Researchers have shed new light on a possible mechanism whereby bacterial spores are killed by wet heat, potentially paving the way to more effective ways of killing spores.

The study by a team at University of Connecticut Health Center is described in ‘Expression of the 2Duf Protein in Wild-Type Bacillus subtilis Spores Stabilises Inner Membrane Proteins and Increases Spore Resistance to Wet Heat and Hydrogen Peroxide’ which appears in the Journal of Applied Microbiology, an Applied Microbiology International publication.


Spores of bacteria of Bacillus and Clostridium species are major agents of food spoilage, foodborne disease and are vectors for some nasty consequences such as botulism, tetanus, gas gangrene, uncontrolled chronic diarrhoea and anthrax, lead author Professor Peter Setlow says.  

“As a consequence, there are ongoing searches for the best way to safely inactivate spores without destroying the environment or turning foodstuffs into mush, which is obviously a concern of the food industry, for which judicious use of high temperature treatments is still the most common way to deal with the threat of spores,” he says.

“Work, largely from my lab over the past 40 or so years, has indicated a number of ways that spores are NOT killed by wet heat, and some data indicating that spore killing by wet heat is accompanied by some protein denaturation and a defect in generating ATP – the latter, of course, is essential for bacteria to grow and do nasty things. 

“However, the specific proteins, denaturation of which leads to spore death and defects in ATP synthesis are not known.”

Wet heat

The paper provides new evidence for a possible mechanism whereby spores of bacteria are killed by wet heat. 

“Spore eradication is a problem that has been around for more than 50 years, which is how long I have been working on spores, but as yet there is no answer as to ‘how’! Professor Setlow says.

“Perhaps knowing the ‘how’ would suggest ways to improve spore killing in applied settings. Note that spores are throughout our environment, are metabolically dormant and extremely resistant to just about everything, although thankfully, not completely so.”

The study used primarily Bacillus subtilis spores of two strains, A and B, that were essentially identical, except that strain B’s spores had a protein, likely in the spore inner membrane (IM), which can be called H for heat, and the H protein made spores of strain B much more wet heat resistant - even to boiling water - than A spores. 

The B spores and the role of the H protein were discovered and worked out a few years ago in elegant work by a group at the University of Groningen in the Netherlands. 

“When we started the work with the A and B spores, the H protein was only predicted to be an IM spore protein. However, late in our work with the A and B spores, I and my colleague Bing Hao analysed published spore proteomic data and found that, as expected, the H protein was indeed a spore IM protein and not in growing cells,” Professor Setlow said.

“The hypothesis we started with in the work was that wet heat was killing spores by damaging one or more IM proteins essential for spore ATP production and thus cell growth, and the H protein somehow stabilised IM proteins.” 

Major findings

Major findings supporting the hypothesis were that spore IM germination proteins were indeed greatly stabilised by the H protein in spores, and the H protein seemed to make the IM more rigid, such that IM proteins would be less likely to thrash about and denature at high temperatures than in a more fluid IM.

“The third observation was one that has been known for years but we duplicated it with both A and B spores - that wet heat generates so called “damaged spores” that can come back to life and make ATP and grow on a rich medium, in particular one with glucose (G), but not on a medium without G and with only amino acids (AA) to metabolise,” Professor Setlow said.

“This difference is extremely dramatic on an otherwise poor medium that is + AA, + G or with both AA and G, as heat-treated wild-type spore viability is 10 to 100-fold higher on G alone compared to AA alone, and almost identical to that with AA + G.  

“The problem faced in catabolism of G or AA to make ATP is that both require reactions that take electrons from the substrate to reduce NAD to NADH. However, unless there is a place for the NADH’s electrons to go then catabolism will stop. 

“In catabolism of glucose, this is not a problem, as the end product of glycolysis, pyruvate, can be reduced by NADH to lactate regenerating NAD to keep glycolysis producing ATP via substrate-level phosphorylation. 

“However, ATP from AA catabolism is via the utilisation of NADH in oxidative phosphorylation, catalysed by spore IM enzymes, and if these enzymes are damaged NADH cannot be dealt with in AA catabolism. 

Hydrogen peroxide

“In addition, spores treated with hydrogen peroxide (H2O2), exhibit the same behaviour as wet heat treated spores on the various recovery media, suggesting that this agent also inactivates IM enzymes involved in oxidative phosphorylation - and we know it does not kill by DNA damage!”

The next step will be to determine whether one or more enzymes of oxidative phosphorylation are indeed among the ‘trigger’ proteins for wet heat killing of spores, Professor Setlow says.

He hopes that upon learning how wet heat and H2O2 kill spores, others may be motivated to work out how to accelerate this process to make spore killing much faster, and with lower energy expenditures.

‘Expression of the 2Duf Protein in Wild-Type Bacillus subtilis Spores Stabilises Inner Membrane Proteins and Increases Spore Resistance to Wet Heat and Hydrogen Peroxide’ by George Korza, Sarah DePratti, Daniel Fairchild, James Wicander, Julia Kanaan, Hannah

Shames, Frank C. Nichols, Ann Cowan, Stanley Brul and Peter Setlow is published in the Journal of Applied Microbiology. [LINK]