Quorum sensing in Gram negative and Gram positive bacteria

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RNPP family

The second quorum sensing mechanism of Gram positive bacteria is based on the direct binding of the oligopeptide to the cytoplasmic response regulator in the responder cell, particularly in bacteria from the Firmicutes phylum. These systems belong to the RNPP family – named from the key regulator members – Rap, NprR, PlcR and PrgX. Even if these proteins regulate various processes in different bacterial species, they share two main features: the intracellular interaction with a linear processed oligopeptide (Phr, NprX, PapR, cCF10, respectively) that is re-imported by oligopeptide permeases (Opp), and a similar structure of the regulators, which contain tetratricopeptide repeat (TPR) motifs (Figure 12) (Declerck et al., 2007; Perez-Pascual et al., 2016; Rocha-Estrada et al., 2010).
Figure 12. Structure of RNPP protein. Regulators of the RNPP family contain multiple TPR repeats. TPR: tetratricopeptide repeat; HTH: helix-turn-helix, a DNA binding-domain (Perchat et al., 2011). The TPR domain is a structural motif present in a wide range of proteins, identified in diverse organisms from bacteria to humans and involved in different biological processes such as cell cycle regulation, transcriptional control and protein folding. The TPR motifs comprise three to sixteen tandem repeats of degenerated sequences of 34 amino acids residues and adopt a structural arrangement of two-antiparallel α-helices. This arrangement is essential to its role in mediating protein-protein or protein-peptide interactions or in assembling multiprotein complex (D‘Andrea & Regan, 2003).
In addition to these main features, the genes coding for the precursor of the signaling oligopeptide is generally located downstream from genes coding for the response regulator and can be found on the chromosome or plasmids. When the oligopeptide binds to its cognate receptor it induces a conformational change, activating or inhibiting its activity. Except from the Rap phosphatase, the RNPP regulators possess a helix-turn-helix (HTH) DNA binding domain in the N-terminal region (Figure 12), allowing them to act as transcriptional factors. The similar characteristics of the RNPP systems suggest that they derive from a same ancestor (Figure 13) (Declerck et al., 2007; Do & Kumaraswami, 2016; Perchat et al., 2016b; Perez-Pascual et al., 2016; Rocha-Estrada et al., 2010).

Rap-Phr systems

Response regulator aspartate phosphatases (Rap) are found in genome of Bacillus species. These proteins were extensively studied in B. subtilis, in which they were shown to regulate sporulation, competence, transfer of genetic mobile elements (plasmids or insertion sequence), production of extracellular proteases and biofilm formation (Perego, 2013). The main described roles of Rap are in sporulation and competence with two distinct modes of action: (i) as phosphatase, by dephosphorylating the intermediate response regulator of the phosphorelay, Spo0F, thus inhibiting indirectly the phosphorylation of Spo0A and consequently, the sporulation process (Perego et al., 1994); or (ii) by binding to a cytoplasmic regulator, such as the ComA response regulator, in which Rap binds to the HTH DNA-binding domain, inhibiting the transcriptional activator activity of ComA over genes from its regulon, related to competence (Bongiorni et al., 2005; Core & Perego, 2003). The Rap proteins are inhibited by the Phr oligopeptide (Perego & Brannigan, 2001; Perego & Hoch, 1996). In the B. cereus group species, Rap-Phr systems were already shown to regulate sporulation in B. anthracis (Bongiorni et al., 2006) and B. thuringiensis (Fazion et al., 2018). Rap proteins are structurally organized in an N-terminal three helix bundle and a C-terminal domain containing TPR motifs (six canonical and one non-canonical). Phr binding induces a conformational change where the entire protein consists of one single domain containing nine TPR-like folds (Figure 12) (Parashar et al., 2011; Perego, 2013). Due to the lack of the HTH domain (Figure 12) and the highly diversity among the RNPP regulators, Rap proteins are suggested as the most ancestral system of the RNPP family (Figure 13) (Declerck et al., 2007).

NprR-NprX system

In bacteria from the B. cereus group, the neutral protease regulator (NprR) was primarily described to regulate the expression of the metalloprotease NprA (also known as NprB or Npr599), which is the main constituent of the secretome of these bacteria during the stationary phase in sporulation medium (Chitlaru et al., 2006; Perchat et al., 2011). NprR is active as a transcriptional regulator in the presence of its cognate oligopeptide NprX (Perchat et al., 2011). The gene encoding the NprX precursor is located downstream from the nprR gene, and both genes are co-transcribed. The expression from the nprR promoter is repressed by the global regulator CodY during the exponential growth and activated by the PlcR quorum sensor (described below) at the onset of the stationary phase. Moreover, the transcription of the nprX gene is also independently activated by two promoters, located upstream the nprX gene, within the nprR gene and related to the sporulation specific sigma factors σH and σE. These independent expressions result in higher concentration of active NprX in the late exponential phase. Differently from some RNPP systems, the transcription of the nprR-nprX cassette is not auto-regulated (Dubois et al., 2013).
In fact, NprR is a bifunctional regulator (Figure 14), depending on the presence of NprX that modify its conformational structure (Perchat et al., 2016a; Zouhir et al., 2013).

PrgX & cCF10/iCF10

PrgX is a sex pheromone receptor of Enterococcus faecalis, which is a commensal bacterium but also act as opportunistic human pathogen, associated to nosocomial infections (Bae et al., 2000; Perez-Pascual et al., 2016). The prgX gene is encoded in the tetracycline resistant plasmid pCF10 and participates in the transcription regulation of conjugative transfer gene (prgQ operon), which controls the conjugation capacity of pCF10. The repressor activity of PrgX is controlled by two antagonistic peptides (Figure 16): the inhibitor iCF10, located in the plasmid pCF10, and the activator cCF10, encode by a chromosomal gene (Do & Kumaraswami, 2016). Both cCF10 and iCF10 are heptapeptides corresponding to the C-terminal end of their precursor lipoprotein (Mori et al., 1988; Nakayama et al., 1994). Moreover, both peptides bind to the same pocket but induce different conformations in C-terminal part of PrgX. The two PrgX-peptide complexes adopt a tetrameric form that induce a particular conformation (Figure 16) (Chen et al., 2017). When iCF10 binds to PrgX regulator, they attach to two sites of the DNA sequence and form a loop that prevents activity of RNA polymerase on the prgQ operon. By contrast, when bonded to cCF10, this repression is disrupted and the transcription of conjugation genes could take place (Neiditch et al., 2017). Donor and recipient cells express cCF10, which is neutralized by iCF10 in cells containing pCF10 plasmid (Rocha-Estrada et al., 2010). When the density of recipient cells increases, concentration of cCF10 overcome iCF10 concentration and release the transcription of conjugation genes repressed by PrgX, enabling conjugation.

Expansion of the RNPP family

New findings in Gram positive bacteria quorum sensing systems had led to the description of new RNPP-like systems, as the Rgg-SHP system from Streptococci, given rise to the new RRNPP family (Monnet & Gardan, 2015; Parashar et al., 2015). Rgg (regulator gene of glucosyltransferase) transcription factors are widespread in Firmicutes, but it is particularly associated to a signaling peptide in Streptococcus species. Rgg proteins represents a large family of receptors that regulate genes with diverse functions, such as commensalism and production of virulence factors, and are regulated by small hydrophobic peptides (SHP) (Neiditch et al., 2017; Parashar et al., 2015; Perez-Pascual et al., 2016). Rgg protein, as well as PrgX, do not contain detectable TPR motif but adopt TPR-like folds (Neiditch et al., 2017). The ComR-XIP system, initially described as member of the Rgg family, was shown to be another new member of the RNPP family (Talagas et al., 2016). ComR is a transcription factor that positively controls competence in Streptococci and is directly activated by XIP (the active form of the ComS peptide).

Regulation of the infectious cycle in B. thuringiensis

The three RNPP systems present in the B. cereus group – PlcR, NprR and Rap – have been shown to regulate the infectious cycle of B. thuringiensis in the insect model Galleria mellonella. These quorum sensing systems are sequentially activated during the lifestyle of B. thuringiensis throughout the different phases of infectious process, namely: pathogenic, necrotrophic and spore formation (Figure 17) (Slamti et al., 2014).

Rap-Phr systems in B. subtilis

The first studied Rap protein was described as a phosphatase that acts on the sporulation phosphorelay by dephosphorylating Spo0F. This activity gives its name: response regulator aspartate phosphatase (Perego et al., 1994). Rap relevance as phosphatase relies on the fact that it represents some additional regulatory points to the signal transduction system that regulates the sporulation, and contribute to the phosphorelay complexity (Perego & Brannigan, 2001). Thereafter, it was shown that some Rap proteins regulate other developmental pathway of the stationary phase, such as the competence (ability to DNA uptake) via the ComA response regulator. The release of the B. subtilis complete genome sequence reveals that its type strain (str. 168) possesses 11 rap genes (rapA to rapK) and eight of which are followed by a downstream short coding sequence, the phr gene (Kunst et al., 1997). The phr genes slightly overlap the rap gene sequences forming transcript units: the rap-phr signaling cassettes (Perego & Hoch, 1996). The mature form of the Phr peptide inhibits its cognate Rap activity, excepted for RapB, RapD and RapJ that are orphans. However, the PhrC, also known as competence and sporulation factor (CSF), is able to inhibit RapB and RapJ phosphatases (Parashar et al., 2013a; Perego, 1997).
RapA, RapB, RapE, RapI and RapJ regulates sporulation by specifically acting on Spo0F (Figure 20, Table 2) (Jiang et al., 2000a; Parashar et al., 2011; Perego et al., 1994; Singh et al., 2013). Likewise, RapC, RapD, RapF and RapK inhibit competence by binding to the response regulator ComA and preventing its functions as transcription factor (Auchtung et al., 2006; Bongiorni et al., 2005; Ogura & Fujita, 2007; Solomon et al., 1996). RapH is the unique protein of the Rap family that was demonstrated to have dual specificity on Spo0F and on ComA (Smits et al., 2007). RapH was shown to play a role in the temporal separation of competence and sporulation pathways which are mutually exclusive. Thus, RapH prevents sporulation initiation in competent cells and later enables the cell to escape from the competent state to enter into the sporulation pathway (Smits et al., 2007). In addition, the other B. subtilis chromosomal Rap proteins were shown to act on other bacterial pathways. RapG regulates negatively the activity of the response regulator DegU which activates the expression of extracellular alkaline protease encoded by aprE (Ogura et al., 2003). RapI is encoded on the mobile genetic element ICEBs1 which is an integrative and conjugative element (ICE) (Auchtung et al., 2005). These conjugative transposons are able to excise, transfer to recipient cells through conjugation and integrate to the chromosome. RapI regulate the ICEBs1 expression, excision and transfer via the inhibition of the immunity repressor ImmR activity. When the cell is surrounded by bacteria that also harbor ICEBs1, RapI is inhibited by the PhrI produced by these bacteria, preventing the expression of ICEBs1 when there are no potential recipient cells (Auchtung et al., 2005).

Plasmid-borne Rap-Phr (QS) systems

Plasmids and other genetic mobile elements (such as ICE) represent important elements for genes spreading and diversification. Furthermore, rap-phr genes were found in various plasmids and some of them were further evaluated with functional studies which have shown the relevance of these plasmid Rap-Phr systems for bacterial development.
The first described plasmid Rap-Phr system from B. subtilis was the Rap60-Phr60, harbor on plasmid pTA1060. This system regulate the production of extracellular proteolytic enzymes (Koetje et al., 2003). The quorum sensing control of the production of protease allows the bacteria to better exploit the nutrients. When in just a small number of bacteria, in which there are sufficient resources to their development, Rap60 inhibit protease production, since it is not required. Once in a high cell density, when the nutrients were already depleted, the Phr60 binds to Rap60 and activates expression of the extracellular protease to improve energy sources. Lately, the Rap60-Phr60 couple was demonstrated to also control the phosphorelay and ComA activity by a non-canonical mechanisms, resulting in modification of diverse process, such as sporulation, cannibalism, biofilm formation and genetic competence (Boguslawski et al., 2015). In addition to the usual role as phosphatase on Spo0F, Rap60 inhibit the autophosphorylation of kinase KinA. Concerning ComA, Rap60 inhibits its activity as transcription regulator by forming a ternary complex with ComA and the DNA promoter region of its target.

Multilocus sequencing type (MLST) tree

According to the scheme of Tourasse et al. (2006), sequences of MLST housekeeping genes of all selected genomes were obtained from the ‗University of Oslo‘s Bacillus cereus group MultiLocus and MultiData Typing website (http://mlstoslo.uio.no)‘. Sequences of adk, glpT, glpF, panC, pycA, ccpA, and pta genes were downloaded already concatenated. Alignment and phylogenetic tree development were performed as described for Rap proteins.

Plasmids construction and growth conditions

To assess the effect of Rap proteins on sporulation, seven plasmid rap genes (rap6-BtHD1, rap8-BtHD1, rap10-BtHD1, rap6-Bt407, rap7-Bt407, rap8-Bt407 and rap7-BtHD73) and three chromosomal rap genes (rap1-BcATCC14579, rap2-BcATCC14579 and rap5-BtHD73) were cloned in the plasmid pHT315-PxylA, a multi-copy vector with xylose-inducible promoter (Grandvalet et al. 2001). All genes were amplified by PCR using primers listed in Online Resource Table 2 and ligated to the plasmid pHT315-PxylA using the appropriate restriction sites. For cloning steps, these plasmids were transformed in Escherichia coli K-12 strain TG1 and then in the Dam- Dcm- E. coli strain ET12567 (Stratagene, La Jolla, CA, USA) by thermal shock.
Finally, each constructed plasmid was transformed by electroporation (Lereclus et al. 1989) in the acrystalliferous (Cry-) B. thuringiensis var. kurstaki HD73 strain (Wilcks et al. 1998). Luria Bertani (LB) medium was used to cultivate E. coli and B. thuringiensis at 37°C for DNA preparation. The medium HCT was used to optimize the sporulation of B. thuringiensis (Lereclus et al. 1982). Antibiotics were used at the following concentration: ampicillin 100 μg/mL for E. coli and erythromycin 10 μg/mL for B. thuringiensis.

Table of contents :

1 INTRODUCTION
1.1 Bacillus cereus group
1.1.1 The Bacillus genus
1.1.2 The Bacillus cereus group species
1.1.2.1 Bacillus cereus sensu stricto
1.1.2.2 Bacillus thuringiensis
1.1.2.3 Bacillus anthracis
1.1.3 Genome & taxonomy
1.1.4 Plasmids
1.2 Quorum sensing systems – RNPP Family
1.2.1 Quorum sensing in Gram negative and Gram positive bacteria
1.2.2 RNPP family
1.2.2.1 Rap-Phr systems
1.2.2.2 NprR-NprX system
1.2.2.3 PlcR-PapR system
1.2.2.4. PrgX & cCF10/iCF10
1.2.2.5 Expansion of the RNPP family
1.2.2.6 Regulation of the infectious cycle in B. thuringiensis
1.3 Rap-Phr Systems
1.3.1 Sporulation in Bacillus
1.3.2 Rap-Phr systems in B. subtilis
1.3.3 Rap characteristics
1.3.4 Phr: sequence and maturation
1.3.5 Plasmid-borne Rap-Phr (QS) systems
2 OBJECTIVES
3 ARTICLE 1 Diversity of the Rap-Phr quorum-sensing systems in the Bacillus cereus group
Abstract
Introduction
Materials and Methods
Bacterial genomes
Construction of the rap-phr database
Rap proteins clustering
Multilocus sequencing type (MLST) tree
Plasmids construction and growth conditions
DNA manipulation
Sporulation assay
Statistical analyses
Results
Genomic overview of the B. cereus group strains
rap-phr genes distribution
Rap protein clustering
Phr peptides
The relationship between MLST phylogenetic tree and Rap distribution
Sporulation activity prediction
Discussion
Acknowledgements
Tables
Figures
References
Online Resources
4 ARTICLE 2 Rap-Phr systems from pAW63 and pHT8-1 plasmids act synergistically to regulate sporulation in the Bacillus thuringiensis kurstaki HD73 strain
Abstract
Introduction
Materials and Methods
Bacterial strains and growth conditions
DNA manipulations
Plasmid and strains constructions
RT-PCR experiment
Synthetic oligopeptides
In vitro sporulation assays
In vivo sporulation assays
Aggregation kinetics
Microscopy
Results
Transcriptional analysis of the rap and phr genes
Rap63 inhibits sporulation
Rap63 delays expression of Spo0A-regulated genes
Auto-aggregation phenotype linked to sporulation
The ΔPhr8ΔPhr63 mutant strain negatively affects the commitment to sporulation
Determination of the Phr63 active form
A crosstalk between Rap-Phr63 and Rap8-Phr8 systems?
Discussion
Acknowledgements
Figures
References
Supporting Information
5 DISCUSSION
5.1 Overview of Rap-Phr systems in B. cereus group
5.2 Sporulation activity prediction
5.3 Rap63-Phr63 regulates sporulation
5.4 Synergistic activity of Rap63-Phr63 and Rap8-Phr8 on regulation of the commitment to sporulation
5.5. The Phr63 peptide and the specificity of Rap-Phr systems
6 CONCLUSION
BIBLIOGRAPHY

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