Identification of a quorum sensing pheromone posttranslationally farnesylated at the internal tryptophan residue from Bacillus subtilis subsp. natto

Abstract

Bacillus subtilis subsp. natto produces poly-γ-glutamic acid under the control of quorum sensing. We identified ComX_natto pheromone as the quorum-sensing pheromone with an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu and the tryptophan residue posttranslationally modified by a farnesyl group. ComX_natto pheromone is unique in the sense that the 5th tryptophan residue from the C-terminal is farnesylated.

Key words:

• Bacillus subtilis
• isoprenylation
• posttranslational modification
• quorum sensing
• tryptophan

Quorum sensing is the system involved in the activation of specific gene expressions dependent on cell density.¹⁾ Bacillus subtilis secrets ComX pheromone that stimulates natural genetic competence controlled by quorum sensing.^2,3) ComX pheromone is a posttranslationally modified oligopeptide synthesized from ComX. The modification is catalyzed by an isoprenyl diphosphate synthase-like protein, ComQ, and is essential for the function of the pheromone.⁴⁾ Previous studies have demonstrated that striking polymorphism is exhibited in the amino acid sequence of the ComX pheromone variants in 6 strains, but each variant possesses a tryptophan residue at either the 3rd or 4th residue from the C-terminal.^3–6) Our studies revealed the precise nature of the ComX modification as follows: the tryptophan residue is modified with either a geranyl group or a farnesyl group at its γ-position, resulting in the formation of a tricyclic structure, including a newly formed proline-like five-membered ring.^7–10) In contrast, many details of processing remain to be clarified.

B. subtilis subsp. natto is closely related to B. subtilis laboratory strains and known as a starter strain for a traditional Japanese fermented food made from soybeans, called natto in Japanese. Molecular genetic analysis of B. subtilis subsp. natto strain BEST195 demonstrated that gene homologs, ComQ and ComX, are also present in its genome cluster.^11–13) The most obvious phenotypical distinction of B. subtilis subsp. natto from the laboratory strains is biofilm formation, which mainly involves the highly sticky polymer poly-γ-glutamic acid (γ-PGA). γ-PGA biosynthetic gene cluster involves the ComQX_natto gene.¹²⁾ This result suggests that the precursor ComX_natto is modified by ComQ_natto, and resulting ComX_natto pheromone activates the gene expression for γ-PGA biosynthesis. However, there is no tryptophan residue at either the 3rd or the 4th residue from the C-terminal, but one tryptophan residue at the 54th among 73 amino acid residues in ComX_natto. Furthermore, our recent studies have demonstrated that the tryptophan residue must be located between the 2nd and the 4th residues from the C-terminal in order to be modified by ComQ.^14–16)

First, we prepared ComX_natto pheromone expression plasmid integrated with a ComQX gene cluster directly amplified from B. subtilis subsp. natto, and then the plasmid was introduced to Escherichia coli. We also prepared alanine-substituted mutant of the 54th residue tryptophan in ComX_natto ([W54A] ComX_natto) based on the constructed plasmid. Next, we analyzed these fermentation liquids of the transformants by LC-MS and compared resulting chromatograms at various values of m/z. As expected, only the sample prepared from the fermentation liquid ComQ_natto and ComX_natto overexpressing transformant contained the characteristic compounds with a molecular ion at m/z 973.7 (Fig. 1(A)). Additionally, the MS at 35.2 min showed a fragment ion at m/z 769.5, 204.2 Da less than the value of the molecular ion (Fig. 1(B)). The difference in ions corresponds to farnesylation (C₁₅H₂₄ addition). In LC-MS analysis, no other modified peptides derived from ComX_natto were observed at all. Subsequently, MS/MS analysis was carried out (Fig. 1(C)), and the MS/MS of the compound at m/z 973.7 ([C₅₄H₈₁N₈O₉]⁺) was consistent with that of the six-residue-modified peptide that possessed the amino acid sequence, Lys-Trp-Pro-Pro-Ile-Glu, and the 2nd tryptophan residue-modified with a farnesyl group (Fig. 1(D)). The amino acid sequence of the compound matched that from the 53rd to the 58th residues in ComX_natto. It is notable that ComQ_natto can catalyze farnesylation of the tryptophan residue up to at least the 5th position from the C-terminal, though it is not clear which biosynthetic step occurred first, farnesylation or truncation of amino acid residues. Based on the phylogenetic resemblance, the tryptophan residue of ComX_natto pheromone is probably similar to that of the ComX_RO-C-2 pheromone.⁸⁾ Unfortunately, since ComX_natto pheromone is much more labile than the ComX pheromones previously isolated or synthesized, it was difficult to purify enough ComX_natto pheromone for NMR analysis.^{7–9,17–20)} We are now attempting to improve the chemical synthetic method for ComX pheromone,^17–20) therefore, the precise chemical structure of ComX_natto pheromone will be determined in the near future.

Fig. 1. LC-MS and MS/MS analyses of ComX_natto pheromone secreted by transformants.

Notes: (A) Extracted ion chromatograms at m/z 973.7 for liquid culture broth: blue, ComQ_natto and ComX_natto overexpressing transformant; black, ComQ_natto and [W54A] ComX_natto overexpressing transformant, (B) MS spectrum at 35.2 min for liquid culture broth of ComQ_natto and ComX_natto overexpressing transformant. A bidirectional arrow represents the difference (204.2) between a parent ion and a fragment ion, (C) Enlarged MS/MS spectrum of ComX_natto pheromone. An asterisk that placed above the observed ion at m/z 973.7 represents a parent ion. Marks (b_n, y_n, and–Far) that placed above observed fragment ions represent ion series indicated from cleavage of a peptide bond or a farnesyl group. The intencity of m/z 455.3 (y₄) was 50 kcps, and (D) Schematic representation of calculated fragment ions of ComX_natto pheromone derived from cleavage of a peptide bond (b_n and y_n) or a farnesyl group (–Far).

Subsequently, we investigated the effect of the modified ComX_natto peptide on γ-PGA production of B. subtilis subsp. natto in liquid culture. The addition of the modified ComX_natto peptide at nearly 10 nM increased the yield of γ-PGA more than 1.5 times than that of water, liquid culture broth of [W54A] ComX_natto, or a plain hexapeptide, Lys-Trp-Pro-Pro-Ile-Glu, at 1 μM (Fig. 2). Unfortunately, since the modified ComX_natto peptide could not be detected in the liquid culture of B. subtilis subsp. natto by LC-MS, the concentration would be estimated to be less than 10 nM. These findings were consistent with previous molecular genetic researches.^3,6,12) It was thus confirmed that the modified ComX_natto peptide plays a crucial role in γ-PGA production as a quorum-sensing pheromone, ComX_natto pheromone, in B. subtilis subsp. natto.

Fig. 2. Effects of ComX_natto pheromone on γ-PGA production of B. subtilis subsp. natto.

Notes: Water (white), the fermentation liquid of ComQ_natto and ComX_natto overexpressing transformant containing ComX_natto pheromone at nearly 10 nM (dark gray), the fermentation liquid of ComQ_natto and [W54] ComX_natto overexpressing transformant (gray), or Lys-Trp-Pro-Pro-Ile-Glu at 1 μM (light gray) was added to liquid culture medium. After static incubation of B. subtilis subsp. natto. for 3 days, the obtained γ-PGA was mesured. Error bars represent standard deviation of triplicate samples.

In conclusion, it has been shown that ComX_natto pheromone stimulates γ-PGA production, which is a hexapeptide having an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu, and the 2nd tryptophan residue is posttranslationally modified with a farnesyl group by ComQ_natto. ComX_natto pheromone is unique in the sense that the 5th tryptophan residue from the C-terminal is modified with a farnesyl group, which corresponds to the 54th residue among 73 amino acid residues in ComX_natto. For industrial application, ComX_natto pheromone will attract attention as a promising additive for improving the production of γ-PGA by B. subtilis subsp. natto.

Additional information

Funding

This work was supported by JSPS KAKENHI [grant number 24688011], and MEXT KAKENHI [grant number 25108724].