Polynucleotide Phosphorylase is Required for Escherichia coli O157:H7 Growth at Low Temperature

Introduction: The growth of Escherichia coli O157:H7 in contaminated dairy and other food products due to temperature fluctuation post a major food safety threat. To effectively control or inhibit the growth of E. coli O157:H7 depends on our understanding of mechanisms regulating its low temperature growth.

Purpose: Polynucleotide phosphorylase (PNPase) is needed for cold adaption of commensal E. coli. We hypothesized that PNPase has a critical role in the growth of E. coli O157:H7 at low temperature.

Methods: The PNPase gene deletion mutant (Δpnp) of E. coli O157:H7 was generated using l Red recombinase system. pBAD was used to construct the pnp complementation plasmid. Swimming motility was assayed using 0.3% agar plate. The growth curve was conducted in 96 well microplates.  The growth and survival of E. coli O157:H7 during cold storage were analyzed every other day by enumeration.

Results: Growth of Dpnp and wild type (WT) strains in both LB broth and agar did not differ at 37°C, but Dpnp strain has impaired growth at 22°C and 10 °C. During 14 days of 10°C storage in both LB and milk, WT grew and reached >8 Log₁₀CFU/ml after 4 days of 10°C storage, while Dpnp was gradually dying off with a more pronounced effects in milk. In addition, pnp deletion impaired the swimming motility of E. coli O157:H7.The observed differences were completely or partially complemented by overexpression of pnp plasmid, indicating a pnp specific effect.

Significance: PNPase is required for the growth of E. coli O157:H7 at low temperature, which provides a molecular target to control the growth of E. coli O157:H7 at low temperature, having important practical applications to food industry. (USDA-AFRI2010-65201-20599, Agricultural Experiment Station at University of Wyoming)