Background: Dolutegravir (DTG)-based antiretroviral therapy (ART) rarely leads to virological failure (VF) and drug resistance in integrase strand transfer inhibitor (INSTI)-naïve persons living with HIV (PLWH). As a result, limited data are available on INSTI-associated drug resistance mutations (DRMs) selected by DTG-containing ART regimens. Methods: We reviewed studies published through July 2023 to identify those reporting emergent major INSTI-associated DRMs in INSTI-naïve PLWH receiving DTG and those containing in vitro DTG susceptibility results using a standardized assay. Results: We identified 36 publications reporting 99 PLWH in whom major nonpolymorphic INSTI-associated DRMs developed on a DTG-containing regimen and 21 publications containing 269 in vitro DTG susceptibility results. DTG-selected DRMs clustered into four largely non-overlapping mutational pathways characterized by mutations at four signature positions: R263K, G118R, N155H, and Q148H/R/K. Eighty-two (82.8%) viruses contained just one signature DRM, including R263K (n = 40), G118R (n = 24), N155H (n = 9), and Q148H/R/K (n = 9). Nine (9.1%) contained ≥1 signature DRM, and eight (8.1%) contained just other DRMs. R263K and G118R were negatively associated with one another and with N155H and Q148H/K/R. R263K alone conferred a median 2.0-fold (IQR: 1.8–2.2) reduction in DTG susceptibility. G118R alone conferred a median 18.8-fold (IQR:14.2–23.4) reduction in DTG susceptibility. N155H alone conferred a median 1.4-fold (IQR: 1.2–1.6) reduction in DTG susceptibility. Q148H/R/K alone conferred a median 0.8-fold (IQR: 0.7–1.1) reduction in DTG susceptibility. Considerably higher levels of reduced susceptibility often occurred when signature DRMs occurred with additional INSTI-associated DRMs. Conclusions: Among INSTI-naïve PLWH with VF and treatment emergent INSTI-associated DRMs, most developed one of four signature DRMs, most commonly R263K or G118R. G118R was associated with a much greater reduction in DTG susceptibility than R263K. Among integrase strand transfer inhibitor (INSTI)-naïve persons living with HIV (PLWH), antiretroviral (ARV) therapy (ART) with the second-generation INSTIs dolutegravir (DTG) and bictegravir has been associated with low rates of virological failure (VF) and emergent INSTI-associated drug resistance mutations (DRMs) . In the Given that resistance to DTG is determined genotypically, we conducted a rapid review of all studies reporting the emergence of INSTI-associated DRMs in INSTI-naïve PLWH experiencing VF while receiving a DTG-containing regimen and of in vitro susceptibility data associated with DTG-selected DRMs. Our group previously published a systematic review of DTG-selected DRMs in 2019 that summarized data from 14 publications reporting 29 cases of DTG-selected DRMs in previously INSTI-naïve PLWH and 12 publications reporting the results of in vitro DTG susceptibility testing using the PhenoSense assay for 179 distinct HIV-1 isolates . In the We reviewed PubMed using the search phrase “(Dolutegravir) AND ((“Drug Resistance”[Mesh] OR Resistance) OR “Clinical Trial” [Publication Type] OR (“Mutation”[Mesh] OR Mutation(s)) OR (“in vitro susceptibility”))”, last updated July 18, 2023, to identify studies containing data pertinent to the following two types of information: (1) integrase mutations emerging in INSTI-naïve persons receiving DTG; and (2) the effect of integrase mutations on in vitro DTG susceptibility.We included studies that provided complete integrase sequences as well as studies that reported only those integrase mutations noted by study authors. Nonpolymorphic major INSTI-associated DRMs were defined as H51Y, T66A/I/K, E92G/Q, G118R, F121Y, E138A/K/T, G140A/C/S, Y143C/H/R/S, S147G, Q148H/R/K, S153Y/F, N155H, S230R, and R263K [When HIV-1 isolates containing INSTI-associated DRMs were reported in more than one publication describing the results from a single clinical trial or cohort, we linked the isolates to just one of the publications. For the few PLWH with more than one isolate containing an INSTI-associated DRM, we recorded the isolate containing the largest number of DRMs. When sequences were available, we analyzed them for their sequence quality and for the presence of APOBEC-mediated G-to-A hypermutation. Two sequences, including one with multiple sequencing errors and another with G-to-A hypermutation, were excluded from analysis. To determine the effect of INSTI-associated DRMs on DTG susceptibility, we analyzed all published in vitro DTG susceptibility results performed using the PhenoSense assay because this is the most commonly used assay and because it is highly reproducible . ReductiIf a tested HIV-1 isolate was a site-directed mutant and the list of mutated integrase residues was provided, the isolate’s mutation list was considered to be complete. If a tested HIV-1 isolate was derived from a clinical sample, the mutation list was considered to be complete only if the nucleic acid sequence or a complete list of amino acid differences from a reference sequence was provided. Replication capacity values were obtained from published studies of isolates in the Stanford HIV Drug Resistance Database (HIVDB) that underwent susceptibility testing using the PhenoSense method . ReplicaWe summarized the results for the most common patterns of INSTI-associated DRMs and performed a least squares regression (LSR) analysis in which DRMs were included as explanatory variables and the log fold change in susceptibility was the response variable. Ten repetitions of 5-fold cross-validation were performed to estimate the variance among the fitted coefficients. p value ≤ 0.05. For this analysis, mutations present as part of a mixture with wildtype were excluded. We also created a list of positively correlated mutation pairs defined as those (1) co-occurring in at least two individuals, (2) having a nonparametric Spearman’s correlation coefficient (ρ) ≥ 0.20, and (3) having a As of 18 July 2023, 861 publications were retrieved from PubMed . FollowiThirty-six publications reported 99 one-per-person viruses with one or more major nonpolymorphic INSTI-associated mutations from a previously INSTI-naïve individual treated with a DTG-containing regimen . Eleven The PLWH developing INSTI-associated DRMs included 49 (49.5%) ART-experienced viremic individuals receiving DTG plus two NRTIs, 11 (11.1%) ART-experienced individuals with virological suppression receiving DTG monotherapy, 15 ART-naïve viremic individuals receiving DTG plus two NRTIs or DTG plus 3TC, and 24 individuals with other combinations of ART history, virological status, and DTG ART . The geon = 40), G118R (n = 24), N155H (n = 9); and Q148H/R/K (n = 9). Nine virus sequences contained more than one signature DRM including two sequences in which a mixture was present at one or more of the signature positions. Eight sequences contained just other  INSTI-associated DRMs including S230R (n = 3), T66I, E138K, H51Y + S147G, E138A + G149A, E138K + D232N.DTG-selected INSTI-associated DRMs clustered into four largely non-overlapping mutational pathways characterized by amino acid mutations at four signature positions: (1) R263K; (2) G118R; (3) N155H; and (4) Q148H/R/K. Indeed, 82 (82.8%) of 99 virus sequences contained just one of the signature mutations including R263K . Therefore, G118R would be expected to result from a single G→C transversion at its first nucleotide rather than by double nucleotide mutations at its first and third codon positions. Indeed, of the eight isolates containing G118R for which sequences were available, seven were encoded by CGC or CGT at position 118, suggesting that these resulted from a G→C transversion at its first codon position. Although sequences were not available from any individual prior to receiving DTG, it was possible to infer from the available post-DTG-containing sequences that R263K resulted from a single G→A transition, while the remaining signature DRMs usually resulted from a single nucleotide transversion. Nearly all previously published wildtype sequences indicate that position 118 is usually encoded by GGC/T (rather than GGG/A), regardless of subtype  . With ≥1Of the DRMs included in the least-squares regression model, 26 mutations occurred ≥5 times in the dataset . The regn = 13), Q148H/R/K (n = 23), N155H (n = 13), and R263K (n = 11)  . The medThe primary objectives of this study were to identify the INSTI-associated DRMs emerging in previously INSTI-naïve persons with VF while receiving a DTG-containing regimen and to estimate the impact of these DRMs on in vitro DTG susceptibility. Among the 99 individuals reported in 36 publications who developed INSTI-associated DRMs while receiving DTG, more than 90% developed one or more of four signature INSTI-associated DRMs, including R263K, G118R, N155H, and Q148H/R/K. R263K and G118R represented distinct non-overlapping mutational pathways, as they were unlikely to occur with each other or with N155H and Q148H/R/K. Emergent DTG resistance was reported in 40 PLWH in 11 clinical trials for which the total number of individuals receiving DTG was known. However, most individuals with emergent DTG resistance were reported in uncontrolled studies for which the total number of individuals receiving DTG was not known. Additionally, our search identified only those studies that contained cases of emergent DTG resistance and did not identify studies in which DTG was received, but INSTI-associated DRMs did not arise. For these two reasons, a different systematic review would be required to determine the prevalence of emergent DTG resistance in different clinical scenarios.R263K is the only signature mutation that results from a single nucleotide transition . Transitions arise more readily in viruses in general and in HIV-1 in particular . In contThe different mutational pathways can be explained in part by the reduced replication capacity and integration by viruses containing certain combinations of signature DTG-associated mutations. R263K is associated with reduced strand transfer activity and reduced infectiousness in cell culture when it occurs in combination with several of the DRMs associated with the first-generation INSTIs including Q148R . In addiOur in vitro susceptibility results are consistent with published biochemical data showing that DTG dissociates more rapidly from integrase DNA complexes containing G118R than those containing R263K . HomologThere are few data on the clinical significance of reduced DTG susceptibility. Data from the VIKING trials evaluated the clinical efficacy of 50 mg DTG twice daily plus an optimized background regimen on treating ART-experienced PLWH with VF and INSTI resistance after receiving a first-generation INSTI ,7. AmongOther than the increased likelihood of Q148H/R/K and N155H developing in PLWH receiving DTG monotherapy, we were unable to determine which clinical factors  were associated with particular signature DRMs. It was also not possible to determine whether HIV-1 subtype influenced which signature DRMs emerged because the absence of sequences from more than 80% of individuals made it impossible to determine the subtype of most viruses with INSTI-associated DRMs.The presence of four mutational pathways associated with DTG resistance suggests that point-of-care assays for detecting mutations at four signature DRM positions might be useful in identifying which PLWH with VF while receiving DTG have developed DRMs that might preclude a response to continued DTG ART and adherence counseling . HoweverThe second-generation INSTI cabotegravir has been reported to select for three of the signature DRMs selected by DTG: Q148R, N155H, and R263K. However, the frequency of these mutations differs from those selected by DTG. Of 18 PLWH with emergent INSTI-associated DRMs while on cabotegravir, 11 had Q148R, six had N155H, one had R263K, and none had G118R . ComparaTwo mechanisms of INSTI resistance involving mutations outside of integrase have been reported, including one caused by mutations in and near the 3′ polypurine tract (3′PPT) and one caused by mutations in the envelope glycoprotein. 3′PPT mutations have been selected during in vitro passage experiments with DTG  and haveIn conclusion, our review provides an initial assessment of the patterns of INSTI-associated DRMs that have been reported in INSTI-naïve PLWH receiving DTG. Future studies in low and middle income countries with rapidly expanding access to DTG-containing ART are required to determine the risk of VF and of emergent DTG resistance in the clinical scenarios in which DTG-containing regimens will most commonly be used. Additionally, studies are required to determine the clinical significance of emergent DTG resistance, particularly when arising from DRMs that are not associated with high levels of reduced DTG susceptibility.


Penicillium oxaliucm is a typical fungus that produces cellulolytic enzymes. In this study, we reported the biological function of eight putative methyltransferases (PoMtr23C/D/E/F/G/H and PoMtr25A/B) containing a methyltransf_23 or methyltransf_25 domain, with a focus on their roles in the production of cellulolytic enzymes. In P. oxalicum, various methyltransferase genes displayed different transcriptional levels. The genes Pomtr23C and Pomtr25A exhibited high transcriptional levels, while Pomtr23D/E/F/G/H and Pomtr25B were transcribed constantly at low levels. The gene deletion mutants (Δmtr23C/D/E/F/G/H and Δmtr25A/B) were constructed. Various mutants have different patterns in cellulolytic enzyme production. Compared to the WT, the largest increase in filter paper activity  was observed in the Δmtr23G mutant, the only mutant with a cellulolytic halo surrounding the colony. Three mutants (Δmtr23C/D and Δmtr25A) also showed increased cellulolytic enzyme production. The Δmtr23E and Δmtr25B mutants displayed decreased FPA activity, while the Δmtr23F and Δmtr23H mutants displayed similar patterns of cellulolytic enzyme production compared with the WT. The assay of transcriptional levels of cellobiohydrolase gene Pocbh1 and β-1,4-endoglucanase Poeg1 supported that higher cellulolytic gene transcription resulted in higher production of cellulolytic enzymes, and vice versa. The transcriptional levels of two transcription factors, activator XlnR and repressor CreA, were measured. The high transcription level of the PoxlnR gene in the Δmtr23D mutant should be one reason for the increased transcription of its cellulolytic enzyme gene. Both XlnR and CreA transcriptional levels increased in the Δmtr23G mutant, but the former showed a more significant increase than the latter, indicating that the activation effect predominated. The PoMtr25A is localized in the nucleus. The catalytic subunit SNF2 of the SWI/SNF chromatin-remodeling complex was found as one of the interacting proteins of PoMtr25A via tandem affinity purification coupled with mass spectrometry. PoMtr25A may affect not only the transcription of repressor CreA but also by recruiting SWI/SNF complexes that affect chromatin structure, thereby regulating the transcription of target genes.Putative methyltranferase LaeA and LaeA-like proteins, conserved in many filamentous fungi, regulate fungal growth, development, virulence, the biosynthesis of secondary metabolites, and the production of cellulolytic enzymes.  Trichoderma, Aspergillus, and Penicillium, have been developed for the industrial production of plant biomass-based biofuels and biochemicals due to their adequate production capacity of extracellular cellulolytic enzymes [Penicillium species are regarded as enzyme factories because they create a high representation of extracellular enzymes, including cellulases and hemicellulases involved in the degradation of plant cell walls, which are valuable in white biotechnology. Penicillium oxalicum (previously Penicillium decumbens), which produces a variety of lignocellulolytic enzymes, has a high potential for use in lignocellulose hydrolysis. Its cellulase hyperproducing mutant has been used to produce lignocellulolytic enzymes on an industrial scale [Plants make up the overwhelming majority of biomass on Earth. Filamentous fungi, such as  enzymes . Some Peal scale ,3.The production of fungal lignocellulolytic enzymes is tightly controlled at the transcriptional level. The majority of cellulolytic enzyme genes are controlled by the coordinated activity of transcription factors (TFs), including activators  and repressors  ,6,7,8.aflR expression) was initially identified as a secondary metabolic global regulator in Aspergillus nidulans. The lack of A. nidulans AnLaeA inhibited the expression of the TF AflR, which controls the secondary metabolic gene cluster in sterigmatocystin formation [Aspergillus spp., Penicillium spp., Fusarium spp., and Trichoderma spp. [laeA significantly reduced sporulation in Aspergillus fumigatus, Trichoderma reesei, and P. oxalicum [laeA gene deletion have been found to display reduced virulence [The LaeA/Lae1 , specifically the genes for cellulolytic enzymes. In addition to being a virulence factor, the insect pathogenic fungus annanase . T. reesn strain . LaeA wamarkably ,16. LaeA. reesei . The traA. nidulans genome [P. oxalicum contained 11 LLMs with SAM sites similar to PoLaeA [A. nidulans AnLimF and AnLlmG, Cochliobolus heterostrophus Llm1 (the ortholog of AnLlmF), and P. oxalicum LLM PoMtr23B, the regulatory functions of these LLMs were both similar to and different from those of LaeA [P. oxalicum Mtr23B controls the production of extracellular GHs; deleting the Pomtr23B gene inhibited the expression of cellulolytic genes, including five cellulase genes and the xylanase gene [Pleurotus ostreatus decreased cellulase production [LaeA is considered a methyltransferase because it harbors a methyltrasf_23 domain and a conserved S-adenosylmethionine (SAM) binding site . Proteins genome . P. oxalo PoLaeA . Accordi of LaeA ,18,19,20 of LaeA ,19. Addioduction . Thus, tA. nidulans, the lack of LaeA leads to the silent sterigmatocystin gene cluster, which includes the occupation of the heterochromatin protein-1 (HepA) and is characterized by repressive histone H3 lysine (K) 9 trimethylation (H3K9me3) [M. thermophila LaeA [laeA deletion mutants have changed chromatin modification, it does not prove that LaeA and histone modification are directly related. It was even found that T. reesei Trlae1 expression was unrelated to H3K9me3, H3K4me2, or H3K4me3 in regions where the carbohydrate-active enzyme coding gene was present [A. nidulans LLMs (AnLlmA to AnLlmJ) roles in regulating fungal development and the synthesis of secondary metabolites have been investigated [LaeA is a cryptic methyltransferase. It has been shown that in H3K9me3) . Modulatila LaeA . However present . Althoug present . Hence, stigated ; it is uP. oxalicum, emphasizing their effects on cellulolytic gene transcription and cellulolytic enzyme synthesis. Different methyltransferases have various effects on the expression of cellulolytic genes and differ in terms of the underlying mechanisms that regulate this expression.In this study, we investigated the fundamental biological roles of eight putative methyltransferases in P. oxalicum 114-2 (CGMCC 5302), which was previously categorized as P. decumbens [3Citrate·2H2O, 250 g KH2PO4, 100 g NH4NO3, 10 g MgSO4·7H2O, 5 g CaCl2·2H2O, 0.25 mg biotin, and 5 mL trace element solution 2(SO4)2·6H2O, 0.25 g CuSO4·5H2O, 0.05 g MnSO4·1H2O, 0.05 g H3BO3, and 0.05 g Na2MoO4·2H2O).The wild-type (WT) strain https://www.uniprot.org/blast, accessed on 10 August 2019), using the protein sequence of P. oxalicum PoLaeA  as a query with E-Threshold 0.0001, restrict by taxonomy P. oxalicum (strain 114-2/CGMCC 5302). Pfam search  and SMART (a Simple Modular Architecture Research Tool)  were used to analyze the domain architectures of LaeA-like methyltransferases [Putative LaeA-like methyltransferases were identified in Uniprot database via BLASTp  from plasmid pME2892 [mtr23C gene and ptrA) were fused with fusion PCR. The PCR product was then amplified using nested primers mtr23C-NF/mtr23C-NR and was transformed into the WT to obtain the Δmtr23C mutant. P. oxalicum protoplast preparation and transformation were performed as previously described [All primers used for genetic manipulations are listed in  pME2892 . Then, tmtr23C mutant, the other gene deletion mutants were created with the exception that different primers were used. For Pomtr23D gene deletion, primer pairs mtr23D-UF/mtr23D-UR and mtr23D-DF/mtr23D-DR were used to amplify the 5′- and 3′-flanking regions of the Pomtr23D gene; nested primers were mtr23D-NF/mtr23D-NR. For Pomtr23E gene deletion, primer pairs mtr23E-UF/mtr23E-UR and mtr23E-DF/mtr23E-DR were used to amplify the 5′- and 3′-flanking regions of the Pomtr23E gene; nested primers were mtr23E-NF/mtr23E-NR. For Pomtr23F gene deletion, primer pairs mtr23F-UF/mtr23F-UR and mtr23F-DF/mtr23F-DR were used to amplify the 5′- and 3′-flanking regions of the Pomtr23F gene; nested primers were mtr23F-NF/mtr23F-NR. For Pomtr23G gene deletion, primer pairs mtr23G-UF/mtr23G-UR and mtr23G-DF/mtr23v-DR were used to amplify the 5′- and 3′-flanking regions of the Pomtr23G gene; nested primers were mtr23G-NF/mtr23G-NR. For Pomtr23H gene deletion, primer pairs mtr23H-UF/mtr23H-UR and mtr23H-DF/mtr23H-DR were used to amplify the 5′- and 3′-flanking regions of the Pomtr23H gene; nested primers were mtr23H-NF/mtr23H-NR. For Pomtr25A gene deletion, primer pairs mtr25A-UF/mtr25A-UR and mtr25A-DF/mtr25A-DR were used to amplify the 5′- and 3′-flanking regions of the Pomtr25A gene; nested primers were mtr25A-NF/mtr25A-NR. For Pomtr25B gene deletion, primer pairs mtr25B-UF/mtr25B-UR and mtr25B-DF/mtr25B-DR were used to amplify the 5′- and 3′-flanking regions of the Pomtr25B gene; nested primers were mtr25B-NF/mtr25B-NR. Then, diagnostic PCR and Southern blot analysis were used to confirm the construction of gene deletion strains. The DIG Easy Hyb kit  was used according to the manufacturer’s protocol. The primer pairs Soumtr23C-F/Soumtr23C-R, Soumtr23D-F/Soumtr23D-R, Soumtr23E-F/Soumtr23E-R, Soumtr23F-F/Soumtr23F-R, Soumtr23G-F/Soumtr23G-R, Soumtr23H-F/Soumtr23H-R, Soumtr25A-F/Soumtr25A-R, and Soumtr25B-F/Soumtr25B-R were used to amplify probes from genomic DNA to verify the Pomtr23C gene deletion (Δmtr23C), the Pomtr23D gene deletion (Δmtr23D), the Pomtr23E gene deletion(Δmtr23E), the Pomtr23F gene deletion (Δmtr23F), the Pomtr23G gene deletion(Δmtr23G), the Pomtr23H gene deletion (Δmtr23H), the Pomtr25A gene deletion (Δmtr25A), and the Pomtr25B gene deletion (Δmtr25B) mutants, respectively. The strategy and the results of Southern blotting analysis are in Similar to the construction of the Δmtr25A fused with the GFP sequence, selection marker gene hygromycin B (hph) from the plasmid Psilent1 [mtr25A gene to produce the Mtr25A–GFP strain. To construct the strain (Mtr25A–TAP) for tandem affinity purification, we used primer pairs TAP-UF/TAP-UR, TAP-MF/TAP-MR, and TAP-DF/TAP-DR to amplify the promoter region and the gene of Pomtr25A fused with the HA-FLAG sequence, selection marker gene hph, and terminator sequence, respectively. The PCR fragments were fused and amplified using nested primers TAP-NF/TAP-NR. The PCR product was directly transformed into the WT to displace the native Pomtr25A gene to produce the Mtr25A–TAP strain. The proper integration of GFP tag and HA-FLAG tag was verified via DNA sequencing.To construct the strain (Mtr25A–GPF) of PoMtr25A fused with a green fluorescent protein (GPF), we used primer pairs GFP-UF/GFP-UR, GFP-MF/GFP-MR, and GFP-DF/GFP-DR to amplify the promoter region and the gene of PoPsilent1 , and terw/v) (VSG), 2% glycerol (VSGly), or 1.0% ball-milled cellulose (VGC) as the sole carbon source, was used to monitor the fungal colony. Additionally, 2 μL conidial suspension (106 conidia mL−1) was spotted onto each plate and then grown at 30 °C for 3 to 5 days. For the conidiation test, 5 × 106 conidia were spread out on 9 cm VSG plates and cultivated at 30 °C for 5 days. Then, colony agar plugs with a 5 mm diameter were taken from the plates. Conidia were then harvested by flooding the plugs with sterile distilled H2O containing 0.02% Tween 80. Conidial concentrations were determined using a hemocytometer. The biomass of all mutants was measured as previously described [Vogel’s salt agar, supplemented with 2% glucose , cel7B/eg1 (PDE_07929) and xyn10A (PDE_08094). Primers xlnR-QF/xlnR-QR and creA-QF/creA-QR were used to amplify transcription factor gene creA (PDE_03168) and xlnR (PDE_07674), respectively. Primers ACT-QF/ACT-QR were used to amplify housekeeping gene actin (PDE_01092). The copy number of unambiguous transcripts for each gene was normalized to the expression ratio using the transcripts of the actin gene. The primers used for qRT-PCR are listed in The WT strain was pre-grown in VSG liquid at 30 °C, 200 rpm for 24 h. Then, 0.3 g filtered mycelia was transferred to 100 mL of VSG liquid freshly prepared for 9 and 24 h to determine the transcription levels of various methyltransferase genes. The WT and the mutants were cultivated according to the method described in section “Determination of enzyme activities” to determine the transcription levels of the cellulolytic gene and transcription factor gene. The mycelia were collected via vacuum filtration and ground to powder in liquid nitrogen. Total RNA was extracted with RNAiso Plus reagent . cDNA synthesis and quantitative reverse transcription-PCR (qRT-PCR) were performed as previously described . PrimersThe hyphae of the Mtr25A-GFP strain were examined using a high-sensitivity laser scanning confocal microscope (ZEISS LSM780) . The nuclei were stained in the dark for 15 min by Hoechst 33342 . The green fluorescence of Mtr25A-GFP was visualized using excitation light at 488 nm, while Hoechst 33342-stained blue nuclei were visualized using excitation light at 405 nm.Detailed steps of tandem affinity purification coupled with mass spectrometry (TAP-MS) were described previously . The WT P. oxalicum. Among the 11 LaeA-like methyltransferases (LLMs), only the protein PDE_00555 (UniProt accession: S7ZAB0) has been named PoMtr23B and characterized [A. nidulans FGSC A4 (AnLlmA/B/C/D/E/F/F/G/I/J) as queries, the most identical proteins are observed within these seven proteins, except that the ortholog of AnLlmJ is another protein PDE_06987 (UniProt accession: S8AZY5), which contains a methyltransf_25 domain  using qPCR analysis when the P. oxalicum WT was cultivated in VMG liquid. In order to compare these methyltransferases with the already characterized PoLaeA and PoMtr23B [laeA and Pomtr23B. The results showed that PolaeA and Pomtr23B were transcribed at a high level. The transcriptional levels of Pomtr25A and Pomtr23C were the top two highest among the eight methyltransferase genes. Pomtr23E/F/G/H and Pomtr25B were transcribed at low levels . These data came from the different cultivation conditions when P. oxalicum WT was cultivated on different carbon sources, including glucose (GSE106558) [mtr23B, Pomtr25A, and Pomtr23C have the top three highest transcriptional levels among the ten methyltransferase genes under the glucose condition  are transcribed at high levels, whereas some genes (Pomtr23D/E/F/G/H and Pomtr25B) are transcribed constantly at low levels under different culture conditions.Furthermore, we reviewed the transcriptome data retrieved from the Gene Expression Omnibus (GEO) database of NCBI (E106558) , starch E106558) , celluloE106558) , and celE106558) . Pomtr23ondition B. Pomtr2ondition C,D. Pomtondition E. Both rmtr23C/D/E/F/G/H and Pomtr25A/B) deletion mutants (Δmtr23C/D/E/F/G/H and Δmtr25A/B) were constructed. The mutants were verified using diagnostic PCR and Southern blot analysis. The strategies and the results of the Southern blot are presented in In order to ascertain the functions of the various methyltransferases, the gene (Pomtr23C/D/E/F/G/H and Δmtr25A/B) displayed identical colony morphologies to those of the WT. When grown on VMC agar, the seven mutants (Δmtr23C/D/E/F/H and Δmtr25A/B) showed identical colony morphologies to those of the WT, with the exception that the mutant Δmtr23G displayed a clearer cellulolytic halo around the colony than the WT, implying a higher cellulolytic enzyme secretion than WT  reached their peak biomass at 48 h, whereas Δmtr25G and Δmtr25H reached their peak biomass at 24 h. However, the maximum biomass they could reach was similar despite a slight difference in the temporal trend of biomass growth  gene expression [In addition to the function in regulating fungal conidiation, the LaeA also controls the expression of cellulolytic genes and then affects the synthesis of cellulolytic enzymes in various fungi such as . reesei , M. therrmophila , and Plestreatus . P. oxalpression ,18. Thermtr23C/D/G and Δmtr25A increased compared to the WT. Among them, the Δmtr23G mutant showed the most significant increase, with its FPA being 2.7, 2.8, 1.6, and 4.1 times higher than that of the WT on days 2, 3, 4, and 5 of culture. The FPA of Δmtr23E and Δmtr25B decreased compared to that of the WT. On the fourth day, the FPA of Δmtr23E and Δmtr25B were 58.7% and 62.2%, respectively, of those of WT, suggesting that proteins PoMtr23E and PoMtr25B are required in normal cellulolytic enzyme production. The FPA of Δmtr23F and Δmtr23H showed a similar pattern to the WT , CMCase activity , and xylanase activity (indicating hemicellulase activity) were determined. Various mutants have different patterns in cellulolytic enzyme production. The FPA of mutants Δo the WT A.mtr23C/D/G and Δmtr25A increased compared to the WT. Among them, the most significant increases were seen in the Δmtr23G and Δmtr25A mutants: on days 2, 3, 4, and 5 of culture, the Δmtr23G mutant had CMCase activity that was 2.3, 1.8, 1.4, and 1.4 times that of WT, and the Δmtr25A mutant had CMCase activity that was 2.1, 2.0, 1.4, and 1.4 times that of WT. The CMCase activities of Δmtr23E and Δmtr25B decreased compared with that of the WT. On the fourth day, the CMCase activities of Δmtr23E and Δmtr25B were 37.2% and 38.5% of those of WT, respectively. The CMCase activities of the Δmtr23F and Δmtr23H mutants showed similar patterns compared with the WT , and the key hemicellulase gene Poxyn10A (PDE_08094) in WT and mutants was determined using qPCR. The genes Pocbh1, Poeg1 and Poxyn10A encode cellobiohydrolase, β-1,4-endoglucanase, and β-1,4-xylanase, respectively. Their products are the most abundant cellulase and hemicellulase produced by fungal cells [Since the production of cellulolytic enzymes in fungi is tightly regulated at the transcriptional level, we investigated whether the altered production of cellulolytic enzymes in various mutants was caused by the up- or downregulated expression of the corresponding genes. The transcriptional level of two key cellulase genes, Pocbh1 (PDE_07945) and Po1 PDE_0799, and thcbh1 gene was significantly increased in the Δmtr23C/D/G and Δmtr25A mutants but significantly decreased in the Δmtr23E and Δmtr25B mutants and remained essentially unchanged in the Δmtr23F and Δmtr25H mutants. For example, at the 24th hour of cultivation, the transcriptional levels of the Pocbh1 gene in Δmtr23C/D/G and Δmtr25A mutants were 85.7, 22.0, 241.4, and 28.6 times higher than those in the WT, respectively. The transcriptional levels of the Pocbh1 gene in the Δmtr23E and Δmtr25B mutants reduced to 9.6% and 11.9% of the WT, respectively , including activators and repressors. The activator XlnR and repressor CreA are key dose-dependent regulators of cellulolytic gene transcription ,28. We amtr23D and Δmtr23G mutants displayed elevated PoxlnR gene expression; these mutants showed 6.2- and 15.3-fold increases in PoxlnR gene expression compared to WT. The PoxlnR gene expression levels in other mutants were similar to the WT , the transcription level of Pomtr25A gene is the highest under four of the five detection conditions   A–D, and llulose) E. Even tP. oxalicum (We fused the PoMtr25A-coding sequence with the GFP sequence and introduced it to the WT to obtain the strain Mtr25A–GFP. The overlap of green fluorescence (green dots) and nuclear staining (blue dots) on the merged image indicated the nuclear localization of PoMtr25A in oxalicum A.A. nidulans, AnLaeA and the two LLMs AnLlmF and VipC (VipC is AnLlmB) have respective interactions with VeA, a velvet domain protein, and play a role in regulating fungal development and secondary metabolism via the interaction [In eraction . Therefoeraction , which wSaccharomyces cerevisiae 60S ribosomal protein L20 (66.7% identity) exhibited the highest emPAI. PDE_01194, an ortholog of Schizosaccharomyces pombe glutathione S-transferase Gst2 (40.2% identity), and PDE_09363, an ortholog of catalytic subunit SNF2 (56.7% identity) of S. cerevisiae SWI/SNF chromatin-remodeling complex exhibited the second and the third-ranking emPAI, respectively. Other proteins were only detected in one of the Mtr25A-TAP triplicates, so they were not considered credible.Among the proteins identified via LC-MS/MS, it is expected that PoMtr25A is the top protein according to emPAI. In addition to PoMtr25A itself as the bait, PDE_02916, an ortholog of P. oxaliucm is a typical fungus that produces cellulolytic enzymes, we focused on the functions of methyltransferases (PoMtr23C/D/E/F/G/H and PoMtr25A/B) in cellulolytic enzyme gene expression. In different methyltransferase gene deletion mutants, higher cellulolytic gene transcription resulted in a higher production of cellulolytic enzymes, and vice versa. The results were expected because cellulolytic enzyme production in fungi is always regulated at a transcriptional level. However, there may be differences in the mechanisms by which these methyltransferases regulate the transcription of cellulolytic enzyme genes.LaeA/Lae1 and LaeA-like methyltransferases (LLMs) play essential roles in fungal growth, development, virulence, and the production of secondary metabolite and cellulolytic enzymes in various fungi ,17,18,19mtr23G was the only mutant that displayed a clearer cellulolytic halo around the colony than did the WT. This result is consistent with the highest FPA and CMCase results shown by Δmtr23G in liquid culture  showed a significant increase in cellulolytic gene expression compared to the WT. For the Δmtr25D mutant, the high transcription level of PoxlnR gene should be one reason for the increased transcription of its cellulolytic enzyme gene, as TF XlnR is not only the key transcriptional activator of cellulolytic and xylanolytic genes in P. oxalicum [T. reesei, N. crassa and A. niger [mtr25A mutant, the downregulation of the PocreA gene may be the main reason for the upregulation of cellulolytic gene transcription since CreA, a TF involved in carbon catabolite repression (CCR), is a crucial repressor of cellulolytic enzyme gene [Four mutants  with low cellulolytic gene expression exhibited significant differences in the expression levels of the PoxlnR and PocreA genes compared with the WT. Evidently, PoMtr23C, PoMtr23E, and PoMtr25B do not regulate cellulolytic genes through TF PoXlnR and PoCreA. Except for XlnR and CreA, many other TFs intricately regulate the expression of the cellulolytic gene. In P. oxalium, along with XlnR and CreA, TFs ClrB and AraR can positively regulate the expression of cellulolytic genes directly, while TF AmyR negatively does so in an indirect way [M. thermophila caused a significant reduction in the expression of Cre1, Clr-1, Hac1, and Vib-1 and an increase in the expression of AmyR and Ace1 [P. oxalicum PoLaeA and PoMtr23B affect the expression of 21.3%  and 14.1%  of the genome, respectively [T. reesei and A. fumigatus, respectively [P. oxalicum. Therefore, PoMtr23C, PoMtr23E, and PoMtr25B may affect TFs other than XlnR and CreA or other regulators to affect the transcription of cellulolytic genes.Neither the Δrect way ,30,43. Mrect way . The absand Ace1 . Accordiectively . Lae1/Laectively ,45. In aectively ,47, whicA. nidulans AnLaeA and AnLlmF interact directly with VeA, a fungus-specific velvet domain protein [T. reesei orthologue of A. nidulans VeA, is a positive regulator of the cellulolytic gene. The deletion of the vel1 gene completely impaired the expression of cellulases, xylanases, and the transcription activator Xyr1 (the ortholog of XlnR) on a cellulase-inducing medium [vel1 overexpression enhanced the production of cellulases and xylanases [A. nidulans VeA interacts with at least four methyltransferases, including AnLaeA, AnLlmF, and the methyltransferase heterodimers VipC-VapB (VipC is LlmB), but not all methyltransferases interact with VeA [P. oxaliucm PoLaeA interacted with PoVeA, whereas PoMtr23B did not [T. reesei activator Xyr1 recruits the SWI/SNF complex to remodel chromatin at cellulase gene promoters, activating cellulase gene expression [The proteins that interact with LaeA and LLMs must also be considered.  protein ,48. Vel1g medium . The velylanases . A. niduwith VeA . P. oxal did not . PoVeA w did not . T. reespression . Therefomtr23F and Δmtr23H, neither fungal growth, spore production, nor cellulase production were found to be significantly different from the WT. Similarly, the absence of the AnLlmD (the ortholog of PoMtr23F) and AnLlmI (the ortholog of PoMtr23H) in A. nidulans did not lead to any noticeable phenotypic changes [mtr23F and Pomtr23H might be redundant genes in the genome.For the mutants Δ changes . AdditioP. oxalicum PoLaeA and PoMtr23B both alter colony morphology. However, the underlying mechanisms are inconsistent: PoMtr23B primarily regulates conidial pigment formation, whereas PoLaeA is crucial for conidiation but has little impact on conidial pigment formation [A. nidulans AnLaeA, localized in the nucleus, positively regulates sterigmatocystin production [P. ostreatus, designated PoLaeA1, PoLaeA2, and PoLaeA3, also demonstrated functional differentiation. Silencing PoLaeA1 reduced the intracellular polysaccharide, whereas silencing PoLaeA2 or PoLaeA3 did not. Silencing PoLaeA2 and PoLaeA3 decreased cellulase production by interfering with intracellular Ca2+ signaling, whereas silencing PoLaeA1 did not [This study showed that different methyltransferases have different effects on the expression patterns of cellulolytic genes from different mechanisms, indicating their functional differentiation. According to previous studies, different LLMs demonstrated significant differentiation via various mechanisms in their effects on fungal growth, development, and SM formation, in addition to controlling the expression of cellulolytic genes. The absence of ormation . A. niduoduction , whereasoduction . Three L did not . TherefoP. oxalicum. The absence of each protein has different patterns in cellulolytic gene transcription and cellulolytic enzyme production. The underlying mechanisms are also different. This study can improve our knowledge of the role of the LaeA-like protein in fungal development, growth, and cellulolytic enzyme production.The biological roles of eight LaeA-like proteins were investigated in


Today, geopolymers are an emerging building material generated by the chemical activity of inorganic molecules without the Portland Cement addition. The most common alternative cementitious agents used in the cement industry are blast-furnace slag and fly ash. In the present work, the effect of 5 wt.% µ-limestone in mixtures of granulated blast-furnace slag and fly ash activated with sodium hydroxide (NaOH) at different concentrations was studied to evaluate the physical properties in the fresh and hardened states. The effect of µ-limestone was explored through XRD, SEM-EDS, atomic absorption, etc. The addition of µ-limestone increased the compressive strength reported values from 20 to 45 MPa at 28 days. It was found by atomic absorption that the CaCO3 of the μ-limestone dissolved in NaOH, precipitating Ca(OH)2 as the reaction product. SEM-EDS analysis showed a chemical interaction between C-A-S-H- and N-A-S-H-type gels with Ca(OH)2, forming A-S-H- and C-(N)-A-S-H-type gels, improving mechanical performance and microstructural properties. The addition of μ-limestone appeared like a promising and cheap alternative for enhancing the properties of low-molarity alkaline cement since it helped exceed the 20 MPa strength recommended by current regulations for conventional cement.Concrete is the most used construction material, needing large quantities of Portland cement. Unfortunately, Ordinary Portland Cement production is one of the main generators of CO CO2, as the main greenhouse gas, is responsible for global warming, and the cement industry causes around 8% of CO2 emissions worldwide [2 emissions from cement production are generated by the combustion essential to attain temperatures of about 1450 °C (30–35%), limestone decarbonation to produce clinker (50–60%), and materials handling (around 10%) [2 emissions and conserve energy [2 emissions, and (ii) clinker production with no cement content.Portland cement (PC), a traditional cementitious material, is the most predominant binder used in construction with a large consumption worldwide . Howeverorldwide . CO2 emiinker 50–0%, and me energy . Two praIt is common in the building materials sector to find traditional binders replaced by alternative secondary products such as silica fume, blast-furnace slag, fly ash, biomass, ash, red mud, and natural, and artificial puzzolans. The diversity of chemical compositions in these by-products results in new hydrated phase formation  ,9.Otherwise, the alkaline activation of powders obtained from by-products based on aluminosilicates (precursors) can generate an inorganic binder when some alkaline compounds react with those precursors. The alkaline-activated binder’s interest is due to its extreme-temperature tolerance, minor permeability, stable bonding, favorable durability, attractive chemical corrosion resistance, immobilization of toxic waste, and environmental friendliness, among other benefits ,21,22,23Raw materials used for geopolymer synthesis frequently come from industrial aluminosilicate wastes, natural aluminosilicate minerals, or their mixtures ,24. A geLarge quantities of ground granulated blast-furnace slag (GGBFS) and fly ash (FA) are still available in the trading market. This fact motivates a part of the scientific community and the cement industry to focus on new geopolymer development through GGBFS and FA utilization based on their probable high hydraulic potential and monetary and atmospheric benefits ,26,27,28Fly ash is a powder by-product with fine spherical particles ranging from <1 mm to more than 100 mm and produced in thermal power plants during coal burning, whose annual production is now difficult to estimate due to the general crisis of energy products (mainly oil and gas), but a few years ago, it was estimated to be about 900 million tons .Alkaline activation of fly ash with high hydroxide concentrations leads to a reaction product (N-A-S-H gel) that may develop more than 60 MPa when thermally cured.On the other hand, ground granulated blast-furnace slag is another by-product material with a high fraction of glassy phase rich in silica, alumina, and amorphous calcium, making it suitable for use as a precursor for synthesizing GP . The ann3+, including Na in its structure. The other hydration product is alkaline aluminosilicate hydroxide with a three-dimensional structure [The synergistic effect of binary ash/slag composites (with a 50/50 ratio) activated with NaOH and cured at 25 °C, which have reached compressive mechanical strength values of about 50 MPa at 28 days of reaction, has also been studied ,43,44. Atructure ,43,44.2SiO3 solution. Compressive strength increased at higher GGBFS/Class F fly ash ratios. The highest compressive strength (51 MPa) was reached at 20% slag and 80% fly ash with a 40% NaOH and Na2SiO3 solution and cured at 20 °C. Xu et al. [Wang et al.  studied u et al.  synthesi3) addition. It has been reported that limestone powders homogenize and broadly distribute clinker particle size when limestone particle sizes below 8 μm are used in a range of 10% to 40% [On the other hand, many studies have demonstrated improvements of the properties of Portland cement by limestone  addition effect on the formation of hybrid hydration gels of alkaline-base cement (slag and ash) at different curing conditions and their influence on mechanical properties.Based on the above, there is much interest in the scientific community to study the effect of CaCOThe precursors used to elaborate the geopolymers were: (i) granulated blast-furnace slag from Lazaro Cardenas steel-making plant , (ii) Class F fly ash (FA) from Nava thermoelectric plant , and (iii) µ-limestone from mountain deposits in Nuevo León, México. Sodium hydroxide from Jalmek with 98.5% purity , bi-distilled water, and water glass  were used for the alkaline solution preparation.Before using GGBFS, FA, and limestone as precursor raw materials to prepare alkali-activated cements, a 1 h grinding process was performed on them in a vibro-energy grinding ball mill  using 80 kg of 20 mm steel balls and 5 kg of raw material charge in the mill.The hydraulic activity of slag was 1.23, which represents good hydraulic behavior, according to the literature ,50,51,52FA(MFA)/slag-based binary pastes with and without µ-limestone addition were mixed, following the proportions in The L/S ratio was determined according to ASTM C230-14 to obtain good workability; this value was set between 0.22 and 0.40 see .After mixing, the consistency test was carried out according to the ASTM C230-14 standard. The paste was poured into the molds to create cylindrical samples of 25.4 mm × 50.8 mm, allowing it to be set at an ambient temperature for 24 h. The hardened specimens were removed from the molds and then subject to a specific curing process.The specimens were classified and treated under three groups of curing methods: (1) saturated curing (immersed in water): water-saturated state at 100% relative humidity for 28 days; (2) controlled curing at 25 °C: permanent water spray cured at controlled temperatures of 25 °C for 28 days; and (3) controlled curing at 80 °C: water steam cured at controlled temperatures of 80 °C during the first 24 h and then set at room temperature until 28 days.The samples were evaluated by compressive strength using an INSTRON universal mechanical testing machine . The evaluation was set to a 200 kg/s loading rate for 28-day-old specimens.The final reported values were calculated as the mean value of six specimens for each paste system.After the compression test, the samples were collected, immersed in an acetone solution for 48 h, oven-dried for 24 h to stop hydration and aging, and then crystallographiccally and microstructurally analyzed.The phase composition of the raw material and experimental mixtures was determined using an X-ray diffractometer. For X-ray diffraction analysis, a D8 advanced Bruker model equipped with a Vantec detector  was used at an excitation voltage of 40 kV, current of 40 mA, scan rate of 0.05 (2θ/sec), and a 2theta (2θ) angle between 10° and 70°. The powders  and controlled curing mixture systems were analyzed at 28 days of age by XRD.A scanning electron microscopy technique (SEM) was utilized to determine the microstructure of the experimental specimens using a JEOL microscope JSM-6490LV equipped with an Oxford energy dispersive detector (EDS) for qualitative and quantitative microanalysis .2+ ions were dissolved. μ-limestone powders were also analyzed by X-ray diffraction after treatment in the alkaline solution.On the other hand, the μ-limestone’s stability was studied in NaOH alkaline solutions with separate molarities of 4 M and 8 M. μ-limestone powders were added to both alkaline solutions and stirred for 24 h. Then, the resulted solution was analyzed by atomic absorption to determine if CaThe binder’s alkaline activation process would be accelerated with the L/S ratio’s diminishment due to the mixture’s consistency decrement . In thisThe compressive resistance results of controlled curing at 25 °C are also shown in It has been found that C-A-S-H- and N-A-S-H-type gels can exist together when slag/fly ash composites are mixed. A hybrid-type gel (N-C)-A-S-H is also identified in slag systems as a part of the calcium released by the dissolution of the slag and its incorporation into the N-A-S-H-type gel because of fly ash activation ,64.Regarding the mechanical strength of controlled curing pastes at 80 °C see , most paThe optimal mixtures of controlled curing at 25 °C are shown in In addition, there was a tendency where µ-limestone added to the pastes accelerated the reactions at 7 days, reaching early mechanical strength increments similar to those attained at 28 days. Taking into account the obtained results, the paste with 40% slag and 60% milled fly ash with 5% of µ-limestone addition, L/S ratio = 0.30, and 8 M of NaOH (40S60MFA5C 8M-0.30 paste) could be proposed as the most favorable mixture for compressive resistance, reaching 30 MPa at 28 days.As observed, the addition of µ-limestone could undoubtedly modify the mechanical responses of slag-based alkali-activated binders. As communicated in the literature, the mechanisms promoted by limestone that are favorable to the resistance development of slag-based paste are: (i) producing nucleation sites ,70,71,  and some crystalline phases, such as quartz, mullite, hematite, and magnetite, can be found in FA-based-activated pastes activated with NaOH. When <5% of additive was added to these pastes, the same crystalline phases remained present, i.e., no new phase formation was observed except for calcite. These non-reactive phases mostly acted as fillers or micro-aggregates embedded in the matrix.Low µ-limestone content in FA-based alkali-activated pastes can result in a more packed and denser microstructure ,81,84,852+) and did not present new phases [Meanwhile, the effect of µ-limestone on the compressive strength of binary pastes exhibits positive trends ,86,87,88w phases ,89. Regaw phases .In binary pastes, µ-limestone can increase strength because of the filler effect . The µ-lThe 80S20MFA5C pastes developed the highest strength among the saturated curing pastes due to possibly extensive formation of C-S-H and C-A-S-H gels induced by µ-limestone. The acceleration and intensification of the reaction in this paste could be explained by the fact that µ-limestone grains worked as seeding-like sites, facilitating the precipitation and growth of reaction products. Furthermore, a small amount of µ-limestone might have refined the pore structure. This phenomenon might have reduced the porosity and densify the microstructure of the pastes matrix, resulting in a strengthened effect.The 60S40MFA5C pastes reached the highest compressive strength of all controlled curing at 25 °C pastes. In this case, μ-limestone might have interacted with the slag aids as a nucleation agent to C-S-H and C-A-S-H gels formation. Meanwhile, C-S-H or calcium hydroxide, N-A-S-H formation, and low N-(C)-A-S-H gels formation could be found due to the dissolution of µ-limestone within fly ash systems. Additionally, a filler effect and better packing density might have been present in this paste.The 40S60MFA5C pastes reached the highest compressive strength of all controlled curing at 80 °C pastes. However, a hindrance in compressive strength development could be found when compared to that of the other systems, mainly by a high L/S ratio (high liquid content) used in a high-alkali solution (8 M), which negatively affected gel formation and subsequent increment of poor gel reaction products as N-A-S-H gels. However, there was a tendency where µ-limestone added to the pastes accelerated the reactions at 7 days, reaching early mechanical strength increments similar to those reported at 28 days. In this case, μ-limestone might have interacted with the fly ash as a nucleation agent for N-A-S-H gels formation.Both pastes with an L/S = 0.25 ratio reported Ca/Si values between 1.5 and 1.9. These ratios favor the C-A-S-H gels and the hydrotalcite phase formation since the Mg/Al ratio with values between 1 and 1.2. Additionally, 2 particles leaching and beginning to react with the N-A-S-H gels, according to the variation of the stoichiometric ratios forming (N) C-A-S-H gels.2 resulting from the dissolution of the μ-CaCO3 modifies the pH and can influence the decrease of MFA dissolution and N-A-S-H gel formation [3 may show polymorphism into vaterite, calcite, and Ca(OH)2 by the dissolution through the gel formation process [The amounts of Ca(OH)ormation . The dom process .The curing temperature influenced the formation of zeolites , which w2 particles were closer to the interface of the N-A-S-H gels, the presence of C-S-H-type gels calculated by Ca/Si ratios >1.50 was observed.In N-A-S-H-type gels’ formation occurred at the point farther away from the μ-limestone particle interface. It should be noted that the 100MFA5C pastes showed a better reaction of the MFA particles thanks to the particle size combined with the extreme alkalinity of the activating solution and curing at a temperature of 80 °C.2+ was not a part of the structural polymerization chain.The joint hydration of these compounds resulted in hybrid-gel formation, such as C-S-H, C (N-A)-S-H, N(C-A)-S-H, N-A-S-H, C-A-S-H, C-A-S-H, with the type of hybrid gel mixture depending on the type of activation.2+ freed by slag partition and its incorporation into an N-A-S-H-type gel. This gel can coexist with forming C-A-S-H gels by slag activation and N-A-S-H gels by ash activation, which is more distinguishable at earlier ages [Pastes with a higher Ca/Si ratio develop a hybrid C-N-A-S-H gel in response to the Caier ages .2+ was calculated by microanalysis.The microstructure of the ternary slag-MFA-μ-limestone pastes was analyzed in detail in 2 particles was observed in response to the high alkalinity of the activating solution. At the μ-limestone/paste interface, the registered Ca/Si ratio was about 1.7–2.2. Meanwhile, the N-A-S-H gels around the μ-limestone were at average values of an Si/Al ratio = 2.6.2 compounds improves the mechanical characteristics of FA-based geopolymer gels. Ca(OH)2 is considered a more reactive additive than CaO. Ca(OH)2 compound addition results in the precipitation of C-S-H gels or hydrated calcium aluminosilicate phases. Ca(OH)2 was obtained in the present work by dissolving CaCO3 from the μ-limestone in an alkaline NaOH-4 M solution. This alkaline compound contributes to slag activation in binary paste mixtures.As reported by J. Temuujin et al. , the addCaO content seems to strengthen the geopolymers, forming a structured Ca-Al-Si gel, showing a slight positive effect on the physical properties .The stoichiometric ratio Si/Al = 2.8 indicated that most of the gel formed was an N-A-S-H-type gel. There was N-A-S-H-type gel formation with some areas of C-A-S-H gel formation due to the Ca/Si = 1.1 value ratio shown in The XRD results of raw material are plotted in 3 addition influenced the reaction kinetics of hydration gel formation. Hydrotalcite formation (H) (JCPDS 41-1428) shown by the appearance of a peak between 11° and 13° (2θ) was established. C-S-H(C) gel formation was found in these pastes at 30° to 32° (2θ) peaks (JCPDS 29-0373). The angles of the “halos” match those reported in the literature [terature ,83.In the diffractogram, there were also peaks corresponding to calcite (Cc) JCPDS 83-0578) and merwinite (W) (JCPDS 84-1205) phases from the slag. For fly ash, no hydrated or unreacted phases such as mullite (M) (JCPDS 89-2644), quartz (Q) (JCPDS 89-1962), and ferrite (F) (JCPDS 80-2377) could be observed. The diffractogram of the water-saturated 60S40MFA5C between 15° and 25° (2θ) showed an amorphous halo attributed to an amorphous N-A-S-H gel reaction. The possible reaction of limestone with the alumina from the mullite may form calcium carboaluminates since this crystalline phase can occur at low angles in small proportions (10° and 15°) -0578 and.It is worth mentioning that the 60S40MFA5C paste showed 20% of the mechanical strength increment of 60S40MFA. This behavior was possibly associated with the more intensive and accelerated formation of crystalline phases of the hydrated calcium silicates attributed to the µ-limestone addition, since limestone can act as a nucleation site for the reaction products, improving their precipitation. µ-limestone content can lead to a more compact and denser microstructure that eventually improves the strength of alkali-activated pastes.The analysis showed an amorphous halo between 25° and 35° (2θ), derived from the amorphous phase of the slag. The appearance of a C-S-H(C) signal at 31° (2θ) was shown in the systems with the presence of slag. In the water-saturated 100S pastes, hydrotalcite (H) formation between 11° and 13° (2θ) and C-S-H(C) gel were shown as new phases that also had high intensity between 50° and 52° (2θ). In the 100MFA5C pastes, an amorphous halo was observed between 15° and 25° (2θ). This phenomenon was attributed to the limestone addition (Cc) and the alumina present in the mullite phase from the FA. In the 100MFA5C cured at 80 °C, a zeolitic phase was observed at 37° (2θ), corresponding to a phase called chabazite (JCPDS 46-1427).Alexander M. Kalinkin et al. 2020  also repXRD analysis indicated that slag as a raw material presented crystalline phases such as calcite, merwinite, and quartz ,95. AddiAfter slag activation, the same crystalline phases before activation were detected , as well as hydrotalcite and calcium silicate hydrate as new crystalline phases. The slag alkaline activation promotes the development of an Mg/Al ratio that matches the hydrotalcite phase. This formation depends on the raw material nature and the activator’s alkalinity . HydrotaAs was reported in the literature, the formation of the calcium silicate hydrate gel was influenced by the amount of alumina in the slag. After slag alkaline activation, calcium silicate hydrate was detected at 29.5° (2θ) .The μ-limestone addition in the slag acts as a nucleating agent and a C-S-H gel precursor , besidesXRD analysis of fly ash as a raw material presents crystalline phases such as mullite, quartz, and hematite. The glassy phase is also evident in fly ash ,98,99. AOn the other hand, the degree of geopolymerization in the activated fly ash is directly related to the intensity of the amorphous silica peak around 30° ,99.2) and calcium carbonate (CaCO3) of the μ-limestone were indicated. It was found that there was a reaction between the activating solution and the addition of limestone, this addition mineral being one more ingredient for the effective activation of the binary compounds (slag and fly ash). This analysis coincides with the studies of Nicholas E. Pingitore et al. [e et al.  on the d3  activation in a Na(OH) solution using fine (62 to 125 μm in size) and coarse (250 to 500 μm in size) lime particles. They found CaCO3 in any mineralogical phase dissolved in calcium hydroxide-Ca(OH)2.Nicholas E. Pingitore et al.  analyzed3) and sodium hydroxide (NaOH) is the formation of calcium hydroxide Ca(OH)2 in the form of portlandite and sodium carbonate (Na2CO3).The product of the reaction between calcium carbonate (CaCO3 by Ca(OH)2 that matched Nicholas E. Pingitore and co-workers’ research.X-ray diffraction results see  showed a2 caused by the CaCO3 reaction in aqueous and alkaline solutions. The μ-CaCO3 particle’s agglomeration and reacted Ca(OH)2 particles were also observed. The mapping showed the distribution of the compounds where a contrast between the Ca(OH)2, Ca (green), and Na (blue) plates was shown.The µ-limestone addition on alkali-activated binders was investigated in terms of the mechanical, microstructural, and crystallographic properties. Afterwards, the most favorable mixtures were suggested. According to the results, the principal conclusions can be listed as follows:The compressive strength of alkali-activated binders increased significantly with (i) µ-limestone addition, (ii) slag content increment, (iii) NaOH molarity, and (iv) L/S ratio reduction.The slag-based alkali-activated pastes exhibited compressive strength higher than that of fly ash-based alkali-activated pastes. It was proposed that this mechanical characteristic was reached by reaction intensification (C-A-S-H or C-S-H gels formation), porosity reduction, matrix densification, nucleation sites availability, and pore refinement through µ-limestone addition.Low µ-limestone content in FA-based alkali-activated pastes could result in a more packed and denser microstructure that eventually improves the strength of alkali-activated pastes. This phenomenon might be attributed to a filler effect of unreacted particles and more gel products due to the nucleation sites generated by limestone and other Ca-containing phases.The effect of µ-limestone on the compressive strength of binary pastes exhibited positive trends. A C-(A)-S-H-type gel may be present in fly ash-slag systems. The µ-limestone addition benefited the strengthening mechanisms by pore size control and seeding-like sites simultaneously with minor chemical modifications. Additionally, the µ-limestone addition might improve strength by a dissolution mechanism on the activator, yielding better packing.The paste with 60% slag and 40% milled fly ash with 5% of µ-limestone addition, an L/S ratio of 0.30, and 4 M of NaOH (60S40MFA5C 4M-0.30 paste) could be proposed as the most favorable mixture regarding the performance criteria of compressive strength, reaching 42 MPa at 28 days. μ-limestone might interact with the slag aids as a nucleation agent for C-S-H and C-A-S-H gels formation. Meanwhile, the formation of C-S-H or calcium hydroxide, N-A-S-H, and low polymerization N-(C)-A-S-H gels could be due to the dissolution of µ-limestone within fly ash systems. A filler effect and better packing density might also be present in this paste.


C-diazeniumdiolate group in theamino acidgraminine is emerging as a new microbially produced Fe(III) coordinatingligand in siderophores, which is photoreactive. While the few siderophoresreported from this class have only been isolated from soil-associatedmicrobes, here we report the first C-diazeniumdiolatesiderophores tistrellabactins A and B, isolated from the bioactivemarine-derived strain Tistrella mobilis KA081020-065.The structural characterization of the tistrellabactins reveals uniquebiosynthetic features including an NRPS module iteratively loadingglutamine residues and a promiscuous adenylation domain yielding eithertistrellabactin A with an asparagine residue or tistrellabactin Bwith an aspartic acid residue at analogous positions. Beyond the functionof scavenging Fe(III) for growth, these siderophores are photoreactiveupon irradiation with UV light, releasing the equivalent of nitricoxide (NO) and an H atom from the C-diazeniumdiolategroup. Fe(III)-tistrellabactin is also photoreactive, with both the C-diazeniumdiolate and the β-hydroxyaspartate residuesundergoing photoreactions, resulting in a photoproduct without theability to chelate Fe(III).The  Siderophores are small-molecule microbial natural productsthat have evolved to facilitate iron sequestration and microbial ironuptake. Biological competition for available iron has driven the evolutionof a large selection of siderophores, with hundreds identified thusfar, yet siderophores with a C-diazeniumdiolate ligandhave only just begun to be uncovered.17 Up to this point, graminine-containing siderophores have only beenisolated from soil-associated microbes within the related Burkholderiaceaefamily, including gramibactin lose two equivalents of NO under physiological conditions,19 while C-diazeniumdiolates aremore thermally stable. We recently demonstrated that C-diazeniumdiolate siderophore Gbt releases the equivalent of NO andan H atom for each Gra residue when irradiated with UV light, producinga mixture of E and Z oxime isomersas the photoproduct.14 Additionally, peroxidase-mediatedNO release from Gbt has been reported.15 Given the relevance of NO and oximes in physiological signalingpathways20 and defensive responses,22 this ligand system may serve multiple functions within the microbebeyond Fe(III) scavenging.The N–N bond in the diazeniumdiolate moietyis particularlyintriguing in the context of nitric oxide (NO) release.l-Gra have been identified in thebiosynthetic gene cluster(BGC) for gramibactin as grbD and grbE through gene knockout studies with the strain P. graminis DSM 17151.15 GrbE shares sequence homologyto several Arg hydroxylases , while GrbDshares sequence homology to the cupin domain of the N–N bond,forming enzyme SznF7/StzF4, which carries out the oxidative rearrangement of Nδ-hydroxy-Nω-hydroxy-Nω-methyl-l-Arg.Targeted discovery of new C-diazeniumdiolate siderophoresis achievable by searching microbial genomes containing a nonribosomalpeptide synthetase (NRPS) adenylation (A) domain with the specificitycode for Gra (DVHRTGLVAK) and FASTA sequences of grbD and grbE as queries. With this strategy, genomemining efforts reveal a widespread prevalence of l-Gra innatural products from a variety of environments, including clinicalisolates, plant pathogens, and, as described in this study, a marine-derivedmicrobe.The biosynthetic genes encoding theenzymes that synthesize Tistrella mobilis KA081020-065,isolated from the Red Sea, produces C-diazeniumdiolatesiderophores tistrellabactins A and B. The tistrellabactins are photoreactive,losing NO in UV light, including in actively growing cultures of T. mobilis. Surprisingly, the tistrellabactin NRPS enzymesload glutamine residues iteratively via an unknown mechanism, whichis also observed in the biosynthesis of the anticancer didemnin naturalproducts produced by T. mobilis KA081020-065.26 The characterization of tistrellabactins A andB reveals other unusual biosynthetic features, including a promiscuousNRPS A domain, a 22-membered macrolactone ring, and a 3-hydroxybutyricacid group appended to N-hydroxy-l-ornithine.We report herein grbD and grbE, consistent with incorporationof l-Gra, was identified on plasmid 2 .26 For targeted discoveryof the metabolite, the FASTA sequence of the A domain specific forGra was used as the query in NCBI BLASTP . Hits were then narrowed to microbes that included biosyntheticgene clusters with siderophore-related genes, an A domain with thespecificity code for l-Gra, and sequence homologues of theproposed Gra biosynthesis genes, grbD and grbE, from the BGC of gramibactin.13A putative siderophore BGC with gene analogs of C-Diazeniumdiolate siderophorescurrently reportedin the literature are all produced by soil-associated microbes, whichraises the question if they are also produced by microbes in otherenvironments.15 To investigate the extent ofthis siderophore type beyond the terrestrial soil environment, thefamily Burkholderiaceae was excluded from the genome search. The presenceof GrbD and GrbE homologues, alongside an NRPS protein containingan A domain with a specificity code for Gra, indicated marine-derived T. mobilis KA081020-065 should incorporate Gra with a C-diazeniumdiolate for Fe(III) coordination . Given the mass similarityof the metabolites and the presence of only one siderophore gene clusterin the genome of T. mobilis KA081020-065, the compoundsappear to be congeners. The first-eluting peak corresponds with theprotonated molecule m/z 1092.4 [M+ H]+ and the latter to m/z 1093.4 [M + H]+, which will herein be referred to astistrellabactin A and tistrellabactin B, respectively . The structures of tistrellabactins A and B wereelucidated by complete assignment of all 1H, 13C, and 15N resonances . Overlapping methylene 1H resonancesand several sets of diastereotopic 1H resonances were differentiatedusing 2D NMR experiments, primarily multiplicity edited HSQC and TOCSY.The empirical formulas of tistrellabactins A andB were assignedby high-resolution mass spectrometry (HR-MS) to be Cectively S2 and S31H spin systems wereattributed to theGln side chain amine protons in tistrellabactins A and B.27 An additional discrete 1H spin systempresent in tistrellabactin A only was attributed to the Asn side chain.27 Through-bond 1H–13C HMBC correlations connect the side chain amine 1H resonancesto methylene protons of three Gln and one Asn in tistrellabactin Aonly . Observed in the TOCSY spectrum wasa 1H spin system assigned to a 3-hydroxybutyric acid group(Hbu). Correlations between the hydroxylated 15N on theOrn side chain and the Hbu group established the 3-hydroxybutyricacid is appended to the l-Orn residue, forming a hydroxamateligand. This rare acyl group has been seen in the siderophore structuresof cupriachelin and imaqobactin.29 The presenceof the Gra residue, which has a distinct 1H–15N HMBC fingerprint, was confirmed with the presence of correlationsbetween the Gra Cδ methylene protons to both nitrogens in thediazeniumdiolate group and Cγ methylene protons to the hydroxylatednitrogen only . The diastereotopic Cβ 1H’s on the Ser residue are deshielded in comparison to thetypical ppm range observed, consistent with the cyclization due tothe proximity to the ester group.The Asn in tistrellabactin A is replaced by an Asp in tistrellabactinB, consistent with the mass difference of 1 Da between them. Connectivityof the peptidic backbone was determined based on Figures S21–S24). Linearizationwas achieved with mild conditions by incubation in pH 8 Na2HPO4 buffer for 48 h, at which point the linear form oftistrellabactin A was observable by UPLC-MS analysis at m/z 1110.4 [M + H]+ and tistrellabactinB at m/z 1111.4 [M + H]+ . Mass fragmentation of bothlinear and cyclic tistrellabactins A and B is consistent with theNMR-assigned structure. Mass losses of NH3 and H2O from b and y fragments were observed, as would be expected withhighly ionizable side chain amines and hydroxyl groups on Asn, Asp,and Gln residues (S24).Due to the cyclized and chargedstructures of tistrellabactinsA and B, optimized mass fragmentation was obtained by linearizationof tistrellabactins A and B via hydrolysis of the Ser ester linkageS21–S24. C-diazeniumdiolate ligands. TheUV–vis spectrum of Fe(III)-tistrellabactin A shows an absorptionband at 340 nm and a broad shoulder centered at 420 nm. The peak at340 nm is consistent with a ligand to metal charge transfer band (LMCT)for the α-hydroxycarboxylate ligand, β-OH-Asp, coordinatedto Fe(III), while the broad shoulder centered at 420 nm correspondsto the LMCT band for the Fe(III)-hydroxamate group31 .TistrellabactinsA and B coordinate Fe(III) with the β-OH-Asp,hydroxamate, and d-configured(S26–S28). Thesecond A domain selective for Gln in MobF is not followed by an Edomain; however only d-Gln was detected . Therefore, we propose that MobG may beworking with the last module of MobF to epimerize the two Gln residuesloaded iteratively at this position . The 15N-enriched-tistrellabactins were prepared by culturing T. mobilis KA081020-065 in an artificial seawater (ASW)medium using 15NH4Cl in place of NH4Cl as the sole nitrogen source. UPLC-MS analysis of purified 15N-enriched tistrellabactins A and B showed both had an isotopicmass of m/z 1107.4 [M + H]+, indicating 15 nitrogens in the structure of tistrellabactin A and14 nitrogens in tistrellabactin B . Corresponding ionization-induced mass losses of 31 Da are observedfor both tistrellabactins A and B, consistent with ionization of 15NO instead of 14NO.l-Arg,given the presence of protein homologues of GrbD and GrbE in the tistrellabactin’sBGC, 14N-l-Arg was supplemented into the 15N-ASW T. mobilis KA081020-065 culture. Purifiedtistrellabactins A and B grown under these conditions both have anisotopic mass of m/z 1102.4 [M +H]+, indicating five nitrogens in each structure are unenriched.Also notable is the mass ion fragment showing a loss of 30 Da (m/z 1072 [M + H]+). A mass lossof 30 rather than 31 indicates the distal nitrogen on the diazeniumdiolatein Gra is unenriched. As part of the urea cycle,32 Arg is converted to Orn, which then may get incorporatedbefore the microbe initiates de novo biosynthesisof Orn from 15NH3. Therefore, the most plausiblenitrogens to be unenriched would be the three in the Gra and two inthe Orn residue, which matches the MS analysis . Moreover, 1H–15NHMBC correlations confirm the unlabeled nitrogens are in both Graand Orn residues and therefore originate from Arg, in agreement withthe isotopic enrichment of gramibactin in P. graminis DSM 17151 .14To determine if theGra residue in the tistrellabactins originates from T. mobilis KA081020-065 encodesaccessory genes consistent with the biosynthesis of three differentFe(III) ligands within the structure. Along with the C-diazeniumdiolate biosynthesis genes, tailoring enzymes MobD, MobI,and MobJ would be responsible for the modification of l-Aspand l-Orn to yield β-hydroxyaspartate and the Orn-hydroxamateligand groups, respectively (asp) and an A domain specific for activation of l-Aspin MobH followed by a TE domain (GGDSI motif) directs thestereospecific 3R hydroxylation of the Asp producing the l-erythro diastereomer.33 Modification of l-Orn by an N-acetyltransferase(MobI) and a N-monooxygenase (MobJ) yields the hydroxamategroup as the third bidentate Fe(III) ligand. The predicted NTN hydrolase,MobM, suggests an N-terminal fatty acyl group may be present duringthe biosynthesis; however it was not detected in the culture workup.34The putative siderophore BGC identifiedin the genome of ectively 2. A stan15 NRPS modules minimallyconsist of a condensation (C), adenylation (A), and thiolation (T)domain; however mobG is missing a C and A domain.35 At the level of genome mining alone, it is unclearif the C domain at the end of MobF followed by an unannotated gapmay be a sequencing error or biosynthetically relevant.The core peptidic structure is assembledby the concerted action of three NRPS modules: MobF, MobG, and MobH.The third NRPS module in MobF contains an A domain with specificitycode DVHRTGLVAK, matching that of the A domains of Gra in the reportedGra-containing metabolites.15N-isotopic labeling demonstrate that tistrellabactins A and B differby an Asn and Asp at the analogous position. These results indicatethat the A domain in the first NRPS module of MobH can accept eitherresidue. The ability of an A domain to accept both Asn and Asp asa substrate is not entirely surprising given the similarity of thetwo amino acids in size and polarity. The ratio of the peaks in theHPLC chromatogram of the extracted T. mobilis supernatant(215 nm) shows incorporation of Asn is favored over Asp .Promiscuous NRPS A domains have been observed previously, for examplein pacifibactin (Ser/Gly),29 xenemetides(Trp/Phe),37 moanachelins (Ala/Gly),38 and pseudoalterobactin (Asn/Asp) among others.40NMR assignments, MSMS fragmentation, and over Asp 3A. Addit41 The promiscuity of the A domain substrate allows for explorationof the fitness landscape without the loss of the siderophore’sactivity.42 In tistrellabactin B, the second Asp isnot hydroxylated. If tistrellabactin B was hydroxylated, this conformationmay be unfavorable and would be unnecessary for Fe(III) coordinationgiven the presence of three bidentate ligands to provide hexadentatecoordination to Fe(III). Assignment of the absolute configurationof the amino acid constituents of the tistrellabactins shows thatthe Asn/Asp is d-configured, which is in agreement with theNRPS module including an E domain following the A domain that activatesthe Asn/Asp. The presence of a TE domain with a standaloneβ-hydroxylase correlates with a hydroxylation event; howeverthe TE also functions to position the amino acid for epimerization.43The production of two products from the tistrellabactin BGCmay be indicative of NRPS evolution, as functional divergence andenzyme promiscuity are often related.C-diazeniumdiolateundergoes transformation to E and Z oxime isomers in the same manner as occurs for apo-gramibactin (S33–S35).14 UPLC-ESIMS analysisof the apo-tistrellabactin A photoproduct shows a new mass of m/z 1061.4 [M + H]+ and completeloss of the apo-tistrellabactin A m/z 1092.4 [M + H]+ species . Also present is a mass showing Fe(III) coordinated to the photoproductwith mass m/z 1114.3 [M –2H + Fe]+, demonstrating tistrellabactin A retains itsability to coordinate Fe(III) with the remaining hydroxamate and β-OH-Aspligands (S33).Irradiationof apo-tistrellabactin A with UV light diminishes the intensity ofthe absorption band at 246 nm as the ligands 6C, S33.T. mobilis with a positive chrome-azurol-S assay (CAS) response,indicatingsiderophore production, were also photolyzed in UV light. After 1h of irradiation, the cells were pelleted and the resulting supernatantwas analyzed by UPLC-MS . Theresulting sample shows a mass loss of 31 from the peaks correspondingto tistrellabactins A and B, consistent with the photoproduct observedfor the purified and concentrated sample. In comparison, a nonirradiatedaliquot from the same culture shows no change to the apo-tistrellabactinsand no trace of their respective photoproducts . These results demonstrate the biological relevanceof the photoreactivity of these siderophores.Actively growing cells from a culture of 44 When Fe(III)-tistrellabactin A (m/z 1145.4 [M – 2H + Fe]+) is photolyzedselectively with 254 nm UV light (Hg(Ar) Oriel No. 6035), the intensityof the LMCT band for the α-hydroxycarboxylate and the hydroxamateto Fe(III) both decrease, while a band at 270 nm evolves and increasessteadily with a bathochromic shift to 277 nm over 13.5 h of irradiation. Aliquots taken along thephotolysis time course were analyzed by MS and show the decrease ofthe Fe(III)-tistrellabactin protonated molecule and the appearanceof several new protonated molecules. The β-OH-Asp and the graminineresidues undergo photoreactions, as evinced by the presence of doublycharged protonated molecules at m/z 550.1 and 557.6 [M + 2H]2+, consistent with the decarboxylationof the β-OH-Asp (46 Da) or transformation to E/Z oxime isomers from the C-diazeniumdiolategroup (31 Da), respectively, while retaining coordination to Fe(III).At later time points in the photolysis the ion count of the protonatedmolecule at m/z 508.2 [M + 2H]2+ increases further, consistent with both the graminine andβ-OH-Asp ligands having undergone photoreactions with the lossof the ability to coordinate Fe(III) .Less is understoodabout the photoreactivity of the Fe(III)-bounddiazeniumdiolate complex. It is well established that the Fe(III)-β-OH-Aspwill undergo a radical decarboxylation reaction when irradiated, potentiallyplaying an important role of iron cycling in the marine ecosystem.TistrellamobilisKA081020-065produces not only the bioactive didemnins,26 but also, as presented here, the two depsipeptides tistrellabactinsA and B, with rare biosynthetic features. The BGC of the tistrellabactinswas identified in a previous study,26 althoughthe expression of these metabolites was not observed when culturedin iron-rich media, highlighting the importance of mimicking certainenvironmental cues for laboratory culturing. To induce siderophoreproduction, T. mobilis KA081020-065 was culturedunder Fe-poor conditions, yielding tistrellabactins A and B.The marine-derived microbe C-diazeniumdiolate,an α-hydroxycarboxylate, and a hydroxamate. This report is thefourth account of a C-diazeniumdiolate siderophorewith Gra, but the first to be produced by a marine-derived microbe.In the process of characterizing the structure, several unexpectedfeatures were uncovered, which could not have been predicted a priori by genome mining, including an iterative additionof Gln, a promiscuous A domain, the incorporation of a 3-hydroxybutyricacid group, and a 22-membered macrolactone ring. As a result of thepromiscuous NRPS adenylation domain, two siderophores arise from asingle BGC, differing only by a d-Asn in tistrellabactinA and a d-Asp in tistrellabactin B. The identification ofpromiscuous enzymes, including A domains, is of interest, as theyhave been leveraged for bioengineering efforts.46 A hydroxylated ornithine appended with a 3-hydroxybutyric acid groupwas elucidated, providing a hydroxamate-type ligand for bidentateFe(III) coordination. The third Fe(III) ligand was identified as the C-diazeniumdiolate, and isotopic feeding study results furthersubstantiate the origin of Gra is indeed Arg.14 The mixed-ligand tistrellabactins are atypical siderophores, withthree different Fe(III)-binding ligand types.The tistrellabactins are mixed ligand siderophores composed ofthree Fe(III) ligand types: a T. mobilis was isolated.The tistrellabactins lose an equivalent of NO and H+ fromthe Gra residue to give an E/Z oximeisomer photoproduct following the same photoreactivity as the siderophoregramibactin.14 In contrast to the photoproductof apo-gramibactin, the apo-tistrellabactins are still able to maintaintheir ability to coordinate Fe(III) with the presence of two bidentateligands remaining postphotolysis as opposed to one in the photoproductof apo-gramibactin.The most intriguingfeature of the tistrellabactins is their photoreactivity,given the environment from which C-diazeniumdiolate graminine-containingsiderophores.15 Unlike the previously reportedsiderophores in this family, whichare all isolated from rhizospheric microbes,15T. mobilis KA081020-065 originates from the RedSea, establishing a much wider distribution of this recently identifiedsiderophore type. The BGC encoding the tistrellabactins was identifiedon a plasmid; consequently the genes may have been acquired via horizontalgene transfer. This gene cluster’s presence on a plasmid ratherthan within a chromosome is also appealing for potential bioengineeringof this BGC.47 Natural products containingN–N bonds are often found to have biological activity;1 therefore the understanding of how those bondsare formed can open up doors within synthetic biology to take advantageof this functionality. We propose l-Arg is oxidatively rearrangedto yield Gra, utilizing at least the two protein homologues of GrbEand GrbD, which are putatively assigned as an Arg hydroxylase anda nonheme iron enzyme capable of oxidative rearrangement to form theN–N bond. Further investigations will deduce whether the putativearginine hydroxylase fused to an NRPS domain in the tistrellabactinsBGC . NMR spectra for characterizationwere collected in DMSO-d6, and spectraof the photoproduct were collected in 50 mM Na2HPO4-buffered D2O (pD 8.0). Spectra were indirectlyreferenced by the residual solvent peak or 2H lock. MSanalysis was carried out on a Waters Xevo G2-XS QTOF with positivemode electrospray ionization coupled to an AQUITY UPLC-H-Class systemwith a Waters BEH C18 column. Samples were run with a linear gradientof 0% to 100% acetonitrile (0.1% formic acid) in ddH2O(0.1% formic acid) over 10 min. IR data were collected on a BrukerAlpha FTIR spectrophotometer. CD data were collected on a JASCO J-1500CD spectrometer on tistrellabactin A (71 μM) and tistrellabactinB (52 μM) in Na2HPO4 (pH 8). CD data wasreferenced to the buffer and collected with a scan rate of 20 nm/min,DIT 8 s bandwidth of 1 nm, and data pitch of 0.5 nm. Deionized waterwas dispensed from a Milli-Q IQ 7000 water purification system (Resistivity18.2 MΩ). All glassware was acid-washed with 4 M HCl and subsequentlyrinsed with Milli-Q water.UV–visible spectrawere obtained on an Agilent Technologies Cary 300 UV–vis spectrometer.NMR spectroscopy was carried out at 25 °C on a Bruker 500 MHzspectrometer equipped with a Prodigy cold probe -CAS response.48The strain Culture supernatants were obtained bycentrifugation at 6000 rpm for 30 min at 4 °C . To extract the tistrellabactins, the culture supernatantwas decanted and shaken with 100 g of Amberlite XAD-4 resin. The XAD-4resin was prepared by washing with methanol and then equilibratingwith Milli-Q water. The resin and supernatant were allowed to equilibratefor 4 h at 120 rpm. The resin was filtered from the supernatant andwashed with 0.5 L of Milli-Q water. The adsorbed organics were elutedwith 40% aqueous methanol. The eluent was concentrated under vacuumand stored at 4 °C. Tistrellabactins A and B were purified fromthe eluent by semipreparative HPLC on a YMC 20 × 250 mm C18-AQcolumn, with a linear gradient of 10–55% methanol (0.05% trifluoroaceticacid) over 45 min, yielding pure tistrellabactins A (8 mg) and B (5mg) from 1 L of culture.15NH4Cl was used in place of NH4Cl. An additional experiment was completed with 10 mM 14N-Arg added to the 15N-enriched culture medium to followpotential utilization of l-Arg in the biosynthesis.The ASWmedium used for culture growth only contains one nitrogen source.To isotopically label the full structures of tistrellabactins A andB, 49 Amino acid standards  were derivatizedunder the same conditions.Aliquots (1 mgin Milli-Q water) of tistrellabactin A and tistrellabactin B werecombined with 12 M HCl or with 57% HI for final concentrations of6 M HCl or 45% HI. The acidified solution was transferred to a glassampoule, blanketed with Ar(g), and sealed under flame. The ampoulewas heated at 108 °C for 21 h. After heating, the ampoule wasbroken, and the crude solution was evaporated and redissolved in 0.7mL of Milli-Q water five times to ensure acid was removed from thehydrolysate. A 100 mL aliquot of the hydrolysate was reacted withMarfey’s reagent following standard conditions.l-erythro hydroxyasparticacid, in positive ion mode.The FDAA-hydrolysate was analyzedby UPLC-MS and RP-HPLC (250 × 4.6 mm YMC C18-AQ column). HPLCsamples were monitored at 340 nm using a linear gradient of 15% to50% acetonitrile in 50 mM triethylamine phosphate (pH 3.0) over 50min. The hydrolysate and standards were also analyzed by UPLC-MS (15%to 50% acetonitrile with 0.1% formic acid in Milli-Q water) to identifythe graminine and 2HPO4 prepared in D2O (99.9% purity)) with the pD adjustedto 8.0. For the Fe(III)-tistrellabactin A photolysis, an Edmund Opticsfilter was used to selectively irradiate at 253.7 nm.Photolysisof apo-tistrellabactin A was carried out in a quartz NMR tube. AnOriel Instruments Hg(Ar) (No. 6035) pen lamp was used as the UV source.Samples were dissolved in aqueous buffer  followed by UV–vis spectroscopy. The solution wasequilibrated for 30 min after each addition of Fe(III). The Fe(III)stock was standardized spectrophotometrically with 1–10 phenanthroline and hydroxylamine as the reducing agent. Titration of apo-tistrellabactinA with Fe(III) yields extinction coefficients calculated at the 1:1Fe(III)-siderophore breakpoint: 1479 ± 173 M–1 cm–1 at 420 nm, 2959 ± 300 M–1 cm–1 at 340 nm, and 9013 ± 774 M–1 cm–1 at 246 nm over three trials .Apo-tistrellabactinA  was titrated with a standardizedFe(III) stock solution (FeClPhotolysis ofFe(III)-tistrellabactin A was carried out in a quartz cuvette witha 75 mm stir bar. An Oriel Instrument Hg(Ar) (No. 6035) pen lamp wasused as the UV source equipped with a bandpass filter .max (log ε) 247 nm (3.95) ; ECD  λmax (Δε) 204 nm (+16.53), Figure S38; IR, 1655 cm–1 , 1423 cm–1 , 1265 cm–1 , Figure S39; 1H, 13C, and 2D NMR data, Table S2; HRESIMS m/z 1114.4377[M + Na]+ .white solid; UV–vis, λ2HPO4, pH 8.0); ECD  λmax (Δε) 204 nm (+17.65), Figure S38; IR, 1654 cm–1 , 1447 cm–1 , 1262 cm–1 , Figure S39; 1H, 13C, and 2D NMR data, Table S5; HRESIMS m/z 1093.4380[M + H]+ .white solid (Na


To uncover novel factors responsible for the nucleosome positioning, we analyzed potential involvement of the histone N-tails in this process. To this aim, we reconstituted the H2A/H4 N-tailless nucleosomes on human BRCA1 DNA (~100 kb) and compared their positions and sequences with those of the wild-type nucleosomes. In the case of H2A tailless nucleosomes, the AT content of DNA sequences is changed locally at superhelical location (SHL) ±4, while maintaining the same rotational setting as their wild-type counterparts. Conversely, the H4 tailless nucleosomes display widespread changes of the AT content near SHL ±1 and SHL ±2, where the H4 N-tails interact with DNA. Furthermore, a substantial number of H4 tailless nucleosomes exhibit rotational setting opposite to that of the wild-type nucleosomes. Thus, our findings strongly suggest that the histone N-tails are operative in selection of nucleosome positions, which may have wide-ranging implications for epigenetic modulation of chromatin states.The precise mechanisms governing sequence-dependent positioning of nucleosomes on DNA remain unknown in detail. Existing algorithms, taking into account the sequence-dependent deformability of DNA and its interactions with the histone globular domains, predict rotational setting of only 65% of human nucleosomes mapped  Organization of eukaryotic DNA in chromatin is essential for many cellular processes such as gene expression, DNA replication and repair . The basHistone interactions with DNA involve both the histone globular domains and the tails. For the histone domain-DNA interactions, previous studies have identified 14 highly conserved ‘sprocket’ arginine residues deeply buried in the DNA minor groove , 9, 10. Sequence-dependent bending anisotropy dictates how DNA is wrapped around a histone octamer –5. PreviIn addition, the alternative DNA sequence pattern known as anti-WW/SS was described, in which the WW and SS profiles are in counterphase with the corresponding profiles observed in canonical WW/SS nucleosomes , 26. In in vivo  along nucleosomal DNA across different eukaryotic genomes. In addition to the periodically oscillating sequence patterns observed earlier , we founin vitro with that of the wild-type (W-T) NCPs. We found that overall, the H2A N-tailless NCPs are positioned in phase with the W-T NCPs . By contrast, a substantial number of the H4 N-tailless NCPs are in counterphase with the W-T NCPs . Thus, our results suggest that preferential interactions of the histone N-tails with AT-rich minor groove may provide a novel mechanism for stabilization of anti-WW/SS nucleosomes observed in vivo. Implications for epigenetic control of nucleosome positioning are also discussed.To elucidate a possible effect of the H2A and H4 N-tails on the sequence-specific formation of nucleosomes, we reconstituted the N-tailless NCPs and compared their positioning E. coli with TAR/BAC containing full length hBRCA1 gene (BRCA1-TAR/YAC/BAC) was obtained from V. Larionov (Transformed Larionov . It was Xenopus histone octamers with wild-type (W-T) and H2A/H4 N-tailless histones obtained from Histone Source, Protein Expression and Purification Facility at Colorado State University. Reconstitution was performed as described earlier . Nucleosomal DNA fragments were subjected to paired-end sequencing on Illumina platform.Reconstituted chromatin (1 μg in 60 μl) in buffer containing 50 mM NaCl, 10 mM Tris 0.025% NP-40, 20% Ficoll, 0.7 mM CaClcutadapt , using the sequence of the BRCA1 gene (from human genome hg19) as a reference. Fragments with samtools ; rather, they are inherent in a wide spectrum of eukaryotes.To elucidate the sequence specificity of these off-peaks, we calculated the high-resolution profiles of mononucleotides A and T as well as the dinucleotide WW in nucleosomal DNA obtained from various species see . Note thversus T  coincide with locations where the N-tails of H2A and H4 histones are in close contact with the DNA minor groove . Since tin vitro, we compared the local AT content in nucleosomes reconstituted with the wild-type (W-T) and the H2A/H4 N-tailless histones (see Methods). For brevity, the N-tailless NCPs are denoted H2A, H4-glob and H4-del. First, we analyzed the combined frequencies of occurrences of adenines and thymines in the W-T nucleosomes and H2A constructs  frequency upon transition from the N-tailless to the W-T nucleosomes . The difversus T described above in vivo  profiles shows that they are negatively correlated , 5B. Indn+5 bp . Therefore, one can expect that a similar shift of ~10n+5 bp would be observed between the W-T and H4-tailless nucleosomes  profile resembles the anti-WW/SS profile studied earlier  – both hleosomes , 5B.dist’ between the NCP dyads is calculated, and the occurrences of all distances are summed up (dist=20) is calculated as 5 × 10 = 50.To describe relative positions of nucleosomes in two populations we used the distance cross-correlation (DCC) function introduced earlier . For eacummed up . Note thA comparison of the W-T and H2A nucleosome subsets revealed strong DCC peaks at ~10, ~20 and 30 bp , indicatn + 5 bp .In the case of W-T and H4-glob nucleosomes, the DCC profile has an additional peak at 15 bp , suggestAccording to our model, a nucleosome is stabilized by two types of histone-DNA interactions . The firThe second type of interactions involves the H2A and H4 histone N-tails, which are in close contact with the minor groove facing out at SHL ±4 and SHL ±2/±1, respectively . Our datversus T strand asymmetry near SHL ±4 by Cole et al.  simulations performed by Shaytan et al. . The hiset al. . In partet al.  gave esset al. . Finallyet al.  accompanNote, however, that the H4 N-tail is sufficiently long to penetrate the minor groove close to SHL 0 . Our reset al. . Nevertheless, we can safely summarize that removal of the histone tails can lead to noticeable alteration of the sequence-dependent nucleosome positioning in vitro.Fan et al.  analyzedet al. . This si . These results are consistent with the above assumption that the H2A and H4 N-tails are involved in selection of the optimal NCP sequences at SHL ±4 and SHL ±1/±2, respectively. As to the putative interactions between the H4 N-tail and DNA in the vicinity of the dyad (at SHL 0), further structural studies are needed to clarify this question.To test this hypothesis, we reconstituted the H2A- and H4-tailless NCPs on human BRCA1 gene Furthermore, we obtained direct evidence for the distinctive repositioning of nucleosomes induced by truncation of the histone N-tails. Importantly, the H2A-tailless nucleosomes remain in phase with the W-T nucleosomes, whereas a significant number of H4-tailless nucleosomes are out of phase with the W-T nucleosomes, suggesting that the N-tails of H2A and H4 histones have different impact on the nucleosome positioning.The strongest differences between the AT content of the H4-tailless and the W-T nucleosomal sequences are found at SHL 0/±1/±2, where the DNA minor groove faces out . OverallWhile the structural details of sequence readout by histone tails remain unclear, our data strongly suggest that histone tails preferentially select certain nucleosome positions over other positions, which may have important implications for epigenetic modulation of nucleosome positioning. Certain epigenetic marks, such as acetylation of lysines on histone N-tails, decrease affinity of the tails to the DNA minor groove. This, in turn, may change nucleosome positions and even shift the equilibrium between different chromatin conformation states. Obviously, more studies are needed to elucidate the role of epigenetic marks in positioning of nucleosomes.Supplement 1Supplementary Figure S1. Histone H2A and H4 N-tails in the DNA minor grooves.The ventral (A) and dorsal (B) halves of the nucleosome X-ray crystal structure  are showSupplementary Figure S2. A representative region of BRCA1 gene where the nucleosome occurrences have two strong peaks separated by 20–21 bp.The dominant NCP positions in the W-T and H2A populations of nucleosomes are shifted by 20–21 bp one from the other, so that the NCP positions are in the same phase.Supplementary Figure S3. A representative region of BRCA1 gene where the nucleosome occurrences have two strong peaks separated by 15 bp.in vivo. This periodicity does not hold for the whole BRCA1 gene, however (data not shown).The nucleosome occurrences in the region 21,500 – 22,500 bp of BRCA1 gene are presented for the four NCP subsets . Note that in the W-T and H2A subsets, the nucleosomes shown in red represent the dominant NCP positions, whereas in the H4-glob and H4-del subsets, these are the positions shown in green that are dominant. Intriguingly, in the region 21,500 – 22,200 bp there are four peak clusters separated by 190–200 bp, which is remarkably close to the nucleosome spacing