tobacco recombinant dna technology antimicrobial enterococcus faecium  (ATCC)

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: Antimicrobial specificity of nucleobase analogues against Gram-positive priority pathogens

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Analogues

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Structure of ibezapolstat (IBZ) with the guanine nucleobase moiety in light blue. b Structure of ACX-801 with the position of R1 ( N 2 -subtitution), R2 and R3 marked in coloured squares, and the blue arrow indicating displacement of the ring nitrogen from position 9 to 8 in the ACX scaffold compared to guanine and IBZ. c MIC distribution of 46 compounds from the ACX library for different bacterial species. Only MIC values below 16 mg/L for VRE were used. Values ≥ 64 mg/L are grouped together. Abbreviations: VRE (vancomycin-resistant E. faecium ), Efa ( E. faecalis) , MRSA (methicillin-resistant S. aureus ), Sau (susceptible S. aureus ), PRSP (penicillin-resistant S. pneumoniae ), and Eco (E. coli) . The size of the circles corresponds the number of compounds, from 1 (smallest) to 46 (largest). d Gel-based primer extension assay showing DNA polymerase activity of E. faecium wild-type (WT) and exonuclease-inactivated PolC (Exo null ; D431A + E433A), but not for polymerase-inactivated PolC (Pol null ; D972A + D974A). Schematic of non-extended primer:template DNA substrate is shown at the bottom right and fully extended primer:template above. A complementary exonuclease assay is shown in Supplementary Fig. . A representative gel of multiple runs with reproducible results is shown. e Real-time assay (Supplementary Fig. ) measuring inhibition of polymerase activity by IBZ and 4 representative compounds from the ACX library using exonuclease-inactivated E. faecium PolC, with derived IC 50 values. Individual data points for each replicate ( n = 3) are shown. A normalized dose-response (three parameter) fit was used to determine IC 50 values and, where an IC 50 could be determined ( < 100 μM), the standard error of the mean ( n = 3) is given. f Real-time assay measuring susceptibility of DnaE-type polymerases E. coli Pol IIIα and E. faecium DnaE to IBZ and ACX-801. Individual data points for each replicate ( n = 3) are shown. A normalized dose-response (three parameter) fit was used to determine IC 50 values and, where an IC 50 could be determined ( < 100 μM), the standard error of the mean ( n = 3) is given.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Primer Extension Assay, Activity Assay, Inhibition, Derivative Assay

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Apo structure (PDB-9QRN) of exonuclease-inactivated E. faecium PolC with a 3-nucleotide ssDNA bound to the exonuclease domain (Exo). Other domains are labelled OB (oligonucleotide/oligosaccharide-binding) and PHP (polymerase and histidinol phosphate) as in 2c. The flexible N-terminal domain (N-term), not resolved in the density map and structure, is indicated in grey. b Structure of exonuclease-inactivated E. faecium PolC (yellow) bound to DNA (grey) and ACX-801 (blue) (PDB-9QPC). c Schematic representations of the domains of E. faecium PolC. The PHP domain is interrupted by the Exo domain, and the palm domain is split by the thumb (T) subdomain. The position of the catalytic residues of the Exo domain (D431 and E433) are indicated with an asterisk. d Ligand interaction map of IBZ as derived from the structure (PDB-9QRL). An alternative map containing further details is provided in Supplementary Fig. . Conserved interacting residues are highlighted. e Ligand interaction map of ACX-801 as derived from the structure (PDB-9QPC). An alternative map is provided in Supplementary Fig. . Residues uniquely identified as interacting with ACX-801 are indicated with a stroke. Conserved interacting residues are highlighted. f Base-pairing (represented by dashed lines) between the dCMP (grey) and dGTP (yellow, from PDB-3F2C), IBZ (pink, PDB-9QRL) and ACX-801 (blue, PDB-9QPC). Stick representations are coloured by atom but with different backbone colours. g Close-up of the binding pocket in the IBZ-bound PolC structure (PDB-9QRL). PolC is shown in blue with specific residues in yellow and IBZ as sticks with a purple backbone. h Close-up of the binding pocket in the ACX-801-bound PolC structure (PDB-9QPC). PolC is shown in yellow with specific residues in pink and ACX-801 in blue. Water is represented as a blue spheres and the dashed lines indicate interactions with residue Y1274. i Displacement of residues in the ACX-801-bound structure (yellow; PDB-9QPC) compared to the ligand-free, apo structure (purple; PDB-9QRN). Polymerase catalytic residues (D972 and D974) are annotated. The arrows highlight the rotation and displacement of F1276, and minor displacement of Y1274 and Y1284 to accommodate the inhibitor.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Binding Assay, Derivative Assay, Residue

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Sequence conservation plotted on the surface of PolC (PDB-9QPC), showing the highest conservation in the DNA binding cleft and exonuclease active site. The inset on the left shows the conservation of PolC sequences plotted on an enlargement of the surface of the inhibitor binding pocket, with relevant amino acid residues indicated. b Structure-based sequence alignment of C-family sequences from Gram-positive and Gram-negative bacteria. Species names are coloured according to PolC-type (black) and DnaE-type (blue) polymerase sequences, with a + or – indicating Gram-positive or -negative bacteria, respectively. The secondary structures (coils representing helices numbered according to PDBs) are shown above for E. faecium PolC (PDB-9QPC) and below for E. coli PolIIIα (PDB-5M1S). The residues that are part of the inhibitor-binding pocket of E. faecium PolC are indicated, with residues that are displaced in the inhibitor-bound conformation in pink. The red asterisk (*) marks the truncated helix in DnaE-type polymerases. c Superposition of E. faecium PolC in ACX-801-bound form (in yellow) and three DnaE-type polymerases (in three tones of grey): E. coli PolIIIα (PDB-5M1S), M. tuberculosis DnaE1 (PDB-7PU7), and Thermus aquaticus Pol IIIα (PDB-3E0D). The two arrows indicate the movement of residues required to create a full inhibitor binding pocket. The red asterisk (*) marks the end of the helix in DnaE-type polymerases; this helix is extended by half a turn in PolC-type polymerases and positions the fourth aromatic residue of the PolC inhibitor binding pocket, which has no structural equivalent in DnaEs. Numbering of residues is based on E. faecium PolC.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Sequencing, Binding Assay, Bacteria, Residue

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Close-up of the inhibitor binding pocket in exonuclease-inactivated E. faecium PolC (PDB-9QPC) with two residues involved in resistance to PolC inhibitors (F1276 and A1281) highlighted in purple. ACX-801 is shown in blue and other residues that make up the binding pocket are shown in grey. b Polymerase activity inhibition of F1276 mutants compared to wild-type PolC protein by IBZ (left) and ACX-801 (right), with derived IC 50 values. All proteins were exonuclease-inactivated. Individual data points for each replicate ( n = 3) are shown with a normalized dose-response (three parameter) fit and, where an IC 50 could be determined, the standard error of the mean is given. c Susceptibility of C. difficile carrying different plasmid-based polC alleles to IBZ and ACX-801. Cells were spotted onto BHI agar with increasing amounts of IBZ (left) or ACX-801 (right). Plasmids carrying the wild-type polC gene ( polC ), or mutant alleles polC p .F1258L (F1258L), polC p .F1258I (F1258I), polC p .F1258S (F1258S) or polC p .A1263T (A1263T) are shown. d DNA polymerase activity of different PolC variants in the absence of inhibitor. The PolC A1281T (A1281T) mutant shows an ~10-fold reduction in activity compared to wild-type PolC and two different F1276 variants. The activity is shown as an average of three replicates; the standard error is omitted as it is obscured by the size of the symbols.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Binding Assay, Activity Assay, Inhibition, Derivative Assay, Plasmid Preparation, Mutagenesis

Journal: Indian journal of medical microbiology

Article Title: A new approach of real time polymerase chain reaction in detection of vancomycin-resistant enterococci and its comparison with other methods.

doi: 10.4103/0255-0857.108721

Figure Lengend Snippet: Figure 1: Construction of standards.(a) Bold line (-) shows the result of experiment in which serial dilutions (105-101) containing known quantities of the E. faecium ATCC 51559 strain plasmid deoxyribonucleic acid (DNA) containing vanA fragment were subjected to real time quantitative polymerase chain reaction (Q-PCR) while dotted line (….) shows the results of three represented clinical isolates. The amplifi cation curve shifted to right as the quantity of DNA was reduced. The system was sensitive enough to detect as few as 10 copies of vanA DNA. (b) Plot of threshold cycle (ct) against the target quantity with the later plotted on a common logarithm scale. The linearity of graph demonstrated the large dynamic range and the accuracy of Q-PCR assay

Article Snippet: Construction of standards and calculation of fold change of vanA For calculation of threshold cycle (Ct) value, a standard curve was generated using 10-fold dilutions of vanA positive E. faecium ATCC 51559 DNA varying from 105-101 copies.

Techniques: Plasmid Preparation, Real-time Polymerase Chain Reaction