ORIGINAL ARTICLE Lytic enzyme-assisted germination of Bacillus anthracis and Bacillus subtilis spores B.G. Blankenship 1 , J.D. Heffron 2 and D.L. Popham 1 1 Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA 2 Novozymes Biologicals, Inc., Salem, VA, USA Keywords anthracis, Bacillus, cortex, germination, germination-specific lytic enzyme, SleB, spore, subtilis. Correspondence David L. Popham, Biological Sciences, Virginia Tech, 970 Washington ST SW, Life Sciences I - MC0910, Blacksburg, VA 24061, USA. E-mail: dpopham@vt.edu 2015/0518: received 11 March 2015, revised 30 April 2015 and accepted 3 May 2015 doi:10.1111/jam.12839 Abstract Aims: The goal of this work was to determine conditions under which external application of a spore germination-specific lytic enzyme (GSLE) can increase the germination efficiency of spore populations. Methods and Results: The Bacillus anthracis GSLE SleB was applied to native and coat-disrupted B. anthracis and Bacillus subtilis spores. SleB was inactive on native spores but was able to trigger rapid germination of coat-disrupted spores. Using spores lacking their GSLEs or their germinant receptors to model poorly germinating spores, SleB application was able to increase colony- forming efficiency 100-fold for native spores and >1000-fold for coat-disrupted spores. SleB effects on GSLE-deficient spores were greater than on germinant receptor-deficient spores. Conclusions: SleB treatment can increase spore germination efficiency. The greater effect of SleB on coat-disrupted spores is presumably due to the greater access afforded to the cortex. However, SleB apparently gained access to the cortex of native spores after they responded to nutrients and completed stage I of germination, which may result in the disruption of coat structure. Significance and Impact of the Study: Treatment of spore populations with a GSLE can increase germination efficiency. Such a treatment might be utilized to increase the rapid activation of industrial spore-based products. Introduction Bacillus, Clostridium and some other Gram-positive gen- era have the ability to produce endospores, which dem- onstrate long-term dormancy, stability and extreme resistance properties (Setlow 2006). Endospores have a modified cell structure, including a relatively dehydrated spore core (cytoplasm), an immobile inner spore mem- brane, a cortex peptidoglycan wall and multiple coat pro- tein layers (Setlow 2006). Upon interaction with germinants, the endospore releases solutes, including a large amount of Ca 2+ -dipicolinic acid (Ca 2+ -DPA) and the dehydrated core experiences an influx of water (Setlow 2003; Moir 2006). For the completion of germi- nation, germination-specific lytic enzymes (GSLEs) must degrade the cortex peptidoglycan (Popham et al. 1996b), which allows core swelling, a further influx of water, and resumption of metabolism (Setlow et al. 2001). Spore populations can exhibit a great deal of heterogene- ity in the per cent of spores that germinate in the presence of nutrients and the rate at which they initiate germination (Ghosh and Setlow 2009; Wang et al. 2011). This can reduce the activity of industrially important spore prod- ucts, and thus methods for increasing spore germination efficiency can potentially increase the effectiveness of such products. Bypassing the normal nutrient-germinant sensing apparatus via the application of a cortex lytic enzyme has the potential for increasing germination efficiency within a spore population. Bacillus species possess a major GSLE known as SleB (Popham et al. 2012). SleB is produced in the forespore and is located outside the inner spore mem- brane, adjacent to the cortex of the dormant spore. Puri- fied SleB has previously been shown to act on spore cortex PG, but only following chemical or physical disruption of the spore coat layers (Makino et al. 1994; Heffron et al. 2011; Li et al. 2012, 2013; Mundra et al. 2014). Journal of Applied Microbiology 119, 521--528 © 2015 The Society for Applied Microbiology 521 Journal of Applied Microbiology ISSN 1364-5072