Saccharomyces cerevisiae, it is controlled by a system of four methyltransferases  and four demethylases . While the histone targets for these enzymes are well characterized, the connection of the enzymes with the intracellular signaling network and thus their regulation is poorly understood; this also applies to all other eukaryotes. Here we report the detailed characterization of the eight S. cerevisiae enzymes and show that they carry a total of 75 phosphorylation sites, 92 acetylation sites, and two ubiquitination sites. All enzymes are subject to phosphorylation, although demethylases Jhd1p and Jhd2p contained one and five sites respectively, whereas other enzymes carried 14 to 36 sites. Phosphorylation was absent or underrepresented on catalytic and other domains but strongly enriched for regions of disorder on methyltransferases, suggesting a role in the modulation of protein–protein interactions. Through mutagenesis studies, we show that phosphosites within the acidic and disordered N-terminus of Set2p affect H3K36 methylation levels in vivo, illustrating the functional importance of such sites. While most kinases upstream of the yeast histone methylation enzymes remain unknown, we model the possible connections between the cellular signaling network and the histone-based gene regulatory system and propose an integrated regulatory structure. Our results provide a foundation for future, detailed exploration of the role of specific kinases and phosphosites in the regulation of histone methylation.Histone methylation is central to the regulation of eukaryotic transcription. In  Histone methylation is a widespread and dynamic post-translational modification (PTM) that is central to the regulation of eukaryotic transcription . It reguSaccharomyces cerevisiae retains the transcriptionally activating lysine methylation sites on H3K4, H3K36, and H3K79 (S. cerevisiae is controlled by four methyltransferase (MTase) and four demethylase (DMase) enzymes. The SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain MTases Set1p, Set2p, and Set5p methylate histone proteins on their N-terminal tails  and two mass spectrometry fragmentation methods  and electron transfer/HCD (EThcD)), applied to homologously overexpressed and purified versions of enzymes. Seven of all eight of the histone methylation proteins could be analyzed; Set1p was refractory to overexpression in via different methods, thus providing confirmatory evidence for the existence or absence of modification sites. For example, a total of 47 phosphorylation sites were identified in multiple independent mass spectrometry analyses  and LysargiNase-generated  peptides. Similarly, novel phosphorylation at serine 30 of Dot1p was confirmed by exemplar HCD and EThcD fragmentation spectra of chymotryptic  and Asp-N-generated peptides , respectively.To establish the extent to which histone MTase and DMase enzymes are connected to the intracellular signaling network, mass spectrometry data were searched for the presence of phosphorylation. Putative phosphosites were filtered using stringent score and localization probability cutoffs, thus generating a high-confidence library of modification sites. From all analyses, we identified 180 non-redundant phosphopeptides  mapping Fig. S2e.g., Set5p, Rph1p, and Gis1p) are potential integrators of signaling information, while enzymes with few sites  are likely to be regulated through other means.With respect to the number of phosphorylation sites present on each enzyme, some striking trends emerged. Histone MTases Set1p, Set2p, Set5p, and Dot1p are all phosphorylated to similar degrees, harboring between 14 and 26 sites each . By contWe then explored the relative stoichiometry of phosphorylation for the enzymes, as purified from the yeast cell. This was done by label-free quantification of phosphopeptides and their unmodified counterparts . Our quaIn regard to the relative amount of phosphorylation that is present at specific sites, we identified some modest but appreciable differences between histone MTase and DMase enzymes. Rph1p and Gis1p, histone DMases with the largest number of phosphosites , also haHaving identified and collated a large number of phosphorylation sites on yeast histone MTase and DMase enzymes, we next examined their potential regulatory role by contextualization within relevant sequence and structural features. Firstly, phosphosites were mapped onto known protein domains  and disotop panel). Although histone MTases are still mostly ordered, their phosphorylation sites reside predominantly within disordered stretches. This enrichment of phosphorylation within disordered sequences on histone MTase enzymes is of functional relevance as such regions are known to modulate protein–protein interactions  (bottom panel). Consistent with total protein values, the majority of phosphorylation sites (>70%) on DMase enzymes are located in ordered sequence contexts.Our analysis, importantly, revealed that histone MTases and DMases have demonstrably different disorder profiles; in terms of both total protein disorder and phosphosite distribution. This may reflect distinct modes of action and/or regulation. With the exception of Set5p, histone MTases are predicted to be more disordered than their DMase counterparts, ranging between 29% (Dot1p) and 47% (Set1p) sequence disorder  . Of its ; Set1C) , 46 and S. cerevisiae , have, at least partially, been resolved (A). Given that phosphorylation is known to regulate multimerization of some yeast protein MTases . This phosphosite is surface exposed and located on a spatially accessible protrusion distal to the catalytic JmjC domain.To determine whether phosphorylation is associated with interaction interfaces, phosphosites were projected onto three-dimensional crystal structures of histone MTases and DMases. Structures for four enzymes from resolved . The strresolved A. Given ., Hmt1p ), phosph., Hmt1p B. This pC). Notably, two phosphorylation sites previously reported on Set1p . The region on Swd1p that physically contacts Set1p is rich in negatively charged aspartate and glutamate residues, thus Set1p phosphosites could impede Swd1p binding and subsequent Set1C assembly.Set1p is the catalytic constituent of the multi-subunit COMPASS, or Set1C, H3K4 MTase complex, for which the molecular stoichiometry and three-dimensional configuration are known C. Notablon Set1p , at threon Set1p C, inset.A), with the exception of the yeast-specific MTase, Set5p. Strikingly, of the 19 phosphorylated amino acids in yeast that are conserved at the sequence level in human, six are known to be phosphorylated in vivo (A) (B).We explored the evolutionary conservation of phosphoresidues on histone MTase and DMase enzymes in eukaryotic orthologs. To determine the degree of conservation in mammals, pairwise sequence alignments were performed between all yeast histone MTases and DMases and their equivalent human proteins A, with t in vivo A 49, 50, 50A, wivivo (A) . More brvivo (A) B.Figure There is a paucity of kinases known to phosphorylate specific sites on histone MTase and DMase enzymes in yeast. As such, our understanding of the biological function of phosphosites and the network context of their cognate proteins is fragmentary. Using publicly available phosphoproteomic datasets, interaction databases, and prediction tools, we modeled possible connections between the histone methylation enzymes and cellular signaling pathways . Our drae.g., Rim15p, Sch9p, and Tpk2p). Interestingly, our network analysis revealed a highly interconnected and integrated regulatory structure whereby kinases may phosphorylate multiple sites on the same or different enzymes. For example, seven observed phosphosites on Set5p are predicted to be regulated by the sphingolipid signaling kinase, Pkh1p, and five sites on Set1p are likely phosphorylated by Hsl1p, a mitogen-activated protein kinase , that may function cooperatively with phosphorylation to regulate Set2p activity through an electrostatic mechanism. Accordingly, multiple phosphorylation sites at the N-terminus of mammalian heterochromatin protein 1α are known to regulate its recruitment to chromatin . By comparison, a set2Δ negative control showed complete loss of all H3K36 methylation states , the overall H3K36 methylation of each SET2 mutant yeast strain was compared with the wild-type control. This revealed significant differences for triple phosphonull (p = 2.0 × 10−16), triple phosphomimetic (p = 2.2 × 10−9), and S8D (p = 1.1 × 10−4) Set2p phosphomutants when compared with the wild-type control . These changes were characterized by a lower level of H3K36 trimethylation and a higher level of unmodified H3K36. Other mutants, however, did not show any statistically significant differences in H3K36 methylation.For functional characterization of Set2p, phosphonull  and phosphomimetic (serine-to-aspartate) mutations of phosphosites were engineered into the bination . Six strtrometry . In yeastrometry B. By comB). This observation suggests that there is a minimum charge requirement within this region for Set2p to retain wild-type levels of activity. With respect to phosphomimetic substitutions, each confers a single, but not double negative charge. Accordingly, single-site mutations  did not markedly affect H3K36 methylation, while the triple phosphomimetic substitution  reduced the relative abundance of H3K36 trimethylation to ∼12% . This model highlights the likely importance of charge in the N-terminus of Set2p, as the measured methylation of H3K36 in triple phosphomimetic and triple phosphonull mutations corresponded to the capacity for these mutant proteins to carry negative charge.Given that the degree of negative charge in the N-terminal region of Set2p may regulate its interaction with histones, we examined the net possible charge on amino acids 1 to 15 of Set2p, with and without mutations, and its association with H3K36 methylation. We considered one positive charge each for the N-terminus and lysine 3, six potential negative charges from phosphorylation sites at serine residues 6, 8, and 10, and one positive and four negative charges within acidic patch EDEKE. From this, wild-type Set2p can carry a net charge of −7 in its N-terminal region, while Set2p phosphomutants have a reduced capacity to hold negative charge . With reFig. 7Having identified extensive post-translational phosphorylation of yeast histone MTases and DMases, we sought to establish the extent to which enzymes are subject to other modification types. Akin to our systematic approach to phosphosite discovery, combinatorial mass spectrometry data were queried for the presence of acetylation, methylation, ubiquitination, SUMOylation, ADP-ribosylation, and crotonylation. Altogether, we identified 92 acetylation sites across all enzymes that were analyzed and discovered two ubiquitination sites on Jhd1p . Our anaS. cerevisiae is modest. Similar to the distribution of phosphorylation across enzymes, different histone MTases and DMases are also acetylated to markedly different extents. Three-quarters of all acetylation sites are found on just three enzymes: Set2p, Set5p, and Jhd2p, which harbor 37, 23, and 15 sites, respectively. Mapping of sites onto linear protein sequences revealed that acetylation generally occurs outside of MTase and DMase domains. Of the 101 acetylation sites identified to date, 27 (26.7%) are located within catalytic or regulatory domains  confirms previous reports; however, the number of proteome-scale studies of protein acetylation in  domains . It is i domains  but show domains . Conversin vivo, suggesting that there may be interplay between two competing modifications of the same residue. More broadly, it will be interesting to determine whether closely positioned modifications of different types participate in crosstalk on histone MTase and DMase enzymes. For example, three acetylation sites are proximal to phosphorylation at serine 572 of Set2p and may affect kinase recognition . PhosphHistone methylation enzymes are phosphorylated to strikingly different extents. They are thus likely to be regulated through different mechanisms. This was particularly apparent for the two evolutionarily related pairs of histone DMases in yeast. The paralogous H3K36 DMases, Rph1p and Gis1p, are heavily phosphorylated, harboring 36 and 26 phosphosites, respectively, suggesting regulation at the post-translational level by upstream signaling pathways. Rph1p and Gis1p also have the highest average relative occupancy of phosphorylation across their cognate sites, thus furthering evidence for their extensive phosphoregulation. By contrast, the Jumonji domain-containing DMases, Jhd1p and Jhd2p, receive relatively little information from the intracellular signaling network, containing just one and five phosphosites, respectively. We note that the small number of sites was not just due to differences in protein overexpression or small protein size. The phosphosites on Jhd1p and Jhd2p may function as molecular switches to activate/inactivate enzyme function in response to a specific signal, or alternatively, transcriptional  and poste.g., Rad53p, Tpk2p, Cdc28p, and Rim15p) may have the capacity to transmit information to multiple histone methylation enzymes, raising the possibility of coordinated regulation. While evidence for kinases acting on multiple MTases and/or DMases in the histone methylation network has not been demonstrated in yeast, there are several instances of this in the human cell. For example, Akt kinase phosphorylates H3K36 MTase NSD2   , 71. Morusculus) , 73. TheThe molecular interactions of histone MTases may be regulated by phosphorylation. Sites on MTases were strongly enriched within disordered regions and at interaction interfaces but largely absent or under-represented on domains. This is functionally important as intrinsically disordered regions are known to mediate protein–protein interactions  and assoGiven the aforementioned differences in the extent to which histone methylation enzymes are phosphorylated, their cognate phosphosites are likely to have different functional roles. In the case of extensively phosphorylated proteins, such as Set5p, Rph1p, and Gis1p, an obvious question emerges: do individual phosphosites carry out distinct functional roles (switch), or does the cumulative effect of multiple phosphosites fine-tune enzyme activity ? There is evidence emerging in support of both regulatory processes. Several targeted studies into histone DMases Rph1p and Gis1p have shown that specific phosphorylation events can have switch-like effects on function. For example, phosphorylation at serine 652 of Rph1p triggers its dissociation from chromatin in response to DNA damage , while pin vivo. Charge-based interactions are known to regulate the nucleosomal association of S. cerevisiae histone methylation enzymes (Chaetomium thermophilum Set2 have shown that Set2 makes extensive contact with histone proteins H2A and H3 (via phosphorylation. This model contrasts with the downstream acidic patch in Set2p (residues 31–39) that is sequence encoded and thus not tuneable nor signal responsive.Through mutagenesis studies, we have shown that a phosphorylation cluster at serine residues 6, 8, and 10 of Set2p, within an acidic and intrinsically disordered N-terminal region, affects its H3K36 MTase activity  enzymes , 82. An  enzymes . Our res enzymes , that stA and H3 ; howeverin vivo  between phosphosites reported here and those identified previously in large-scale phosphoproteomic screens, which is strong evidence that our sites reflect those in natively expressed proteins. For our novel phosphorylation sites, many of these were likely not reported previously as they are either of low phosphorylation stoichiometry, making them difficult to detect in native phosphoproteomic analyses, or because they were found using proteases other than trypsin. We also note that there is a precedent for using overexpression to identify functionally important PTMs; our previous systematic analysis of overexpressed MTases   as per the manufacturer’s instructions, except that SapI  endonuclease digestion was uncoupled from the T4 DNA ligase (New England Biolabs) reaction (GAL1 promoter and the URA3 selectable marker for auxotrophic selection in yeast (S. cerevisiae haploid strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) using the lithium acetate method with URA3 selection   were a keviously  into thereaction . All ovein yeast . Resultain yeast  and tranelection .SET2 was amplified from S. cerevisiae genomic DNA and cloned into the p426 shuttling vector using the Gibson Assembly cloning kit (New England Biolabs). Site-directed mutagenesis of p426-SET2 was performed using relevant mutagenic oligonucleotides  supplemented with glucose (0.68% (w/v) yeast nitrogen base with ammonium sulfate , 0.4% (w/v) synthetic dropout medium without uracil (Sigma Aldrich), 2% (w/v) D-glucose) were grown at 30 °C and 200 rpm shaking to an optical density at 600 nm of 0.8. To alleviate repression of GAL1 by glucose, cells were harvested by centrifugation, washed twice with sterile deionized water before resuspension in 150 ml SC-ura medium. Recombinant protein expression was then induced by the addition of 75 ml of 3× yeast extract peptone galactose medium (3% (w/v) yeast extract, 6% (w/v) bacteriological peptone, 6% (w/v) galactose) and carried out overnight at 30 °C and 200 rpm shaking. Following induction, yeast cells were harvested and stored at −80 °C until lysis and purification. Protein extraction and affinity purification of hexahistidine-tagged enzymes were performed as per  using Amicon Ultra-4 10K centrifugal filters  to a volume of 80 μl.Hexahistidine-tagged proteins were overexpressed in eviously , with thd as per , with th4HCO3), LysargiNase , Asp-N , or chymotrypsin , 10 mM CaCl2) for 16 h in 100 μl reactions. Digests with trypsin, LysargiNase, and Asp-N were at 37 °C, while chymotryptic digestion was at 25 °C. Resultant peptides were extracted from gel bands and dried down in a SpeedVac  before resuspension in 0.1% (v/v) formic acid, as described previously (Lysates (10 μl) and eluates  were separated by SDS-PAGE and prepared for liquid chromatography mass spectrometry according to , with thargiNase  .Peptide samples were separated and ionized by nano-electrospray liquid chromatography using established methods  and thenRS node was incorporated into processing workflows after database searching to provide localization probabilities for putative modification sites in the search output (ptmRS Best Site Probabilities column). False discovery rates (FDRs) for peptide-spectrum matches (PSMs) were determined by a decoy database search, and PSMs for modified peptides (FDR < 0.01) were filtered by ion score (≥30), expectation value (p < 0.05), and site localization probability (≥95%) and tabulated in To identify putative modification sites on histone MTases and DMases, mass spectrometry data were analyzed on Proteome Discoverer 2.2 (Thermo Fisher Scientific) using basic PTM analysis processing and consensus workflows with primarily default settings . Data wem/z of observed phosphorylated peptides, and their unmodified counterparts, were used to generate extracted ion chromatograms (XICs) in the Thermo Xcalibur Qual Browser 2.2, within a mass range window of 10 ppm. The ICIS algorithm was used to determine the area under the curve of XIC peaks, corresponding to either phosphorylated or unmodified versions of a peptide. The relative amount of phosphorylation present at a given site was then calculated by dividing the abundance of its cognate phosphopeptide by the total amount of peptide detected (phosphorylated + unmodified). Where phosphosites occur proximally to one another, isobaric phosphopeptides were differentially quantified by their unique chromatographic retention times. Specific details regarding phosphorylation quantification are summarized in For phosphopeptide abundance measurements, label-free quantification was performed as described previously . Brieflyhttps://visquant.cmri.org.au/). PTMs reported in the literature and on modification databases are summarized in http://www.interactivepeptidespectralannotator.com/PeptideAnnotator.html, (http://bioinf.cs.ucl.ac.uk/psipred/). For three-dimensional contextualization of phosphosites, the following yeast protein structures were retrieved from the Protein Data Bank (RCSB): Dot1p  from peptide sequences identified in database searches using an in-house script and visualized in GraphPad Prism . Modification sites were mapped, to scale, onto linear protein representations using PTM VisQuant , while D: 1U2Z, ), Rph1p D: 1U2Z, ), and SeD: 1U2Z, ). StructCaenorhabditis elegans), common fruit fly (Drosophila melanogaster), zebrafish (Danio rerio), African clawed frog (Xenopus laevis), and red junglefowl  were retrieved from UniProt (2020_04 release) and aligned by multiple sequence alignment in Geneious Prime using an identity cost matrix. Phosphorylation sites on mammalian enzymes were collated from the PhosphositePlus metadatabase  using a global alignment with free end gaps. For extended conservation analysis across Eukarya, sequences for relevant homologues of yeast enzymes in nematode  were included in the regulatory network. Kinase–substrate interaction data from Ptacek et al.  were collated from the Yeast Kinase Interaction Database  was use .SET2 mutant yeast strains were grown in triplicate 15 ml cultures to an optical density at 600 nm of 0.8 in yeast extract peptone dextrose medium (2% (w/v) D-glucose, 2% (w/v) bacteriological peptone, 1% (w/v) yeast extract). Cells were lysed in 300 μl HEPES lysis buffer (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesolfonic acid (HEPES), 100 mM NaCl, 2 mM DTT, 2 mM EDTA, 0.5% (v/v) Triton-X-100, pH 7.5), supplemented with 1× protease inhibitor cocktail (Roche), by glass-bead homogenization, as done previously  using collision-induced dissociation as per established methods K(propionyl)PHR, m/z = 539.97; monomethylated, sequence K(propionyl)SAPSTGGVK(methyl+propionyl)K(propionyl)PHR, m/z = 544.65; dimethylated, sequence K(propionyl)SAPSTGGVK(dimethyl)K(propionyl)PHR, m/z = 530.64; trimethylated, sequence K(propionyl)SAPSTGGVK(trimethyl)K(propionyl)PHR, m/z = 535.31. Label-free quantification was performed using the area under the curve of XIC peaks, as described above for phosphopeptide abundance measurement. The relative amount of methylation present at H3K36 was calculated by dividing the abundance of each methylation state by the total amount of peptide detected (unmodified + monomethylated + dimethylated + trimethylated).To quantify H3K36 methylation from mass spectrometry data, XICs were generated in the Thermo XCalibur Qual Browser 2.2 using the thylated , the expSET2 mutant was compared with the wild-type control using an ordinal logistic regression model with the four possible methylation states as response variables and the SET2 mutation as a categorical covariate (A). This regression makes the proportional odds assumption, assuming that the odds of moving between adjacent categories  are equal. Using a likelihood ratio test, we found that fitting a more complex adjacent category model (B), which does not make the proportional odds assumption, explained significant variation in the data (p = 0.01). However, a comparison of the two regression models revealed that their respective outputs were not sensitive to the proportional odds assumption, and thus we have reported the common odds ratio and its statistical significance, per mutant, from the ordinal logistic model in Figure 7B for ease of interpretability.For statistical analysis, the overall H3K36 methylation level for each ovariate A. This rry model B, which via the PRIDE (All data described in this study are contained within the article and its supporting information, with the exception of the mass spectrometry proteomics data, which has been deposited to the ProteomeXchange Consortium he PRIDE  partner The authors declare that they have no conflicts of interest with the contents of this article.


Escherichia (E.) coli EPEC, Shigella (Sh.) sonnei, Salmonella (S.) typhimurium, Klebsiella (K.) pneumoniae and Clostridioides (C.) difficile were determined. Investigated products were a yeast mono strain probiotic containing Saccharomyces (Sac.) boulardii, bacterial mono strain probiotics containing either Lactobacillus (L.) rhamnosus GG or L. reuteri DSM 17938, a multi strain probiotic containing three L. rhamnosus strains , and a multi strain synbiotic containing nine different probiotic bacterial strains and the prebiotic fructooligosaccharides (FOS). Inhibition of pathogens was moderate by Sac. boulardii and L. rhamnosus GG, medium by L. reuteri DSM 17938 and the L. rhamnosus E/N, Oxy, Pen mixture and strong by the multi strain synbiotic. Head-to-head in-vitro pathogen growth inhibition experiments can be used to differentiate products from different categories containing probiotic microorganisms and can support the selection process of products for further clinical evaluation.A variety of activities potentially contribute to the beneficial effects of probiotic bacteria observed in humans. Among these is a direct inhibition of the growth of pathogenic bacteria in the gut. The present study characterizes head-to-head the in-vitro pathogen growth inhibition of clinically relevant infectious bacterial strains by different types of probiotics and a synbiotic. In-vitro growth inhibition of  The invention of antibiotic therapy some 100 years ago was a major step forward in medical practice, allowing medical practitioners to manage otherwise deadly infections caused by pathogenic bacteria. However, in the recent past, the rapid increase of bacterial antibiotic resistance has become a pressing problem of global healthcare [C. difficile. Pathogenic bacteria are omnipresent in the human gut. However, as long as the gut microbiota is well balanced and diverse, no pathogen overgrowth will take place [Every orally taken antibiotic is leading to alterations of the gut microbiota, in some patients with symptoms , in others without symptoms . While ake place . Disturbke place .E. coli EPEC, Sh. sonnei, S. typhimurium, K. pneumoniae and C. difficile by representatives of different categories of products containing probiotic microorganisms are studied. The evaluated probiotics are the clinically well-established mono strain probiotics containing either Sac. boulardii [L. rhamnosus GG [L. reuteri DSM 17938 [L. rhamnosus strains , the other being a complex multi strain synbiotic containing six lactobacilli , three bifidobacteria strains (Bifidobacterium (B.) bifidum, B. longum, B. lactis) and the prebiotic FOS.Today, a huge variety of products containing probiotic microorganisms are available. These products can be categorized according to their composition: (i) yeast and bacterial products, (ii) mono strain and multi strain products or (iii) probiotic and synbiotic products. In the present study, the inhibitions of five bacterial pathogens, oulardii , L. rhamnosus GG  or L. reMost published in-vitro growth inhibition studies focused on the characterization of only one pathogenic bacterium and one or a limited number of products containing probiotic microorganisms. The present study evaluated the antagonistic activity of a range of products in head-to-head in-vitro growth inhibition experiments with a number of different bacterial pathogens, which can support the selection of products for future, more in-depth investigations.Sac. boulardii probiotic Enterol®  contains in each capsule 4.5 × 109 colony forming units (CFU) of the Sac. boulardii strain CNCM I-745. Dicoflor®  contains 6 × 109 CFU of L. rhamnosus GG ATCC© 53103 per capsule. BioGaia®,  contains 108 CFU L. reuteri DSM 17938 per 5 drops. Lakcid®  contains a total of 2 × 109 CFUs as mixture of the L. rhamnosus strains E/N (40%), Oxy (20%) and Pen (40%), [® Baby  is a freeze-dried powder. Each sachet contains a total of 109 CFUs as a mixture of equal CFU amounts of L. acidophilus LA-14; L. casei R0215; L. paracasei Lpc-3; L. plantarum Lp-115; L. rhamnosus GG, L. salivarius Ls-33, B. lactis Bl-04, B. bifidum R0071, B. longum R0175 and 1.43 g of the prebiotic ingredient FOS.The yeast E. coli EPEC , Sh. sonnei ATCC© 9290TM , S. enterica subsp. enterica serovar typhimurium ATCC© 14280TM  and K. pneumoniae subsp. pneumoniae ATCC© 700603TM  the respective pathogen was inoculated on Columbia agar with 5% sheep blood  and incubated at 37 °C under aerobic conditions for 24 h [Sac. boulardii, L. rhamnosus GG, L. reuteri DSM 17938, the multi strain L. rhamnosus E/N, Oxy, Pen and the complex multi strain synbiotic) each containing 106 CFU were inoculated on MRS agar  and incubated for 48 h in the presence of 5% CO2 [For the in-vitro pathogen inhibition experiments with f 5% CO2 . After tC. difficile ATCC© 9689TM , the pathogen was cultivated under anaerobic conditions at 35–37 °C for 24–48 h on Schaedler agar  [6 CFU of the five evaluated products were inoculated on MRS agar and incubated for 48 h in the presence of 5% CO2. 10 mm diameter bars were transferred to a Mueller–Hinton agar with 5% horse blood and 20 mg/L NAD  and incubated under anaerobic conditions for 24 h.For the in-vitro pathogen inhibition studies with Germany) . SuspensFor testing a potential pathogen growth inhibitory effect of FOS, 100 μL of a solution containing 14.3 mg/mL FOS  was applied to a 10 mm filter disk that was then administered to respective pathogen testing plates. The multi strain synbiotic was tested on the same plates as a positive control.p-value < 0.01 was achieved.At the end of the incubation, measurements of inhibition zones around the tested colonies were taken from the outer edge of the colonies to the outer edge of the clear zones. Each test was performed in triplicate and the arithmetic means of the radii measuring from the edges of the colonies to the edges of the clear zones were calculated as well as the standard deviations SD . Independent T-test statistical analyses of datasets were conducted with GraphPad Prism software version 8.2 , Datasets were considered as significantly different when a In-vitro growth of all five tested pathogenic bacteria were inhibited by all evaluated products. However, the extent of in-vitro growth inhibitions by the products varied significantly for all tested pathogens .E. coli EPEC and K. pneumoniae were similar. The antagonistic effects of the tested products ranked from weak to strong inhibition as follows: L. rhamnosus GG < Sac. boulardii < L. reuteri DSM 17938 = multi strain probiotic L. rhamnosus E/N, Oxy, Pen < complex multi strain synbiotic. The inhibitory patterns found for Sh. sonnei and S. typhimurium an inhibition ranking from weak to strong of Sac. boulardii = L. rhamnosus GG < L. reuteri DSM 17938 = multi strain probiotic L. rhamnosus E/N, Oxy, Pen < complex multi strain synbiotic was found. While there was a significant difference  between the inhibitions of Sh. sonnei by Sac. boulardii or L. rhamnosus GG and the L. reuteri DSM 17938 probiotic or the multi strain probiotic L. rhamnosus E/N, Oxy, Pen, the difference of the inhibitions by these two groups of probiotics of S. typhimurium was not significant .For the inhibition of C. difficile by the tested products resembled that of Sh. sonnei and S. typhimurium. The weakest inhibitions were found for Sac. boulardii and L. rhamnosus GG. Inhibitions by L. reuteri DSM 17938 and the multi strain probiotic L. rhamnosus E/N, Oxy, Pen were intermediate and the best inhibitory effect was found for the complex multi strain synbiotic.The inhibition of the gram-positive E. coli EPEC. FOS also had no inhibitory effect on the growth of the other four tested pathogens (data not shown).As shown in A large number of studies have been published which are characterizing the in-vitro pathogen growth effects of individual probiotic microorganisms. There are also studies which compare the growth inhibitory effects of individual strains in head-to-head experiments. Less data are available for the in-vitro growth inhibitory effect of individual multi-strain products. To our knowledge there is no study published which compares the in-vitro pathogen growth inhibitory effects of probiotic products from different categories  head-to-head. The present study compares the in-vitro pathogen growth inhibitory properties of products containing probiotic microorganisms commonly used by physicians in Poland. The in-vitro pathogen growth inhibition experiments of the present study are not aiming to identify the underlying mechanisms of growth inhibition or to identify the causes of potential synergistic effects of probiotic bacteria in the multi-strain products, but to focus primarily on establishing a ranking of efficacy in this one particular experimental set-up. Inhibition of pathogen growth is only one potential effect exhibited by probiotic bacteria in the gut and other activities  are also potential contributors to their efficacy. However, in-vitro pathogen growth inhibition testing is a helpful measure that can be used to support the selection of products for further investigation, e.g., in clinical trials.For the in-vitro growth inhibition testing, five bacterial pathogens were selected which physicians encounter in their day-to-day practice. All five selected bacterial pathogens represent major health care concerns. As outlined in the following, infections with these pathogens are not always requiring antibiotic therapy. In addition, the increasing rate of developing antibiotic resistance in these bacteria has stimulated the interest in probiotics as prophylactic, alternative or adjuvant therapy for infections caused by them.Enteropathogenic E. coli EPEC is a major cause of infant diarrhea [E.coli against antibiotics [E. coli infections [E. coli growth antagonism by the yeast Sac. boulardii [E. coli have been described for a number of mono strain bacterial probiotics, among them L. rhamnosus GG [L. reuteri DSM 17938 [diarrhea . As longibiotics , probiotfections . In-vitroulardii . In contnosus GG , L. reutSM 17938  and multSM 17938 ,18,19.Shigella infections are a major public health problem in areas of poor sanitation with especially high incidence, morbidity and mortality in children [Shigella bacteria can already cause clinical disease. Most patients recover from Shigellosis without antibiotic treatment within 5−7 days. Various antimicrobial agents are effective in the treatment of severe cases of Shigellosis, however, a globally emerging antibiotic resistance is observed [Sac. boulardii has been shown to interfere with Shigella pathogenesis [Sh. sonnei by Sac. boulardii has not been published. Inhibition of in-vitro growth of Sh. sonnei has been demonstrated for L. rhamnosus GG [L. reuteri [lactobacilli [Sh. sonnei have not been demonstrated.children . Shigellobserved . Consequogenesis , howevernosus GG , for L.  reuteri  and a nuobacilli . For theS. typhimurium. Antibiotic therapy is not advised for most of the patients with S. typhimurium infection, as the infection is often self-limiting and antibiotic resistance of the bacterium is on the rise [Sac. boulardii has potential in the prevention and treatment of infections with S. typhimurium [S. typhimurium by Sac. boulardii with that of bacterial probiotics. In-vitro growth inhibition of S. typhimurium has been shown for a number of individual lactobacilli strains, [lactobacilli strains with growth inhibitory potential against S. typhimurium [S. typhimurium was more strongly inhibited by a multi strain synbiotic (different from that investigated in the present study) when compared with the in-vitro growth inhibition caused by its individual bacterial strains [Most food-borne bacterial gastroenteritis is caused by the rise . Therefothe rise , non-antthe rise . There ihimurium . Howeverstrains, ,29,30. Thimurium ,31. Our K. pneumoniae is responsible for an alarming increase in hospital infections, especially in intensive care units. More and more antibiotics have lost their efficacy against K. pneumoniae, and strains of K. pneumoniae have emerged that are resistant against most of the presently available antibiotics [K. pneumoniae has emerged. Few studies have investigated the effect of probiotics on the in-vitro growth of K. pneumoniae [ibiotics ,33. Conseumoniae ,35,36, hC. difficile infection (CDI), the exposure to antibiotics is the most important risk factor [C. difficile, production of toxins and disease development. Characteristic symptoms are diarrhea and abdominal pain. In severe cases, the CDI can result in a life-threatening pseudomembranous colitis. Recurrence of CDI is a not uncommon observation. First step in the treatment of CDI is discontinuing the therapy with the inciting antibiotic as soon as possible. Depending on severity of the CDI, three antibiotics can be considered for therapy: metronidazole, vancomycin and fidaxomicin [For an initial k factor . In thisaxomicin . In addiaxomicin .C. difficile by a number of mono strain probiotics, among them L. rhamnosus, and bifidobacteria has been described [C. difficile by representatives from different categories of products containing probiotics.In-vitro growth inhibition of escribed ,41. To oSac. boulardii and L. rhamnosus GG, intermediate for L. reuteri DSM 17938 and the L. rhamnosus E/N, Oxy, Pen mixture and strongest inhibition is found for the multi strain synbiotic. This finding is in line with the hypothesis that multi strain probiotics exhibit superior growth inhibitory effects towards pathogenic bacteria because they are combining a broader range of independent antibacterial activities, some of which might even act synergistically [The products evaluated in the present study can be categorized by the following criteria: (i) yeast/bacteria, (ii) mono strain/multi strain and (iii) probiotic/synbiotic (containing probiotic and prebiotic components). While the in-vitro pathogen growth inhibitory effects vary among the tested products, the inhibitory effects seem not to be pathogen-dependent. Weakest pathogen inhibition is observed for stically ,43. HoweAn obvious limitation of the present study is that it is using only one experimental set-up to establish an in-vitro efficacy ranking among the evaluated products. No efforts have been undertaken to investigate the underlying mechanisms of the observed pathogen inhibitions or the underlying mechanisms of potential synergies in the multi strain products. Dedicated human clinical studies will be necessary to evaluate if these in-vitro findings will translate into clinical benefits for patients.In-vitro growth inhibition of a variety of pathogens is helpful to differentiate products and product categories containing probiotic microorganisms. Based on the results of the present study, multi strain probiotics should be preferred in case a strong in-vitro growth inhibition of a broad range of pathogens is desired. Our study results can support the product selection for future clinical investigations.


Since the discovery of biological antifreeze glycoproteins (AFGPs), which can inhibit ice nucleation, there has been considerable interest in understanding their mechanisms and mimicking them in synthetic polymers. In this study, we used molecular dynamics simulations of modified polyvinyl alcohol (PVA) compounds to show that the hydroxyl (OH) group distance is a key factor in whether certain compounds promote or inhibit ice nucleation. A hydroxyl distance smaller than ~2.8 Å but greater than ~7.1 Å in modified PVA (MPVA) compounds was associated with the promotion of ice nucleation, while a hydroxyl group separation distance of approximately ~5.0 Å was correlated with a delay in ice nucleation, owing to changes in the energy of the system. Thus, these results may help explain some of the mechanisms of current known anti-freeze compounds and may have implications for designing new anti-freeze compounds in the future. Various methods have been presented for preventing ice formation, including low ice adhesion surfaces and anti-freeze agents ,2,3,4,5.Boreogadus saida) produce specific types of AFPs known as antifreeze glycoproteins (AFGPs), which limit the formation of ice in their veins, allowing them to inhabit waters ranging from 0 °C to −4 °C [In order to survive in sub-freezing waters, animals such as the Antarctic Notothenioids  10 mer molecules that were constructed from a base PVA structure. Each compound was created with varying distances between the hydroxyl (OH) groups, as shown in n = 10) resulted in the nucleation of hexagonal ice, with notable differences. MD nucleation simulations showed a clear promotion of ice nucleation in the presence of PVA (1), MPVA (3), and MPVA (4) compared to the nucleation of pure water, regardless of ice embryo constraint , are shown in nstraint a–j. Ice nstraint a,b.By contrast, MPVA (2), which had an average hydroxyl distance of ~5 Å, extended the initiation of nucleation compared to pure water by a significant margin e,f. NuclThe average hydroxyl distances in all PVA molecules changed after nucleation was complete, with MPVA (2) exhibiting an increase of 0.68 Å, and MPVA (4) decreasing by 0.63 Å. The average hydroxyl distances after and before nucleation are shown in When measured as a function of minimum hydroxyl distance, only MPVA (2) with a final average hydroxyl distance of 5725 Å  was corrx-axis, in both the constrained and unconstrained simulations. In simulations with PVA and modified PVA molecules, PVA (1), MPVA (3), and MPVA (4) molecules adsorbed on the 001 basal plane of the ice crystal after ice crystallization. By contrast, MPVA (2) adsorbed on the 100 plane  molecules produced entirely different results . The watice–Oice distances between the water molecules in the nucleated pure ice with no PVA model revealed an average of 2.689 Å, which is very close to initial/final hydroxyl distances of PVA (1) and MPVA (3): . The presence of carbonyl groups in MPVA (3) appeared to only minimally affect the initiation of nucleation. Additionally, upon nucleation, PVA (1), MPVA (3), and MPVA (4) did not significantly disturb the hexagonal crystalline structure of the ice  to occur, it appears that the hydroxyl distance (HD) for anti-freeze molecules must therefore be approximately greater than 2858 Å but less than 7117 Å: 2858Å < IN HD < 7117 Å.The MD simulations showed drastic differences in nucleation activity depending on the hydroxyl distance along the polymer backbone among the various PVA and MPVA structures in this study. An increase or decrease in the distance beyond a critical value was shown to promote or accelerate ice nucleation. It can therefore be inferred that an ideal hydroxyl distance is required to delay or inhibit ice nucleation. For nucleation inhibition  and MPVA (3) in this study.Indeed, the small differences in hydroxyl distance and their effects on ice nucleation could help to explain the IRI activity of other well-known anti-freeze compounds, such as ethylene glycol and propylene glycol. The hydroxyl distances in ethylene glycol and propylene glycol, when measured using MD, are approximately 3832 Å and 2911 Å, respectively, when measured from O⋯O. Furthermore, the hydroxyl distances in AFGP-8 ranged between ~3455 Å to ~5518 Å, when measured using MD software. This distance can be correlated to the unit cell dimensions of hexagonal crystal ice, which has a lateral lattice spacing of 4518 Å, and 7356 Å (14–20), and in our study the average Oice⋯Oice distance supports the notion that additives such as PVA can promote homogeneous crystallization by destabilizing the liquid phase when an ideal set of structures is present. Furthermore, the hydroxyl distance in MPVA (2) mimics that of liquid water, and the bending and twisting motion of the MPVA (2) polymer backbone may provide a small increase in the local kinetic energy of the system, which could act to stabilize the liquid phase. As the temperature decreases, the intramolecular motion decreases, until longer-lasting hydrogen bonds start to form between water and hydroxyl groups on the PVA backbone. These hydrogen bonds may “lock” the hydroxyls in place at 0.63 Å farther apart than on the native backbone, which induces a net strain on the MPVA (2) molecule. The combination of supplemental kinetic energy due to polymeric intramolecular motion and the additional energy required to strain the MPVA (2) molecule therefore creates an energy barrier to crystallization that inhibits nucleation. This is especially noticeable when more molecules of MPVA (2) were present, which delayed or possibly inhibited ice nucleation altogether, as shown in It can be speculated that the similarity of the OIn contrast, when the hydroxyl distance on the PVA backbone is either smaller or larger than that of liquid water, as in MPVA (4), the intramolecular motion of the PVA chain may create a local hydrogen repulsion zone (as hydrogen moves around its oxygen host), which can increase the local potential energy of the system and promote the formation of a lower energy crystalline phase away from the PVA molecule. Once the crystalline network has enveloped an MPVA (4) molecule, the hydroxyl groups are forced to move 0.68 Å closer and are again “locked” into position on the backbone. However, the strain induced in the molecule in this case is negligible due to the increase in the length of the polymer chain.To observe the effects of PVA on ice nucleation, a series of numerical ice nucleation simulations, with and without PVA molecules, were conducted using Materials Studio molecular dynamics (MD) software. Three different MPVA (10 mer) molecules (MPVA (2–4)) were constructed from a base PVA structure (PVA (1)) using Block Copolymer Builder in Materials Studio, and each MPVA compound was created by varying the distances between the hydroxyl (OH) groups, as shown in Two sets of simulations were performed for each PVA and MPVA type, with and without a constrained ice embryo present. Molecular dynamics simulations were constructed by manually placing the PVA on a pre-constructed 84.183 Å diameter water droplet, which consisted of 1705 water molecules. The water droplet used in the ice nucleation simulations was constructed by using a pre-constructed ice crystal from the Materials Studio library. A small section of the ice crystal, approximately 16 × 5 × 20 Å in size , in red n, volume V, and temperature T) at 373 K for a total of 5000 picoseconds (ps), using the COMPASS II forcefield in Materials Studio. Hydrogen bonding distance interactions were set to 19 Å to allow for the surrounding water molecules to coalesce and form a water droplet approximately 84 Å in diameter, as shown in Melting of the ice crystal was accomplished by running Forcite Dynamics NVT  for 5.000 picoseconds (ps), followed by NVT for a total of 15,000 picoseconds (ps). Temperature was held constant at 250 K, with an undercooling of 23 K, and quasi Newton–Raphson was utilized for all Forcite Dynamics nucleation simulations. Molecular dynamics simulations of the water droplet were conducted first to ensure nucleation occurred consistently with just pure water, followed by simulations containing the PVA and MPVA compounds.All nucleation simulations utilized the COMPASS II forcefield and were run using Forcite Dynamics: the module that controls the classical molecular dynamics simulations in Materials Studio. Bonding distance was set to 19 Å, and all simulations were run at standard atmospheric pressure. These simulations were first run at NVE  was conducted following the results of the initial nucleation simulations. This four-molecule model was included because ice nucleation inhibition is directly affected by the concentration of IRI compounds, and the additional nucleation simulation was therefore conducted to account for MPVA compound concentration ,23,24,25The promotion, delay, or even inhibition of ice nucleation was shown to be greatly affected by the presence of ice embryos, as well as PVA and MPVA structure, concentration, and hydroxyl group separation distance. Ice nucleation was initiated sooner when a constrained ice embryo was present in all cases. However, MPVA (2), which had an initial hydroxyl distance of 5045 Å, delayed ice nucleation by a much greater margin compared to other PVA and MPVA molecules. When a greater concentration of MPVA (2) molecules was simulated, ice nucleation did not occur in the simulation timeframe of 15,000 ps, indicating possible nucleation inhibition activity of MPVA (2). However, further investigation will be required to determine the extent of delay in ice nucleation and whether the system reached equilibrium without nucleating.Therefore, because IRI activity was found to occur when the hydroxyl group distance was between 2858 Å and 7.5117 Å, new anti-freeze compounds could be constructed based upon this example, as long as the underlying backbone composition or overall structure remains flexible.


Glycine max L.) seeds, possesses insect resistance and anti-tumor properties. But its specific mechanisms of action are not yet known. This article reports an efficient method to produce recombinant SKTI (rSKTI) in Escherichia coli, reveals some biochemical properties of rSKTI, and discusses the inhibition mechanism of SKTI. The rSKTI was expressed as inclusion body in E. coli BL21 (DE3). After refolding, the active rSKTI was obtained and was further purified with anion-exchange chromatography (DEAE-FF) efficiently. There were similar biochemical properties between SKTI and rSKTI. The optimum pH and the optimum temperature were pH 8.0 and 35 °C, respectively, being stable during pH 7.0–11.0 and below 37 °C. The activity against trypsin was inhibited by Co2+, Mn2+, Fe3+, Al3+, and epoxy chloropropane. Inhibition kinetic assay of SKTI against trypsin as Lineweaver-Burk plots analysis both showed an unchanged Km and a decreased Vmax with N-benzoyl-l-arginine ethyl ester (BAEE) as substrate. Molecular modeling showed Arg63 of SKTI (active residue of SKTI) that interacts with four residues of trypsin, including three catalytic site  and one binding site (Asp189), forming five interactions. These provide reference for understanding the inhibition mechanism of such kind of Kunitz trypsin inhibitors.Soybean Kunitz trypsin inhibitor (SKTI), extracted from soybean (The online version of this article (10.1007/s12010-020-03254-5) contains supplementary material, which is available to authorized users. Glycine max L.) is one of the most important and widely consumed legume crops in the world. SKTI, a member of the serine protease inhibitor family, is a major anti-nutritional factor in soybean seeds that can inhibit the activity of both trypsin and chymotrypsin  is the concentration of inhibitor; and KI is the inhibition constant.The kinetic parameters including Michaelis constant ((http://swissmodel.expasy.org/) was used for homologous modeling of the SKTI and trypsin. AutoDock Vina (http://vina.scripps.edu/) was used to protein-protein molecular docking and screens the feasible results. All PDB files of protein structures were visualized on PyMol (http://pymol.sourceforge.net/).SWISS-MODEL E. coli BL21 (DE3) showed a significant protein band of about 20.1 kDa, corresponding to the theoretical molecular weight of rSKTI  was dissolved in denaturation buffer  for 2 h and then diluted in refolding buffer  with a ratio of 1:10 (v:v) at 20 °C for 20 h.DEAE-FF anion-exchange chromatography was used to concentrate and purify active rSKTI successfully. The eluents were highly pure, as shown by 15% SDS-PAGE analysis  increased followed by decreased and reached the maximum at 37 °C. Meanwhile, the activity of trypsin from treatment group (trypsin containing SKTI or rSKTI) was almost consistent. The optimum temperature of inhibitor including SKTI and wild type was 35 °C Fig. . It was The SKTI and rSKTI were quite stable below 37 °C Fig. . After iUsing BAEE as the substrate, the optimal pH was determined to be pH 8.0 for SKTI and rSKTI Fig. . With thThe rSKTI was stable in pH 7.0–11.0; above 90% activities were kept, while in pH 3.0 and pH 4.0 buffers, 50% and 30% activity was lost, respectively. Therefore, we concluded that rSKTI was stable in neutral and alkaline conditions Fig. . At the 2+, Mn2+, Fe3+, and Al3+ , while it was less affected by other metal ions , which might be that the hydrolysis reaction of trypsin mainly occurred in aqueous phase instead of in organic phase . Perhaps, higher purity for rSKTI attributed to a lower KI value compared with SKTI.Kinetic parameters of trypsin in the presence and absence of inhibitors (SKTI or rSKTI) were determined, and the results are shown in Fig. nt Table . This inKm and decreased Vmax value of trypsin. The inhibition mechanism of rSKTI against trypsin might be non-competitive even if more amounts of substrates existed. In addition, the KI value of rSKTI was 2.29 μM, which was far less than Km value of trypsin that was 0.02 mM. All above suggested that the inhibition of rSKTI to trypsin was more likely to be irreversible type instead of non-competitive type. It was contrary to the previous report that the SKTI was a competitive inhibitor to trypsin [In this study, the results of inhibition kinetic assay as Lineweaver-Burk plots analysis of rSKTI against trypsin showed an unchanged  trypsin .For the inhibition mechanism between inhibitor and enzyme, it was generally accepted that the side chain of Arg63 residue in SKTI carried the positive charges, forming strong electrostatic interaction with the negative charges of the side chain of Asp189 in trypsin, significantly contributing to the binding of inhibitor to the active center of trypsin . Here, tKI value of inhibitor was far less than Km value of enzyme suggested that SKTI had better affinity and more easily bound to trypsin compared with substrate. Rühlmann et al. [Combining above three-dimensional structure analysis with inhibition kinetics behavior gave evidences that the inhibition mechanism of SKTI to trypsin should be irreversible. The fact that n et al.  found thn et al.  discoverE. coli with optimized refolding technology. Biochemical properties of rSKTI studied here should be useful for the better application of rSKTI in production. Furthermore, the specific study on inhibition kinetic behavior and molecular structure modeling of complex gave new insight into inhibition mechanism of SKTI against trypsin. These results provide reference to further research on the inhibition of other Kunitz trypsin inhibitors to their target protease.In this research, an efficient method was established to produce rSKTI in ESM 1(DOCX 52 kb)


In Europe, ticks are major vectors of both human and livestock pathogens . Agricultural landscapes, where animal breeding is a major activity, constitute a mosaic of habitat types of various quality for tick survival and are used at different frequencies by wild and domestic hosts across seasons. This habitat heterogeneity, in time and space, conditions the dynamics of these host-vector-pathogen systems and thus drives acarological risk (defined as the density of infected ticks). The principal objective of the OSCAR project (2011-2016) was to examine the links between this heterogeneity and acarological risk for humans and their domestic animals. Here, we present the data associated with this project.I.ricinus ticks - the most common tick species in French agricultural landscapes - and the prevalence of three tick-borne pathogens  in two sites in north-western (“Zone Atelier Armorique”: ZA site) and south-western  France. The distribution and density of ticks along a gradient of wooded habitats, as well as biotic variables, such as the presence and abundance of their principal domestic (livestock) and wild hosts , were measured from forest cores and edges to more or less isolated hedges, all bordering meadows. Ticks, small mammals and information on local environmental conditions were collected along 90 transects in each of the two sites in spring and autumn 2012 and 2013 and in spring 2014, corresponding to the main periods of tick activity. Local environmental conditions were recorded along each tick and small mammal transect: habitat type, vegetation type and characteristics, slope and traces of livestock presence. Samples consisted of questing ticks collected on the vegetation (mainly I.ricinus nymphs), biopsies of captured small mammals and ticks fixed on small mammals. In the VG site, livestock occurrence and abundance were recorded each week along each tick transect.This paper reports a database on the distribution and densities of I.ricinus) and trapped small mammals (N = 908) were analysed for three pathogenic agents: A.phagocytophilum, Borrelia spp. and Babesia spp.A total of 29004 questing ticks and 1230 small mammals were captured during the study across the two sites and over the five field campaigns. All questing nymphs (N = 12287) and questing adults (N = 646) were identified to species. Ticks from small mammals (N = 1359) were also identified to life stage. Questing nymphs (N = 4518 I.ricinus nymphs for A.phagocytophilum, Borrelia spp. and Babesia spp. were, respectively 1.9% [95% CI: 1.2-2.5], 2.5% [95% CI: 1.8-3.2] and 2.7% [95% CI: 2.0-3.4]. In small mammals, no A.phagocytophilum was detected, but the prevalence for Borrelia spp. was 4.2% [95% CI: 0.9-7.5]. On this site, there was no screening of small mammals for Babesia spp. In ZA site, the average prevalence in nymphs for A.phagocytophilum, Borrelia spp. and Babesia were, respectively 2.2% [95% CI: 1.6-2.7], 3.0% [95% CI: 2.3-3.6] and 3.1% [95% CI: 2.5-3.8]. In small mammals, the prevalence of A.phagocytophilum and Borrelia spp. were, respectively 6.9% [95% CI: 4.9-8.9] and 4.1% [95% CI: 2.7-5.9]. A single animal was found positive for Babesiamicroti at this site amongst the 597 tested.In the VG site, the average prevalence in  Borreliaburgdorferi s.l., the agent of Lyme disease) and livestock pathogens , with Ixodesricinus being the most commonly-involved vector.In agricultural landscapes, where livestock production occupies a large proportion of the surface area, pastures often adjoin different semi-natural ecosystems . This type of landscape mosaic implies that areas exploited by livestock are also frequently used by a diverse range of wild fauna. Many parasites and pathogens are shared amongst these animal species, even in the absence of direct contact and some may be transmitted between agricultural and semi-natural systems via common arthropod vectors. In France, ticks are major vectors for both human . While larvae and nymphs may feed on a range of different-sized hosts, adult ticks require a bloodmeal from a larger host, like roe deer Capreoluscapreolus or domestic animals  was to explore the relationships between landscape structure and acarological risk. The study was carried out in two agricultural sites that are part of the International Long-Term Ecological Research (ILTER) network (Zones Ateliers network in France http://www.za-inee.org/en/node/804) and encompass the intrinsic diversity of agricultural landscape features: one LTER site - the “Zone Atelier Armorique” (“ZA site” hereafter) - in north-western France and the second in south-western France in the region of “Vallées et Coteaux de Gascogne” . Before conducting analyses, the initial task of the OSCAR project consisted of mapping the distribution of ticks, pathogens and the principal domestic (cattle) and wild  hosts, along a gradient of landscape fragmentation, from forest cores and edges to more or less isolated hedges, all bordering meadows. This paper describes the collected datasets  on q on qOutiLaboratories involved: § BIOEPAR, # CEFS, ¶ MIVEGEC, ‡ EPIA, | ECOBIO, 1 UMR CBGP MontpellierCoordinator of the project: Plantard O. §Task managers of the project: Vourc’h G. ‡ , McCoy K.D. ¶ , Hoch T. § Site managers and contacts for samplings: Verheyden H. # for the VG  LTER site and Butet A. | for the ZA (“Zone Atelier Armorique”) LTER site.Data management and Geographic Information System (GIS): Agoulon A. §, Bastian S. §, Dorr N. ‡, Lebert I. ‡, Lourtet B. #, Mahé H. §, Rantier Y. |Sample collectionVG site: Angibault J. #, Bailly X. ‡, Bard E. ‡, Bastian S. §, Cargnelutti B. #, Cebe N. #, Chastagner A. ‡, Delrue B. §, Lebert I. ‡, Léger E. ¶, Lourtet B. #, Mahé H. §, Masseglia S. ‡, McCoy K.D. ¶, Merlet J. #, Noël V. ¶, Perez G. §,|, Picot D. #, Pion A. ‡, Poux V. ‡, Quillery E. §, Toty C. ¶, Vaumourin E. ‡, Verheyden H. #, Vincent S. ‡, Vourc'h G. ‡ZA site: Agoulon A. §, Al Hassan D. |, Armand F. §, Audiart J.-Y. §, Bastian S. §, Billon D. §, Bouju-Albert A. §, Boullot F. §,|, Bruneau A. §, Butet A. |, Daniel J. §, de la Cotte N. §, Delrue B. §, Faille F. §, Gonnet M. ‡, Hermouet A. §, Hoch T. §, Jambon O. |, Jouglin M. §, Lemine-Brahim M. §, Mahé H. §, Moreau E. §, Navarro N. §, Pavel I. §, Perez G. §,|, Plantard O. §, Quillery E. §, Rantier Y. |, Renaud J. §, Roy P. §Identification of small mammalsVG site: Bastian S. §, Butet A. |, Cèbe N. #, Chastagner A. ‡, Cosson J. 1, Léger E. ¶, Masseglia S. ‡, McCoy K.D. ¶, Noël V. ¶, Perez G. §,|, Vaumourin E. ‡, Vourc'h G. ‡ZA site: Butet A. |, Perez G. §,|, Agoulon A. §, Bastian S. §, Bouju-Albert A. §, Gonnet M. ‡, Hermouet A. §, Moreau E. §, Pavel I. §, Plantard O. §Tick identificationVG site: Pion A. ‡, Poux V. ‡ZA site: Agoulon A. §, Bouju-Albert A. §, Hermouet A. §, Plantard O. §Laboratory analysisVG site: Chastagner A. ‡, Masseglia S. ‡, McCoy K.D. ¶, Noël V. ¶, Léger E. ¶ZA site: Bouju-Albert A. §, Daniel J. §, Faille F. §, Hermouet A. §, Jouglin M. §, Léger E. ¶, McCoy K.D. ¶, Noël V. ¶, Perez G. §,|, Quillery E. §Livestock survey: (VG site only): Angibault J. #, Cargnelutti B. #, Lourtet B. #, Sevila J. #, Verheyden H. #LTER site “Vallées et Coteaux de Gascogne” (VG site)https://pygar.omp.eu/), located 75 km from Toulouse in south-western France  , either individually or in mixed groups. The roe deer density has been estimated at around 6 roe deer/km2 in open areas and more than 30 roe deer/km2 in one of the forest areas  site https://osur.univ-rennes1.fr/za-armorique) . The area includes a wide array of agricultural landscape features, a forest of about 1000 ha and many woods smaller than 50 ha. The southern part of the site is a fine-grain heterogeneous landscape with a complex network of hedgerows (160 m/ha) enclosing small fields. At the northern part of the site, agricultural intensification has led to a more homogeneous coarse-grain landscape with fewer hedgerows per hectare (70 m/ha) enclosing larger fields. The proportion of grassland is greater in the southern part, whereas fields of maize and cereal dominate the northern part. Small woods are disseminated within both northern and southern areas of the site (The ZA site (ue) Fig.  is a labthe site .The study was performed in the two LTER sites (ZA and VG) from 2012 to 2014. Questing ticks and small mammals were sampled during five field campaigns: spring and autumn 2012, spring and autumn 2013 and spring 2014. The sampling design is presented in Fig. Low Hedgerow network density (LH); Agricultural landscapes with a High Hedgerow network density (HH); Forest Edge (FE); and Forest Core (FC)  were located in 4 landscape types: Agricultural landscapes with a FC) Fig. . Small mFC) Fig. . Small m2 each (10 m length x 1 m width), with a space of 20 m between sub-transects . In each zone, one or two transect-lines were defined: 1) a single transect-line was sampled when found along hedgerows and in FC; 2) two transect-lines were run when situated at wood and forest edges (i.e. on either side of the ecotone: one in the meadow and one in the forest) Fig. . This rects Fig. .The design was fully applied (60 sampling zones) in four campaigns , but only 36 transect lines from the 24 zones used to quantify small mammal presence were sampled during autumn 2013, corresponding to an optimisation of the sampling effort during a less favourable period of tick activity.Recording local environmental conditionsGeoreferencing of sampling locations of ticks Table  and smalSampling of questing ticks2) across the lower vegetation and leaf-litter  protocol outlined in Borrelia species, positive samples were re-amplified using nested PCR protocols for the FlaB and OspC genes   . The detRNA gene . PositivRNA gene  or SNP lRNA gene .In small mammals .Livestock abundance was measured in the VG site on the pasture adjoining each tick transect-line in 2012 and 2013 Table . The numDataBaseAll the data of Tables The data presented in this dataset are detailed by campaign and by site in Table Variables not included in the datapaperhttp://eurodeer.org/ or upon request to the CEFS.Information on the infection rate and movement of roe deer in some of the studied habitat types were recorded at the VG site  and VG  sites. Additional weather data were measured near the VG site at the meteorological weather station (INRAE in SAMAN), located at the UMR DYNAFOR (INRAE-INPT) in Saint-André (F-31420) or near the ZA site at the COSTEL meteorological weather station (CNRS in COSTEL), located in the LEGT RENNES. According to the location, the weather stations were equipped with sensors to measure air and ground temperatures, air humidity, pluviometry, wind speed and direction, relative humidity, atmospheric pressure and light intensity. The data (2011-2014) are available upon request to the corresponding author.Additional variables were calculated to measure landscape heterogeneity around the sampling locations. These data and their production  are presented in VG site (19004 ha):top left 43°22'11,59''N, 0°43'59,17''E;bottom right: 43°11'41,25''N, 0°59'15,61''EZA site (14203 ha):top left 48°34'20,83''N, 1°19'21,26''W;bottom right: 48°25'20,46''N, 1°29'56,85''WOtherCreative Commons CC-BY 4.0Data from ANR OSCAR Projecthttps://data.inrae.fr/Portail Data INRAE, 4Field description of tick datasetstab1UTF-8https://data.inrae.fr/dataset.xhtml?persistentId=doi: 10.15454/93LPP7The data concerning questing tick sampling are presented in the 3 following tables.Table Table Table The date format ISO 8601 (YYYY-MM-DD) was used.Description of small mammal datasetstab1UTF-8https://data.inrae.fr/dataset.xhtml?persistentId=doi: 10.15454/93LPP7The data concerning small mammal sampling are presented in the 3 following tables.Table Table Table The date format ISO 8601 (YYYY-MM-DD) was used.Description of the livestock datasettab1UTF-8https://data.inrae.fr/dataset.xhtml?persistentId=doi: 10.15454/93LPP7Field description for the livestock dataset Table  We provide a quick description of the results in the following section. A total of 29004 questing ticks and 1230 small mammals were collected during the study at the two sites and over the five campaigns. All questing nymphal (N = 12311) and adult ticks (646) were identified to species. Ticks from small mammals (N = 1359) were also identified to the stage.I.ricinus nymphs, 646 I.ricinus adults and 24 Ixodesfrontalis nymphs were collected on the vegetation (Table During the five campaigns (from spring 2012 to spring 2014), 16047 larvae, 12287 on Table .I.ricinus nymphs, according to landscape type and field campaign. Densities were generally higher in the ZA site than in the VG site, regardless of the campaign or landscape type. However, large heterogeneities were found amongst the five campaigns in both sites.Fig. Apodemussylvaticus) were the dominant species, accounting for 75% of the captured individuals. Bank vole (Myodesglareolus) was the second most frequently-encountered species in both sites (VG: 11% and ZA: 24%).Over the study, 335 small mammals were trapped in the VG site Table  and 895 In the VG site, the forest type was mainly deciduous (N = 41) with one mixed forest (including coniferous trees). In the ZA site, collections were performed in 33 deciduous forest type and eight mixed forests. Table The livestock survey was performed in the VG site: livestock occurred on 28 of the 90 questing tick transect-lines, cattle being the main species present in pastures Table . Median I.ricinus) and 908 trapped small mammals (N = 300 in VG site and N = 608 in ZA site) were analysed for the three pathogenic agents: A.phagocytophilum, Borrelia spp. and Babesia spp.  were identified in nymphs in the two sites  amongst 597 tested was positive for Babesia (ls Table . A.phagls of ZA . Small merraneus . In the  Babesia .


Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β-lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β-lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β-lactamase from  The β-lactams are amongst the most important antibiotics , but theClinicalTrials.gov). TAN has been shown to successfully inhibit SBLs and MBLs in vitro  (k2/K’)K’ representing the equilibrium coefficient of AmpCEC inhibition in the presence of FC-5 in the set concentration of 5 µM.Plotting the resultant tion [I]  and fittMK) of FC-5 for AmpCEC and the FC-5 concentration [S] according to Equation (4) to give the second-order rate constant (k2/K):This can then be corrected using the Michaelis constant :The same data were used to determine the apparent inhibition constant Kiapp . The reck-2 or koff) were measured using the jump-dilution method . C. CEC-CB31 pocket d. These lsewhere . Such Am[E. coli , which iEC with TAN clearly reveals a second TAN molecule at the interface between chain A and symmetry related molecules , this molecule of TAN was thus modelled in a tricyclic form, as shown in recedent . An analrecedent . Overlayemblance c; howeveEC active site . Further comparisons suggest that the other SBLs, for which crystallographic studies are reported in complex with the bicyclic form of TAN  compared to acyclic boronic acids.EC reveal remarkable similarity between the binding modes of the 6,6-core ring system of the bicyclic boronates to SBLs from class A , class CTX-M-15 ,24) and ruginosa  as well hia coli . The reshia coli ,21; thisn as TAN , possiblS)-isomer indicates that stereoselective synthesis might further benefit AmpCEC inhibition. Further optimization will need to address the relatively low potency of CB3 in cells, which could reflect potential poor cell penetration of CB3 due to its lipophilicity. CB3 has a predicted logP of 4.42; studies have shown that compounds with relatively low logPs  manifest improved penetration of Gram-negative bacteria [Together with previous reports showing enhanced activity of CB3 against class D SBLs, as well as certain B2 and B3 MBLs compared to TAN and CB2 , our resbacteria , leavingbacteria .EC is of interest from a boronate chemistry perspective as it potentially further exemplifies the ability of boron-based inhibitors to interchange between different forms in aqueous solution. It should be noted that, in both the cases where a tricyclic boronate form has been observed crystallographically, i.e., with AmpCEC and the MBL NDM-1, the crystallization conditions were acidic  can bind a tricyclic form at its active site  [The observation of an apparently weakly bound tricyclic form of TAN on the surface of AmpC and 5.8 , respect and 5.8 . Compari and 5.8 . Althougrystals) , thus th


Pseudomonas aeruginosa from bathroom and kitchen sink drains in nine households. Growth inhibition of these P. aeruginosa by bacteriocins, known as pyocins in this species, was measured using pairwise inhibition assays. Carbon source usage of these isolates was measured, and genetic distance was estimated using multilocus sequencing. We found that as the distance between sites of isolation increased, there was a significantly higher probability of inhibition, and that pyocin inhibition and susceptibility vary greatly among isolates collected from different houses. We also detected support for other mechanisms influencing diversity: inhibition outcomes were influenced by the type of drain from which isolates were collected, and while we found no indication that carbon source utilization influences inhibition, inhibition was favoured at an intermediate genetic distance. Overall, these results suggest that the combined effects of dispersal limitation among sites and competitive exclusion within them maintain diversity in pyocin inhibition and susceptibility phenotypes, and that additional processes such as local adaptation and effects of phylogenetic distance could further contribute to spatial variability.Nearly all bacteria produce narrow-spectrum antibiotics called bacteriocins. Studies have shown that bacteriocins can mediate microbial interactions, but the mechanisms underlying patterns of inhibition are less well understood. We assembled a spatially structured collection of isolates of  For example, P. aeruginosa has the ability to produce three different types of pyocins: S, R and F. S-type pyocins are similar to the bacteriocins of Escherichia coli, called colicins, and are typically composed of a toxin and immunity gene. On the other hand, R- and F-type pyocins resemble phage tails and resistance is thought to be mediated through receptor incompatibility [P. aeruginosa that may or may not come into contact in the field. The observation of inhibition in these assays can be a result of the production of any type of pyocin  and indicates that processes occurring in the environment contribute to the maintenance of diversity in pyocin inhibition and susceptibility.A central goal of evolutionary biology is understanding the underlying mechanisms that generate and maintain diversity. One factor thought to promote diversity in microorganisms is the production of bacteriocins, which are narrow-spectrum antimicrobials produced by nearly all species of bacteria. These toxins differ from traditional antibiotics in that their targets are presumably restricted to members of the same species or closely related species, although there is some diversity among the phylogenetic killing range of these toxins –3. The btibility . Furthertibility –6. We usP. aeruginosa isolates collected at varying levels of spatial separation: isolates from the same drain in a human house, from different drains from the same house, and from different houses in a metropolitan region. We predict a pattern of increased inhibition outcomes as the sites of isolation become more spatial distant, based on likely dispersal limitations among these drains and the large diversity of pyocins described among P. aeruginosa, including among household isolates [One process believed to maintain diversity in bacteriocins is the presence of spatial structure in the environment, particularly when there is limited dispersal. We investigate the capacity to inhibit among isolates ,6. We alisolates .P. aeruginosa is most common in the house [P. aeruginosa recovery between bathroom and kitchen sink drains, with some studies showing P. aeruginosa to be more common in bathrooms [Another factor that can promote diversity at a global scale is local adaptation to different environments . This sthe house ,9. Previathrooms ,10 whileathrooms . This poAlthough there are indications that bacteriocin-mediated inhibition may not be directly predictive of competitive outcomes , there aP. aeruginosa by Schoustra et al. [Second, a hump-shaped relationship such that bacteriocin-mediated inhibition is maximized when two individuals are genetically related at an intermediate level  is also a et al. , but otha et al. ,13,16.P. aeruginosa, where the high production of pyocins [The goal of this study was to elucidate the underlying mechanisms that drive diversity in bacteriocin-mediated inhibition. Relationships among spatial scale, metabolic dissimilarity, genetic distance and bacteriocin inhibition have been examined in different microbial systems with differing results. Furthermore, these patterns have not yet been studied in household isolates of  pyocins ,6 sugges2.(a)P. aeruginosa [Seventy houses in the Louisville, Kentucky metropolitan area were sampled from June 2016 to May 2017. Bathroom and kitchen sink drains were sampled using a sterile cell scraper. Samples from all 70 houses were collected in sterile 1X PBS and stored at 4°C. Samples were plated onto cetrimide agar [ruginosa .P. aeruginosa was identified using a species-specific PCR reaction [For the first 12 houses, up to 13 clonal picks were performed for each drain sample to maximize likelihood of recovery. Based on results of this sampling, a standardized protocol was used in sampling of houses 13–70. In these latter houses, the minimum of all colonies on a plate or up to seven colonies were isolated and reaction .P. aeruginosa isolated  were stored in 20% glycerol at −80°C. Of these, three isolates were randomly chosen from each of the two drain types (bathroom sink drain and kitchen sink drain) in all nine houses to be used in pairwise inhibition assays. This resulted in a total of 54 P. aeruginosa isolates for the pairwise inhibition assays.Across all 70 houses, nine houses yielded a total of 105 isolates. All (b)et al. [The 54 (three isolates × two drain types × nine houses) isolates were tested for ability to inhibit and resist inhibition in all pairwise combinations according to the protocol described by Fyfe et al. . Each pa−1. The second, uninduced (‘indicator’) culture was also incubated alongside the producer culture.Briefly, individual clones of the 54 isolates were picked and used to inoculate 4 ml of LB broth. Each culture was grown for about 18 h at 28°C with shaking at 250 r.p.m. After the initial incubation period, 10 µl of this overnight culture was used to inoculate two tubes of 4 ml LB broth. Cultures were incubated with shaking at 28°C for 2–3 h. Pyocin production was then induced in one culture (the ‘producer’ culture) using mitomycin C, a DNA damaging agent that has been shown to induce bacteriocin expression , to a fi595 reading among the set of 54. Once standardized in this way, 100 µl of each indicator culture was used to inoculate 6 ml of soft agar, which was vortexed gently and poured over the agar surface of plates that had been spotted with killed producer cultures. Plates were left to incubate for 16–18 h at 28°C. Zones of inhibition of the indicator isolates were identified by visual inspection. Cases of inhibition presented as either a clear, defined zone of inhibition (indicating R or F-type pyocin) or diffuse zone of inhibition (indicating S-type pyocin inhibition); both inhibition phenotypes were considered cases of inhibition for the purposes of data collection.After 16–18 h incubation at 28°C, 5 µl of the producer cultures were spotted onto LB plates and incubated for 6 h at 28°C. 1.5 ml of chloroform was added to each plate to kill the producer colonies and plates were allowed to dry for 30 min. Producer colonies were killed prior to soft agar overlay to avoid the observation of inhibition due to a mechanism other than pyocin production, such as contact-dependent or secretion-mediated killing. In each experimental block, the indicator cultures were standardized to the lowest observed OD(c)ascA, aroE, guaA, trpE, mutL, nuoD, ppsA) used in a P. aeruginosa multilocus sequence typing scheme developed by Curran et al. [Genomic DNA was extracted from isolates using 1 ml of overnight culture and Wizard Genomic DNA purification kit . Genetic distances were estimated using Illumina whole genome sequencing and ARIBA  which wan et al. . These gn et al.  on RStudn et al. .(d)P. aeruginosa isolates to use 31 carbon sources was measured in triplicate using EcoPlates  and a protocol adapted from Schoustra et al. [595 of 0.8. Cultures were diluted by adding 10 µl of overnight culture to 10 ml of minimal salt media . After a starvation period of two hours, plates were inoculated with 150 µl of sample. The OD595 of all wells in the plate were measured immediately after inoculation (T0) and again after 48 h of incubation at 28°C. The absorbance measures from the 48-hour time point were used to calculate pairwise Bray–Curtis dissimilarity indices with R package vegan [The ability of the 54 a et al. . Culturege vegan .(e)P. aeruginosa from each drain was analysed using McNemar's test in the set of 58 houses which were sampled using a standardized sampling technique.To assess the differences in recovery from bathroom and kitchen sink drains, presence or absence of Four generalized linear mixed models were performed with the SAS v. 9.4 TS1M3 GLIMMIX procedure . Models We examined the importance of spatial scale by running a generalized linear mixed model with inhibition as a binary response variable and spatial scale as a categorical predictor variable. House of isolation was not included in this model as a predictor variable because it is confounded with spatial scale, but was evaluated in all other models. This was done by including the house of the producer, the house of the indicator, as well as the interaction between the two as random effects. We also tested the effect of drain type by running a generalized linear mixed model with inhibition as a response variable and producer drain type, indicator drain type, and the interaction between the two terms included as fixed predictor variables.We explored the hump-shaped relationship between metabolic dissimilarity and inhibition by running a generalized linear mixed model with metabolic dissimilarity and the square of metabolic dissimilarity as predictor variables and call as the response variable. Similarly, we ran a generalized linear mixed model using genetic distance and the square of genetic distance as predictor variables and call on inhibition as the response variable.3.(a)P. aeruginosa recovery. Of these 58 houses, 22 yielded P. aeruginosa from at least one drain with an overall recovery of roughly 37%. Pseudomonas aeruginosa was collected from the bathroom sink drain only in 11 houses (19%), from the kitchen sink in only five houses (9%) and from both the bathroom and kitchen sink drain in six houses (10%). Despite slightly higher recovery from bathroom sink drains than from kitchen sink drains, this analysis identified no significant difference in recovery from the two drain types .The bathroom and kitchen sink drains of 70 houses were sampled in the Louisville metropolitan area. 58 of these 70 houses were sampled using a standardized sampling protocol; this subset of houses was used for analysis of (b)We tested an isolate's ability to inhibit using an agar overlay method from a well-established protocol , which up < 0.0001; Self-on-self interactions, as expected, never resulted in inhibition P. aeruginosa [p = 0.05). In particular, when indicators from kitchen sink drains were challenged with producers from bathroom sink drains, significantly less inhibition occurred in comparison to interactions between any other combination of isolation sources, and none of the other combinations resulted in differences in inhibition  is likely driven by this single combination of drain types.Previous studies have shown that bathroom and kitchen sink drains often differ in their recovery of ruginosa ,10. Basehibition . The det(d)p = 0.4905 and 0.7486, respectively). Therefore, we found no evidence of a hump-shaped relationship between metabolic dissimilarity and inhibition. These results indicate that competition mediated by differential use of these common carbon sources may not play an important role in determining the outcomes of inhibitory interactions.To determine if metabolic dissimilarity is an important predictor of the outcome of inhibition, we measured resource use of 31 different carbon resources using EcoPlates  and calculated pairwise metabolic dissimilarities. We then ran a generalized linear mixed model with inhibition as the response variable and metabolic dissimilarity and the square of metabolic dissimilarity as predictor variables . Neither(e)P. aeruginosa genome. We looked to see if inhibition occurs most frequently at an intermediate genetic distance by running a generalized linear mixed model with inhibition as the response variable and genetic distance and the square of genetic distance as the predictor variables (p < 0.0001) and fitted a negative parameter estimate for the effect of the square of genetic distance (p = 0.0006). These estimates model the predicted hump-shaped relationship for genetic distance and bacteriocin-mediated interactions in which these interactions are predicted to occur most frequently when two isolates are intermediately genetically related P. aeruginosa than in previous sampling studies done by our group (37% as opposed to 15 and 28%) [P. aeruginosa was expected to be higher than in similar studies, for two reasons. The culture-based sampling employed the more species-specific medium cetrimide rather than Pseudomonas Isolation Agar [P. aeruginosa has previously been shown to most frequently inhabit, and despite higher recovery in this study from each drain type than in those previous studies, we found that the proportion of houses from which P. aeruginosa was recovered from both bath and kitchen sink drains was still relatively low (10%). This suggests that, in addition to dispersal into the house, within-house forces such as dispersal limitation or selective differences between bathroom and kitchen sink drains limit the distribution of this species. This is notable in part because it runs counter to the traditional characterization of this species as a ubiquitous generalist [In this study, we found a greater percentage of drains containing and 28%) . The senion Agar , and morneralist ,30.(b)P. aeruginosa may have eliminated diversity with respect to this phenotype at the within-drain level. Isolates within the same drain have either co-evolved as to not be susceptible to the particular pyocins being produced within a single drain, or pyocin sensitive isolates have been competitively excluded by producer isolates. In contrast, isolates taken from different houses that were less likely to have interacted showed greater diversity in pyocin phenotypes and were able to inhibit one another. The positive relationship among inhibition and increasing spatial scale is consistent with those of previous studies in Pseudomonas fluorescens and in species of the Xenorhabdus genus [et al. [Pseudomonas species and species in the Xenorhabdus genus [P. aeruginosa, and particularly in environmental isolates of P. aeruginosa [In our assays, we found that inhibition increases with the separation in the isolation sites of the producer and indicator . This suus genus ,16. Alth [et al. , the scaus genus ,13,16, wEscherichia coli, it has been demonstrated that in unstructured environments such as a broth culture, bacteriocin production will lead to a decrease in diversity as one strain will competitively exclude the other. However, in a structured environment such as that of an agar plate, competitive interactions occur more locally, which allows for the coexistence of multiple bacteriocin phenotypes [Previous studies have shown that spatial structure in microbial populations may lead to a decrease in local population diversity, but an increase in overall diversity across larger spatial scales ,13,16. Ienotypes .et al. [P. aeruginosa has increased fitness relative to a non-pyocin producer under conditions of local competition. This is because, when competition is localized, individuals which are genetically related to the bacteriocin producer are close by and are likely to benefit as bacteriocin production frees up resources. High rates of dispersal, however, will cause competition to occur more globally and bacteriocin production will provide less of a fitness benefit. Based on the results of our sampling study, it appears that P. aeruginosa dispersal among houses, and, to a lesser extent, within the houses, could potentially be limited. Moreover, even accounting for differences among drain type, metabolic similarity and genetic distance, significant variability in pyocin phenotype was detected among P. aeruginosa isolated from different houses. This further supports our conclusion that dispersal limitations across different houses leads to the maintenance of diversity in inhibitory interactions. Further investigation into the relationship between highly patchy environments, like the built environment, and diversity in bacteriocin inhibition in this species and others, is warranted.Similarly, Inglis et al.  also fou(c)P. aeruginosa recovery from bathroom and kitchen sink drains, with either greater recovery from bathroom drains [Previous sampling studies have yielded varying results regarding the difference in m drains ,10 or nom drains ,11; our P. aeruginosa isolates. For example, there could be differences in available resources or in microbial community compositions, either of which could affect selection through competitive interactions among P. aeruginosa. Future studies in this area could work to clarify the differences in drain types with respect to both abiotic and biotic factors.While we have not explored the particular ecological differences between bathroom and kitchen sink drains, there may be both abiotic and biotic components of these drain types that could contribute to local adaptation of (d)et al. [P. aeruginosa isolates. Differences between our results and that study could be due the fact that we used only environmental isolates, not clinical isolates and laboratory strains. Kraemer et al. [et al. [et al. [et al. [et al. [Pseudomonas species rather than within one species, and Bruce et al. [P. aeruginosa [We looked for differences in resource use among isolates to test the prediction that resource dissimilarity may help predict inhibitory interactions –14, but r et al.  found me [et al.  similarl [et al.  and Krae [et al.  could be [et al.  were like et al.  calculatruginosa .et al. [Additionally, the carbon resources in Biolog EcoPlates are biased toward plant-based carbon sources, which are probably different from the carbon sources available in sink drains. Future work addressing metabolic similarity could explore other resources over which competition might be occurring such as alternative carbon sources, nitrogen sources, and other bacterial nutritional requirements that might be more available to isolates in sink drains. It can also be noted here that Bara et al.  found th(e)et al. [et al. [et al. [et al. [We examined the relationship between genetic distance and patterns of inhibition among the isolates in this study and found strong evidence to support a hump-shaped relationship. These results are consistent with the predicted hump-shaped relationship that was observed by Schoustra et al. , though  [et al. , Kraemer [et al.  and Bruc [et al. , which eP. aeruginosa, particularly in household isolates [Pseudomonas and bacterial species [Because the measure of genetic distance that we used is based on sequence comparison of conserved housekeeping genes, it measures genetic similarity based on phylogenetic history, and is not mechanistically driven by selection on pyocin use. We know that bacteriocin production in isolates , is much species . Thus, t species ,33,34. S5.In this study, we explored many potential driving forces of diversity in pyocin-mediated inhibition using isolates collected from spatially structured bathroom and kitchen sink drains across nine different houses. Differences in the outcome of inhibition among isolates from different drain types and among isolates differing in their genetic distance suggest roles for local adaptation and/or limited local dispersal, and a signature of phylogenetic history in maintaining pyocin diversity. However, we also found strong patterns indicating an important role for dispersal limitation at a landscape level in driving patterns of pyocin-mediated inhibition. When combined with competitive exclusion within a drain, dispersal limitation can explain an increase in inhibition with spatial scale and random house-to-house variation in patterns of inhibition.