Rap Protein Paralogs of Bacillus thuringiensis: a Multifunctional and Redundant Regulatory Repertoire for the Control of Collective Functions

Abstract

Quorum sensing (QS) is a mechanism of synthesis and detection of signaling molecules to regulate gene expression and coordinate behaviors in bacterial populations. In Bacillus subtilis, multiple paralog Rap-Phr QS systems (receptor-signaling peptides) are highly redundant and multifunctional, interconnecting the regulation of differentiation processes such as sporulation and competence. However, their functions in the Bacillus cereus group are largely unknown. We evaluated the functions of Rap proteins in Bacillus thuringiensis Bt8741, which codes for eight Rap-Phr systems; these were individually overexpressed to study their participation in sporulation, biofilm formation, spreading, and extracellular proteolytic activity. Our results show that four Rap-Phr systems (RapC, RapK, RapF, and RapLike) inhibit sporulation, two of which (RapK and RapF) probably dephosphorylate Spo0F from the Spo0A phosphorelay; these two Rap proteins also inhibit biofilm formation. Four systems (RapC, RacF1, RacF2, and RapLike) participate in spreading inhibition; finally, six systems (RapC, -F, -F2, -I, and -I1 and RapLike) decrease extracellular proteolytic activity. We foresee that functions performed by Rap proteins of Bt8741 could also be carried out by Rap homologs in other strains within the B. cereus group. These results indicate that Rap-Phr systems constitute a highly multifunctional and redundant regulatory repertoire that enables B. thuringiensis and other species from the B. cereus group to efficiently regulate collective functions during their life cycle in the face of changing environments. IMPORTANCE The Bacillus cereus group of bacteria includes species of high economic, clinical, biological warfare, and biotechnological interest, e.g., B. anthracis in bioterrorism, B. cereus in food intoxications, and B. thuringiensis in biocontrol. Knowledge about the ecology of these bacteria is hindered by our limited understanding of the regulatory circuits that control differentiation and specialization processes. Here, we uncover the participation of eight Rap quorum-sensing receptors in collective functions of B. thuringiensis. These proteins are highly multifunctional and redundant in their functions, linking ecologically relevant processes such as sporulation, biofilm formation, spreading, extracellular proteolytic activity, and probably other functions in species from the B. cereus group.

Multiple and Overlapping Functions of Quorum Sensing Proteins for Cell Specialization in Bacillus Species

“…In contrast, B. thuringiensis is a soil-inhabiting, insect-pathogenic, and saprophytic bacterium (87) that may use its Rap repertoire to regulate virulence, sporulation, Cry toxin production, nutrient scavenging, and dispersal. Accordingly, we recently found that the lab strain B. thuringiensis Bt8741 has expanded the regulation of extracellular proteases to six out of the eight Rap proteins encoded in its genome (80). This contrasts acutely with only one Rap protein involved in the regulation of extracellular proteases in B. subtilis (83).…”

“…First, although several Rap protein paralogs are encoded in both B. subtilis and the B. cereus group genomes, they have diversified independently after speciation, with no known examples of horizontal transfer between the two groups; therefore, no Rap homolog is shared between these groups. That is, all Rap proteins from B. subtilis share a common ancestor, while all Rap proteins for the B. cereus group share a different common ancestor (23,78,80); second, NprR, which targets Spo0F in the B. cereus group, is not found in B. subtilis. Additionally, not all of the cell differentiation processes from B. subtilis are shared in the B. cereus group species; for example, homologs of the ComP-ComA two-component system are largely absent in species from the B. cereus group (79).…”

“…Furthermore, Spo0F-binding Rap proteins are likely involved in the regulation of multiple functions, as the Spo0A phosphorelay is a signal integration pathway that modulates many differentiation programs (28). Another level of complexity is the presence of a gene coding for a putative precursor of a Phr signaling peptide in most of the rap paralogs of the B. cereus group (78,80,81), which indicates that they retain a QS capability that directs their functions.…”

Diferenciación de Bacillus thuringiensis HD73 en medio líquido y superficial: células especializadas para esporulación, virulencia, necrotrofismo y formación de bio-película

“…De hecho, sólo existe un estudio sobre la interacción bacteria-insecto en Bt (Verplaetse et al 2015), en donde se logró observar que el principal gen que se expresan sobre el cadáver del insecto es el regulador de virulencia PlcR, además se presenta heterogeneidad en su expresión, es decir que se generan subpoblaciones durante esta interacción. Proponemos que nuestras cepas reporteras son un recurso valioso para estudios futuros sobre la regulación genética en diferentes aspectos del ciclo de vida de Bt, que es un tema de interés en nuestro grupo de investigación en CIAD-CIDEA (Rocha et al 2010: Rocha et al 2012Cabrera et al 2014;Cabrera et al 2016;Gastélum et al 2020;Verdugo et al 2020). Además, pueden ser útiles para seguir caracterizando la fisiología bacteriana en sistemas de fermentación de Bt para su uso biotecnológico, como también se ha hecho en el grupo de investigación en UAM-I (Lima et al 2019).…”

“…Además, NprR regula la transcripción de genes involucrados en la formación de bio-película (Slamti et al 2014) y regula el inicio de la esporulación (Cabrera et al 2014). Además de NprR, 6 proteínas Rap de Bt regulan la producción de proteasas extracelulares, y por lo tanto son importantes para la regulación del desarrollo del necrotrofismo (Gastélum et al 2020).…”