Levels of germination proteins in Bacillus subtilis dormant, superdormant, and germinating spores.

Yan Chen,Stephen B Melville,Richard F Helm,W Keith Ray,David L Popham,Daniel Paredes-Sabja

doi:10.1371/journal.pone.0095781

Abstract

Bacterial endospores exhibit extreme resistance to most conditions that rapidly kill other life forms, remaining viable in this dormant state for centuries or longer. While the majority of Bacillus subtilis dormant spores germinate rapidly in response to nutrient germinants, a small subpopulation termed superdormant spores are resistant to germination, potentially evading antibiotic and/or decontamination strategies. In an effort to better understand the underlying mechanisms of superdormancy, membrane-associated proteins were isolated from populations of B. subtilis dormant, superdormant, and germinated spores, and the relative abundance of 11 germination-related proteins was determined using multiple-reaction-monitoring liquid chromatography-mass spectrometry assays. GerAC, GerKC, and GerD were significantly less abundant in the membrane fractions obtained from superdormant spores than those derived from dormant spores. The amounts of YpeB, GerD, PrkC, GerAC, and GerKC recovered in membrane fractions decreased significantly during germination. Lipoproteins, as a protein class, decreased during spore germination, while YpeB appeared to be specifically degraded. Some protein abundance differences between membrane fractions of dormant and superdormant spores resemble protein changes that take place during germination, suggesting that the superdormant spore isolation procedure may have resulted in early, non-committal germination-associated changes. In addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein may contribute to heterogeneity of spore germination rates. Understanding the reasons for superdormancy may allow for better spore decontamination procedures.

Highlights

Bacterial endospores are metabolically dormant and resistant to a variety of anti-microbial treatments due to their protective structures and dehydrated spore core [1,2]
In an effort to provide additional insight into the mechanisms of germination, we developed a multiple-reaction monitoring (MRM) mass spectrometry assay [29] to quantify 11 germination proteins believed to be associated with the spore inner membrane
Spore germination starts at the spore inner membrane with the interaction of germinant with Ger receptor proteins and progresses through core rehydration and cortex breakdown

Summary

Introduction

Bacterial endospores are metabolically dormant and resistant to a variety of anti-microbial treatments due to their protective structures and dehydrated spore core [1,2]. These spores can survive for decades in the absence of nutrients. They are able to return to a metabolically active state through a series of events termed spore germination. Once spores lose many of their resistance properties during germination, they can be eliminated by routine decontamination methods [3]. Since the spores of Bacillus and Clostridium species cause food spoilage and are infectious agents in several human diseases [4], the development of methods or reagents that stimulate highly efficient germination across a spore population could greatly simplify decontamination efforts and reduce morbidity and mortality

Methods

Results

Conclusion