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The bacteriophage T4 MotB protein, a DNA binding protein, boosts the level of T4 late gene expression

Thursday, September 14, 2017 — Poster Session III

12:00 p.m. – 1:30 p.m.
FAES Terrace
NIDDK
MOLBIO-15

Authors

  • J Patterson-West
  • M Arroyo-Mendoza
  • L Iyer
  • D Hinton

Abstract

Condensation and organization of genomic DNA is crucial in all cells for orderly replication and gene expression. In bacteria, histone-like proteins such as the abundant H-NS and its less abundant homolog StpA, are DNA binding proteins that form higher-order nucleoprotein complexes needed for DNA condensation. H-NS targets AT-rich DNA sequences and condenses genomic DNA through a proposed looping mechanism that typically represses transcription at the affected region. As phage genomes and xenogeneic sequences acquired from horizontal gene transfer often display a high AT content, H-NS can protect bacteria from the expression of foreign genes by preferentially binding these sequences. Bacteriophage T4 (65.5% AT) is a lytic virus that infects E. coli, (45% AT) resulting in cell lysis after ~20 min. The T4 genome is expressed temporally from early, middle and late promoters. The T4 late promoter, TATAAATA, is strikingly similar to the H-NS binding motif, TCGATAAATT. Thus, it is not surprising that T4 may need to overcome H-NS repression. T4 Arn, a protein that structurally mimics DNA has been shown to bind H-NS, preventing its interaction with DNA and formation of higher order structures. However, Arn is not essential. The T4 motB gene encodes a highly conserved early protein whose function has not been characterized previously. We find that expression of plasmid-borne motB is highly toxic to E. coli, resulting in decondensation of host DNA, cell lengthening, significant reduction in actively dividing cells compared to a vector control, and cell lysis. MotB co-purifies with DNA, H-NS, and StpA. Electrophoresis mobility shift assays indicate that H-NS and MotB bind similar AT-rich sequences. However, MotB binds with 10-100-fold higher affinity than H-NS depending on the sequence. Although a T4 motB amber mutant has no noticeable phenotype in a plaque assay, RNA-seq indicates that the expression of several T4 late genes are significantly reduced. These findings are consistent with MotB relieving H-NS binding to and repression of late promoters and/or a direct activation of late gene expression by MotB. We hypothesize that the interaction of MotB with either H-NS (and StpA), DNA, or both is part of a mechanism used by T4 to disrupt H-NS dependent repression leading to optimal expression of its late genes. Determining why and how MotB is toxic can provide paths to the formation of anti-bacterials that work by a mechanism not previously targeted by antibiotics.

Category: Molecular Biology and Biochemistry