Structural Analysis of the Essential Resuscitation Promoting Factor YeaZ Suggests a Mechanism of Nucleotide Regulation through Dimer Reorganization

Extent: 8p.Background: The yeaZ gene product forms part of the conserved network YjeE/YeaZ/YgjD essential for the survival of many Gram-negative eubacteria. Among other as yet unidentified roles, YeaZ functions as a resuscitation promoting factor required for survival and resuscitation of cells in a viable but non-culturable (VBNC) state. Methodology/Principal Findings: In order to investigate in detail the structure/function relationship of this family of proteins we have performed X-ray crystallographic studies of Vibrio parahaemolyticus YeaZ. The YeaZ structure showed that it has a classic actin-like nucleotide-binding fold. Comparisons of this crystal structure to that of available homologues from E. coli, T. maritima and S. typhimurium revealed two distinctly different modes of dimer formation. In one form, prevalent in the absence of nucleotide, the putative nucleotide-binding site is incomplete, lacking a binding pocket for a nucleotide base. In the second form, residues from the second subunit complete the nucleotide-binding site. This suggests that the two dimer architectures observed in the crystal structures correspond to a free and a nucleotide-bound form of YeaZ. A multiple sequence alignment of YeaZ proteins from different bacteria allowed us to identify a large conserved hydrophobic patch on the protein surface that becomes exposed upon nucleotide-driven dimer re-arrangement. We hypothesize that the transition between two dimer architectures represents the transition between the ‘on’ and ‘off’ states of YeaZ. The effect of this transition is to alternately expose and bury a docking site for the partner protein YgjD. Conclusions/Significance: This paper provides the first structural insight into the putative mechanism of nucleotide regulation of YeaZ through dimer reorganization. Our analysis suggests that nucleotide binding to YeaZ may act as a regulator or switch that changes YeaZ shape, allowing it to switch partners between YjeE and YgjD.Inci Aydin, Yumiko Saijo-Hamano, Keiichi Namba, Connor Thomas and Anna Roujeinikov

Genotypes and phenotypes of Shiga toxin producing-Escherichia coli isolated from healthy cattle in Thailand

Shiga toxin producing-Escherichia coli (STEC) has not yet been identified as an important aetiologic agent of human disease in Thailand. To evaluate the potential for STEC to contribute to human disease in Thailand, 139 fecal samples were collected from healthy cattle from five different provinces and analysed by genotypic and phenotypic methods for STEC. Of 139 samples, 27 (19.4%) were positive for stx1 and/or stx2 by multiplex polymerase chain reaction, or for O157 lipopolysaccharide (LPS) by immunoassay. Isolates positive for stx and/or O157 were subdivided into 49 strains that varied in the presence of the virulence determinants stx1<SUP>+</SUP>/stx2<SUP>+</SUP> (22 strains), stx2<SUB>+</SUB> (22 strains), stx1<SUP>+</SUP> (4 strains), and O157 LPS (1 strain). Within these 49 distinguishable strains, other virulence determinants varied as follows: hlyA+ (77.6%), eae<SUP>+</SUP> and tir<SUP>+</SUP> (4.1%), and katP<SUP>+</SUP> (6.12%). The most predominant profile (22 isolates) was stx1<SUP>+</SUP>/stx2<SUP>+</SUP>, eae<SUP>-</SUP>, tir<SUP>-</SUP>, etpD<SUP>-</SUP>, hlyA<SUP>+</SUP>, katP<SUP>-</SUP>. For further characterization of the isolated strains by two molecular typing assays, plasmid profiles and ERIC PCR were performed. The results suggest that the genetic and phenotypic profiles of STEC associated with human disease are not prevalent at this time in cattle in Thailand