Komagataella yeasts, many of which are known to colonize plants and insects involving yet unknown adhesion mechanisms. Here, we report the functional and structural analysis of two of its members: KpFlo1 (=Cea1), that is highly specific for terminal N-acetylglucosamine moieties, and KpFlo2, which represents an orphan lectin with intact binding site but unknown specificity. Crystal structures of the Cea1 adhesion domain complexed to N-acetylglucosamine and N,N′-diacetylchitobiose reveal a Ca2+-dependent binding mode that differs from other members of the PA14/Flo5 adhesin family. Heterologous expression of Cea1A in Saccharomyces cerevisiae promotes cellular adhesion to non-reducing ends of non-crystalline chitin. Overall, our data suggest that high-affinity recognition of β-GlcNAc-capped glycans by Cea1 enable Komagataella species to interact with surface cues present in fungi and insects.Cell–cell and cell-substrate based adhesion of yeasts are major determinants of their adoption of different life styles. Genome-mining of ascomycetous GPI-anchored cell wall proteins with lectin-like PA14 domains identified a unique class of putative adhesins in the clade of methylotrophic  Saccharomyces species, up to 30% of the cell’s dry weight consists of wall material composed of β1,3- and β1,6-glucans, β1,3-glucan-chitin complexes and many heavily glycosylated mannoproteins including GPI-anchored cell wall proteins (GPI-CWP)  . In Saccdrolases . Many ofI domain , specifiN-terminal A domain that is followed by a region carrying a highly variable number of serine/threonine-rich tandem repeats (B region). The C-terminal domain (C domain) carries the GPI-anchor that confers covalent fixation to the cell wall by transglycosylation to β1,6-glucans. The A domain that is crucial for adhesion comprises the binding site, which, in the case of PA14 domain-related adhesins, recognizes disaccharidic ends of glycans in a C-type lectin-like manner. Binding is achieved via a Ca2+-ion being complexed by a unique DcisD motif and further amino acid residues in two calcium binding loops . For example, the Flo-5 A domain of S. cerevisiae (ScFlo5A) mediates specific adhesion to Man-α1,2-Man moieties presented on the surface of other yeast cells and thereby causes floc formation by cell–cell interactions , Lg-Flo1A (4GQ7), ScFlo1A (4LHN), Epa1A (4ASL), and Epa6A (4COU). Despite revealing the common PA14-like β-sandwich and DcisD motif these structures showed different modes of carbohydrate recognition , binds to N-acetylglucosamine or at non-reducing ends of chitinous polymers with high affinity in vitro and in vivo. Furthermore, K. pastoris A domains share features from both previously characterized Flo and Epa subgroups. Among them Cea1A represents a novel subgroup of adhesive PA14 domains, which mediate high affinity recognition of chitinous poly- and oligomers.In this study, we mined the genomes of ascomycetous fungi for hitherto unknown PA14/Flo5-like adhesin domains and identified such domains in several putative GPI-CWP adhesins of methylotrophic yeasts from the  unknown . Compare insects . Here, wScFlo5 (Uniprot: P38894) and Epa1 (Uniprot: Q6VBJ0) were used as seeds to identify further non-annotated homologs using BLAST tools on ascomycetous genomes (tax id: 4890). Based on the revised genome data of Komagataella species by K. pastoris and seven in the related K. phaffii strain. In a second step designated A domains sequences were aligned by T-Coffee  domain belongs to a predicted adhesin with PA14 domain ; likewise the KpFlo2A domain resides at the N-terminus of another GPI-CWP . Gene fragments coding for these A domains were amplified from genomic DNA using primers 5′-CAGTCGACATATGGATGACAGTGGAAATGG/5′-GCTCGAGTTATTCATGGCAGGAGTTCTC for Cea1A and 5′-CAAGTTACATATGCAGGAAAGTGGTGATGG/5′-GCTCGAGTTATGCTTGGCATTGTTCTTC (Metabion) for KpFlo2A, respectively. These fragment were subcloned into pET28a (Novagen) using NdeI and XhoI restriction sites (underlined in primer sequence) to encode N-terminally His6-tagged A domain fusions. Heterologous overexpression was performed according to Escherichia coli SHuffle T7 Express (New England Biolabs). Cells were grown at 37°C in TB medium to an OD600 = 0.2 and were subsequently cooled to 12°C. Overexpression was induced by addition of 10 μM IPTG when the culture reached OD600 = 0.6. After harvesting, cell pellets were resuspended in AM-buffer , frozen in liquid N2 and stored at -80°C. After thawing and addition of lysozyme, PMSF, EDTA and DNAseI cell disruption was performed using a French press (Aminco). After centrifugation the supernatant was applied to a Ni-NTA column (Macherey Nagel); the recombinant KpFlo A domains were eluted with AM-buffer containing 150 mM imidazole. Eluate fractions were checked by 12% SDS-PAGE and fractions accordingly pooled and concentrated using a 10 kDa cut-off Amicon Ultra concentrator (Millipore). Finally, size exclusion chromatography with a Superdex 200 column  and SEC-buffer  was used to yield pure monomeric KpFlo A domains. Protein solutions concentrated to 10 mg/ml  were stored at 4°C.In KpFlo1A (=Cea1A) and KpFlo2A were fluorescently labeled using an Alexa Fluor 488 THF kit (Invitrogen) and applied to CFG array V5.1 chips at protein concentrations of 20 μg/ml and 200 μg/ml, respectively. Chip surfaces were repeatedly washed and remaining fluorescence was measured and quantified. Data and the exact procedure can be found under cfg_rRequest #2555 at the webpage of the Consortium for Functional Glycomics2. The pie charts for the Cea1A domain were generated using the quantity of carbohydrates belonging to a specific group. All ligands showing ≥4% relative fluorescence units (RFU) of the best binder (GlcNAc directly linked to a spacer) were used. Given that we found no binder for KpFlo2A, we tested a few sugars  by fluorescence spectroscopy using W119 near the putative glycan binding site  as reporter, but failed likewise to identify any significant fluorescence quench indicating that none of these carbohydrates bound (data not shown).Recombinant 2SO4 or NaCl/PEG 8000 containing conditions lacked any diffraction. After identification of GlcNAc as cognate ligand co-crystallization was performed using 5 mM GlcNAc or 5 mM N,N′-diacetyl-chitobiose and 5 mM CaCl2 in the protein solution. Orthorhombic crystals were obtained in conditions containing MgCl2 and 15–30 mg/ml Cea1A. Optimization was done using the hanging drop vapor diffusion method in 24-well format and streak seeding. After 3–5 days crystals for data collection were obtained in conditions containing 100 mM MgCl2, 100 mM NaCl, 100 mM sodium citrate pH 3.5 and 12% PEG 4000 at 4°C. Well diffracting monoclinic crystals of the KpFlo2A domain were obtained in 30 mg/ml protein, 20 mM MgCl2, 200 mM sodium cacodylate and 50% PEG200 at 18°C.Crystallization was done with a Cartesian robotic system (Genomic Solutions) using commercially available screens (Qiagen) in sitting drops containing 300 nl protein solution with 300 nl reservoir solution at either 4°C or 18°C. First Cea1A crystals grown in LiKpFlo2A structure was likewise solved using instead the Cea1A domain as template. Data processing was performed with XDS, XSCALE, PHENIX, and CCP4 (Rfree was possible and no unexplained difference electron density was left for interpretation (For statistics see Table Bave: 15.3 vs. 19.4 Å2) due to comparably poorer electron density in the latter for the N-terminus, A89-V99, that takes part in molecules A/B in formation of the neck region, and the stretch K205-A217. Accordingly, the neck region defined for molecules A/B is partly missing in C/D because of a lack of the disulphide linkage between C207 and C294. The latter cysteine belongs to a C-terminal stretch that is not defined in molecules C/D (D290-E296) by electron density. Similarly, the KpFlo2A structure was refined with phenix.refine , soaked in mother liquor containing 15–20% glycerol as cryoprotectant and frozen in liquid nitrogen before X-ray datasets were recorded at 100 K. The Cea1A structures were solved by molecular replacement using PHASER  and a trand CCP4 . The Ceaand CCP4  and Cootand CCP4  and NCS x.refine . Figuresx.refine  and UCSFx.refine .N,N′-diacetylchitobiose complex. For both simulations, three different disaccharide conformers have been used for producing independent trajectories (Supplementary Table -1). Finally, constant pressure, restraint-free equilibration to one atmosphere was carried out for 50 ps , before each trajectory was allowed to run for 20 ns as NpT ensemble for allowing final convergence. After equilibration 100 ns production trajectories were generated. Overall, we obtained a total of 600 ns overall simulation time from 6 trajectories á 100 ns for analysis. One snapshot was extracted every 0.1 ns, resulting in 1000 snapshots per trajectory, which were combined into 3000 snapshots per studied glycan complex. cpptraj (Molecular dynamics (MD) simulations used the Amber 17 software  with its cpptraj  and dbsc200-System  using a 200 μl cell. A 450 μM solution of Cea1A in SEC-buffer was used as sample. The ligands GlcNAc and N,N′-diacetyl-chitobiose were dissolved at 5 mM in SEC-buffer. ITC experiments were performed at 10°C with the first injections being 0.4 μl and 29 injections each with 2 μl. Every injection was applied over a period of 4 s and individual injections were separated by breaks of 360 s. To subtract dilution heat of the carbohydrate, a measurement with the same protocol was performed using only SEC-buffer in the sample cell. Analysis was done using the ITC-data plugin  for Origin 7.0 (Origin-Lab) with the one-binding-site model. Three individual measurements were made per ligand.Isothermal titration calorimetry measurements were performed with the ITCS. cerevisiae cells to chitin beads was investigated using the non-adhesive S. cerevisiae strain RH2520 (CEA1A domain on ScFLO11BC) or BHUM1964 (ScFLO11BC without A domain as a control). The CEA1 A domain was isolated by PCR using the primer pairs 5′-AAAAAACCGCGGGATGACAGTGGAAATGGCG/5′-AAAAAAGAGCTCTTCATGGCAGGAGTTCTCATC for Cea1A and chromosomal DNA from K. pastoris strain DSMZ 70382 as a template Plasmid BHUM2297 was constructed by integration of the CEA1A fragment into plasmid BHUM1964  to 10 OD of exponentially growing cells in 1 ml culture. Cells were mixed and washed with 200 μl of CaCl2-supplemented (10 mM) SC-4 medium before resuspension in the same medium. The cell suspension was mixed in a 3:1 ratio with chitin beads (New England Biolabs), which were treated with chitinase  to produce non-reducing terminal GlcNAc ends. 1 ml of SC-4 (+10 mM CaCl2) was added to 200 μl of this mixture, and the suspension was gently mixed by inversion followed by 2 min of incubation. The suspension was again mixed by inversion and shortly incubated for 10 s before the supernatant with unbound cells was removed. This washing step was repeated once. Microscopy was performed on beds of 1% agarose with a Zeiss Axiovert 200 M microscope. The cells were examined using differential interference microscopy (DIC) and a GFP filter set for detection of the DIOC6-fluorescence . Cells were photographed with a Hamamatsu Orca ER digital camera (Hamamatsu) and pictures were processed and analyzed using the Volocity software (Perkin Elmer). The amount of free or adherent cells was quantified using 10 comparable image sections of each strain and the program ImageJ  detect the Cea1A domain at the cell surface using a rhodamine filter set . Cells were photographed with a Hamamatsu Orca ER digital camera (Hamamatsu) and pictures were processed and analyzed using the Volocity software (Perkin Elmer).Exposure of the Cea1A domain on the t medium  to an opN,N′-diacetylchitobiose and 6HOS for KpFlo2A•glycerol, respectively. The numbering scheme for Cea1A used in the following refers to Uniprot entry A0A1B2J5V1 and is hence offset by +55 relative to PDB entries 5A3L and 5A3M. The shown glycan V5.1 profiles are deposited at the Consortium for Functional Glycomics (CFG) with the assigned identifier cfg_rRequest #2555 and consistent with earlier profiles from less complete CFG array V4.1 chips (cfg_rRequest #2079).The coordinates and structure factors have been deposited at the Protein Data Bank (PDB), Research Collaboratory for Structural Bioinformatics (RCSB), with accession codes 5A3L for Cea1A•GlcNAc, 5A3M for Cea1A•K. pastoris and K. phaffii genome sequences ; here the PA14 domain is part of a GH3-type glycoside hydrolase, which removes glycosides from the non-reducing ends of glycans.To search for new, uncharacterized PA14 domain containing adhesins, we used genome mining on the revised equences . By usinPSIBLAST  nine ortglabrata . In contresidues . InteresK. pastoris A domains to a phylogenetic analysis by including PA14 domain-containing Flos and adhesins as well as A domains of the PA14_2 type from the Pfam database. The maximum likelihood phylogenetic tree shows clustering of these GPI-CWP A domains into six different subgroups  and are closely related to each other, while the Epa A domain paralogs from C. glabrata and related genera are highly diverse (blue) within their separate cluster. In contrast, the Pwp A domains from C. glabrata (Kluyveromyces species (light red). The sixth cluster (brown) belongs to putative adhesins, which miss the conserved CC-motif for linking the N- and C-terminal ends to the PA14 core, but comprise the DcisD motif for Ca2+-binding unlike KpFlo4a-KpFlo4f. This cluster is found in fission yeasts and filamentous fungi , whereas the pairwise identities within the Flo, Epa, and Komagataella clusters are in the range of 50–77%  harbors in its B-region 25 agglutinin-like (ALS) repeats between the A domain (D78-V299) and the C-terminal GPI-anchor motif  sharing a high pairwise sequence identity of 59% with KpFlo1A. Unlike KpFlo1 and KpFlo3, the stalk-like B-region of KpFlo2 is not made up of any agglutinin-like repeats. Alignments of KpFlo1A, KpFlo2A and the not further characterized KpFlo3A domain with other fungal adhesins show conservation of both features required for Ca2+-binding, the DcisD motif in CBL1 and an asparagine in CBL2. These KpFlo A domains also have disulfide bridges that have counterparts in both the Flo and Epa subgroups  besides minor contributions from a few glycans bearing terminal galactosyl moieties , given its affinity and specificity toward β-GlcNAc capped glycans including chitinous polymers.To examine ligand specificity, we performed large-scale glycan array screening  by usingt Figure , we founs Figure . While tKpFlo2A. However, crystallization of the former without any ligand failed to yield well-diffracting crystals. Addition of Ca2+ ions with either GlcNAc or the disaccharide N,N’-diacetylchitobiose  led to orthorhombic Cea1A crystals, which diffracted to 1.7 and 1.8 Å, respectively  using a search model that was based on the structure of the Epa1A domain  and shows a glycerol molecule coordinated to the Ca2+ ion of the glycan binding site  as wellKpFlo A domains have a conserved β-sandwich, which consists of a six-stranded and a four-stranded β-sheet  lacks a hydrogen bond between a tyrosine from β12  and the DcisD-backbone. In KpFlo1A and KpFlo2A, this residue is replaced by a phenylalanine . Consequently, only the remaining hydrogen bond to the backbone A262-R263 (KpFlo2A: A219-V220) stabilizes the cis-peptide , mediate the majority of interactions with the GlcNAc moiety. While A258 and L259 form a hydrophobic part of the binding pocket and interact with C6 of GlcNAc, the rest of the pocket adopt a more polar character. E260 directly interacts with the nitrogen atom of the N-acetyl moiety, while R261 contributes, together with W147 from L2, to a hydrogen bond with the acetyl group. Interestingly, in molecule B of the asymmetric unit, R261 adopts an alternative rotamer, where it directly interacts with the N-acetyl-group of the ligand. The indole group of W147 forms a hydrophobic patch facilitating van der Waals interactions with the methyl-group of the N-acetyl moiety. K227 from L3 above the pocket interacts not only directly with the 6-OH group, but also indirectly via a water-bridged hydrogen bond. Finally, N230, that protrudes from loop L3 acts like a lid above the pocket by contributing to a complex water network above and on the back of the monomeric ligand.The binding pocket of Cea1A shows a complex network of hydrogen bonds and electrostatic interactions and the bound GlcNAc moiety Figure . As expeScFlo5A and Lg-Flo1A, the glycan binding site is much wider and shallow than in Epa1A and Cea1A. These differences are reflected by the interaction surfaces between the glycan cap and the different adhesins with values of 192 Å2 for Cea1A•GlcNAc, 164 Å2 for Epa1A•galactose and only 133 Å2 for ScFlo5A•Man.A striking feature of the Cea1A binding pocket are its numerous interactions with the terminal GlcNAc to enclosure it fully. Epa1A also contains a small and deep pocket, that binds to Gal-capped glycans with μM affinity due to a similar number of interactions including a sterically demanding tryptophan from L3 situated above the pocket. In the Flos, N-acetyl moiety of the second GlcNAc moiety, we used instead glucose as second glycoside. Further bias from initially set coordinates was avoided by running three independent 100 ns production dynamics replicas for both systems by employing different start values for the dihedral angles Φ and Ψ, which define the 1,4-glycosidic bond  or other PA14/Flo5-like adhesins. In summary, these data show that while Cea1A shares some of the structural characteristics found in Flos and epithelial adhesins, it confers ligand binding by a clearly distinct mode not observed in other PA14/Flo5-like adhesins so far.Comparison with Epa1A shows a different orientation of the terminal hexose moiety Figure . CompareN,N′-diacetylchitobiose  than by GlcNAc . However, KD values of 7.6 μM for GlcNAc and 13.1 μM for N,N′-diacetylchitobiose, respectively, are similar  failed to bind under analogous ITC conditions  and 7 (K. phaffii) PA14/Flo5-like domain containing GPI-CWP adhesins, respectively. Three of them KpFlo1-KpFlo3 are well conserved also in K. phaffii given pairwise sequence identities of 89, 86, and 94% for their A domains. In this study, we characterized the A domains of two of them. KpFlo2 represents currently an orphan lectin, as we found no target glycan when screening CFG glycan arrays, which are biased toward mammalian glycans by omitting microbial and plantal glycan structures. In the case of the related Epa A domains from C. glabrata 5 of 17 analyzed A domains likewise lacked any specificity . Indeed, our MD simulations show that Cea1 position III is often contacted by the reducing ends of both β- and α-linked disaccharides. While the interaction with the β-linked glucose moiety is strong (46.1% interaction probability), the α-glycosidic bond initially appears to produce an even more interaction (82.1%, Figure However, our study uncovers Cea1 as the first known fungal cell wall adhesin that strictly recognizes β-GlcNAc caps of chitinous polymers and other glycans with μM affinity and is able to mediate specific cell adhesion, e.g., to non-crystalline chitin. Soluble lectins specific for GlcNAc moieties have been so far discovered in basidiomycetes . AlthougN- and C- termini to the core of the A domain and forms an elongated stalk for the linkage with the B region. The B region of GPI-CWPs is predicted by de novo modeling of Candida albicans Als-adhesins to consist of small β-sheets and has been shown to be functionally important in S. cerevisiae for the presentation of A domains on the cell wall surface. In Cea1, the neck-like subdomain projects the repetitive B region into a direction of nearly 45° relative to the A domain (Figure ScFlo5A, the rest of this loop completely differs from Cea1A and adopts a different conformation (Figure ScFlo5A exhibit clearly distinct ligand binding modes, even though the orientation of the primary carbohydrate is similar. The small pocket and binding mode of Cea1A cause restricted ligand specificity for terminal GlcNAc, whose high affinity is sufficient to confer binding to non-crystalline chitin in vivo.Our data show that Cea1A harbors features distinct from other characterized PA14/Flo5-like adhesins. One feature of Cea1A not found in other PA14/Flo5-like adhesins is the neck-like subdomain that fixes the n Figure  (Verstren Figure . A seconn Figure . Accordiin vivo? An interesting observation of our study is the formation of small flocs by S. cerevisiae strains, which present Cea1A on their cell surfaces (Figure S. cerevisiae and other yeasts, the total amount of chitin corresponds to only 1–2% of the dry weight of unstressed cells (ScFlo5A/α-mannoside interactions. Interestingly, KpFlo1 does not belong to the GPI-CWPs, because it is anchored to the plasma membrane by an N-terminal transmembrane helix. An alternative function may be hence a stabilization of the fungal cell wall by Cea1, because its location would foster an interaction with the inner chitin layer of the fungal cell wall. However, such a cell-wall stabilizing function is likely restricted to K. pastoris, because the Flo1 ortholog of K. phaffii corresponds to a conventional GPI-CWP, which are commonly exposed on the outer cell wall.Can we delineate a biological function from the characteristics of Cea1A to recognize terminal β-GlcNAc caps s Figure . Here, Ced cells  and is mK. pastoris (Figure K. phaffii indicate that these proteins might confer adhesion in processes other than flocculation, such as the efficient binding to solid substrate surfaces or host cells. It is interesting to note, that decomposing wood has been suggested to represent the natural habitat of yeasts of the genus Komagataella (S. cerevisiae, social wasps have for example been found to act as environmental vectors (Pichia clade were indeed found in the guts of the passalid beetle Odontotaenius disjunctus and in specialized organelles used for myco-symbionts, where they support the digestion of complex carbohydrates (K. pastoris was initially found to be closely associated with the fruit fly Drosophila melanogaster (Komagataella subfamily of PA14/Flo5-like adhesins might be crucial for different yeast-insect interactions. This hypothesis is further supported by the fact that ascomycetes possess not only free but also secreted chitinases, which are capable to generate free β-GlcNAc ends from polymeric or crystalline chitin on host surfaces (The large set of putative PA14/Flo5-like adhesins present in s Figure  and K. pgataella . Furthergataella . In the  vectors . Yeasts hydrates . K. pastnogaster . Given tsurfaces .Komagataella provides insights into the structural and functional complexity and evolution of fungal adhesins and their ability to specifically recognize a wide variety of carbohydrates. In contrast to the Epa family, whose broad phylogenetic relationships indicate high variability, the cluster of PA14/Flo5-like adhesins from Komagataella is relatively small, and in this regard comparable to the Flos (Figure Candida species have a significantly higher number of genes for Epa-like adhesins than non-pathogenic strains (KpFlo genes Komagataella species apparently follow this trend for functional specialization of adhesins (Figure In summary, our discovery of a novel subgroup of PA14/Flo5-like adhesins from s Figure . Further strains . Given ts Figure .in vivo experiments. MM-R performed the molecular dynamics analyses. L-OE, H-UM, MM-R, and MK wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.L-OE, MK, and MV designed the research. MK, SB, JS, and NW performed the research. MK, SB, and L-OE analyzed the data. MK and SB contributed to The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The transcription factor USF2 is supposed to have an important role in tumor development. However, the regulatory mechanisms contributing to the function of USF2 are largely unknown. Cyclin-dependent kinase 5 (CDK5) seems to be of importance since high levels of CDK5 were found in different cancers associated with high USF2 expression. Here, we identified USF2 as a phosphorylation target of CDK5. USF2 is phosphorylated by CDK5 at two serine residues, serine 155 and serine 222. Further, phosphorylation of USF2 at these residues was shown to stabilize the protein and to regulate cellular growth and migration. Altogether, these results delineate the importance of the CDK5-USF2 interplay in cancer cells. The upstream stimulatory factors (USFs), USF1 and USF2, belong to the basic helix-loop-helix leucine zipper (bHLH-Zip) family of transcription factors. They can bind as homodimers or heterodimers . USFs arAlthough it is known that USF2 is a phosphoprotein and that CDK5 has an abnormal expression and activity in several human cancers, it is unknown whether USF2 is a substrate for CDK5. To examine this, we expressed full-length USF2 and several deletion variants as GST-fusion proteins in bacteria A and useTo determine whether USF2 is phosphorylated by CDK5 in vivo we performed immunoprecipitation assays (IPs) in PC3 cells which were edited by the CRISPR-Cas9 technique to eliminate CDK5 (ΔCDK5). When endogenous USF2 was precipitated from the cells and probed with anti-phosphoserine and anti-phosphothreonine antibodies we found that serine- and threonine-phosphorylated USF2 was present in the WT cells C. IntereIn order to map the exact CDK5 phosphorylation sites we performed bioinformatic sequence analyses of the USF2 protein with PhosphoMotif Finder , and PhoSince the phosphorylation sites could be important for USF2 transactivity, we performed transactivity assays with USF2 constructs in which the DNA binding domain (aa 232–346) was replaced with the Gal4 DNA binding domain and where the two CDK5 phosphorylation sites were substituted with alanine , respectively. Only Gal4 DNA binding domain containing constructs were used as a control. The respective GAL4-USF2-WT and S155A/S222A mutant constructs were then cotransfected with a luciferase (Luc) reporter regulated by 5 Gal4 response elements (RE). Thus, changes in Luc activity reflect the changes in the transactivity of USF2. While CDK5 increased Luc activity with the USF2-WT construct, no changes could be detected with the USF2-S155A/S222A double mutant C. Thus, Next we aimed to study the functional importance of CDK5-dependent USF2 phosphorylation in the broader cellular context in the presence and absence of CDK5. To do this, we used, in addition to the prostate cancer cell line PC3, the hepatoma cell line HepG2 and the breast cancer cell line MDA-MB 231 in which we eliminated CDK5 by using the CRISPR-Cas9 technique A. IntereThe above data suggest that CDK5-dependent USF2 phosphorylation directly affects the stability of the USF2 protein. Hence, we next investigated whether lack of CDK5 affects USF2 protein stability. To do this, we treated respective control (Sc) and ΔCDK5 PC3, ΔCDK5HepG2 and ΔCDK5 MDA-MB-231 cells with the protein synthesis inhibitor CHX and monitored the level of USF2 by western blotting. The USF2 half-life in cells lacking CDK5 was estimated to be significantly reduced  when compared to the half-life of USF2 in the respective control cells  . These dNext we aimed to determine what is the biological significance of CDK5-dependent USF2 phosphorylation. By measuring the proliferation rate A,B, BrdUIn this study, we explored the CDK5-mediated phosphorylation of USF2 as a regulatory mechanism, which can cause functional alterations of USF2 in cancer. We identified USF2 as a novel CDK5 target that is phosphorylated at two serine residues, Ser155 and Ser222. The CDK5-mediated USF2 phosphorylation promotes USF2 protein stability at least in three different cancer cell lines—PC3, HepG2, and MDA-MB-231. Additionally, expression of USF2 Ser155D/Ser222D, the USF2 phospho-mimicking mutant, in a CDK5 null background confers a proliferative and a migration advantage, which suggests that phosphorylation of USF2 by CDK5 can play a role during carcinogenesis. Collectively, these results establish a novel mechanism by which USF2 protein levels, and hence its function, can be regulated in cancer cells.USF2 as a transcription factor plays important roles in processes, such as embryogenesis, metabolism and cancer development. Interestingly, USF2 has been reported to have a dual role as either tumor-suppressor or tumor-promoter. USF2 is observed in various human cancers but what mechanisms cancer cells could use to suppress or activate its function is largely unknown. Thus, understanding the mechanism(s) regulating USF2 function will help to reveal its role in the progression of cancer. In that respect, the current findings identifying USF2 as a CDK5 target provide novel insights into those mechanisms. However, CDK5 is not the only kinase having the ability to convert USF2 into a phosphoprotein and need to be seen in conjunction with earlier findings showing that also PKA  and GSK3Interestingly, the phosphorylation signal detected was stronger with the USF2 protein containing only the USF-specific region  than with the USF protein containing the N-terminal domains (1–199 and 1–161) . Given tThe analysis of the data obtained by the phosphorylation prediction software revealed four putative CDK5 phosphorylation sites within the mapped domains: Ser155 within the N-terminal activation domain and Thr201, Ser222, and Thr230 within the USF-specific region (USR). The kinase assays showed that Ser155 in the N-terminal activation domain (1–161) and Ser222 in the USR domain (200–231) are then the only CDK5 phosphorylation sites, which were also in line with the IP data using the residue-specific phospho-antibodies where only serine-phosphorylated USF2 was undetectable in ΔCDK5 cells The preferred substrate recognition motif of CDK5 was determined to be S/T-P-X-K/H/R, whereby S or T are the phosphorylatable serine or threonine residues, P is the obligatory proline residue in the +1 position, X is any amino acid and K (lysine), H (histidine), and R (arginine) are the preferred basic residues in the +3 position ,51,52. FInterestingly, our study also revealed that lack of CDK5 and hence lack of CDK5-mediated phosphorylation of USF2 dramatically reduces the protein levels  and the p < 0.001) as well as the presence of genetic alterations in both genes   together with the observed enhanced proliferation and migration of all cell lines expressing the phosphomimicking USF2 Ser155D/Ser222D but not the non-phosphorylatable USF2 Ser155A/Ser222A mutant favor the tumor-promoting role of USF2. These functional data strongly imply that USF2 might be an integral component of proliferation and migration processes in prostate, hepatocellular and breast cancer cells. Our data with PC3, HepG2, and MDA-MB-231 cells with CRISPR-Cas9-mediated CDK5 knockout strongly confirmed that CDK5 affects the cellular proliferation and migration in line with previous reports using CDK5 knockdown by siRNA or inhibition by roscovitine ,56. The All biochemicals and enzymes were of analytical grade and purchased from commercial suppliers. Active recombinant GST-tagged full-length human CDK5/p35 was purchased from SignalChem . The generation of plasmids allowing bacterial expression of GST-tagged full-length human USF2 and USF2 deletion fusion proteins in pGEX-5X-1 , as well as the plasmid pcDNA6A-Gal4-USF2, have been already described . The expPC3, HepG2, and MDA-MB-231 cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM , supplemented with 10% fetal bovine serum , 1% non-essential amino acids (Sigma-Aldrich), and 0.5% ciprofloxacin , at 37 °C in an atmosphere of 16% O2, 5% CO2, 79% N2 and 97% humidity. Transient transfections and luciferase assays were performed as described .Isolation of total RNA was performed using the Qiagen RNeasy® Mini Kit  following the manufacturer’s instructions. One μg of total RNA was used for cDNA synthesis with the iScript™ cDNA synthesis Kit . Quantitative real-time PCR was performed in an Applied Biosystems 7500 Real-Time PCR System  by using an Applied Biosystem Power SYBR® green PCR master mix (Life Technologies). The following primers were used: USF2 forward 5′-GCGTTCGGCGACCACAATA-3′, USF2 reverse 5′-GACTACGCGGTATGTCACCT-3′, ActB forward 5′-GTTGTCGACGACGAGCG-3′, ActB reverse 5′-GCACAGAGCCTCGCCTT-3′, Hprt forward 5′-CCTGGCGTCGTGATTAGTGAT-3′, Hprt reverse 5′-AGACGTTCAGTCCTGTCCATAA-3′. All primer sets used were validated for their product and amplification efficiency using standard dilution analysis and melting curve analysis. β-Actin (ActB) and hypoxanthine-guanine phosphoribosyltransferase (Hprt) were used as internal controls to normalize the variability in expression levels. The experiments for each data point were carried out in triplicate. The relative quantification of gene expression was determined using the ΔΔCt method ,59.www.genome-engineering.org); a non-targeting, scrambled sequence 5′-GCACTACCAGAGCTAACTCA-3′ (OriGene) was used as a negative control. gRNA-encoding oligonucleotides (Sigma-Aldrich) were cloned into the vector SpCas9(BB)-2A-GFP  using standard procedures as described [A 20-bp guide sequence targeting the second exon of human CDK5  with the sequence 5′-CCGGGAGACTCATGAGATCG-3′ was designed online using Zhang’s laboratory web resource , incubated for 20 min on a lab-roller at 4 °C and then centrifuged at 13,000× In half-life studies, 10 µg/mL cycloheximide  was added to the cell culture medium of WT and ΔCDK5 cells for the indicated periods. After harvesting the cells, endogenous USF2 protein levels were measured by western blot analyses and quantified with Fiji (NCBI) software.For immunohistochemistry, cells were placed on coverslips and fixed by ice-cold methanol/acetone (1:1) for 10 min, blocked with 3% BSA in PBS, and incubated with an anti-USF2 mAb  at room temperature for 1 hr. Subsequently, cells were washed in PBS three times for 10 min before incubation with FITC-conjugated goat anti-rabbit IgG secondary Ab . Thereafter, cells were washed three times again with PBS for 10 min. DAPI (1 µg/mL) was added in the final wash. For immunoprecipitation experiments, protein samples from WT and ΔCDK5 PC3 cells were prepared as already described  and subjFor the analysis of proteins by western blotting, cell extracts were denaturated with a sample buffer  by incubating at 95 °C for 10 min. Then, the proteins were resolved at SDS-PAGE and blotted to a nitrocellulose membrane. As primary antibodies polyclonal antibodies against CDK5 , USF-2 , phospho-serine , HA-tag  as well as monoclonal antibodies against phospho-threonine , V5 tag , and α-tubulin  were used. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgGs  were used. The enhanced chemiluminescence (ECL) system  was used for detection.E.coli BL21 (DE3) at 30 °C and purified with glutathione sepharose beads as described previously [GST-tagged full-length human USF2, USF2 deletion, and USF2 double point mutation fusion proteins were expressed in eviously . Radioaceviously . In brie24 h post-transfection, the lentivirus containing medium was collected and filtered. Prior infection of cells using a single infection with 6 mL virus, polybrene was added (1 µg / mL). Infected cells were subjected to selection with 0.5 μg/mL blasticidin for 2–3 weeks before harvesting and analyses. The overexpression of USF2 was verified by western blot analysis. Proliferation assays were performed by counting viable cells using a hemocytometer as well as by measuring BrdU incorporation. For counting, cells were seeded in triplicate in 6-well plates at a density of 30,000 cells/well. Cells were trypsinized and counted each day for 4 days at the same chronological time (as indicated in the results section). The BrdU assay  was performed according to the manufacturer’s instructions and the absorbance was measured at dual wavelengths of 450–540 nm.Cell migration was measured by using medium-treated 24-well Transwell chambers  with 8.0 µm polycarbonate membranes. The bottom chamber was filled with 600 µL medium containing 10% fetal calf serum; cells were seeded into the top chamber at a density of 1 × 104 cells per well in 100 µL serum-free medium. After incubation in a humidified incubator with 5% CO2 at 37 °C for 4 h, cells were fixed with 4% paraformaldehyde and the non-migratory cells were scraped off from the top of the Transwell with a cotton swab. The migrated cells attached to the bottom chamber were stained with 0.0075% crystal violet. Crystal violet absorption at 595 nm was measured in a Tecan Infinite® M1000Pro microplate reader. For each migratory condition, two identical replicates were performed.Cell apoptosis was measured by Annexin-V-FLOUS staining kit  according to the manufacturer‘s protocol. Apoptotic cells were analyzed by flow cytometry (BD FACSAria™ III cell sorter).p < 0.05 to be significant.Each experiment was performed at least three times and representative data are shown. Data in bar graphs are given as mean values ± standard deviation (SD). Statistical differences were calculated by using the Student t-test with error probabilities of The findings described in this study demonstrate the important role for CDK5-mediated phosphorylation of USF2 and provided a mechanism by which USF2 protein levels can be regulated in cells.


Piezoelectric materials based on lead zirconate titanate are widely used in sensors and actuators. However, their application is limited because of high processing temperature, brittleness, lack of conformal deposition and, more importantly, intrinsic incompatibility with biological environments. Recent studies on bioorganic piezoelectrics have demonstrated their potential in these applications, essentially due to using the same building blocks as those used by nature. In this work, we used piezoresponse force microscopy (PFM) to study the domain structures and polarization reversal in the smallest amino acid glycine, which recently attracted a lot of attention due to its strong shear piezoelectric activity. In this uniaxial ferroelectric, a diverse domain structure that includes both 180° and charged domain walls was observed, as well as domain wall kinks related to peculiar growth and crystallographic structure of this material. Local polarization switching was studied by applying a bias voltage to the PFM tip, and the possibility to control the resulting domain structure was demonstrated. This study has shown that the as-grown domain structure and changes in the electric field in glycine are qualitatively similar to those found in the uniaxial inorganic ferroelectrics. Currently, inorganic ferroelectrics such as lead zirconate titanate (PZT), lithium niobate (LNO) and barium titanate (BTO) are widely used as efficient piezoelectrics, pyroelectrics, memory cells, and electrooptic modulators . They poIn this context, piezoelectric or pyroelectric materials made from the same building blocks as those used by nature  may be used, e.g., for bone repair or axonal regeneration after nerve injury. Until recently, biomolecular piezoelectrics had properties which were by far inferior to their inorganic counterparts, thus making their applications unfeasible. Recent studies have revealed that the softness of hydrogen bonds in some representative classes of biomolecular polar materials may be the origin of strong piezoelectric and pyroelectric effects at room temperature. For example, piezoelectric coefficients in self-assembled short peptides ) are practically the same as in the popular transducer material LNO .2 . Th. Th25]. t = 15, 30, and 60 s) with the probe withdrawal, wedge-shaped domains with a length above 7 μm were formed  with probe withdrawal, short (up to 2.5 μm) needle domains grew in the same direction as after the local polarization reversal by a positive pulse  with probe withdrawal, a flat charged tail-to-tail wall was formed at the probe location due to the displacement of the existing domain wall  with probe withdrawal, a zigzag-shaped (head-to-head) wall  wall e formed ad) wall c,d. Switt = 30 s) without probe withdrawal partial or full backswitching and formation of reverse domains were observed (After the local polarization reversal by a pulse of positive U > 0 and negative voltage U < 0 (U = ±100 V; observed .a was 15 nm, and about 200 nm for the head-to-head walls. This also confirms the earlier assumption that the charged tail-to-tail walls are conductive, which accelerates the bulk screening of the depolarizing fields created by the bound charges, and that the head-to-head walls are not conductive, which leads to a change in the shape of the domain wall to reduce the density of bound charges.The two types of charged domain walls written by the probe differed in morphology. The tail-to-tail walls were straight and smooth, while the head-to-head walls were jagged and zigzag-shaped . To quanA significant backswitching effect was demonstrated: The domains obtained by local polarization reversal slowly became short and narrow. It is shown that the backswitching was carried out by the motion of macrosteps along the neutral domain walls . BackswiA detailed study of the as-grown domain structure in β-glycine allowed us to identify three domain types with charged domain walls, the formation of which is attributed to the influence of growth layers and pyroelectric fields. The formation of nanodomains, observed in β-glycine for the first time, is attributed to the cyclic motion of the charged macrosteps caused by the influence of the alternating pyroelectric field that appeared during the heating–cooling cycle. The performed measurements showed that the domain structure changes with slight heating and/or cooling. The observed significant unipolarity of the local polarization reversal in β-glycine was attributed to the existence of an internal bias field. The effects of spontaneous backswitching after external field removal, and “anomalous” growth of the domains with polarization direction that did not coincide with the direction of the applied field, were detected. For the first time in organic ferroelectrics, we show that the formation in-field domains grown on nonpolar cuts, as well as their reduction after the field removal occur by the motion of kinks in the polar direction and the formation of macrosteps. It was shown that as-grown domain structure and its change under electric field in β-glycine are qualitatively similar to those found in uniaxial inorganic ferroelectrics. The control of the domain structure by an external local electric field provided by the PFM tip was demonstrated.


Puroindoline a (Pina) and characterized PINA’s effects on kernel hardness, total flour yield and dough mixing properties in durum wheat. The medium-hard kernel texture is potentially useful for exploring culinary applications of durum wheat. In the present study, we examined the milling parameters and flour attributes of the transgenic lines, including particle size distribution, damaged starch and water binding capacity. PINA expression results in increased break and reduction flour yield but decreased shorts. PINA expression also leads to finer flour particles and decreased starch damage. Interestingly, PINA transgenic lines showed increased peak viscosity and breakdown viscosity but leave other flour pasting parameters generally unaltered. PINA transgenic lines were associated with increased small monomeric proteins, appearing to affect gluten aggregation. Our data together with several previous results highlight distinct effects of PINs on pasting properties depending on species and variety. The medium-hard kernel texture together with improved pasting parameters may be valuable for producing a broader range of end-products from durum wheat.Durum wheat has limited culinary utilizations partly due to its extremely hard kernel texture. Previously, we developed transgenic durum wheat lines with expression of the wildtype  Triticum aestivum L.; 2n = 42, AABBDD) and ∼7% by allotetraploidy durum wheat  locus, which is located at the distal end of chromosome 5DS and harbors two causal genes for kernel hardness, Puroindoline a and b , while specific Pina and/or Pinb alleles of deletions or mutations result in hard kernel  is due to lack of the D genome and the hardness , whereas the starch-bound PINA levels were slightly lower than that of CS possibly due to lack of PINB. Due to its higher expression level of Pina, we designated the transgenic lines as Pina-overexpressing lines in order to be coherent with previous studies. The flour quality and dough mixing properties have been studied in Pina-overexpressing lines and their progeny crossed with 1Ax1-expressing lines in durum wheat. PINA expression in durum wheat results in medium-hard kernel, increased flour yield, decreased dough resistance of extension, and minor effects on dough strength  depends on species or varieties. However, it needs to emphasize that durum wheat is a different genetic material compared to common wheat because it lacks the D genome and several QTLs of pasting properties  and amylopectin (AP), that vary in structure. The effects of AMY/AP on pasting properties of wheat flour have been illustrated via genetic or transgenic materials . Previous reside . The eff to characterize pasting properties in Pina were generated by particle bombardment with plasmids pUbi-pinA and pCa-neo using cultivar Luna as donor variety (Pina-D1a gene (cloned from Chinese Spring) driven by the maize Ubiquitin promoter, while plasmid pCa-neo contains the selection marker neomycin phosphotransferase II gene (npt II) driven by cauliflower mosaic virus 35S (CaMV 35S) promoter. The presence, expression of the transgenes Pina and nptII and the selection of Pina non-segregating lines from independent transgenic events were described elsewhere  and the null-segregant line N-2 were used in the present study. Protein levels of PINA have been characterized in our previous paper  compared to CS due to lack of PINB. The T7 generation of the transgenic lines was grown in a randomized complete block design with duplicates in Wuhan . The agronomic performance, molecular characterization, grain texture and other kernel characteristics of the transgenic and control lines have been reported  4100  on samples of 300 seeds harvested from each plot. After tempered to 16% (w/w) moisture content for 24 h (including 1 h of agitation), seed samples per genotype and plot were milled with a Chopin CD1 mill. The milling fractions were first sieved by an 800-μm sieve followed by a 160-μm sieve after milling. The milling fraction held by the 800-μm sieve was called large brans, while the milling fraction that can pass the 800-μm sieve but not the 160-μm sieve was middlings. The milling fraction passed both sieves was break flour. The middlings were milled once again followed by passing the two sieves. After the second milling and sieving process, the milling fraction that passed both sieves was reduction flour, while the fraction hold by the 160-μm sieve were shorts. Straight-grade flour yield is calculated from the total amounts of break and reduction flour divided by amounts of all millstream . Flour characteristics were obtained by near-infrared reflectance spectroscopy.Seed protein was extracted from wheat flour first by 50% propan-1-ol (v/v) extraction according to Wet gluten was washed from straight-grade flour by using a Perten 2200 Glutomatic System , following by the AACC 38-12-02 method . Both thStarch damage was determined in duplicate by the amperometric method with SDmatic from Chopin Technologies . Straighg for 10 min. The supernatant was discarded, and the retained residue was weighed and expressed as the weight of flour plus the water absorbed  with a polydisperse analysis mode and a 300 mm lens. Dry samples were dispersed in isopropyl alcohol in the circulation unit to attain an obscuration of 15–20%. The sample was first mechanically agitated to be circulated in the equipment, ultrasound for 1 min to dissolve flour clots, and then measured with 2 min interval. Size distribution was determined from duplicated samples with four measurements per sample.The pasting properties were determined using straight-grade flour with a Rapid Visco Analyser  containing 0.5% (w/v) SDS. Then the mixed system was sonicated for 15 s and centrifuged for 10 min at 13,000 p < 0.05).Data were analyzed using one-way analysis of variance (ANOVA) and significant differences between lines were determined using the least significant difference pairwise comparison and displayed by letters  and a null-segregant line (N-2) had been generated previously, with the expression of PINA and kernel hardness phenotypes characterized (Pina expression led to a medium-hard kernel texture (HI ranged from ∼56 to ∼64), while the null-segregant line and non-transgenic Luna had extremely hard kernel (HI > 95) .Three cterized . PINA ovcterized . Here, wHI > 95) . Consistcv. Luna . This recv. Luna . Howeverng lines . In the Pina-overexpressing and control lines. Pina-expressing lines had smaller particle size compared to those of the control lines , the higher breakdown viscosity values in Pina-overexpressing lines could be explained by their higher peak viscosity compared to the control lines , are located in this locus and Pins play major roles in kernel hardness and Gsp-1 has minor effects (Pina. Nevertheless, it is time to unleash the kernel-hardness limitation to expand the utilization of durum wheat.Recently, soft-kernel texture was first achieved in durum wheat cultivar Svevo and then was introgressed into a series of durum varieties. Systematic investigations on these soft-durum wheat lines show several traits improvement compared to traditional durum wheat, including soft kernel texture, less damaged starch, smaller flour particle size. The transgenic lines described here have several advantages. First, they can achieve a medium-hard kernel texture through PINA expression, together with intermediate levels of starch damage, flour particle size and distinct millstream patterns compared with soft- and extremely hard- durum wheat. These intermediate phenotypes may not be fulfilled by recombination of the  effects . The tra effects . Third, The soft-durum wheat has been exemplified as a paradigm shift in terms of the grain and food processing qualities of traditional durum wheat cultivars ,b. In paYL, GY, and GH designed the experiments. QW, YL, FS, XL, and PW performed the experiments. QW and YL analyzed the data. QW, YL, and GH wrote the manuscript. GY edited the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium bovis isolates since 2000 and whole genome sequencing was implemented in 2013. The program has been highly successful, yet as bTB prevalence has reached historic lows, a small number of new bTB-affected cattle herds occur annually. Therefore, understanding the epidemiology of bTB transmission is critically important, in order to target limited resources for surveillance and achieve eradication. This evaluation described the diversity and epidemiology of M. bovis isolates identified in the USA livestock. Isolates from animals within the bTB endemic area of Michigan were excluded. Broad diversity was found among 1,248 isolates, collected from affected cattle and farmed cervids herds and fed cattle during 1989–2018. Nearly 70% of isolates from 109 herds/cases during 1999–2018 were European clonal complex 1 and 30% were European clonal complex 2. The sources of infection based on the herd investigation were known for 41% of herds/cases and 59% were not epidemiologically linked to another USA origin herd. Whole genome sequencing results were consistent with the investigation findings and previously unrecognized links between herds and cases were disclosed. For herds/cases with an unknown source of infection, WGS results suggested several possible sources, including undocumented cattle movement, imported cattle and humans. The use of WGS in new cases has reduced the time and costs associated with epidemiological investigations. Within herd SNP diversity was evaluated by examining 18 herds with 10 or more isolates sequenced. Forty percent of isolates had not diverged or accumulated any SNPs, and 86% of the isolates had accumulated 3 or fewer SNPs. The results of WGS does not support a bTB reservoir in USA cattle. The bTB eradication program appears to be highly effective as the majority of herds/cases in the USA are unique strains with limited herd to herd transmission.The United States official bovine tuberculosis (bTB) eradication program has utilized genotyping for  Mycobacterium bovis (bTB) has a broad host range, causing economic loss to beef and dairy production and infecting humans and wildlife. Therefore, most developed countries and many developing countries have national bovine tuberculosis eradication programs in livestock. The United States (USA) began a national eradication program for M. bovis infection in cattle in 1917. At the program's inception, the apparent prevalence of bTB was 5% of cattle, as estimated by positive responses to the caudal fold tuberculin skin test (CFT) (st (CFT) , 3.M. bovis, in an estimated cattle population of 87 million head on 913,000 operations , Ravenel (Biosample: SAMN04448492), BCG (Biosample: SAMN06847294), AN5 (Biosample: SAMN04448491), and 94-1MIDNRdeerAlp (Biosample: SAMN04386752) (the index Michigan deer isolate). See Supplemental File Because all official bTB eradication program laboratory diagnostics were performed at the United States Department of Agriculture (USDA), National Veterinary Services Laboratories (NVSL), all isolates archived and maintained at that facility up to May 2018 were sequenced. Prior to 2000, there were no procedures in place to permanently archive isolates, consequently a limited number of isolates prior to 2000 were available. After 2000, nearly all Official program activities include ongoing slaughter surveillance, live animal testing, and investigations of bTB-affected herds. These activities are described in the USDA, Bovine Tuberculosis Eradication, Uniform Methods and Rules , and areWhen bTB is suspected, an official investigation is conducted by state and federal animal health regulatory officials . This inAdult cattle were defined as sexually intact animals >2 years of age, whereas fed cattle are defined as castrated or spayed animals without regard to age that are raised for the purpose of beef production. Another type of cattle are those animals used for roping and other performance events. Castration status has precedence over age, for example, a 4-year-old castrated steer is classified as a fed animal. Slaughter surveillance targets culled adult cattle because these are more likely to exhibit lesions suspicious of bTB . bTB-affM. bovis was either a histologic diagnosis of compatible for mycobacteriosis with a positive polymerase chain reaction (PCR) test performed on formalin fixed tissue using primers for IS6110 to identify M. tuberculosis complex, or bacteriological isolation of M. bovis. Affected herds are confirmed when an animal from within the herd is confirmed with bTB. When this criterion cannot be met, the singleton animal  was defined as a case. The source of infection for affected herds was based on the results of epidemiological investigations as being either unknown, or another USA herd. The latter classification was applied when there was documented animal movement or the potential for fence line or other direct contact. Outbreaks were defined as two or more bTB-affected herds or cases with a documented exposure, such as animal movement between premises.The case definition used for classifying an animal as confirmed infected with To identify the likely source,  we conservatively estimated that a USA origin strain would not be tlikely to be exported and established in another country after the USA's national bTB prevalence was below 0.5%, which occurred around 1960. Consequently herds that could have shared a common ancestor within the last 60 years would more likely be internal transmission rather than importation. Using the average reported SNP mutation rate of 0.3 SNP/year , suggestDuring slaughter inspection or when bTB test positive animals are euthanized and necropsied, granulomatous-appearing lesions are collected and submitted to the laboratory for histologic examination, PCR testing and mycobacterial culture . For somhttps://github.com/USDA-VS). Reads were aligned to the reference genome AF2122/97, NCBI accession number NC_002945  using 2 × 250 paired-end chemistry and the Nextera XT library preparation kit . FASTQ files were put through the NVSL in-house pipeline  in the herd. Then, the most closely related isolate was selected from the NVSL WGS database, based on having the fewest number of single nucleotide polymorphisms (SNP) differences, when compared to the herd or case isolate. A pairwise comparison of SNPs to the MRCA was recorded. For herds with M. bovis isolates from multiple bTB confirmed infected animals, the tip with the shortest branch length to the tree root was used for the comparison. The most closely related isolates were grouped based on information about the animal or human from which the isolate was obtained. For example, whether the isolate was from imported cattle or a confirmed bTB-affected herd in the USA.We examined WGS results for M. bovis isolates separated into 24 major phylogenetic groups . There were four M. caprae isolates from three non-human primates and one rhinoceros residing in zoological collections.Sequencing was performed on 1,248 isolates, this included 154 that were published previously . Of thesThere were 83 bTB-affected herds, and 26 cases in infected adult domestic cattle during 1999–2018  of herds/cases  and 64 were not epidemiologically linked to another USA origin herd. There was only one documented case of transmission from Mexico, in a roping steer newly imported from Mexico that was found to be infected during a herd test for interstate movement. These 45 herds with a known infection source were associated with 14 outbreaks. Eight outbreaks involved two herds each, two outbreaks involved three herds, one outbreak involved five herds , one invThe source of infection could not be determined epidemiologically for 64 herds/cases.The most recent common ancestor was within 20 SNPs of a Mexican origin animal for 22 (34.9%) of USA herds/cases suggesting Mexico may be the source  were represented in fed cattle cases but not bTB-affected herds and cases, and one group was found in a farmed cervid herd but not in fed cattle (group 8). The five largest groups are 7, 13, 16, 17, and 23, and contain 72.3% of isolates from fed cattle. In comparison, the five largest groups are 6, 7, 14, 17, and 23, containing 65.4% of affected herds and cases during 1989–2018.A comparison of spoligotyping and whole genome sequencing results are shown in Figure Consistent with previous reports, broad diversity exists among USA bTB isolates detected in cattle and farmed cervids . Not uneIn all cases, WGS results corroborated investigative evidence of herd-to-herd transmission events. Not unexpectedly, there was less SNP diversity between epidemiologically linked herds, almost half these herds had no unique SNPs and the maximum number of unique SNPs was four (Supplemental Table M. bovis occurred because of independent introductions from sources outside the system (WGS results also discovered previously unrecognized links between herds and cases. Isolates from 12 herds/cases with an unknown source of infection were within three SNPs of other USA herds/cases during 2009–2018, including six herds/cases clustering within the group 14 outbreak. In another example, the isolate from a 2010 bTB-infected Holstein cow from an Ohio dairy had one additional SNP from a single isolate obtained from a 2008 New Mexico dairy cow (Supplemental Table Most of the isolates recovered from imported fed cattle are not closely related to isolates from USA herds, with only one of the 109 herds/cases within 5 SNPs of an imported fed steer, despite having nearly complete representation of fed cattle and affected herds during 1999–2018. This suggests there may be other vectors transmitting bTB to the USA national herd such as humans or even imported dairy products , 30. It The most closely related isolates to some affected herds are from fed cattle that occurred earlier in time than the affected herds, many prior to 1999. We hypothesized that if imported cattle were the source of infection for affected herds, we would find closely related isolates among imported fed cattle and affected herds/cases during 1999–2018. That pre-1999 fed cattle cases are the closest match to some post-1999 affected herds/cases is noteworthy, because the pre-1999 isolates from fed cattle represent only a small fraction of the cases that occurred . There wLimited diversity occurred within most herds, with the majority of isolates having three or fewer SNPs. This may be useful in guiding epidemiologic investigations. For example, additional unique SNPs may indicate that an intermediary herd exists. The estimated SNP occurrence per genome per year ranges from 0.147  to 0.53 The use of WGS in new cases has focused epidemiological investigations and significantly reduced time and costs associated with these investigations and reduced the burden on livestock producers. Whole genome sequencing results for a 2013 case in a California Holstein heifer without animal identification enabled resources to focus on testing an epidemiologically linked herd (Supplemental Table bTB prevalence in dairies is approximately twice that of beef herds . The risM. bovis isolates from Baja California, Mexico found that 155 isolates from cattle and 17 from humans clustered into seven major groups (M. bovis for humans. In these examples from the USA and Mexico, direct or indirect contact between the human and animal subjects occurred; however, as noted previously, closely related isolates should not be used to imply transmission events, in the absence of epidemiologic linkages.Transmission from human workers to cattle has been hypothesized, especially for dairy cattle operations because of intensive management practices . In 2013r groups . The humAll isolates were sequenced for some herds, particularly those with a small number of bTB infected animals, but because of resource limitations, a smaller proportion of available isolates were sequenced for larger herds with dozens or hundreds of bTB infected animals. This limitation may bias these results by potentially under reporting the number of SNPs in herds with large numbers of infected animals, or conversely, fail to document if limited SNPs are present despite extensive within herd transmission.Whole genome sequencing has become a cost effective, essential tool for the USA official bTB eradication program, providing information that increases the success and efficiency of the extensive investigations that occur when bTB is confirmed in cattle and farmed cervids. The isolates that occur among USA livestock are diverse, and the lack of diversity within herds support that a reservoir does not exist in the USA cattle population, although transmission of one strain has continued to occur from farmed cervids to cattle herds. The source of infection is unknown for approximately half of bTB-affected herds, and WGS results suggest several possible sources, including undocumented cattle movement, imported cattle, as well as humans or unpasteurized dairy products.SR-A and TS performed WGS and provided summarized results. KO and SR-A compiled and analyzed the summarized WGS results. KO and SR-A wrote the paper. BH and MS provided epidemiological information. BH and MS reviewed/edited the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AD and the handling Editor declared their shared affiliation.