Toxin‐Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms

Environmental Microbiology (2016) 18(12), 5032–5047 doi:10.1111/1462-2920.13471 Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms Zohar Bloom-Ackermann, † Nitai Steinberg, Gili Rosenberg, Yaara Oppenheimer-Shaanan, Dan Pollack, Shir Ely, Nimrod Storzi, Asaf Levy † and Ilana Kolodkin-Gal* Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel. Summary Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromo- somes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient lim- itation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the tran- scription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms. Introduction Toxin2antitoxin (TA) systems are specific genetic modules, and are ubiquitous in bacterial chromosomes and plas- mids. Each module consists of a pair of genes that encodes for two components: a stable toxin and an unsta- ble antitoxin that interferes with the lethal action of the toxin. Found first in Escherichia coli on low copy number plasmids, TA systems are responsible for what is called Received 29 April, 2016; revised 07 July, 2016; accepted 20 July, 2016. *For corresponding. E-mail ilana.kolodkin-gal@weizmann.ac. il; Tel. 972-8-934 6981; Fax 972-8-934 4108. Present addresses: Broad Institute of MIT and Harvard, Boston, USA; ‡ US DOE Joint Genome Institute, Walnut Creek, CA 94598, USA. C 2016 Society for Applied Microbiology and John Wiley & Sons Ltd V the post-segregational killing effect. When bacteria lose these plasmid(s) (or other extrachromosomal elements), the cured cells are selectively killed because the unstable antitoxin is degraded faster than the more stable toxin [reviewed in (Hayes, 2003; Engelberg-Kulka et al., 2006; Hayes and Van Melderen, 2011). The cells are ‘addicted’ to the short-lived antitoxin product, because its de novo synthesis is essential for cell survival. TA systems, some of which are homologous to these extrachromosomal ‘addiction modules’, are widespread in prokaryote chromo- somes, with species frequently possessing tens of plasmid and chromosomal TA loci (Hayes, 2003; Engelberg-Kulka et al., 2006; Hayes and Van Melderen, 2011). The complexes are categorized into five types based on genetic organisation, the nature of the antitoxin and the mechanism of action (Markovski and Wickner, 2013). TAs classified as type I and II systems are probably the most abundant and the most extensively studied (Hayes and Sauer, 2003; Fozo et al., 2010). In these TA systems, the toxins are proteins directed against specific intracellular targets. The antitoxins are either small RNAs [Type I TA systems (Fozo et al., 2010)] or proteins [Type II TA sys- tems (Unterholzner et al., 2013)] that inhibit toxin synthesis or alternatively, neutralize the toxin (Hayes, 2003). As a result of TA systems activity in a subpopulation of the cells, key resources are conserved, ensuring the sur- vival of individual cells and, consequently, of the bacterial population (Kolodkin-Gal et al., 2007; Kolter, 2007; Moll and Engelberg-Kulka, 2012; Bayles, 2014). The toxin effects can be transient and reversible, permitting a set of dynamic, tunable responses that reflect environmental conditions. In Bacillus subtilis the EndoA toxin, encoded by the ndoAI/ndoA TA system (Park et al., 2011), was shown to have an effect on cell counts during vegetative growth and to play a role in stress resistance and tolerance (Wu et al., 2011). A direct role for TA systems in controlling the transforma- tion from planktonic to biofilm cells has long been debated: The MqsR toxin in E. coli was shown to be involved in the production of curli, the amyloid structural component of E. coli biofilms (Soo and Wood, 2013). In addition, deletion of the mazEF and dinJ-yqfQ TA systems in E. coli decreased biofilm formation (Kolodkin-Gal et al., 2009). Last, the HipA-HipB TA system promoted biofilm formation