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  • DNA methylation assay To test

    2019-07-12

    DNA methylation assay. To test the biological activity of purified A1S_0222, a methylation assay was performed using the Int1 DNA. As an alternative DNA substrate, Seq3 (located in gene A1S_0965 of A. baumannii ATCC 17978) was amplified from Acinetobacter baumannii 29D2 using the oligonucleotides Seq3-for: 5′-GAAGTCACTGATACCAAGGAAGGTATTCATTTTG-3′ and Seq3-rev: 5′-GTCTGGAAAATGCTGTGTTTCTAATGCTAG-3’ (801 bp). Each fragment contains one EcoRI restriction site (G↓AATTC). For the methylation reaction 1 μg of Int1 or Seq3, 2 μL B1 buffer, 4 μL of 800 μM SAM, 8 μL RNase free water and 3 μL A1S_0222 (2.46 μg/μL) or 40 units EcoRI methyltransferase (New England BioLabs) were mixed and incubated for up to 1 h at 37 °C. After incubation A1S_0222 or EcoRI methyltransferase was inactivated by 95 °C for 5 min and Int1 or Seq3 were purified with Hi Yield® Gel/PCR DNA Fragment Extraction Kit (SLG®) according to the manufacturer\'s instructions and eluted in 10 μL RNase free water. Subsequently, an EcoRI restriction reaction was performed (5 μL purified Int1 or Seq3, 1 μL EcoRI, 2 μL EcoRI buffer, 12 μL RNase free water) for 2 h at 37 °C. For visualization, agarose electrophoresis was carried out at 110 V for 50 min in TAE buffer (agarose concentration: 1.25% (w/v)). GelRed™ (GeneON) was used for gel staining. GelRed™ stock solution was diluted 1:5000 into the agarose gel solution. As a size standard GeneRuler DNA Ladder Mix (Thermo Fisher Scientific) was used. The methyltransferase activity was calculated based on the definition that 1 unit (U) of enzyme converts 1 μmol of substrate per minute at 37 °C. Small angle X-ray scattering (SAXS). Synchrotron SAXS measurements (I(s) vs s, where s = 4πsinθ/λ; 2θ is the scattering angle and λ = 0.125 nm) were performed at the EMBL-P12 bioSAXS beam line at the PETRA III storage ring (DESY, Hamburg) as described in Ref. [26] under continuous-flow batch mode operations at 10 °C utilizing an automated robotic sample changer [27]. The scattering intensity data were recorded from 25 μl aliquots of A1S_0222 (2.4 mg/mL) and a corresponding matched solvent blank (150 mM NaCl, 10 mM Tris, 1 mM DTT and 5% glycerol, the person 7.4) as a series of sequential 50 m s 2D-data frames for a total exposure time of 1 s (Pilatus 2 M photon counting detector). The resulting individual 50 m s 2D-frames underwent radial averaging to produce unsubtracted 1D I(s) vs s scattering profiles [28]. Additional statistical checks were employed through the data reduction process so as not to include any sample (or buffer) frames affected by radiation damage (or systematic scaling errors) in the final, reduced 1D scattering profiles [29]. The scattering from the buffer was subtracted from the sample scattering to generate the final SAXS profile measured across an s-range of 0.013–3.8 nm. From these data, several structural parameters were extracted using the ATSAS 2.8 software package [30]. First, the number of Shannon channels and maximum working s was assessed using SHANUM (Shannon channels = 14; smax = 3.2 nm−1 [31]). The extrapolated forward scattering intensity at zero angle, I(0) and the radius of gyration R, were determined from both the Guinier approximation (lnI(s) vs s2, in the s-range of 0.22 < sR < 1.3, defining a working smin of 0.07 nm−1 [32]) and from the probable real-space distance distribution (p(r) vs r profile) calculated from the indirect inverse Fourier transform of the data using GNOM [33]. The latter was also used to evaluate the maximum particle dimension, D. The molecular weight (MW) estimate of A1S_0222 was assessed using both concentration-dependent and concentration-independent methods. The concentration-independent MW methods included that of [34], the ‘volume’ of correlation, V, from Ref. [35] and the empirical correction to the Porod volume, V, as described in Ref. [36] as implemented in the respective ATSAS dattools DATMOW, DATVC and DATPOROD. The concentration-dependent MW estimate was performed relative to a bovine serum albumin (BSA) standard using the extrapolated I(0) and the sample concentration (described in Ref. [37]). The final spatial representation of A1S_0222 – classified as compact using DATCLASS with an AMBIMETER shape ambiguity score of 0.9, i.e., as potentially unique [38] was obtained using ab initio bead modelling implemented in the program DAMMIN [39]. Ten individual bead models were generated that fit the data (assessed using the reduced χ2 test and the Correlation Map, or CorMap, p-value. A χ2 of 1 and p > 0.01 indicate no systematic discrepancies between the data-model fit [29]). The individual models were spatially aligned using SUPCOMB where the normalized spatial discrepancy (NSD) of the 10 model ensemble was assessed at 0.5 (aligned models with NSD < 1 are considered similar [40]). The models were subsequently averaged and bead occupancy-corrected using DAMAVER [41] and refined against the SAXS data using DAMMIN/DAMSTART to produce a final average 3D-spatial representation of A1S_0222 at an estimated resolution of 2.5 nm [42]. The SAXS data, p(r) vs r profile, as well as the individual, averaged and refined DAMMIN models and the associated fits are deposited in the Small-angle Scattering Biological Data Bank with the accession code SASDD32 (SASBDB: www.sasbdb.org [43]).