Fundamentals of Microbiology

Lesson 13. STRUCTURE AND GERMINATION OF ENDOSPORE, MICROBIAL LOCOMOTION

Module 4. Structure and function of prokaryotic cells

Lesson 13

STRUCTURE AND GERMINATION OF ENDOSPORE, MICROBIAL LOCOMOTION

13.1 Introduction

The spore (i.e. endospore), discovered in 1876 by Cohn, Koch and Tyndall independently, is one of the hardiest dormant life forms on earth formed during the resting stage in the life cycle of spore-forming genera. Endospores are produced within cells and are refractile - light cannot penetrate them so that they are very easy to see in the phase microscope. They are resting structures, meaning that there is little or no metabolism inside the spore and it is a real form of suspended animation. Spores can survive for a very long time, and then re-germinate. Spores that were dormant for thousands of years in the great tomes of the Egyption Pharohs were able to germinate and grow when placed in appropriate medium. There are even claims of spores that are over 250 million years old being able to germinate when placed in appropriate medium.

Bacterial endospores are complex structures whose basic architecture is conserved across species. Endospore enables an organism to resist extreme environmental conditions such as

• Temperature
• Drying
• UV radiation
• Strong acids and bases
• Oxidizing agents
• Extremes of both vacuum and ultrahigh hydrostatic pressure

The resistance of spore is attributed to following factors:

• The presence of high content of calcium-dipicolinate which stabilizes and protect the DNA
• DNA-binding proteins protect DNA from heat, drying, chemicals, and radiation
• Dehydration that results in resistance to heat and radiation
• DNA repair enzymes are able to repair damaged DNA during germination

These resistant structures survive heating to 150°C although mostly killed at 121°C in moist medium. They can remain dormant for immense periods of time, perhaps even millions of years.

13.2 Endospore Forming Bacteria

Spore formers generally found in soils as their ability to form spores as advantageous for soil microorganisms. Spore formers are distinguished on the basis of cell morphology, shape and cellular position of endospore (Fig. 13.1). Key spore forming genera are Bacillus, Clostridium, Sporosarcina, Heliobacterium. Sporosarcinae are unique among endospore formers because cells are cocci instead of rods and are strictly aerobic, spherical cells. Other spore forming genera are Paenibacillus, Sporolactobacillus, Desulfotomaculum, Thermoanarobacter, Sporomusa, Sporohalobacter, Amphibacillus, Heliophylium, Heliorestis, Syntrophosphora, and Desulfitobacterium.

Fig. 13.1 Endospore forming bacteria

13.3 Endospore Structure

It is more complex than of vegetative cell. Inside spore there is a core (Spore protoplast), containing cytoplasm, nucleoid and ribosomes (Fig. 13.2). The core of the mature endospore has only 10-25 % of the vegetative cell water content what increases its resistance to heat and chemicals. The pH of core is one unit lower than that of vegetative cell cytoplasm and the core contains a high level of small acid-soluble proteins, SASPs, able to bind DNA and to protect it from potential damage. The coat contains 50-78% of total spore protein (Cot) which can be divided into two groups - alkali soluble and alkali insoluble.

Fig. 13.2 Structure of endospore

13.4 Endospore Formation

The formation of a spore is an expensive and complex process for the bacterial cell. Spores are only made under conditions where cell survival is threatened such as starvation for certain nutrients or accumulation of toxic wastes. Regulation of sporulation is tight and the first few steps are reversible. This helps the cell conserve energy and only sporulate when necessary. Sporulation is a seven step process (Fig. 13.3). When a bacterium detects environmental conditions are becoming unfavourable it may start the process of endosporulation, which takes about eight hours. The first stages of sporulation are involved in forming a separate compartment for the spore in the mother cell. Once this occurs, sporulation is irreversible. The DNA is replicated and a membrane wall known as a spore septum begins to form between it and the rest of the cell. The plasma membrane of the cell surrounds this wall and pinches off to leave a double membrane around the DNA, and the developing structure is now known as a forespore. Calcium dipicolinate is incorporated into the forespore during this time. The next stages involve laying down the various layers of the spore. The peptidoglycan cortex forms between the two layers and the bacterium adds a spore coat to the outside of the forespore. Both, the spore and the mother cell, play a role in this process. In the final stages, the spore dehydrates its cytoplasm and is released from the cell. Research on sporulation in Bacillus subtilis is intense. It is a wonderful model system for development, since the microbe is genetically tractible, allowing the isolation of many mutants in each stage of sporulation.

Endospores are resistant to most agents that would normally kill the vegetative cells they formed from. Household cleaning products generally have no effect, nor do most alcohols, quaternary ammonium compounds or detergents. Alkylating agents however, such as ethylene oxide, are effective against endospores. While resistant to extreme heat and radiation, endospores can be destroyed by burning or by autoclaving. Endospores are able to survive boiling at 100°C for hours, although the longer the number of hours the fewer that will survive. An indirect way to destroy them is to place them in an environment that reactivates them to their vegetative state. They will germinate within a day or two with the right environmental conditions, and then the vegetative cells can be straightforwardly destroyed. This indirect method is called Tyndallization. It was the usual method for a while in the late 19th century before the advent of inexpensive autoclaves. Prolonged exposure to high energy radiation, such as x-rays and gamma rays, will also kill most endospores.

13.5 Reactivation/Germination

Reactivation of the endospore occurs when conditions are more favourable and involves activation, germination, and outgrowth. Even if an endospore is located in plentiful nutrients, it may fail to germinate unless activation has taken place. This may be triggered by heating the endospore. Germination involves the dormant endospore starting metabolic activity and thus breaking hibernation. It is commonly characterised by rupture or absorption of the spore coat, swelling of the endospore, an increase in metabolic activity, and loss of resistance to environmental stress. Outgrowth follows germination and involves the core of the endospore manufacturing new chemical components and exiting the old spore coat to develop into a fully functional vegetative bacterial cell, which can divide to produce more cells.

13.6 Significance

Endospore formation can be considered a primitive system of cell differentiation and has become a paradigm for the study of this phenomenon in prokaryotes. As a simplified model for cellular differentiation, the molecular details of endospore formation have been extensively studied, specifically in the model organism Bacillus subtilis. These studies have contributed much to our understanding of the regulation of gene expression, transcription factors, and the sigma factor subunits of RNA polymerase.

Endospores of the bacterium Bacillus anthracis were used in the 2001 anthrax attacks. The powder found in contaminated postal letters was composed of extracellular anthrax endospores. Inhalation, ingestion or skin contamination of these endospores, which were technically incorrectly labelled as ‘spores’, led to a number of deaths.

The presence of endospore formers can be problematic as they can not be inactivated by heat treatments normally applied in food industry and cause food poisoning (Bacillus cereus, Clostridium botulinum) and spoilage of even heat treated foods (B. subtilis, B. coagulans).

Geobacillus stearothermophilus endospores are used as biological indicators when an autoclave is used in sterilization procedures.

On positive note, Endospore preparations derived from Bacillus thuringiensis and Paenibacillus popillae are commercially available as biological agents for control of insects. Popularly known by one of its trade names, Botox or Dysport, a preparation prepared from Botulinum toxin is used for various cosmetic and medical procedures.