, 2006; Abram et al., 2008; O’Byrne & Karatzas, 2008), thus it seemed logical to Selleck Omipalisib assess if SigB function contributed to the development of L. monocytogenes GASP. When examined for long-term survival in culture, a ΔsigB mutant exhibited the expected death and long-term stationary growth phases during the course of a 12-day incubation in BHI at 37 °C (Fig. 4a). Similar to the prfA* mutant, ΔsigB long-term stationary phase cultures exhibited final stable bacterial CFU numbers that were approximately twofold lower than those maintained by wild-type L. monocytogenes (Fig. 4a). SigB is thus
required for the optimal fitness of L. monocytogenes during the long-term stationary growth phase. ΔsigB mutant bacteria from 12-day-old cultures were added to 1-day-old mutant cultures at a final ratio of 1 : 100. Over 10 days, bacteria from the 12-day-old culture outcompeted bacteria of the 1-day-old culture such that the ratio at day 10 was 1 : 1 (Fig. 4b), indicating that the ΔsigB mutant
retained its ability to express the GASP phenotype. However, similar to the phenotype expressed by the prfA* mutant, the GASP phenotype exhibited by the ΔsigB strain was not as robust as that exhibited by wild-type L. monocytogenes (Fig. 4b). Although bacteria derived from 12-day-old wild type cultures increased 1000-fold in comparison to 1-day-old wild-type bacteria Selleckchem Ibrutinib (Fig. 4b), the bacterial numbers of a 12-day-old ΔsigB culture increased approximately 100-fold in comparison to those of the 1-day-old ΔsigB culture (Fig. 4b). Similar to the situation described above for prfA* strains, the failure of the ΔsigB mutant to express a robust GASP phenotype could reflect an impaired ability to develop GASP or may indicate that the loss of SigB contributed to a partial GASP phenotype for 1-day-old cultures. To distinguish between these two possibilities, the CI between wild type 12-day-old cultures and 1-day-old wild type or ΔsigB cultures was assessed. If the ΔsigB mutant expresses a partial GASP phenotype as the
result of the loss of SigB, then the competitive advantage of a wild-type 12-day-old culture should be less in comparison to 1-day-old ΔsigB than in comparison to Selleckchem Etoposide 1-day-old wild type. Interestingly, the difference in the competitive advantage of wild-type 12-day-old cultures observed vs. 1-day-old wild-type or 1-day-old ΔsigB was minimal (Fig. 4c). SigB contributes to L. monocytogenes fitness in broth culture, based on the competitive advantage of 1-day-old wild-type strains vs. 1-day-old ΔsigB mutants (Fig. 4c). Thus, in spite of ΔsigB mutants exhibiting a broth culture fitness defect, the overall magnitude of the competitive defect observed between 12-day-old wild-type L. monocytogenes and 1-day-old wild-type strains and ΔsigB mutants was similar rather than exacerbated for ΔsigB, suggesting that the loss of SigB may indeed contribute to the development of the GASP phenotype. Taken together, these data indicate a role for SigB in L.