Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Summary

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

Abstract

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

Introduction

The goal of this work is to provide a protocol for the separation of bacterial endospores from vegetative bacterial cells in environmental samples. The formation of bacterial endospores is a survival strategy, usually triggered by starvation, found in a number of bacterial groups belonging to the phylum Firmicutes¹. Endospore-forming bacteria are well studied, mainly because a number of strains are pathogens and hence of medical importance (e.g., Bacillus anthracis or Clostridium difficile). Environmental strains of endospore-forming bacteria have been isolated from virtually every environment (soil, water, sediment, air, ice, hum....

Protocol

1. Preparation of Chemicals and Equipment 

1. Make 500 ml of a 1% sodium hexametaphosphate (SHMP) (NaPO₃)_n solution and sterilize by autoclaving.
2. Sterilize nitrocellulose (NC) filters (pore size 0.22 µm, diameter 47 mm) by autoclaving in closed glass Petri dishes.
3. Sterilize NC filters (pore size 0.22 µm, diameter 25 mm) by autoclaving in closed glass Petri dishes.
4. Weigh and note the empty weight of sterile 50 ml tubes (with cap on) (one tube per sample).
5. Prepare Tris-EDTA-buffer (TE-buffer) 1x: make solution of 10 mM Tris (tris(hydroxymethyl)aminomethane) and 1 mM EDTA buffe....

Results

The results presented here have been published earlier^10,21. Please refer to those articles for the environmental interpretation and discussion of the data.

The overall procedure is summarized in Figure 1 and corresponds to three main steps: first, the separation of biomass from sediment or any other environmental matrix; second, the destruction of vegetative cells; and third, the downstream analysis of the separated endospores. Downstream analysis could consist, fo.......

Discussion

The resistance of endospores to external aggressive physicochemical factors (e.g., temperature or detergents) was used to devise a method to separate bacterial endospores from vegetative cells in environmental samples. This is the first comprehensive method to isolate endospores from environmental samples in a non-destructive manner. Previous methods to quantify, detect or analyze endospores in samples were based on the measurement of specific proxies for endospores such as dipicolinic acid or specific marker ge.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Whatman nitrocellulose membrane filters, 0.2 μm pore size, 47 mm diameter	Sigma-Aldrich	WHA7182004 Aldrich
Tris(hydroxymethyl)aminomethane	Sigma-Aldrich	252859 Sigma-Aldrich	CAS 77-86-1
EDTA	Sigma-Aldrich	E9884 Sigma-Aldrich	CAS  60-00-4
Lysozyme from chicken egg white	Sigma-Aldrich	62971 Fluka	powder, CAS  12650-88-3
Ultra-Turrax homogenizer T18 basic	IKA	3720000
Glass filter holders for 47 mm membranes, Pyrex glass	EMD Millipore	XX10 047 00
Chemical duty vacuum pump	Millipore	WP6122050	220 V/50 Hz
Manifold sampling filtration for 25 mm membranes	Millipore	1225 Sampling Manifold	polypropylene
Dnase	New England Biolabs	M0303S	Rnase free
NaOH	Sigma-Aldrich	S5881 Sigma-Aldrich	CAS  1310-73-2
Sodium dodecyl sulfphate (SDS)	Sigma-Aldrich	L3771 Sigma
Whatman nitrocellulose membrane filters, 0.2 μm pore size, 25 mm diameter	Sigma-Aldrich	WHA7182002 Aldrich