Protein Folding Failure Sets High-Temperature Limit on Growth of Phage P22 in Salmonella enterica Serovar Typhimurium

Abstract

The high-temperature limit for growth of microorganisms differs greatly depending on their species and habitat. The importance of an organism's ability to manage thermal stress is reflected in the ubiquitous distribution of the heat shock chaperones. Although many chaperones function to reduce protein folding defects, it has been difficult to identify the specific protein folding pathways that set the high-temperature limit of growth for a given microorganism. We have investigated this for a simple system, phage P22 infection of Salmonella enterica serovar Typhimurium. Production of infectious particles exhibited a broad maximum of 150 phage per cell when host cells were grown at between 30 and 39°C in minimal medium. Production of infectious phage declined sharply in the range of 40 to 41°C, and at 42°C, production had fallen to less than 1% of the maximum rate. The host cells maintained optimal division rates at these temperatures. The decrease in phage infectivity was steeper than the loss of physical particles, suggesting that noninfectious particles were formed at higher temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a decrease in the tailspike adhesins assembled on phage particles purified from cultures incubated at higher temperatures. The infectivity of these particles was restored by in vitro incubation with soluble tailspike trimers. Examination of tailspike folding and assembly in lysates of phage-infected cells confirmed that the fraction of polypeptide chains able to reach the native state in vivo decreased with increasing temperature, indicating a thermal folding defect rather than a particle assembly defect. Thus, we believe that the folding pathway of the tailspike adhesin sets the high-temperature limit for P22 formation in Salmonella serovar Typhimurium.

The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures

“…The availability of such cell proteins for recombinant protein folding can be alternatively enhanced by reducing the input of the target protein through limiting gene dosage or weakening recombinant gene expression (Hoffman et al, 1995;Weickert et al, 1997). On the other hand, the use of low growth temperatures in production processes usually results in higher yields and improved biological activity of the soluble protein, even for folding-reluctant species (Baldwin, 1986;Betts and King, 1998;Ferrer et al, 2004;Pope et al, 2004;Schellman, 1997;Strandberg and Enfors, 1991;Scharnagl et al, 2005). However, the conformational status of the remaining insoluble fraction has been hardly explored.…”

Molecular Cloning and Expression Analysis of COX II from <em>Sitophilus zeamais</em>, a Potential Action Site of AITC

“…Therefore, improving solubility is a major goal in studying protein production processes. Recently, the use of low growth temperatures to increase soluble protein yields has been reported [41][42]. In present study, most of the COXⅡ protein expressed in E.coli was as inclusion bodies.…”

Enhancement of chicken macrophage cytokine response toSalmonellaTyphimurium when combined with bacteriophage P22

“…In the present study, the effects of P22 on the host (LT2) and the production of four different cytokines in chicken macrophage cells were evaluated by adherence and uptake assays as well as ELISA and qRT‐PCR. The specific ability of P22 for killing LT2 has been reported in several studies (Pope et al ., ; Toro et al ., ). The phage utilized in this study was able to initiate killing of extra‐ and intracellular S. Typhimurium within a few minutes after infection and completed bacterial lysis within 16 h.…”