PhD Seminar: Increased activity of the phosphorelay triggers sporulation during growth in a gut strain of Bacillus subtilis

ITQB PhD SEMINAR

Title: Increased activity of the phosphorelay triggers sporulation during growth in a gut strain of Bacillus subtilis

Speaker: Cláudia Serra

Laboratory: Microbial Development, ITQB

Abstract

Sporulation in Bacillus subtilis is generally regarded as a last resource adaptation to extreme nutritional stress, triggered only upon entry into the stationary phase of growth. We have found that a wild (undomesticated) gut isolate of B. subtilis forms spores with high efficiency during exponential growth, and hence bypassing the cell density, nutritional, cell cycle and other signals that normally delay sporulation. High-level sporulation during growth occurs because of increased activity of Spo0A, the key regulatory protein which, when activated by phosphorylation governs entry into sporulation. We used fluorescence microscopy to quantify the activity of Spo0A in individual vegetative cells across the population, and found that compared to a laboratory strain, more cells of the gut strain accumulate Spo0A above a certain threshold level which is required for the initiation of sporulation. Moreover, by replacing the auto-regulatory Spo0A promoter by an inducible promoter, we show that normal sporulation can take place at nearly undetectable levels of Spo0A. We infer that Spo0A is also more active per cell of the gut strain, and is not normally the limiting factor for sporulation. We show that the genes for three response regulator aspartyl phosphatases (Rap) are absent from the genome of the gut strain. These phosphatases act on a cascade of regulatory proteins, known as the phosphorelay. The phosphorelay is served by five sensor kinases, that transfer phosphoryl groups to Spo0A via two intermediate proteins, Spo0F, a modified response regulator, and the phosphotransferase Spo0B. The Rap phosphatases act on Spo0F, and negatively regulate the phosphorelay during exponential growth, preventing entry into sporulation. They are however inactivated at the end of growth by a quorum-sensing mechanism involving import of extracellular inhibitory peptides. Increased activity of the phosphorelay caused by the absence of the three Rap proteins thus explains the elevated activity of Spo0A both across the population and per cell of the gut strain, and shows that the activity of the phosphorelay and not the levels of Spo0A (above) is the limiting factor for entry into sporulation. We speculate that in the gut strain, the phosphorelay is tuned to favor high-level sporulation during growth. Because spores but not vegetative cells, survive exposure to bile salts and low pH, this is a characteristic that may favor survival of the population in the gut environment.

Short CV

Since April 2007: PhD student (SFRH / BD / 29397 / 2006) at Microbial Development group, ITQB- UNL, Oeiras, Portugal. Research Project: Analysis of the regulatory circuits involved in sporulation initiation in undomesticated Bacillus subtilis. Supervisor: Prof. Adriano O. Henriques.
2006/2007 - Post-graduate employee. DSM Nutritional Products, Basel, Switzerland.
2003/2006 - Scientific Initiation Grant (BIC). Microbial Development Group ITQB- UNL, Oeiras, Portugal.
2001/2002 - Research assistant. Institute for Animal Science and Health (ID- Lelystad), Lelystad, The Netherlands.
Graduation (Licenciatura) in Engenharia Zootécnica, Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal