When nutrients are scarce, Bacillus species form metabolically dormant and extremely resistant spores that enable survival over long periods of time under conditions not permitting growth. The presence of specific nutrients triggers spore germination through interaction with germinant receptors located in the spore's inner membrane. Bacillus licheniformis is a biotechnologically important species, but it is also associated with food spoilage and food-borne disease. The B. licheniformis ATCC 14580/ DSM13 genome exhibits three gerA family operons (gerA, gerK, and ynd) encoding germinant receptors. We show that spores of IMPORTANCETo ensure safe food production and durable foods, there is an obvious need for more knowledge on spore-forming bacteria. It is the process of spore germination that ultimately leads to food spoilage and food poisoning. Bacillus licheniformis is a biotechnologically important species that is also associated with food spoilage and food-borne disease. Despite its importance, the mechanisms of spore germination are poorly characterized in this species. This study provides novel knowledge on germination of B. licheniformis spores. We characterize the germinant recognition profiles of the three germinant receptors present in B. licheniformis spores and demonstrate that the GerA germinant receptor cooperates with the Ynd and GerK germinant receptors to enable an effective germination response to L-amino acids. We also demonstrate that GerK is required for germination in response to the single germinant glucose. This study demonstrates the complex interactions between germinant receptors necessary for efficient germination of B. licheniformis spores. E ndospore formation is a phenotypic adaptation to unfavorable environmental conditions, which allows bacteria to persist in the environment in a dormant and extremely resistant state. Nevertheless, spores are able to continuously monitor the environment for conditions favorable for growth. Many members of the bacterial orders Bacilliales and Clostridiales are able to survive starvation by forming endospores, which are much more resistant to heat, chemicals, irradiation, and desiccation than the vegetative cells (1). However, upon exposure to nutrient germinants, spores can return to active growth within minutes in the process of germination (2-5).The nutrient-induced germination is initiated when specific nutrients are recognized by their cognate germinant receptors (GRs) located in the spores inner membrane (6-8). Multiple GR isoforms, with distinct nutrient specificities, have been characterized in different spore-forming species of the genera Bacillus and Clostridium (4, 9-15).The genetic organization and knowledge gained from functional studies suggest that the GRs are heterotrimeric complexes consisting of A, B, and C subunits, and at least in Bacillus, they are all required for the formation of a functional receptor (3, 16).Genes encoding GR homologs are often organized in polycistronic so-called gerA family operons, encoding the A, B...

BackgroundNatural transformation enables acquisition of adaptive traits and drives genome evolution in prokaryotes. Yet, the selective forces responsible for the evolution and maintenance of natural transformation remain elusive since taken-up DNA has also been hypothesized to provide benefits such as nutrients or templates for DNA repair to individual cells.ResultsWe investigated the immediate effects of DNA uptake and recombination on the naturally competent bacterium Acinetobacter baylyi in both benign and genotoxic conditions. In head-to-head competition experiments between DNA uptake-proficient and -deficient strains, we observed a fitness benefit of DNA uptake independent of UV stress. This benefit was found with both homologous and heterologous DNA and was independent of recombination. Recombination with taken-up DNA reduced survival of transformed cells with increasing levels of UV-stress through interference with nucleotide excision repair, suggesting that DNA strand breaks occur during recombination attempts with taken-up DNA. Consistent with this, we show that absence of RecBCD and RecFOR recombinational DNA repair pathways strongly decrease natural transformation.ConclusionsOur data show a physiological benefit of DNA uptake unrelated to recombination. In contrast, recombination during transformation is a strand break inducing process that represents a previously unrecognized cost of natural transformation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-0953-2) contains supplementary material, which is available to authorized users.

Bacillus and Clostridium species form spores, which pose a challenge to the food industry due to their ubiquitous nature and extreme resistance. Pressurization at Ͻ300 MPa triggers spore germination by activating germination receptors (GRs), while pressurization at Ͼ300 MPa likely triggers germination by opening dipicolinic acid (DPA) channels present in the inner membrane of the spores. In this work, we expose spores of Bacillus licheniformis, a species associated with food spoilage and occasionally with food poisoning, to high pressure (HP) for holding times of up to 2 h. By using mutant spores lacking one or several GRs, we dissect the roles of the GerA, Ynd, and GerK GRs in moderately HP (mHP; 150 MPa)-induced spore germination. We show that Ynd alone is sufficient for efficient mHPinduced spore germination. GerK also triggers germination with mHP, although at a reduced germination rate compared to that of Ynd. GerA stimulates mHP-induced germination but only in the presence of either the intact GerK or Ynd GR. These results suggests that the effectiveness of the individual GRs in mHP-induced germination differs from their effectiveness in nutrient-induced germination, where GerA plays an essential role. In contrast to Bacillus subtilis spores, treatment with very HP (vHP) of 550 MPa at 37°C did not promote effective germination of B. licheniformis spores. However, treatment with vHP in combination with elevated temperatures (60°C) gave a synergistic effect on spore germination and inactivation. Together, these results provide novel insights into how HP affects B. licheniformis spore germination and inactivation and the role of individual GRs in this process.IMPORTANCE Bacterial spores are inherently resistant to food-processing regimes, such as high-temperature short-time pasteurization, and may therefore compromise food durability and safety. The induction of spore germination facilitates subsequent inactivation by gentler processing conditions that maintain the sensory and nutritional qualities of the food. High-pressure (HP) processing is a nonthermal food-processing technology used to eliminate microbes from food. The application of this technology for spore eradication in the food industry requires a better understanding of how HP affects the spores of different bacterial species. The present study provides novel insights into how HP affects Bacillus licheniformis spores, a species associated with food spoilage and occasionally food poisoning. We describe the roles of different germination receptors in HPinduced germination and the effects of two different pressure levels on the germination and inactivation of spores. This study will potentially contribute to the effort to implement HP technology for spore inactivation in the food industry.KEYWORDS Bacillus licheniformis, spore germination, germination receptor, endospores, high pressure, high-pressure processing, spore inactivation, flow cytometry

Germination of Bacillus spores is triggered by the binding of specific nutrients to germinant receptors (GRs) located in the spore’s inner membrane. The GRs typically consist of A, B, and C subunits, encoded by tricistronic ger operons. The Bacillus licheniformis genome contains the gerA family operons gerA, ynd, and gerK. In contrast to the ABC(D) organization that characterizes gerA operons of many Bacillus species, B. licheniformis genomes contain a pentacistronic ynd operon comprising the yndD, yndE3, yndE2, yndF1, and yndE1 genes encoding A, B, B, C, and B GR subunits, respectively (subscripts indicate paralogs). Here we show that B. licheniformis spores can germinate in the absence of the Ynd and GerK GRs, although cooperation between all three GRs is required for optimal germination with amino acids. Spores carrying an incomplete set of Ynd B subunits demonstrated reduced germination efficiencies, while depletion of all three Ynd B subunits restored germination of the spore population to levels only slightly lower than those of wild-type spores at high germinant concentrations. This suggests that the presence of an incomplete set of Ynd B subunits exhibits a dominant negative effect on germination and that the A and C subunits of the Ynd GR are sufficient for the cooperative functionality between Ynd and GerA. In contrast to the B subunits of Ynd, the B subunit of GerA was essential for amino acid-induced germination. This study provides novel insights into the role of individual GR subunits in the cooperative interaction between GRs in triggering spore germination. IMPORTANCE Spore-forming bacteria are problematic for the food industry, as spores can survive decontamination procedures and subsequently revive in food products, with the risk of food spoilage and foodborne disease. The Ynd and GerA germination receptors (GRs) cooperate in triggering efficient germination of Bacillus licheniformis spores when nutrients are present in the surrounding environment. This study shows that the single B subunit of GerA is essential for the cooperative function between Ynd and GerA, while the three B subunits of the Ynd GR are dispensable. The ability of GRs lacking individual subunits to stimulate germination together with other GRs could explain why ger operons lacking GR subunit genes are maintained in genomes of spore-forming species.

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