Bacillus anthracis (Ba) and human infection-associated Bacillus cereus (Bc) strains Bc G9241 and Bc 03BB87 have secondary cell wall polysaccharides (SCWPs) comprising an aminoglycosyl trisaccharide repeat: →4)-β-d-ManpNAc-(1→4)-β-d-GlcpNAc-(1→6)-α-d-GlcpNAc- and Côte d’Ivoire (Bc CI). The SCWPs of Bc CA/CI contained the identical HexNAc trisaccharide backbone and Gal modifications found in Ba, together with the α-Gal-(1→3) substitution observed previously at ManNAc residues only in Bc G9241/03BB87. Interestingly, the great ape derived strains displayed a unique α-Gal-(1→3)-α-Gal-(1→3) disaccharide substitution at some ManNAc residues, a modification not found in any previously examined Ba or Bc strain. Immuno-analysis with specific polyclonal anti-Ba SCWP antiserum demonstrated a reactivity hierarchy: high reactivity with SCWPs from Ba 7702 and Ba Sterne 34F2, and Bc G9241 and Bc 03BB87; intermediate reactivity with SCWPs from Bc CI/CA; and low reactivity with the SCWPs from structurally distinct Ba CDC684  and non-infection-associated Bc ATCC10987 and Bc 14579 SCWPs. Ba-specific monoclonal antibody EAII-6G6-2-3 demonstrated a 10–20 fold reduced reactivity to Bc G9241 and Bc 03BB87 SCWPs compared to Ba 7702/34F2, and low/undetectable reactivity to SCWPs from Bc CI, Bc CA, Ba CDC684, and non-infection-associated Bc strains. Our data indicate that the HexNAc motif is conserved among infection-associated Ba and Bc isolates (regardless of human or great ape origin), and that the number, positions and structures of Gal substitutions confer unique antigenic properties. The conservation of this structural motif could open a new diagnostic route in detection of pathogenic Bc strains. Bacillus anthracis is a Gram-positive, spore forming bacterium that causes anthrax. It is considered a high threat bioterrorism agent because of the relative ease with which its highly resilient spores can be weaponized and dispersed [B. anthracis (Ba) vegetative cell have structural or immunochemical properties suitable for the development of new and improved medical counter-measures, such as improved diagnostics, vaccines and targeted immunotherapies.ispersed –4. Renewispersed ,6. We soB. anthracis is a member of the B. cereus (Bc) group of related species that also includes B. thuringiensis. These species show high DNA sequence similarities, but display an array of pathogen and non-pathogen associated phenotypes [enotypes –9. The cenotypes .Ba vegetative cells are not dependent on plasmid content [Ba and antigenic [Ba spores react with the HF-SCWP from Ba, but not with the HF-SCWPs from Bc ATCC 10987, a dairy isolate, or from the type strain Bc ATCC 14579 [Ba HF-SCWPs [Bc strains capable of causing severe or fatal illness in humans (e.g. Bc G9241 and 03BB87 causing severe pneumonia) but not with closely related Bc cells and cell walls from non-infection-associated strains, such as Bc ATCC 10987 and Bc ATCC 14579. This observation suggested a degree of antigenic structure conservation between SCWPs from infection-associated Ba and Bc strains. The repeating unit structures for HF-SCWPs from all currently examined Bc group members are summarized in Fig 1.We have previously reported that the structure and composition of HF-released secondary cell wall polysaccharides (HF-SCWPs) from  content , are spentigenic –14. For CC 14579 –14. In aBc strains, strain Bc CI from Côte d’Ivoire and strain Bc CA from Cameroon designated as B. cereus biovar anthracis, that caused “anthrax-like” disease in great apes [B. cereus strains have recently been identified as the causative agents of severe outbreaks of anthrax-like disease among a diversity of great apes in the African tropical rain forest environment . We compared these structures to those from infection-associated and non-infection-associated Bc group members. Our findings show that the HF-SCWPs of both great ape strains contained the identical HexNAc trisaccharide backbone core structure and Gal modifications known from Ba HF-SCWP. In addition, their SCWP contains the additional Gal-substitution at 50% of ManNAc residues originally identified in human infection-associated Bc strains G9241/03BB87/03BB102. Interestingly, both great ape-derived strains contained an additional, unique disaccharide substitution at some of the ManNAc residues that varied in percentage between them. The data from strains Bc CI and CA support the hypothesis of a conserved structural core motif of SCWPs in virulent, infection-associated Bc group strains. We also describe a comparison of the immunoreactivity of these SCWPs with both Ba SCWP polyclonal antiserum (pAb) and monoclonal antibody (mAb) EAII-6G6-2-3 specific for Ba [Bc group of related species.We investigated the SCWP structures of two recently isolated eat apes –17. Thesc for Ba ,19. The Table 1.The strains and isolates used in this study are listed in ad libitum and environmental enrichment was provided e. g. with treats and toys. Rabbits were ultimately euthanized by exsanguination under anesthesia. There was no direct use of non-human primates per se in our current study; all Bacillus strains used in this study (Table 1) originally isolated from the great apes were previously described by Klee et al., [All animal procedures were carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Specifically, procedures for antisera production in rabbits were approved by the Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention (protocol #1420SAIRABC-A1). The rabbits were housed in stainless steel cages. Room temperature was maintained in a range from 20 to 23°C; the light/dark cycle was approximately 12 hours each per d. Water was available to the animals  et al., ,16.Cell walls were prepared from cultured cells as previously described ,14 folloTable 1and Fig 1(adapted from [Bc isolates from great apes (strains Bc CA and Bc CI) were treated with 48% HF at 4°C for 48 h. The reaction mixtures were rotated slowly in an end-over end mixer to assist solubilization of the cell wall material. The reaction mixtures were then adjusted to pH 6.5 by rapidly transferring the mixtures directly into an ammonium hydroxide solution (20% v/v) pre-cooled to -20°C. The solutions were then dialyzed at 4°C vs. deionized water for 3 d using 1,000 MWCO dialysis tubing (Spectrapor). Dialysates were concentrated by centrifugal vacuum-evaporation then subjected to ultracentrifugation  to remove HF-insoluble material, yielding a supernatant containing the solubilized HF-SCWPs (crude SCWP preparation). The HF-insoluble residue consisted of peptidoglycan fragments and protein (amino acids) as determined by GC-MS analysis of the heptafluorobutyrate esters [The SCWPs examined in this study, and their parent strains, are summarized in ted from ,13,26,27ted from ,14,26,27e esters  and the S12 Fig). The eluent was monitored by UV absorbance (210 nm/260 nm/280 nm) and refractive index, and fractions containing the SCWP were identified by glycosyl analysis. The HF-SCWP fractions were dialyzed  against water, lyophilized and used for NMR, linkage, and immunological analyses. Typical yields were 5 mg of purified SCWP per 40 mg of cell wall material.Crude HF-SCWP was dissolved in deionized water, filtered (0.45 μm cellulose acetate) , then purified by size exclusion chromatography on Superose-12 FPLC column  in 50 mM ammonium acetate pH 7.8  were dissolved in water (200 μl) and mixed with 0.5 mg sodium borodeuteride (NaBD4) in water (200 μl) and reduced for 1 h at room temp. The reaction was neutralized by adding methanol containing dilute acetic acid, then evaporated several times from methanol in a centrifugal vacuum-evaporator , before dialyzing (1000 MWCO membrane) for 1 d vs. deionized water. The reduced-HF-SCWP (red-HF-SCWP) was rechromatographed on Superose-12, isolated as described above, and subjected to NMR and linkage analyses as for the native materials.Portions of the 1H spectra and all two-dimensional homo- and heteronuclear spectra of the great ape derived HF-SCWPs were recorded at 25°C on a Varian Inova 600 MHz or Varian 800 MHz spectrometer, each equipped with a 3-mm cryogenic probe, using Varian software . The SCWPs released by HF treatment, and purified by Superose-12 chromatography were exchanged with D2O  and dissolved in 200 μl D2O (“100%”) yielding clear solutions at approximately 7 mg/ml in 3 mm tubes. The 1H-spectra/axes were referenced to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (0.1% DSS in D20) at δH 0.00 ppm, yielding the HOD resonance at 4.78 ppm. Certain samples, available only in small amounts  were dissolved in 90 μl of D2O and analyzed in 3 mm Shigemi tubes  using the cryogenic probe. The 13C axis in 2D analyses was then referenced to the calibrated proton axis, using the Varian software command “setref1(‘c13)”, an accepted method which uses the referenced proton dimension to calculate the 2nd dimension (13C) according to a mathematical relationship. This procedure yields most reproducible and consistent referencing in biomolecular NMR, where the natural abundance of 13C and the % of DSS is too low to allow reliable detection of the DSS carbon signal, see e.g., Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., and Sykes, B. D., "1H, 13C and 15N Chemical Shift Referencing in Biomolecular NMR," J. Biomol. NMR 6, 135–140 (1995).1H-1H COSY [1H-1H zTOCSY [1H-1H NOESY experiments, collected with a 150 ms mixing time and matrix size identical to that used for zTOCSY, with 64 scans per increment. Carbon chemical shifts and carbon-proton one-bond correlations were determined with a gradient-selected 1H-13C HSQC [1H-detection mode. The acquisition time was 0.2 s, and the matrix size was typically 256 x 2,000 complex points, at 88 to 144 scans/increment with multiplicity distinction for primary/secondary protons. The coupling constant (j1ch) was set to 150 Hz and the carbon spectral width was 110 ppm, transmitter offset 65 ppm. Gradient 1H-13C-H2BC spectra [1H) x 140 (13C) complex points with acquisition times of 0.2 s (1H) and 22 ms (13C) respectively. Phase-sensitive 1H-13C HMBC spectra were acquired with 256 x 2048 complex points at 96 to 144 scans/increment. The carbon and proton sweep widths were identical to HSQC values, and the acquisition time (t2) was 0.27 s. The HMBC transfer delay was set to 50 ms (10 Hz), and 1-bond J-filter was 150 Hz. Anomeric configurations of the glycosyl linkages were assigned from carbon-proton coupling constants  measured for the native SCWP by 1H-13C HSQC analysis without 13C decoupling. The acquisition time was 0.6 s, collecting 128 scans per increment; the j1ch was set to 160 Hz and carbon sweep width was 80–120 ppm.-1H COSY  data werH zTOCSY  was recoH zTOCSY  was perf13C HSQC  collecte spectra  were acqUsing the GLYCAM Webtool (39), and the associated Glycam06 force field parameters for carbohydrates  we generet al., 2009 [To facilitate immobilization to polystyrene microtiter immunoassay plates and to make polyclonal antibody, the HF-SCWP was conjugated to either bovine serum albumin  or keyhole limpet hemocyanin  as described by Leoff l., 2009 .Ba [Ba Ames HF-SCWP-KLH conjugate ). Serum used in this study was collected 14 d after the final immunization. All animal work was conducted with CDC IACUC approvals.Monoclonal antibody (mAb) EAII-6G6-2-3 specific for Ba  was obtaMonoclonal (mAb) and polyclonal (pAb) immuno-analyses were done using HF-SCWP conjugated to BSA  in electrochemiluminescent (ECL) immunosorbent assays . Each SCWP-BSA conjugate was adjusted to a concentration of 2 μg/mL total carbohydrate content in 0.01 M PBS pH 7.4. 25 μL of the concentration adjusted SCWP-BSA conjugates and 25 μL of 1 μg/mL of BSA protein (negative control) were incubated at +4°C for 16–18 h. For use, plates were washed, aspirated, and 150 μL of blocker  was added to each well . Plates were washed three times with wash buffer  on an automated plate washer .Monoclonal antibody EAII-6G6-2-3 was evaluated over a range of 93.6 μg/mL to 0.0731 μg/mL in the blocker buffer . After washing, bound EAII-6G6-2-3 was detected with MSD sulfo-tagged goat anti-mouse IgM detection antibody at 2.5 μg/mL and incubated at room temperature for 1 h with shaking. The conjugate was detected using MSD Read Buffer and plates were analyzed using a MSD SECTOR imager for emission at 620 nm.Rabbit pAb was detected with 2.5 μg/mL of sulfo-tagged goat anti-rabbit IgM . To assess the reactivity of each HF-SCWP sample to polyclonal anti-Ames SCWP-KLH, antiserum was added in two fold serial dilutions starting at 1/100 for each strain to generate a full dilution curve. Response curves were fitted to a 4 parameter logistic (4-PL) nonlinear regression model for quantitative analysis .50) and the effective dilution of rabbit pAb generating a 50% response (ED50). The EC50 and ED50 values were determined as the reciprocal of the dilution corresponding to the inflection point (‘c’ parameter) of the 4 parameter logistical (4-PL) model to fit a dose-response curve to the data as described [50 and EC50 values from three independent experiments. GM reactivity for each of the individual strains were compared using Student’s t-test with a P = 0.05 level of significance. Low EC50 values and high ED50 values indicated a high level of reactivity against specific HF-SCWP.The quantitative endpoints for serological analysis were the effective concentration of mAb EAII-6G6-2-3 generating a 50% response (ECescribed . ReportaBa derived SCWPs [Bc derived HF-SCWPs [Bc G9241) are reported in Table 2, along with those from the great ape isolate B. cereus Cameroon (Bc CA). Our initial 1H-NMR analysis of the HF-SCWPs isolated from the Bc CA and Bc CI strains indicated that their structures were probably similar to those of the human infection-associated Bc G9241 and Bc 03BB87 strains, although with some interesting and significant differences in their 1H spectra . A number of points were noted in these proton spectra: (i) HF-SCWPs from both great ape isolates appeared to contain all of the NMR anomeric signals as found in the Bc G9241 HF-SCWP [Bc G9241; (ii) in addition to these “identical” components, both of the great ape isolates displayed two additional “new” signals in the anomeric region, not previously observed in the Bc G9241 and Bc 03BB87 human infection-associated strains. These new components were designated residues J and K, and their NMR parameters are listed in Table 2, along with the NMR parameters of all other repeating unit components.We have previously reported detailed NMR analysis of ed SCWPs , 26, andHF-SCWPs . For com HF-SCWP , howeverBa strains examined to date, including Ba Ames, Ba Pasteur, Ba Sterne 34F2, Ba 7702, and Ba CDC684 [Bc G9241 and Bc 03BB87 [pNAc-(1→4)-β-D-GlcpNAc-(1→6)-α-D-GlcpNAc-Bβ(1→4)Cβ(1→6)Aα(1→ to conform with previous nomenclature (26). With the exception of Ba strain CDC684, these polysaccharide backbones are highly galactosylated with α- and β-Gal residues, and the galactosylation pattern  is species/strain specific. HF-SCWPs from all examined Ba strains share the same overall galactosylation pattern yielding the hexasaccharide repeating unit shown in Fig 2A. Thus, the ratio of Gal substitution to HexNAc backbone residues is 1:1, which represents a high density of side chain attachment for a polysaccharide. In these Ba derived HF-SCWPs, a small percentage of the repeating units typically lack one or more of the Gal substituents, contributing to signal heterogeneity in their NMR spectra. This heterogeneity in Gal side residue attachment is observed in all the examined Ba and Bc derived HF-SCWPs.All of the a CDC684 , 26, as c 03BB87  share anBc derived HF-SCWPs, a single additional Gal substitution is introduced at 50% of the backbone ManNAc residues, yielding the repeating structure and spectrum shown in Fig 2B. These Bc derived HF-SCWPs essentially consist of two types of repeating units, one with, and one without the Gal-substituted ManNAc. The introduction of this additional Gal residue modifies the magnetic environment of adjacent residues, such that their anomeric signals split, yielding twin signals, where one member of each pair arises from repeats which lack the Gal-ManNAc, and the other member signal arises from repeats which carry the Gal-substituted ManNAc [Bc derived HF-SCWPs results in polysaccharides having a side residue/backbone ratio of > 1:1. An extensive NMR comparison of the Ba-derived and human infection-associated Bc-derived HF-SCWPs was previously described [S1 Fig).In the human infection-associated d ManNAc . Other rescribed  and reprBc strain CA HF-SCWP is compared with Ba and human infection-associated Bc derived HF-SCWPs in Fig 2C. The “new” signals are indicated, and their associated spin systems and residue identification are described in the following sections. Signals originally appearing in the Ba samples are again split into “twin signals”, but the splitting ratio is not 50/50, reflecting the fact that the Gal-substitution on ManNAc is not 50% in the great ape-derived SCWPs. This feature, a lower percentage Gal-substitution at the backbone ManNAc residue, is observed to an even greater degree with the great ape Bc strain CI HF-SCWP. The 1H-anomeric regions of Bc CI, Bc CA, the human infection-associated strain Bc G9241, and Ba Sterne 34F2 are all compared in S2 Fig, demonstrating their relationship and differences. The 1H-1H TOCSY spectra of the great ape Bc CA HF-SCWP and the previously analyzed human infection-associated Bc G9241 HF-SCWP are compared in S3 Fig, demonstrating the similarity of the conserved residues, and the presence of new glycosyl systems in the great ape isolate. The 1H-1H TOCSY spectra of SCWPs from the two great ape isolates Bc CA and Bc CI are compared in S4 Fig demonstrating their overall similarity.In our present study, the NMR anomeric region of the great ape derived Fig 3, we compare the regions of the 1H-NMR spectra where the new, additional signals (J and K) are observed in the great ape derived HF-SCWPs compared to Bc G9241. This region also reveals the percentage of ManNAc residues (B′) that are substituted with an α-Gal residue (G), originally identified in the human infection-associated Bc G9241 HF-SCWP [Fig 3A, the HF-SCWP from Bc G9241 demonstrates the near 50% substitution of the ManNAc residue (B +B′) with this Gal(α1→3) substituent (G). In comparison, the HF-SCWPs from both Bc strains CA and CI  appeared to show a lowered ratio of ManNAc substitution by residue G, as calculated by integration of the anomeric signals for residues G, J, and (B+B′+B′′). Thus, area integration suggested that substitution of ManNAc by G occurs at a lower percentage in the great ape derived SCWPs, compared to the Bc G9241.In  HF-SCWP . In Fig K, H1 δH 5.13), actually had the same chemical shift as a low-abundance resonance previously observed in all Ba and Bc HF-SCWPs, and identified as the free reducing end  of the polysaccharides [K in the great ape isolates. To remove this interference, the Bc CA and Bc CI HF-SCWPs were reduced at the reducing end with sodium borodeuteride, yielding the reduced-HF-SCWPs (red-HF-SCWP). In Fig 4, the effect of reduction on the α-reducing end signal is indicated by a decrease in the relative signal area at K+α, in the Bc CA red-HF-SCWP. Although reduction was incomplete, the area of K+α was decreased substantially to the point where it was roughly equivalent to the area of residue J, due to removal of mostthe α-reducing end contribution. To further clarify the effect of reduction on the SCWPs, the 1H-13C-HSQC spectra of the anomeric regions, before and after reduction, were examined . These HSQC spectra demonstrate that the NaBD4 treatment was effective in removing both the α- and β- reducing end anomeric signals and it did not appear to alter the repeating anomeric signals of the HF-SCWPs in any manner. A comparison of the TOCSY spectra of this region before and after reduction is also provided . Having removed interference by the α-reducing end, the identification and location of the new residues K and J in the red-HF-SCWP samples were investigated.We also initially found that one of the new anomeric signals , indicating that both residues were probably of α-anomeric form. 1H-1H-COSY and 1H-13C HSQC analysis identified H2 for each residue, indicating that neither was a 2-amino sugar. 1H-1H COSY, and TOCSY analysis  revealed weak magnetic transfer beyond position 4 for both residues, consistent with the galacto-configuration. New, hydroxy-methylene protons, not observed in the Bc G9241 spectrum, were identified in the HSQC spectra of Bc CA (and Bc CI) for residue J at δ 3.63/3.54/63.18 , and TOCSY analysis demonstrated connectivity to J H5/C5 , and subsequently to H4/C4 (δH/δC 4.18/65.84) of residue J . Additional HSQC and TOCSY spectra comparing relevant regions from the great ape Bc CA and human infection-associated Bc G9241 HF-SCWPs are shown in S8and .The K system was defined with the assistance of HSQC and H2BC one and two-bond couplings for assignment of positions 6 and 5. H2BC revealed methylene protons (δH 3.73/3.73) which coupled to C6 (δC 61.6) and C5 (δC 71.52), with assignment of H5 (δH 4.09) from a new HSQC signal, not present in the Bc G9241 spectrum. Partial TOCSY spectra defining residues J and K are shown in Fig 5A. The galacto-configuration was assigned to both J and K on the basis of weak scalar coupling at H4, the relative downfield shift at H4, and intra-residue NOEs between H3-H4 and H5-H4 in addition to H3-H5. As described below, GC-MS analysis of the TMS-methyl glycosides and partially methylated alditol acetates detected elevated levels of Gal in the great ape HF-SCWPs, compared to the Bc G9241, and failed to detect any other “new” hexoses.The Bc G9241 and Bc 03BB87, the attachment of the α-Gal (here assigned residue G) to the 3-position of 50% of the ManNAc residues was clearly demonstrated by inter-residue NOEs and trans-glycosidic HMBC connectivities [B′ H3/C3 (δH/δC 4.24/80.28) which differs from that of the unsubstituted ManNAc residue B (Table 2), and is unique in the HSQC spectrum. For both great ape derived HF-SCWPs, a certain percentage of the backbone ManNAc residues were found to be substituted at O3 by an α-Gal residue (G) . Inspection of the 1H-1H NOESY spectrum for Bc CA red-HF-SCWP revealed an inter-residue NOE between G1 (δH 5.05) and B′3 (δH 4.24), Fig 5B, indicating that Gal is linked α1→3 to ManNAc in these great ape isolates as well. Most important, further inspection of the NOESY spectra indicated that the new residue J anomeric proton (δH 5.13) showed only two strong NOEs, to a proton at δH 3.91 (identified as H2 from COSY) and an NOE to a new proton at δH 4.27 , subsequently identified as H3 of a new variant of the ManNAc residue (assigned B′′). These results indicated that the α-Gal residue J is also linked to the 3-position of a percentage of ManNAc residues, however, the residue J spin system differed from that of residue G. Inspection of the J spin system defined above suggested that it may itself be substituted at H3/C3 (δH/δC 4.18/77.20) by an unknown residue. This substituent was readily identified as the new α-Gal residue K, having a strong inter-residue NOE between K1 (δH 5.13) and J3 (δH 4.18),  and NOEs to protons J2 (δH 3.91) and (intra-residue) to K2 (δH 3.85). The resulting glycosidic sequence, Kα1→3Jα1→3B′′, was substantiated by a trans-glycosidic 1H-13C HMBC connectivity between JH1 (δH 5.11) and B′′C3 (δC 80.09) . The linkage Gα(1→3)B′ was also confirmed by a trans-glycosidic HMBC between GH1 (δH 5.05) and B′C3 (δC 80.09), as previously observed for the human infection-associated Bc strains G9241 and 03BB87. Additional confirmation for intra-residue assignments defining the new residues in the Bc CA red-HF-SCWP were confirmed by 2-bond 1H-13C H2BC connectivities observed for JH1/JC2 (δH 5.11/δC 67.03); KH1/KC2 (δH 5.13/δC 68.90), and KH2/KC1 (δH 3.85/δC 95.33) inter alia. shown in S10 Fig.In the SCWPs from human infection-associated strains tivities . This suBc G9241 HF-SCWP. Only the great ape derived HF-SCWP yielded low levels of a 3-linked Gal PMAA derivative, not previously observed with any of the human pathogen derived strains . Together these results indicated that HF-SCWPs from great ape derived CA and CI strains exhibit a nearly identical structure as that from Bc G9241, with one interesting and rather unusual difference. All contained a portion of their backbone ManNAc residues substituted with α-Gal at the 3-position (a feature that is lacking in the Ba derived HF-SCWPs). However, the great ape isolates exhibited a further modification, in which a percentage of the ManNAc residues carried a Galα1→3Galα1→3 disaccharide substituent. Table 3summarizes the relative percentage of these substituents, estimated from the NMR signal areas of anomeric resonances , for these human infection-associated and great ape-isolated Bc strains.To further clarify the nature of the new residues and their linkages, chemical linkage (methylation) analysis was performed and the resulting PMAA derivatives were compared for great ape derived HF-SCWPs and the previously analyzed human infection-associated Table 4). The HF-SCWPs from all examined isolates reacted with the polyclonal (pAb) anti-Ames HF-SCWP antiserum. The highest reactivity was observed with the HF-SCWPs from strains Ba Sterne 34F2 and Ba 7702 (Table 4). There were no significant differences in the reactivity of the pAb serum with HF-SCWPs from Ba Sterne 34F2, Ba 7702 and infection-associated Bc 03BB87 and Bc G9241 strains (p = 0.17). There was, however, an approximately 35% reduction in pAb binding to the HF-SCWPs from the great ape disease causing strains Bc CI and Bc CA compared to the infection-associated Ba and Bc strains (p <0.05). There was an approximately 70% reduction in pAb binding to the HF-SCWP from the avirulent Ba CDC684 strain and the non-infection-associated strain Bc ATCC 10987 as well as an approximately 50% reduction of pAb binding to the HF-SCWP from strain Bc ATCC 14579 when compared to the infection-associated Ba and Bc strains (p <0.05). The ED50 values obtained with HF-SCWPs from the two non-infection-associated strains Bc ATCC 14579 and 10987 were statistically different from each other (p <0.05). However, while there was a significant difference between the HF-SCWP reactivities from the non-infection- associated type strain Bc ATCC 14579 and Ba CDC684 (p <0.05), there was no significant reactivity difference between dairy isolate Bc ATCC 10987 and the highly attenuated, Gal-substitution negative Ba CDC684 strain (p = 0.24).For comparative analysis, strains were grouped based on their structural similarity in the aminoglycosyl backbone and its galactosyl modifications (Table 4). HF-SCWPs from the strains Ba Sterne 34F2 and Ba 7702  had similar immuno-reactivities to the human infection-associated strains Bc G9241 and Bc 03BB87 . The immuno-reactivities of these two groups were not significantly different from each other . HF-SCWPs from the Bc CI  and Bc CA  were not statistically different from each other (p = 0.32). The lowest reactivities were observed with the HF-SCWP from Ba CDC684 , and from the non-infection-associated strains Bc ATCC 10987 and Bc ATCC 14579 .The HF-SCWP pAb reactivities separated into distinct groups  with the examined HF-SCWPs (Table 4). The HF-SCWPs from Ba Sterne 34F2 and Ba 7702 strains had the highest reactivities , and were not statistically different from each other (p = 0.48). This reactivity was significantly lower (p < 0.05) for all Bc strains and isolates evaluated (e.g. the dairy-isolate strain Bc ATCC 10987 had an EC50 from 141.89 μg/mL and the Bc ATCC 14579 type strain of 223.45 μg/mL). However, all infection-associated Bc strains displayed distinctly higher reactivity. For example, the human infection-associated strains Bc G9241 and Bc 03BB87, Structural Group 2, had approximately 10- to 30-fold lower EC50 values and the great ape isolates, Structural Group 3, approximately 2- to 3-fold lower EC50 values compared to the non-infection-associated Bc strains (p < 0.05). The variance in the EC50 values between Structural Group 2  and between Structural Group 3  were not statistically significant. The HF-SCWP from the highly attenuated Ba CDC684 was of particular interest. This SCWP comprised the core HexNAc trisaccharide backbone but was devoid of α- and β-Gal substitutions. This HF-SCWP displayed a significantly higher EC50 (lower reactivity) compared to HF-SCWP from Ba Sterne 34F2 and Ba 7707 (p < 0.05), and from the infection-associated Bc G9241, Bc 03BB87 and Bc CA and CI (p < 0.05). The reactivity of the Ba CDC684 isolate was not significantly different to that of the non-infection-associated and structurally unrelated HF-SCWP from Bc ATCC 14579 and ATCC 10987 . Essentially, the HF-SCWP from Ba CDC684, the great ape Bc CA and Bc CI isolates, and the Bc ATCC 14579 and ATCC 10987 environmental isolates may be considered non-reactive with the mAb  although the two great ape isolates did show a slightly elevated reactivity compared to the environmental isolates. Molecular models, generated using the Glycam Webtool [Ba Ames/Sterne), an intermediate mAb binder (human-isolate Bc G9241), and a non-binder (Bc ATCC 10987) . Several minimal energy conformers were generated including gt-gt and gg-gg forms; only the gt-gt conformers are shown. The possible role of these conformers, including the relative importance of individual Gal residues in EAII-6G6 recognition is discussed in Fig 8and in the Discussion.There was a hierarchy of mAb reactivity  indicate that mAb reactivity can distinguish between the polysaccharide backbones of Ba and Bc based on the galactosylation pattern of the →4-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-1→ backbone. The absence of Gal residues such as in the Ba CDC684 SCWP results in a dramatic loss of binding activity with an EC50 value of 356 μg/mL compared to a strain with full galactosylation such as Ba Sterne 34F2 with EC50 0.56 μg/mL. The addition of one α-Gal to O3 of the ManNAc residue in Bc G9241 SCWP resulted in a statistically significant reduction in binding (EC50 13.6 μg/mL). The addition of a second α-Gal, i.e., as the disaccharide substituent [Gal(α1→3)Gal in the SCWP of the Bc CI and Bc CA isolates resulted in further reductions in binding  of the monoclonal antibody when compared to a monosaccharide α-Gal substitution .Comparisons of HF-SCWP structures  trisaccharide backbone repeating unit that contains the common structural core motif: →4)-β-D-ManpNAc-(1→4)-β-D-GlcpNAc-(1→6)-α-D-GlcpNAc- to cell septa.Structural investigations of SCWPs from pNAc-, but not by GlcNAc. This observation suggested to us that the epitope for this antibody may be localized in the SCWP, with particular emphasis on the presentation and arrangement of Gal residues carried on the HexNAc backbone. Modifications in SCWP structures of the Bc group strains may therefore be correlated to EAII-6G6-2-3 binding. The recently isolated and phylogenetically well-defined Bc CA and Bc CI causing anthrax-like symptoms in great apes [Bc.Previous studies by Ezzell et al.  with mAbeat apes ,16 were Bc CA and Bc CI isolates had SCWP primary structures almost identical to the SCWP from human infection-associated strain Bc G9241 [Table 3). Apart from this disaccharide, the SCWP structures from the Bc CA and CI strains contain the structural features previously identified in infection-associated Ba and Bc strains, i.e. the conserved aminoglycosyl trisaccharide backbone that is substituted with strain-specific galactosylation modifications at conserved locations.The results presented here reveal that the SCWPs isolated from great ape infection- associated Bc G9241  except tBc CA and CI strains compared to the SCWP from Ba raised the question whether these modifications affect SCWP immuno-reactivity compared to the those of SCWPs from other infection-associated and non-infection-associated Bc group strains [Ba Ames and human infection-associated isolates Bc G9241 and Bc 03BB87. The HF-SCWPs from both Bc CA and Bc CI had similar reactivity, with approximately a 35% reduction in binding of the pAb relative to HF-SCWPs from the Ba Ames and human infection- associated isolates Bc G9241 and Bc 03BB87 . There was a 50% reduction in pAb reactivity to the non-infection-associated strain Bc ATCC 10987, and an approximately 80% reduction in reactivity to HF-SCWP from the non-infection-associated strain Bc ATCC 14579 and the nongalactosylated aminoglycosyl repeating motif from Ba CDC684.The structural modifications discovered in the SCWPs from the great ape infection- associated  strains . The datBa strains and the pathogenic Bc isolates, is a predominant antigenic determinant for the pAb, and that their strain specific, α- and β-Gal substitutions at GlcNAc and at ManNAc, confer additional, sometimes unpredictable, polyclonal response. This is emphasized by the fact that a significant response, well above base line, was observed between the polycolonal sera and the HF-SCWP from Ba CDC684 . The polyclonal serum was also somewhat reactive with HF-SCWPs having a structurally different HexNAc backbone, such as those from the nonpathogenic strains Bc ATCC 10987 and the type strain Bc 14579. These non-infection- associated environmental isolates have HexNAc backbone structures that differ from those shared by the pathogenic Ba and Bc isolates , specifically, [→4)-β-D-ManpNAc-(1→4)-β-D-GlcpNAc-(1→6)-α-D-GalpNAc--β-D-ManpNAc-(1→4)-α-D-GlcpNAc- involved in pAb binding.These data suggest that the conserved aminoglycosyl repeating unit backbone, common only to the e . There was approximately a 100-fold reduction in binding of the mAb to HF-SCWPs from both Bc CA and Bc CI, relative to HF-SCWPs from the Ba Sterne 34F2 or Ba 7702, and a 20-fold reduction in reactivity compared to human infection-associated isolates Bc G9241 and Bc 03BB87. Small differences in mAb reactivity between isolates Bc G9241 and Bc 03BB87 were also detected, however, these differences could simply result from experimental variability, for instance, differences in purity of the two preparations. All of the 2-D NMR signals arising from each of these two Bc human infection-associated SCWPs were essentially indistinguishable [Fig 7). Reactivity of the mAb to the non-galactosylated aminoglycosyl repeating motif from Ba CDC684 was reduced more than 600-fold compared to Ba Sterne 34F2 or Ba 7702. Reactivity to the differing HF-SCWP structures from Bc ATCC 10987 and Bc ATCC 14579 was reduced by 250- and 400-fold, respectively. Thus, the observed reactivity of EAII-6G6 with the examined HF-SCWPs  suggests that four main structural groups can be distinguished with this mAb: 1) non-reactive  low reactive (both great ape isolates (Bc CA and Bc CI), 3) intermediate reactivity (human infection-associated Bc G9241 and Bc 03BB87), and 4) highly reactive . The structural basis of this reactivity is suggested to reside in the presentation of multiple Gal residues in the proper 3-dimensional conformation. This 3-dimensional arrangement (secondary structure) is optimal only in the Ba derived strains, . The introduction of an additional Gal residue, at O-3 of the backbone ManNAc residue , disrupts this secondary structure of the polysaccharide sufficiently, such that EAII-6G6 binding is reduced. The attachment of further additional Gal,  causes further deviation from the optimal secondary structure necessary for mAb recognition, yielding the weak binding observed with this structural group. One possibility is that approach of the mAb to the multiple Gal epitope presented by the SCWP is simply hindered by the presence of the new disaccharide (and monosaccharide) Gal substituents, at ManNAc in these strains, as visualized by molecular models . Alternatively, the presence of these new substituents could also result in a change in the overall secondary conformation presented by the SCWP, compared to that of Ba Ames, Pasteur, Sterne, and related Ba strains. A combination of these factors may also account for the observed binding differences. It is interesting to note that HF-SCWPs derived from the nonpathogenic strains , which do not contain the conserved HexNAc trisaccharide backbone of the infection-associated strains, elicit binding essentially at baseline levels, similar to BSA. The Ba CDC684 HF-SCWP, which does contain the pathogenicity-associated HexNAc backbone but lacks all Gal substituents, also shows this same lack of reactivity with the EAII-6G6 mAb. Thus, both the pathogenicity-associated HexNAc trisaccharide backbone, together with the optimal pattern of α/β- attached Gal residues (found only in Ba strains), is required to yield the maximum binding of EAII-6G6. Molecular models generated using the Glycam web tool [Ba (Ames), Bc G9241, and Bc ATCC 10987  as compared in Fig 8. These models reveal how additional Gal substituents located at ManNAc could perturb mAb binding and accessibility to the correct Gal epitope. For instance, Fig 8shows identical views of a minimal energy gt-gt conformer for an eleven (or twelve) residue segment for both the Ba Sterne 34F2 and the Bc G9241 HF-SCWPs. The backbone segments depicted are of the sequence: C-A-B-C-A, with ManNAc (residue B), depicted in the middle of the segment since this ManNAc residue is the focal point of the structural difference. Of interest is the close proximity of the α-Gal (E) and β-Gal (F) substituents at the 6-linked GlcNAc (residue A) to each other, as well as their close proximity to the α-Gal monosaccharide substituent (residue G) at ManNAc in the Bc G9241 segment. We propose that accessibility of these two Gal residues (E and F) to the mAb could be reduced by the presence of a monosaccharide Gal substituent at ManNAc (as in the human isolate Bc G9241), and further reduced by the presence of a Galα1→3Gal disaccharide at ManNAc, resulting in the weaker binding for the great ape isolates . The particular low energy conformer depicted in Fig 8also suggests the possibility that exposed portions of the two closely located Gal residues (E and F) might contribute, perhaps equally, to the EAII-6G6 epitope. Note also that the SCWP from Bc ATCC 10987 does indeed carry a β-Gal substituent, yet is essentially non-reactive with the mAb EAII-6G6 (Table 4). This observation is consistent with the hypothesis that the Ba-conserved “pathogenicity motif” backbone is essential in presenting the Gal substituents to the mAb, by providing the proper conformational scaffolding. Although not proven here, we suggest the possibility that the combined arrangement of both Gal residues E and F may contribute to the EAII-6G6 epitope. The HexNAc backbone itself probably does not interact directly with the mAb, , however, this pathogenicity-specific backbone is necessary to provide the proper scaffolding for presentation of the multiple Gal epitopes which do interact with the mAb. The models provide a visual assist to our central hypothesis, that the correct number of Gal residues, carried on the proper HexNAc backbone at specific locations, are required for optimal mAb EAII-6G6 recognition. Removal or addition of Gals or any change in the Gal attachment pattern, from that displayed in Ba strains, results in variable degrees of attenuation of mAb binding.In contrast to the polyclonal reactivity, the mAb EAII-6G6 had significantly greater ability to discriminate between subtleties in SCWP structuruishable , suggestweb tool  with staBc isolates. In contrast to the conserved core repeating trisaccharide backbone of the infection-associated Ba and Bc strains, the HF-SCWPs of Bc ATCC 10987 and the Bc type strain ATCC 14579 possessed entirely different backbone repeating structures [50 values when compared to the Ba Sterne 34F2 and Ba 7702 strains.The aminogycosyl backbone is also likely to be of importance for the SCWP immunoreactivity with the mAb, as shown with the nearly abrogated reactivity of SCWP samples from the non-infection-associated ructures , 42. PreBc group strains. De et al. analyzed 230 Ba isolates with a two component direct fluorescent antibody (DFA) assay using fluorescein labeled EAII-6G6-2-3 against the cell wall polysaccharide antigen and a monoclonal antibody against the capsule antigen [Ba isolates tested, 228 were positive for reactivity with EAII-6G6. The assay specificity for EAII-6G6-2-3 was only 78.6%, and almost 100% of the false-positive isolates were Bc or Bacillus thuringiensis strains. Ten of 23 (43%) B. cereus strains were reported positive for the cell wall antigen. Further DFA analysis of B. cereus strains G9241 and 03BB87 were also positive for cell wall polysaccharide antigen [Bc ATCC 14579 was negative [The EAII-6G6-2-3 antibody has been used in diagnostic tests and to probe for the distribution of the EAII-6G6-2-3 epitope in SCWPs of  antigen . Of the negative .Ba CDC684) causes loss of binding and the addition of Gal to ManNAc reduces binding , we do not yet know the specific contributions of each of the three Gal residues in the Ba structure that provides optimal binding. Our minimal energy models  suggest a preliminary possibility that Gal residues E and F may together contribute to the EAII-6G6 epitope. A detailed analysis of the minimal Ba SCWP epitope and Gal positions required for EAII-6G6-2-3 binding is being investigated by preparing synthetic structural analogs. Further, while we know that the terminal repeat unit is pyruvylated, O-acetylated, and de-N-acetylated [Ba strains, the Ba CDC684 non galactosylated strain, the human isolates Bc G9241 and Bc 03BB87, the great ape isolates Bc CA and CI, and both ATCC environmental strains), carry a pyruvate residue at identical location on the terminal repeating unit as reported here (S5and ) and discussed in (26), however, only the galactosylated strains containing the pathogenicity-associated HexNAc backbone demonstrate significant EAII-6G6 binding.We suggest that the mAb antibody is recognizing a specific epitope in the cell wall polysaccharide antigen, which is impacted by number and position of galactose modifications. While we have shown that removal of all Gal residues  galactofuran chain [B. cereus group may be useful in development of rapid diagnostic tests for inhalation anthrax.It has been established that cell wall polysaccharides can be virulence factors in both Gram-positive and Gram-negative pathogens –45 and aan chain . As suchB. anthracis [Bc group is based on the number and position of galactosyl modifications around a secondary cell wall polysaccharide backbone. So far, in Bacillus species capable of causing infection this polysaccharide was comprised of →4)-β-D-ManpNAc-(1→4)-β-D-GlcpNAc-(1→6)-α-D-GlcpNAc--β-D-ManpNAc-(1→4)-β-D-GlcpNAc-(1 →6)-α-D-GlcpNAc-Bc G9241; (B)B. anthracis Sterne 34F2. For complete details of this structural comparison see [Glycobiology (2011) 21:934–948. The anomeric signals circled in red are unique to the human infection-associated Bc strains, and absent from Ba Sterne, Ames, and Pasteur strains. Their presence reflects an alteration in magnetic environment due to 3-O-substitution of 50% of the ManNAc residues (B′) with αGalρ residue (G) in the Bc G9241 SCWP; the α/β-GlcNAc signals arise from the reducing-end residue of the HF-released SCWPs [ison see  Forsberged SCWPs .(TIF)Click here for additional data file.S2 FigJ and K) are indicated. Signals having a unique downfield shift, diagnostic for 3-substituted ManNAc  are also labeled and are present only in the Bc infection-associated strains (refer to Table 2).New anomeric signals (from residues (TIF)Click here for additional data file.S3 FigComparison of1H-1B. cereus CA (Cameroon), and (B) human infection-associated B. cereus G9241.H-TOCSY spectra for HF-SCWPs from (A)  Consistent with S2, S4, S7and  the TOCSY spectra from these two strains are virtually identical and superimposable, except for the presence of the additional residues J and K in Bc CA SCWP. In panel A, the (red box) shows the TOCSY region expanded in S6A Fig. In panel B, the human isolate Bc G9241 lacks the J and K spin systems. All other TOCSY connectivities detected in the great ape Bc CA and Bc CI isolates are virtually identical to those from the human infection-associated HF-SCWPs; these signals arise from the conserved residues that are shared with, and previously identified [Bc G9241/Bc 03BB87/Bc 03BB102), as represented in panel B. The chemical shifts of these conserved residues are remarkably similar in these HF-SCWPs. For example, all examined HF-SCWPs show connectivities arising from the α- and β- reducing end GlcNAc residue on each polysaccharide (labeled α/β). These weak spin systems are listed in the Table 2footnote: “Additional Signals”. Positional assignments from COSY analysis (not shown) have been previously published for this reducing end residue from Ba and Bc strains [1H-13C-HSQC in S7 Fig, which shows the H2/C2 correlation for the reducing end residue. Other substoichiometric signals—Residue C′ is a 3-O-acetylated variant of C, which also occurs at a specific location in each HF-SCWP molecule from all examined strains [S5 Fig, showing the 1H-13C HSQC (H3/C3 δH/δC 5.09/74.2). Residue C′ was first identified in the B. anthracis strain CDC684 HF-SCWP [Ba Sterne 34F2, Bc G9241 [Bc strains. Of further interest, in all examined HF-SCWPs the attached Gal substituents D, E, and F all display signal heterogeneity, which appears to arise from nonstoichiometric Gal attachment in some of the repeating units. The weak system at δH 5.39 (*) arises from the anomeric proton of 4-linked α-glucose residues, due to variable amounts of a contaminating glucan produced by the bacteria. The majority of this glucan was removed prior to NMR analysis by Superose-12 chromatography  as previously described for Ba and Bc SCWPs [entified  for the  strains ,27. Thei strains . Shown i HF-SCWP  and was Bc G9241 , and in Bc SCWPs , 27.(TIF)Click here for additional data file.S4 FigA)B. cereus CA (Cameroon), and (B)B. cereus CI (Côte d’Ivoire). These TOCSY spectra are virtually superimposable, reflecting the structural similarity of the SCWPs from these two great ape isolates. This is consistent with the 1H anomeric NMR spectra  which indicated differences only in signal area for these two strains, with identical chemical shifts.((TIF)Click here for additional data file.S5 FigA, native Bc CA strain HF-SCWP, and B, the same sample reduced with borodeuteride to remove the anomeric signal from the reducing end  allowing elucidation of the K residue system. Arrows show the location of both α- and β- reducing end (GlcNAc) anomeric signals prior to reduction; note their absence in the red-HF-SCWP (panelB). These spectra demonstrate that reduction did not appear to affect any other residues, only the free reducing end. The locations of new residues J and K  and all other anomeric signals from the repeating units were unchanged. Note also the presence the of δH downfield-shifted O-acetyl “reporter” signal, which arises from the H3 proton on a 3-O-acetylated β-GlcNAc residue (C′); this O-acetylated GlcNAc residue occurs at a specific, non-repeated location in all Ba and pathogenic Bc derived HF-SCWPs examined to date [S3 Fig. A detailed description of the location and identity of this and other substoichiometric modifications is presented in Forsberg et al., [ to date . The sca et al., .(TIF)Click here for additional data file.S6 FigComparison of1H-1Bc CA HF-SCWP showing the region containing residues J, K, and G, before (A), and after (B) reduction of the reducing end of the SCWP with borodeuteride.H TOCSY spectra for the  In panel A, α = α-reducing end GlcNAc anomeric proton. Certain scalar correlations arising from this α-reducing end anomeric proton overlapped with some of the K residue protons; removal of these α-reducing end signals by borodeuteride reduction allowed definitive assignment of the K ring system (panel B). The β-GlcNAc reducing end anomeric is found at δH 4.74 .(TIF)Click here for additional data file.S7 FigPartial1H-13Bc strains: (A) human isolate Bc G9241; (B) great ape isolate Bc CA (Cameroon).C HSQC spectra comparing HF-SCWPs from  Proton multiplicity is indicated by peak polarity, i.e., orange/red gradient signals (positive polarity) arise from methyl or methine ring protons, and blue/pink gradient signals (negative polarity) arise from methylene (-CH2-) protons. Distinct signals arising from position H6/C6 of residue J (“J6”) were observed in Bc CA , which were absent in the human Infection-associated Bc G9241 and Ba strains . Pyruvate "reporter signals" and the location of pyruvate in HF-SCWPs from representative Ba and Bc strains is discussed in detail in Forsberg et al., 2012 [B′. This substitution precludes further chain polymerization. At B′ positions 4, 5, and 6, unique δH/δC result from this pyruvate substitution . A 3-bond 1H-13C HMBC coupling was also observed between the pyruvate C2 carbon, and a favorably oriented H6 proton of B′ for all examined HF-SCWP [Ba and Bc.ublished ,27). In l., 2012 . Briefly HF-SCWP . These a(TIF)Click here for additional data file.S8 FigComparison of the1H-13Bc HF-SCWPs from: (A) Bc G9241; (B) Bc CA (Cameroon).C HSQC anomeric regions of  The spectra are virtually superimposable, with the exception of "new" anomeric signals from residues J and K in the Bc CA sample. Additional heterogeneity of residue B (residue B′′) is also observed in Bc CA (and Bc CI) SCWPs. The α- and β- labels designate the anomeric signals arising from the reducing-end GlcNAc residue of the respective HF-SCWPs.(TIF)Click here for additional data file.S9 FigJ to the 3-position of residue B′′, and of residue G to 3-position of B′.These 3-bond connectivities prove the linkage of residue (TIF)Click here for additional data file.S10 FigPanel A, HSQC, and panel B, H2BC of the Bc CA red-HF-SCWPs.(TIF)Click here for additional data file.S11 Fig(A) Total ion chromatogram (TIC) of the neutral sugar PMAA derivatives from Bc G9241 SCWP; (B) TIC of neutral sugar PMAAs from Bc CA HF-SCWP; (C) electron impact mass spectrum of the 3-linked Gal PMAA derivative from the Bc CA sample. The GC-MS analysis of the neutral sugar PMAA derivatives demonstrates the presence of 3-linked Gal in the "great ape" strain HF-SCWP, but not in Bc G9241. Only the great ape derived SCWPs (from Bc CA and Bc CI) yield this derivative, which arises from the K1→J3 disaccharide substituent unique to these strains. The results of this chemical analysis (1.86 residues/PS for Bc CA) are in close agreement with NMR signal integration (refer to Table 3). The 4-linked-Glc derivative arises from traces of a contaminating α-glucan, most of which is removed by SEC .(TIF)Click here for additional data file.S12 Fig→6 linked dextrans. A, great ape isolate B. cereus CA (Cameroon) (Kav 0.410). B, B. anthracis Sterne 34F2 (Kav 0.433). C, type strain B. cereus ATCC14579 . The HF-SCWP from the great ape isolates Bc CA/CI and Bc strains all elute at approximately the same location, between the 25 kDa and 10.5 kDa dextran standards, having calculated mass of approx. 12,000 Da for Bc CA (Kav 0.410) and for Bc G9241 . The elution profiles of human isolates Bc G9241 and Bc 03BB87 HF-SCWPs are all essentially identical to that of Bc CA . Interestingly, the structurally unrelated B. cereus type strain ATCC14579 HF-SCWP elutes slightly after the 25 kDa dextran standard suggesting a mass around 20 kDa. The Bacillus SCWP isolates, in particular those from Ba and pathogenic Bc, contain variable amounts of a high molecular weight glucan which elutes at the void volume (Vo) (panels A and B). Glycosyl composition (GC-MS) and NMR analysis  indicated that this void volume polysaccharide is composed exclusively of glucose in predominately α1→4 linkage. This glucan may correspond to a glycogen polysaccharide previously reported in certain B. anthacis and related pathogenic Bacillus strains. Proteins  also elute at this void region. Column total inclusion volume = (Vi). In addition to the 35–40 kDa dextran profile, the elution positions of the 25 kDa and 10.5 kDa dextrans are indicated by arrows .SCWP were released from cell walls by treatment with hydrofluoric acid, dialyzed, then chromatographed on a Superose-12 FPLC size exclusion column with comparison to commercially available α1f Bc CA Click here for additional data file.


Synthesis and scission of phosphodiester bonds in DNA and RNA regulate vital processes within the cell. Enzymes that catalyze these reactions operate mostly via the recognized two-metal-ion mechanism. Our analysis reveals that basic amino acids and monovalent cations occupy structurally conserved positions nearby the active site of many two-metal-ion enzymes for which high-resolution (<3 Å) structures are known, including DNA and RNA polymerases, nucleases such as Cas9, and splicing ribozymes. Integrating multiple-sequence and structural alignments with molecular dynamics simulations, electrostatic potential maps, and mutational data, we found that these elements always interact with the substrates, suggesting that they may play an active role for catalysis, in addition to their electrostatic contribution. We discuss possible mechanistic implications of this expanded two-metal-ion architecture, including inferences on medium-resolution cryoelectron microscopy structures. Ultimately, our analysis may inspire future experiments and strategies for enzyme engineering or drug design to modulate nucleic acid processing.  •Basic residues in the active site of two-metal-ion enzymes are structurally conserved•These residues are also conserved in evolution•Mutagenesis suggests these residues may exert an effect on DNA- and RNA processing•Our work offers insights into CRISPR/Cas9, spliceosome, and DNA/RNA polymerases Genna et al. reveal structural conservation of basic amino acids and monovalent cations in the active site of two-metal-ion enzymes and ribozymes, which catalyze the vital biological reactions of DNA and RNA synthesis and scission. The work offers insights into fundamental two-metal-ion machineries such as CRISPR/Cas9, the spliceosome, and DNA/RNA polymerases. A) stabilizes the activated nucleophile, while the second metal  assists the release of the leaving group. Together, MA and MB stabilize the trigonal bipyramidal intermediate formed at the transition state , while a basic amino acid replaces the missing MB for catalysis  facilitate product release . HoweverA-MB and surrounding conserved nucleotides, two potassium ions (K1 and K2) located in the vicinity of the active site are essential for catalysis  structures in the PDB having an integer metal ion center for at least one enzyme of every considered class . This seOceanobacillus iheyensis group II intron with those of substrate-bound BamHI. Two BamHI lysines match the group II intron K1 and K2 ions  in other orthodox PD-(D/E)XK restriction endonucleases ucleases . Such seucleases  and partucleases   structure that displays the MA-MB center, but no substrates (PDB: 4CMQ), with a SpyCas9 structure that displays the substrate DNA but no divalent metals (PDB: 5F9R) (Actinomyces naeslundii (AnaCas9), Francisella tularensis (FtuCas9), and Staphylococcus aureus (SauCas9), and in RNase-H, which is evolutionarily related to the RuvC domain of Cas9  . Such suB: 5F9R) C. Lys974B: 5F9R) C. Intere of Cas9  , which catalyzes 5′ to 3′ exonucleolytic cleavage in dsDNA  and Exo-I of the extremophilic host Pyrococcus horikoshii .Finally, K1- and K2-like residues are also persistently conserved among exonucleases. in dsDNA , presentin dsDNA D. Both rin dsDNA , abolishin dsDNA  , are viral RNA-directed RNA polymerases (RdRps)  and Y-faCaliciviridae and from other single-stranded positive RNA viruses  and to the reverse transcriptase of retroviruses , i.e., aviridae  and Flaviviridae . In otheA-MB in a position analogous to the K1 ion, while Lys224 orients the phosphate backbone in the proximity of the cleavage site in a position analogous to K2  classical molecular dynamics (MD) simulations of human wild-type DNA Pol-η in complex with the substrate dsDNA and the incoming dATP, and of three Pol-η mutants derived from such a wild-type system . Also, to react  and S3.  ∼300 ns  and S3. Our work suggests that K1- and K2-like residues are a common feature of nucleases and polymerases. Some K1- and K2-like residues play idiosyncratic roles in specific enzyme classes. For instance, K1-like Lys144 of BglI and Lys131 of Exo-λ activate the reaction nucleophile , while KA-MB center, and all K2-like residues localize within a 3.5-Å-radius sphere at about 8.0 Å from the MA-MB center  that display all reactants and metal ions in the enzymatic active site. By multiple-sequence and structural alignments, molecular modeling, MD simulations, and electrostatic potential maps, we have identified two second-shell and positively charged elements  that are located in the vicinity of the two-metal-aided active site. In all the analyzed structures, despite the different size, shape, composition, and biological function of the respective enzymes, we have found that these elements share four common characteristics: they occupy analogous structural positions, they help modulating both the electrostatics in the surroundings of the active site and substrate stability, they are evolutionarily conserved, and they are associated to functional defects if mutated see , and S2.A-MB center are missing in some structures , and that enzymatic assays on mutants are not available yet.Also, these common properties of the K1- and K2-like residues can serve as a guide to identify analogous residues in other classes of two-metal-ion enzymes, particularly those for which structures can be determined only at medium-low resolution (>3 Å). To exemplify the predictive potential of our analysis of K1- and K2-analogs, we have analyzed the recent 3.9–4.0 Å resolution X-ray and cryoelectron microscopy structures of DNA-directed RNA polymerases (DdRp), which synthetize RNA in eukaryotic cells , and theS. cerevisiae RNA polymerase II (Pol-II) (∗ complex (We suggest that in (Pol-II) , Lys752 (Pol-II) F. Mutati(Pol-II) . Moreove(Pol-II)  (Data S1(Pol-II) . Althoug(Pol-II) . In addi complex , seem to complex .Figure 5A-MB  or by other non-basic residues (A-MB-binding acidic residues are not coordinated by residues near the putative K1-site (Arg183 or Arg149), but rather by the K2-like residue . In the first and faster reaction, the non-coding DNA strand is cleaved with support of MA and MC, while in the second and slower reaction, the coding DNA strand is cleaved with support of MB and MC. Our analysis shows that functionally crucial Lys120 , but no K1 counterparts, the role of the latter possibly being replaced by conserved lysines that directly coordinate Mresidues . DNA pol Arg254; . A thirdl Lys120  occupiesl Lys120 . Insteadl Lys120 . The asyl Lys120 , is thusA-MB, while also correctly orienting the substrates into the active site, as suggested by our MD simulations .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Marco Marcia  we performed a confidence interval (C.I.) analysis. The null hyphothesis is expressed by μX=μY, where μ represents the mean of the distribution, while X and Y are two different systems among the mutated. In order to test the null hypothesis, we computed the distributions of the d-newbond differences (dX-Y) for all possible cases and evaluated the mean μ and the associated standard error σE. Values are reported in in the d-newbond in DNA Pol-η K231A, K224A, and K224A/K231A were 90001, 90001, 83540, respectively, and were obtained by sampling the MD trajectory every 10 ps, for a total of 1.5 μs.To determine whether the M.M. and M.D.V. conceived and supervised the project. M.M. performed sequence and structural alignments. V.G. performed the MD analyses and calculated the electrostatic potential maps. M.C. performed the analyses of K1 and K2 mutants. M.M. and M.D.V. wrote the manuscript with contributions from all authors.


Escherichia coli to medium chain carboxylic acid -induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid ) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of  Escherichia coli or Saccharomyces cerevisiae [E. coli [Metabolic engineering allows us to produce a variety of carboxylic acids from renewable biomass compounds such as glucose, xylose, glycerol, and fatty acids. C8 to C18 carboxylic acids can be produced from glucose or glycerol by engineering the fatty acid synthesis pathways of revisiae . C5 to C[E. coli   in ty acids , remainsE. coli BL21 is a known and often used host for recombinant protein production [P. putida KT2440 and P. fluorescens DSM50106 were produced in a functional form [E. coli BL21 is more susceptible to various stresses including acid-induced stress when compared to E. coli K-12 strains   see . TherefoE. coli BL21(DE3) and MG1655 to n-heptanoic acid were intensively examined. The observed differences allowed us to identify factors that influence bacterial tolerance against n-heptanoic acid. Implementing the strategy observed in MG1655 i.e., dsrA and rcsB over-expression, allowed BL21(DE3) to increase viability in the presence of n-heptanoic acid and subsequently resulting in higher productivity of n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4) from the C18 fatty acid, ricinoleic acid (1)  .E. coli BL21(DE3) and K-12 MG1655 were cultivated in Lysogeny broth (LB) and Riesenberg medium [E. coli cultures were incubated at 37°C with shaking at 250 rpm . When the E. coli cultures (50 mL in 500 mL baffled flask) reached the exponential growth phases , n-heptanoic acid was added to different concentrations (0–10 mM) into the cultivation broth to conduct the growth experiments, in silico carbon flux analysis, transcriptome analysis, and measurement of glutamic decarboxylase (GadA/B) activity. E. coli B, W3110, W, and C strains were grown in the Riesenberg medium containing different concentrations (0–15 mM) of n-heptanoic acid for measurement of specific growth rate and GadA/B activity.Strains and plasmids used in this study are listed in g medium . The lat2SO4 at a constant rate of 0.6 mL/min at 50°C. A refractive index detector and a UV dual absorbance detector  were used for detection of the metabolites. UV detection was carried out at 210 nm.Extracellular concentrations of glucose, acetic acid, lactic acid, and ethanol were measured by high performance liquid chromatography (HPLC)  using an HPX-87H column . The column was eluted with 5 mM HE. coli metabolic network model, which consisted of 57 metabolites  and 58 biochemical reactions. Concentrations of all measured nutrients and products  were used to calculate their specific consumption or production rates, which were then specified as the capacity constraints in the model. The cellular objective of the cell growth rate during the exponential growth phase was maximized using linear programming (LP), thereby resulting in a set of metabolic flux distribution corresponding to the optimal phenotype [in silico model to validate results, as previously reported [E. coli BL21(DE3) and MG1655 strains at the specific glucose uptake rates and carbon fluxes.The constraints-based flux analyses were carried out by using the henotype . In the henotype . The spereported . The fluThe cellular maintenance energy was calculated according to earlier studies , 32. In E. coli_chip; Agilent Design ID: 063245). Total of 4,267 coding sequences of K12 and 4,153 coding sequences of BL21(DE3) were obtained from NCBI Database and 1 probe was designed for each coding sequence using the eArray software according to standard Agilent probe design criteria. We successfully designed 60-mer probes for 8,238 genes using eArray software (http://earray.chem.agilent.com/earray/). Microarrays were manufactured by Agilent Technologies (USA).Microarray interrogations were performed using a custom-designed, Agilent-based microarray platform with 4 × 44 K probes per slide , 3 or 10 mM heptanoic acid was added to the cultures of BL21(DE3) and K-12 MG1655. After 3 h of incubation, the cells were harvested for RNA purification. Total RNA was isolated by using the Qiagen RNeasy kit . For each RNA, the synthesis of target cRNA probes and hybridization were performed using Agilent’s Low Input Quick Amp WT Labeling Kit  according to the manufacturer’s instructions. The hybridized microarrays were washed according to the manufacturer’s washing protocol . The hybridization images were analyzed using the Agilent DNA microarray Scanner  and the data quantification was performed using the Agilent Feature Extraction software 10.7 . The average fluorescence intensity for each spot was calculated, while local background was subtracted. All data normalization and selection of fold-changed transcripts were performed using GeneSpringGX 7.3.1 . Normalization for the Agilent one-color method was performed, which including ‘Data transformation: Set intensity values which was less than 5.0 to 5.0’ and ‘Per chip Normalization: Each intensity measurement on a microarray was divided by the median intensity of all measurements included in the microarray. The averages of normalized ratios were calculated by dividing the average of control normalized signal intensity by the average of test normalized signal intensity. The categorization of data quality was according the Agilent manual: genes were labeled following the Agilent manual; genes were labeled “present (P)”, “marginal (M)” or “absent (A)” and filtered by selecting only “P” in each experiment. The intensity values lower than the background value were excluded. Genes showing differences in expression levels of ≥2- or ≤0.5-fold were considered to be differentially expressed genes. The microarray data have been deposited in the GEO database (www.ncbi.nlm.nih.bo/geo) and are accessible through GEO Series accession number GSE73640 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73640).When E. coli cells were harvested 3 h after n-heptanoic acid treatment by centrifugation at 5000 ×g for 15 min at 4°C. The cells were resuspended in 5 mM potassium phosphate buffer (pH 7.3) and sonicated on ice with 10 pulses for 20 sec (10 sec pauses between pulses). Concentration of total protein in each sample was measured by the Bradford assay (Bio-Rad). 50 μL of the cell lysate were then incubated with 100 mM phosphate—citrate buffer (pH 4.6) containing 20 mM L-glutamate, 1 mM pyridoxal 5’-phosphate, and 100 mM sodium chloride at 37°C for 60 min as described previously with some modifications [+, and 0.13 U/ml of GABase) for 80 min at 25°C. After the GABase reaction, the amount of NADPH produced was determined by measuring absorbance at 340 nm.ications . The rea600 of 1. Cultures were diluted 1:10 in M9 medium containing the stressor and were exposed for 1 h. Serial dilutions were plated on LB agar before and after exposure to each stress condition and survival frequency was calculated as survival (%) = colony forming units (CFU) (post stress) / CFU(prior stress)×100.Tolerance to n-heptanoic acid and low-pH was tested as described . BrieflyE. coli K12 MG1655 grown in LB medium by using Gspin™ Genomic DNA Extraction Kit . The dsrA and rcsB genes were PCR-amplified from genomic DNA and subcloned into the BamHI-EcoRI (MCS1) and NdeI- XhoI (MCS2) of the pCOLAduet vector, respectively. Amplified genes were digested using appropriate restriction enzymes and ligated into the vector using T4 DNA ligase . Ligated plasmid was introduced into E. coli DH5α. Transformants were inoculated in 3 mL LB medium and cultivated overnight. Recombinant plasmids were prepared from cultured E. coli cells using Exprep plasmid purification kit .Genomic DNA was extracted from E. coli were cultivated in Riesenberg medium at 37°C. The expression of the target genes was induced at 20°C with 0.1 mM IPTG. After cell growth reached stationary phase, the culture pH was adjusted to 8.0 with NaOH. Afterwards, the biotransformation was initiated by adding 15 mM ricinoleic acid and 0.5 g/L tween80. The reaction was performed in a 250 ml flask (working volume: 20 ml) in a shaking incubator at 35°C and 200 rpm.The whole-cell biotransformation was carried out on a basis of our previous work . BrieflyThe concentrations of remaining carboxylic acids in the medium such as ricinoleic acid, n-heptanoic acid, and ω-hydroxyundec-9-enoic acid were determined as described previously . The reaE. coli BL21(DE3) and K-12 MG1655 were cultivated in a glucose mineral medium with and without n-heptanoic acid to examine the effects of medium chain carboxylic acids on cell growth and carbon metabolism  in order to afford the increased requirement of the cellular maintenance energy. One of the major reasons may include the enhanced requirement of ATP and maintenance of the proton motive force. The toxicity of weak organic acids because proton transport against the proton gradient is well established [With an aim to investigate the effects of n-heptanoic acid on carbon metabolism of  studies , 36. The studies . Overallablished .icd, sucAB, gltA, and icd) were markedly increased as was the carbon flux. This result confirmed that the TCA cycle is involved in stress metabolism of E. coli BL21(DE3) under aerobic conditions.The transcriptional changes of the central carbon metabolic pathways were rather consistent with the carbon fluxes . In partE. coli BL21(DE3) to medium chain carboxylic acid . First, the oxidative electron transport chain that is implicated in proton efflux during mild acid stress under aerobic growth conditions [bo oxidase (CBO) complex were upregulated at least 2-fold in the presence of n-heptanoic acid in both strains  [gadC, glsA or gltB, gabT and gabD, hdeA/B), which products are involved in the GDAR system (E. coli MG1655 [Another mechanism to remove protons inside cells would be to use proton-consuming acid resistance systems . We havedA/GadB) , 46, wasR system , was alsi MG1655 , confirmE. coli [Blocking and/or reducing proton and carboxylic acid import could also be an important mechanism in acid tolerance. The first barrier in transport of carboxylic acids may include the porin proteins  and theE. coli , was muccfa, which catalyzes the cyclopropanation of unsaturated lipids in bacteria [E. coli, cyclopropanation is thought to be involved in long-term survival of non-growing cells and often associated with environmental stresses [cfa was increased with n-heptanoic acid only in the MG1655 , whereas expression of the ibpA and hchA, which products belong to the Hsp31 system that stabilizes unfolded proteins at low pH, was increased 2.5 and 3.4-fold respectively in the MG1655 only.Besides, expression behavior of the molecular chaperones was different between the strains . ExpressE. coli strains , B, MG1655, W3110, W, C) appeared to be dependent upon their GadA/B activities . Here, we have investigated activation of one of the acid resistance systems  in the BL21(DE3), because the acid resistance of tivities . The fir of RpoS , which i of RpoS , 55, is rcsB and dsrA of the MG1655 strain with their native promoters were introduced into E. coli BL21(DE3), the recombinant cells  pCOLA-RcsB-DsrA) showed a GadA/B activity, which is much higher than E. coli BL21(DE3) pCOLA empty vector at pH 3 or pH 4  pCOLA-RcsB-DsrA and E. coli MG1655 pCOLA was also observed when n-heptanoic acid was added into the culture broth (pH 6.7) (When the  or pH 4 . In fact(pH 6.7) . This reE. coli BL21(DE3) pCOLA-RcsB-DsrA was comparable to that of E. coli MG1655 pCOLA at pH 3 or pH 4, which was 10 (p<0.05) and 3 (p<0.01) times higher than E. coli BL21(DE3) pCOLA  pCOLA-RcsB-DsrA was markedly higher than E. coli BL21(DE3) pCOLA in the presence of n-heptanoic acid (We have next examined whether the activation of the GDAR system in BL21(DE3) increases acid tolerance. The colony forming unit-based survival frequency was evaluated as previously reported . The sur3) pCOLA . This reoic acid , indicatE. coli BL21(DE3). The recombinant E. coli BL21(DE3) pCOLA-RcsB-DsrA and E. coli BL21(DE3) pCOLA were grown at the conditions identical to the experiments shown in E. coli BL21(DE3) pCOLA-RcsB-DsrA was markedly lower than that of E. coli BL21(DE3) pCOLA at carboxylic acid concentrations of 3 or 5 mM ) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4) (E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA or E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA-RcsB-DsrA expressing the alcohol dehydrogenase (ADH) of Micrococcus luteus and the Baeyer-Villiger monooxygenase (BVMO) of Pseudomonas putida, the final product formation rates of the both cells were similar to at t < 4 h (E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA ceased resulting in an accumulation of the reaction intermediate (2) in the culture medium at t > 4 h, when the product concentration and bioconversion yield reached over 6 mM and 45%, respectively (E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA-RcsB-DsrA was further maintained resulting in a final product concentration of over 10 mM , which was 1.6-fold higher as compared to the E. coli BL21(DE3) pACYC-ADH, pET-BVMO, pCOLA. This value was also over three-folds greater than that of E. coli MG16655-based biocatalysts expressing the ADH and the BVMO. This result might be ascribed to low expression level of the BVMO in a functional form in the MG16655 strain. Overall, this is consistent with acid tolerance of the cells and functional production of the key enzymes are core factors to influence the fatty acid biotransformation activity.The effect of the GDAR system engineering on the whole-cell fatty acid biotransformation activity was investigated by conducting the bioconversion of ricinoleic acid  , which w t < 4 h . Howeverectively . This miectively . In contE. coli BL21(DE3); the specific glucose uptake and acetate production rates were substantially increased (p<0.05) by two- and eighteen-folds, respectively, in the presence of 5 mM n-heptanoic acid  [The metabolic responses to heptanoic acid of nchanged . A previexanone) , 58. Addexanone) . It was E. coli BL21(DE3) seemed to be related with acid tolerance. Activation of the GDAR system in BL21(DE3) allowed to reduce the specific glucose uptake and acetate production rates by ca. 26% , B, MG1655, W3110, W, C) were reduced depending on the n-heptanoic acid concentration in the culture medium  decreased most. Another interesting point was the positive correlation between specific growth rate in the presence of acid and the GadA/B activity  genome, were introduced to restore a functional the GDAR system in BL21(DE3). Activation of the GDAR system by expressing the dsrA and rcsB genes with their native promoters in BL21(DE3) allows to enhance tolerance not only to low pH but also to n-heptanoic acid , anpoS mRNA , 63, 64)poS mRNA , 65. Dsroic acid . The acetionally  Figs. ThE. coli BL21(DE3) is more susceptible to acid stress than E. coli K-12 MG1655. Activation of the GDAR system by introducing the rcsB and dsrA of MG1655 into BL21(DE3) led to increased acid tolerance not only to low pH but also to n-heptanoic acid. Therefore, it was concluded that the GDAR system played a key role in acid tolerance of E. coli. The higher ricinoleic acid biotransformation activity of the engineered E. coli BL21(DE3) also indicated that acid tolerance is one of the factors that influence fatty acid biotransformation activity. This study contributes to a deeper understanding of acid tolerance of E. coli and presents an engineering strategy for acid tolerance applicable for productive whole-cell biotransformation to synthesize carboxylic acids.S1 FigE. coli BL21(DE3) (blue), B (cyan), MG1655 (red), W3110 (dark green), W (dark yellow), C (pink)) was cultivated in a glucose mineral medium containing different concentrations of n-heptanoic acid.(TIF)Click here for additional data file.S2 FigE. coli BL21(DE3) (blue), B (cyan), MG1655 (red), W3110 (dark green), W (dark yellow), C (pink)) was cultivated in a glucose mineral medium containing different concentrations of n-heptanoic acid.(TIF)Click here for additional data file.S3 FigE. coli BL21(DE3), whereas added to zero (solid lines) or 10 mM (dashed lines) into the culture broth of E. coli MG1655. The cultures were incubated in a shaking incubator (200 rpm and 37°C).n-Heptanoic acid was added to zero (solid lines) or 3 mM (dashed lines) into the culture broth of (TIF)Click here for additional data file.S4 FigE. coli BL21(DE3) and K-12 MG1655 strains growing in the absence of n-heptanoic acid. The second upper values and lower values indicate the internal carbon flux distribution in E. coli BL21(DE3) and K-12 MG1655 strains growing in the presence of n-heptanoic acid. The BL21(DE3) was cultivated with 3 mM n-heptanoic acid, while the MG1655 was with 10 mM n-heptanoic acid. The flux data were normalized based on the specific glucose uptake rates.The cells were grown in the presence of different concentrations of n-heptanoic acid, as shown in (TIF)Click here for additional data file.S5 FigE. coli BL21(DE3), whereas added to zero (solid lines) or 10 mM (dashed lines) into the culture broth of E. coli MG1655. The cultures were incubated in a shaking incubator (200 rpm and 37°C).n-Heptanoic acid was added to zero (solid lines) or 3 mM (dashed lines) into the culture broth of (TIF)Click here for additional data file.S6 FigE. coli BL21(DE3), whereas added to zero, 5mM, and 10 mM into the culture broth of E. coli MG1655 during the exponential growth phase. The culture was incubated in a shaking incubator (200 rpm and 37°C). Values are the mean of more than three independent samples. Bars represent standard error of the mean.n-Heptanoic acid was added to zero, 3 mM, and 5 mM into the culture broth of (TIF)Click here for additional data file.S7 FigE. coli BL21(DE3) pCOLA, E. coli BL21(DE3) pCOLA-RcsB-DsrA, and E. coli MG1655 pCOLA growing in the absence of n-heptanoic acid. The second upper values and fourth upper values indicate the internal carbon flux distribution in E. coli BL21(DE3) pCOLA and E. coli BL21(DE3) pCOLA-RcsB-DsrA growing in the presence (3 mM) of n-heptanoic acid. The lower values indicate the internal carbon flux distribution in E. coli MG1655 pCOLA growing in the presence (10 mM) of n-heptanoic acid. Carbon flux distribution was estimated based on stoichiometric constraints using a metabolic network model implemented in MetaFluxNet [The upper values, third upper values, and fifth upper vlaues indicate the internal carbon flux distribution in aFluxNet .(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S1 Scheme1) is converted into ω-hydroxyundec-9-enoic acid (4) and n-heptanoic acid (5).Ricinoleic acid ((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


In mammals, neural circuits are formed based on a genetic program and further refined by neuronal activity during the neonatal period. We report that in the mouse olfactory system, the glomerular map is not merely refined but newly connected to second-order neurons by odorant-receptor-derived neuronal activity. Here, we analyzed a pair of molecules, Sema7A, expressed in olfactory sensory neurons (OSNs) in an activity-dependent manner, and PlxnC1, localized to dendrites of mitral/tufted (M/T) cells in the first week after birth. In Sema7A or PlxnC1 knockout (KO) mice, initiation of synapse formation and dendrite selection of M/T cells were perturbed. Reconstitution and rescue experiments demonstrated that Sema7A–PlxnC1 interaction is essential to form the post-synaptic assembly. Pharmacological blocking experiments indicated that synaptic transmission triggers primary dendrite selection by synaptic competition. We conclude that Sema7A signaling is key to inducing activity-dependent post-synapse events and dendrite selection in M/T-cells during the neonatal period. The molecular mechanisms underlying synapse formation in the olfactory bulb are not fully understood. Here the authors demonstrate that semaphorin 7A on olfactory sensory neurons, and its receptor plexin C1 expressed on mitral and tufted cells, is required for correct synapse formation. Each olfactory sensory neuron (OSN) expresses only one functional OR gene2, and OSN axons expressing the same OR species converge to a specific set of glomeruli in the olfactory bulb (OB)3. Since a given odorant activates multiple OR species4, odor signals detected in the olfactory epithelium (OE) are converted to a unique combination of activated glomeruli, enabling the mammalian brain to discriminate a variety of odorants5. The odor information is further processed by local circuits in the OB and conveyed by mitral/tufted (M/T) cells for decision making to various areas in the olfactory cortex (OC)6.The mouse olfactory system detects a variety of odorants using a repertoire of ~1000 odorant receptors (ORs)8. After OSN axons reach their approximate destinations in the OB, further refinement of an olfactory map occurs through fasciculation of OSN axons in an activity-dependent manner9. Although most of these processes take place by axon–axon interactions of OSNs10, proper connections are needed between OSN axons and the dendrites of M/T cells. In contrast to the primary projection of OSN axons, little is known about synapse formation with M/T cells. For example, what mediates the synapse formation between OSN axons and M/T-cell dendrites? How do they find their partners for proper synapse formation? What are the synaptic events involved in these processes? In an effort to address these questions, we searched for a pair of ligand and receptor molecules that are involved in synapse formation and dendrite selection of M/T cells.During embryonic development, a coarse olfactory map is generated by a combination of dorsal–ventral (D–V) patterning based on anatomical locations of OSNs, and anterior–posterior (A–P) patterning based on the OR-derived cAMP signals9.To determine what kinds of molecules are essential for initiating the synapse formation in glomeruli, we searched for a receptor and ligand pair each of which was expressed by OSN axons or M/T-cell dendrites. We examined 26 different genes of axon guidance, cell adhesion, and signaling molecules for their expression in the OE and OB Table . Among t11. Since the CNG-A2 gene is carried on the X chromosome, mosaicism can occur among OSNs for CNG-A2 activity due to stochastic X-chromosome inactivation in the female12. In the CNG-A2+/− mice, duplicated glomeruli are formed: one is for CNG-A2+ axons and the other for CNG-A2−. When rI7 glomeruli were analyzed at P5, Sema7A levels in the CNG-A2− glomerulus were much lower than in that of CNG-A2+  was expressed, Sema7A expression was diminished . When one naris was occluded, Sema7A expression in the OE was markedly reduced on the occluded side Fig.  left, shA2+ Fig.  middle. hed Fig.  right, b16. We, therefore, examined whether these receptors are expressed in M/T cells by in situ hybridization (ISH) of OB sections. At early developmental stages, PlxnC1, but not Integrin β1, is transcribed in M/T cells  stainingely Fig. . Each KO14 after crossing with Tg MOR29B18 or MOR28 knock-in mice19. Since the KOs of other semaphorin molecules, e.g., Sema3A and Sema3F, severely affect OSN projection to the OB8, we examined glomerular map formation in the Sema7A KO. Whole mount staining of the OB from the Sema7A KO at P8 did not reveal any notable defects in OSN projection or in glomerular formation  was injected into the glomerular structure for EYFP-tagged rI7 and connecting M/T-Cell dendrites were analyzed by immunostaining with anti-GFP and LY antibodies. We counted the numbers of M/T-cells with primary dendrite as mature and with multiple dendrites as immature. In the Sema7A KO, dendrite selection of M/T cells was perturbed, although dendrite extension to the glomerular layer (GL) was normal  was generated for PlxnC1 using the Pcdh21-Cre mousehigh-type glomeruli , synapse formation and dendrite selection occur a few days earlier than in the Sema7Alow-type . This was shown by immunostaining of synaptic markers and LY staining of M/T-cell dendrites at P4  was found to be co-localized with those of myc (PlxnC1) when the full-length PlxnC1 was expressed with the secrete type of Sema7A in HEK cells , rescued the phosphorylation of PAK, but not of ERK, in the Sema7A KO mice  and the effect of Sema7A–PlxnC1 signaling (synapse maturation vs. elimination) are opposite to what is found in the olfactory system. It will be interesting to study how Sema7A/PlxnC1 signaling functions in the two synaptic systems in different ways.In the mouse olfactory system, Sema and Plxn molecules have been extensively studied for their roles in targeting OSN axons, where both are produced in the OE for axon-axon interactions of OSNs28, which may allow the recruitment and assembly of PSD molecules. Another possibility is that Sema7A–PlxnC1 interactions activate signal transduction pathways. Recent studies revealed that some transmembrane molecules initiate PSD formation using their own kinase activity, and not by recruiting PSD components via PSD domains34. Our study is the first to demonstrate that Sema7A signaling activates the Rac1/Cdc42-PAKpathway that regulates vesicle trafficking, cytoskeletal dynamics and gene expression necessary for the post-synaptogenesis in M/T cells.In the mouse olfactory system, interaction of Sema7A with PlxnC1 triggers post-synaptic assembly. How does Sema7A signaling recruit PSD molecules? One possibility is that binding of Sema7A to PlxnC1 induces the conformational change in PlxnC111. In the CNG-A2 KO, other ion-channels in OSNs may compensate the CNG-derived activity and induce basal expression of Sema7A for triggering the post-synaptic events in M/T cells -ires-gap-YFP mice8, OSN-specific cKO of Nrp142, and MOR28-knockin mice19 were described previously. To generate cell-type-specific Sema7A and PlxnC1 knockout in the Cre driver line, we cloned exon 5 of the Sema7A and exon 5 of the PlxnC1 gene into the double-floxed, inverted open-reading frame plasmid DT-A/Conditional FW (http://www.cdb.riken.jp/arg/cassette.html). The conditional KOs were generated according to the published protocol (http://www.cdb.riken.jp/arg/tg.html). The Pcdh21-Cre mouse (RBRC02189) was obtained from RIKEN24. (http://www.cdb.riken.jp/arg/tg.html).CNG-A2 KO and Sema7A KO mice were purchased from The Jackson Laboratory (Nos. 002905 and 005128). BAC Tg mice containing the All animal experiments were approved by the Animal Care Committee in the University of Fukui and Tokyo and RIKEN institute in Kobe.Antibodies against Sema7A , PlxnC1 , and CNG-A2 , vGlut2 , GluR1 , GFP , Lucifer yellow , and OMP , were purchased from R&D Systems, Abcam, Alomone Labs, Millipore, Invitrogen, Abcam, Thermo Fisher Scientific, Thermo Fisher Scientific, and Wako Chemicals, respectively. Antibodies against PAK, p-PAK, ERK, and p-ERK  were obtained from Cell Signaling. Antibodies against FLAG  and c-myc  were from Abcam.9. Primer sequences used for PCR are as follows: sema7A, 5′-gctccattgcagaaggtttc-3′ (forward) and 5′-gctcacagctctgttccaca-3′ (reverse); plxnC1, 5′-gggactttcaagcgactgag-3′ (forward) and 5′-agtgtcttgcggagatgctt-3′ (reverse); integrinβ1, 5′-gaaggtggctttgatgcaat-3′ (forward) and 5′-tgttggaacactttcgtcca-3′ (reverse).To prepare cRNA probes, DNA fragments of 500–3000 bp were amplified by PCR from the OB cDNA of C57BL/6 mice. Only unique sequences were amplified for each gene. PCR products were subcloned into pGEM-T vector (Promega) and used as templates to make cRNA probes. Hybridization was performed according to the standard method9. Sections were photographed with a fluorescence microscope, Model IX70 (Olympus), coupled to a cooled CCD camera, C4742-95-12ERG (Hamamatsu Photonics).For immunohistochemistry, OB sections were perfused and treated with the primary and secondary antibodiesMice were transcardially perfused with 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) in 0.1 M phosphate buffer (PB), immersed in the same buffer at 4 °C for 1–2 h, and then sectioned transversely into 50-μm slices with a vibratome. The sections were rinsed in 0.1 M PB, incubated in 0.1 M PB with 2% normal serum for 1 h, and then incubated in primary antibody/ 1% normal serum over-night at 4 °C. The sections were rinsed in 0.1 M PB, incubated with Vector Labs biotinylated secondary immunoglobulin for 60 min, rinsed in 0.1 M PB, incubated in the Molecular Probes Nanogold for 90 min, and again rinsed in 0.1 M PB. The samples were then incubated in 0.1% GA for 10 min, and in Molecular Probes Silver Enhance Kit for 3–5 min. For electron microscopy, the sections were further treated as described in the previous section.Unilateral naris occlusion was performed at P0 by electrical cautery and continued to P6. Mice were killed at P6 and OB sections were analyzed by immunohistochemistry for the expression of synaptic markers.23 was performed as described below. Under a water-immersion ×40 objective lens, a micropipette was inserted into a given glomerulus. After labeling, slices were incubated with a blocking solution containing 5% serum and 0.5% Triton-X in PBS. Samples were incubated with rabbit polyclonal anti-Lucifer yellow antibodies and chicken anti-GFP antibodies, rinsed with PBS, and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) conjugate and Alexa Fluor 555-conjugated goat anti-chicken IgG (H+L) . Mature (single dendrite) and immature (multiple dendrites) M/T cells were counted.Intracellular injection17.To generate fusion proteins, cDNAs corresponding to the ectodomain were introduced into the APtag-5 (GenHunter Corporation) of AP fusion vectors. DNA fragments of the ectodomain were obtained by PCR. This enables the ectodomain to be fused to the N terminus of AP. AP fusion proteins were produced in the transfected HEK293T cells and concentrated using Centriprep (Millipore). Fresh-frozen OB sections were postfixed at −20 °C for 20 min in 100% methanol. AP staining was performed following the standard methodSema7A cDNA (pCMV-AP-secret type Sema7A mut)17. The cytosolic domain sequence in the PlxnC1 gene was deleted by PCR to generate pCMV-c-myc-ΔC PlxnC1. Co-transfection of HEK293 cells was performed as described previously with minor modification17. Two days after transfection, HEK cells were fixed on micro slides and processed for immunocytochemistry with antibodies against FLAG, myc, and Sema7A.Coding sequences of mouse SAP90, PlxnC1, and ectodomain of Sema7A were cloned into pCMV-SC-NF (STRATAGENE), APtag-5 (GenHunter Corporation), and pCMV-SC-NM (STRATAGENE), respectively. FLAG-tag and AP-tag were attached to the N-terminal of SAP90 and Sema7A, respectively. Myc-tag was inserted between the signal peptide and semaphorin domain of PlxnC1. The mutant Sema7A, Y213S, was generated by PCR on the MK-801 (Sigma–Aldrich) was dissolved in normal saline to achieve a final concentration of 0.02 mg/kg immediately before use. MK801/saline injection was performed with a stainless steel needle (30 gauges), which was directly inserted into the dorsal OB, twice a day from P2 to P5 in two groups of littermates.For fluorescent signals of immunostaining, digital images were captured with a digital CCD camera, C4742-95-12ERG (Hamamatsu Photonics), and two-photon microscopy (Olympus). Tone was reversed and a monochrome image was used for the measurement. For staining of the OB sections, digital images were captured with a digital CCD camera, Model DP70 (Olympus). To quantitate the staining level of each glomerulus, the mean pixel intensity within the region surrounded by the periglomerular cell nuclei was measured using Scion Image (Scion Corp.).All statistical analyses were performed using Excel 2003 (Microsoft) with the Statcel2 add-on (OMS).The data that support the findings in this study are available from the corresponding author on request.Supplementary InformationPeer Review File