The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis

Abstract

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication

“…S4 C-F). However, data were also in agreement with a previous study which deciphered the biosynthetic pathway of a Pse isomer: legionaminic acid (Leg) [ 95 ]. As this isomer should display the same fragmentation patterns under collision-induced dissociation (CID) as Pse, we considered m/z 333 in mutants as Leg.…”

“…S4 G, H). Finally, PEP depletion in pykA T>D likely result from Leg production as PEP is a precursor of Leg [ 95 ] and as metabolome data suggested that PEP concentration inversely correlates with Leg concentration (Fig. 3 C, D).…”

“…Strains constructed using PCR products and plasmid DNA as transforming material are described below. The DGRM1173 strain containing the markerless spsABCDEF deletion was constructed as previously [ 95 ]. Genotypes of constructed plasmids and strains were checked by phenotypic analyses, endonuclease restriction, PCR analysis, and/or Sanger sequencing (Eurofins Genomics, Germany).…”

Identification of Native Cross-Links in Bacillus subtilis Spore Coat Proteins

“…Both of these proteins are involved in spore coat formation. 24 Supplementary Table 4 shows the fragmentation pattern of the SpsB–CotR cross-link.…”

“…Nevertheless, we have identified a ε-(γ)-glutamyl-lysine cross-link between SpsB (K 139 ) and CotR (Q 165 ) in wild-type spores, and as expected, it is absent in tgl spores. Both of these proteins are involved in spore coat formation Supplementary Table 4 shows the fragmentation pattern of the SpsB–CotR cross-link.…”