detection-of-live-escherichia-coli-o157h7-cells-by-pma-qpcr

Detection of Live <em>Escherichia coli</em> O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Food and Drug Administration

Selection of the division site in Escherichia coli is regulated by the min system and requires the rapid oscillation of MinD between the two halves of the cell under the control of MinE. In this study we have further investigated the molecular basis for this oscillation by examining the interaction of MinD with phospholipid vesicles. We found that MinD bound to phospholipid vesicles in the presence of ATP and, upon binding, assembled into a well-ordered helical array that deformed the vesicles into tubes. Stimulation of the MinD ATPase by addition of MinE led to disassembly of the tubes and the release of MinD from the vesicles. It is proposed that this MinE-regulated dynamic assembly of MinD underlies MinD oscillation.

Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE.

In Escherichia coli, the min system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC.

Recruitment of MinC, an inhibitor of Z-ring formation, to the membrane in Escherichia coli: role of MinD and MinE.

MinD is a key component of an oscillatory system that spatially regulates cell division in Escherichia coli. It is a peripheral membrane ATPase that recruits MinC and oscillates between the two halves of the cell in a MinE dependent manner. In vitro MinD binds to phospholipid vesicles in an ATP-dependent manner and is released through MinE-stimulated ATP hydrolysis. In this study we examined the function of the conserved C-terminus of MinD. Short truncations of three and ten amino acids dramatically decreased the ability of MinD to localize to the membrane and spatially regulate division. These truncations bound MinC but were deficient in targeting MinC to the septum. In vitro they dimerized, but were deficient in binding to phospholipid vesicles and undergoing MinE stimulation. We suggest a model in which the ATP-dependent dimerization of MinD affects the conformation of the C-terminal region, a potential amphipathic helix, triggering membrane binding.

A conserved sequence at the C-terminus of MinD is required for binding to the membrane and targeting MinC to the septum.

The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.

Analysis of MinD mutations reveals residues required for MinE stimulation of the MinD ATPase and residues required for MinC interaction.

Detection of RNAs on microarrays is rapidly becoming a standard approach for molecular biologists. However, current methods frequently discriminate against structured and/or small RNA species. Here we present an approach that bypasses these problems. Unmodified RNA is hybridized directly to DNA microarrays and detected with the high-affinity, nucleotide sequence-independent, DNA/RNA hybrid-specific mouse monoclonal antibody S9.6. Subsequent reactions with a fluorescently-labeled anti-mouse IgG antibody or biotin-labeled anti-mouse IgG together with fluorescently labeled streptavidin produces a signal that can be measured in a standard microarray scanner. The antibody-based method was able to detect low abundance small RNAs of Escherichia coli much more efficiently than the commonly-used cDNA-based method. A specific small RNA was detected in amounts of 0.25 fmol (i.e. concentration of 10 pM in a 25 microl reaction). The method is an efficient, robust and inexpensive technique that allows quantitative analysis of gene expression and does not discriminate against short or structured RNAs.

An antibody-based microarray assay for small RNA detection.

The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C.

Genetic analysis of the roles of agaA, agaI, and agaS genes in the N-acetyl-D-galactosamine and D-galactosamine catabolic pathways in Escherichia coli strains O157:H7 and C.

The monoclonal antibody S9.6 binds DNA-RNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. A single-chain variable fragment (scFv) of S9.6 was produced, and its affinities for various synthetic nucleic acid hybrids were measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNA-RNA and, surprisingly, 2.7 nM for RNA-RNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNA-RNA hybrid. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Zonglin Hu

Center for Food Safety and Applied Nutrition,
Division of Molecular Biology,
Center for Food Safety and Applied Nutrition, Division of Molecular Biology

Food and Drug Administration

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Zonglin Hu

Center for Food Safety and Applied Nutrition,Division of Molecular Biology,Center for Food Safety and Applied Nutrition, Division of Molecular Biology

Food and Drug Administration

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

Center for Food Safety and Applied Nutrition,
Division of Molecular Biology,
Center for Food Safety and Applied Nutrition, Division of Molecular Biology