Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the resting conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two similar P domain conformations were also found simultaneously in the human norovirus GII.3 capsid, although the mechanism of the conformational change is not yet clear. These results provide new insights into the mechanisms of non-enveloped norovirus transmission that invades host cells, replicates, and sometimes escapes the hosts immune system, through dramatic environmental changes in the gastrointestinal tract. The capsid structure of caliciviruses has been reported to be classified into two different types, according to the species and genotype. One is the rising type of P domain conformation as shown in human norovirus GII.10 and rabbit hemorrhagic disease virus (RHDV), where the P domain rises from the S domain surface. The other is the resting type of P domain conformation as shown in human norovirus GI.1, sapovirus and San Miguel sea lion virus (SMSV), where the P domain rests upon the S domain. Here, we demonstrate that the P domain of the murine noroviruses changes reversibly between the rising and resting P domain conformation types in response to aqueous conditions. We also found the similar two P domain conformations in human norovirus GII.3 VLPs at the same time. Our findings provide new insights into the mechanisms of viral infection of caliciviruses. Human norovirus (HNoV) is a major cause of epidemic nonbacterial gastroenteritis worldwide . HNoV ofCaliciviridae family  A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Ralf BartenschlagerGuest EditorPLOS PathogensGuangxiang LuoSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The manuscript “Dynamic rotation of the protruding domain enhances the infectivity of norovirus” by Song et al. is generally well written and of high interest to the readers of Plos Pathogens. It provides a link between two previously noted conformations in noroviruses and clearly links the resting conformation to higher infectivity. It also clearly shows that metal binding is beneficial to infectivity. While of broad interest, the manuscript requires a major revision including discussion of results by some other groups prior to publication.Reviewer #2: Here Song, Takai-Tokada et al provide 8 cryo-EM reconstructions (7 at middle to low resolution) of norovirus capsids : 4 of infectious murine norovirus for two strains (MNV-1 and MNV-S7) in 2 conformations each, 2 of a human norovirus (genotype II.3) VLP again in 2 conformations, 1 of MNV-S7 VLP in complex with the soluble part of the murine norovovirus receptor CD300lf, and finally a high resolution (3.5 A) VLP of MNV-S7. There are now numerous structures of norovirus capsids and several have been solved to high enough resolution that atomic models could be built: One X-ray structure for a human GI.1 VLP  and several cryo-EM structures in recent years . Previously, depending on the strain, the spike domains were found in two distinct conformations  and 'resting' (Re)). It has long been suspected in the field that all norovirus capsids can actually assume both conformations (e.g. indirect evidence from the T.J. Smith and G. Hansman labs). Here Song et al. establish this fact for MNV-1 and MNV-S7 particles and for GII.3 VLP . This, and the triggers used by Song et al., are major findings and should be better emphasized in the paper. In general, the paper should be carefully rewritten following the suggestions below.Reviewer #3: This paper by Murata, Katayama, and coworkers describes Cryo-EM based analysis of MNV capsids at different pH values and in the presence and absence of bivalent metal ions. The experimental data appear to be sound and the presentation of the results is adequate. The study has led to interesting new results that further our understanding of the MNV infection process. Essentially, the authors demonstrate that the „rising“ and „resting“ conformations of the P-domain can be switched by altering pH and the concentration of bivalent metal ions. They hypothesize that this conformational mimicry is linked to disassembly or assembly of virus capsids as an essential step during the infection process. Rising and resting conformations of Calicivirus capsids, including Norovirus, have been described before, but this is the first time that interconversion between the conformational states has been observed as being a direct result of a changing chemical environment. Importantly, the study links these structural features to binding and infection assays. As a major result, the „resting“ conformation apparently binds better to target cells and leads to more efficient infection. The topics addressed certainly match the audience of PLOS Pathogens very well. Therefore, I definitely support publication.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: Major:https://www.biorxiv.org/content/10.1101/693143v1), 5 months before this study appeared on biorxiv. Here, also elevated P domains were observed in MNV, which rotated compared to the resting state. The study used heat treatment instead of pH. The results should be linked to and discussed with those presented here. In light of these results, l. 73/74 require rephrasing.- On July 04 2019 Snowden,..., Stonehouse published a study on biorxiv . This was shown to be influenced by slight sequence variations as observed here for the two MNV strains (p. 10). This should be mentioned.- Jung et al. 2019 also observed rising and resting conformations in huNoV-like particles. Even more relevant to the here presented study Koromyslova et al. observed in 2015 structural alterations in presence of citrate (Treatment of norovirus particles with citrate), which resemble a lot the micrographs of the rising domain in Fig. S1. Do the authors consider these structures similar? It would present alternative strategies to achieve the rising conformation in huNoVs, which are not known to bind cations. What is the fraction of broken/disassembled particles observed?- The authors only touch briefly on VP2 and genome. It should be stated more clearly if in any of the conformations of MNV-1 or MNV-S7 virions extra density was observed in asymmetrical reconstructions that could be attributed to VP2 or genome-protein contacts.- DMEM is rather complex compared to PBS. It would be important to also show particles in PBS +/- EDTA at pH 8 and also at lower pH to more clearly pinpoint the effect to pH.- The structural conversion is super slow with 2-8 h and also the mixed species that are observed are super-interesting. Have the authors a theory for the slow kinetics? Could the P domain connections stabilize a given conformation causing hysteresis? While these aspects are discussed, they warrant a more thorough reflection in terms of potential mechanisms, timeline of infection . I hence recommend extending the discussion of these aspects.- What was the reasoning to use such closely related MNV strains? A more distant MNV would have been of higher interest to establish a general relationship between rising/resting conformation and infection.- 40-50 nm VLP of MNV-1 suggests assembly defects or formation of T=4 particles. Those have been observed in huNoVs previously, e.g. in GII.4 strains by Devant et al 2019. While the larger particles can be the result of suboptimal production conditions, it would be of interest to show those micrographs and explain what the larger particles are.- Upon lifting and rotation, are there any structural changes within the P domain backbone? This should be mentioned explicitly.- Have the authors an idea why CD300lf binds so poorly? Have they tested whether bile acids or glycans enhance the binding of the protein receptor? This could be discussed in a bit more detail. How would the reconstruction look without imposing symmetry? Maybe the receptor would be less blurred out?- Fig. S9C: electron density seems to indicate a subunit interaction that is not well reflected in the model, i.e. Ser-Gln maybe positioned lower or are in fact other residues interacting? The two lower interactions lack density entirely. How certain are these?- How different are the positions of the rising P domain from each other in the three different strains/particles? An overlay would be useful.As side note, with the wealth of supplementary data and the caption being given after the references, it was difficult to review the manuscript. I would have appreciated two files and captions closer to the figures.Reviewer #2: (No Response)Reviewer #3: None**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: Minor:- last sentence of abstract is difficult to understand and requires rephrasing- l. 221 vs. l. 227 appear contradictory. The fine structure discussion is not fully clear from the phrasing.- l. 500 A/B dimer not diner- Fig. S1 caption says PBS(-)-EDTA whereas figure says +EDTA. The latter makes more sense to the referee. The authors should check whether this has been correctly stated throughout text.- Fig. S6: comparing MNV sequences to huNoV GII.3 would be more useful to estimate how different these are. What is the rationale for showing the Norwalk sequence?- Fig. S10: please also label the other residue present in B and C. Is it an Arg and how far is its functional group away from the putative Ca2+? There is an additional residue, presumably from the helix, overlaying with Asp 231. If this is not close to the metal binding, consider taking this out to increase clarity of the view.Reviewer #2: 1) I suggest merging figures 1 and 5 so the reader grasps at once that the two conformations are found in both MNV-1 (for which only Ri was reported so far) and GII.3 VLPs.2) Count of particles in Ri and Re conformations: This is another important piece of evidence. Both conformations are found on the same grids and depending on buffer conditions their relative abundance changes . Furthermore a time course of going from Ri to Re is actually given for MNV-1, with indication that mixed particles  can be detected .a) Most important: How are the particles counted ? I could not find that information in the paper. I am guessing from e.g. S3 Fig. that during image processing 2D classes are used to assign particles to Ri, Re or mixed. Or was this done by eye on random subsets of extracted particles ? If so, which set of particles was used to draw the subsets ? Please give a clear and detailed description of the process.b) On a related note, very few particles per image are actually used in the 3D reconstructions. For instancel. 448 'For MNV-1 infectious particles suspended in DMEM and PBS(-)-EDTA (pH 8.0), 6,708 and 4,704 particles were selected from 1,046 and 2,188 images, respectively'.Yet e.g. the inserts from micrographs in S1 Fig. show 6-12 seemingly good particles, so most are rejected during image processing. Please give numbers: How many 'not junk' particles (seemingly good particles) don't make it to the final 3D reconstruction ? Discuss in terms of heterogeneity .c) Make a separate, main figure from appendix figures S1 I, S4 K and S3. This could be new Fig. 2.3) l. 345 'HNV GII.3 VLPs also showed that the rising P domain conformation easily collapsed due to surface tension of thin vitreous ice, thus leaving most of the particles with a resting P domain conformation in the thin ice of cryo-EM (S14 Appendix).'l. 896 'The VLPs having the rising P domain conformation easily collapsed due to the surface tension in thin vitreous ice, and only the stable VLPs with the resting P domain conformations were left.'I do not understand the basis of these statements. Do the authors suggest that the spikes collapsed between sample deposition and plunge freezing ? Why ?4) l. 291-314 in the discussion section actually belong in the results.5) l. 276 'metal ions binding to the region between the P and S domains, may help to stabilize the resting277 conformation (S10 Appendix).'The evidence for this is that EDTA-containing buffers seem to favor Ri, while DMEM favors Re. However, pH seems to be the most important factor.a) What is the metal ion content of DMEM ?b) It could be worth mentioning that the GI.1 crystal structure, until very recently the only Re conformation, was obtained at pH4.8.c) l. 279 'we identified density between the two carboxylates (Glu223 and Asp231) in the flexible hinge of the resting P domain conformation, where a metal ion is expected to bind to these residues (S10 Appendix)'. The density shown in S10 Fig is not convincing. At this threshold the map shows other unexplained features, suggesting that it is contoured too low. Please remove this statement.6) The CD300lf data are not convincing.a) l. 875 'rCD300lf is fitted to the extra density using the docking simulation with a parameter set shown in Materials and Methods'. If I understand correctly, the authors perform ab initio docking, find a pose that roughly matches the extra density, and conclude that they have the approximate position of the receptor binding site. However, the extra density is clearly not that of rCD300lf and the docking pose does not match the crystal structure of the complex of the receptor with the spike . This part should be discarded entirely.b) Discussion of the accessibility to rCD300lf in Ri vs Re is relevant here but should simply match the available crystal structure to the Re and Ri reconstructions.c) l. 329 'The resting P domain conformation showed higher accessibility of the cellular receptor CD300lf than the rising P domain conformation, causing greater viral infectivity.' As I remember Kilic et al. did not find any hindrance to binding to the MNV (Ri) conformation. But even then the 'causing greater viral infectivity' would be speculative.7) l. 218 'The flexible N-terminals formed various interactions with each other between adjacent S domains at the twofold, threefold, pseudo-threefold, and fivefold axes (S8 Appendix B-F)'This is a secondary point in the paper, but S8 Fig. is a very nice recap of the N-terminal arm's differing positions in Caliciviridae (same subunit vs domain-swapped). The authors should point out that the 5-fold (molecule A) vs 3-fold  positions of the arms are determinants of quasi-equivalence between pentamers (5 'A' arms) and hexamers (3 'B' arms), with the 'C' arms always more disordered.8) Minor corrections/clarificationsa) l. 148 'homology' shoud be 'sequence identity'b) l. 390-392 'two days after infection. MNV was precipitated using ultracentrifugation with a 30% sucrosecushion''diluted with DMEM without any additional supplements and precipitated again to remove CsCl.'Replace 'precipitated' with 'pelleted'.c) S2 Table define 'Ri' 'Re'd) Fig. 1 '(red arrows in A and C)' ... '(red arrows in B and D)' are almost invisible at printed size e) S2 Fig. '(B) The cells' please indicate 'HEK293T/CD300lf cells'. In general label the figures so that it is immediately clear which of the two cell types was used.f) l. 429 '(C) The early genome replication in each cell incubated for 0, 2, 4, 6, 8, 10, and 12hours post-infection.' Please describe the protocol. I could not find it in the methods. Presumably cells were pelleted and lysed to separate intracellular from extracellular RNA?g) l. 771 'MNV-S7 pretreated with PBS(-)-EDTA (pH 8) produced slower propagation of the virus thanthose pretreated with DMEM, though the difference was small compared to that of MNV-1 .' All points are actually within error bars. The PBS(-)-EDTA (pH 8) curve is systematically below though, so an appropriate test (Mann-Whitney rank test ?) may be helpful here.h) l. 815 'Asn49 and Asp52 of each C monomer form a charged interaction as shown by the dotted lines'. None of the interactions shown are actually charge-charge (at most one charged residue involved). Please change all 'charged' to 'polar' or 'likely hydrogen bond'.Reviewer #3: There are points that should be addressed by the authors before publication. It has been described by others that Ca2+ improves the binding affinity of MNV P-domains for CD300lf by about one order of magnitude, with bile acid, i.e. GCDCA adding another factor of two . The respective crystal structure has well defined binding sites for Ca2+. The authors of that paper discuss the possibility that binding fo MNV to CD300lf is linked to conformational changes of the A’-B’ and the E’-F’ loops, which exist in an open and closed conformation. From this, two questions arise immediately. First, have the present experiments been performed in the absence or in the presence of GCDCA? Second, could the improvement of cell attachment of the resting conformation be the consequence of a combined effect, i.e. „loop conformation“ plus „overall conformation“?There is no information about the concentrations of bivalent metal ions, except that binding behavior changes upon addition of EDTA. It would be very helpful to have that information somewhere in the Materials & Methods section.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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A member of our team will be in touch with a set of requests. In addition, during this process, please take again a close look at your manuscript to remove the typos that are still contained in your manuscript.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.Best regards,Ralf BartenschlagerGuest EditorPLOS PathogensGuangxiang LuoSection EditorPLOS PathogensKasturi HaldarEditor-in-ChiefPLOS Pathogensorcid.org/0000-0001-5065-158XMichael MalimEditor-in-ChiefPLOS Pathogensorcid.org/0000-0002-7699-2064***********************************************************Reviewer Comments :Reviewer's Responses to QuestionsPart I - SummaryPlease use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.Reviewer #1: The authors have satisfactorily addressed my comments in their revision, I hence recommend publication in PLOS Pathogens.As a side note, the revision still contains some typos.**********Part II – Major Issues: Key Experiments Required for Acceptanceabsolutely required to validate study conclusions.Please use this section to detail the key new experiments or modifications of existing experiments that should be Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".Reviewer #1: (No Response)**********Part III – Minor Issues: Editorial and Data Presentation ModificationsPlease use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.Reviewer #1: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: No


Archaea to Eukaryota. An additional N-terminal glycine–arginine-rich (GAR) domain is present in the FBL of eukaryotes. Here, we demonstrate that the GAR domain is involved in FBL functioning and integrates the functions of the nuclear localization signal and the nucleolar localization signal (NoLS). The methylation of the arginine residues in the GAR domain is necessary for nuclear import but decreases the efficiency of nucleolar retention via the NoLS. The presented data indicate that the GAR domain can be considered an evolutionary innovation that integrates several functional activities and thereby adapts FBL to the highly compartmentalized content of the eukaryotic cell.Fibrillarin (FBL) is an essential nucleolar protein that participates in pre-rRNA methylation and processing. The methyltransferase domain of FBL is an example of an extremely well-conserved protein domain in which the amino acid sequence was not substantially modified during the evolution from  Eukaryotic cells contain numerous organelles with or without a membrane(s) that are effectively compartmentalized the cellular processes. Nuclear proteins are translated in the cytoplasm and, therefore, sophisticated mechanisms of nuclear import have evolved. Proteins larger than ~40–60 kDa are selectively transferred by an energy-dependent mechanism that requires additional transport factors, called karyopherins, which recognize the nuclear localization signals (NLSs) of their cargo proteins. Inside nuclei, proteins can accumulate inside membrane-free organelles, which are usually referred to as nuclear bodies. Some nuclear bodies have complex organization. For example, the nucleolus is composed of three subcompartments, namely, the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The nucleolus demonstrates vectoral organization: transcription of pre-rRNA occurs at the border of the FCs and DFC, pre-rRNA processing and pre-ribosome biogenesis occur in the DFC and GC. The origin and evolution of the mechanisms that lead to effective compartmentalization of nuclear proteins  are not obvious. Here, we investigate the possible mechanisms that enabled proteins to adapt to the highly compartmentalized content of eukaryotic cells, focusing on essential nucleolar protein fibrillarin (FBL) as an example.FBL localizes preferentially in the DFC of the nucleolus, where active ribosomal DNA transcription and the early stages of rRNA processing take place . FBL catArchaea to human  that consists of three domains—an N-terminal glycine–arginine-rich (GAR) domain (80 a.a.), an RNA-binding domain (~90 a.a.) and a C-terminal α-helical domain (~33 a.a.) . The RNA Xenopus , Arabidobidopsis  (but notrahymena ). FBLs cnop1 gene) indicated that all major posttranscriptional activities in ribosome synthesis, such as pre-rRNA processing, pre-rRNA modification and ribosome assembly, were dependent on NOP1  (barley stripe mosaic virus (BSMV) Triple Gene Block 1 (TGB1) protein  of s (PSLV) , barley  protein  and p2 ope virus  interactmovement .Archaea homologs of eukaryotic FBL (FBL-like proteins) (The GAR domain is missing in roteins) , indicatfbl gene (Invitrogen) and EGFP-N1 .The following plasmids were used for the plasmid construction and site-directed mutagenesis: TagRFP-FBL , EGFP-NPFor FBL-EGFP, GAR-EGFP and ΔGAR-EGFP plasmid construction, the region encoding human FBL and its domains (GAR and ΔGAR) was amplified from cDNA using the primers presented in For pFBL-LacZ-EGFP plasmid construction, the region encoding human FBL was amplified from cDNA . The PCRTo generate the pLacZ-EGFP plasmid, the region encoding β-galactosidase was amplified by PCR from the pCMV-LacZ plasmid . The resSV40-EGFP plasmid, we designed adapters encoding the NLS from the T antigen of SV40 virus with the necessary restriction sites  according to the manufacturer’s instructions. Some amino acid residues were combined into loci to facilitate mutation . Mutationop1 gene under the control of a regulatable galactose promotor GAL1, we designed primers in which the 5′-ends (~40 nucleotides) were complementary to the desired target gene sequence (NOP1) and the 3′-ends (~20 nucleotides) were annealed to the selected marker gene (His3MX6) and regulatable promotor (pGAL). We used plasmid pFA6a-His3MX6-pGAL1  and mutant forms of FBL, in which all arginine residues in the GAR domain were replaced by alanine residues (R⇒A) or lysine residues (R⇒K). Human FBL cDNA and the mutated forms of FBL were used as templates. The amplified products were purified, digested by BamHI and HindIII and ligated into the p416GPD yeast vector . The resnop1 gene was amplified with PCR using total yeast genomic DNA as a template. The amplified product was purified, digested by BamHI and HindIII and ligated into the p416GPD yeast vector. The resulting plasmid was referred to as pGPD-NOP1. GAL-NOP1 cells were transformed with pGPD-FBL, pGPD-ΔGAR, pGPD-GAR, pGPD-GAA-FBL, pGPD-GAK-FBL or pGPD-NOP1 plasmid; the transformed cells were spread onto the selective medium YNB-Ura, Gal. All newly generated strains were verified by PCR with independently designed primers (The  primers .6 cells/ml). Suspensions of each strain were spotted on YNB-Ura (D) and YNB-Ura  with 10× dilutions and grown for 2–5 days at 23 °C. The number of colony forming units (CFUs) in each spot was counted and the obtained data were statistically analyzed. To set a baseline of 100%, we took the number of CFUs on the YNB-Ura  medium. Yeast survival was evaluated as the ratio of the CFUs on a medium with glucose to the CFUs on a medium with galactose.The obtained yeast strains  were grohmt1 by homologous recombination. The transformed cells were spread on selective medium YPGal + G418. Generated strains were verified by PCR. Verified yeast strain pGAL-NOP1 hmt1Δ was transformed with earlier obtained pGPD-FBL; the transformed cells were spread onto the selective medium YNB-Ura, Gal.Disruption of HMT1 in GAL-NOP1 yeast strain was performed as described by Gueldener with coathors . A PCR sRNA was isolated from yeast cells using the hot formamide extraction method . RNA quaElectrophoresis of PCR products was performed in 1.5% agarose gel. The gels were visualized in ChemiDoc Gel Imaging System .Human carcinoma cells  were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum , 2 mM L-glutamine  and antibiotic-antimycotic solution . To inhibit RNA transcription, the cells were grown in medium with 0.1 μg/ml actinomycin D (ActD). Cellular transfection was performed using the Lipofectamine 2000 reagent  according to the manufacturer’s instructions.2, 1 mM MgCl2 and 20 mM HEPES, pH 7.4) containing 10 mM sodium azide and 6 mM 2-deoxy-D-glucose and incubated for 50 min. Live cell imaging was performed with a Nikon C2 confocal microscope and a 60× Plan-Apo objective (NA 1.4) at 37 °C; focus stabilization was performed using the PFS system .ATP depletion was carried out as described by http://rsbweb.nih.gov/ij/). The mean fluorescence intensity in the nucleolus (Fn), nucleoplasm (Fnuc) and cytoplasm (Fcyt) were estimated to measure the concentration of each fusion protein. The ratios of the nucleoplasmic EGFP concentration to the cytoplasmic EGFP concentration (Fnuc/Fcyt) and the nucleolar EGFP concentration to the nucleoplasmic EGFP concentration (Fn/Fnuc) were used to measure the nuclear and nucleolar accumulation, respectively. Statistical analysis and preparation of the graphs were performed using Prism 6 software .To evaluate the nuclear and nucleolar accumulation of the protein, images of at least 50 living HeLa cells expressing EGFP-fused proteins were taken from two different experiments using a Nikon C2 confocal laser scanning microscope. Cells expressing different EGFP-fused proteins were imaged under identical conditions in all experiments. The regions of interest in the nucleolus, the nucleoplasm and cytoplasm were selected manually and the mean intensity of the fluorescence was measured using ImageJ software  was calculated as RFI = To inhibit methylation, the transfected cells were grown in medium with 2 μg/ml AdOx. The cells were analyzed the next day with a Nikon C1 confocal microscope.HEK 293 cells were cotransfected with the created lentivector construct coding FBL-FLAG and the lentiviral packaging plasmids using Lipofectamine 2000 reagent  according to the manufacturer’s instructions. 48 h after transfection, the supernatant containing the lentiviruses was collected, filtered through a 0.2 µm filter and immediately used to infect recipient HeLa cells. To increase the efficiency of the transduction, Polybrene (8 µg/ml) was added to the medium with pseudoviral particles. After infection, the cells were selected with puromycin (1 µg/ml).3 of the Coomassie-stained gel were destained twice with 40% acetonitrile, 20 mM NH4HCO3 solution, dehydrated with 200 μl of 100% acetonitrile and rehydrated with 5 μl of the 20 mM NH4HCO3 solution (pH 7.5) containing 15 µg/ml enzyme. The proteins were digested either with trypsin for 6 h at 37 °C or with Lys-C for 4 h at 37 °C. The resulting peptides were extracted with 10 μl of 20% acetonitrile, 0.5% TFA solution and subjected to MALDI-MS analysis. Aliquots of 1 μl of the samples were mixed on a steel target with an 0.5 μl of 2,5-dihydroxybenzoic acid  solution . Spectra were recorded on an UltrafleXtreme MALDI-TOF/TOF mass spectrometer  equipped with an Nd laser. For tryptic maps, the monoisotopic MH+ were measured in the reflection mode; the accuracy of mass peak measurement was within 30 ppm. For a number of tryptic fragments, MS/MS analysis was performed. Fragmentation spectra were obtained in the LIFT mode, the accuracy of daughter ion measurement was within 0.5 Da. Spectra of N-terminal peptides after Lys-C proteolysis were obtained in a linear mode; the accuracy of the measured average masses was within 1 Da. Peptide identification was carried out on the combined MS + MS/MS data using the Mascot search service (www.matrixsciene.com) in the SwissProt database. Candidate proteins having a confidence parameter of score >80 were considered certain reliably (p < 0.05).FBL-FLAG was immunoprecipitated using FLAG immunoprecipitation kit . Pieces of about 2 mmXenopus   —under thWhile growing on galactose medium, cells simultaneously expressed both NOP1 and human FBL or its mutants (GAR or ΔGAR) , and theLacZ) gene and EGFP (FBL-LacZ-EGFP with molecular weight of 177.25 kDa). The LacZ protein fuzed with EGFP predominantly localized to the cytoplasm (It was previously suggested that the GAR domain has NLS function . ProteinTo determine which FBL domain contains the NLS(s), we analyzed the localization of FBL-EGFP, ΔGAR-EGFP and GAR-EGFP in living HeLa cells . FBL-EGFSV40-EGFP). To estimate nuclear accumulation, the ratio of nucleoplasmic fluorescence to cytoplasmic fluorescence (Fnuc/Fcyt) was measured. ATP depletion did not affect the localization of EGFP, while a twofold decrease in nuclear accumulation (Fnuc/Fcyt) was observed for NLSSV40-EGFP , and therefore, it can potentially diffuse through nuclear pore complexes. In this situation, nuclear accumulation can be driven either by an active NLS-dependent import mechanism or by nuclear retention via interactions with intranuclear components. NLS-dependent nuclear import is an energy-dependent process , and ATPV40-EGFP . A signiInside nucleoli, both FBL-EGFP and ΔGAR-EGFP were preferentially localized in small dot-like or ring-like intranucleolar structures, which most likely corresponded to DFC . It shouIt seems that the double localization of FBL (in the DFC and the GC) is driven by (i) the MT domain (ΔGAR), which is responsible for the accumulation inside the DFC and (ii) the GAR domain, which is responsible for the accumulation inside the GC. These two types of nucleolar accumulation can be driven by different types of interactions and therefore, we used FRAP for the investigation of FBL binding with the DFC and the GC in the nucleoli. After photobleaching, the fluorescence of FBL-EGFP was rapidly recovered in the GC of the nucleolus, and then it was slowly recovered in the DFC (t1/2) of FBL-EGFP was ∼8 s , indicating a slower exchange of protein between the nucleolus and nucleoplasm compared to that of FBL-EGFP  and found a gradual decrease in the accumulation of the mutant GAR domains with an increase in the number of the substituted arginine residues . The morAccording to published data, arginine residues in the GAR domain undergo posttranslational asymmetric dimethylation . To idenSaccharomyces cerevisiae, NOP1 is methylated by HMT1 (heterogeneous nuclear ribonucleoprotein MT)  . TherefoΔ strain . It shoumt1 gene . This obmt1 gene  and 4C, mt1 gene .Thus, arginine methylation in the GAR domain seems to be involved in FBL functioning.To investigate the role of arginine methylation on the nuclear import of FBL, HeLa cells were treated with the methylation inhibitor AdOx, which inhibits all SAM-dependent enzymatic reactions, including protein arginine methylation .To estimate the efficiency of the AdOx treatment, we used cells stably expressing FBL-FLAG. FBL-FLAG was immunoprecipitated from control cells and cells which were treated with AdOx. The bands containing FBL-FLAG were visible both in Coomassie-stained gels  and withSV40-EGFP (nuc/Fcyt) of GAK-EGFP was lower than that of GAR-EGFP in the AdOx-treated cells, and ATP depletion did not decrease the nuclear accumulation of GAK-EGFP  increased twofold compared to the efficiency of the untreated cells  the presence of a semipermeable barrier between the cytoplasm and the nucleus, which consists of a nuclear envelope with embedded nuclear pore complexes and (ii) the formation of nuclear bodies—membrane-free nuclear structures inside which intranuclear processes are compartmentalized. The data presented here indicate that the MT domain cannot effectively accumulate inside nuclei or correctly localize inside nucleoli. It is likely that the conservativity of the MT domain could not adapt FBL to the eukaryotic cell organization; therefore, an additional GAR domain evolved.The MT domain of FBL is an example of an extremely conserved protein domain, in which the amino acid sequence was not substantially modified during evolution from Here, we demonstrate that the GAR domain is necessary for FBL functioning and, additionally, integrates two activities necessary for proper localization inside eukaryotic cells. It should be noted that functional activity of GAR domain was investigated using yeast complementation assay. In some works this experimental approach was used to demonstrate the conservation of FBL function . Here, wSV40-EGFP decreased to the level of EGFP after inhibition of active nuclear import (ATP depletion), the accumulation of GAR-EGFP was slightly decreased, indicating that only some of the nuclear FBL accumulation relied on energy-dependent nuclear import. The important feature of this energy-dependent accumulation is that the methylation of arginine residues in the GAR domain was necessary.Nuclear accumulation of small proteins (<40–60 kDa), which can diffuse through nuclear pore complexes, can be mediated by either active nuclear import or by interactions with nuclear components (nuclear retention). The presented data indicate that, in the case of FBL, both mechanisms are used. Indeed, if nuclear accumulation of NLSThe additional interaction of GAR with nuclear structures  can lead to energy-independent nuclear retention. The results from the FRAP experiments demonstrate that the retention of the GC was mediated by the extremely dynamic interactions of the GAR domain with GC components. The mechanism of this retention may be similar to those of NoLS-mediated nuclear accumulation. It has been demonstrated that protein accumulation in the GC of the nucleoli can be driven by protein regions enriched with positively charged amino acids (NoLS(s)), which electrostatically interact with nucleolar components . The accS. cerevisiae . If this supposition is correct, then the additional FBL domain contributes to effective functioning not only by accumulating inside nuclei but also by accumulating in the GC of the nucleoli. These activities of the GAR domain may lead to the effective accumulation of dynamic proteins in the limited area of the DFC.Archaea to Eukaryota, FBL-like proteins acquired additional domain that functions as NLSs and NoLSs, leading to the evolution of the eukaryotic FBL. The possible principle of integration of different activities is summarized in Thus, during the transition from Thus, the GAR domain effectively adapted FBL to function inside highly compartmentalized eukaryotic cells and methylation of the GAR domain enables the optimization and integration of the different activities of this protein.10.7717/peerj.9029/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.9029/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.9029/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.9029/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.9029/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.9029/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.9029/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.9029/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.9029/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.9029/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.9029/supp-11Supplemental Information 11Cell viability of the yeasts expressing GPD-FBL, GPD-ΔGAR, GPD-GAR, GPD-NOP1 or the empty vector p416-GPD after growing on YPD plates. Colony forming units (CFUs) were counted for each yeast strain. Yeast survival was estimated as the ratio of the cells on the glucose medium to the ratio of the cells on the galactose medium (CFU D/CFU GAL). The comparison was performed using the Mann–Whitney test.Click here for additional data file.10.7717/peerj.9029/supp-12Supplemental Information 12SV40-EGFP) was used as a positive control. Box plots show the Fnuc/Fcys ratios. Horizontal lines represent the median. n.s, not significant; *p < 0.05; **p < 0.001 . Bars, 5 µm.ATP depletion decreased the accumulation of GAR-EGFP in the nucleus, revealing the energy-dependent nature of the nuclear import of this protein. EGFP was used as a negative control, and NLS from the T antigen of the SV40 virus fuzed with EGFP . The FRAP curve represents an average of ~15 cells.Click here for additional data file.10.7717/peerj.9029/supp-14Supplemental Information 14Recovery curves of FRAP experiments with FBL-EGFP, ΔGAR-EGFP, and GAR-EGFP (HeLa cells). The FRAP curve represents an average of ~15 cells.Click here for additional data file.10.7717/peerj.9029/supp-15Supplemental Information 15Recovery curves of FRAP experiments with FBL-EGFP, ΔGAR-EGFP and GAR-EGFP (HeLa cells). The FRAP curve represents an average of ~15 cells.Click here for additional data file.10.7717/peerj.9029/supp-16Supplemental Information 16n/Fnuc) of the GAR domain and the mutated forms of the GAR domain, in which different numbers of arginine residues were substituted with alanine residues.Nucleolar accumulation  were counted for each yeast strain. Yeast survival was estimated as the ratio of the cells on the glucose medium to the ratio of the cells on the galactose medium (CFU D/CFU GAL).Click here for additional data file.10.7717/peerj.9029/supp-18Supplemental Information 18Cell viability of the yeast cells expressing GPD-NOP1, GPD-FBL or GPD-FBL in dHMT1 strain after growth on YPD plates. Colony forming units (CFUs) were counted for each yeast strain. Yeast survival was estimated as the ratio of the cells on the glucose medium to the ratio of the cells on the galactose medium (CFU D/CFU GAL).Click here for additional data file.10.7717/peerj.9029/supp-19Supplemental Information 19nuc/Fcyt) or the ability to react to ATP depletion of NLSSV40-EGFP. In contrast, AdOx treatment reduced the nuclear accumulation (Fnuc/Fcyt) of GAR-EGFP, and after this treatment, ATP depletion did not substantially influence the nuclear accumulation of GAR-EGFP, indicating that arginine methylation of the GAR domain is necessary for nuclear import. The substitution of arginine residues with lysine residues (R⇒K) also decreased nuclear accumulation. The GAK domain was insensitive to ATP depletion, similar to the GAR domain, in AdOx-treated cells. Box plots show the Fnuc/Fcys ratios. Horizontal lines represent the median. The comparisons were performed using the Mann–Whitney test. n.s. – not significant; **p < 0.001 (n > 50).Arginine methylation influences the nuclear import of FBL. Treatment with AdOx did not influence nuclear localization  accumulation of GAR-EGFP, and the unmethylated variant of the GAR domain (GAK domain) was accumulated to higher levels inside the nucleoli compared to the accumulation level of the methylated GAR domain, findings that indicate that arginine methylation impairs the NoLS function of the GAR domain. Box plots show the Fn/Fnuc ratios. Horizontal lines represent the median. The comparisons were performed using the Mann–Whitney test. n.s. – not significant; **p < 0. 001 (n > 50).AdOx treatment increased the nucleolar (FClick here for additional data file.


Protein oligomerization is a fundamental process to build complex functional modules. Domains that facilitate the oligomerization process are diverse and widespread in nature across all kingdoms of life. One such domain is the Phox and Bem1 (PB1) domain, which is functionally well-studied in the animal kingdom. However, beyond animals, neither the origin nor the evolutionary patterns of PB1-containing proteins are understood. While PB1 domain proteins have been found in other kingdoms including plants, it is unclear how these relate to animal PB1 proteins. To address this question, we utilized large transcriptome datasets along with the proteomes of a broad range of species. We discovered eight PB1 domain-containing protein families in plants, along with four each in Protozoa and Fungi and three families in Chromista. Studying the deep evolutionary history of PB1 domains throughout eukaryotes revealed the presence of at least two, but likely three, ancestral PB1 copies in the Last Eukaryotic Common Ancestor (LECA). These three ancestral copies gave rise to multiple orthologues later in evolution. Analyzing the sequence and secondary structure properties of plant PB1 domains from all the eight families showed their common ubiquitin β-grasp fold, despite poor sequence identity. Tertiary structural models of these plant PB1 families, combined with Random Forest based classification, indicated family-specific differences attributed to the length of PB1 domain and the proportion of β-sheets. Thus, this study not only identifies novel PB1 families, but also provides an evolutionary basis to understand their diverse functional interactions. Initially, the two animal proteins, p40Phox and p67Phox, were shown to interact through a novel motif that contains a stretch of negatively charged amino acids4. In the same study, it was also shown that the yeast CELL DIVISION CONTROL 24 (Cdc24) protein contains the same motif as in p40Phox, and hence named as PC motif4 (for p40Phox and Cdc24). Later, the BUD EMERGENCE 1 (Bem1) protein in yeast was also found to have this motif, after which it has been renamed as PB1 domain (for Phox and Bem1). The PB1 domain of Bem1 in yeast is required for the interaction with Cdc24 to maintain cell polarity5. Later, in mammals, many protein families were identified that contain a PB1 domain6. In plants, the PB1 domain was initially recognised as domain III/IV in the auxin response proteins AUXIN/INDOLE-3-ACETIC-ACID (Aux/IAAs)7. Later, the domains III/IV were found to form a similar fold as (and hence renamed) the PB1 domain, in multiple gene families in plants10.Protein-protein interaction is a basic and important mechanism that brings proteins together in a functional module and thus allows the development of higher-order functionalities. One of the versatile interaction domains that brings this modularity through either dimerization or oligomerization is the PB1 domain12. In general, the β-grasp fold is widespread in nature with its presence in more than 60 distinct protein families across eukaryotes13. The first half of the PB1 domain represents a positively charged face, with a conserved lysine (K) in β1. The latter half of the domain represents a negatively charged face, with D-x-D/E-x-D/E as core12 (OPCA motif). Based on the presence or absence of these important residues/motifs, the PB1 domains are divided into three types. If the PB1 domain contains only the conserved OPCA motif but not the lysine, it is considered as a type-1 (or type-A) PB1 domain. If there is only lysine but not an OPCA motif, it is a type-2 (or type-B) domain. If the PB1 domain contains both the lysine and the OPCA motifs, it is referred as type-1/2 (or type-AB). Various proteins that harbour a PB1 domain undergo either dimerization or oligomerization, where the positive face of one PB1 domain interacts with the negative face of another in a head-to-tail fashion11. Hence, depending on the type of PB1 domain they interact with, there can be either homotypic or heterotypic PB1 interactions.The PB1 domain ranges from 80–100 amino acids in length and exhibits a Ubiquitin-like β-grasp fold with five β-sheets and two α-helices15. PB1 domain-containing proteins have been relatively well studied in Animalia, when compared to the other kingdoms. At least nine gene families have been shown to encode a PB1 domain6. Animal genomes encode proteins that contain all three types of PB1 domains: type-1 - NEUTROPHIL CYTOSOL FACTOR 4 (NCF4/p40Phox), MITOGEN-ACTIVATED PROTEIN KINASE KINASE 5 (M2K5) and NEXT TO BRCA 1 (NBR1); type-2 - NEUTROPHIL CYTOSOL FACTOR 2 (NCF2/p67Phox), PARTITIONING DEFECTIVE 6 (Par6) and MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 2/3 (M3K2/3); type-1/2 - SEQUESTOSOME-1 (SQSTM1/p62), ATYPICAL PROTEIN KINASE C (aPKC) and TRK-FUSED GENE (TFG). A systematic analysis through yeast two-hybrid and pull-down assays revealed various homotypic and heterotypic interactions among these PB1 domains3. The p67Phox upon its interaction with p40Phox activates the phagocyte NADPH oxidase that is important for innate immunity in mammals16. The Par6-aPKC complex establishment through PB1 is essential for cell polarity in mammals and insects17. This complex, along with Par3, also regulates the formation of junctions through apical-basal polarity in mammalian epithelial cells18. p62 acts as a crucial scaffolding protein playing important roles in autophagy, apoptosis and inflammation19.All eukaryotes are divided into five kingdoms: Protozoa, Chromista, Fungi, Animalia and Plantae20. TFG PB1 domain is involved in transforming activity by forming the TFG-TrkA (Tyrosine Kinase A) fusion21. NBR1 interacts with p62 through PB1 which is required for targeting p62 to sarcomeres3. Few non-canonical PB1 interactions were also observed, for example, in p40Phox PB1 and PX domains undergo intramolecular interaction, disruption of which is required to activate the NADPH oxidase22. In yeast, interaction of both the PB1 domain containing proteins, Bem1 and Cdc24 is critical for the cell polarity establishment at both budding and mating5. The NADPH OXIDASE REGULATOR (NoxR) plays a central role in fungal morphogenesis, growth and development through NADPH oxidation pathway23.The PB1 domain of M3K2/3 interacts with M2K5 to activate EXTRACELLULAR-SIGNAL-REGULATED KINASE 5 (ERK5) mediated signalling in response to growth factors and osmotic stress24. The structural basis for these interactions has also been scrutinized in detail25. Both ARFs and Aux/IAAs are involved in auxin-dependent gene regulation through the Nuclear Auxin Pathway, that controls various growth and developmental processes26. Another PB1 domain containing protein, AtNBR1, an Arabidopsis ortholog of animal NBR1, is involved in autophagy and was shown to homopolymerize through its PB1 domain8. Joka2, an AtNBR1 orthologue of tobacco, can also homodimerize through its PB1 domain10. Moreover, the same study also revealed non-canonical interaction of the PB1 domain with the C-terminal UBA domain within the same protein10. Homotypic interactions through PB1 domains of NIN-LIKE PROTEINS OF PLANTS (NLPs) are required to induce nitrate-dependent gene expression28. Interestingly, like AtNBR1/Joka2, the PB1 domain of NLP also undergoes non-canonical interaction with the HQ domain of TEOSINTE BRANCHED 1, CYCLOIDEA, PCF DOMAINS CONTAINING PROTEIN 20 (TCP20)27. Another study identified a novel unclassified PB1 domain-containing protein PAL OF QUIRKY (POQ) that undergoes non-canonical interaction with QUIRKY (QKY)29. However, the structural or mechanistic basis of these non-canonical interactions are yet to be elucidated.The best-studied PB1 domains in plants belong to the AUXIN RESPONSE FACTOR (ARF) transcription factors and their AUXIN/INDOLE-3-ACETIC-ACID (Aux/IAA) inhibitors. Both the homotypic and heterotypic interactions among and between these gene families is relatively well established30, their evolutionary histories are essentially unknown. Moreover, it is unclear how many PB1 domain-containing gene families are present in other kingdoms. Deep evolution has been relatively well studied for ARF and Aux/IAA gene families31 and to a certain extent for NLPs32, but the presence and the evolution of other PB1 domains, if any, in plants and unicellular eukaryotes is obscure. Hence, in this paper we address several important questions related to the distribution and ancestry of PB1 domains in the eukaryotic tree of life: (1) How many PB1 domain-containing gene families are present in the kingdoms Protozoa, Chromista, Fungi and Plantae? (2) What is the origin of the PB1 domain? (3) How many copies of PB1 were present in the Last Eukaryotic Common Ancestor (LECA)? (4) How have PB1-containing proteins diversified/multiplied in evolution across multiple kingdoms? (5) What are the sequence/structural patterns specific to each PB1 family and how to classify them?Even though PB1 domain proteins are well defined and their mechanistic basis is relatively well established in animalsTo answer these questions, we have utilized the large transcriptome datasets available in Chromista and Plantae and the  complete proteomes from Fungi and Animalia. We found that the PB1 domains have a deep evolutionary origin with at least two copies in LECA. Moreover, we find that the PB1 domain is associated with a variety of domains, ranging from DNA-binding domains to Kinases and membrane-binding domains. Further, a detailed sequence analysis of PB1 domains in Plantae revealed that these are poorly conserved among various families in general, with few residues being specific to each family. Taken together, this study provides the first evolutionary framework of the PB1 domains across eukaryotes.Phox and NCF4/p40Phox both contain SRC Homology 3 (SH3) and PB1 domains in the C-terminus, where NCF2 contains Tetratricopeptides and NCF4 contains Phox homologous domain (PX) in their N-terminus  and animal (Human and Mouse) PB1 sequences were used to query the database, and processed the data with a pipeline developed earlier31. Two gene families, Kinase and NBR1 were identified in Chromista, with a similar domain architecture, being orthologous to the respective gene families in Animalia  group and Cryptophyta, all the transcriptomes from the MMETSP database were used , especially in Fungi, Animalia and Plantae . Monophyletic grouping, as well as the presence in multiple kingdoms, support the notion that there would have been at least two common ancestral copies of PB1 domains, each corresponding to Kinase and Phox orthologues across eukaryotes. Even though the Plantae NBR1 PB1 domains, along with the animal orthologues (and the similar proteins p62) are not monophyletic in origin, they are still placed in the phylogeny as sister clades , mosses (Physcomitrella patens), basal angiosperms (Amborella trichopoda) and a core eudicot . All PB1 domain protein sequences that belong to the eight families were aligned, and an individual sequence logo was derived for each family  just before the OPCA motif in Kinase and Kinase-derived PB1 domains Fig. . InteresSince there are no clear patterns in the PB1 domains that set apart the protein it belongs to and because it is also not possible to identify important features of a specific PB1 domain based on the sequence alignment, one might detect patterns based on the secondary structure composition along with the amino acid properties. Random forest (RF) based classification was performed with 28 amino acid descriptors as variables. After bootstrap aggregating (bagging) all the decision trees from the RF, the mean out-of-bag (OOB) error rate is only 6% which indicates the high reliability of the RF model Fig. . The claThe importance of each variable is evaluated through the mean decrease in accuracy (MDA) and the mean decrease in Gini (MDG). Higher values of both MDA and MDG indicate the most important variables. In this case, the top 10 important variables are shown in Fig. Arabidopsis thaliana, encodes more than 80 PB1 domain copies grouped into eight families , and functions in cell polarity by forming oligomers in plants and animals. In a similar way, the PB1 orthologs across multiple kingdoms may share common functionality in similar pathways. One such is the PB1 domain-containing protein NBR1, which serves as an autophagy cargo receptor in both plants and animals, where homodimerization of the PB1 domain is also conserved as a part of its function35. Phox orthologues in animals (p67Phox) and fungi (NoxR) play a key role in NADPH oxidation pathway by interacting with the membrane associated proteins (gp91Phox and NoxA/B respectively) as well as through PB1 domain with p40Phox and Cdc24 respectively36. Similarly, in Arabidopsis, Phox4 was shown to interact with membrane-associated proteins KNOLLE, SYP22 and PEN1, which belong to the SNARE family37. However, it is unknown which PB1 domain protein interacts with Phox4-PB1 in plants. In another study, Phox proteins (referred as MadB) were shown to be involved in the myosin-driven interactions, preferably through their PB1 domain38. Hence, discovering these unknown and novel interactions may provide a link to the existence of common pathways across kingdoms controlled by PB1-dependent interactions.The evolutionary patterns of the PB1 domain showed that there are multiple families shared across multiple kingdoms Fig. . Kinase 26. NLPs are master regulators of nitrate-inducible gene regulation in higher plants28. On the other hand, animal Par6 and aPKC PB1 domains are known to interact with each other playing a key role in epithelial cell polarity39. Thus, as far as can be inferred, these kingdom-specific PB1 domain-containing proteins appear to regulate processes that are specific to that kingdom.Apart from the proteins that are shared across multiple kingdoms, some are specific to each group Fig. . ARF, Au29. The PB1 domain of NLP interacts with HQ domain of TCP2027. In animals, the M2K5 PB1 interacts with ERK5, among many others6. However, the structural and/or mechanistic basis of any of these non-canonical interactions is currently unknown.It is interesting to see that the key interacting PB1 domains have also evolved in pairs. Some such pairs are: aPKC-M2K5 , Bem1-Cdc24 (Fungi) and ARF-Aux/IAA (Plantae)  a single copy gene, where ARFs and Aux/IAAs are represented by large gene families with more than 20 copies (Supplementary Table 40. The classification error rate is as low as 2% (in ARFs), but up to 14% in Kinase-derived, which could be due to the broader distribution of all three most important variables . RF has been very successful in classification with highly correlated variables at low error rateles Fig. . This clles Fig. . How thi13. The DIX domain is one the recently studied families of β-grasp fold domains that is conserved across multiple kingdoms and structurally highly similar to PB1 domain41. However, despite their highly homologous structural fold, they appear to be evolved independently from a common ancestor in eukaryotes34 . To find other PB1 domains, we then used ten proteomes from the kingdom across various phyla  and animal (Human) species. BLASTP module in NCBI BLAST 2.7.1+42 was employed for this search and InterPro domain database v5.30–69.0 (https://www.ebi.ac.uk/interpro/) was used for domain identification in the BLAST hits. All the sequences that have a PB1 domain have been used for further phylogenetic analysis.To study the PB1 domains in the kingdom Animalia, based on the literature, we first extracted all the PB1 domain sequences of Human proteome from the UniProt database (https://mycocosm.jgi.doe.gov). For the Protozoa, we have used the six reference proteomes from UniProt  and Fungi. However, the proteomes of twelve fungi across multiple phyla have been obtained from MycoCosm database at JGI (43 (www.onekp.com). Out of nearly 1300 transcriptomes in the database, we have used 485 transcriptomes in this study, covering all the phyla in the kingdom Plantae. We have adapted a protocol that was developed earlier31. In brief, the query PB1 sequences from Arabidopsis were searched against each transcriptome, where the resulting scaffold hits were translated using TransDecoder . All these translated sequences were checked for the presence of PB1 domains using InterProScan44 and only those protein sequences with a PB1 domain identified were used for further analysis. In a similar way, for SAR group in Chromista, we have employed another transcriptome dataset, Marine Micro Eukaryote Transcriptome Sequencing Project (MMETSP) database45. We have used all the available transcriptomes and adapted a similar protocol as mentioned above  database46). Alignment was cleaned up further, where the positions with more than 20% gaps were removed with trimAl, prior to phylogeny construction47. ModelFinder (accessed through IQ-TREE) indicated ‘LG’ as the best model of evolution, of all the 462 models tested48. Further, the Maximum Likelihood (ML) method, employed in the IQ-TREE program was used for the phylogenetic tree construction, with 1000 rapid bootstrap replicates and tree branches tested by SH-aLRT method49. The resulting tree was manually curated further for some misplaced taxa. An unrooted tree was visualized in iTOL v4 (https://itol.embl.de/shared/dolfweijers). In a similar way, another phylogenetic tree was generated using the PB1 sequences only from three kingdoms .Using all the PB1 sequences that were identified in all the five kingdoms of eukaryotes, we performed the phylogenetic analysis , aligned them using ClustalOmega50. After the alignment, the domains from each family were separated and a sequence logo was generated. All the gene identifiers from these four species are available in Supplementary Table https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/logoddslogo/proteins.cgi). Amino acid groups ‘PAGFLIMV’, ‘KRH’ and ‘DE’ were shown in purple, blue and red colours respectively. All other amino acids were shown in black colour.To understand the PB1 domains in the plant kingdom further, we have taken the PB1 sequences of all the eight families from four species . We have used 28 amino acid descriptors (variables) calculated either with ‘protr’ or ‘peptides’ R packages53. Among these, 20 variables correspond to the composition of 20 amino acids, and the remaining eight correspond to the general parameters such as length, molecular weight, hydrophobicity, net charge, isoelectric point (pI), aliphatic index, hydrophobic moment of alpha and beta sheets. We used ‘RandomForest’ R package to build a maximum of 500 decision trees with 5 variables being tried at each step (www.r-project.org54). Confusion matrix and variable importance plots showing mean decrease in accuracy and gini were obtained. Descriptive plots and other graphs shown were obtained using ‘ggplot2’ R package. Additional File The Random Forest (RF) method was used to identify key amino acid descriptors to differentiate and classify each of the PB1 domains into eight plant PB1 families55. The identifiers of the PB1 sequences used were: ARF5 (PDB: 4CHK), IAA17 (PDB: 2MUK), At2G36500 (CBS), At1G04700 (Kinase), At2G01190 (Kinase-derived), NBR1, NLP9 and Phox2. Obtained homology models were visualized in PyMol software .Homology modelling for the eight selected members in Arabidopsis, one each from each plant PB1 family were performed using Phyre2 webserver ‘normal’ modeSupplementary Information.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during replication. These pathways include translesion synthesis and template switching. These pathways are regulated largely through post-translational modifications of proliferating cell nuclear antigen (PCNA), an essential replication accessory factor. Mono-ubiquitylation of PCNA promotes translesion synthesis, and K63-linked poly-ubiquitylation promotes template switching. This article will discuss the mechanisms of how these post-translational modifications of PCNA control these bypass pathways from a structural and biochemical perspective. We will focus on the structure and function of the E3 ubiquitin ligases Rad18 and Rad5 that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA, respectively. We conclude by reviewing alternative ideas about how these post-translational modifications of PCNA regulate the assembly of the multi-protein complexes that promote damage bypass pathways. Ionizing radiation, ultraviolet radiation, and a wide range of chemical agents damage DNA . The resCells have evolved multiple DNA damage bypass pathways that mitigate the effects of DNA damage during replication ,6,7. TheAn underappreciated aspect of the regulation of these DNA damage bypass pathways is the role played by the E3 ubiquitin ligases that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA. A substantial amount of evidence suggests that the Rad18 protein, which is the E3 ubiquitin ligase responsible for PCNA mono-ubiquitylation . Th. Th44]. The RING domain of Rad18 binds to Rad6 via a characteristic RING-E2 interface consisting of both hydrophobic and electrostatic interactions . The RIN2HC zinc-binding domain comprised of three cysteine residues  and one histidine residue (corresponding to residue 206 of yeast Rad18). NMR titrations show that the Rad18 UBZ domain binds to ubiquitin with a dissociation constant in the low micromolar range [The NMR structure of the human Rad18 UBZ domain (corresponding to residues 187 to 210 of yeast Rad18) shows that this domain has an overall β-strand/β-strand/α-helix fold  45]. It. It45]. Interestingly, mutations in the UBZ domain do not block the ability of Rad18 to function as a ubiquitin ligase during the mono-ubiquitylation of PCNA (47). This suggests that this domain may be involved in a function of Rad18 other than translesion synthesis. In fact, the UBZ domain is essential for Rad18 auto-ubiquitylation , which lThis small domain (residues 278 to 312) is named after the three proteins in which it was first identified: SAF-A/B, Acinus, and Pias . The strThe R6BD is a small domain (residues 371 to 410) near the C-terminus of Rad18 that binds Rad6. This domain has a helix–loop–helix fold  42]. Th. Th42]. Yeast Rad18 contains a SIM (residues 139 to 142) in the predicted disordered region between the RING domain and the UBZ domain . ConservPol η is a non-canonical DNA polymerase that carries out translesion synthesis through thymine–thymine dimers and 8-oxoguanine lesions ,22. HumaAs described above, Rad18 interacts with Rad6, pol η, and PCNA. In addition to these proteins, Rad18 interacts with several other proteins involved in DNA damage bypass including RPA (replication protein A), Rev1, and Rad5.Replication Protein A (RPA) is a single-stranded DNA-binding protein involved in DNA replication, repair, and recombination . It has Rev1 has two functions in DNA damage bypass. First, it is a non-canonical DNA polymerase that catalyzes translesion synthesis of minor-groove and exocyclic guanine adducts as well abasic sites ,24,25. SRad5 is the E3 ubiquitin ligase that is necessary for the template-switching pathway of DNA damage bypass (see below). Rad18 and Rad5 interact with each other . This inUbc13–Mms2–Rad5 is the E2 ubiquitin-conjugating enzyme/E3 ubiquitin ligase that catalyzes the K63-linked poly-ubiquitylation of PCNA . Ubc13 iYeast Rad5 is a 134 kDa protein that has two functions ,67. FirsCurrently, there is no high-resolution structural information about Rad5. Our limited structural understanding of Rad5 has been obtained from homology modeling of its structured domains. These domains include the HIP116 Rad5p N-terminal (HIRAN) domain, the helicase domain, and the RING domain.The HIRAN domain of Rad5 (corresponding to residues 170 to 293 of yeast Rad5) is located near the N-terminus of the protein and is flanked by intrinsically disordered regions. While there are no high-resolution structures of this domain, the HIRAN domain of Helicase-Like Transcription Factor (HLTF), a human homolog of Rad5, has been determined  68,69,7,769,70. The HIRAN domain of HLTF is required for unwinding forked DNA substrates containing double-stranded DNA on both the leading and the lagging strand of the fork without single-stranded gaps at the fork junction . By contRad5 is a member of the Swi/Snf subfamily within the SF2 superfamily of helicases ,73. The The helicase domain is largely responsible for the ATPase and fork-remodeling activity of Rad5 . The ATPIn the case of human HLTF, footprinting experiments suggest that translocation occurs in the 3′–5′ direction, with the helicase domain positioned on the lagging strand template of the parental duplex in front of the fork . For Rad3HC4 zinc finger-type domain comprised of seven cysteine residues  and one histidine residue (residue 934) that coordinate zinc ions [Swi/Snf helicases often contain a small, folded domain between the two RecA-like folds of the helicase domain . In the inc ions . There iinc ions . Though Rad5 has several structured domains, approximately 30% of it is likely unstructured. There are two large intrinsically disordered regions: one at the N-terminus (starting at residue 1 and ending at residue 169 in yeast Rad5), and one between the HIRAN domain and the helicase domain (starting at residue 294 and ending around residue 525 in yeast Rad5). The role of these unstructured regions is likely to provide conformational flexibility to Rad5, allowing the HIRAN domain and the helicase domain to reorient themselves with respect to one another. In addition, these regions likely mediate protein–protein interactions. A recent structural model of full-length Rad5 has been proposed on the basis of a combination of molecular simulations and small-angle X-ray scattering (SAXS) data . The molAs described above, Rad5 interacts with Ubc13 and Mms2. In addition to these proteins, Rad5 interacts with several other proteins including PCNA, Rev1, and Rad18.Rad5 binds PCNA through its N terminus (amino acids 1–430) . The preLike Rad18, Rad5 interacts with Rev1, the non-canonical polymerase that helps to organize the structure of the complex that carries out DNA damage bypass . Based oAs described above, Rad5 interacts with Rad18, the E3 ubiquitin ligase that is necessary for translesion synthesis . This inThe coordination of these DNA damage bypass pathways is not well understood. A common view of the role of Rad6–Rad18 and of Ubc13–Mms2–Rad5 in regulating DNA damage bypass is as follows. When replication forks stall upon encountering DNA damage, Rad6–Rad18 catalyzes the mono-ubiquitylation of PCNA. This signals for the recruitment of non-canonical polymerases such as pol η, Rev1, and pol ζ. If translesion synthesis fails, then Ubc13–Mms2–Rad5 catalyzes the poly-ubiquitylation of PCNA. This signals for fork reversal and template switching.The protein–protein interactions discussed above and several observations in the literature cast doubt on the traditional model. For example, pol η interacts with the Rad6–Rad18 complex, and this interaction is essential for the recruitment of these proteins to stalled replication forks ,78,79,80These pieces of evidence suggest that the mono-ubiquitylation and poly-ubiquitylation of PCNA do not merely act as signals to recruit the non-canonical polymerases and fork-remodeling helicase. Instead, they suggest that the ubiquitylation machinery plays a more direct role, recruiting these other proteins. According to this alternative view, DNA damage bypass is carried out by the coordinated activity of three multi-protein factors . The firWhether or not there is a bypassosome, the evidence reviewed here provides a compelling case for the presence of three bypass factors. According to this model, Bypass Factor 1 is directed to the stalled replication fork via its Rad6–Rad18 components. In this way, its pol η component rides piggyback on its Rad6–Rad18 components. If this model is correct, then pol η is already present at the stalled replication fork when the ubiquitin moiety is transferred to PCNA. Next, Bypass Factor 2 is directed to the stalled fork via interactions of its Rev1 component with ubiquitin-modified PCNA or with the pol η component of Bypass Factor 1. Bypass Factor 2 may also be directed to stalled replication forks via interactions of its pol ζ with ubiquitin-modified PCNA.Finally, Bypass Factor 3 is directed to the stalled replication fork via the E2 ubiquitin-conjugating and E3 ubiquitin ligase activities of Ubc13–Mms2–Rad5. Because Rad5 also possesses a fork-remodeling helicase activity, this activity rides piggyback on the ubiquitylation activity. This factor may also be recruited via interactions of its Rad5 component with mono-ubiquitylated PCNA, with the Rad18 component of Bypass Factor 1, or with the Rev1 component of Bypass Factor 2. Because poly-ubiquitylation of PCNA likely requires the mono-ubiquitylation of PCNA, the recruitment of Bypass Factor 3 likely occurs after the recruitment of Bypass Factor 1.In this review, we have discussed the regulation of translesion synthesis and template switching by post-translational modifications of PCNA. We have focused on the structures, functions, and interactions of the E3 ubiquitin ligases Rad18 and Rad5, which facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA. We have focused on these E3 ubiquitin ligases because they play an important and arguably more direct role in orchestrating the steps of translesion synthesis and template switching than was initially thought. Unfortunately, we still know very little about the structures, functions, and interactions of these proteins.One reason we know so little about these E3 ubiquitin ligases is that they contain extensive regions of intrinsic disorder conferring to them a high degree of conformational flexibility. Another reason is that they are components of large, dynamic, multi-protein complexes that likely change their composition during the course of the DNA damage bypass process. Consequently, these are extremely difficult proteins to study from a structural and biochemical perspective because traditional experimental approaches, such as X-ray crystallography, NMR, and even cryo-electron microscopy, do not have the capability to tackle such conformationally and compositionally flexible systems. Fortunately, new approaches, both experimental and computational, are emerging that should allow us to gain valuable new structural and biochemical information about such conformationally flexible, multi-protein complexes. For example, hybrid approaches, such as full ensemble methods that combine small-angle X-ray scattering data and molecular dynamics simulations, should be extremely useful in studying such systems . Similar


Streptococcus suis is a major cause of meningitis, arthritis, and pneumonia in pigs worldwide, and an emerging pathogen in humans. In Sweden, S. suis has previously received little attention but has in recent years become increasingly recognized as affecting the pig production. The aim of the present study was to investigate the occurrence, serotypes and antimicrobial susceptibility of S. suis in Swedish grower pigs from herds with and without reported S. suis associated disease, as well as possible associations between S. suis associated disease and selected environmental and production factors. Swab samples were taken from the tonsils of clinically healthy 8–13-week-old grower pigs from ten case herds and ten control herds. Isolates were cultured, identified using MALDI–TOF MS, and serotyped using latex agglutination. The antimicrobial susceptibility of 188 isolates was tested using broth microdilution. Production data was gathered and environmental parameters were measured on the farms.Streptococcus suis was isolated from 95% of the sampled pigs in both the case and the control herds. Serotypes 3, 4, 5, 7, 9, 10, 11, 15, 16, and 17–34 were detected, although a majority of the isolates (81.5%) were non-typeable. There was less diversity among the serotypes isolated from the case herds than among those from the control herds; four and nine different serotypes, respectively. Isolates resistant to penicillin (3.8%) were reported for the first time in Sweden. Tetracycline resistance was common (88.4%). No association was noted between the production and the environmental factors investigated, and the carriership of S. suis.S. suis was found to be higher in clinically healthy Swedish pigs than previously estimated, and for the first time, the presence of Swedish isolates resistant to penicillin was reported. Many of the most commonly disease-associated serotypes, e.g. serotypes 2, 9, 3, and 7, were detected in healthy grower pigs although further studies are needed to investigate the virulence of these isolates.The carriership of  Streptococcus suis is considered one of the most important pathogens affecting pig production worldwide and is also an emerging zoonotic agent in humans. In both humans and pigs, S. suis may cause meningitis, sepsis, arthritis, pneumonia, endocarditis, and acute death [te death , 2. Pigste death , 4, and te death , and frote death .S. suis carriership, as have production factors such as crowding and continuous production systems [S. suis-infections [Environmental factors such as higher outside temperature, excessive temperature fluctuations, and high air relative humidity have been associated with a higher proportion of  systems , 8. Furtfections .S. suis is a phenotypically and genetically diverse species with a complex taxonomy and may be challenging to accurately identify in the laboratory. Based on the antigenicity of the polysaccharide capsule, 35 serotypes were initially described, although sequencing of the 16S rRNA and cpn60 genes has more recently indicated that six of these belong to other species [Presumptive diagnosis in pigs is based on clinical signs and should be confirmed by necropsy and isolation of the pathogen.  species –12. The  species , and ind species , 15. RelS. suis-infections in pigs and humans were published in the mid-1980s [S. suis-infection. Therefore, the aim of this study was to investigate the occurrence, serotypes and antimicrobial susceptibility of S. suis in Swedish grower pigs from farms with and without reported disease, as well as possible associations with selected environmental and production factors.Although the first Swedish reports of id-1980s , 18, thiid-1980s , and vetThe study was approved by the Ethics Committee for Animal Experimentation, Uppsala, Sweden (Dnr 5.8.18-15404).S. suis-infections and clinical signs had been noted in several batches of weaned pigs during the year preceding the sampling. An equal number of herds where no such diagnoses had ever been made by the herd health veterinarians were used as controls. No selected herds opted out of participating. For the purposes of this study, clinical signs indicative of S. suis-infection were defined as grower pigs (from weaning to 30 kg body weight) circling or exhibiting seizures, lateral recumbency with paddling leg movements, or severe pneumonia in herds where S. suis pneumonia had previously been laboratory confirmed.A case–control design was used. Herds were chosen based on the referrals of herd health veterinarians from the Swedish “Farm & Animal Health” service that covers 80% of the Swedish pig herds. The case group included ten pig herds where the health-service veterinarian had previously diagnosed The samples were collected during 2018 and 2019 from 20 farms located in the south and central parts of Sweden, in the counties with the highest pig density. The herds included piglet producing, farrow-to-finish, and gilt producing conventional herds, as well as piglet producing and farrow-to-finish organic herds, and piglet producing and farrow-to-finish sow pool satellites . In accIn each farm, environmental measurements were collected from the room housing the targeted pigs. Temperature and air relative humidity were measured at the height of approximately 1 m in the middle of the room using a Testo 625 thermohygrometer . The air velocity was measured at the height of approximately 0.1 m in 2–6 randomly distributed pens per room, adjacent to the solid and to the slatted floor areas , using a Testo 405-V1 thermal anemometer (Testo SE & Co. KGaA). Carbon dioxide was measured at the height of approximately 1 m in the middle of the room and ammonia was measured at the height of approximately 0.1 m adjacent to the slatted floor area in 4–6 randomly distributed pens per room, , using colorimetric detector tubes and a manual GV-100 air sampling pump .Information on management practices and production data covering 1 year before sampling was obtained from the farmers through interviews and from the farm management software PigVision . All data collection was performed by the first author. Mean values for the case and control group were compared using a two-tailed t-test, and P < 0.05 was considered significant. Statistical analysis was performed in R version 3.6.2 .From each farm, samples were taken from ten clinically healthy grower pigs from the same batch, if possible from one pig per pen. The pigs were 8–13 weeks of age and had not been subjected to any treatment for at least 1 month before sampling. The pigs had all been weaned at between 4 and 6 weeks of age . A sampl2 overnight. From each sample, 5–12 small, translucent colonies exhibiting α- or β-hemolysis [2 overnight. Following incubation, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to identify the bacterial colonies to the species level. Material from 1 to 3 pure-cultured colonies was smeared directly onto a polished steel target , covered with 1 μL of α-cyano-4-hydroxycinnamic acid (HCCA) matrix , prepared according to the manufacturer’s instructions, and allowed to dry at room temperature. MALDI-TOF MS analysis was performed using a Microflex LT System running version 4.1.60 of the BDAL database . A positive species identification was defined as a MALDI-TOF MS score ≥ 2.00 [S. suis isolates per pig were preserved at − 70 °C, and later one isolate per pig was randomly selected for serotyping and antimicrobial susceptibility testing.The swabs were streaked directly onto streptococcal selective colistin-oxolinic acid-blood agar (COBA) plates , and incubated at 37 °C in 5% COemolysis  were sube ≥ 2.00 . Isolatee ≥ 2.00 , where 1S. suis kit  according to the manufacturer’s instructions. The test identifies serotypes 1 through 16 separately, and groups serotypes 17 through 34 together. Briefly, 1 μL of colony material, pure-cultured on 5% horse blood agar at 37 °C in 5% CO2 overnight, was suspended in 250 μL of sterile saline before mixing 10 μL of the suspension with 10 μL of latex reagent. Agglutination that occurred within 60 s was interpreted as a positive reaction.Isolates were serotyped by latex agglutination , 29 usin2 overnight. Colonies were suspended in a 0.9% sterile saline solution to obtain an inoculum density of 5 × 105 colony-forming units per mL (CFU/mL), which was added to cation-adjusted Mueller–Hinton broth  supplemented with 3% lysed horse blood . Each of the 96 wells of a VetMIC GP-mo plate was inoculated and incubated aerobically at 37 °C for 18–20 h. S. suis ATCC 43765 and Streptococcus pneumoniae ATCC 49619 were used as control strains and minimum inhibitory concentrations (MIC) were within the accepted quality control ranges. The MIC was recorded as the lowest concentration of an antimicrobial inhibiting visible bacterial growth. MICs of cefoxitin, cefalotin, ciprofloxacin, clindamycin, chloramphenicol, enrofloxacin, erythromycin, gentamycin, penicillin, tetracycline, trimethoprim, and trimethoprim/sulfamethoxazole were determined. The results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) breakpoints for S. suis [Antimicrobial susceptibility testing was performed by broth microdilution using commercially available VetMIC GP-mo panels , according to the manufacturer’s instructions. Isolates were cultured on 5% horse blood agar plates and incubated at 37 °C in 5% CO S. suis , where aThe case group consisted of seven farrow-to-finish herds and three piglet-producing herds. Out of the ten case herds, three were sow pool satellites from different sow pools. The control group consisted of eight farrow-to-finish herds, one gilt-producing, and one piglet-producing herd. Out of the ten control herds, two were specific pathogen-free (SPF) herds  and threS. suis. All herds had recorded at least one case during the month preceding the sampling. At the interviews, three control herds reported that single pigs with clinical signs consistent with an S. suis-infection had been observed by the farmers on single occasions. These potential cases had however not been clinically confirmed by the herd health veterinarians.Clinical signs indicative of meningitis predominated in eight out of ten case herds, while two herds reported respiratory signs and acute deaths due to There were no significant differences between case and control herds in the environmental parameters measured Table . Taking No significant differences were found between the case and control herds regarding the production data parameters Table . HoweverThe total number of sows in the sow pools ranged from 1000 to 1700, with between 40 and 55 sows farrowing in each batch at the sampled satellite farms.Streptococcus suis was isolated from at least eight out of ten  of the sampled animals in each control herd, and from at least eight out of ten  of the sampled animals in each case herd. In total, S. suis was isolated from 95% (190 out of 200) of the sampled animals included in the study.Latex agglutination was performed on 189 out of 190 isolates . A majority of the isolates  exhibited insufficient or no agglutination and were therefore serologically non-typeable. The percentage of non-typeable isolates did not differ  between case and control herds. The distribution of serotypes is shown in Fig. Serotype 5 was detected only in one case herd, whereas serotypes 3, 4, 7, 9, 10, and 16 were detected only in the control herds. The isolates of serotypes 3, 4, 7, 10, and 16 were from one herd each, whereas serotype 9 was found in two herds. Serotypes 11, 15, and 17–34 were detected in both case and control herds.S. suis-infections as case herds, resulted in one isolate from each of the serotypes 3, 7, 9, 15, and 16 being included in the case group.Reclassification of the three previously mentioned control herds with occasional single cases of suspected Antimicrobial susceptibility testing Table  was perfSeveral isolates  were classified as intermediate, and 3.8% (7 out of 184) were resistant to penicillin. A majority of these not susceptible isolates , including all of the resistant isolates, originated from the case herds. The percentage of isolates not susceptible to enrofloxacin was 27.7% in the case group and 21.3% in the control group while the percentages of isolates not susceptible to tetracycline were similar in the two groups, at 100% and 98.9%, respectively.S. suis was isolated from a higher percentage than expected of clinically healthy Swedish grower pigs in both case and control herds. There was no difference in the percentage of carriers between the groups, and interestingly, the conventional herds did not differ in this respect from the closed SPF-herds or the organic herds with lower stocking density and outdoor access.Compared to the limited historical data available , 33, S. S. suis is generally reported to be uncommon [In contrast to previous studies on Swedish isolates , 34 the uncommon , and sinuncommon . In Sweduncommon . A majorDespite the low Swedish sales of tetracyclines, as compared to sales in other European countries , 37, theS. suis infections is questionable.Using the CLSI breakpoints defined for enrofloxacin , 73.4% oAll of the isolates investigated originated from clinically healthy pigs, and several previous studies have also shown that resistance to several classes of antibiotics is more common in non-clinical isolates than in isolates from diseased pigs –42. It mS. suis in clinically healthy pigs [S. suis. Temperature logging, outside and in the pig barn, over a longer period of time and in a greater number of herds may be considered in the future when assessing possible disease-triggering factors.Environmental factors such as higher outside temperature, temperature fluctuations, and a relative humidity of > 70%, have previously been associated with a higher carriership of thy pigs . In the S. suis-infections were reclassified as case herds, the number of sows farrowing per batch was significantly higher in this group. Since the number of routes of transmission increases with an increasing number of individuals this might indicate that the group size is of importance. However, production data was not available for some herds, and care should be taken when interpreting these results.In addition, none of the investigated production factors differed significantly between case and control herds. However, if the three herds that had experienced single clinical cases of presumptive S. suis are less common problem in these herds. However this interesting observation needs further investigations.The herds in the study were included in the case or control group solely based on their herd health veterinarian’s assessment, as judged by the clinical picture and laboratory results. The few participating SPF- and organic farms were all found in the control group, which could indicate that clinical problems with S. suis serotypes, e.g. serotypes 9, 3, and 7, were detected in clinically healthy pigs this study. S. suis is often considered part of the normal flora of the tonsils, and although certain serotypes are more often associated with disease, virulence can also vary within serotypes [S. parasuis, S. orisratti, and S. ruminantium [Several of the most commonly disease-associated erotypes . The diverotypes , 43–45. erotypes . The serinantium , 12, 47.S. suis-infection may be underreported or misdiagnosed, and that the pathogen might be a more common cause of disease than previously acknowledged. Further, the possibility of the MALDI–TOF MS method generating false-positive results cannot be excluded.The perceived low incidence of clinical disease in Sweden may be due to low virulence of the strains present, or it may be related to other factors such as a high weaning age, a legislated minimum space allowance for growing pigs that is higher than the EU minimum , 48, or S. suis to be more common in Swedish pig herds than previously estimated, and for the first time reports the presence of Swedish isolates resistant to penicillin. Several of the most commonly disease-associated serotypes were isolated from clinically healthy grower pigs, although a large number of isolates were serologically non-typeable using latex agglutination. Further studies are needed to investigate the serotypes and virulence of these isolates. No association was noted between the environmental factors investigated and the carriership of S. suis in clinically healthy grower pigs.This study shows