Cystoisospora suis (C. suis) are considered two important pathogens associated with diarrhoea in piglets during the suckling and early post weaning periods. To obtain an overview on the prevalence of porcine rotavirus and C. suis in piglet producing herds with solid floors and age segregated rearing, faecal sampling of 791 litters in 81 farms was performed.Piglet diarrhoea is considered a worldwide problem resulting in animal welfare problems and financial losses for pig farmers. Porcine rotavirus and the coccidian parasite C. suis, faecal samples were analysed using sedimentation. The overall prevalence of C. suis in the examined herds was 11.9 ± 15.1% at 2 weeks of age, 10.7 ± 16.7% at 4 weeks and 8.7 ± 15.3% at 6 weeks of age and the accumulated prevalence was 56, 76 and 85%. The number of empty days between farrowing batches did influence the shedding of rotavirus at 2 weeks of age but not later. Regarding C. suis, no difference in prevalence was correlated to the number of days between consecutive farrowing batches.For porcine rotavirus, faecal samples were analysed using a sandwich ELISA. The overall prevalence of rotavirus in the examined herds was 11.4 ± 17.7% at 2 weeks, 56.8 ± 30.7% at 4 weeks and 71.1 ± 29.1% at 6 weeks of age and the accumulated prevalence was 49, 97 and 100%. To detect C. suis frequently occurred in pig herds and that the number of infected litters was rather stable from two to 6 weeks of age. Consequently, both rotavirus and C. suis may play a role in intestinal disturbances in piglets during the suckling and post weaning periods despite age segregated rearing, at least in systems with solid floors. However, as this study was carried out in herds without reported problems with diarrhoea or poor weight gain, the role of these pathogens should not be overestimated.Our study confirmed that rotavirus should be regarded as an ubiquitous virus that can be expected to be present in almost every pig herd in Sweden. The study also demonstrated that the number of infected litters increased from birth to 6 weeks of age. Secondly, it showed that  Cystoisospora suis (C. suis) [Diarrhoea in piglets during the suckling and post weaning periods is recognised as a major problem across the world, resulting in animal welfare problems and financial losses for pig farmers –3. PigleC. suis) .Reoviridae family that may cause diarrhoea in the young of many mammals, including pigs [Rotavirus is an ubiquitous virus belonging to the ing pigs . Porcineing pigs . Diarrhoing pigs , 5. Rotaing pigs , 5. Rotaing pigs –6.Cystoisospora suis (previously known as Isospora suis) is a protozoan parasite belonging to the phylum apicomplexa. It is found worldwide, and infections can occur in all types of management systems [C. suis may predispose the piglet to other infections and co-infections with either viruses or bacteria are not uncommon [C. suis and Clostridium perfringens type A may worsen clinical signs and infected piglets may develop necrotic enteritis [C. suis may cause more extensive lesions in the intestine and a synergistic effect is suspected [ systems –9. Just  systems . Infecte systems , 10, 11. systems , 8, 11. uncommon –14. Expenteritis . Other sC. suis are globally considered as important gastrointestinal pathogens of suckling piglets [C. suis has previously been demonstrated in around 20% of piglets in Sweden [Both rotavirus and  piglets . A previ piglets . In the  piglets . C. suisn Sweden .C. suis in Swedish piglets, delivered by sows reared in deep litter systems and raised in pens with at least 70% solid floor, during the suckling and early weaning period.In 1989 a new animal welfare law was implemented in Sweden which states that, for pigs, a maximum of 30% of the pen area may be slatted and, consequently, 70% of the floor of must be solid. Sows must also, by law, always be kept loose  and dry By Swedish law, pigs are to be kept loose at all times including at farrowing. The slatted part of the floor must not exceed 30% of the total pen area, tails are non-docked and routine use of growth promotors has been banned since 1986 . Dry sowIn total, 81 sow herds were randomly selected from a register at the Swedish Board of Agriculture. The selected herds were sampled and a questionnaire regarding management routines was filled in. All selected herds had more than 80 sows and all but one of these herds conducted age segregated production from birth, thus emptying and cleaning each unit before entrance of new animals. The empty time between batches was 5.5 ± 4.5 days in the farrowing units and 6.2 ± 4.1 days in weaner units. Herd sizes and production results are shown in Table Escherichia coli (E. coli), they were individually treated following written instructions from the herd veterinarian. However, in 12 of the herds  all piglets were orally medicated with either antibiotics or 2500 ppm zinc oxide (ZnO) for one to 2 weeks following weaning. According to the antimicrobial policy of Sweden, laboratory tests confirming post weaning diarrhoea must have been made prior to initiating such treatments. In 14 of the herds  all piglets were treated with 20 mg per kg body weight of the anti-protozoal agent toltrazuril  during the first week of life. Before initiating such treatment, the antimicrobial policy of Sweden states that C. suis must first be verified in the herd. Four of the integrated herds treated their piglets with both toltrazuril during the first week of life and antibiotics and/or ZnO post weaning. All herds in the study weaned the piglets at no earlier than 28 days and the mean time for weaning was 36.5 ± 4.4 days.None of the herds used growth promotors and in 59 of the herds (73%), no routine treatments of pigs were carried out whatsoever. When sporadically piglets were diagnosed with diarrhoea, most often associated with In each of the 81 herds, ten litters were randomly selected. Ten faecal samples were collected from the pen floor of each selected litter. These ten samples were pooled into one sample per pen and kept cool until transported to the National Veterinary Institute in Sweden by mail. The samples were not cooled during the postal transport, which had a duration of less than 24 h. The same litters were sampled as the piglets were two, four and 6 weeks of age.Rotavirus was detected in the faecal samples using a sandwich enzyme-linked immunosorbent assay (ELISA), demonstrating virus antigen according to the instructions of the manufacturer . The ELISA was based on polyclonal antibodies to rotavirus type A.C. suis was diagnosed using sedimentation according to Telemann. In short, one gram of faeces was suspended in 5 mL of a 5% acetic acid solution and shaken. The suspension was allowed to settle for one minute and then filtered through a sieve into a centrifuge tube. An equal amount of ether was then added, and the mixture was shaken before being centrifuged for one minute. The sediment was examined for coccidian oocysts and nematode eggs using light microscopy [croscopy .P-values < 0.05 were considered statistically significant.All results are presented as mean ± standard deviation (SD) of the prevalence (%) of the herds or animals sampled. Paired student t tests and Spearman rank correlation tests between prevalence on herd levels were carried out . All herds emptied and cleaned farrowing pens before introducing new sows. The 80 herds that effectuated age segregated rearing, emptied and cleaned whole units between batches. Out of these, 38 herds also disinfected the unit always or almost always, 22 herds disinfected regularly, and 20 herds disinfected never or almost never. All herds had extra heating in the farrowing pens . Bedding was also used in all herds (straw in 79 herds and peat in two herds), occasionally complemented with saw dust. The quantity of bedding was medium to rich in all but two herds. The hygienic standard was assessed to be high in 74 herds and medium in seven herds.At two weeks of age, 81 herds and a total of 791 litters were sampled. At four weeks of age, 74 herds and a total of 727 litters were sampled and, at six weeks, 72 herds and a total of 683 litters were sampled. In 72 out of the 81 farms, samples were retrieved on all three occasions. At every farm, the same litters were sampled on each occasion.The overall prevalence of rotavirus in the examined herds was 11.4 ± 17.7% at 2 weeks, 56.8 ± 30.7% at 4 weeks and 71.1 ± 29.1% at 6 weeks of age. Rotavirus was found at all three sampling occasions in 30 out of the 72 herds (41.7%) and no herd was free from the virus at all three sampling occasions  tested positive, with 23.1 ± 18.9% positive litters. At four weeks of age, 66 of 74 herds (89%) tested positive with 63.7 ± 24.6% positive litters. Finally, at six weeks of age, 69 of 72 herds (96%) tested positive for rotavirus with 74.2 ± 25.3% of the sampled pens being positive , and in 11 of the 81 herds (15%) it was never detected  tested positive with 21.8% ± 17.6% positive litters in these herds. Finally, at six weeks of age, 27 out of 72 herds (38%) tested positive with 23.8 ± 16.4% of the sampled litters being positive  tested positive for Balantidium coli (B. coli) was found in 9.2% of the sampled litters at two weeks, 10.7% at four weeks and 25.8% at six weeks of age, respectively. Eggs from the parasitic nematodes Ascaris suum (A. suum), Trichuris suis (T. suis) and Strongylida species were also found in some of the samples from two weeks of age but these were considered transient intestinal passengers since patent infections of pigs at this young age are unlikely.C. suis was demonstrated in 10, 10 and 40% of the litters at two, four and six weeks of age, respectively. Rotavirus was, in this herd, demonstrated in 0, 50 and 60% of the litters at two, four and six weeks of age, respectively. When dividing herds into piglet producers (n = 29), satellites in multisite production (n = 9), and farrow to finish herds (n = 43), no significant difference in prevalence of rotavirus nor C. suis was found (data not shown). As seen in Table C. suis.All but one herd employed age segregated rearing. One farrow-to-finish herd with 144 sows had a continuous production system. In that herd cers n = 9, satelln = 16; 17.1 ± 19.9%, p = 0.007) to herds with three to four empty days  and to herds with five to seven empty days . P-values for herds with five to seven empty days were 0.093 and 0.065 compared to herds with less than three and three to four empty days, respectively. By merging all herds with less than five empty days , they differed significantly to herds with five to seven empty days (p = 0.020) and to herds with eight to 14 empty days (p < 0.001). At the age of four and six weeks, no difference in prevalence of litters shedding rotavirus was observed. Regarding C. suis, no difference in prevalence of positive litters was correlated to the number of days between consecutive farrowing batches , which was significantly (p < 0.001) lower than in the non-treated herds. At the age of four weeks, C. suis was found in four herds , which was also significantly (p = 0.038) lower than in the non-treated herds. Finally, at six weeks of age C. suis was demonstrated in six of the 14 herds that treated their pigs with toltrazuril , which did not differ from non-treated herds. No significant difference between toltrazuril treated and non-treated piglets was seen regarding shedding of rotavirus, weaning age, weaning weight or age at 25 kg body weight. In total, 12 of the 81 herds administered either antimicrobials or ZnO in the feed for 14 days post weaning. At no occasion did the shedding of rotavirus or C. suis nor weaning age, weaning weight or age at 25 kg body weight differ significantly from non-treated piglets.In total, 14 of the herds administrated toltrazuril to the piglets during the first week of life. In one of these herds (7.1%), C. suis frequently occurred in pig herds and that the number of infected litters was rather stable from two to six weeks of age. Thereby both these pathogens may play a role in intestinal disturbances during the suckling and post weaning periods.This study confirmed that porcine rotavirus must be regarded as an ubiquitous virus that can be expected to be present in almost every pig herd in Sweden. The study also demonstrated that the number of infected litters increased from birth to six weeks of age. Secondly, this study showed that Rotavirus has been a known cause of diarrhoea in young animals since the 1960s, with a worldwide distribution. It is considered an important cause of both impaired animal welfare and financial loss for pig farmers in Europe and porcine rotavirus was significantly more often demonstrated in neonatal piglets with diarrhoea than in control pigs in a recently published report , 24. TheC. suis was first demonstrated in pigs in 1934, but it was not until the 1970s that the clinical importance of this pathogen was really recognised [C. suis was common among piglets during the suckling and early weaning period. Despite that the number of herds where C. suis was demonstrated decreased from around 60% at two weeks of age to about 40% four weeks later, the prevalence of infected litters within a herd that remained infected, persisted over time. Still, the overall prevalence was around 50% lower when compared to earlier studies done in Sweden [C. suis shed oocysts in a cyclic fashion and shedding may vary greatly between individual animals in a litter [C. suis a challenge and it has been recommended that at least two samplings ought to be carried out. A negative sample should also be followed with at least one new sampling one week later, as clinical signs and shedding of oocyst do not always occur simultaneously [C. suis in our study, compared to the studies in the 1980s and 1990s, was supported by our study design where pooled samples collected at three separate occasions, two weeks apart, reduced the risk of false negative results at herd level. In our study, we used sedimentation and light microscopy to detect oocysts from C. suis in the faecal samples. Several diagnostic methods are available for the detection of C. suis and although the one used in this study was chosen for practical reasons it is not the most sensitive method. Highly sensitive and specific molecular diagnostic tools are available but are not practical or cost-effective to use in the field. A recent study comparing various methods for C. suis diagnosis found that autofluorescence microscopy of faecal smears is the most reliable, practical and economical diagnostic method to use in pig practice [cognised . Since tcognised , 9, 28. cognised , 29. In cognised . In thisn Sweden , 17. Thin Sweden . Pigletsa litter , 31. Thianeously . A trulypractice .C. suis oocysts which under the right conditions may become infective in 24 h [C. suis oocysts infecting piglets [C. suis oocysts spreading between batches. Still, the prevalence of C. suis positive litters was not correlated with the number of days between consecutive farrowing batches which was surprising given that longer empty periods between batches ought to reduce the load of infective oocysts as has been previously described [C. suis. It should also be mentioned that cleaning and disinfection of the farrowing pens need to be done in such a way that it is effective against coccidia. Not all disinfectants used routinely by pig farms are effective against coccidia [C. suis.Something else that supports age segregated rearing could be a reduced risk of piglets becoming infected by ingesting sporulated oocysts in a contaminated farrowing pen. This despite high ambient temperatures in farrowing pens greatly favouring sporulation of  in 24 h . Several piglets , 16, 31. piglets , 34. Thuescribed . Howevercoccidia  and fromB. coli, another protozoan parasite, was found in 25,8% of the samples at six weeks of age. This parasite is generally considered non-pathogenic in pigs and infections are usually subclinical [A. suum, T. suis and Strongylida species found in piglets aged two weeks were defined as transients due to patent infections of these parasites being unlikely in piglets at this young age.clinical . As mentE. coli infections, were in more than 70% of the herds sporadic and treated at an individual level. The reduced incidence of C. suis following treatment with toltrazuril during the first week of life, as recorded in piglets aged two and four weeks in the 14 herds (17%), proved the efficacy of the drug. Toltrazuril has been shown to be highly effective against cystoisosporosis by reducing the risk of diarrhoea, improving piglet health and reducing oocyst shedding. This has been seen under both experimental and field conditions [C. suis. Instead this highlighted the importance of good hygiene and good management routines. This has also been shown in other studies [E. coli associated diarrhoea at weaning, treated weaners with either antibiotics or ZnO in the feed. These treated herds performed equal to non-treated herds, indicating an effectiveness of the treatment. As expected, neither antibiotics nor ZnO had any influence at all on the shedding of C. suis or rotavirus, which emphasises the importance of having made a proper diagnosis before initiating treatment with such drugs [Intestinal disorders in piglets, mainly nditions –39. Howech drugs .C. suis was common in piglets with a remained prevalence of infected litters from birth to six weeks of age in the herds that tested positive for this parasite. Consequently, each of these pathogens may play a role in intestinal disturbances in piglets during the suckling and post weaning periods despite age segregated rearing, at least in systems with solid floors. Still, as this study was carried out in herds without reported problems with diarrhoea or poor weight gain, with high hygienic standard and with feed free from antibiotics, their role ought not to be overestimated. However, other factors such as concurrent infections, biosecurity and hygiene may influence the intestinal health in piglets. Hence, shortcomings in management may increase a negative impact of porcine rotavirus and C. suis with respect to health and productivity in pig herds.Porcine rotavirus was a common virus in piglets with an increased prevalence of infected litters from birth to six weeks of age. Likewise,


The volume fraction of total deformation texture is relatively independent of the thickness reduction for Δt/t0 ≤ 30%, and becomes an approximately linearly increasing function of the thickness reduction for Δt/t0 > 30%. Increasing the thickness reduction causes the increase of the Vickers hardness of the cross-section of the cold-rolled sheets, which exhibits a similar increase trend to the volume fraction of total deformation texture for Δt/t0 > 30%. A simple relation between the Vickers hardness and the thickness reduction is established and is used to curve-fit the experimental results.Using cold rolling, we plastically deform AA6061 sheets at room temperature and investigate the variations of the microstructures, textures and local deformation of the cold-rolled AA6061 sheets as functions of thickness reduction (Δt/t The demand for producing light vehicles with reduced fuel consumption has led to an increasing interest in the applications of AA6xxx aluminum (Al) alloy instead of steel for automotive industry due to reasonable strength, good formability, and corrosion resistance of AA6xxx Al alloy ,2,3,4,5.It is known that the texture, a spatial distribution of specific crystallographic orientations, plays an important role in the appearance of ridging or roping in sheet metals ,9,10, anAA6061 Al alloy is a precipitation-hardenable Al-Mg-Si alloy, with unique mechanical properties, including medium strength, formability, and corrosion resistance in comparison with other Al alloys ,17, and 0 = 10%, t0 and t are the thicknesses of the AA6061 sheet before and after the cold rolling) was achieved per rolling pass. Five different thickness reductions of 10%, 30%, 50%, 70%, and 90% were achieved. It took nine rolling passes to reach 90% thickness reduction. The resultant thickness reductions are 10%, 30%, 50%, 70%, and 90%, corresponding to the thicknesses of 5.72, 4.45, 3.18, 1.91 and 0.64, respectively. The AA6061 Al sheets of 300 mm × 300 mm × 6.35 mm were purchased from Alfa Aesar, Ward Hill, MA, USA. 4 and 950 mL DI water (deionized water) at a DC voltage of 18 V for 60 s. After the etching, the grain structures of the specimens were analyzed on an optical microscope  under polarized light. A scanning electron microscope (SEM) (JEOL 5900 LV) was used to observe the particle structures in the specimens. The as-annealed and cold-rolled samples were cut to obtain the specimens with the surface being parallel to the plane consisting of rolling direction and normal direction for the analyses of microstructures and the indentation tests. The surfaces of the specimens were manually ground and polished to obtain mirror-like surface. For the optical microscopy analysis, the mirror-like surface was electrochemically etched in a solution of 50 mL HBFα radiation for each specimen. The alpha rotation angle was in a range of 15–90° with the angle increment of 5°. The Tex Tools software  was used to calculate the 3D ODFs (orientation distribution functions) and the volume fractions of texture components. A triple of Euler angles  was used to represent the orientation intensity, following Bunge’s notation. The texture measurement of all the specimens was performed on the rolling surface, which was mechanically polished to reduce surface residual stresses and limit the stress effect on X-ray diffraction. The {111}, {200}, {220} and {311} pole figures were measured by the Schulz back-reflection method using CuKThe local deformation behavior of the polished surface (transverse cross-section) of the cold-rolled specimens was measured on a Microhardness Tester . The maximum normal load was 200 mN. The unloading time was the same as the loading time, and both were 30 s. There was no intermediate pause between the loading stage and the unloading stage. After unloading, the diagonal lengths of the indentation mark were measured on an optical microscope, and used to calculate the Vickers hardness. The area enclosed in the loading–unloading curve and the displacement axis was used to calculate the energy dissipation. According to 2Si), and the other consists of Al, Fe and Si Si). The cold-rolled specimens exhibit the β-fiber rolling texture, which extends from the {112} <111> Copper component through the {123} <634> S component to the {011} <211> Brass component. Such a result is in accordance with the results given by Wen et al. . There iThe texture effect on the deformation of metals is dependent on the types of textures . It is of great importance to examine the variation of the total components of both the recrystallization texture (softening orientations) and the deformation texture (hardening orientations). 0, as shown in Different from the work by Yang et al. , we are VH, which represents the resistance to local plastic deformation, can be calculated as:F is the normal load, and D is the diagonal length of the impression on the indented surface. The Vickers hardness, 0 > 30%. For Δt/t0 ≤ 30%, the trends are different. Such behavior may imply the possible connection between the deformation texture and the dislocation density, i.e., the variation of the deformation texture  may be associated with the increase of the dislocation density (the rate of work hardening) and the dislocation motion in FCC (face-centered cubic) metals with severe deformation. From 0 is the flow stress at the reference state of εeff = 0, εeff is the effective strain, α is a constant, μ is the shear modulus, n is strain exponent, and b is the magnitude of Burgers vector. For a cold-rolled AA6061 Al sheet, the effective strain can be calculated from the thickness reduction as:t being the thickness of the cold-rolled AA6061 Al sheet, t0 being the thickness at the state of εeff = 0, and ∆t/t0 being the thickness reduction. Substituting Equation (3) into (2) and using the relationship of V =H 3σ, we obtain:It is known that there exists strain hardening associated with multiplication of dislocations and the increase of dislocation density, which leads to the increase in the resistance to the dislocation motion. Generally, the strain dependence of plastically deformed metal, σ, can be expressed as (2)σ=σ0+n = 0.5. This result confirms the dominant role of the dislocation motion in controlling the local deformation of the cold-rolled AA6061 alloy. Such a result together with the trend shown in plasticE, as:max is the maximum indentation depth, δris the residual indentation depth, and unF is the normal load applied to the indenter for the unloading process. It is known that the plastic energy dissipated during mechanical deformation is associated with the ductility of the material. Using the loading–unloading curves shown in plasticE is a nonlinear decreasing function of Δt/t0. The specimen with Δt/t0 = 0 has the largest energy dissipation and ductility, and the cold-rolled specimen of 90% in the thickness reduction has the least energy dissipation and ductility. Such a result is due to that plastic deformation causes the increase in dislocation density. The larger the thickness reduction, the more severe is the plastic deformation, and the higher is the dislocation density. Note that plastic deformation is a function of the microstructure  in the materials, which needs to be considered in the calculation of the energy dissipation for the plastic deformation of crystallized metals.AA6xxx Al alloys have potential as structural materials in the applications of automobile and aerospace industries. It is of great importance to investigate the effect of plastic deformation on the microstructure and mechanical behavior of AA6xxx Al alloys for analyzing the structural integrity of the mechanical structures made from AA6xxx Al alloys. 0 likely due to the compression deformation of grains during the rolling. A slight difference in the grain sizes exist between the material near the free surface and the material around the center portion for a small reduction in the thickness. Increasing the thickness reduction (plastic deformation) leads to the decrease of the volume fraction of the recrystallization texture and the Cube component as well as the increase of the volume fraction of the deformation texture due to the transform of the Cube component and random texture to the components of the deformation texture. Using the cold-rolling processing, plastic deformation was introduced in AA6061 Al sheets. Grains elongate along the rolling direction during the cold rolling, which causes the changes of textures. The grain elongation is an increase function of Δt/tMicroindentations of the as-annealed and cold-rolled AA6061 Al sheets were performed. The Vickers hardness is an approximately linearly increasing function of the thickness reduction, and displays almost the same increase trend as the volume fraction of the deformation texture for the thickness reduction larger than 30%. Such behavior may imply that the variation of the deformation texture is dependent on the dislocation density and the dislocation motion in FCC metals with severe deformation. A simple relation between the Vickers hardness and the thickness reduction is established and used to curve-fit the experimental results. The curve fitting gives the strain exponent of 0.5, which confirms the dominant role of the dislocation motion in controlling the local deformation of the cold-rolled AA6061 alloy.


Competence domain through the analysis of ComEC proteins from known naturally competent species. Then, we followed this observation to identify Competence domain containing proteins (CDCPs) from all bacteria and used CDCPs as putative ComEC proteins for evolutionary analysis. A near universal presence of CDCPs was revealed, with 89% of the proteomes and 96% of the genomes encoding a single CDCP or a CDCP-like fragment. Two domains, DUF4131 and Lactamase_B, were found to commonly co-occur with the Competence domain. Ancestral state reconstruction of CDCPs over the bacterial species phylogeny suggested an origin of a Competence-only domain profile, while multiple gains and losses of the DUF4131 and Lactamase_B domains were observed among diverse bacterial lineages.Natural transformation enables the incorporation of exogenous DNA into host genomes and plays a fundamental role in the evolution of microbial populations. At the center of the natural transformation machinery, the ComEC protein mediates DNA import and serves potential functions in DNA recognition and single strand degradation. Despite its importance, the evolution of ComEC is not fully understood. Here, we aim to fill this knowledge gap by surveying putative ComEC proteins across 5,574 bacteria that span diverse phyla. We first derived the presence of a universal, core  Natural transformation (NT) is a process by which exogenous DNA is imported and incorporated into a genome. It is an important mechanism of horizontal gene transfer and as such enables DNA repair and the acquisition of new functions, such as antibiotic resistance and nutrition utilization . Due to Streptococcus pneumoniae  uptake into the periplasmic space involves proteins related to components of the type IV pilus, the type II secretion system , or the eumoniae  or by a subtilis . The sinsubtilis . In a fosubtilis .The composition of NT machinery varies among naturally competent species . SeveralB. subtilis contains three Pfam domains, a domain of unknown function (DUF4131), a transmembrane competence domain (Competence), and a metallo-beta-lactamase domain (Lactamase_B), ordered accordingly from the N-terminus to the C-terminus of the protein  among 5,574 bacteria and provide a first ancestral state reconstruction of the protein among diverse bacterial lineages.hmmscan function in the HMMER package, version 3.1b2  were identified as CDCPs following the above-mentioned criteria of domain mapping, and the CDCPs were further analyzed to enumerate the co-occurrence of additional domains. Detailed information of all CDCPs were enlisted as individual rows in Supplementary Table Escherichia coli with an occurrence of 9, 13, and 177 strains for each profile. Hence, a relative count of 0.045, 0.065, and 0.889, respectively, were assigned to each of the profiles in E. coli.A set of 5,574 complete bacterial genomes and their corresponding proteomes were downloaded from the NCBI RefSeq database  on OctobCompetence domains were searched against the database of 5,574 complete genomes using the blastn function in the BLAST+ package, version 2.2.31 , and Lactamase_B (Pfam accession PF00753), were considered in the ancestral state reconstruction. A total of five different states were assigned to each leaf of the species phylogeny based on the presence or absence of each domain, these included (1) No CDCP (NC); (2) Competence only (C); (3) Competence and Lactamase_B (CL); (4) DUF4131 and Competence (DC); and (5) DUF4131, Competence, and Lactamase_B (DCL). Ancestral state reconstruction was performed with the ‘ace’ function from the APE package, version 5.1 . While the Competence domain was universally conserved, the DUF4131 and Lactamase_B domains appeared in some but not all ComEC proteins. Overall, four domain profiles were identified , followed by the DC profile (14 occurrences). The least abundant were the CL (7 occurrences) and the C profiles (6 occurrences). The great diversity in domain compositions and sequence lengths of ComEC proteins prevents the global identification of the protein from comparison of full length sequences alone. However, due to the universal presence of the Competence domain among all known ComEC proteins, here we study the Competence domain containing proteins (CDCPs) and use them as a proxy to infer the distribution, domain profiles, and evolution of putative ComEC proteins among a diverse range of fully sequenced bacterial genomes.A list of 51 naturally competent bacterial strains were identified from the literature . Three dd Figure . The mosCompetence domains in the genomic DNA  appeared to be a truncated copy of the other (WP_058932395.1), as they share a sequence identity of 94% and the former is 330 amino acids shorter than the latter. In addition, all identified CDCPs were encoded in the chromosomes except for the strains Xanthomonas sacchari R1 and Prevotella sp. oral taxon 299 F0039, where the CDCPs were encoded in plasmids. The occurrence of CDCPs varied among different taxa. For example, in the phyla Thermotogae, Fusobacteria, Chlorobi, and Chloroflexi, a CDCP was found in every strain. In contrast, a CDCP was missing among 10.7% and 36.6% of the strains in Actinobacteria and Tenericutes, respectively.When present, only a single CDCP was found in every proteome except for the DUF4131 and Lactamase_B domains found in known ComEC proteins, five additional domains were found to co-occur with the Competence domain through the global search of bacterial proteomes  and Lactamase_B_3 (Pfam accession PF13483), the alpha/beta hydrolase families Abhydrolase_5 (Pfam accession PF12695) and Abhydrolase_6 (Pfam accession PF12697), and the SLBB domain (Pfam accession PF10531) of the beta-grasp fold  and the Lactamase_B domain was in 52.3% . In contrast, all other domains only had from one to 97 occurrences , Helicobacter pylori (83 distinct proteomes), and B. subtilis (45 distinct proteomes). Substrains of these species may contain CDCPs with distinct domain profiles. For example, of the 199 distinct E. coli proteomes analyzed, nine encoded no CDCP (NC), 13 encoded CDCPs of the CL profile, and 177 encoded CDCPs of the DCL profile  in Aquificae, Campylobacterota, Thermotogae, Deinococcus-Thermus, Chlamydiae, and Spirochaetes. The DC profile, one of the most abundant among all profiles, was the main representative in Cyanobacteria, Bacteroidetes, and Alphaproteobacteria. The DCL profile occurred in multiple taxa, but was only dominant in the Gammaproteobacteria. Additionally, a dominance of the CL, NC, and Other profiles was not observed in any taxa, and the main occurrences of these profiles were in Actinobacteria, Firmicutes, and Gammaproteobacteria.The analysis of domain distributions Figure  revealedC profile as the most parsimonious ancestral state among all bacteria, while it also revealed multiple gains and losses of the DUF4131 and Lactamase_B domains  were available, and the slope of their linear models are 0.75 and 0.67, respectively. Finally, for the Lactamase_B domain, slopes of 0.93 and 0.80, respectively, were observed for the CL and DCL profiles. All domain distances had a positive offset as compared to the species distances. The offset ranges from 0.16 to 0.25, with only an exception in the DC profile of the Competence domain, which had an offset of 0.096 . Some of the variations indicated the lateral acquisition of particular domains. For example, in the above-mentioned case studies, the acquisition of  Figures .T. azotonutricium ZAS-9  or among selected subgroups  profile appears to be the most ancient and most widely distributed among bacterial phyla (Figure C profile, and subsequently modified through multiple gains and losses of the DUF4131 and Lactamase_B domains over diverse lineages.Two domains, a Figure . This oba Figure , S2, whiCompetence domain evolve at a rate sixfold higher than the corresponding species (Competence domain of DC, DCL, and CL proteins appeared to maintain a near 1:1 slope, and that of the C profile had a lower slope of 0.61 (Figures DUF4131 and Lactamase_B in CDCPs among diverse lineages. Interestingly, no obvious HGT has been found for the DUF4131 domain among proteins that carry the DCL profile (Figure DUF4131 generally proceeds the acquisition of Lactamase_B (Supplementary Figure DCL profile are unlikely to lose the DUF4131 domain and then regain it into the resulting CL proteins.An earlier study of naturally competent strains and the ComEC proteins encoded by these strains suggests the  species . This ph Figures –D. Furthe Figure . Since tYZ conceived the project and led the data analysis and interpretation. ZP contributed to data acquisition, analysis and interpretation. All authors composed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spodoptera frugiperda ascovirus (SfAV-1a) virions contain a large unique DNA-binding protein  with a novel architecture proposed to condense its gDNA. Here we used physical, biochemical, and transmission electron microscopy techniques to demonstrate that P64’s basic C-terminal domain condenses SfAV-1a gDNA. Moreover, we demonstrate that only P64 homologs in other ascovirus virions are unique in stably binding DNA. As similar protein families or subfamilies were not identified in extensive database searches, our collective data suggest that ascovirus P64 homologs comprise a novel family of atypical large viral gDNA condensing proteins.Eukaryotic dsDNA viruses use small basic protamine-like proteins or histones, typically <15 kDa, to condense and encapsidate their genomic (g)DNAs during virogenesis. Ascoviruses are large dsDNA (~100–200 kbp) viruses that are pathogenic to lepidopteran larvae. Little is known about the molecular basis for condensation and encapsidation of their gDNAs. Previous proteomic analysis showed that  The condensation and packaging of viral genomes into capsids are essential steps in virogenesis ,2,3,4. IAutographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes a basic protamine-like protein, P6.9 (6.9 kDa), rich in arginine and serine, that condenses viral gDNA for encapsidation [A/S]RR) are present in the C-terminal domain . To deteThe ability of rP64, rN-term and rC-term to condense SfAV-1a gDNA was further analyzed by TEM of negative stained samples of protein-gDNA complexes using well established methods . TEM revIn database searches, we identified P64 homologs with a similar 2-domain architecture ; these hIn our extensive database searches, we did not identify proteins that contained similar distinct N-terminal and C-terminal domains and motifs that conformed to the architecture of the ascovirus P64 homologs.In the present study, we demonstrate that cationic P64 and its structural domains and basic motifs independently bind, precipitate, and condense SfAV-1a gDNA. As P64 (i) is a structural component of the virogenic stroma ; (ii) isWhereas arguably it is expected that the highly basic arginine/serine-rich C-terminal domain of P64 is primarily involved in DNA condensation, the role of the N-terminal domain is less clear, as it precipitates SfAV-1a gDNA comparable to the C-terminal domain B, likelypI of >9; (ii) one or several vs2C-ad motifs; and (iii) one or several kinds of motifs repeated in tandem and rich in arginine and serine residues. Two groups of proteins matched these criteria and were specific to members of Ascoviridae and Iridoviridae, which interestingly, are considered to be ancestral to AVs [Iridovirus and the Chloriridovirus, and two representatives of a single species of unclassified crustacean iridoviruses [Diadromus pulchellus toursvirus  [Iridovirus , in the two unclassified crustacean iridoviruses [Ranavirus genus. No relatives were found in genomes of IIV3 [With regard to the evolutionary origin of the P64 family of proteins, comparative analysis of the conserved motifs of homologs in SfAV-1a, TnAV-2c, and the HvAV isolates  suggestsl to AVs ,32. No ml to AVs . The firoviruses ,35. It i ORF008) , in two oviruses ,35, and  of IIV3 , IIV22 [ of IIV3 , IIV25 [ of IIV3 , IIV30 [ of IIV3 , and IIV of IIV3 . As expeFinally, although little has been published on the molecular biology of ascoviruses, in comparison to other insect viruses such as baculoviruses, it is clear that these entomopathogens are unique among all known viruses with regard to their ultrastructure, pathobiology, and cytopathology ,20. For Our study shows that, invariably, ascoviruses encode a novel basic protein composed of a bipartite architecture in which the N-terminal domain contains multiple copies of a virus-specific 2-cysteine adaptor (vs2C-ad) motif  with an intervening stretch of basic amino acids between two vs2C-ad, and multiple tandem repeats of an arginine/serine-rich motif in the C-terminal domain. This protein, first identified and reported in SfAV-1a and named P64 ,24, and


Active manipulation of spin waves is essential for the development of magnon-based technologies. Here, we demonstrate programmable spin-wave filtering by resetting the spin structure of pinned 90° Néel domain walls in a continuous CoFeB film with abrupt rotations of uniaxial magnetic anisotropy. Using micro-focused Brillouin light scattering and micromagnetic simulations, we show that broad 90° head-to-head or tail-to-tail magnetic domain walls are transparent to spin waves over a broad frequency range. In contrast, magnetic switching to a 90° head-to-tail configuration produces much narrower and strongly reflecting domain walls at the same pinning locations. Based on these results, we propose a magnetic spin-wave valve with two parallel domain walls. Switching the spin-wave valve from an open to a closed state changes the transmission of spin waves from nearly 100 to 0%. Active control over spin-wave transport through programmable domain walls could be utilized in magnonic logic devices or non-volatile memory elements. 3 film with abrupt rotations of uniaxial magnetic anisotropy.Magnon-based spintronic devices crucially rely on the capability of spin wave manipulation. Here the authors achieve active control of spin wave transmission by programming a pinned 90 degree Néel domain wall in a continuous CoFeB/BaTiO High group velocities2, long decay lengths3, and the ability to tailor the wavelength of spin waves down to the nanoscale6 offer fascinating prospects for magnonics. Logic devices based on a Mach–Zehnder spin-wave interferometer have been proposed and realized10. In this geometry, spin waves are launched into the interferometer branches, and their phase or amplitude is controlled by an Oersted field9 or spin-wave current from a third magnetic terminal10. Interference of spin-wave signals after manipulation determines the logic output. Other logic concepts exploit reprogrammable magnonic crystals12. Here, spin-wave transmission is controlled actively by a spatial modulation of magnetic properties using, for instance, electric currents13 or optical pulses14. Besides these dynamic approaches, field-induced toggling between ferromagnetic and antiferromagnetic states in magnetic stripe arrays has been shown to modify spin-wave transport in a non-volatile way16.Wave-like computing based on spin waves has generated interest as a potential low-power and parallel computing alternative for conventional CMOS technologies18. It has been analytically calculated that spin-wave transport through an infinitely extended one-dimensional Bloch wall induces a finite phase shift without reflection19. In contrast, interactions between spin waves and 180° Néel walls or magnetic domain walls in confined geometries are more complex20. Dynamic stray fields in such domain walls reduce the transmission of spin waves if their wavelength exceeds the wall width22. Additionally, domain-wall resonances limit the transmission of spin waves at specific frequencies26. This effect, known as resonant reflection, relates strongly to the spatial inhomogeneity of the effective magnetic field inside the wall. Thus, because of dynamic stray fields and resonance modes, narrow domain walls reflect spin waves more than broad walls. In many studies, the interaction between spin waves and magnetic domain walls is investigated as a new method to drive walls into motion29. It has been shown that the direction and velocity of wall motion depend strongly on the spin-wave transmission coefficient. For coefficients close to unity, transfer of angular momentum causes domain walls to move against the spin waves28. In contrast, strong spin-wave reflection induces forward domain-wall motion26.Non-collinear spin structures, such as magnetic domain walls, can also be exploited to control the amplitude or phase of spin wavesIn this paper, we propose the use of programmable domain walls for active spin-wave manipulation Fig. . Periodi31. The head-to-tail domain walls in these experiments act as barriers for spin-wave transport. To realize active switching between such narrow domain walls and broader 90° walls with a head-to-head or tail-to-tail structure, we couple a 50 nm thick ferromagnetic CoFeB film to a ferroelectric BaTiO3 substrate with regular ferroelastic stripe domains , X-ray photoemission electron microscopy (XPEEM), and micromagnetic simulations on CoFeB/BaTiO3 and related systems have previously shown that the width (δ) of 90° head-to-head/tail-to-tail domain walls increases sharply with CoFeB film thickness (t) because of their large magnetostatic energy (δ ∝ t)38. In contrast, the width of 90° head-to-tail domain walls is almost entirely determined by a competition between ferromagnetic exchange and the strength of magnetic anisotropy and, thus, varies much less with t36. Consequently, the difference in domain wall width is more pronounced in thick ferromagnetic films. In our experiments, we focus on 50-nm-thick CoFeB because it combines full imprinting of magnetic stripe domains  and 1.6 μm . Spin waves were excited locally by an out-of-plane sinusoidal magnetic field at the center of one of the domains, i.e., 10 μm from the pinned domain wall. More details on micromagnetic simulations are given in the Methods section.We performed micromagnetic simulations in MuMax3Figure x = ±20 nm surround this peak. The non-uniform field profile produces a resonance mode that is characterized by two oscillatory out-of-phase antinodes on opposite sides of the domain-wall center  and spin-wave scattering time (τ), mz(x) ∝ exp(−x/vgτ), where vgτ corresponds to the spin-wave decay length (ld). Since the spin-wave scattering time is approximated by τ = 1/2παf41, we can write mz,DW/mz,NoDW ∝ exp). Because the spin-wave group velocity is enhanced inside the head-to-head domain wall , this expression predicts an exponential increase of mz,DW/mz,NoDW with frequency. We emphasize that Fig. mz,DW/mz,NoDW ratio rather than the amplitude of spin waves after passing the head-to-head domain wall . The amplitude of spin waves decreases with frequency because of an overall reduction of the spin-wave decay length, as illustrated by a comparison of the dashed orange curves in Fig. Figure f = 11 GHz is shown in Fig. 36, the configuration with broad domain walls is fully transparent for propagating spin waves  measurements, we derived a magnetic damping parameter of 0.007. μ-BLS measurements, with and without phase resolution, were performed by scanning a laser beam spot with a diameter of about 235 nm35 and36. Previous work on high-resolution experimental characterization of pinned 90° magnetic domain walls has shown that micromagnetic simulations provide a good estimate of their width38. After selection of the domain-wall type, spin waves were excited locally by a 100 mT out-of-plane sinusoidal magnetic field. The ac field was applied to a one-cell-wide line at the center of one of the domains. To visualize propagating spin waves, we recorded the z-component of magnetization after reaching steady-state excitation. It took about 9 ns to reach this state. Besides information on propagating spin waves, the simulation signal also contained a dc component caused by ~2° out-of-plane magnetization tilting inside the domain walls. For clarity, we subtracted this dc component from the simulation data.We performed micromagnetic simulations using open-source GPU-accelerated MuMax3 software. Before spin-wave excitation, the simulation geometry was initialized by aligning the magnetization along the uniaxial anisotropy axes and letting the system reach its ground state in zero magnetic field. The domain-wall widths were calculated for this ground state using the same procedure as in Refs. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


Serratia marcescens acts as a pH-dependent translocase to deliver its effector into host cells. Based on evolutionary footprinting of LCT-T homologs, we further define an evolutionarily conserved translocase region that we show is an autonomous translocase capable of delivering heterologous cargo into host cells. Our work uncovers a broad class of translocating toxins and provides insights into LCT translocation.Large Clostridial Toxins (LCTs) are a family of six homologous protein toxins that are implicated in severe disease. LCTs infiltrate host cells using a translocation domain (LCT-T) that contains both cell-surface receptor binding sites and a membrane translocation apparatus. Despite much effort, LCT translocation remains poorly understood. Here we report the identification of 1104 LCT-T homologs, with 769 proteins from bacteria outside of clostridia. Sequences are widely distributed in pathogenic and host-associated species, in a variety of contexts and architectures. Consistent with these homologs being functional toxins, we show that a distant LCT-T homolog from  Serratia marcescens.Large Clostridial toxins infiltrate host cells using a translocation domain (LCT-T). Here, using a genomics-driven approach and functional assays, the authors uncover the presence of distant LCT-T homologs in bacteria outside clostridia and provide evidence for a toxic effector function in the gammaproteobacterium  TcdA and TcdB are the major causative agents of C. difficile infection, the leading cause of hospital-acquired diarrhea in developed countries4, while other LCTs are implicated in gas gangrene, enterocolitis and toxic shock syndrome8. Although LCTs vary in their clinical manifestation, they all have highly similar structure and function. LCTs are high molecular weight (>200 kDa) single-chain polypeptides, sharing between 36 and 90% sequence identity1 and inactivate GTPases in the Ras superfamily by glycosylation9. In order to gain entry into cells and access cytosolic GTPases, LCTs utilize their multi-domain architecture10, much like other AB toxin families, including diphtheria toxin (DT)11 and botulinum neurotoxin (BoNT)12. In brief, using their central translocation and receptor-binding domain (herein referred to as T-domain), LCTs bind cell-surface receptors and undergo receptor-mediated endocytosis. Low-pH mediated conformational changes in acidified vesicles culminates in insertion of regions of the T-domain into the endosomal membrane, resulting in formation of a translocation pore. The translocation pore facilitates passage of the LCT glycosyltransferase (GTD) and cysteine protease (CPD) into the cytosol, where the GTD is proteolytically released.Large Clostridial Toxins (LCTs) are a family of bacterial toxins comprised of six proteins 10. The LCT T-domain is much larger than the T-domain of other similar toxins , and has a unique structural fold at high pH13. The LCT T-domain at high pH is mostly composed of extended β-sheets, with a hydrophobic α-helical region that extends and wraps around the β-sheet structures. Within the β-sheet enriched region of the T-domain, four different LCT receptors have been identified  that all bind within the C-terminal region of the T-domain, with one receptor (CSPG4) binding partially to the C-terminal repeating region (CROPS) of TcdB20. The dual functionality of the LCT T-domain to bind receptors and facilitate translocation has made it difficult to disentangle receptor-binding from translocation, although several studies have concluded that the N-terminal region of the T-domain is important for pore formation and translocation. We and others have identified a pore-forming region between residues 956–111523, which maps to the hydrophobic α-helical stretch in the T-domain, and important pore formation and translocation residues clustered between residues 1035–110722. Recently, the structure of full-length TcdB was solved at endosomal pH with 3 neutralizing VHHs24. Conformational changes can be observed within the pore-forming region, although binding of a VHH within the pore-forming region and lack of a membrane prevent a complete understanding of the toxin structure at low pH and in the membrane. Outside 956–1115, the functional significance of the N-terminal region of the T-domain remains unclear. Comparison of the six LCT T-domain sequences does not reveal any striking patterns in conservation or hydropathy, and by extension, obvious clues into important functional regions10.While much is known about the enzymatically active LCT domains, the function(s) of the LCT T-domain have remained much more elusive25. Although homologs of major AB-toxins such as BoNT29, DT30, and others32 have been identified using bioinformatic approaches, there have been no genomics-driven approaches to uncover and characterize LCT homologs. For BoNT and DT, most studies have focused on identification and characterization of homologs conserving the full toxin architecture, such as the BoNT-like toxin in a commensal strain of Enterococcus faecium28. Among multi-domain homologs, the receptor (which in other toxins is distinct from the T-domain) and effector domains are the most extensively analyzed, since these domains contain well-characterized functionally important residues. Compared to receptor and effector domains, less is known about AB toxin translocation, and to date, no study has used genomics to elucidate AB toxin translocation. Since homologs have been critical to understand the function of countless other proteins, we contend that T-domain homologs have the potential to make significant strides in our understanding of toxin translocation. Understanding the process of translocation is not only critical for a complete understanding of toxin entry and uptake into cells, but also has numerous applications, both in therapeutic interventions of toxin-mediated diseases, and in biotechnology applications, such as bacterial toxin-mediated drug delivery35.In the past 5 years, genomics-driven approaches have facilitated the discovery of hundreds of bacterial toxin homologs, providing fundamental insights into toxin evolution and diversitySerratia marcescens and show that it causes changes in cell morphology and has pH-dependent translocation activity. We also leverage the LCT-T homologs to define an evolutionarily conserved translocation apparatus, which is present in all homologs. In addition to uncovering hundreds of toxins, which are linked through a shared mechanism of protein delivery into host cells, our results provide fundamental insights into translocation of the medically relevant LCTs.Here, we take a genomics-driven approach to uncover distant LCT-T homologs. We report hundreds of LCT-T homologs that are present in pathogenic and host-associated bacterial species outside of clostridia. We characterize an LCT-T homolog from C. difficile, C. perfringens, C. novyi, and Paeniclostridium sordellii, previously C. sordellii), and 769 sequences in species outside of clostridia (hereafter referred to as LCT-T homologs)  for 1023 of the 1104 sequences. LCT-T homologs are mostly distributed among the class Gammaproteobacteria (688 sequences across 32 genera); of the 32 genera, sequences are most common in Pseudomonas (419 sequences), followed by Vibrio (72 sequences), and Providencia (67 sequences)  within the Genbank database, and retrieved all sequences containing an LCT-like T-domain. To this end, a PSI-BLAST search was performed using the putative TcdB T-domain as a query . After removing partial and truncated hits 1,104 sequences were uncovered, including 335 LCT sequences found in various clostridia (gs) Fig. . Similares) Fig. . The pat37, with genes for toxin regulation  or toxin export (tcdE) ; these bacteria are generally opportunistic pathogens, and are associated with severe diseases in immunocompromised individuals43. Interestingly, the majority of remaining LCT-T homolog sequences occurred in species associated with pathogenicity in non-human hosts, including species of Vibrio, Pseudomonas, Xenorhabdus, and Photorhabdus, which are known pathogens of aquatic organisms, insects and fungi45. Notably, species of Pseudomonas, Xenorhabdus and Photorhabdus produce insecticidal toxins FitD and Mcf, which have previously noted homology to the TcdA/TcdB T-domain46. Although the association of LCT-T containing proteins with disease or infection is not known, their presence in pathogenic species strengthens the claim of a putative toxin functionality.In line with their toxin-like genomic signatures, 75% of LCT-T containing proteins were found in organisms with evidence of pathogenicity . Interestingly, in ~40% of LCT-T containing proteins, the region N-terminal to the translocase-like domain is unannotated. These unannotated regions could be explained by the presence of known LCT-related domains that fall below detection thresholds, or else potentially represent uncharacterized domains. By comparison, the putative effector types associated with DT and BoNT translocation domains were predominantly ADP-ribosyltransferases (ADPR) and peptidases, respectively, the well-known effectors of DT and BoNT . In support of a putative function as a toxin, most LCT-T homologs contain one or more LCT-like domain, with ~30% of sequences containing a glucosyltransferase (GTD-containing), ~20% with a glucosyltransferase and cysteine protease  and ~10% with a cysteine protease (CPD-containing) Fig. . We alsooNT Fig. .Fig. 2AnWith respect to the size of the translocated cargo, we found that the LCT-Ts on average translocate much larger products: LCT effectors average 965 amino acids, compared to 479 amino acids for BoNT-T homologs and 218 amino acids DT-T homologs Fig. . MoreoveSerratia marcescens (WP_073532240.1). Demonstrating even partial functionality for the S. marcescens (Sm) homolog, which lacks an annotated effector domain and shares only 20.2% sequence identity TcdB T-domain, would support the notion that the many remaining homologs that are more closely related to TcdB are also functional translocases , containing the entire sequence upstream of the suspected domain boundary with CPD; and, (Sm 36–600) in which the amino-terminal 35 largely hydrophobic residues were removed. In both cases, intracellular expression of Sm putative effector sequences resulted in profound cell-rounding compared to the empty plasmid control Fig. . Next, t22. Within the ECT there are three distinct regions  that share a remarkably similar pattern of hydropathy—one small peak, followed by two larger peaks—that map to putative membrane-insertion regions21  motif was identified as being strongly conserved in LCT-T homologs.Next, we leveraged the greatly expanded number and diversity of identified LCT-T homologs to uncover conserved molecular features of the T-domain. Alignment of the TcdB T-domain and LCT-T homologs revealed a shared core region, with a distribution of start and end sites at amino acids 815 (±6 residues) and 1514 (±99 residues), respectively Fig. ; hereafts21 Fig. . Further 22 Fig. . In addiviz., residues 800 and 851), and a variable C-terminus   in LCT-T homologs led us to hypothesize that such smaller-sized forms of the larger LCT T-domain might comprise the core machinery that is necessary and sufficient for pore-formation and translocation. To address this directly, we used the relative distribution frequency of start and end sequence coverage sites as guides to design a series of T-domain truncations in the most well-characterized LCT homolog, TcdB, generating TcdB truncations with two different N-terminal start sites (38) Fig. . In ordeWe subjected the chimeras to two rounds of experimental testing, first testing their ability to form pores, and then, their ability to translocate. To probe pore formation, we measured dye release from pre-loaded liposomes, and to assess translocation, we evaluated intoxication (indirect measure of ADPR translocation into the cytosol) and protein synthesis inhibition (direct measure of ADPR translocation into the cytosol). All TcdB truncations formed pores in our dye release assay Fig. ; howeverTo further interrogate the evolutionarily conserved  translocase, we also produced TcdB 851–1473 in a hybrid TcdB-DT system, with the GTD and CPD of TcdB and the receptor-binding domain of DT, such that the chimera was GTD-CPD-[TcdB(851–1473)]-DTR Fig. . To evalIn context of the entire TcdB T-domain, the ECT extends from one end of the T-domain to the other, is a mixture of both helical and β-sheet content and generally does not look like an independently folded protein domain Fig. ; furtherIn this work, we conducted a targeted search to identify proteins that have homology to the T-domain of TcdB—the best characterized member of the small LCT family, of which there was previously only 6 total members . Querying just the T-domain of TcdB, rather than the entire toxin, enabled identification of LCT-T homologs in bacteria outside of clostridia, and outside the conventional LCT domain architecture . Despite different genomic contexts and diverse upstream effectors, LCT-T homologs have—and are linked—by an ECT.The ECT has important implications for our understanding of LCT translocation. Primarily, the ECT is a functional translocation unit that exists within the larger scaffold of the T-domain. Within the LCT T-domain, the ECT is entwined with receptor binding moieties, which is an unexpected positioning of a functional domain. Although it is not yet clear how the ECT functions in context of the rest of the T-domain at low pH or a membrane, it is evident that the ECT does not require the rest of the T-domain to facilitate translocation. The ECT therefore reflects the minimal necessary and sufficient requirements for translocation.Within the ECT, LCT-T homologs clarify important translocation features. Principally, important residues for LCT translocation are strongly conserved in distant LCT-T homologs, indicating an essential role of these residues in translocation, that seems to persist independent of the organism and the translocating effector. Analysis of LCT-T homologs also reveals strongly conserved residues—and therefore, potentially important translocation residues—that are not apparent when only comparing sequences of the LCT family. Importantly, the conservation of residues and hydropathy pattern within the ECT suggests that LCTs and LCT-T homologs have highly similar membrane-inserted structures and translocation mechanisms, and that homologs could be used in mechanistic studies of translocation.22, we contend that the large majority of the proteins described here have the capacity to function as toxins. To demonstrate this, in part, we selected a distant LCT-T homolog from S. marcescens with an unannotated effector and low sequence identity within the ECT and showed that it was capable of inducing cell rounding by intracellular delivery of its effector. Although our work supports the claim of Sm as a toxin, it is important to note that we do not know the relevance of Sm toxin to S. marcescens infections. As is the challenge for many bioinformatically identified toxins, clarifying the toxin role in virulence is key to understanding toxin functionality. Clarifying the role of Sm toxin—and other LCT-T homologs—requires identifying a relevant host, and fulfillment of Falkow’s molecular Koch’s postulates47, which require that toxins (or virulence factors) exist only in a pathogenic strain, with mutation or deletion of the virulence factor resulting in loss of pathogenicity. We hope our work provides a starting point and framework to further interrogate the function and ecological significance of these hundreds of putative toxins.On the basis of the overall architectures of the hundreds of identified LCT-T homologs, many of which contain both a known cytotoxic effector domain and a toxin-derived autoprocessing domain that are upstream of an ECT that is replete with key sequence motifs that are essential for pore-formation and translocation48  against the NCBI non-redundant protein database (nr) on 13 June 2019. A total of 1573 protein sequences were retrieved, 1216 of which yield E-values less than 1e-5 after a single BLASTP search. Proteins labeled as ‘partial’ or otherwise truncated, as well as any proteins with <100 amino acids upstream of the translocase were removed from the dataset, leaving a final set of 1104 translocase sequences. In order to verify the relationship of the query to these sequences, pairwise comparisons between TcdB to target translocases shuffled 10,000 times were performed using the FASTA3 package (v. 36.3.8), 1023 of which produce E-values less than 1e-5.The TcdB T-domain  was used as a query for two iterations of PSI-BLAST36. Proteome-level Pfam annotations from the AnnoTree database were used to determine the domains most correlated with an annotated LCT pore-forming domain model (Pfam model PF12920), the top 100 of which are available in Supplementary Table http://www.ebi.ac.uk/metagenomics) using the TcdB translocase  as the query.AnnoTree was used for phylogenomic visualization of species containing LCT-T homologs across the bacterial tree of lifeThe association of bacterial organisms with pathogenicity was estimated based on where the organism was isolated and reviewing the literature, where possible. Broadly, an organism’s level of pathogenicity was categorized into one of four possibilities: no known pathogenicity or host association, host-associated with no known pathogenicity, known pathogen of non-human hosts, and known pathogen of humans. A detailed spreadsheet can be found in the source data file .49. Effector types were assigned using InterProScan (v5.33-72)54.The effector domains from different toxin families were retrieved by searching with each toxin’s translocase domain as a query  against the NCBI non-redundant protein database with two iterations of PSI-BLAST. For DT and BoNT, the entire portion N-terminal to the translocase hit region was extracted and treated as the effector region. For the LCT family, the effector region was more difficult to define because it contains the glucosyltransferase domain as well as the autoproteolytic cysteine peptidase domain, and not all LCT-T homologs have detectable peptidase domains. Thus, the entire region N-terminal to the translocases in proteins lacking a peptidase, and the regions N-terminal to peptidases in peptidase-containing sequences, were extracted separately to yield the set of LCT effectors. The putative effector regions from BoNT, DT, and the LCTs were clustered at increments of 5% cluster sequence identity between 50 and 100% using USEARCH49 (v10.0.240) at 90% identity. These sequences were aligned with the L-INS-I algorithm of the MAFFT package (v7.407)55, and a maximum likelihood tree was inferred using RAxML (v8.2.4)56 with automatic evolutionary model selection (LG), 4 gamma-distributed rate categories, automatic bootstrapping with autoMRE, and a thorough ML search. Protein domains were annotated using InterProScan (v5.33-72)54. AnnoTree was used for phylogenomic visualization of species containing LCT-T homologs across the bacterial tree of life36.The curated set of translocase sequences was further reduced to a set of 203 non-redundant sequences by clustering with USEARCH57. The structural context of these sites was depicted on the TcdA structure (PDB 4R04) using PyMol (https://pymol.org). Transmembrane helix prediction was estimated using the TMHMM2.0 server58. Motifs were depicted using WebLogo59.Conservation scores were calculated for residues in the reduced set of 203 translocase sequences using the ConSurf web serverE. coli (GenScript) and fused into a pET28a vector using In-Fusion HD cloning (Clontech). Sm toxin residues 1–35 were removed to improve solubility during purification. The source data file (tab Sm toxin) contains the Pfam domain annotations and hydropathy plot for Sm toxin. Regions of the TcdB T-domain were amplified from a codon-optimized TcdB gene for expression in E. coli (GenScript) and fused into a pET28a vector using In-Fusion HD Cloning (Clontech). For ADPR-[truncated TcdB T-domain]-DTR chimeras, TcdB T-domain was fused into a vector containing the diphtheria toxin ADP-ribosyltransferase (ADPR) (defined here as residues 1–201) with an intact furin cleavage site and diphtheria toxin receptor binding region (DTR) (defined as in DT as amino acids 378–535). For GTD-CPD-[truncated TcdB T-domain]-DTR chimeras, the TcdB T-domain was fused into a vector contained the TcdB GTD and CPD  and DTR. For regions of the TcdB T-domain truncation beginning at 851 or 881, a short linker (four glycine followed by one serine (G4S)) was added between the truncated TcdB T-domain, and the CPD or ADPR.Full-length Sm protein (WP_073532240.1) lacking the first 35 amino acids was synthesized and codon-optimized for expression in E. coli BL21 DE3 competent cells and expressed with N-terminal H6-SUMO and C-terminal Strep tags  or with an N-terminal H6-SUMO tag (TcdB 851–1473). For all proteins, a total of 20 mL of overnight culture was inoculated into 1.0 L of TB with 50 μg/mL kanamycin and induced at OD600 ~ 0.6–0.8 with 1 mM isopropyl-β-thiogalactopyranoside (IPTG) at 25 °C (toxin chimeras) for 4 h or 18 °C overnight (Sm toxin and TcdB 851–1473). Cells were harvested by centrifugation and resuspended with lysis buffer  and lysed by an EmusliFlex C3 microfluidizer (Avestin) at 15,000 psi. Whole cell lysates were then centrifuged at 15,000 × g for 20 min and the supernatants were passed through a 0.2-μm filter. Toxin chimeras were purified by Strep-Tactin affinity chromatography using a Strep-Tactin column  and eluted in 20 mM Tris pH 8.0, 150 mM NaCl, 1 mM D-desthiobiotin and 5% glycerol. Sm toxin and TcdB 851-1473 were purified by nickel affinity chromatography using a Ni-Penta column (Marvelgent biosciences) and eluted with an imidazole gradient. The H6-SUMO tag of Sm toxin and TcdB 851-1473 was removed by adding 1U of Sumo protease (Life Sensor), 1 mM DTT (Thermo Scientific) and incubating at 25 °C for 2 h. Sm toxin and TcdB 851–1473 were dialyzed to 20 mM Tris pH 8.0, 500 mM NaCl to remove imidazole and purified using His-Pure Ni-NTA resin (Thermo Scientific) to remove the H6-Sumo protease and H6-Sumo tag from the purified protein samples. Sm toxin was further purified by Strep-Tactin affinity chromatography as detailed above. All proteins were verified by SDS-PAGE, concentrated with a 30,000 MWCO ultracentrifugation device. Protein concentration was calculated by densitometry using ImageJ software.Sm toxin, toxin chimeras and TcdB 851–1473 were transformed into In all, 5 μg of Sm toxin in 50 mM Tris pH 8.0 was incubated with 5 mM DTT ±500 μM Insp6 (Thermo Scientific) for 20 min at 37 °C before stopping with Laemmli loading buffer with beta-mercaptoethanol (Bio-Rad). Insp6 induced autoprocessing was assessed by electrophoresing the samples on SDS polyacrylamide gels and staining with Coomassie Blue R250.2 overnight. The next day, Sm toxin was added to HCT-116 cells in a serial dilution of 1/3 and incubated at 37 °C and 5% CO2. After 24 h, light microscopy images were taken to assess cell morphology.HCT-116 cells  were cultured in McCoy’s 5 A medium (Wistent) with 10% FBS (Wisent) and 1% penicillin/ streptomycin (Wisent). HCT-116 cells were seeded at a density of 8000 cells per well in 96-well plates (Corning) and cultivated at 37 °C and 5% CO2 overnight. The next day, PiggyBac Dual Promoter PB5131B-1 vector (System Biosciences) with the effector region of Sm toxin co-expressing GFP was transfected into HeLa cells using FuGENE HD Transfection Reagent (Promega). To assess changes in cell morphology from the transfection reagent and the plasmid, an empty PiggyBac vector lacking the Sm effector was transfected as a control. Transfection efficiency was assessed after 24 h by GFP fluorescence, and light microscopy images were taken to assess the morphology of transfected cells.HeLa cells  were cultured in DMEM (Wisent) with 10% FBS (Wisent) and 1% penicillin/streptomycin (Wisent). HeLa cells were seeded at a density of 8000 cells per well in 96-well plates (Corning) and cultivated at 37 °C and 5% CO23. In brief, liposomes were prepared with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids), with 0.8% 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]  (DGS-NTA[Ni]) (Avanti Polar Lipids). After drying down with N2, the lipid film was resuspended in 20 mM Tris, 150 mM NaCl, pH 8.0, 35 mM 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and 50 mM p-xylene-bis-pyridinium bromide (DPX) (Thermo Fischer). Lipid vesicles were subjected to 10x freeze-thaw cycles and extruded using a 200 μm filter. To get rid of un-encapsulated dye, the lipid vesicles were then subjected to gel filtration and eluted in 20 mM Tris, 150 mM NaCl. To assess fluorophore leakage, protein were added in a ratio of 1:10,000 with liposomes, such that the final liposome concentration was ~400 μM. The fluorescence was monitored in a 96-well opaque plate (Corning)  in high pH buffer  or low pH buffer , or with citrate-phosphate buffers ranging from pH 4.0-pH 7.5 in 0.5 pH increments. To determine total HPTS fluorescence, Triton X-100 was added to each well to a final concentration of 0.3%. All spectra were normalized to 100% dye release by 0.3% triton. Quantification of dye release data . Fluorescence was read on a Spectramax M5 plate reader (Molecular devices). For each toxin condition, the data were blank subtracted  and normalized  and converted to fraction viable cells. From these data, dose response curves and half-maximal effective concentrations  were generated using Prism software. Individual EC50 values  were cultured in DMEM (Wisent) with 10% FBS (Wisent) and 1% penicillin/streptomycin (Wisent). Vero cells were seeded at a density of 4000 cells per well in 96-well plates (Corning) and cultivated at 37 °C and 5% CO2 overnight. Toxin chimeras were added to Vero NLuc cells in a serial dilution of 1/3 and incubated at 37 °C and 5% CO2 for 24 h. Nano-Glo Luciferase Assay substrate and buffer (Promega) were added to cells as per the manufacturer’s instructions, and luminescence was read on a Spectramax M5 plate reader at (Molecular Devices). For each toxin condition, the data were blank subtracted  and normalized  and converted to fraction protein synthesis inhibition. From these data, dose response curves and half-maximal effective concentrations  were generated using Prism software. Individual EC50 values  stably expressing NanoLuc (Nluc) Luciferase (Promega) were cultured in DMEM (Wisent) with 10% FBS (Wisent) and 1% penicillin/ streptomycin (Wisent). Vero Nluc cells were seeded at a density of 4000 cells per well in 96-well plates (Corning) and cultivated at 37 °C and 5% COues Fig.  are prov2 overnight. The next day, media was exchanged with serum-free media and cells were intoxicated by adding toxin chimeras at 1 nM. After 3 h, light microscope images were taken to assess rounding of cells.Vero cells  were cultured in DMEM (Wisent) with 10% FBS (Wisent) and 1% penicillin/ streptomycin (Wisent). Vero cells were seeded at a density of 8000 cells per well in 96-well plates (Corning) and cultivated at 37 °C and 5% CO2 overnight. The next day, media was exchanged with serum-free media and cells were intoxicated by adding toxin at 1 nM. After 1 hr, media was aspirated from cells, cells were washed with PBS and lysed by addition of Laemmli loading buffer with beta-mercaptoethanol (Bio-Rad) to each well. Samples were heated to 90 °C before immediately loading on an SDS-PAGE gel. Following electrophoresis, samples were transferred to nitrocellulose using standard wet transfer protocols, blocked with 5% milk/ Tris-buffered saline (TBS) and probed for total Rac1 (1:1000 dilution) with Anti-Rac1 antibody 23A8  or for non-glucosylated Rac1 (1:1000 dilution) with Anti-Rac1 Mab102 . Anti-α-tubulin (1:5000 dilution)  was used as the loading control. Following overnight incubation with the primary antibody, the blot was washed with TBS/0.1% Tween20 and incubated with  with Anti-mouse conjugated horseradish peroxidase  for 60 min. After the final washes in Tris-buffered saline with Tween20, chemiluminescent detection was carried out using Clarity Western ECL Substrate (Bio-Rad) and exposing to BioMax MR film (Kodak). The uncropped Western blots  were cultured in DMEM (Wisent) with 10% FBS (Wisent) and 1% penicillin/ streptomycin (Wisent). Vero cells were seeded at a density of 100,000 cells per well in 6-well plates (Corning) and cultivated at 37 °C and 5% COots Fig.  are in tg for 5 min to pellet aggregates (but not detergent). The supernatant was removed from each sample, and mixed 1:1 with Laemmli loading buffer with beta-mercaptoethanol (Bio-Rad) and boiled for 2 min. Samples were then loaded onto an SDS-PAGE gel and stained with Coomassie Blue R250. The uncropped SDS-PAGE gels  was incubated in citrate-phosphate buffers ranging from pH 4.0-pH 7.5 in 0.5 pH increments at room temperature in the presence and absence of 20 mM dodecylphosphocholine (DPC). After 30 minutes, samples were spun at 5000 × els Fig.  are in tg for 5 min to pellet aggregates but not detergent. The supernatant was removed for each sample, and CD spectra were acquired from 250 to 190 nm at 50 nm/min, with a data pitch of 0.1 nm and three accumulations. Spectra were then averaged, blank subtracted and converted to mean residue ellipticity using standard formulas.Far-UV CD spectra were recorded at room temperature using a J-810 spectropolarimeter (Jasco) with 0.1 cm path length cuvettes. Protein was added to a final concentration of 5–10 μM in presence or absence of 20 mM dodecylphosphocholine (DPC) in citrate-phosphate buffers ranging from pH 4.0–7.5 in 0.5 pH increments. After 30 min at room temperature, all samples were spun down at 5000 × Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Streptococcus agalactiae, the antitoxin AbiEi negatively autoregulates abiE expression through positively co-operative binding to inverted repeats within the promoter. The human pathogen Mycobacterium tuberculosis encodes four DUF1814 putative toxins, two of which have antitoxins homologous to AbiEi. One such M. tuberculosis antitoxin, named Rv2827c, is required for growth and whilst the structure has previously been solved, the mode of regulation is unknown. To complete the gaps in our understanding, we first solved the structure of S. agalactiae AbiEi to 1.83 Å resolution for comparison with M. tuberculosis Rv2827c. AbiEi contains an N-terminal DNA binding domain and C-terminal antitoxicity domain, with bilateral faces of opposing charge. The overall AbiEi fold is similar to Rv2827c, though smaller, and with a 65° difference in C-terminal domain orientation. We further demonstrate that, like AbiEi, Rv2827c can autoregulate toxin-antitoxin operon expression. In contrast with AbiEi, the Prv2827c promoter contains two sets of inverted repeats, which bind Rv2827c with differing affinities depending on the sequence consensus. Surprisingly, Rv2827c bound with negative co-operativity to the full Prv2827c promoter, demonstrating an unexpectedly complex form of transcriptional regulation.Toxin-antitoxin systems play key roles in bacterial adaptation, including protection from antibiotic assault and infection by bacteriophages. The type IV toxin-antitoxin system AbiE encodes a DUF1814 nucleotidyltransferase-like toxin, and a two-domain antitoxin. In  Toxin-antitoxin (TA) systems are encoded by genetic loci that are widely distributed throughout prokaryotic genomes. They can play pivotal roles in bacterial physiology and in managing stress responses, helping bacteria to survive nutrient limitation, immune system attack, antibiotic treatment and predation by bacteriophages . TA systStreptococcus agalactiae V/R 2603, has been shown to act as a type IV TA system ) (1 mg/ml), 1 µl of diluted protein or buffer control and water to a final volume of 10 µl. Native 0.5× TBE polyacrylamide gels  were pre-run at 150 V and 4°C for 2 h. Binding reactions were titrated at protein concentrations from zero to an appropriate upper limit, and incubated at 20°C for 30 min. Non-specific binding controls used an additional excess of 2.5 µM TRB1108 template DNA amplified with TRB1109 as forward primer, and non-labeled reverse primer. Specific binding controls used additional excess of 2.5 µM unlabeled specific probe DNA. Samples were then separated by native polyacrylamide gel electrophoresis at 200 V and 4°C for 45 min.Kd) can also be extracted from the Hill plot as Kd = 10X − intercept. Mean and standard error of the mean values are derived from at least three independent experiments.Native polyacrylamide gels were then visualized using the Amersham Biosciences Typhoon 9400 on variable mode image in fluorescence mode, emission filter 526 SP. Sensitivity was set to normal. Band intensities were calculated using the grid scan feature and triplicate data processed in Prism (GraphPad Software). Fractional saturation curves were produced with fractional saturation, Y, varying from 0–1.0. Y values are calculated by (Y/(Y + (1 − Y))) and plotted against protein concentration. Data were converted to the Hill plot to analyse the degree of co-operativity in the binding events, characterized by the Hill coefficient (slope of the plot at log(θ) = 0). The Hill plot is constructed by plotting logθ against log[protein], with θ defined as (θ = (Y/(1 − Y))). Dissociation coefficients , pTRB484 (Prv2827c) or pTRB486 (PabiEi), and the IPTG-inducible pTA100-antitoxin plasmids pPF656 (Rv1044), pPF658 (Rv2827c) or pTRB481 (AbiEi). Transformants were re-seeded from overnight cultures and grown in 37°C at 200 rpm in LB supplemented with Tc, Sp, and with/without IPTG until mid-log phase, then 80 µl of cells were added to 120 µl master mix ) in corresponding wells of a 96-well plate. This was then placed in a SPECTROstar Nano absorbance plate reader (BMG LABTECH) set to 30°C with shaking at 500 rpm, wherein OD600 and OD420 readings were taken every 90 s for 1 hour. Data analysis was performed in the MARS Data Analysis software package (BMG LABTECH). The kinetic OD420 readings were converted into the slope of OD420 over time (OD420/min). These values were multiplied by 5000 and divided by the OD600 reading from the first time point to generate Miller Units (mU). Plotted data are the normalized mean and standard deviation obtained from three independent experiments.Promoter regions were cloned into the promoterless d, pRW50 . Antitoxd, pRW50 ,46. BrieS. agalactiae [We had previously hypothesized that there was structural similarity between the biochemically characterized antitoxin AbiEi from alactiae  and the alactiae . We sougalactiae , we concabiE promoter, whilst the C-terminal domain alone is sufficient for antitoxicity against the effects of AbiEii [AbiEi contains an N-terminal winged helix-turn-helix DNA-binding domain and a C-terminal antitoxin domain, connected by a short linker . Mutagenf AbiEii . The N-tf AbiEii . When AbWhen AbiEi and Rv2827c are aligned via the N-terminal winged helix-turn-helix domain, the respective C-terminal domains differ in position relative to the N-terminal domains by ∼65° . We propWhen the N-terminal domain helices and C-terminal domains from AbiEi and Rv2827c are separated and structurally superposed, it is possible to see an approximate overlay between corresponding regions, with RMSDs of 3.04 Å for the N-terminal helices and 3.41 Å for the C-terminal domains . The N-tIt has been shown that the AbiEi C-terminal domain is required for negative autoregulation and likely contributes to positive co-operativity through C-terminal domain interactions . The conabiEi, which are separated by 3 bp [rv2827c revealed two pairs of 23 bp inverted repeats within the region −1 to −131 bp from the rv2827c start codon, that also overlap the promoter  within the promoter of P by 3 bp  , closely followed by IR4 (Kd of 0.121 µM), then IR2 (Kd of 0.862 µM), and finally IR3 (Kd of 11.0 µM)  . This derv1044–rv1045 locus, two, slightly overlapping, 70 bp probes were generated to cover the 131 bp region upstream of the rv1044 translational start site, and used to test Rv1044 binding , we have demonstrated structural conservation across species. As sequences diverge, structure is conserved , 2, whicn tested  and absen tested . Furthersearches . Therefosearches , howeverM. tuberculosis are known to be more complex than those of E. coli; they can stretch to 2000 bp from the start site and lack canonical elements such as the conserved −35 sequence [M. tuberculosis, allowing for greater promoter sequence variation. The −10 sequence for rv2827c–rv2826c is a predicted recognition site for principle M. tuberculosis sigma factor SigA, which is usually maintained at a constant level for cellular ‘housekeeping' [rv2827c cause a growth defect [Promoters of sequence . Transcrsequence  and envisequence  present keeping' . SigA alkeeping'  and regukeeping' . Deletioh defect ,26, suggh defect . One stuh defect .abiEi and Prv2827c  have been co-opted to form different regulatory modules. A greater understanding of how these nuances of regulation are applied in the cognate hosts may provide greater insight into the control of bacterial growth. Understanding these systems and how they regulate bacterial behavior is thereby an important step in developing a means to control TA systems towards utilizing them for their potential therapeutic value.The crystal structure of AbiEi has been deposited in the Protein Data Bank under accession number 6Y8Q.


Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP–ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO–PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet  Prion disease is a fatal, incurable neurodegenerative disease caused by misfolding of the prion protein (PrP) . AntisenEscherichia coli and purified from inclusion bodies by fractionation, denaturation, and refolding on a Ni-NTA column as described [Recombinant full-length human PrP (HuPrP23-230) was expressed in escribed . The vecASOs were prepared and purified as described . HeparinITC was performed on a MicroCal Auto-ITC200 instrument . HuPrP23-230 and ASOs were prepared in dialysis-matched  buffer containing 20 mM phosphate at pH 7.0. Protein was placed in the cell and compound in a syringe, and each experiment was paired with a control experiment injecting the compound into dialysis buffer. Cell temperature was set to 25 °C. Each experiment involved 40 injections of 2 s duration , at 120 s spacing, with a 5 s filter period, 10 µcal/s reference power, 750 rpm stirring speed, and high gain feedback mode. Data were processed, background-subtracted and curves fit in “single set of sites” mode in the manufacturer’s Origin software to obtain thermodynamic parameters.15N-labeled recombinant HuPrP23-230 was prepared by growing the E. coli in 15N-enriched cell growth medium  supplemented with Overnight Express Autoinduction Medium . 1H-15N TROSY spectra were acquired on a 600 MHz Bruker Avance III spectrometer  equipped with a 5 mm QCI cryoprobe. Samples contained 50 μM HuPrP23-230 in 20 mM sodium phosphate, pH 7.0, with 90% H2O/10% D2O.The HuPrP23-230 and active ASO 1 hydrodynamic radii (Rh) were measured using a DynaPro Plate Reader II  with five 3 s acquisitions. HuPrP23-230 and ASO were used at 50 μM in a 20 mM sodium phosphate pH 7.0 buffer, and all measurements were performed in a 384-well format (25 μL sample/well) at 25 °C. Regularization analysis was used for curve fitting, and Rayleigh spheres were used as the model to determine the Rh.ScN2a cells  , a generv/v) 2-mercaptoethanol . RNA was isolated with an RNeasy 96 Kit (Qiagen) that included in-column DNA digestion with 50 U of DNAse 1 . Prnp mRNA in cells was quantified as previously described [Ppia) and then to the level in saline-treated cells. Primers and probes were as follows: Prnp forward primer TCAGTCATCATGGCGAACCTT, reverse primer AGGCCGACATCAGTCCACAT, and probe CTACTGGCTGCTGGCCCTCTTTGTGACX; Ppia forward primer TCGCCGCTTGCTGCA, reverse primer ATCGGCCGTGATGTCGA, and probe CCATGGTCAACCCCACCGTGTTCX.Cells were lysed in 300 µL of RLT buffer  containing 1%  to determine protein concentration. Part of the supernatants were saved as PK-sensitive material for total PrP. For PK-resistant PrPSc, 25 µg total protein was subjected to PK digestion (P6556) using 1 µg/mL PK for 5 min. Digestion was stopped by adding 1 µL of 25 mM Pefabloc. Then, 20 µg total protein for each sample was run on 4–12% Bis-Tris gels (Thermo Fisher Scientific NP0322) with MES buffer (Thermo Fisher Scientific NP002), probed with 1:500 6D11 antibody (Biolegend 808001) in 5% milk TBS with 0.1% Tween overnight, detected with IRDye 680RD goat-anti-mouse IgG, imaged, and quantified using LI-COR Odyssey.Cells were lysed at 72 h with lysis buffer containing 0.5% Triton X-100 and 0.5% DOC in PBS and spun at 3000× Studies on human samples were approved by the Broad Institute’s Office of Research Subjects Protection (ORSP-3587). Experiments used previously reported  human CShttps://github.com/ericminikel/aso_in_vitro.Data were analyzed in R 3.5.1. Dose-response curves were fit using the drc package  with a fKd) values were similar, in the low- to mid-nanomolar range  to estimate the thermodynamic parameters of interactions between full-length recombinant human prion protein HuPrP23-230) and eight previously described 3-230 and,13 ASOs ar range C, for al1H–15N transverse relaxation-optimized spectroscopy [15N-labeled HuPrP23-230, with or without 50 µM ASO. While the control PrP spectrum appeared as expected, virtually all peaks representing PrP backbone amides disappeared when ASO was added  as added A. Inspecas added B. SubseqThe foregoing results suggested that the PrP–ASO interaction observed in vitro might arise from aggregation. As dependence upon ionic strength has been observed for other aggregators , and giv50 values observed for RNase H1-mediated activity of potent ASOs either after transfection or in cells exhibiting productive free uptake of ASOs [Sc, consistent with previous reports, albeit with substantial inhibition only at near-micromolar concentrations (Sc has a longer half-life than PrPC, and its clearance by oligonucleotides is time-dependent [Given that the biophysical parameters of the ASO–PrP interaction appeared similar across ASOs, we sought to replicate, with ASOs recently tested in vivo , the pre of ASOs ,25. In ctrations C. PrPSc ependent , so the Measurement of PrP concentration in CSF has been proposed as a pharmacodynamic biomarker for clinical trials of PrP-lowering ASOs ,16, meanPhosphorothioate oligonucleotides have been reported to bind PrP in vitro in a sequence-independent manner with nanomolar affinity and to antagonize misfolded PrP accumulation in cell culture with nanomolar efficacy ,11,12, yHowever, we also found evidence, by NMR, DLS, and observation by the naked eye, that the in vitro interaction between otherwise monomeric ASOs and monomeric PrP involves the formation of large aggregates, apparently containing both the ASO and PrP. Although we have not deeply characterized these aggregates to determine whether they are protease-resistant and/or represent liquid–liquid phase separation ,29, our In a small-molecule drug-discovery campaign, a compound found to exhibit or trigger aggregation would generally be labeled a false positive hit and removed from consideration. The fact that ASOs do exhibit sequence-independent antiprion activity in cell culture argues that the binding event may not be purely an in vitro artifact, but might correspond to some genuine interaction at the surface of cultured cells, in line with previous reports ,32. NeveKd values for PrP binding for more than a few hours or days. In contrast, cytosolic and nuclear concentrations of ASO remain above the effective concentration for RNase H1-mediated activity for months following bolus dosing [Alternatively, even if ASO–PrP binding as characterized here does occur in vivo, our characterization of the biophysical parameters of this interaction suggests that pharmacokinetics might also be sufficient to explain why non-PrP-lowering ASOs do not extend survival in prion-infected mice. Following bolus injection into CSF, ASOs are rapidly either absorbed into CNS tissue or cleared into plasma . In clins dosing .Importantly, despite the interaction between ASO and PrP and despite the ability of ASOs to trigger recombinant PrP precipitation, we find that the presence of ASO in CSF, even at concentrations well above those likely to be sustained in human dosing, does not interfere with quantification of endogenous PrP by ELISA. The fact that ELISA reactivity of CSF PrP is apparently unaffected by the presence of ASO may be due in part to the low concentration of PrP in CSF, the presence of other proteins in CSF, and the presence of protein and detergent in the blocking buffer used for ELISA. Measurement of CSF PrP should be able to report on the PrP-lowering effects of an ASO in the human CNS in clinical trials.Our data support the further development of ASOs for prion disease on the basis of RNase H1-mediated lowering of PrP RNA as a mechanism of action and the measurement of PrP in CSF as a pharmacodynamic biomarker to monitor this effect.