Journal: bioRxiv

Article Title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

doi: 10.1101/2021.12.19.473356

Figure Lengend Snippet: Panel a: Seven genes within the BREX locus encode for proteins ranging in size from 22 kD (BrxB, 191 residues) to 134 kD (PglX, 1173 residues). A transcription start site analysis shows that BREX is expressed via a single long transcript (upper arrow and dashed line) in logarithmically grown Acinetobacter394 cells in the absence of a phage challenge. Panel b: PacBio sequencing of Acinetobacter NEB394 and E. coli genomes (harboring the endogenous BREX operon and the same operon introduced via transformation with plasmid pACYC-BREX) identifies the host target site and base (5’-GTAG A T-3’) methylated by the BREX system via action of the PglX methyltransferase. Panel c: Systematic deletion of each BREX gene from the pACYC-BREX plasmid and additional PacBio methylation analysis in transformed E. coli cells demonstrates that four BREX genes (BrxA, BrxB, BrxC and PglZ) are required for genome modification in addition to the PglX methyltransferase. Panel d: Growth curves of NEB2683 E. coli cells transformed with pACYC-BREX plasmids in which each BREX gene has been systematically deleted. Three deletions (ΔPglZ, ΔBrxB and ΔBrxR) appear to display lags or reductions in growth rates. These observations are reflected in reduced transformation efficiencies and small or irregular colony size for the same constructs .

Article Snippet: Experiments were performed both in E. coli NEB 5-alpha cells (New England Biolabs) and in E. coli strain ER2683 (New England Biolabs).

Techniques: Sequencing, Transformation Assay, Plasmid Preparation, Methylation, Modification, Construct

Journal: bioRxiv

Article Title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

doi: 10.1101/2021.12.19.473356

Figure Lengend Snippet: E. coli ER2683 cells transformed with empty vector, vector encoding the WT BREX operon, or the same operon harboring precise deletions of each BREX protein factor. Reduced colony size and transformation efficiency is observed for ΔBrxL, ΔBrxB and ΔPglZ.

Article Snippet: Experiments were performed both in E. coli NEB 5-alpha cells (New England Biolabs) and in E. coli strain ER2683 (New England Biolabs).

Techniques: Transformation Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

doi: 10.1101/2021.12.19.473356

Figure Lengend Snippet: Panel a: BREX transcription start site analysis in Acinetobacter 394. A single long transcript is detected beginning 23 base pairs upstream of the BrxR start codon. The top track shows BREX ORFs. The bottom track shows PacBio SMRT capable seq showing coverage (gray). This shows a strong TSS just before BrxR and the BREX operon, with very low levels of internal operon reads, indicating one TSS for the entire operon. See Supplementary Figure S2 for additional detail. Panel b: Restriction by BREX visualized in a plaque formation assay. Restriction was assayed using a plaque formation assay with λ vir phage deployed against E. coli strain ER2683. For each construct, serial 10-fold dilutions of phage were spotted on a bacterial lawn, and individual plaques were counted. All assays were repeated in biological triplicate. The intact BREX operon generates similar (30- to 50-fold) reductions in plaquing efficiency, relative to the empty pACYC vector, when preceded by either a constitutive tet promoter (‘tet’) or by the upstream putative promoter and regulatory region from Acinetobacter (‘native’). Panel c:) . Restriction by the same BREX constructs in liquid culture. Bacterial cultures were challenged with λ vir phage at the indicated MOI and culture density (OD 600 nm) was monitored over time.

Article Snippet: Experiments were performed both in E. coli NEB 5-alpha cells (New England Biolabs) and in E. coli strain ER2683 (New England Biolabs).

Techniques: Plaque Formation Assay, Construct, Plasmid Preparation

Journal: bioRxiv

Article Title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

doi: 10.1101/2021.12.19.473356

Figure Lengend Snippet: Panel a: Growth of E. coli strain NEB5α (New England Biolabs) transformed with pACYC184-BREX and challenged with λ vir phage at MOI’s ranging from 0.1 to 0.001. BREX - cells display lysis within five hours of the challenge, versus continued growth and saturation of the cells that harbor the BREX system. The level of protection conferred by the intact BREX system displayed a dependence on the phage MOI; cells containing BREX eventually crashed at the highest MOI (0.1) but continued to grow at MOI’s of 0.01 and 0.001.

Article Snippet: Experiments were performed both in E. coli NEB 5-alpha cells (New England Biolabs) and in E. coli strain ER2683 (New England Biolabs).

Techniques: Transformation Assay, Lysis

Journal: bioRxiv

Article Title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

doi: 10.1101/2021.12.19.473356

Figure Lengend Snippet: Panel a: Fold change in phage plaque formation using λ vir phage and E. coli strain ER2683 as a function of the presence or absence of each gene in the BREX operon. Cells transformed with the indicated pACYC constructs, growing at log phase, were mixed with 0.5% top agar, plated on chloramphenicol plates to form a lawn, then spotted with 10-fold serial dilutions of λ vir phage. The plaque formation efficiency of λ vir phage on a ‘cells only’ (no BREX) control is normalized to 1; the corresponding fold reduction in plaquing formation efficiency of the same phage is then indicated for WT BREX and for BREX harboring a precise deletion of each gene. All seven genes appear to be involved in BREX restriction function. Panel b: Growth of E. coli strain ER2683 (New England Biolabs) challenged with the λ vir phage at an MOI of 1.0. The intact BREX system conferred robust protection against λ vir phage, whereas individual deletions of each BREX gene led to a significant reduction in phage restriction at both high MOI (shown here) and a lower MOI of approximately 0.01 .

Article Snippet: Experiments were performed both in E. coli NEB 5-alpha cells (New England Biolabs) and in E. coli strain ER2683 (New England Biolabs).

Techniques: Transformation Assay, Construct

Journal: bioRxiv

Article Title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

doi: 10.1101/2021.12.19.473356

Figure Lengend Snippet: E. coli strain ER2683 transformed with pACYC-BREX constructs containing the indicated single deletions were challenged with λ vir phage at an MOI of 0.01. All BREX ORF’s were required for restriction.

Article Snippet: Experiments were performed both in E. coli NEB 5-alpha cells (New England Biolabs) and in E. coli strain ER2683 (New England Biolabs).

Techniques: Transformation Assay, Construct

Journal: bioRxiv

Article Title: Identification and characterization of BrxR as a regulatory gene in the BREX phage restriction system

doi: 10.1101/2021.12.19.473356

Figure Lengend Snippet: Panels a-c : BrxR containing a single point mutation in its DNA binding domain (R47A) was expressed and purified to homogeneity; it behaves similarly to the wild-type protein dimer in solution as indicated by its elution profile on size exclusion chromatography (SEC) and displays similar thermal stability and unfolding behavior as the wild-type protein. Panel d : Electrophoretic mobility shift assays (EMSA) demonstrate a significant reduction in DNA target binding by purified BrxR (R47A) protein. Panel e: Phage restriction plaque assays indicate that unlike the effect of a precise deletion of the BrxR gene ( ; which causes significant toxicity and reduction in BREX restriction activity), the disruption of the BrxR gene by incorporation of an early stop codon or by introduction of a mutation that blocks BrxR DNA binding activity has little effect on cell growth or phage restriction in the same assays. Panel f: SDS PAGE analysis of whole cell lysates of E. coli ER2683 cells transformed with the following pACYC-BREX constructs: cells only (lane 1); pACYC vector only (lane 2); WT BREX (lane 3); a precise deletion of the BrxR gene (ΔBrxR; lane 4); an epitope-tagged version of BrxC that runs slightly higher than untagged BrxC (BrxC-TST; lane 5); a precise deletion of the BrxC gene (ΔBrxC; lane 6); BREX expressed from its native promoter (lane 7). Transformation with pACYC harboring wild-type BREX results in the appearance of a novel band at approximately 150 kD (arrow), and deletion of the BrxR gene results in significant increase in its intensity (compare lanes 3 and 4). The same band disappears altogether when BrxC gene is deleted from the BREX operon (lane 6), and shifts slightly upward when BrxC is fused to a 35 amino acid twin-strep tag (lane 5), indicating that the upregulated protein product is BrxC. This same band is present in when the BREX operon is expressed from the native promoter (lane 7), albeit at a reduced level compared to when BREX is expressed from the constitutive tet promoter (lane 3).

Article Snippet: Experiments were performed both in E. coli NEB 5-alpha cells (New England Biolabs) and in E. coli strain ER2683 (New England Biolabs).

Techniques: Mutagenesis, Binding Assay, Purification, Size-exclusion Chromatography, Electrophoretic Mobility Shift Assay, Activity Assay, SDS Page, Transformation Assay, Construct, Plasmid Preparation, Strep-tag