Biology subjects

Paton, A. W.

Publications and source records attributed to Paton, A. W..

2 recordsLinked to original sources

bioRxiv2017

SMRT sequencing reveals differential patterns of methylation in two O111:H- Shiga toxigenic Escherichia coli isolates from a historic hemolytic uremic syndrome outbreak in Australia

Shiga toxigenic Escherichia coli (STEC) are important food-borne pathogens and a major cause of haemorrhagic colitis and haemolytic-uremic syndrome (HUS) worldwide. In 1995 a severe HUS outbreak in Adelaide occurred. A recent genomic analysis of STEC O111:H-strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes although the structure of the Stx2-converting prophages could not be fully resolved due to the fragmented assembly. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) long read sequencing to characterise the complete epigenome (genome and methylome) of 95JB1 and 95NR1. Using long reads we completely resolved the structure of two, tandemly inserted, stx2-converting phage in 95NR1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5-CTGCm6AG-3) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethalyted in the 95JB1, including at least 180 potential promoter regions that could explain regulatory differences between the strains. We identified a Type IIG methyltransferase encoded in both genomes in association with three additional genes in an operon-like arrangement. IS1203 mediated disruption of this operon in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the enormous potential of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

genomics↗

bioRxiv2017

Intermolecular Disulfide-Bond Formation In Human FICD Modulates The Activity Of The Hyperactive E234G Mutant

Endoplasmic reticulum (ER) stress that leads to the accumulation of misfolded proteins in the ER initiates the unfolded protein response (UPR). This homeostatic response activates signaling pathways that seek to reinstate a proper ER protein folding balance or induce apoptosis if ER stress persists. Recently, we and others identified human FICD (Filamentation induced by cyclic AMP domain-containing protein), an enzyme with adenylyltransferase (aka AMPylation) activity, as a new UPR target. Here, we demonstrate that FICD is functionally linked to the UPR, as evidenced by the finding that the adenylyltransferase activity of the protein induces ER stress, while FICD silencing increases sensitivity to ER stress. We identify BiP, an abundant ER chaperone and key regulator of the UPR, as the main substrate of FICD AMPylation in ER-derived microsomes, further emphasizing close functional connection of FICD to the UPR and in line with recent reports that AMPylation inactivates BiP. Notably, BiP overexpression increased the levels of BiP AMPylation as well as FICD auto-AMPylation, suggesting a homeostatic response that balances the pool of active BiP to modulate its functions in protein folding as well as UPR signaling. Finally, we show that overexpressed FICD forms a disulfide-bonded homo-dimer through Cys51 and Cys75 and demonstrate that mutation of these two cysteines in the context of a hyperactive FICD mutant leads to increased BiP AMPylation. This latter finding opens up the possibility that FICD activity is redox regulated and closely connected with ER redox homeostasis.

molecular biology↗