 
 cxsam for modification of cytosine instead of 5-hydroxy uridine it 
 might employ a different mechanism for carboxymethyl transfer . in 
 contrast to cmob , mccbctd has 
 high specificity to cxsam since no alkyl transfer was detected in 
 the presence of sam . genes encoding cmoa homologues without neighboring cmob homologues are common and our work suggests that such 
 enzymes may produce cxsam for nucleobase modification in bioactive 
 compounds unrelated to trna . mccbctd appears to lack specificity toward 
 the peptide part of peptide - cytidylates , which allowed us to produce 
 carboxymethylated cytidylate of e.  coli mcca . the modification is functionally important for while 
 it has a marginal effect on target inhibition , it inactivates one 
 of the mechanisms of resistance to mcc - like compounds , clearly a beneficial 
 trait for the producing organism . nonhydrolyzable aminoacyl - adenylates 
 are potent inhibitors of trna 
 aminoacylation reactions that proceed through an obligatory activated 
 aminoacyl - adenylate intermediate . thus , it was an expected result 
 that the common product of proteolytic processing of various peptide - adenylates 
 related to e.  coli microcin c inhibits asp - rs . processed peptide - cytidylates studied in this 
 work also inhibit asp - rs . the result is not a consequence of general 
 promiscuity of asp - rs to the nucleotide moiety of aspartyl - nucleotides , 
 since introduction of uridine or guanosine at the terminus of the 
 mcca peptide had no inhibitory effect either in vivo or in vitro . the specificities of other aminoacyl - trna synthetases may be different , 
 since a study of chemically synthesized isoleucyl - sulfamoyl nucleoside 
 analogues attributed the highest inhibitory activity toward ile - rs 
 to uridylated followed by adenylated- and cytidylated compounds . while the group of organisms naturally targeted 
 by b.  amyloliquefaciens dsm7 producing mcc is presently unknown , one may hypothesize that the asp - rs 
 from these bacteria is inhibited more efficiently by aspartyl - cytidylates 
 than by the adenylate . several other natural pyrimidine - containing 
 compounds that bind 
 to the catalytic center of aminoacyl trna synthetases are known . one 
 is a natural trojan - horse antibiotic , albomycin 2 , produced by streptomyces sp . it comprises of the 
 vehicle siderophore part and the inhibitor part , an n4-carbamoyl-3-methylcytosine 
 attached to diseryl - thioxylofuranosyl moiety . processed albomycin 2 inhibits ser - rs , though 
 its structure is completely different from that of processed mcc . bioinformatics analysis predicts that mcc operons 
 from evolutionary distant bacteria likely produce cytidylated , rather 
 than adenylated ripps . some of these operons also contain additional 
 genes , distinct from functionally characterized mcc genes of e.  coli and the b. amyloliquefaciens genes characterized in this work . the 
 products of these genes may be carrying out additional decorations 
 that increase the activity of basic peptidyl - nucleotides and/or decrease 
 resistance of targeted bacteria . the reasons why a particular operon 
 evolves to produce a particular kind of peptide nucleotide 
 and additional decoration sets presents an interesting evolutionary 
 and functional problem that remains to be solved . in the interim , 
 the ability to attach various modified nucleotides to peptides capable 
 of facilitated transport into cells presents an attractive strategy 
 for design of novel antibacterial compounds . b.  amyloiquefaciens dsm7 was purchased from german collection of microorganisms and 
 cell cultures ( dsmz ) . b.  subtilis 168 was used as a heterologous 
 host for b.  amyloliquefaciens mcc cluster expression . protein expression and in vivo mcc activity tests were done in e.  coli b strain . e.  coli bw25113 
 rimi riml rimj was used for in vitro 
 asp - rs inhibition assay . m9 minimal medium complemented with 
 0.1% casamino acids , 0.5% glycerol , and either 0.01% yeast extract 
 ( for e.  coli ) or 5 mg / l of l - tryptophan 
 ( for b.  subtilis ) was used where indicated . b.  amyloliquefaciens mcc gene cluster including its 
 5 intergenic region was amplified with phusion dna polymerase 
 and primers bam_f and bam_r . pcr product was cloned into pht01 shuttle 
 vector between kpni and bamhi to 
 create pht - mcc . this removes plasmid - borne pgrac 
 promoter and introduces the mcc natural promoter . pht - mcc plasmid was transformed into b.  subtilis 168 by electroporation and clones were selected on lb agar plates 
 with 10 g / ml chloramphenicol . mcc production 
 by the heterologous b.  subtilis host was tested 
 by maldi ms of cells grown for 8 h ( od600 = 1 ) in lb broth 
 supplemented with 10 g / ml chloramphenicol . full length 
 mccb from b.  amyloliquefaciens and mccb from e.  coli were expressed 
 as fusion proteins with n - terminal leaderless male . for this , the 
 corresponding pcr products were introduced between bamhi and xhoi restriction sites of pet22mbp . dna fragments spanning 1348 codons and 348612 codons 
 of b.  amyloliquefaciens mccb were cloned into bamhi and xhoi restriction sites of pet22mbp to obtain mbp - fused 
 mccbntd and mccbctd correspondingly . coding sequences of e.  coli cmoa and b.  amyloliquefaciens mccs were cloned 
 into pet28 between bamhi and xhoi sites to create 
 n - terminally fused his tag . e.  coli asps and pros were cloned into pbad - his between ncoi and xhoi sites 
 to create pbad - asprs and pbad - prors plasmids . for protein expression e.  coli bl21(de3 ) 
 were transformed with the proper plasmids , grown on lb medium supplemented 
 with either kanamycin or ampicillin at 37 c until od600 reached 0.6 and induced with 1 mm isopropyl -d-1-thiogalactopyranoside . cells were allowed to grow for an additional 1116 h at 20 
 c before harvesting . pelleted cells were washed with loading 
 buffer ( 20 mm tris - hcl ph 8.0 , 150 mm nacl , 5 mm dtt ) and resuspended 
 in the same buffer supplemented with 0.2 mm phenylmethane sulfonyl 
 fluoride and 1 mg / ml lysozyme . after sonication the lysates were cleared 
 by centrifugation at 15  000 rcf for 30 min . at this stage for 
 his - tagged proteins purification imidazole his - tagged proteins were loaded to hitrap chelating hp column ( ge 
 healthcare ) precharged with co and equilibrated with 
 the loading buffer . mbp - fused proteins were purified on mbptrap 
 hp ( ge healthcare ) and eluted with 10 mm maltose in the loading buffer . synthetic peptides 
 mcca ( mlkirkvkivraqnghytn ) and mcca ( mrtgnan ) 
 with and without n - terminal formyl group were purchased from genscript 
 ( usa ) . reactions contained 100 m peptide , 2 m of the 
 corresponding enzyme , and 5 mm of one of the ntps in 100 mm tris - hcl 
 ph 8.0 , 150 mm nacl , 10 mm mgcl2 , 5 mm dtt . for the nucleotide 
 selection test all four ntps were added to the reaction at the 2 mm 
 final concentration . reaction mixtures were incubated for 1 to 16 
 h at 32 c depending on the required yield of the peptide  nucleotide . when necessary , reaction was quenched with 0.1% trifluoroacetic acid 
 ( tfa ) , precipitated protein was removed by centrifugation and soluble 
 peptide  nucleotides were purified using eclipse plus c18 column 
 ( 4.6  250 mm , particle size 5 m ) ( agilent technologies ) 
 at flow rate of 1 ml / min . a 10 min ( approximately 2.5 column volumes ) 
 linear gradient from 5 to 15% acetonitrile in 0.1% tfa was applied . fractions 
 corresponding to the peptide  nucleotides were dried in a rotary 
 evaporator and redissolved in water . published extinction coefficients 
 for nucleotide monophosphates were used to calculate peptidyl nucleotide 
 concentrations . for enzymatic cxsam 
 production 1 mm 
 sam and 1 mm prephenate were incubated with either 10 m cmoa 
 or 10 m mccs in 100 mm tris - hcl ph 8.0 , 150 mm 
 nacl , 5 mm dtt for 2 h at 32 c . chemical synthesis of cxsam 
 was performed as described in kim et al . , 2013 . in brief , 30 mg of 2-iodoacetic acid was added to 1 ml 
 of the 5 mg / ml suspension of s - adenosyl - l - homocysteine in 300 mm ammonium bicarbonate and incubated at 37 
 c for 20 h with constant agitation . analytical 
 chromatography was performed on prontosil-1205-c18 aq column 
 ( particle size 5 m , 2.0  75 mm column dimensions ) ( econova , 
 russia ) at a flow rate of 0.2 ml / min with uv detection at 260 nm . cxsam was eluted in a linear gradient from 0 to 25% acetonitrile 
 in the presence of 10 mm na2hpo4 in 8 min . preparative 
 purification was performed using c18 eclipse plus ( 4.6  100 
 mm , 5 m particle size ) ( agilent technologies ) column and eluted 
 using the same buffer system . neutral ph and low acetonitrile content 
 in the eluted cxsam samples allowed their direct use in the carboxymethylation 
 reactions . reaction 
 mixture contained 
 100 m purified mcca - cmp or mcca - cmp peptidyl - cytidylates , 0.5 mm cxsam , and 2 m mccbctd in 20 mm tris - hcl ph 8.0 , 150 mm nacl , 5 mm dtt . reaction 
 was carried out for 6 h at 32 c then quenched with 0.1% tfa , 
 and centrifuged to remove protein precipitate . fractions containing carboxymethylated 
 peptide - cytidylates were dried in a rotary evaporator and redissolved 
 in water . 1-l aliquot of in 
 vitro reaction mixture or 0.2 l of cells were diluted 
 in 10 l of 0.5% tfa . one l of the diluted sample was 
 mixed with 0.5 l of 2,5-dihydroxybenzoic acid solution ( 20 
 mg / ml in 30% acetonitrile , 0.5% tfa ) and left to dry on the stainless 
 steel target plate at room temperature . maldi - tof ms analysis was 
 performed on ultraflexetreme maldi - tof / tof mass spectrometer ( bruker 
 daltonik , germany ) equipped with nd laser . the mh+ molecular ions 
 were measured in reflector mode ; the accuracy of monoisotopic mass 
 peak measurement was within 30 ppm . spectra were acquired by averaging 
 of a minimum 1000 laser shots from  sweet spots  of 
 matrix crystals . spectra of fragmentation were obtained in lift mode , 
 the accuracy of daughter ions measurement was within 1 da range . mass - spectra 
 were processed with the use of flexanalysis 3.2 software ( bruker daltonik , 
 germany ) and analyzed manually . wild type e.  coli b strain and its isogenic 
 deletion mutants yejb and sbma transformed with vector plasmid 
 pbad - his were used as tester strains . for protein target and immunity 
 mechanism verification tests , e.  coli b strain 
 transformed with expression plasmids pbad - asprs , pbad - prors , pbad - mccf , 
 and pbad - mcce were used . cells were grown in lb medium supplemented 
 with 100 g / ml ampicillin and 10 mm arabinose . the overnight 
 culture was diluted 1000-fold with soft agar containing m9 medium 
 supplemented with 1% glycerol , 0.01% yeast extract , 10 mm arabinose 
 and 100 g / ml ampicillin . nucleotides 
 solutions were deposited on the surface of the solidified agar and 
 allowed to dry . kanamycin ( 1 mg / ml ) was used as a control growth inhibitor . e.  coli bw25113 
 rimi rimj riml were grown in 50 ml of lb medium at 37 c until 
 od600 reached 0.8 . cells were collected by centrifugation , 
 washed with 20 mm tris - hcl , ph 8.0 , 10 mm mgcl2 , 100 mm 
 kcl , resuspended in 1 ml of the same buffer supplemented with 1 mm 
 dtt , and disrupted by sonication . the s30 cell lysate was prepared 
 as described earlier . to process the 
 peptide part , 25 m peptidyl - nucleotides were preincubated with 
 s30 cell lysate ( 3 mg / ml final protein concentration ) for 2 h at 25 
 c . sixteen l of the aminoacylation mixture ( 30 mm tris - hcl , 
 ph8.0 , 30 mm kcl , 8 mm mgcl2 , 1 mm dtt , 3 mm atp , 5 mg / ml 
 bulk e.  coli trna , 23 m c - asp ) were combined with 4 l of processed peptidyl - nucleotide 
 samples and incubated for 5 min at 25 c . after washing with ice - cold 
 10% trichloroacetic acid , filters were washed with acetone and dried .
microcin 
 c and related antibiotics are trojan - horse peptide - adenylates . 
 the peptide part is responsible for facilitated transport inside the 
 sensitive cell , where it gets processed to release a toxic warhead  a 
 nonhydrolyzable aspartyl - adenylate , which inhibits aspartyl - trna synthetase . 
 adenylation of peptide precursors is carried out by mccb thif - type 
 nad / fad adenylyltransferases . here 
 , we describe a novel microcin c - like 
 compound from bacillus amyloliquefaciens 
 . the b.  amyloliquefaciens mccb demonstrates an unprecedented 
 ability to attach a terminal cytidine monophosphate to cognate precursor 
 peptide in cellular and cell free systems . 
 the cytosine moiety undergoes 
 an additional modification  carboxymethylation 
 that is 
 carried out by the c - terminal domain of mccb and the mccs enzyme that 
 produces carboxy - sam , which serves as a donor of the carboxymethyl 
 group . 
 we show that microcin c - like compounds carrying terminal cytosines 
 are biologically active and target aspartyl - trna synthetase , and that 
 the carboxymethyl group prevents resistance that can occur due to 
 modification of the warhead . 
 the results expand the repertoire of 
 known enzymatic modifications of peptides that can be used to obtain 
 new biological activities while avoiding or limiting bacterial resistance .