The Product of Yersinia pseudotuberculosis mcc Operon Is a Peptide

Oct 18, 2017 - Upon processing, McCYps leaves the producing cell, is actively taken up by the YejABEF transporter in sensitive cells and demonstrates ...
0 downloads 7 Views 2MB Size
Subscriber access provided by University of Florida | Smathers Libraries

Article

The Product of Yersinia pseudotuberculosis mcc Operon Is a Peptidecytidine Antibiotic Activated Inside Producing Cells by the TldD/E Protease Darya Tsibulskaya, Olga Mokina, Alexey Kulikovsky, Julia Piskunova, Konstantin Severinov, Marina Serebryakova, and Svetlana Dubiley J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07118 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 18, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

The Product of Yersinia pseudotuberculosis mcc Operon Is a Peptide-cytidine Antibiotic Activated Inside Producing Cells by the TldD/E Protease Darya Tsibulskaya†,§, Olga Mokina§,†, Alexey Kulikovsky§,†, Julia Piskunova§,†, Konstantin Severinov§, †,#,*, Marina Serebryakova†,‡, and Svetlana Dubiley§,† †

Institute of Gene Biology, Russian Academy of Science, 34/5 Vavilov str., 119334 Moscow,

Russia; §

Center for Data-Intensive Biomedicine and Biotechnology, Skolkovo Institute of Science and

Technology, 3 Nobel str., 143026 Moscow, Russia; #

Waksman Institute for Microbiology, 190 Frelinghuysen Road, Piscataway, New Jersey 08854-

8020, USA ‡

A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,

Leninskie Gory 1, Bldg. 40, Moscow 119991, Russia.

Running title: Activation of antibiotic production by the Tld protease *Corresponding author: Konstantin Severinov, Waksman Institute, 190 Frelinghuysen Road, Piscataway, NJ 08854. E-mail: [email protected]; phone: 848 445 6095; FAX: 848 445 5735 Darya Tsibulskaya ([email protected]) Olga Mokina ([email protected]) Alexey Kulikovsky ([email protected]) Julia Piskunova ([email protected]) Konstantin Severinov ([email protected]) Marina Serebryakova ([email protected]) Svetlana Dubiley ([email protected])

Keywords: microcin, peptidyl-cytidylate, carboxymethyl cytidine, ribosomally synthesized and post-translationally modified peptides, TldD, TldE

1 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

ABSTRACT Microcin C is a heptapeptide-adenylate antibiotic produced by some strains of Escherichia coli. Its peptide part is responsible for facilitated transport inside sensitive cells where it is proteolysed with release of a toxic warhead - a non-hydrolysable aspartamidyladenylate, which inhibits aspartyl-tRNA synthetase. Recently, a microcin C homolog from Bacillus amyloliquefaciens containing a longer peptide part modified with carboxymethylcytosine instead of adenosine was described but no biological activity of this compound was revealed.

Here,

we

characterize

modified

peptide-cytidylate

from

Yersinia

pseudotuberculosis. As reported for B. amyloliquefaciens homolog, the initially synthesized compound contains a long peptide that is biologically inactive. This compound is subjected to endoproteolytic processing inside producing cells by the evolutionary conserved TldD/E protease. As a result, an 11-aminoacid long peptide with C-terminal modified cytosine residue is produced. This compound is exported outside the producing cell and is bioactive, inhibiting sensitive cells in the same way as E. coli microcin CProteolytic processing inside producing cells is a novel strategy of peptide-nucleotide antibiotics biosynthesis that may help control production levels and avoid toxicity to the producer.

2 ACS Paragon Plus Environment

Page 3 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

INTRODUCTION Microcin C (McC) is a peptide-adenylate produced by some strains of Escherichia coli and other enteric bacteria carrying the mcc gene cluster.1 The ribosomally synthesized McC precursor heptapeptide MRTGNAN, the product of mccAEco gene, is adenylated by MccBEco, a THIF-type adenylyltransferase. The reaction proceeds in two steps: ATP-dependent formation of peptide-succinimide intermediate followed by hydrolysis of succinimide ring and the attachment of AMP to resulting aspartamide through a non-hydrolysable phosphoramidate bond.2 The peptide part of McC is responsible for export from the producing cell through the MccCEco pump and import into sensitive cells through the YejABEF transporter.3 Inside the sensitive cells the peptide part is degraded by aminopeptidases releasing “processed McC” - a non-hydrolysable aspartamidyl-adenylate4 that binds aspartyl-tRNA synthetase (Asp-RS) and inhibits it, leading to cessation of protein synthesis.5 McC is a founding member of a family of antibiotics encoded by numerous mcc-like operons in diverse gram-negative and gram-positive bacteria.6 Most of validated mcc-like gene clusters contain just three genes, mccABC, which is sufficient to produce a toxic adenylated peptide and export it outside the producing cells.7 The E. coli mcc gene cluster contains 6 genes (Fig. 1). The products of the mccDEco and mccEEco genes are jointly responsible for decoration of the phosphate of adenylated MccAEco with aminopropyl group8, increasing the bioactivity.9 MccEEco is a bifunctional protein, which participates in aminopropylation with its N-terminal decarboxylase domain8 and provides self-immunity by acetylating processed McC with its Cterminal domain.10 The product of mccFEco contributes to self-immunity by cleaving the phosphoramidate bond connecting the terminal aspartamide and AMP.11 The mccD, mccE, and mccF homologs are present in only a handful of non-E. coli mcc clusters. On the other hand, some mcc-like operons contain additional genes that are absent from the E. coli operon.7 A group of mcc-like clusters from Bacillus amyloliquefaciens DSM7, Streptococcus bovis JB1, and various strains of Yersinia sp. and Serratia sp. encode MccB homologues extended with a C-terminal methyltransferase domain and also contain an additional gene mccS. In a recent study of B. amyloliquefaciens McC-like compound we have shown that the N-terminal part of B. amyloliquefaciens MccB, MccBNTDBam, performs cytidylation instead of adenylation of the terminal Asn residue of cognate MccABam.12 MccSBam is a carboxy-Sadenosyl-L-methionine (cxSAM) synthetase. The cxSAM is used by MccBCTDBam as a donor of 3 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 26

carboxymethyl group for additional modification of MccABam-CMP. Though no biological activity for McCBam was revealed, modified aspartamidyl-cytidylate that is released after processing of chimeric compound based on E. coli MccA peptide inhibits Asp-RS. The presence of carboxymethyl modification increases the toxicity and inactivates the self-immunity/resistance mechanism mediated by MccEEco.12

Figure 1 Comparison of mcc gene clusters from E. coli, B. amyloliquefaciens DSM7, and Y. pseudotuberculosis IP 32953. Genes are indicated by arrows (letters indicate gene names, i.e., “B” stands for “mccB”), same colors show homologous genes. MccE of E. coli is a bifunctional protein and is encoded by two adjacent genes in the gene Y. pseudotuberculosis IP 32953 clusters. For E. coli and Y. pseudotuberculosis IP 32953 clusters, positions of transcription terminators are schematically shown as hairpins. Known (E. coli, B. amyloliquefaciens DSM7) or predicted (Y. pseudotuberculosis IP 32953) mcc genes products functions are listed at the bottom. RRE - RiPP precursor peptide recognition element; MFS - Major Facilitator Superfamily; cxSAM – carboxy-SAM.

Here, we have investigated McCYps, a product of Y. pseudotuberculosis IP 32953 mcc operon. We show that upon modification with cytidine and propylamine, the 42 aminoacid long peptide part of pro-McCYps is processed inside the producing cell releasing bioactive McCYps whose peptide part is just 11 amino acids long. The processing is carried out by an evolutionary conserved TldD/E protease. Upon processing, McCYps leaves the producing cell, is actively taken up by the YejABEF transporter in sensitive cells and demonstrates toxicity comparable with that of McCEco. Proteolytic processing inside producing cells is a novel strategy of peptide-nucleotide antibiotics biosynthesis that may help control production levels and avoid toxicity to the producer. 4 ACS Paragon Plus Environment

Page 5 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

RESULTS Y. pseudotuberculosis mcc operon encodes a compound with antibacterial activity A putative mcc-like biosynthesis/self-immunity gene cluster present in Yersinia pseudotuberculosis IP32953 combines characteristics of both E. coli and B. amyloliquefaciens operons (Fig. 1). The Y. pseudotuberculosis MccA peptide (MccAYps) is 42 amino acids long and contains a terminal asparagine. The mccAYps gene is located upstream of mccCYps, which encodes a predicted export pump of the Major Facilitator Superfamily (YPTB1884). The next gene is mccBYps (YPTB1885). Recombinant MccBYps was shown to adenylate MccAYps in vitro.7 Similar to MccBBam and unlike MccBEco, MccBYps contains, in addition to a nucleotydyltransferase part, a C-terminal SAM-dependent methyltransferase domain. The mccBYps gene is followed by mccSYps (YPTB1886), a homologue of mccSBam. The following gene, mccXYps, encodes a protein of unknown function (YPTB1887). MccDYps (YPTB1888) is homologous to MccDEco, which uses SAM to transfer carboxylated propylamine onto the phosphate of heptapeptide adenylate produced by MccBEco. The Y. pseudotuberculosis MccE homolog is split into two separate polypeptides encoded by adjacent genes. MccE1Yps (YPTB1889), the homolog of MccENTDEco, is likely a decarboxylase that removes the carboxyl from the carboxylated propylamine transferred by MccD.8 MccE2Yps (YPTB1890) is a GNAT-type acetyltransferase expected to be involved in self-immunity. To check if any McC-like compounds are produced by Y. pseudotuberculosis IP 32953 carrying the mccYps operon in its genome, we monitored the ability of these cells to inhibit growth of McC-sensitive E. coli or Y. pseudotuberculosis #3526 strain that does not carry the mccYps operon in its genome. No growth inhibition zones were observed and we therefore cloned the entire mccYps cluster along with the upstream region that should contain a promoter into a multicopy pDT1 plasmid vector and introduced the resulting pDT1-Ymcc plasmid in Y. pseudotuberculosis #3526 or wild-type E. coli. Mass-spectrometric analysis of pDT1-Ymcccontaining Y. pseudotuberculosis #3526 cells did not reveal any additional peaks compared to control strain transformed with the pDT1 vector. In contrast, in the mass-spectrum of E. coli cells harboring the pDT1-Ymcc plasmid, two mass-peaks that were absent from cells transformed with pDT1 and that corresponded to unmodified full-sized MccAYps peptide and its N-terminal 31-aminoacid fragment were clearly seen (Fig. S1). Despite the presence of MccAYps product in E. coli cells transformed with pDT1-Ymcc, no inhibition of microcin C-sensitive E. coli tester 5 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 26

cells growth was observed (Fig. 2A). Considering the length of the mccYps operon, we wondered if all of its genes were expressed in the heterologous E. coli host. To this end, real-time RT-PCR reactions with primer pairs specific for every gene of plasmid-borne mccYps operon were performed for RNA prepared from wild-type E. coli. Expression levels were normalized to that of pDT1-Ymcc kanamycin-resistance gene aph gene. As can be seen from Fig. 2B, there was strong expression of the mccAYps gene. However, expression of downstream mccCYps and mccBYps genes was very low. Since mccBYps encodes a nucleotydyltransferase responsible for MccAYps modification,7 the result may explain the presence of unmodified MccAYps in E. coli cells transformed with pDT1-Ymcc as revealed by mass-spectrometry. Analysis of RNA prepared from Y. pseudotuberculosis #3526 transformed with pDT1-Ymcc revealed much more uniform mccYps genes expression. However, the level of mccAYps expression was significantly lower than in E. coli, which may explain the lack of formation of growth inhibition zones around these cells (Fig. 2A).

Figure 2. E. coli cells carrying plasmid-borne mcc operon from Y. pseudotuberculosis IP 32953 produce an antibacterial compound(s) in the absence of the hns gene. A. Growth inhibition by Y. pseudotuberculosis #3526 and wild-type or ∆hns E. coli BW25113 cells harboring pDT1-Ymcc or control pDT1 plasmids on E. coli B cell lawns. Results of overnight growth at 30 °C are shown. A growth inhibition zone is only visible around the patch of ∆hns E. coli BW25113 transformed with pDT1-Ymcc.

6 ACS Paragon Plus Environment

Page 7 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

B. Expression levels of individual mccYps genes in Y. pseudotuberculosis #3526 and wild-type or ∆hns E. coli BW25113 cells harboring pDT1-Ymcc. Results of RT-PCR with primer pairs specific for indicated genes are presented. Expression levels were normalized to that of kanamycin resistance aph gene of pDT1-Ymcc.

We tested an E. coli ∆hns mutant lacking a transcription factor known to be involved in global negative control of expression of horizontally-transferred genes as a production host.13 Though expression of mccAYps was decreased several fold compared to levels seen in wild-type E. coli in this host (Fig. 2B), expression levels of mccCYps and mccBYps were strongly increased. Moreover, clear growth inhibition zones on lawns of microcin C-sensitive E. coli B tester cells were observed around patches of E. coli ∆hns cells carrying pDT1-Ymcc (Fig. 2A). Comparison of mass spectra of E. coli ∆hns cells with and without plasmid-borne mccYps operon revealed several difference mass-peaks that were present only in cells transformed with pDT1-Ymcc (Fig. 3A). In addition to m/z 4656 peak matching unmodified MccAYps peptide, there was a m/z 4638 peak matching its succinimide intermediate, and an additional m/z 5076 peak (Fig. 3A). MS-MS analysis of 5076 peak (Supplementary Fig. S2) revealed a major fragmentation mass-ion with an m/z of 4656 matching unmodified MccAYps peptide. Thus, the compound of the 5076 peak consists of the MccAYps peptide, likely modified by the products of the mccYps operon, and this modification(s) adds 420 Da.

7 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 26

Figure 3. Identification of products of full-sized and partial Y. pseudotuberculosis IP 32953 mcc operons accumulating inside E. coli ∆hns cells. A. MALDI-MS spectra of BW25113 ∆hns cells harboring control pDT1 vector (upper panel) and pDT1Ymcc (lower panel) plasmids and the corresponding growth inhibition zones formed on lawns of E. coli B tester cells. Mass-peaks that are common to both control and experimental cells match E. coli ribosomal protein RS22 (m/zcalc 5097) and double-charged ion of acidic stress chaperones HdeA (m/zcalc 4870) and

8 ACS Paragon Plus Environment

Page 9 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

HdeB (m/zcalc 4532). The m/z values of difference mass-peaks of MccAYps peptide and its modified forms are highlighted with red-color font (see text for details). B. Mass-spectra of ∆hns E. coli cells transformed with various pDT1-Ymcc plasmid derivatives harboring indicated truncations of the Y. pseudotuberculosis IP 32953 mccACBXDE1E2 operon and growth inhibition zones on lawns of wild-type E. coli cells formed around patches of same cells are shown. C. The biosynthesis pathway of the product of Y. pseudotuberculosis IP 32953 mcc operon. The part of the pathway that has been reconstituted in vitro is highlighted by green shading. The last two steps without shading are analogous to previously characterized enzymatic maturation steps of E. coli microcin C8 and are carried out by homologous Y. pseudotuberculosis gene products. R=MHQSEIKLTKRLKIKRVDVNKVKEQQKKVLECGAATCGGGS.

The biosynthetic pathway of Y. pseudotuberculosis mcc operon product and identification of genes involved in production of toxic compound To define the roles of individual mccYps genes in MccAYps modification, derivatives of pDT1-Ymcc were constructed by sequentially deleting mccYps operon genes. The E. coli ∆hns cells harboring these plasmids were tested for biological activity and subjected to massspectrometric analysis (Fig. 3B). Cells harboring a plasmid with the largest deletion that left only the minimal three-gene arrangement (mccACB) did not inhibit growth of McC-sensitive E. coli. In addition to unmodified MccAYps these cells accumulated a m/z 4638 and m/z 4961 compounds corresponding to MccAYps-succinimide and cytidylated MccAYps (MccAYps-CMP) (Fig. 3C). The identity of the later compound was confirmed by MS-MS analysis (Supplementary Fig. S2). Thus, while earlier in vitro work showed that MccBYps attaches AMP to its cognate peptide7, in vivo, CMP is attached. A similar situation was observed for MccB encoded by Bacillus amiloliquefaciens.12 A peak with m/z 5019 appeared in cells harboring a four-gene plasmid (mccACBS). This compound corresponds to carboxymethylated form MccAYps-cxCMP (Supplementary Fig. S2), an expected product based on the analysis of Bacillus amiloliquefaciens mcc operon.12 No growth inhibition zones were observed around patches of these cells. The compound of the same mass was synthesized when five genes -- mccACBSX -were present on the plasmid, suggesting that MccXYps does not participate in modifications that lead to a mass-shift of the product MccB/MccS action. There was no biological activity evident 9 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 26

for cells transformed with mccACBSX plasmid. When the plasmid-borne cluster contained mccDYps but lacked the downstream genes, the biological activity was still lacking. A new peak with average m/z of 5120 was observed. The E. coli MccD protein attaches carboxylated propylamine to cognate peptide adenylate, adding extra 101 Da.8 The mass difference between MccAYps-cxCMP (m/z 5019) and the m/z 5120 compound also equals 101 Da, suggesting that carboxylated propylamine was attached to the phosphate group of MccAYps-cxCMP by MccDYps. This interpretation is supported by MS-MS analysis presented in Supplementary Fig. S2. When the plasmid-borne mccYps contained the ACBSXDE1 genes, growth inhibition of E. coli tester cells was finally observed and a mass-ion with an average m/z of 5076 was detected in massspectra of cells. The same mass-ion is detected in cells with pDT1-Ymcc harboring complete mccYps cluster (Fig. 3A). In E. coli, the N-terminal domain of bifunctional MccE protein decarboxylates the carboxypropylamine group, resulting in a loss of 44 Da.8 The mass difference between the 5120 compound present in cells carrying ACBSXD genes and the m/z 5076 compound is the same. Overall, the results of our analysis are consistent with a biosynthesis pathway involving the initial attachment of CMP to the MccAYps peptide followed by carboxymethylation and aminopropylation to produce a fully modified, biologically active compound (Fig. 3C). We attempted to reconstruct the entire biosynthesis pathway in vitro using chemically synthesized MccAYps and recombinant modification enzymes (Supplementary Fig. S3). MccBYps selectively attached CMP to MccAYps when supplied with an equimolar mixture of NTPs, producing MccAYps-CMP (m/z 4961). In the presence of MccBYps, MccSYps, SAM, and prephenate, MccAYps-cxCMP (m/z 5019) was produced. The same product was obtained in the presence of MccBYps only when reactions were supplemented with cx-SAM (Supplementary Fig. S3). Thus, MccSYps role in the MccAYps modification pathway is limited to the production of carboxy-SAM from SAM and prephenate. Further in vitro modification of MccAYps-cxCMP impossible since recombinant MccDYps protein was insoluble. The steps of modification of MccAYps that have been reconstituted biochemically are highlighted by green shading in the scheme of Fig. 3C. The last two steps are proposed based on analogy with the E. coli microcin C maturation pathway8 and are supported by analysis of intermediates produced in vivo (Fig. 3B) and homology between MccD and MccE enzymes of E. coli and Y. pseudotuberculosis.

10 ACS Paragon Plus Environment

Page 11 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

The bioactive product of Y. pseudotuberculosis mcc-like operon is different from the compound that accumulates in producing cells Mass-spectrometric analysis of conditioned media after growth of E. coli ∆hns cells harboring pDT1-Ymcc failed to reveal a mass-peak of m/z 5076 corresponding to fully modified MccAYps. The cultured medium was therefore fractionated by reverse phase HPLC and antibiotic activity in the fractions was followed. A single fraction that inhibited the growth of wild-type E. coli was obtained (Fig. 4A). Two prominent mass-peaks with m/z of 1315 and 1331 were present in this fraction (Fig. 4B, upper panel). Treatment with dithiothreitol led to a mass-shift of 2 Da (to m/z of 1317 and 1333) corresponding to disulfide bond reduction for both peaks (Fig. 4B, lower panel). We were unable to separate these peaks by additional chromatographic steps.

11 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 26

Figure 4. Identification of bioactive Y. pseudotuberculosis IP 32953 mcc operon product. A. HPLC traces of cultured media of cells harboring pDT1-Ymcc (red trace) and control cells with empty pDT1 vector (blue trace). Results of bioactivity testing of indicated HPLC fractions on lawns of E. coli B

12 ACS Paragon Plus Environment

Page 13 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

cells are shown below. “Gm” shows the results of growth inhibition zones formed around drops of gentamicin. HPLC peak with antibiotic activity is labeled “McCYps”. B. MALDI-MS spectra of bioactive HPLC fraction 5 from panel A. The bottom spectrum was obtained after preincubation with 2 mM DTT. C. Fragmentation spectra of m/z 1315.4 and m/z 1331.4 mass-ions from the upper spectrum in panel B and a structure of a compound whose fragmentation pattern is consistent with the top spectrum. “D-NH2” – aspartamide, “cxC” - carboxymethylated cytosine; “Rib.” – ribose, “ap.” – aminopropyl group; “HPO3+ap.” – aminopropylated phosphate, “cxC+O” – oxidized form of carboxymethylated cytosine. On the structure, sites of fragmentation that generate experimentally observed mass-peaks are indicated.

Material from bioactive fraction was subjected to MS-MS analysis. A daughter ion with m/z 897 of the 1315 mass-ion, matched the C-terminal part of the MccAYps peptide, CGAATCGGGSN (Fig. 4C). This assignment was supported by signals resulting from fragmentation of the peptide part. Daughter ions with m/z 1034 and 1148 matched CGAATCGGGSN-aminopropyl-phosphate

and

CGAATCGGGSN-aminopropyl-phosphate-

ribose fragments. The loss of 169 Da during fragmentation corresponds to removal of carboxymethyl-cytidine nucleobase. The fragmentation pattern of the 1331 mass-ion resulted in accumulation of identical daughter ions (Fig. 4C). Thus, the 16 Da difference between 1315 and 1331 compounds resides on the cytidine base. Overall, the results demonstrate that the compound with m/z 1315 is a truncated form of m/z 5076 compound detected in producing cells. It corresponds to the last 11 amino acids of MccAYps, with C-terminally attached CMP, aminopropyl on the phosphate group, and carboxymethyl on the base. We name this compound McCYps (microcin C from Yersinia pseudotuberculosis). High-resolution MS measurements of material from active fraction resulted in m/z values of 1315.4087 and 1331.4042, which is in 0.5 ppm accordance with brutto formulae of [MmonoisoH+] C45H72N16O24S2P1 with the calculated value of 1315.4082. The shift of 15.9955 Da that results in m/z of 1331.4042 is consistent with one oxygen atom addition (Fig S4). Thus, the 1331 compound is thus an oxidized form of McCYps. As is shown in Fig. 2A, Y. pseudotuberculosis strain #3526 with the pDT1-Ymcc plasmid did not cause growth inhibition of E. coli tester cells. However, since there was detectable level of mccYps gene expression (Fig. 2B) we fractionated medium conditioned by growth of these cells 13 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 26

and obtained a fraction with biological activity (Fig. S5). MALDI-MS analysis revealed that both active fractions contained m/z 1315 and m/z 1331 compounds (Fig S5). MS-MS analysis of m/z 1315 ion confirmed its identity to McCYps produced in E. coli. We take this result as a strong indication that the product of Y. pseudotuberculosis mcc operon is endoproteolytically processed in the natural host. The TldD/E protease is responsible for endoproteolytic processing of pro-McCYps inside producing cells The finding that active McCYps is distinct from the compound that accumulates in the producing cells raises a question about the mechanism by which active compound with shorter peptide part is generated. Inspection of mass spectra of ∆hns cells carrying pDT1-Ymcc and its derivatives revealed a prominent peak with an average m/z 3777 that corresponded to N-terminal 31-aminoacid fragment of MccAYps (Fig. 5A, middle panel). The same fragment is detected in wild-type E. coli transformed with pDT1-Ymcc (Fig. S1). This fragment must arise from a proteolytic event that generates McCYps. To identify a protease(s) involved in MccAYps cleavage, a set of mutants in known and putative E. coli protease genes present in the Keio collection of single-gene E. coli mutants14 was transformed with pDT1-based plasmid harboring the mccACB genes and screened for the presence of m/z 3777 mass-ion. Cells lacking either the tldD or tldE genes coding for subunits of TldD/E protease15 did not contain the N-terminal MccAYps fragment, suggesting that TldD/E is involved in McCYps maturation. Indeed, when cells carrying deletions of tld genes and hns were transformed with pDT1-Ymcc and tested for bioactivity, no growth inhibition zones were formed (Fig. 5B). Mass spectrometric analysis of intracellular contents of ∆tldD ∆tldE ∆hns triple mutant transformed with pDT1-Ymcc revealed, compared to ∆hns mutant cells, a strongly increased m/z 5076 signal corresponding to full-sized MccAYps with C-terminal modifications. The m/z 3777 peak corresponding to N-terminal product of cleavage that generates bioactive McCYps was absent from spectra of ∆tldD ∆tldE ∆hns triple mutant cells. We therefore conclude that the TldD/E protease is an essential component of McCYps production. A minor m/z 3880 peak was seen in ∆hns cells with or without tld mutations. This peak matches the N-terminal fragment of MccAYps cleaved at position 32 (Fig. 5A, lower panel). The intensity of this peak increased in tld mutants suggesting that there exist additional proteases capable of off-target cleavage that does not result in biologically active compound. 14 ACS Paragon Plus Environment

Page 15 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Accumulation of unprocessed microcin C-like compound in E. coli ∆hns ∆tldD ∆tldE strain harboring pDT1-Ymcc allowed us to purify pro-McCYps – full length modified MccAYps - and test its activity. The compound was found to be inactive at concentrations when McCYps produced robust growth inhibition zones on lawns of wild-type E. coli (Fig. 5C-D).

Figure 5. The tld genes are required for McCYps production. A. MALDI-MS spectra of E. coli ∆hns cells harboring empty pDT1 vector (top) or the pDT1-Ymcc plasmid (middle), and ∆hns E. coli ∆tldD ∆tldE derivative harboring pDT1-Ymcc (bottom). The peaks of full-length MccAYps modified peptide (m/z 5076) and its N-terminal proteolytic fragments 1-31(m/z 3777) and 1-32 (m/z 3880) are marked with red-color font. Mass-peaks present in control and experimental cells match E. coli ribosomal proteins RL36 (m/zcalc 4364) and RS22 (m/zcalc 5097). B. Growth inhibition zones on the lawn of E. coli B cells formed around patches of Tld+ (E. coli ∆hns) and Tld- (E. coli ∆hns ∆tldE ∆tldE) strains harboring pDT1 or pDT1-Ymcc plasmids. C. An HPLC trace of cellular extract of E. coli ∆hns ∆tldD ∆tldE cells harboring pDT1-Ymcc. HPLC peak labeled “pro-McCYps” contains a major mass ion with m/z 5076 corresponding to fully modified MccAYps peptide. D. 5-µL drops of 40 µM solutions of McCYps and pro-McCYps) purified from Tld- E. coli were deposited on a fresh lawn of E. coli B cells. Growth inhibition zones appearing after 16 h incubation at 30 °C are shown. A 5-µL drop of 0.5 mg/ml gentamycin (Gm) was used as a positive control.

15 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

The TldD/E proteins are highly conserved in eubacteria and Y. pseudotuberculosis TldD and TldE are 85 and 83% identical to E. coli counterparts. Recombinant Y. pseudotuberculosis TldD and TldE, when present together and combined with MccAYps-CMP caused accumulation of a m/z 3777 mass-ion which corresponds to N-terminal fragment present in E. coli cells with intact tld genes (Fig. S6). No corresponding C-terminal peptide-nucleotide fragment was detected, suggesting that it was rapidly degraded. An identical result was obtained with recombinant E. coli TldD/E (Fig. S6). The results may imply that in vivo McCYps is removed from producing cells immediately after processing to prevent further degradation. Be it as it may, we take these results as evidence that the TldD/E protease is responsible for proteolytic maturation of pro-McCYps in the natural host Y. pseudotuberculosis. In principle, two mechanisms of TldD/E involvement in McCYps biosynthesis are possible. Unmodified MccAYps may be proteolytically processed first followed by enzymatic modification of shorter C-terminal peptide. Alternatively, the modified full-length MccAYps may be proteolysed by TldD/E with release of active compound. We tested synthetic peptides corresponding to the full-sized MccAYps and the 11-aminoacid long peptide found in McCYps for ability to be modified by MccBYps in vitro (Fig. S7). The results showed that MccBYps modified only the 42-aminoacid peptide. It is therefore likely that TldD/E role in McCYps production is to cleave pro-McCYps rather than unmodified MccAYps. We considered a possibility that activation of McCYps production in E. coli hns mutants is due to increased expression of tld genes. Indeed, RT-PCR analysis (Fig. S8) showed that there were 10 and 2-fold increases in levels of respectively, tldD and tldE transcripts in ∆hns cells compared to the wild-type. Thus, in addition to direct effects on mccYps genes transcription (Fig. 2B) the absence of H-NS may also stimulate processing of modified MccAYps and formation of bioactive product.

Comparing Y. pseudotuberculosis and E. coli microcins action McCYps was active against both E. coli and Y. pseudotuberculosis. The minimal inhibitory concentration (MIC), at which inhibition zones on lawns of tester cells were visible, was 3 µM for E. coli B, which is 8 times higher than the corresponding value for McCEco. Both compounds showed similar level of activity against Y. pseudotuberculosis (Fig. S9). To compare the 16 ACS Paragon Plus Environment

Page 17 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

mechanism of action of McCYps with that of well-characterized E. coli McC, their ability to inhibit various derivatives of sensitive E. coli strains was tested (Fig. 6). Cells lacking the yejA gene that encodes a component of McCEco transporter YejABEF, were resistant to McCYps. Cells lacking aminopeptidases A, B, and N involved in degradation of McCEco peptide part4 were fully resistant to McCYps. Cells overproducing Asp-RS, the target of McCEco, were also resistant to both McCYps and McCEco while no resistance of cells overproducing prolyl-tRNA synthetase (Pro-RS) was observed. 12.5 μM of McCEco or McCYps was added to E. coli S30 extracts and, after incubation for various times, aminoacylation reaction of tRNAAsp was carried out (Fig. S10). Aminoacylation levels remained unchanged after the first 5-minute of incubation compared to control extracts (no microcin addition), then decreased by 10 minutes of incubation and remained stable afterwards. The kinetics of inhibition suggests that processing of peptide parts of both microcins proceeds at similar rates. However, while reactions containing McCEco retained only 5% of aminoacylation activity after processing was complete, reactions containing McCYps were inhibited only by 50%. Thus, as expected, the McCYps warhead, an aspartamide attached to aminopropylated carboxymethyl CMP is a worse mimic of aspartyl adenylate compared to aminopropylated aspartamidyl-AMP produced after processing of McCEco. Overall, we conclude that both McCYps and McCEc enter sensitive cells through YejABEF transporter and, after intracellular processing of the peptide part by aminopeptidases, inhibit Asp-RS.

17 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 26

Figure 6. Figure 6. Sensitivity of E. coli mutants or cells producing autoimmunity proteins to McCYps and McCEco. 5-µL drops of 25 µM McCYps, 2.5 µM McCEco, and 0.5 mg/mL of gentamicin solutions were deposited on the surface of freshly prepared top agar seeded with indicated E. coli cultures. Growth inhibition zones appearing after 16 h incubation at 30 °C are shown.

Cells expressing the MccECTD acetyltransferase from E. coli, while resistant to McCEco, remained sensitive to McCYps, in agreement with earlier observation that carboxymethyl modification of cytidine base renders the self-immunity mechanism mediated by MccEEco inactive.12 Conversely, cells expressing MccE2Yps were resistant to McCYps but sensitive to 18 ACS Paragon Plus Environment

Page 19 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

McCEco. These results indicate that MccE proteins, which provide self-immunity by acetylating processed McC, are adapted for their cognate warheads.

19 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 26

DISCUSSION In this work, we characterized McCYps, a product of Y. pseudotuberculosis mcc operon, that inhibits susceptible cells growth and determined the role of individual Yersinia sp. mcc genes in its production and immunity. The microcin encoded by Y. pseudotuberculosis mcc operon is an interesting case of a hybrid molecule: it contains both the terminal carboxymethylated cytosine previously observed in B. amyloliquefaciens microcin C-like compound and propylamine at the phosphate group that is characteristic for E. coli McC. The structure of carboxymethylated cytidine remains to be resolved, although one can speculate, based on analogy with post-transcriptionally modified nucleotides in tRNAs, that C5 is the position for alkyl group attachment. This modification would leave the N3 atom as a likely target for subsequent oxidation that we observe in McCYps. Further study of McCYps structure and how its modified nucleobase mimics adenine and fits the active site of Asp-RS is required. Uniquely for peptide-nucleotide antibiotics, McCYps undergoes an additional modification in the production host that is strictly required for biological activity. The 42-aminoacid long MccAYps peptide part is proteolytically processed inside the producing cell releasing mature McCYps whose peptide part is only 11 amino acids long. Upon processing, the compound leaves the producing cell, presumable due to the action of the MccCYps export pump, and is actively taken up by the YejABEF transporter in sensitive cells. The peptide parts of both McCYps and McCEco are processed inside sensitive cells by aminopeptidases A, B, and N, with release of toxic warheads that inhibit Asp-RS. Aminopropylated aspartamidyl-carboxymethylcytidylate that is produced after processing of McCYps appears to be a less potent inhibitor of Asp-RS than aminopropylated aspartamidyl-adenylate released after processing of McCEco. We can assume that aminopropyl decoration that is known to increase affinity of aspartamidyl-adenylates to E. coli Asp-RS also increases antibiotic potency of modified aspartamidyl-cytidylate. In addition, the presence of the carboxymethyl group on cytidine nucleobase strengthens the interaction with a Asp-RS,12 allowing it to compete more efficiently for the binding site normally occupied by the aspartyl-adenylate intermediate. Be it as it may, the fact that mccYps operons are widely distributed in Y. pseudotuberculosis suggests that its products offer advantages to bacteria that carry them at least in their particular ecological niche. The processing of modified MccAYps inside the producing cell is carried by TldD/E. The exact function of this highly evolutionary conserved endoprotease16,

17

remains obscure. Our 20

ACS Paragon Plus Environment

Page 21 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

recent structural work indicates that it may be a general-purpose enzyme capable of degrading unstructured polypeptides.15 In E. coli, TldD/E is essential for production of microcin B (McB), a thiazole-oxazole containing gyrase inhibitor that is unrelated to McC. During the McB synthesis TldD/E removes the leader part of pro-McB after post-translational modifications required for activity have been introduced by the McB synthase.18 There is evidence that TldD/E cleavage of pro-McB is associated with its export outside the producing cell.18 The situation may be similar in the case of McCYps, since we are unable to detect mature McCYps inside the producing cells and during in vitro digestion only the N-terminal MccAYps fragment is detected, while the McCYps is degraded Apparently, the MccCYps pump is unable to act on pro-McCYps but is able to recognize the shorter peptide of McCYps and export it outside the cell. The presence of stable Nterminal 31-aminoacid MccAYps TldD/E cleavage fragment inside the producing cells suggests that this peptide is structured and may have a function of its own, for example in regulating the mccYps expression. The additional proteolytic processing step during the biosynthesis of active compound encoded by the Y. pseudotuberculosis mcc operon may help solve the problem of selfintoxication of producing cells that inevitably arises due to processing by aminopeptidases of peptide-nucleotides accumulating inside the producing cells. In the case of McCEco producers, such toxicity was recently demonstrated directly.19 Relying on a much longer, possibly structured MccA peptide should increase intracellular stability of post-translationally modified MccAYps and prevent accumulation of toxic aspartamidyl-cytidylate that will inhibit protein synthesis in the producer. Though nothing is known about the regulation of TldD/E activity, one can imagine that its activation at specific conditions may allow Y. pseudotuberculosis carrying the mcc operon to produce massive amounts of McCYps from a depot of previously synthesized inactive pro-McCYps. The layout of mcc genes suggests that they form an operon with a single upstream promoter. However, it appears that activity of this promoter is not sufficient for McCYps production. This follows from the observation that McCYps was only produced when the hns gene was deleted. While H-NS is a well-known factor that silences transcription initiation20 its role must be different in the case of McCYps production. This follows from the fact that MccAYps accumulates inside the cells even in the presence of functional hns, indicating that the main promoter of the operon is active in the presence of H-NS. Therefore, H-NS is either essential for 21 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 26

expression of downstream mcc genes or plays a direct role in silencing the enzymes of the pathway. For example, H-NS may serve as a roadblock preventing RNA polymerase transcription elongation through the operon.21 We detected activation of tld genes expression in hns mutants. This effect is likely unrelated to McCYps production, since accumulation of Nterminal MccAYps fragment, presumably generated by TldD/E cleavage, occurs in wild-type cells carrying plasmid-borne mcc operon. However, the finding suggests that the physiological role of TldD/E is connected with stresses that derepress H-NS inhibited genes. Microcins are a class of small ribosomally-synthesized antibacterial peptides produced by Enterobacteriaceae and active against closely related bacterial species.22 With the accumulation of sequenced bacterial genomes it was recognized that E. coli microcin B and microcin J are representatives of vast groups of, respectively, linear azole-containing peptides and lasso peptides23, while microcin C is a member of peptide-nucleotide group antibiotics. Linear azolecontaining peptides, lasso peptides, and peptide-nucleotides are in turn subfamilies of ribosomally-synthetized post-translationally modified peptides (RIPPs). A common theme in RIPP biosynthesis pathways is the presence in the ribosomally-synthesized peptide precursor of a leader part to which modification enzymes bind, and a core part onto which modifications are introduced.24 Once modifications are completed, the leader part is removed releasing mature RIPP. Up to now, peptide-nucleotides appeared to deviate from this scheme. However, the McCYps fully conforms to the RIPP synthesis paradigm: it contains a removable leader peptide that is required for nucleotide attachment by MccBYps to the core part. The N-terminal domain of MccBYps nucleotydyltransferase contains structural similarity to a divergent winged helix-turnhelix RiPP precursor peptide recognition domain (RRE) present in leader binding domains of modification enzymes of RiPPs of other classes.25 We hypothesize that in MccBYps this domain also recognizes the leader peptide. Interestingly MccBEco also contains an RRE , which, however, makes only limited contacts with the short, 7 aminoacid-long MccAEco peptide.26 These observations suggest that mcc operons with longer peptides are ancestral: they interacted with their post-translational machinery in a “standard” way but eventually became shorter, as is now seen in most mcc operons, and acquired a new mode of interactions with cognate modification enzymes. ACKNOWLEDGMENTS

22 ACS Paragon Plus Environment

Page 23 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

We are grateful to Professor M. Skurnik (University of Helsinki, Finland) for extensive help with Yersinia strains handling and valuable advises. We thank Dr. M. Metelev (Uppsala University, Sweden) and Prof. Sergey Borukhov (Rowan University, NJ) for critical reading of this manuscript and Dr. Tatyana Artamonova from Research Center of Nanobiotechnologies, Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia for invaluable help with Hi-Resolution mass-spectroscopy. The work was supported Russian Science Foundation RSF 16-14-10356 to SD and an NIH grant R01 AI117210 (NIAID) to Satish A. Nair and KS. MALDI MS facility became available to us in the framework of the Moscow State University Development Program PNG 5.13.

SUPPORTING INFORMATION Figures S1-S10 and detailed experimental procedures. This information is available free of charge at the ACS website.

23 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 26

REFERENCES 1. Guijarro, J. I.; González-Pastor, J. E.; Baleux, F.; San Millán, J. L.; Castilla, M. A.; Rico, M.; Moreno F.; Delepierre, M. J. Biol. Chem. 1995, 270, 23520-23532. 2. Roush, R. F.; Nolan, E. M.; Löhr, F.; Walsh, C.T. J. Am. Chem. Soc. 2008, 130, 36033609. 3. Novikova, M.; Metlitskaya, A.; Datsenko, K.; Kazakov, T.; Kazakov, A.; Wanner B.; Severinov, K. J. Bacteriol. 2007, 189, 8361-8365. 4. Kazakov, T.; Vondenhoff, G. H.; Datsenko, K. A.; Novikova, M.; Metlitskaya, A.; Wanner, B.L.; Severinov, K. J. Bacteriol. 2008, 190, 2607-2610. 5. Metlitskaya, A.; Kazakov, T.; Kommer, A.; Pavlova, O.; Praetorius-Ibba, M.; Ibba, M.; Krasheninnikov, I.; Kolb, V.; Khmel I.; Severinov, K. J. Biol. Chem. 2006, 281, 1803318042. 6. Severinov, K.; Semenova, E.; Kazakov, A.; Kazakov, T.; Gelfand, M. S. Mol. Microbiol. 2007, 65, 1380-1394. 7. Bantysh, O.; Serebryakova, M.; Makarova, K. S.; Dubiley, S.; Datsenko, K. A.; Severinov, K. MBio. 2014, 5, e01059-14. 8. Kulikovsky, A.; Serebryakova, M.; Bantysh, O.; Metlitskaya, A.; Borukhov, S.; Severinov, K.; Dubiley, S. J. Am. Chem. Soc. 2014, 136, 11168-11175. 9. Metlitskaya, A.; Kazakov, T.; Vondenhoff, G. H.; Novikova, M.; Shashkov, A.; Zatsepin, T.; Semenova, E.; Zaitseva, N.; Ramensky, V.; Van Aerschot, A.; Severinov, K. J. Bacteriol. 2009, 191, 2380-2387. 10. Novikova, M.; Kazakov, T.; Vondenhoff, G. H.; Semenova, E.; Rozenski, J.; Metlytskaya, A.; Zukher, I.; Tikhonov, A.; Van Aerschot, A.; Severinov, K. J. Biol. Chem. 2010, 285, 12662-12669. 11. Tikhonov, A., Kazakov, T.; Semenova, E.; Serebryakova, M.; Vondenhoff, G.; Van Aerschot, A.; Reader, J. S.; Govorun, V. M.; Severinov. K. J. Biol. Chem. 2010, 285, 37944-37952. 12. Serebryakova, M.; Tsibulskaya, D.; Mokina, O.; Kulikovsky, A.; Nautiyal, M.; Van Aerschot, A.; Severinov, K.; Dubiley, S. J. Am. Chem. Soc. 2016, 138, 15690-15698. 13. Navarre, W. W. Adv. Microb. Physiol. 2016, 69, 157-186.

24 ACS Paragon Plus Environment

Page 25 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

14. Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; Datsenko, K. A.; Tomita, M.; Wanner, B. L.; Mori, H. Mol. Syst. Biol. 2006, 2, 2006.0008. 15. Ghilarov, D.; Serebryakova, M.; Stevenson, C. E. M.; Hearnshaw, S. J.; Volkov, D.; Maxwell, A.; Lawson, D. M.; Severinov, K. Structure. 2017, 25, 1549-1561. 16. Hu, Y.; Peng, N.; Han, W.; Mei, Y.; Chen, Z.; Feng, X.; Liang, Y. X.; She, Q. Biosci. Rep. 2012, 32, 609-618. 17. Rife, C.; Schwarzenbacher, R.; McMullan, D.; Abdubek, P.; Ambing, E.; Axelrod, H.; Biorac, T.; Canaves, J. M.; Chiu, H. J.; Deacon, A. M.; DiDonato, M.; Elsliger, M. A.; Godzik, A.; Grittini, C.; Grzechnik, S. K.; Hale, J.; Hampton, E.; Han, G. W.; Haugen, J.; Hornsby, M.; Jaroszewski, L.; Klock, H. E.; Koesema, E.; Kreusch, A.; Kuhn, P.; Lesley, S. A.; Miller, M. D.; Moy, K.; Nigoghossian, E.; Paulsen, J.; Quijano, K.; Reyes, R.; Sims, E.; Spraggon, G.; Stevens, R. C.; van den Bedem, H.; Velasquez, J.; Vincent, J.; White, A.; Wolf, G.; Xu, Q.; Hodgson, K. O.; Wooley, J.; Wilson, I. A. Proteins. 2005, 61, 444-448. 18. Allali, N.; Afif, H.; Couturier, M.; Van Melderen, L. J. Bacteriol. 2002, 184, 3224-3231. 19. Piskunova, J.; Maisonneuve, E.; Germain, E.; Gerdes, K.; Severinov, K. Mol. Microbiol. 2017, 104, 463-471. 20. Schröder, O.; Wagner, R. J. Mol. Biol. 2000, 298, 737-748. 21. Winardhi, R. S.; Yan, J.; Kenney, L. J. Biophys. J. 2015, 109, 1321-1329. 22. Severinov, K.; Nair, S. K. Future Microbiol. 2012, 7, 281-289. 23. Yang, X.; van der Donk, W. A. Chemistry. 2013, 19, 7662-7677. 24. Ortega, M. A.; van der Donk, W. A. Cell. Chem. Biol. 2016, 23, 31-44. 25. Burkhart B. J.; Hudson, G. A.; Dunbar, K. L.; Mitchell, D. A. Nat. Chem. Biol. 2015, 11, 564-570. 26. Regni, C. A.; Roush, R. F.; Miller, D. J.; Nourse, A.; Walsh, C. T.; Schulman, B. A. EMBO J. 2009, 28, 1953-1964.

25 ACS Paragon Plus Environment

Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 26

TOC graphic.

26 ACS Paragon Plus Environment