The 2D gel identifies several proteins with differential
levels of RG7112 order production in these conditions, including S1 and S15 (circled) which are only secreted at 28°C. AZD1390 In vivo and in vitro production of Pam As the identification of highly-secreted Pam occurred at 28°C, a temperature relevant to the infection of insect hosts, we monitored Pam production over time in Galleria mellonella larvae injected with either P. luminescens TT01 (Fig. 2A) or P. asymbiotica ATCC43949 (Fig. 2B). We observed high levels of production in the insect host at 48 h post-injection which continued for a further 11 days, suggesting a possible role of this secreted protein in the occupation of the insect cadaver. It is also possible that Pam is produced in the insect before 48 h and has not been detected with our methods. We were Cilengitide chemical structure unable to isolate tissues within the insect for Pam-specific production patterns due to internal disruption of the cadaver 48 h after infection. In vitro production of Pam was monitored in P. asymbiotica ATCC43949 liquid cultures, and it was first detected in supernatants by Western blot after 6 h 30 min of growth in LB medium at 28°C, corresponding to the exponential phase of the culture (Fig. 3A). Pam continued to be produced throughout growth into
stationary phase (48 h) and up to 6 day-old cultures (data not shown). As expected, no Pam was released at 37°C although cell-associated Pam could be detected, indicating it is synthesized but not released into the surrounding milieu. The fact Dapagliflozin that Pam protein is released only at insect-relevant temperatures and the difficulties with genetic manipulation and transformation of P. asymbiotica strain ATCC43949, led us to make a pam knock-out strain in
the well-characterized P. luminescens TT01. Figure 3B shows a Western blot demonstrating the absence of Pam in the mutant strain TT01pam. For heterologous expression in E. coli, pam was amplified from P. asymbiotica ATCC43949 and cloned in the arabinose-inducible vector pBAD30, under translational control of its native Shine-Dalgarno region. Heterologous production of Pam was confirmed by Western blot (Fig. 3C). The recombinant protein was purified using ion-exchange chromatography for further analysis (Fig. 3D). Figure 2 Detection of Pam in infected G. mellonella. Each insect was injected with (A) P. luminescens TT01 or (B) P. asymbiotica ATCC43949, and was frozen and crushed in 1 ml of buffer at days 1 to 10 and 13 post injection. 10 μl of each sample was used per lane for SDS-PAGE, and Western blot analysis using anti-Pam antibody showed production from the second day after infection. The arrow indicates that Pam is not produced by Photorhabdus in the first day of G. mellonella infection or that it is below the detection limit of the assay. Figure 3 In vitro Pam production. (A) Western blot confirmation of the temperature-dependent secretion of Pam in P.