Vibrio and Proteus species. As wastewater treatment plants accumulate a large and diverse number of such species, we examined raw water samples taken from a municipal wastewater treatment plant initially using SXT/R391 family integrase gene-specific PCR probes to detect the presence of such elements in a directed approach. A positive amplification occurred over a full year period and a subsequent Restriction Fragment Length Polymorphism (RFLP) analysis revealed a very limited diversity in the treatment plant examined. Samples demonstrating positive amplification were cultured using Vibrio and Proteus selective media and PCR amplification tracking was utilized to monitor SXT/R391-ICE family containing strains. This screening procedure resulted in the isolation and identification of a Proteus mirabilis strain harbouring an ICE. Whole-genome sequencing of this ICE containing strain using Illumina sequencing technology revealed a novel 81 kb element that contained 75 open reading frames on annotation but contained no antibiotic or metal resistance determinants. Comparative genomics revealed the element contained a conserved ICE core with one of the insertions containing a novel bacteriophage defence mechanism. This directed isolation suggests that ICE elements are present in the environment without apparent selective pressure but may contain adaptive functions allowing survival in particular environments such as municipal wastewater which are reservoirs for large bacterial phage populations.Integrative Conjugative Elements (ICE’s) of the SXT/R391 family have largely been detected in clinical or environmental isolates of Gammaproteobacteria, particularly  Wastewater influent may also contain many Proteobacteria which normally occur in the environment3. With the abundance and diversity of microorganisms contained in wastewater, many are carriers of Mobile Genetic Elements (MGEs)4.Wastewater treatment plants (WWTP) accumulate large and diverse numbers of pathogens including viruses, bacteria, fungi, protozoans and helminths. The pathogens present may reflect the common diseases in a community and may be present in numbers as great as 100,000,000 per millilitre6. ICEs present in the environment can rapidly spread in bacterial populations and often carry genes that give the ICE an adaptive advantage to that environmental niche. The SXT/R391 family of ICEs is one of the most studied types of MGE with various sub-types of SXT/R391 elements reported7. Type 1 elements have been found in a variety of Gammaproteobacteria species including Vibrio, Providencia, Proteus, Shewanella, Actionbacillus, Alternomonas, Escherichia coli, Enterovibrio, Photobacterium damselae, Idiomarinaceae bacterium and Marinomonas8. They are distinguished by possessing a Type 1 integrase, which allows site-specific integration into the 5′ end of the essential prfC gene9, integration restores a functioning prfC gene and encodes a new hybrid PrfC protein once integrated9. Type 2, 3 and 4 ICEs all insert at the 3′ end of the multi-copy tRNA-Ser gene and have been found solely in Vibrio species10. Another type of SXT/R391 ICE integrates into the pabA (para-aminobenzoate synthase) gene as with ICESh9511.Integrating Conjugative Elements (ICEs) are self-transmissible MGEs that have the ability to integrate into their host’s chromosome, replicate and transfer by conjugation12. Type 1 SXT/R391 ICEs show site-specific integration into the 17 bp integration site at the 5′ end of the prfC gene13 and contain a core of 52 conserved genes that make up the ‘backbone’ of the element with the key functions being integration, excision and regulation14. Type 1 elements contain five hotspot regions and five variable regions which allow for gene or gene segments to be inserted, giving the element an array of potential adaptive functions14. All ICES characterised to date have been isolated serendipitously or identified from genome sequences There have been reports of SXT/R391-like ICEs from wastewater environments, ICEVchMex1 (GQ463143.1) isolated from sewage in San Luis Potosi, Mexico15 and ICESheChnS12 (AXZL01000060.1), isolated from a wastewater treatment plant in China, 201215. However, these were discovered following analysis rather than using a directed approach. ICEVchMex1 contains genes in all its hotspots and in one variable region and encodes ampicillin resistance15. ICESheChnS12 was found to contain genes in all its hotspot but no genes were contained in the variable regions. The hotspots contained accessory genes such as AAA ATPase and a Restriction Modification System16.The integrase encoding gene of the tyrosine recombinase class is a disguising feature of Type 1 elements. Integrase genes from several different SXT/R391 ICE’s were compared to design specific primers to amplify the geneIn this study, a directed approach was used to detect and isolate ICEs of the SXT/R391 family from a municipal wastewater treatment plant and to characterise one of these isolates as the first ICE detected and isolated from Ireland.The bacterial strains and SXT/R391 elements utilised as part of this study are listed in Table Composite 24 hr wastewater samples were collected from the inlet works at a municipal wastewater treatment plant, Ireland in May and November 2018.−2 to 10−8 were prepared and samples spread on selective media. CLED media was used for the detection of Proteus species and TCBS media for the detection of Vibrio species, plates were incubated at 37 °C for 24 hours. Colonies were routinely isolated and inoculated using LB broth, incubated at 37 °C for 24 hours at 200 rpm. The remaining overnight culture was used to prepare glycerol stocks and stored at −80 °C. Replica plating was carried out to identify single colonies containing ICE-like elements via PCR. The streak plate technique was used to isolate a single pure colony from the replica plate.Serial dilutions of the collected and processed samples isolated from wastewater were carried out using a 0.85% Saline solution. Serial dilutions from1012.DNA extractions from isolates were performed using the DNeasy Blood & Tissue extraction kit (Qiagen) under the Gram-Negative bacterial protocol for liquid cultures. A PowerSoil DNA extraction kit (Qiagen) was used for solid materials. PCR was carried out using specifically designed primers Table  to ampli17 (http://www.labtools.us/nebcutter-v3-0/) was used to select restriction endonucleases that could be used to digest modified integrase genes. The nucleotide sequences of sequenced SXT/R391 ICEs present in the ICEBerg database8 and the respective integrase gene sequences were selected to determine what enzymes might be utilised to determine RFLP variation. As a result, three restriction endonucleases were selected, HpaII, ApoII and TauI. An ApoII site was not present in ICER391 (AY090559.1) int amplicons while a TauI site was not present in ICER997 (KY433363), ICESXT or ICEpMERPH (MH974755).PCR-RFLP (Restriction Fragment Length Polymorphism) was carried to determine whether more than one ICE type might be present in the isolated samples. NEB_CutterHpaII and ApoII at 37 °C and TauI at 65 °C. The tubes were incubated for 45 minutes in the thermocycler. To visualize the samples, 3 µl of 5X loading dye was added directly into the digest, 10 µl was loaded onto a 1.5% (w/v) agarose gel in 1X TAE Buffer. The gel was set to run for 1 hour at 100 V and photographed under UV light.PCR, as described above, was carried out on selected isolates containing an amplified integrase gene, ICER391, ICER997 and ICESXT amplicons were used as controls. Additional controls consisted of the use of two different molecular weight markers (NEB 1 kb and NEB 100 kb), uncut PCR product and a control no enzyme “mock” digest. After the PCR, the restriction digest was carried out using 5 µl PCR product, 2 µl Tango Buffer, 0.5 µl of relevant restriction endonuclease and 12.5 µl Milli Q water, bringing the total volume to 20 µl. A cocktail of 15 µl was aliquoted into the PCR tube and 5 µl of the PCR product was then added before the digestion, the tubes were incubated at the appropriate temperatures, E.coli J53 as a recipient. The idea was to mark the ICE with a mini Tn5 (Kanamycin resistance) via pBAM1 and then mobilise the marker ICE into E.coli J53, a Rif resistant strain. Conjugative mating was carried out following the method by Kristensen et al. with some modifications18. The donor, helper and recipient strains were grown overnight. Overnight cultures were then centrifuged for 5–10 minutes at 10,000 rpm to collect cells, and washed with fresh LB broth. The cultures were mixed in a proportion of 5:1:1 (donor population was increased) and 50 µl were applied onto 0.45 µm filter on LB Agar plates. The plates were incubated at 37 °C for 24 hours. The cells were then scraped off the surface of the filter and added to a 0.85% saline solution. The culture was diluted with antibiotics for counter selection of the donor and helper cells.To determine if the identified ICE could undergo conjugation tri-parental mating was carried out using the isolated strain as a host strain, pBAM1 as a helper (pBAM1 reference) and −1) and kanamycin (50 µg.ml−1) supplemented plates and grown at 37 °C for 24 hours. Rifampicin had an MIC of 200 µg.ml−1 for the host strain as it was susceptible at this concentration while E. coli remained resistant. Controls for the donor, helper and recipient were also prepared. Transconjugants were then tested for the presence of the ICE as described above.The dilutions were spread onto rifampicin (200 µg.ml19 and reads were mapped using BWA (Burrows-Wheeler Aligner) mem20 software to assess the quality of the data. De novo assembly of the reads was carried out using SPAdes genome assembler software21 and the reads were mapped back to the resulting contigs using BWA mem software. The genome was identified amongst the contigs by using the BLAST tool to investigate the presence of several different ICER391 (AY090559) and ICESXT (AY055428) core scaffold genes . The sequence data was edited on notepad + + (https://notepad-plus-plus.org/downloads/), submitted to RAST (Rapid Annotation using Subsystem Technology) for annotation (see below), exported as a Genbank and Fasta file and reannotated manually with predicted Orfs blasted via BLASTP to confirm ICE association. Molecular maps were created by inputting the ICE sequence files in GenBank (.gnk) or FASTA (.faa) format into RAST22 (http://rast.nmpdr.org/). Gene Graphics was used to create gene representations of hotspot and variable regions by inputting the ICE sequence in GenBank (.gnk) format23.Full genome sequencing was carried out by MicrobesNG  using 2 ×250 bp paired-end reads HiSeq Illumina Miseq technology giving 30X genome coverage. The genome sequences were analysed using the MicrobesNG’s automated analysis pipeline, the closest available reference was found using Kraken software (Taxonomic Sequence Classification System)24. Bootstrap analysis with 1000 replications was performed to test the reliability of the tree.Phylogenetic analysis of fully sequenced SXT/R391 ICEs was performed based on comparison with the concatenated amino acid sequences of 48 to 52 SXT/R391 core ICE gene-encoded proteins on all previously sequenced whole SXT/R391 elements. An unrooted phylogenetic tree was constructed by the maximum-likelihood method based on the Poisson correction model using the MEGAXComposite 24 hr raw samples isolated from wastewater  were collected at two different times of the year, May 2018 (summer) and November 2018 (winter).Proteus and Vibrio species7. After plates were incubated overnight, colonies of interest were swabbed and inoculated in LB Broth, the overnight cultures were then used for DNA extraction and PCR was performed to detect the presence or absence of an ICE specific integrase gene12 indicating the presence of an SXT/R391 ICE MGE containing host. Screening and re-plating (once PCR positive amplicons were detected) were carried out over several weeks.Samples were plated on genera specific selective media as a significant number of SXT/R391 ICE MGEs have been detected in Proteus species, selectively grown on CLED media. A PCR amplicon was detected at 1378 bp matching the positive control, AB1157ICER391, see Fig. Proteus species based on analysis of its 16S rDNA sequence. The novel ICE was named ICEPmiIre01 as per standard ICE nomenclature25. Proteus isolates isolated in the winter also showed positive amplification for the integrase gene, indicating that the presence of SXT/R391 ICE-like elements is apparently not season dependent.Following multiple screening, and replica plating . HpaII cleaved two types of environmental SXT/R391 ICE integrase genes amplified, one without an HpaII site and one with an HpaII site (each amplicon 1378 bp in length), TauI cleaved one SXT/R391 ICE  as it contained a TauI site and ApoII did not show any cleavage of amplicons from isolates in our case.RFLP was used to determine whether there might be more than one SXT/R391 ICE type present in the wastewater. The integrase specific PCR amplicon generated by IntFor1 and IntRev1 is normally 1378 bp in length. Alignments of numerous ICE integrase genes sequenced from SXT/R391 family members within the ICEberg databaseHpaII site and one that does not.This limited RFLP yield suggested that only a limited number of amplicon types were present, and indeed possibly only two different types  The ICE was found on node 1 (287508 bp in length), this node revealed a novel SXT/R391-like ICE element associated with Proteus mirabilis. 16 s rDNA analysis of the host-based on full genome analysis revealed a 100% match to Proteus mirabilis. Previously SXT/R391 ICE elements have been detected in Proteus mirabilis from China but isolated from clinical and animal samples generally associated with illness27. Proteus species are known to be human and animal opportunistic pathogens, and frequently isolated from urine, wounds and other clinical sites and is present in soil and water habitats possibly from spreading from clinical sources28.ICE6 and is 81 kb in length with prediction of 75 orfs based on RAST annotation. A full genome map can be seen in Fig. PmiIre01 contains 51 core genes, sharing the core molecular backbone and synteny of other SXT/R391 ICEs. A full list of orfs detected is presented in Supplementary Table PmiIre01 are predicted to encode activities for basic ICE functionality such as integration, excision and conjugative transfer29. ICEPmiIre01 is one of the smallest SXT/R391-like ICE MGEs to be discovered thus far, the smallest being ICEPmiChn3 (KY437727) at 57 kb in length also from a Proteus mirabilis, isolated from broiler carcasses in China26.This novel SXT/R391 ICE is of the Type 1 ICE variety based on comparative genomicsPmiIre01 revealed that it contains five hotspot regions and further insertions into one variable region. Some of the hotspot regions contain genes putatively encoding resistance to bacteriophage attack. This is not surprising as there is a continuous arms race between bacteria and phage and such resistance systems include restriction-modification systems and toxin-antitoxin systems30. Interestingly the element contained no antibiotic resistance or metal resistance determinants.Structural analysis of ICEorf27 encoding a putative plasmid-related protein and orf28 encoding a hypothetical protein with 93% and 97% identity to hypothetical proteins found in ICER391 (AY090559.1). The nucleotide sequence of HS1is 99% similar to that of HS1 of ICER99731 but it is unclear what the function of these HS1 predicted genes are.HS1 contains two predicted genes, 32. orf35 encodes a putative AbiEi antitoxin, Type IV TA system with a 96% identity to an Ynd protein (AQT24150.1) found in ICER997 (KY433363.1). orf36 encodes a putative nucleotidyl transferase AbiEii/AbiGii toxin family protein with a 97% identity to an Ync protein (AQT24151.1) found in ICER997 (KY433363.1). Toxin-antitoxin genes are commonly found in HS2 of SXT/R391-like ICEs as revealed through comparative analysis. The nucleotide sequence of HS2 is 100% identical to that of Vibrio cholerae, ICESXT (AY055428).HS2 contains two genes both putatively associated with the Type IV toxin-antitoxin ‘innate immunity’ bacteria abortive infection (Abi) system that protects bacteria from the spread of phage infection. The Abi system, sometimes denoted as phage exclusion, is characterized by a normal start of bacteriophage infection which is then followed by the interruption of phage development leading to the release of few or no progeny particles but can cause premature host cell death. This Abi system has a survival potential for the population rather than the individual by reducing phage bursts at the expense of individuals within the populationorf62 encodes a putative restriction endonuclease, which is 100% identical to a restriction endonuclease (WP_004249392.1) found in Enterobacterial species while orf63 encodes a putative 5-methylcytosine restriction system component which is 100% identical to the mcrBC-5 methylcytosine restriction system of Gammaproteobacteria33. The nucleotide sequence of HS3 is 100% identical to that found in ICEPmiJpn1 (KT894734). These two putative enzymes in HS2 maybe a RMS, which recognizes a specific DNA sequence for cleavage and a cognate methyltransferase that protects from cleavage by methylation of adenine or cytosine bases within the same recognition sequence34 and maybe involved in protection from invading phages35.HS3 contains two genes, none of which show any similarity to ICER391, ICER997 or ICESXT, orf44 encodes a hypothetical protein with 100% identity to a hypothetical protein (AQT24159.1) found in ICER997 (KY433363.1), orf45 encodes a putative Helicase HerA-like protein with 100% identity to a Bipolar DNA helicase HerA protein (AQT24160.1) present in ICER997 (KY433363.1). HerA is a DNA helicase able to utilize either 3′ or 5′ single-stranded DNA extensions for loading and subsequent DNA duplex unwinding36. Lastly, orf46 encodes a putative endonuclease protein with a 94% identity to an endonuclease I precursor protein (AQT24161.1) present in ICER997 (KY433363.1). Endonucleases are thought to generate double-strand breaks in DNA. The biological role associated with this hotspot is unknown37.HS4 contains three predicted genes, PmiIre01. This system has characterised homologs that allow for phage adsorption but blocks phage DNA replication. A similar BREX system was first identified in the genome of Bacillus subtilis H3081.97 (NZ_ABDL01000007.1) and there confers resistance to a broad range of phage both virulent and temperate30. The BREX six gene cassette in Bacillus subtilis includes a putative Lon-like protease, a putative alkaline phosphatase protein, a putative RNA-binding protein, a putative DNA methylase, a putative ATPase domain-containing protein and a protein of unknown function30. The six genes that make up the BREX system in ICEPmiIre01 are orf15, a putative inner membrane protein (BrxA) that is 100% identical to orf23, a hypothetical protein (AAM08040.1) found in ICER391. BrxA shares structural homology with the RNA-binding antitermination protein NusB31. The second gene orf16 is a putative DUF1788 domain-containing protein (BrxB) which is 100% identical to orf24, a hypothetical protein (AAM08046.1) contained in ICER391, the function of this gene is unknown30. The third gene, orf17 a putative BREX system P-loop protein (BrxC), is 100% identical to orf25 a putative ATPase (AAM07998.1) present in ICER391. BrxC contains a putative large ATP binding motif30. The fourth gene orf19 encodes a putative BREX-1 system adenine-specific DNA-methyltransferase (PglX), showing 100% identity to orf26, a hypothetical protein (AAM08017.1) found in ICER391. This gene homolog encodes a protein that has putative DNA modifying functions that enable BREX to differentiate between host and phage DNA30. The fifth gene is orf20 encoding a putative BREX-1 system phosphatase protein , showing 93% identity to orf30, a hypothetical annotated protein (AAM07999.1) present in ICER391. The final gene within the system orf21 encodes a putative a Lon-related BREX system protease (BrxL), showing 97% identity to orf31, a putative ATP-dependent Lon protease protein (AAM08002.1) found in ICER391. Lon proteases are ATP-dependent serine proteases that mediate the selective degradation of mutant and abnormal proteins, as well as certain short-lived regulatory proteins and is required for cellular homeostasis and for survival from DNA damage and development changes induced by stress38. Other genes present in the HS5 include orf18, encoding a hypothetical protein 100% identical to a hypothetical protein (EKA96589.1) found in Proteus mirabilis WGLW6 and orf22 a putative DNA repair protein showing 100% identity to orf32, a hypothetical protein (AAM08000.1) present in ICER391. A similar BREX system appears to be present in ICER391 (AY090559)39 and ICEVpaCan1 (CP028481)7 based on homology reported above but to our knowledge has not been annotated as a putative BREX system previously. Based on the observations above and based on comparative analysis with other SXT/R391 family members it is clear that ICEPmiIre01 is a mosaic of core ICE genes and variable genes located in hotspots and variable regions of the element. These genes are most likely accumulated by ICE encoded recombination systems which generated a mosaic-like genetic structure39. It is interesting to observe elements of this mosaic structure being found in disparate ICE-like elements isolated from different global locations as outlined above.HS5 contains eight predicted genes, six of which make up the novel putative BREX (bacteriophage exclusion) system found in ICEPmiIre01 has an insertion in Variable Region 1 (VR1). Two predicted genes are present orf01, encoding a putative transcriptional regulator of the XRE family, which shows 100% identity to a hypothetical protein (AAM08068.1) present in ICER391 (AY090559.1) and orf02, encoding a putative Serine/threonine-protein kinase HipA, which is 100% identical to a hypothetical protein (AAM08010.1) also present in ICER391 (AY090559.1). The putative transcriptional regulator of the XRE family, orf01 is also present in ICEBs1 and is proposed to repress the transcription of genes required for excision and transfer and prevents the acquisition of additional ICE copies, therefore, conferring immunity40. HipA-like proteins are expressed by various bacterial species and are involved in high-frequency persistence to the lethal effects of inhibition of either DNA or peptidoglycan. When expressed in high amounts alone, HipA is toxic to bacterial cells41 and may also be involved in multidrug tolerance42. These genes are commonly associated with VR1 of several other SXT/R391-family ICEs (see comparative genomics section).ICEPmiIre01 does not have genes present in variable regions VRII, VRIII, VRIV and VRV as occurs in other SXT\R391 elements and this may be reflect its small size relative to other SXT/R391 ICEs.ICEPmiIre01 is MN520463.The designated GenBank accession number for the nucleotide sequence ICEPmiIre01 in the normal way. Therefore a tri-parnetal mating was undertaken using the ICEPmiIre01 containing P. mirabilis host (susceptible to rifampicin), E.coli CC118λ pBAM1, containing a miniTn5 with kanR and E.coli J53 as described above. We isolated putative strains of E. coli J53 containing ICEPmiIre01 containing the transposed miniTn5. Transconjugants were then analysed to validate and confirm the conjugative transfer. Transfer frequency of the element can be seen in Table Due to not detecting a selectable marker associated with the ICE it was not possible to demonstrate conjugation of ICEPmiIre01 is clustered with ICEPmiJpn1, ICER391, ICEIdBUSA1 and ICEPrSt33672. These results illustrate the wide geographic spread of Type 1 SXT/R391 ICE-like elements with these near neighbours being isolated in Japan, South Africa and the USA respectively. Information on SXT/R391 element in Fig. A phylogenetic tree Fig.  was cons35 and BREX systems30. The putative BREX system located in HS5 of ICEPmiIre01 is related to a novel bacteriophage defence system first reported by Goldfarb et al. (2015)30. It consists of a six gene cassette which includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein and a protein of unknown function. This system shows high levels of homology and synteny with the putative ICEPmiIre01 system.As a result of the abundance of bacteriophage present in wastewater and other environmental niches, bacteria have developed various mechanisms to resist their attack. These resistance mechanisms include RMS, toxin-antitoxin (T-A) system, Abi systemsPmiIre01 BREX system it was discovered that a near-identical unannotated system was also located in HS5 of ICER391 the prototype SXT/R391 ICE (showing between 93–100% homology)39. A recently discovered SXT/R391 ICE, ICEVpaCan1, was also found to contain this putative system7 with homology levels between 68–99%. A comparison between the putative HS5 BREX system of ICEPmiIre01 demonstrated high (84–100%) homology to the BREX system found in Bacillus cereus H3081.9. This is most surprising as SXT/R391 ICEs are exclusively found in Gram-negative bacteria and finding a homologous system in a Gram-positive organism with such high levels of homology is unusual 44. This system is found in numerous SXT/R391 ICE elements and is generally also found inserted in HS215. Examples of this system are displayed in Fig. PmiIre01 found in this study.The insertion in HS2 was also compared to ICER391 as it contains a putative T-A system and is commonly associated with this hotspot in other ICEs. T-A systems consist of a stable toxin and its labile antitoxinPmiIre01 was isolated from a local municipal wastewater by using selective media for Proteus and Vibrio strains (common hosts for SXT/R391 ICEs) and monitoring the presence of ICEs using ICE specific int primers amplified by PCR. A Proteus mirabilis ICEPmiIre01 containing strain isolated in this way was subjected to genome sequencing and revealed an 81 kb element with 75 putative orfs. The element contained the 51 core ICE genes and contained insertions in some of its hotspots which indicated the presence of an encoded bacteriophage resistant mechanisms. A novel BREX system was identified in HS5, not previously identified or annotated in other SXT/R391 ICEs. Comparative analysis revealed that this system is also present in ICER391 but was not previously annotated there40 and in ICEVpaCan1 again not previously annotated7. Unusually the nearest BREX homolog to ICEPmiIre01was reported in a Bacillus subtilis isolate which given the differences between Gram-positive and Gram-negative hosts is surprising. ICEPmiIre01 is a novel SXT/R391 ICE-like element with interestingly no antibiotic or metal resistance, the first to be identified in Ireland.Using a directed approach, a novel environmental SXT/R391 Type 1 family ICE, ICESupplementary information.


M. tuberculosis, and its activity is modulated after phosphorylation from its sensor kinase MtrB. Interestingly, many regulatory effects of MtrA have been reported to be independent of its phosphorylation, thereby suggesting alternate mechanisms of regulation of the MtrAB two-component system in M. tuberculosis. Here, we show that RR MtrA undergoes non-enzymatic acetylation through acetyl phosphate, modulating its activities independent of its phosphorylation status. Acetylated MtrA shows increased phosphorylation and enhanced interaction with SK MtrB assessed by phosphotransfer assays and FRET analysis. We also observed that acetylated MtrA loses its DNA-binding ability on gene targets that are otherwise enhanced by phosphorylation. More interestingly, acetylation is the dominant post-translational modification, overriding the effect of phosphorylation. Evaluation of the impact of MtrA and its lysine mutant overexpression on the growth of H37Ra bacteria under different conditions along with the infection studies on alveolar epithelial cells further strengthens the importance of acetylated MtrA protein in regulating the growth of M. tuberculosis. Overall, we show that both acetylation and phosphorylation regulate the activities of RR MtrA on different target genomic regions. We propose here that, although phosphorylation-dependent binding of MtrA drives its repressor activity on oriC and rpf, acetylation of MtrA turns this off and facilitates division in mycobacteria. Our findings, thus, reveal a more complex regulatory role of RR proteins in which multiple post-translational modifications regulate the activities at the levels of interaction with SK and the target gene expression.MtrA is an essential response regulator (RR) protein in  One of the primary signaling systems found in bacteria is two-component signaling (TCS), which contains distinct sensory and regulatory components or proteins. The communication relay between these proteins occurs via a unique phosphorylation design wherein external stimulus causes the autophosphorylation of the sensor kinase (SK) protein, followed by phosphorylation of the response regulator (RR) protein by a phosphotransfer reaction. In this reaction, the phosphoryl group from the SK is transferred to the RR without involving fresh ATP hydrolysis. The phosphorylation of RR has been shown to alter its downstream activity, which generally involves changes in its DNA binding activity . This stM. tuberculosis, which includes TCS proteins and mainly RRs  affects their interaction with SKs, thus, tuning the fidelity of the SK-to-RR phosphotransfer reaction  (oriC)  for its promoter binding ability on the oriC and rpfB promoters. In view of the extensive regulatory role of phosphorylation and acetylation in different physiological processes inside the cell, it is tempting to speculate that the combinatorial effect of both PTMs may control the functionality of the MtrA response regulator, which is what we observed in our studies.Essential RR MtrA  is shownrpfB)  and orig) (oriC) . In Mtb division . RpfA–E ion . Restriction enzymes were from Thermo Fisher (United States). Cloning primers were from Bioserve (India), and radioactive γ32P ATP  was obtained from BRIT-Jonaki .Chemicals, media, biochemicals, and protein reagents were procured from Merck ; the antibiotics and DTT (Dithiothreitol) were from Goldbio (United States); protein markers were from Abcam (United Kingdom); and NiE. coli DH10β and BL21 Arctic ExpressTM   , respect States) . RecombiThe recombinant proteins were expressed and purified as per protocols reported previously , 2016.Autophosphorylation and phosphotransfer assays were performed as per protocols described previously . Brief pAcetylation analysis of the test proteins was performed by Western blotting using anti-acetyl lysine antibodies as previously described .MST measurement to determine the affinity between the SK-RR proteins has been described previously , and theoriC and 440-bp of rpfB promoter region were PCR-amplified from an M. tuberculosis H37Rv genomic DNA template using specific primers as reported previously  as per the manufacturer’s protocol. After labeling, the fragments were purified and used for the EMSA described previously  and 10 μCi [γ32P] ATP in a reaction buffer containing 25 mM Tris-HCl (pH 7.5), 60 mM potassium acetate, and 10 mM MgCl2 for 15 min at 25°C. The reaction mixture was then filtered through a 3 kDa membrane filter to remove the acetate kinase from the reaction mix to obtain labeled AcP. The flow-through containing 32P-AcP was incubated with 150 pmoles of MtrA or DevR as a positive control (Radiolabeled acetyl phosphate was generated using the protocol described previously . In brie control  for 15 mMtrA protein with or without AcP was subject to circular dichroism spectroscopy using a JASCO J-810 Spectropolarimeter . SpectraE.coli K12 for pta and ackA genes were obtained from C. Choudhary (University of Copenhagen) (2-CaCl2 (MC) buffer at 4°C for 3–4 h. Donor phage was transduced in BL21 Arctic cells, and the transduced cells were then washed with 1M sodium citrate buffer to terminate transduction. The transduced cells were revived in LB for 30 min at 37°C and plated on LA containing kanamycin or gentamycin depending on the donor phenotype.The knockout strains of enhagen) . They wein vivo, the phosphotransacetylase (Δpta) and acetate kinase (ΔackA) mutant E. coli BL21 Arctic strain were transformed with an MtrA expression plasmid. MtrA protein expression and purification were performed as per the protocol described above. The acetylation status of the MtrA protein was analyzed by Western blotting as per the protocol described in To determine if MtrA is acetylated by AcP 600 of the culture was recorded at various time points (as shown). For infection studies, the A549 alveolar epithelial cell line was infected with various strains, and colony forming units (cfu) at 4 and 24 h post-infection (hpi) were examined.H37Ra strains carrying the wild type of lysine mutants (described above) of MtrA in pMV261 were grown in Middlebrook 7H9, Tween-80, OADC media. The primary culture was grown to mid-log phase and sub-cultured in either rich (7H9 + OADC) or poor  media with 25 μg/ml kanamycin. The ODrpfB gene was done from various strains by quantitative RT-PCR analysis as per the protocol previously described  RR interaction with SK, phosphotransfer reaction from SK, RR binding to the target DNA, and dephosphorylation of the RR itself. The RR protein’s first activity involves interaction with SK, which is necessary for the phosphotransfer reaction. Here, the phosphoryl moiety from the activated SK MtrB is transferred to the RR MtrA. Previously, it has been shown that enzymatically acetylated MtrA is a better phosphoacceptor from MtrB . To deterculosis   bound to the DNA strongly  affects the DNA binding activity, we generated acetylation defective (KR substitution) and acetylation mimic (KQ substitution) mutant proteins of MtrA. These mutant proteins were tested by EMSA for binding to the promoter  and oriCC region  as the wC region  for MtrApromoter  as well C region  was reco of MtrA .rpfB promoter and oriC DNA similar to the wild-type MtrA protein  viz. MtrA K110R K207R and MtrA K110Q K207Q and analyzed their DNA binding activity. Confirming our hypothesis, acetylation-defective double mutant protein (KR substitutions), bound  protein . There we of AcP . Overallin vivo, we probed for the presence of acetylation on MtrA protein purified from E. coli, where this protein was overexpressed. We modulated the pool of AcP in E. coli cells using pta and ackA mutant strains and then probed for acetylation in the cells vis-à-vis total MtrA expressed in them. In vivo, AcP is generated from acetyl CoA through the activity of enzyme phospho transacetylase (pta) or from acetate through the enzyme acetate kinase (ackA). The ackA enzyme also catalyzes the interconversion of AcP to acetate, which also feeds into the acetyl-CoA pool , AcP is not converted to acetate. But when cells are grown in acetate containing media, the AcP levels are higher in the Δpta strain . Given that MtrAB TCS and, more specifically, RR MtrA regulate the cell division and growth in ium spp. , we initium spp. . This obium spp. , removesium spp. .110 serves as an AcP-sensitive acetylation site, which affects the growth of mycobacterial cells. Given that the AcP pool is higher in nutrition-rich media . The enhanced expression was significantly higher (16-fold) in the strain carrying the MtrA K110QK207Q double mutant  at an moi of 10, and cfu were recorded at 4 and 24 hpi. Given that overexpression of MtrA wild type reduces the infectivity of M. tuberculosis  proposed previously (The dominant effect of acetylation of MtrA is also in agreement with previous reports, which show that mycobacteria can tolerate the deletion of the trA gene . Also, ttrA gene . It has e of rpf  or envireviously . We showThe datasets generated for this study are available on request to the corresponding author.in vivo acetylation, growth curve, and infection experiments. NB performed the MS/MS studies. DS performed the MST experiments. UW performed CD and RT-PCR experiments and analyzed the data. DS conceived the study, analyzed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.KS designed the study, performed the experiments, analyzed the data, and wrote the manuscript. PA performed EMSA, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In numerous fields such as aerospace, the environment, and energy supply, ice generation and accretion represent a severe issue. For this reason, numerous methods have been developed for ice formation to be delayed and/or to inhibit ice adhesion to the substrates. Among them, laser micro/nanostructuring of surfaces aiming to obtain superhydrophobic behavior has been taken as a starting point for engineering substrates with anti-icing properties. In this review article, the key concept of surface wettability and its relationship with anti-icing is discussed. Furthermore, a comprehensive overview of the laser strategies to obtain superhydrophobic surfaces with anti-icing behavior is provided, from direct laser writing (DLW) to laser-induced periodic surface structuring (LIPSS), and direct laser interference patterning (DLIP). Micro-/nano-texturing of several materials is reviewed, from aluminum alloys to polymeric substrates. Ice in its several forms, i.e., frost, glaze, rime, snow, can cause severe problems for locks and dams , solar pIn recent years, great attention has been paid to the anti-icing surfaces, as attested by the increasing number of research papers and patents related to the topic “anti-icing” .It can be difficult to find a common definition of anti-icing, but some ubiquitous features must be exhibited by such surfaces, i.e., (i) inhibition of water condensation on the surface; (ii) inhibition of the incoming water’s freezing; and (iii) weakening of the adhesion strength of ice, when formed, so that it can be easily removed . Different strategies have been employed to prevent ice accretion and to easily remove it, and they can be divided into active and passive approaches. Keeping the surface temperature above the freezing point by means of electro-thermal heating is an efficient active method to reduce the likelihood of ice formation or to enhance its melting . The expThis notwithstanding, the use of chemical agents to prevent ice formation has many drawbacks. First, their effectiveness is short-lived. Therefore, for large surfaces, periodic treatments are required. Furthermore, extensive use of these liquids, besides having high costs, can cause environmental problems .Other active de-icing techniques consist of using mechanical forces to remove ice accretion from surfaces. With this aim, direct scraping or mechanical removal by shock waves, vibrations, or the twisting of conductors are applied. However, these methods often require personnel to reach the lines and towers; furthermore, helicopters or even shotguns must also be exploited when the ice is less accessible . MoreoveIn addition, hybrid solutions consisting of material with a combination of passive anti-icing and active deicing functionalities have also been reported . In 25]25], the Thus, most of the active or hybrid strategies striving against ice formation are apparently inefficient, energy-consuming, expensive, or dangerous to the environment. It is thus crucial to develop innovative, green, cost-effective, and efficient technologies for anti-icing and deicing . A new development perspective for pure passive anti-icing methods is surface micro- and nano-structuring. Recently, several efforts have been devoted towards the modification of surface topography and/or chemistry in order to obtain superhydrophobic properties ,27. A clA comprehensive review of the most relevant works on the use of laser technology to functionalize surfaces with anti-icing properties is still lacking and would be beneficial to assess the latest advances and evaluate how mature is such a technology to be employed at an industrial scale.In the first part of this review, the key concepts regarding wettability, superhydrophobicity and anti-icing properties will be highlighted. Then, the main laser techniques used to fabricate anti-ice surfaces will be illustrated, ranging from Direct Laser Writing (DLW) to Laser-Induced Periodic Surface Structuring (LIPSS) and Direct Laser Interference Patterning (DLIP). The principal laser treatments carried out to obtain superhydrophobic and anti-icing substrates will be discussed in depth either for metallic or polymeric substrates. Wettability is the result of the stability of the three phases, i.e., gas, liquid and solid, which come into play when a liquid lies on a solid surface. In fact, while the force at the solid–liquid interface (adhesive) tends to spread the liquid, that within the liquid itself (cohesive) instead resists spreading. Therefore, the balance between such forces determines the ability of the surface to be wet. The wetting behavior is normally evaluated by considering the contact angle (CA), i.e., the angle θ between a droplet deposited on the surface and the surface itself, as shown in In the early nineteenth century, Young found a relationship relating the intrinsic contact angle θ to the surface tensions of the liquid–gas essed as :(1)γsl=γThis relation, also called the Young equation, is the starting point for all the investigation on the wetting behavior of surfaces.As mentioned before, another important concept to be introduced to investigate the wettability is the adhesion of the water droplet to the substrate, addressable through the work of adhesion Wa  . This caFrom (1) and (2), A low contact angle . Consequently, the apparent contact angle θ*  is consir is ≥1, and it is defined as [In the Wenzel state, the apparent CA depends on the Young’s CA through the Wenzel wetting equation :(4)cosθ*fined as :(5)r=rou surface .The Wenzel Equation (4), perfectly suitable for characterizing hydrophilic and hydrophobic surfaces, reveals that the contact angle s effect  and selfs effect , obtainaIn case of superhydrophobicity, the droplet no longer infiltrates the surface roughness, but a kind of combined phase contact is established between the solid–liquid and gas–liquid interfaces. So they introduced a new relationship for the apparent contact angle :(6)cosθ*henomena . The exihenomena .Besides the apparent contact angle  surface . When a eding θR , definesγ is the surface tension, R and k are length scale and shape constants for describing the droplet profile , respectively, m is the mass of the droplet, and g is the gravitational acceleration. Thus, a small hysteresis corresponds to a small SA, and researchers usually correlate water adhesion to CHA or SA interchangeably.Another angle that characterizes a moving droplet is the rolling angle α see , also cactively) :(8) sinαUsually, the total wetting Wenzel regime characterizes sticky surfaces with high CAH, where the water droplets tend to grip more than to a flat equivalent surface. Conversely, surfaces which behave in the Cassie and Baxter state are slippery and are characterized by a low CAH, which leads the water drop to slide away more easily than an equivalent flat surface. However, the existence of “sticky” surfaces, i.e., with high intrinsic contact angle hysteresis, in the regime of Cassie and Baxter has also been reported ,54.In recent years, scientists have found that a key factor to obtaining anti-icing properties is superhydrophobicity, which can be obtained through low surface energy coatings  or by inTourkine et al. showed that using SHS at subzero temperature made it possible to easily remove a deposited water drop before freezing thanks solely to gravity, thus preventing the formation of an ice layer . Furthericing delay, intended both as a time delay in the solidification of the water and as a reduction in the threshold temperature for freezing. A lower threshold for the formation of ice would, indeed, allow wider range of operating temperature conditions. Similarly, a postponement of freezing would increase the probability of water removal before solidification.interaction between water and the solid surface. That is, if before freezing occurs, water droplets were to roll off the surface, there would be no chance of ice being formed. This phenomenon is strictly correlated with the sliding angle.ice adhesion. Although ice is formed, its adhesion should be minimized, so that a small external force or, e.g., in case of a wind turbine, the centrifugal force of its own blades, is sufficient to remove the frozen layer, facilitating the de-icing process.The main aspects investigated to assess the anti-icing properties of a surface are:Usually, the testing of these three aspects of an anti-icing surface takes place at atmospheric pressure and in static conditions. Consequently, in the aerospace field, these tests are more appropriate for verifying the anti-icing formation on the ground than in the air, where the atmospheric conditions are more extreme. Several authors have tested the anti-icing propension of processed surfaces by evaluating the icing delay time (DT), namely, the time needed for a reference drop to freeze ,66. The T, heterogeneous nucleation occurs at the liquid–solid interface and, according to the classical nucleation theory [R can be expressed as [r is the droplet radius, A suitable explanation for such phenomena and of the anti-icing properties of SHSs when the temperature is kept below 0 °C is provided by the classic nucleation theory, together with the heat transmission between superhydrophobic surfaces and water droplets ,69. In fn theory , the nucessed as :(9)log10Moreover, it should also be considered that for SHS in the Cassie state, being a good thermal insulator, the air trapped between the water droplet and the rough surface contributes to a reduction in the heat transfer between the liquid surface and solid , furtherh by the following equation [The net heat gain in unit time and volume essed as :(11)Δh=heglected . The timequation :(12)tf=ρMinimizing the contact time between a water droplet and a surface at temperatures below the freezing point has been reported to be a fruitful method for preventing ice accretion from incoming water . In factA superhydrophobic surface, characterized by a low sliding angle and low CAH, which would make the overcooled water droplets bounce or roll off, would behave like a good icephobic surface . Bahadur et al.  developeIn Once ice forms on a surface, its interaction with the substrate at the atomic or molecular scale is a combination of short-range interaction forces, namely electrostatic forces, hydrogen bonding and van der Waals forces. In addition, ice infiltration and interlocking with microscopic surface asperities concur to mechanical adhesion ,78. The ice adhesion to the substrate can be measured by different procedures depicted in F to detach the ice column from the icephobic substrate. Here, ice adhesion strength is determined by dividing the maximum breaking force value by the cross-sectional area of the ice–substrate interface.In the Peak force method  Figure a, a testIn the centrifugal force method depicted in In the tensile force method  shown inAnother method to test the ice adhesion based upon the principle of harmonic excitation was developed by Strobl et al.  Figure d. ThrougThe methods reported so far to assess the anti-icing properties are the main ones used by researchers to characterize the anti-icing surfaces fabricated according to the methods described in the next section.The anti-icing properties of surfaces depend on their chemical composition and roughness . Both ch®) and PDMS, seem to be more prone to low ice adhesion [So far, the role of surface roughness has been highlighted, but it is worth noting that non-rough substrates with a low surface energy, such as PTFE (or Teflonadhesion . Chemicaadhesion : (i) seladhesion ,87; and adhesion . Superhydrophobic coatings with anti-icing properties have also been demonstrated using suspension plasma spraying (SPS) of titanium dioxide . Icing wHowever, it has been found that, even conveniently designing the chemistry of the smooth surfaces, it is hard to achieve ice adhesion regimes sufficiently low to be of practical interest . TherefoA reduction up to 50% of the ice adhesion has been also demonstrated exploiting a photolithographic process that introduces sub-structures into smooth polydimethylsiloxane coatings, which stimulates the initiation of macro-cracks . Others A series of micro-cubic arrays were fabricated by Hou et al.  on silicHowever, all these non-laser-based techniques suffer from intrinsic weaknesses, such as complexity, toxicity of the chemical reagent, and/or expensive mask fabrication requiring expensive clean rooms. To overcome these drawbacks, many groups are moving to the laser machining of surfaces to confer them with anti-icing properties.Laser machining is a one-step direct fabrication method that makes it possible to produce features on a micro and/or nano scale on a broad range of substrates such as metals ,95, polyBased on the dimensions of the structures to be obtained, several laser techniques can be exploited for surface modification. Starting from the micro scale to the nano scale, Direct Laser Writing , Direct Laser Interference Patterning , and Laser-Induced Periodic Surface Structuring , and the hatch distance Starting from the micro scale, direct laser writing is a versatile tool for modifying the surface topography of a solid substrate . Laser sDirect laser ablation for reproducing specific topographies on surfaces has been demonstrated by several authors to be a quick and flexible way to obtain the superhydrophobicity of the lotus leaf. Several laser sources have been employed, from nanosecond  to ultraSeveral studies have been concerned with the use of DLW combined with chemical treatments for enhancing the superhydrophobic ,105 and h, Equation (11)) on the micro-nanostructured surface is greater than that on the original sample surface. Therefore, according to Equation (12), the increase of Δh results in the decrease of ft, which essentially delays the water droplet freezing and promotes the anti-icing ability of the laser-textured aluminum alloy surface.Among all the materials, much attention has been directed towards the use of DLW to confer anti-icing properties on metal surfaces. In particular, considering their light weight, aluminum alloys are used extensively in aircraft production, especially for the wings and fuselage, the safety of which is greatly threatened by ice formation . A laserWang et al.  reportedA similar microgrid structure fabricated by fs-laser was investigated in . Here, t2 laser. Micro channel patterns with various spacing from 100 to 500 μm were produced and different laser powers (up to 5 W) and overlapped scan steps were tested. It was reported that an increase in the micro channel distance caused a decrease in the contact angle in both directions . Hierarchical micro-structures were obtained, and the surface micro-roughness was appropriately maximized step by step. A self-made monitoring system  . LIPSS aAnother approach to producing SHS with multi-scaled hierarchical surface structures consists of combining Direct Laser Writing and Direct Laser Interference Patterning (DLIP) . During Milles et al.  studied To characterize the anti-icing properties of the laser-fabricated surfaces, the freezing time of 8 µL water droplets put onto the surface was measured at −20 °C, as shown in A set of design rules for generating superhydrophobic Ti6Al4V (Ti64) surfaces with icephobic properties was defined in . This veRecently, for the first time, Alamri et al.  reportedA final consideration must be taken regarding the scalability of laser processes. None of the above-mentioned laser techniques, from DLW to LIPSS, despite their promising results in anti-icing, appear suitable for large-scale applications. Nevertheless, many large-scale approaches could be involved to obtain a throughput compatible with industrial environment. The on-the-fly method for synchronizing a laser galvanometer scanner and a linear stage for quick and wide area fabrication could be promising in this direction . MoreoveUndesired ice formation poses an increasingly serious threat in many industrial fields, particularly in aeronautics and telecommunications installations. Currently, the main strategies for reducing the problem are still based on active traditional methods such as mechanical, electro-thermal and liquid hybrid approaches. These approaches are definitively inefficient, energy-consuming, expensive, and can cause systems failure. Passive anti-icing coatings have also been proposed as an alternative to reduce or even replace the traditional de-icing methods. However, the existing available techniques present some disadvantages, such as their short-lived effectiveness, high manufacturing costs, and, in the case of chemical methods, their high environmental impact.In recent years, several studies have demonstrated a correlation between superhydrophobicity and anti-icing properties. Based on the surface wetting theory, substrates characterized by surface micro–nanostructures and low surface free energy can well conform to the wetting regime described by the Cassie–Baxter model. Such surfaces exhibit high contact angles (CA), low hysteresis (CHA) and sliding angles (SA) and, consequently, low droplet adhesion. In sub-zero temperature environments, high CAs usually result in an increase of the droplet freezing time, while low CHAs and SAs lead to a short contact time between dripping droplets and the surfaces, thus hindering ice formation. Indeed, low ice adhesion has been reported on SHSs.Among all the technologies for changing surface topography and obtaining SHSs, laser micromachining is considered to have many advantages. It offers extreme flexibility with respect to the morphology of the structures and surface micro-/nano-features that can be created, without posing any restrictions as to the type of material to be treated. The possibility of scaling up such technology to structure large areas with a reduction in process time and cost is also very attractive for industrial implementation.Several approaches  in different ranges, from nano- to microscale, have been reported in the literature to produce SHSs with anti-icing properties. In all cases, a strong influence of the machined structures on the anti-icing behavior was found. Regardless of the materials, a texture smaller than the droplet size may result in an anti-icing behavior with a delay in the droplet freezing time. This can be explained with both ice nucleation theory and thermodynamics. In fact, the nucleation rate decreases when CA increases, delating the ice formation. Moreover, the air trapped between the water droplet and the rough surface, acting as a thermal insulator, contributes to a reduction in the heat transfer between the solid and the liquid surfaces. Similarly, it was demonstrated that the water repellence can induce supercooled water droplets dripped onto the SH material to bounce away rapidly from the surface before completely freezing. Finally, even though an ice film is eventually formed on the functionalized surface, the superhydrophobic wetting regime may induce a smaller contact interface of solid/ice, thus allowing a lower ice adhesion and the pursuit of the aim of preventing ice accumulation.


The concentration of a protein is highly related to its biochemical properties, and is a key determinant for its biotechnological applications. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are structurally diverse macromolecules that are capable of binding to embryonic ice crystals below 0 °C, making them useful as protectants of ice-block formation. In this study, we examined the maximal solubility of native AFP I–III and AFGP with distilled water, and evaluated concentration dependence of their ice-binding property. Approximately 400 mg/mL (AFP I), 200 mg/mL (AFP II), 100 mg/mL (AFP III), and >1800 mg/mL (AFGP) of the maximal solubility were estimated, and among them AFGP’s solubility is much higher compared with that of ordinary proteins, such as serum albumin (~500 mg/mL). The samples also exhibited unexpectedly high thermal hysteresis values (2–3 °C) at 50–200 mg/mL. Furthermore, the analysis of fluorescence-based ice plane affinity showed that AFP II binds to multiple ice planes in a concentration-dependent manner, for which an oligomerization mechanism was hypothesized. The difference of concentration dependence between AFPs and AFGPs may provide a new clue to help us understand the ice-binding function of these proteins. Concentration dependence is a significant factor for all protein studies and their applications. Protein concentration varies from almost completely insoluble to hundreds of milligrams per milliliter ,2. An exLiposetta pinnifasciata) [Brachyopsis rostratus) [Zoarces elongatus Kner) [Eleginus gracilis) [n (n = 4–50), in which Cβ of Thr is glycosylated with a disaccharide β-d-galactosyl-(1→3)-α-N-acetyl-d-galactosamine [n = 4) to 33.7 kDa (n = 50). The AFGP purified from saffron cod consists of at least 18 distinct isoforms, in which a few amino acids are replaced with Arg or Pro [AFPs and AFGP have been purified from various cold-adapted fishes living in northern midlatitude waters . These iasciata) , AFP II stratus) , AFP IIIus Kner) , and AFGracilis) . A technracilis) . For AFPracilis) . The AFGtosamine . Hence tg or Pro . Althougg or Pro .Tm and Tf, respectively), which is controlled according to the types and concentrations of AF(G)Ps [When approximately 10 mg/mL solution of AFP or AFGP is placed in a freezer for example, neither of them turns into a normal ice block, but instead transforms into an assembly of numerous dispersive single ice crystals . The gro AF(G)Ps ,5,6. In  AF(G)Ps . A relat AF(G)Ps . A lower AF(G)Ps . This mu AF(G)Ps .A recent study showed that a native AFP I sample purified from barfin plaice is extremely soluble (~650 mg/mL), and exhibited high TH activity (3.0 °C) at 200 mg/mL . The stuLiposetta pinnifasciata), longsnout poacher (Brachyopsis rostratus), notched-fin eelpout (Zoarces elongatus Kner), and saffron cod (Eleginus gracilis) were collected by Nichirei Corporation. Size exclusion chromatography and anion exchange chromatography were successively performed for the prepared crude sample powders with a Sephadex G-25 size-exclusion column  and DEAE Sepharose anion-exchange column , respectively [The fish species barfin plaice (ectively . The purThe lyophilized powder of each AFP I–III and AFGP was weighed and dissolved with 200 µL of distilled water in a 50 mL centrifuge tube. The respective samples were then placed into NMR tubes (Ø = 5 mm), where they were left overnight at 4 °C, and photographs of the samples were taken . Approxic-axis on a polarizer, each cylindrical ice was half-cut and mounted on a hollow copper tube (Ø = 15 mm), in which −0.8 °C coolant was circulated by a refrigerant pump . The cylindrical ice crystal was then immersed in distilled water for overgrowth to be changed into a hemispherical shape. We prepared many such spherical single ice crystals, and immersed each crystal in a solution of fluorescent AFP II or AFGP, whose concentration was adjusted between 0.01 and 0.1 mg/mL. After 2 h of incubation of each crystal in a solution, the fluorescence emission or the FIPA pattern was observed under UV light. After capturing the snapshots, a six-sided star mark was created in the center of each spherical ice with the ice-pitting protocol [a1–3-axes of each single ice crystal.For the FIPA experiment, several single ice crystals with a cylindrical shape (Ø = 2–3 cm) were initially prepared within the plastic pipes (l = 4 cm) according to a previously described procedure ,28,29. Aprotocol , which dAll methods were carried out in accordance with relevant guidelines and regulations of National Institute of Advanced Industrial Science and Technology (AIST), Japan. All experiments involving animals were conducted with approved methods designated in Guidelines for Proper Conduct of Animal Experiments, Science Council of Japan .https://imagej.nih.gov/ij/) [Sample powders of AFP I–III and AFGP of 20–30% purity were obtained from Nichirei Corporation , and were extracted from the muscle homogenates of barfin plaice, longsnout poacher, notched-fin eelpout, and saffron cod, respectively . Size exgov/ij/)  enabled The consistency between the weight-base and the fluorometer-base concentrations was verified for AFP I–III within an error range of 1–12% . An ultiTm and Tf of an AFP solution [Tf| value is also called freezing-point depression. Differential scanning calorimetry (DSC) [Tf| via detection of the latent heat emission that originates from the number of crystals generated at the moment of freezing. In contrast, a method that uses a photomicroscope system equipped with a cooling stage to determine the growth-initiation temperature and melting temperature of a single ice crystal was also introduced [Tf and Tm of an AFP solution [Tm − Tf) is the temperature range in which an AFP-adsorbed single ice crystal neither grows nor melts. The TH was therefore used as a parameter to evaluate the AFP’s ice-binding ability. It should be noted that the cooling rate (°C/min) and ice crystal size (µm) need to be adjusted before one can compare the TH value between samples. Such a photomicroscopic method was used in this study, for which a key device is shown in TH is defined as the difference between the solution ,34. The ry (DSC)  and sonory (DSC)  were thetroduced ,38. The Tm and Tf, or the TH value, as AFPs bind on to this bipyramid to terminate its further growth. If the lowering of temperature exceeded the Tf point, a bursting growth occurred from a portion that is not strongly protected by AFPs. Although such portions were generally two tips of the ice bipyramid for AFP I–III and AFGP, they were different for the other types of AFPs from insects, fungi, and bacteria. For a reference solution (water), only a slight temperature change affected the growing and melting of a rounded disk-shaped ice crystal, which indicates the equality of Tm and Tf, implying no TH activity [When the temperature of a sample was constantly lowered on this device (−20 °C/min) A, the phactivity .The TH value was determined for the AFP I–III and AFGP samples C,D. The Kinetic analyses suggest that AFPs are associated with the ice surface within the hysteresis gap ,44. Kris(a)2+-dependent AFP II, AFP III, β-helical AFPs—First prism plane;Ca(b)2+-independent AFP II, β-helical AFPs—Second prism plane;Sculpin AFPI, Ca(c)Flounder AFP I, AFP III, β-helical AFPs—Pyramidal plane;(d)Flounder AFP I dimer (Maxi), β-helical AFPs—Basal plane.A technique has been developed to determine the ice-binding specificity of AFPs through the observation of their FIPA on a single ice crystal hemisphere (Ø = 3 cm) ,28. The Each target plane was determined according to the illumination area called “FIPA pattern” on the spherical ice crystal, to which fluorescent AFPs adsorbs. A wider FIPA pattern implies an extensive AFP adsorption, which leads to an effective ice growth inhibition and a high TH activity . Figure c- and a1- to a3-axes) of the spherical ice crystal are indicated in a1- to a3-axes are the second prism planes, and the polar regions correspond to the basal planes [a1–a3 axes symmetrically but not in the polar regions. The data hence indicate that AFP II binds to the second prism plane but not to the basal plane at the concentration of 0.01 mg/mL. The spherical ice is illuminated entirely with the increase in AFP II concentration up to 0.1 mg/mL. This indicates that AFP II expands its target area to the entire ice surface including the basal plane, similar to AFP I [The present study examined the concentration dependence of the FIPA pattern for AFP II C and AFGl planes . As showto AFP I . Note thn = 4–50) whose Thr sidechain is modified with a disaccharide [3+ group, whereas saffron cod AFGP contains Arg [a-axes were illuminated on the ice sphere ccharide . The ordains Arg . This guThe present experiments showed that not only AFP I , but als2+-independent species obtained from longsnout poacher (B. rostratus) (2ZIB.pdb) [2+-dependent species of AFP II from Japanese smelt Hypomesus nipponensis, for which a high-resolution structure was recently determined for both Ca2+-free (6JK5.pdb) and Ca2+-bound states (6JK4.pdb) [The present AFP II is a 127-residue CaZIB.pdb) , in whicZIB.pdb) . The AFPZIB.pdb) . Note thJK4.pdb) . Their oJK4.pdb)  and PymoJK4.pdb)  via mole2+-dependent AFP II from Japanese smelt, only a Ca2+-bound form accompanies such ice-like waters to bind to the ice first prism plane [An IBS of an AFP species generally accompanies the hydration waters in an ice-like arrangement, for which an anchoring role to bring the host protein to a specific ice plane has been speculated ,23,24. Asm plane . If AFP To summarize, the present study determined the solubility limit of native AFPs and AFGP, and evaluated concentration dependence of their TH value and ice-binding property. These proteins are highly soluble in water, and exhibit 2.0–3.0 °C of high TH value at 50–200 mg/mL. When the ice melts to become liquid water, the hexagonal ice structure will collapse ,57. Supp


Actinoalloteichus cyanogriseus WH1-2216-6 is insensitive to inhibition from by-product α-ketoglutarate or pyruvate. Combined with structural and QM/MM studies, we establish the detailed catalytic mechanism for CrmG. Our structural and biochemical studies reveal that the roof of the active site in PMP-bound CrmG is flexible, which will facilitate the PMP or by-product to dissociate from PMP-bound CrmG. Our results also show that amino acceptor caerulomycin M (CRM M), but not α-ketoglutarate or pyruvate, can form strong interactions with the roof of the active site in PMP-bound CrmG. Based on our results, we propose that the flexible roof of the active site in PMP-bound CrmG may facilitate CrmG to overcome inhibition from the by-product.Amine compounds biosynthesis using ω-transaminases has received considerable attention in the pharmaceutical industry. However, the application of ω-transaminases was hampered by the fundamental challenge of severe by-product inhibition. Here, we report that ω-transaminase CrmG from  Xu, Tang et al. show that ω-transaminase CrmG is insensitive to the inhibition from by-products α-ketoglutarate or pyruvate. They find that these by-products cannot interact strongly with the flexible roof of the active site in CrmG. This study provides insights into the rational design of transaminase to eliminate by-product inhibition. The development of effective and broadly applicable methods for amine compound production has been regarded as a research priority in the pharmaceutical industry. Chemical transformation is currently the predominantly used approach for chiral amines synthesis. However, the drawbacks of the conventional chemical method are apparent, including low enantiopurity selectivity, environmental harm, and sometimes the use of precious and even toxic metal catalysts2. In virtue of environmental friendliness, external cofactor independence, extraordinary enantioselectivity and regioselectivity, ω-transaminases have gained considerable attractions in chiral amine synthesis1. ω-transaminases are a subgroup of pyridoxal 5′-phosphate (PLP)-dependent enzymes, which are capable of catalyzing amination of keto acids, aldehydes, and ketones. This catalytic process consists of two half-reactions3. In the first half-reaction, PLP accepts an amino group from amine (amino donor), resulting in the generation of pyridoxamine 5′-phosphate (PMP)-bound enzyme and ketone or aldehyde by-product; in the second half-reaction, the amino group is transferred to an amino acceptor , leading to new amine production and PLP regeneration.Amine moieties are recognized as one of the vital chemical building blocks forming bioactive natural products, agrochemicals, as well as pharmaceutical drugs7. To overcome these challenges, several by-product removal strategies have been developed. Two coupled enzymatic systems were used to convert pyruvate, the corresponding by-product of universal amino donor l-Ala that is accepted by almost all transaminases9, including l-Ala dehydrogenase/glucose dehydrogenase system that converts pyruvate to l-Ala10, and lactate dehydrogenase/glucose dehydrogenase system that converts pyruvate to lactate12. However, the large-scale application of this strategy was limited by the high costs of the coupled enzyme system. Using isopropylamine as an amino donor, the corresponding by-product acetone can be removed by evaporation under reduced pressure14. Unfortunately, isopropylamine is not widely accepted for ω-transaminases9.Despite the tremendous advantage and potential, the widespread application of ω-transaminases for organic synthesis is facing the fundamental challenges of severe by-product inhibition and unfavorable equilibrium15 developed a mutant of ω-transaminase from Vibrio fluvialis JS17 with reduced by-product inhibitory effect. However, it is still difficult to obtain ω-transaminase completely lacking by-product inhibition through enzyme engineering. Interestingly, in 2013, Park et al.16 reported that ω-transaminase from Ochrobactrum anthropi (OA-ωTA) is devoid of inhibition from by-product acetophenone in the transamination between amino acceptor pyruvate and amino donor (S)-α-MBA. However, the mechanism of how OA-ωTA eliminates inhibition from the ketone product is still unknown. Moreover, until now, no ω-transaminase has been reported to circumvent the inhibition from by-product pyruvate, which is derived from the deamination of the universal amino donor l-Ala. Thus, it is highly desirable to discover by-product inhibition-free ω-transaminases and understand the mechanism, which can efficiently facilitate the rational design of ω-transaminases completely devoid of by-product inhibition and eventually eliminate the obstacle for widespread application of ω-transaminases.Aside from by-product removal, protein engineering was developed to overcome the by-product inhibition of ω-transaminase. In 2005, through random mutagenesis, Yun et al.Actinoalloteichus cyanogriseus WH1-2216-6 has been identified20, and the critical role of the (S)-selective ω-transaminase CrmG in the biosynthesis of CRM A has been reported. CrmG catalyzes amino acceptor caerulomycin M (CRM M) transformation to caerulomycin N (CRM N)21. In this study, we report that, without by-product removal in situ, high conversion of CRM M can be achieved by CrmG with amino donor l-Ala, l-Gln, or l-Glu. Moreover, the activity of CrmG is insensitive to inhibition from ketone α-ketoglutarate or pyruvate. To uncover the mechanism on how CrmG eliminates by-product inhibition, we determine the crystal structures of the apo form CrmG, as well as its complexes with amino donor l-Ala, l-Gln, or l-Glu. Combined with detailed structural analysis of CrmG in complex with the cofactor (PLP or PMP), or amino acceptor CRM M, we not only establish the reaction mechanism of CrmG-catalyzed transamination, but also, more importantly, elucidate the mechanism of CrmG overcoming by-product inhibition.Previously, the biosynthetic pathway of the immunosuppressive agent caerulomycin A (CRM A) from the marine actinomycete 7. Vibrio fluvialis JS17 transaminase (Vf-ωTA) was strongly inhibited by the by-product 2-butanone with a Ki of 5.1 mM15; the activity of Vf-ωTA catalyzing acetophenone amination was completely lost in the presence of 5 mM pyruvate11. Previously, we reported that ω-transaminase CrmG can effectively catalyze CRM M amination with some amino acids, especially l-Glu, as amino donors21  of l-Glu, l-Gln, or l-Ala as the amino donor . The calculated molecule weight of CrmG in solution is 114 kDa , β5/α6 loop and α6-α7 helices cover on top of the active site, hereafter we named this region “roof”  binding site and substrate binding site separated by catalytic base K344of” Fig. . Howeverof” Fig.  and apo of” Fig.  is also of” Fig.  indicateof” Fig. . The cloof” Fig. . Residue21. As shown in Fig. π stack, respectively; and I370′ on α11′ helix forms hydrophobic interactions with P222, W223, P226, and F227 on α7 helix. Therefore, the interactions with F207 and K210 and α11′ helix are essential for the formation and stability of α7 helix. Hence, the structural rearrangement of the “roof” is induced by PLP.To further elucidate how PLP binding induces the structural rearrangement of the catalytic pocket, we revisit the crystal structure of PLP-bound form CrmG (PDB code 5DDS)Chromobacterium violaceum (Cv-ωTA), the flexibility of the roof depends on crystallization condition22. This region is disordered in the apo form Cv-ωTA crystallized with PEG3350, but it folds into the active conformation when crystallized from a solution containing poly(acrylic acid). Besides CrmG and Cv-ωTA, the disordered roof region is also observed in one molecule of the apo form structure of catabolic N-succinylornithine transaminase (AstC)23. However, the roof in the other molecule adopts active conformation in the apo AstC structure. This suggests that a certain degree of flexibility in the roof may be a general property of apo ω-transaminase. This flexibility will facilitate the PLP entry to the active site, as PLP sits deeply in the catalytic pocket and is covered by the roof region of ω-transaminase.To our knowledge, although numerous structures of ω-transaminase have been determined, here, we firstly report a closed form catalytic pocket of apo ω-transaminase with the active base inaccessible to the solvent. Nevertheless, the disordered roof of the active site has been reported in other apo form ω-transaminases. In apo form ω-transaminase from 24. A more recent study proposed that the structural change of the later element is triggered by a steric clash between the free catalytic lysine and Y322′ on the interfacial loop, which is induced by the conformational change of catalytic lysine in apo Cv-ωTA24. Moreover, this clash was also proposed to promote the dissociation of apo Cv-ωTA dimer. However, in CrmG, the Y322 is replaced by F375, and the free catalytic lysine moves away from F375′ in the apo CrmG  (PDB code 4E3Q), PMP-bound form ω-transaminase from O. anthropi (OA-ωTA) (PDB code 5GHF). Different from CrmG, both Vf-ωTA and OA-ωTA prefer pyruvate as amino acceptor25. The B-factor distribution analysis clearly shows that the roof region remains well ordered in both PMP-bound Vf-ωTA and OA-ωTA structures , PLP is converted into PMP and then released from K344. This indicates that the interaction of PMP with CrmG is weaker than that of PLP, which potentially leads to the increased flexibility of the roof region in PMP-bound CrmG. Crystallography temperature factor (B-factor) distribution analysis clearly shows that active site in PMP-bound CrmG is less ordered than that in the PLP-bound CrmG Fig. . The fleres Fig. .26, used transaminase to synthesize PMP, indicating that PMP can be released from the active site of some transaminases. We then hypothesized that the PMP can also be easily released from CrmG as a result of the increased flexibility of the roof at the active site in PMP-bound CrmG. To test this notion, we performed the PLP conversion reaction . As shown in Fig. λmax ≈ 414 nm) converted to PMP (absorption λmax ≈ 323 nm). Within 48 h, almost all PLP is converted to PMP using excess amino donor l-Glu, l-Gln, or l-Ala. We should note that the concentration of PLP is 10 times of CrmG in these reactions. This indicates that the synthesized PMP should be released from CrmG, which then allows new PLP to bind to the active site and be converted to PMP. Our structures show that PMP/PLP is deeply buried in the catalytic pocket and covered by the roof , dissociation of geminal diamine to generate external aldimine , and hydrolysis of external aldimine   approach. The energy profile for CrmG catalyzing -3) Fig. . Similarl-Glu deamination is an exothermic reaction with calculated reaction enthalpy of −33.5 kcal/mol, and CRM M amination is endothermic with calculated reaction enthalpy of 11.5 kcal/mol. The QM/MM results suggest that l-Glu deamination is energetically more favorable than CRM N deamination, and CRM M amination is energetically more favorable than α-ketoglutarate amination. This implies that by-product α-ketoglutarate and product CRM N cannot induce severe unfavorable equilibrium for CrmG catalyzing CRM M amination with amino donor l-Glu. This corroborates well with our biochemical results that high conversion of CRM M by CrmG can be achieved using l-Glu as an amino donor.As shown in Fig. 25 and OA-ωTA14, and the acetophenone amination by Vf-ωTA was severely inhibited by pyruvate11. In the docked structure of PMP-bound form OA-ωTA in complex with pyruvate, the carboxyl group of pyruvate was coordinated by hydrogen bonds with residue W58 and R41729, which are conserved in Vf-ωTA. Similarly, in the structure of CrmG in complex with l-Ala (corresponding amino donor for by-product pyruvate), the carboxyl group of l-Ala forms a hydrogen bond with Q318 and a salt bridge with R486  to PLP, generating by-product and PMP. In PMP-bound CrmG, the active site is dynamic and flexible. The by-products converted from l-Ala/l-Glu/l-Gln deamination are easily released from the PMP-bound form CrmG, owing to the flexible roof of the active site and the inability of the by-products to stabilize the flexible roof. Hence, the by-product cannot prevent CRM M from binding to the active site of PMP-bound form CrmG. Furthermore, CRM M binding can stabilize the roof of the active site in the PMP-bound CrmG, which can enable CRM M to form intermediate with PMP, resulting in CRM N formation and PLP regeneration.Taken together, our results indicate that stabilizing the active site in PMP-bound CrmG is essential for amino acceptor amination and by-product inhibition. Thus, we propose a mechanism for the by-product inhibition elimination for CrmG  and 2 µL reservoir . To obtain crystals of l-Gln-bound, l-Glu-bound, and l-Ala-bound CrmG, CrmG was incubated with 5 mM PLP and 5 mM l-Gln, 5 mM PLP, and 5 mM l-Glu, 5 mM PLP, and 5 mM l-Ala prior to crystallization, respectively. The crystals of amino donor-bound CrmG were grown in 0.2 M sodium acetate, 0.1 M Tris pH 8.5, 30% PEG 3350, and 2% glycerol.All crystals of CrmG were grown at 293 K with sitting drop vapor diffusion method using the sparse matrix crystallization kit from Hampton research. The apo CrmG crystal in space group I222 was crystallized with 2 µL protein and 2 µL reservoir ; the apo CrmG crystal in space group C2 was crystallized with 4 µL protein  as the template. The refinement of the structures was conducted using Refmac536. Data collection and refinement statistics are shown in Table l-Gln, 512 residues, 512 residues, 512 residues, and 508 residues were modeled in chain A, B, C, and D, respectively. For structure of CrmG in complex with amino donor l-Glu, 512 residues were modeled in each of the four molecules in the asymmetric unit. For structure of CrmG in complex with amino donor l-Ala, 511 residues, 511 residues, 510 residues, and 512 residues were modeled in chain A, B, C, and D, respectively.All crystals were cryo-protected in mother liquid with addition of 20% glycerol, prior to collecting data at 100 K. Diffraction data for apo form, l-Gln, l-Glu, or l-Ala) at 30 °C. The reaction was analyzed with a CD spectrophotometer after blanked with Tris buffer. For CD spectrophotometer analysis, one milliliter quartz cuvettes were used, and spectra from 500 to 300 nm were recorded.For PLP conversion reaction by CrmG, the enzyme assays were carried out in 1 mL reaction mixture in Tris buffer  by incubating 0.02 mM CrmG or CrmG W223A mutant, 0.2 mM PLP, and 2 mM amino donor , 0.2 mM CRM M, at 30 °C for 0–48 h. HPLC was used to analyze CRM M consumption and CRM N production in the reaction. The HPLC analysis was carried out on a C18 column  with a running program previously described21. Briefly, a gradient was run from 5% CH3CN to 30% CH3CN for 18 min, UV detection was performed at 313 nm.The CRM M conversion reactions were performed in 50 μL assay solution  containing 4 μM CrmG, 80 μM PLP, 0.3 mM amino donor  containing 4 μM CrmG, 80 μM PLP, 100 mM l-Glu, 0–5 mM CRM M, and 0/50 mM/100 mM α-ketoglutarate or pyruvate. Competitive inhibition constant of α-ketoglutarate or pyruvate toward CrmG was calculated by nonlinear regression analysis.The l-Gln, l-Glu, and l-Ala were determined at 30 °C in 50 μL assay solution  containing 4 μM CrmG, 80 μM PLP, 0.2 mM CRM M and amino donor l-Gln/l-Glu/l-Ala.The kinetic parameters of CrmG toward amino donor 37 was adopted to equilibrate the complex structure. The structure in the stable stage of the 50 ns MD simulations was chosen as the initial model for QM/MM simulations. Periodic boundary condition was considered in MM MD, whereas spherical boundary condition was employed in QM/MM simulation. QM atoms were all described by DFT method (B3LYP) at 6–31G* basis set and MM atoms were described by the AMBER99SB force field38. The QM/MM boundary was treated by the improved pseudo bond approach and the 12 and 18 Å cutoffs were employed for van der Waals and electrostatic interactions, respectively. The QM/MM systems were minimized again for several iterations, and more than 5 ps QM/MM MD simulations were performed. The minimal time step of integration (1 fs/step) and equilibration time are adopted to give a more relaxed reaction system. The resulting conformations were used to map out the minimum energy path with the reaction coordinate driving method39. All the QM/MM simulations were run with QChem4.040 and Tinker41 programs. The whole protocol is transplanted from our previous studies and has been carefully validated44. Additional benchmark tests on the computational level , choice of QM region and simulation time are described in Supporting Information  were employed as the initial models for the QM/MM simulation. A multi-step MM MD simulation performed with pmemd.cuda in AMBER12Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummaryPeer Review File


Mitochondrial serine catabolism to formate induces a metabolic switch to a hypermetabolic state with high rates of glycolysis, purine synthesis and pyrimidine synthesis. While formate is a purine precursor, it is not clear how formate induces pyrimidine synthesis.Here we combine phospho-proteome and metabolic profiling to determine how formate induces pyrimidine synthesis.We discover that formate induces phosphorylation of carbamoyl phosphate synthetase (CAD), which is known to increase CAD enzymatic activity. Mechanistically, formate induces mechanistic target of rapamycin complex 1 (mTORC1) activity as quantified by phosphorylation of its targets S6, 4E-BP1, S6K1 and CAD. Treatment with the allosteric mTORC1 inhibitor rapamycin abrogates CAD phosphorylation and pyrimidine synthesis induced by formate. Furthermore, we show that the formate-dependent induction of mTOR signalling and CAD phosphorylation is dependent on an increase in purine synthesis.We conclude that formate activates mTORC1 and induces pyrimidine synthesis via the mTORC1-dependent phosphorylation of CAD. Cells activate metabolic pathways during differentiation and de-differentiation. The activation of one or more biochemical reactions can, in turn, trigger a cascade of metabolic changes leading to a distinct metabolic state. We have recently shown that the mitochondrial-dependent oxidation of the third carbon of serine to formate triggers a metabolic switch . This swNevertheless, how formate induces pyrimidine synthesis is yet unclear. The formate-dependent induction of pyrimidine synthesis is characterized by a dramatic increase of dihydroorotate levels . DihydroCAD maximum activity is regulated by a mechanistic target of rapamycin complex 1 (mTORC1)-dependent phosphorylation , 4. In tCell culture medium and supplements were obtained from the Life Technologies, and all chemicals were purchased from Sigma-Aldrich unless specified otherwise.HAP1-WT were cultured in IMDM medium supplemented with 10% FBS. HAP1-ΔSHMT2 cells were cultured in the same media supplemented with a mix of 16 μM thymidine and 100 μM hypoxanthine (HT). MDA-MB-231-ΔSHMT2 cells -glutamine was added to the media at 4 mM. To harvest, wells were washed with ice-cold PBS and scraped and extracted with freezer cold extraction solvent (acetonitrile/MeOH/H2O (30/50/20)). The extracts were transferred into tubes and centrifuged for 5 min at 18 k G. Supernatants were transferred to LC-MS glass vials and kept at – 80 °C until LC-MS analysis using a pHILIC chromatography and a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Compounds were identified using Tracefinder 4.1 (Thermo Scientific), comparing the exact mass and the retention time against an in-house compound database created with authentic standards.Metabolite extraction and analysis were performed as previously described . BrieflyHAP1-ΔSHMT2/WT and MDA-MB-231-ΔSHMT2 cells were seeded in 60 mm dishes at 350,000 cells/dish and 850,000 cells/dish, respectively. Cells were stimulated the following day with combinations of 1 mM formate, 50 nM rapamycin, 1 μM SHIN1 (MedChemExpress), 50 μM L-alanosine (Santa Cruz Biotechnology) and HT as indicated. Cells were washed twice in ice-cold PBS, scraped and lysed in cold RIPA buffer (ThermoScientific) containing cOmplete phosphatase and protease inhibitors. Total protein concentration was determined by DC protein assay (Bio-Rad), and equal amount of proteins was separated using precast 3–8% or 4–12% NuPage gels (ThermoScientific) or 15% SDS-PAGE gels (for eIF4EBP1 identification) and transferred to nitrocellulose using an Invitrogen XCell II Blot Module. The membranes were incubated overnight at 4 °C using the following primary antibodies at 1:1000: phospho S6-RP Ser235/236 (#4858), total S6-RP (#2217), phospho CAD Ser1859 (#12662), total CAD (#93925), phospho p70 S6K T389 (#9234), total p70 S6K (#2708), 4E-BP1 (#9644)  and β-tubulin (T4026). Secondary antibodies donkey anti-mouse 800CW and goat anti-rabbit IgG (H+L) Alexa Fluor 680  were incubated at 1:5000 dilution. Immunoblots were analysed and protein densities quantified using an Odyssey CLx imager and Image Studio Lite software (Li-COR Biosciences).t test otherwise indicated.Technical replicates were used to calculate statistics in the phosphoproteomics analysis. Biological replicates were used to calculate statistics in the immunoblots metabolomics analyses. Statistical significances were calculated using two-tailed and unequal variance To investigate how formate increases pyrimidine synthesis, we developed a hypothesis based on formate increasing ATP levels . We analIn order to observe dramatic changes in the concentration of dihydroorotate, it is necessary that the maximum activity of synthesis exceeds that of consumption. Therefore, the increase of dihydroorotate levels upon supplementation of formate can be explained by two hypotheses: (i) either the synthesis of dihydroorotate is higher than its turnover before the addition of formate or (ii) the addition of formate induces an increase in dihydroorotate synthesis relative to turnover.Protein phosphorylation can change the activity of metabolic enzymes providing a biochemical mechanism underpinning the second hypothesis that the addition of formate induces an increase in dihydroorotate synthesis relative to turnover (ii). We previously reported that formate induces changes in phosphorylation levels of AMPK and its downstream targets . Here weSHMT2 (HAP1-ΔSHMT2) exhibit a reduction in formate production relative to the parental cell line (HAP1-WT) [The first step in the mitochondrial serine catabolism to formate is catalysed by the mitochondrial serine hydroxymethyltransferase (SHMT2). HAP1 cells genetically engineered to inactivate To investigate the impact of formate supplementation on protein phosphorylation in an unbiased manner, we performed a phospho-proteome analysis of HAP1-ΔSHMT2 cells and HAP1-ΔSHMT2 cells supplemented with formate , inosine monophosphate (IMP), adenosine monophosphate (AMP) and guanosine monophosphate (GMP) (Fig. To investigate the effects of rapamycin and formate on HAP1-ΔSHMT2 cells after longer treatment, we analysed intracellular metabolite levels after 24 h of culture using mass spectrometry. As previously shown [15N incorporation into dihydroorotate and orotate in the first 6 h, and the repression of CAD-Ser1859 phosphorylation by rapamycin (Fig. In contrast, formate had no effect on the levels of most pyrimidines, purines and their precursors when cells were treated with rapamycin. This observation agrees with the finding of rapamycin repression of cin Fig. .There is one noted exception. Formate induced a decrease rather than an increase of AICAR levels, particularly at the highest formate concentrations Fig. e. The sa13C incorporation into ATP up to 6 h (Fig. Based on the bottleneck model of Fig. 6 h Fig. f, and AT6 h Fig. g. In conOur work shows that the formate induction of pyrimidine synthesis is mediated by the mTORC1/S6K1-dependent phosphorylation of CAD at Ser1859. We also provide evidence that these phosphorylation events are just the tip of the iceberg. More than half of the protein phosphorylation changes induced by formate supplementation are suppressed by treatment with rapamycin.Formate induction of mTORC1 signalling is consistent with the formate-dependent increase of purine synthesis and the previously reported mTORC1 activation by purine nucleotides , 9. To cThe extrapolation of these observations to mammalian physiology requires further work. There are mammalian tissues with active purine synthesis, where the formate-dependent regulation of mTORC1 signalling may play a role. The inactivation of mitochondrial formate production genes carries as a consequence neurodevelopmental defects and embryonic lethality, which can be rescued by sodium formate supplementation , 14. mTOTumour metabolism is also characterized by an induction of mitochondrial formate production genes and purine synthesis . In turnFinally, the relationship between mTORC1 signalling and formate availability requires further investigation from the nutritional point of view. Currently, we have a very poor understanding of the nutritional demand of formate or one-carbon units in general , 19. UndFormate activates mTORC1 and induces pyrimidine synthesis through mTORC1-dependent phosphorylation of CAD at Ser1859.Additional file 1. Phosphoproteomics quantifications of HAP1-ΔSHMT2 cells supplemented with formate and controls.Additional file 2. Phosphoproteomics quantifications of HAP1-ΔSHMT2 cells supplemented with formate and controls, with rapamycin treatment.