Curr Genet DOI 10.1007/s00294-015-0479-9

REVIEW

A complex path for domestication of B. subtilis sociality Shaul Pollak · Shira Omer Bendori · Avigdor Eldar 

Received: 4 February 2015 / Accepted: 5 February 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  Microorganisms adapt to the lab environment by eliminating unnecessary genetic systems. In Bacillus subtilis, such adaptation resulted in the lab strain being unable to form complex, matrix-associated structures known as biofilms. We recently showed that the ancestor of the lab strain, which is considered by the research community to be a stereotypical ‘wild’ strain, carries an atypical mutation in the RapP–PhrP quorum-sensing system. We have found that this mutation has profound effects on the biofilm phenotype of the ancestral strain. Here we discuss these recent findings and present more data that focuses on the lessons that can be learned from this work on the domestication of microorganisms. Keywords  Quorum sensing · Biofilm · Bacillus subtilis · Domestication · Peptide signaling · Evolution

Introduction Microorganisms are masters of adaptation and have, over decades of laboratory use, become adapted to the lab environment (Kuthan et al. 2003). Some traits are, therefore, better studied in wild isolates, but beware––even their domestication can be quick and surprising, as we have recently learned while studying the social behavior of Bacillus subtilis (Omer et al. 2015).

Communicated by M. Kupiec. S. Pollak · S. Omer Bendori · A. Eldar (*)  Department of Molecular Microbiology and Biotechnology, Tel Aviv University, 69978 Tel Aviv, Israel e-mail: [email protected]

B. subtilis, the model organism for Gram-positive sporeforming bacteria, has been discovered more than a hundred and fifty years ago and the domesticated lab strain has been used for more than half a century (Zeigler et al. 2008). Domestication led to profound changes in B. subtilis social traits, with a dramatic reduction in its ability to form complex, matrix-adhered colonies known as biofilms, but with a concurrent increase in its competence for genetic transformation (Branda et al. 2001). The reduced capacity to form biofilms may have been inadvertently selected for by picking simpler, smoother and easier to handle colonies, while increased competence for transformation has been specifically selected for (Spizizen 1958). From a genetic perspective, the lab strain, known as strain 168, has undergone considerable genetic variation from its earliest recorded ancestor––strain NCIB3610 (abbreviated 3610) (Zeigler et al. 2008; McLoon et al. 2011). One major genetic change is the loss of a medium-sized (~80 kb) plasmid, called pBS32 (Konkol et al. 2013). pBS32 does not exist in the closest relatives of strain B. subtilis 3610, but is observed in farther related strains (Tanaka and Ogura 1998). The pBS32 plasmid encodes a whole cassette of phage genes (but no conjugation-related genes), therefore, it is most likely a plasmid prophage (Konkol et al. 2013). Together, these two observations support the possibility that this plasmid is horizontally transferred. The realization that growth in biofilms is of great ecological and medical importance in a wide variety of microorganisms (Mathé and Van Dijck 2013; Sepahi et al. 2015) has led the Bacillus community to devote increased attention to biofilm formation and its relation to other, wellcharacterized developmental pathways in B. subtilis, such as sporulation or the development of natural competence for transformation (Lopez et al. 2009; Aguilar et al. 2007; Vlamakis et al. 2013). As the capacity to form biofilms has

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been lost during domestication, strain 168 was obviously not a good model strain, and experiments were diverted from strain 168 to its ancestor, strain 3610, which is studied as an archetypical wild isolate. It was found that the single-cell developmental events of competence and sporulation are intimately linked with biofilm formation (Fig. 1a) (Branda et al. 2001). Specifically, biofilm formation on agar plates requires the production and secretion of an extracellular matrix and of a surfactant. Strikingly, it turned out that matrix production was tightly regulated by Spo0A, the master regulator of sporulation initiation (Kearns et al. 2005), while surfactant production was regulated by ComA, an important regulator which is necessary for competence development (Grossman 1995). The inability of the lab strain to form biofilms has been attributed to genetic modifications in at least five loci (McLoon et al. 2011). Two of these modifications are loss of function mutations in enzymes involved in matrix and surfactant production and three additional modifications are in regulatory proteins. One of the genetic changes that affected sporulation, and the ability to form biofilms, was the loss of the pBS32 plasmid during domestication. Further research has pointed to a specific plasmid-borne locus which is responsible for this phenotypic shift, the rapP– phrP locus (McLoon et al. 2011; Parashar et al. 2013). RapP–phrP is one of nine rap-phr loci appearing in strain 3610, and the only one that changed during the domestication of strain 168. rap-phr loci code for paralogous quorum-sensing systems (Perego 2013). The phr gene codes for a secreted peptide, which is further cleaved extracellularly to form a mature penta- or hexa-peptide signal. The extracellular peptide is then re-imported into the cell, where it binds its cognate Rap receptor (Lazazzera and Grossman 1998). Unlike most characterized quorum-sensing receptors, Rap proteins are functional in their unbound state, and binding of Phr peptides leads to their inactivation. Almost all characterized Rap proteins act as repressors of either the ComA or Spo0A regulators discussed above (Perego 2013) (Fig. 1a). In strain 168, four Raps are known to regulate Spo0A (indirectly, by dephosphorylating its kinase, Spo0F), while four regulate ComA. One Rap protein is known to repress both comA and spo0A. It was found that RapP behaves differently from other Rap-Phr systems in two ways (Parashar et al. 2013). First, deletion of the rapP gene had a very strong effect on biofilm formation, while mutations in most other Raps do not have such an effect. Second, RapP did not respond to its own PhrP signal. In our recent work, we studied the underlying reason for these two events and found it to be dependent on a mutation that is present in strain 3610, which we proposed is an early domestication event (Omer et al. 2015). First, we have used genetics to show that RapP acts independently

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Fig. 1  The role of RapP in the domestication of strain 168. a Two major regulators are controlled by RapP. Spo0A controls sporulation and matrix production and ComA controls competence and surfactant production. Both matrix and surfactant are necessary for the production of biofilms on agar plates and on plant roots. b Hypothetical three-stage evolution of the rapP locus during domestication. Stage I: a phage encoding the rapPN236T infects the plasmid-free ancestor of strain 168 forming a wild isolate containing the pBS32 plasmid. This has low effect on biofilm formation since PhrP represses RapP. Stage II: at early domestication rapP is mutated to its rap3610 allele. RapP becomes constitutively active, significantly reducing biofilm formation. Stage III: later in domestication, pBS32 is lost, leading to the formation of strain 168. This occurs alongside other mutations that block biofilm, saves the cost of plasmid carriage and increase competence. c Competition experiments demonstrate a growth advantage for rap3610 over rapT236N during growth in liquid media. Two strains were constructed on the background of strain 3610 deleted for its rapP3610–phrP locus. One strain encodes rapP3610–phrP while the other encodes rapPT236N–phrP. The strains were marked with different fluorescent markers and allowed to grow in a co-culture with one of them as a minority. As controls, each strain was competed against itself (but with a different fluorescent marker). Competitions were performed in complex (LB) and minimal (SMM) media. The competitive index of the minority is the relative frequency of the minority after 24 h of growth divided by its relative frequency at the beginning of growth

on the Spo0A and ComA pathways, therefore, affecting biofilm formation through both routes. The ability of Rap proteins to repress both pathways has been observed before with RapH, which does not have a profound effect on biofilm formation and, therefore, cannot explain by itself the effects of the rapP–phrP locus (Mirouze et al. 2011). Second, and more importantly, we have found that the rapP allele of strain 3610 (rapP3610) codes for an atypical amino acid substitution, from asparagine to threonine at the signal binding pocket, compared to other rapP homologs. Sequence alignment of all B. subtilis Rap proteins showed

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that the asparagine residue is completely conserved in all other Rap proteins. Moreover, the asparagine residue has been shown to be crucial for backbone alignment of the peptide in homologous peptide binding domains (D’Andrea and Regan 2003). We suspected that this unique mutation is the reason for the inability of phrP to repress the activity of rapP. Indeed, reconstitution of the canonical asparagine into RapP restored the sensitivity of RapP to PhrP. Moreover, this mutation has made the whole RapP–phrP locus irrelevant to biofilm formation––the biofilm phenotype and gene expression pattern of a strain with the corrected rapPT236N– phrP locus was identical to that of a strain that has lost this locus, or indeed––the whole plasmid. We can, therefore, understand the strong effect that RapP has on biofilm formation as a combination of its ability to repress both necessary pathways, without being able to de-repress those as PhrP levels increase. With the mutation reverted, the RapP– PhrP system becomes just “one of many”, behaving like all other Rap–Phr pairs. What is the likely evolutionary sequence of events that led to the current domesticated strain, from the perspective of RapP (Fig. 1b)? Most likely, the ancestor of strain 3610 has been infected by the pLS32 plasmid prior to its isolation from the soil. At this stage, the plasmid encoded the canonical rapP gene. In the early stages of lab domestication, rapP has been mutated into its present form. This mutation may have arisen by direct selection against biofilm formation, or by selection for improved growth due to the reduced production of biofilm-related substances. In agreement with this hypothesis, we found that a strain with the rapP3610 allele grew significantly faster than a strain with the corrected rapPT236N allele in both complex and simple media (Fig. 1c). Finally, at later stages of domestication, maybe after the occurrence of mutations in the structural biofilm-related genes, the plasmid has been lost. This loss may have occurred due to increased fitness resulting from elimination of the plasmid carriage cost. Additionally, as the lab strain was specifically selected for increased competence, the plasmid-free strain may have been selected because it shows improved competence efficiency, as both RapP and an additional locus on the plasmid named comI (Konkol et al. 2013) repress competence. The RapP mutation bears a caution mark to researcher of B. subtilis—strain 3610 is probably not behaving like a “canonical” wild isolate. This is, in fact, not the only atypical mutation in strain 3610. Recently, it was shown that strain 3610 also bears a highly atypical mutation in the D-tyrosyl-tRNA deacylase gene dtd, which prevents it from avoiding D-amino acids incorporation into proteins (Leiman et al. 2013). The domestication of strain 168 also bears

a more general lesson on domestication––it does not need to progress linearly! In this case, a domesticating mutation arises at an early stage only for the whole gene to be eliminated at later stages of domestication. Therefore, not only the current genetics of domesticated strains matters for understanding the history of domestication but also its hidden past.

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A complex path for domestication of B. subtilis sociality.

Microorganisms adapt to the lab environment by eliminating unnecessary genetic systems. In Bacillus subtilis, such adaptation resulted in the lab stra...
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