Mechanism of Thermosonication in Inhibiting Spore of Heat-Resistant Bacillus subtilis via Metabolic Networks and Gene Regulation

Temperature dependence of Bacillus cereus spores: effects on sporulation, germination behavior, and inner membrane protein responses.

Bacillus cereus, a prevalent foodborne pathogen, produces dormant spores that can persist throughout meat preservation. Upon germination and growth, it causes meat spoilage and human illness. Since bacterial spore properties are highly temperature-dependent, this study investigated the effects of temperature on sporulation, germination behavior, and the responses of inner membrane proteins (IMPs). Spores with high germination efficiency were readily formed at 25-30 °C, with the highest efficiency observed at 30 °C. Meanwhile, analysis of spore properties showed that higher sporulation temperatures resulted in lower spore water content and greater resistance to moist heat. Raman spectroscopy further revealed temperature-dependent variations in the molecular characteristics of intact spores, including changes associated with Ca2+-dipicolinic acid (Ca2+-DPA), protein-related, and nucleic acid-related spectral features under different sporulation temperatures. Different heat activation temperatures resulted in distinct spore germination efficiency and induced physicochemical changes inextracted IMPs fractions. Notably, 65 °C represented the optimum heat activation condition across 60-80 °C, resulting in enhanced germination efficiency and moderate conformational rearrangement of IMPs, as characterized by complementary biophysical analyses. These findings highlight the importance of temperature control in regulating spore germination and inactivation during food processing and provide practical insights for the management of bacterial spores in the food industry.

Functional role of the diadenylate cyclase gene cdaA in stress responses of the dairy starter Streptococcus thermophilus.

Streptococcus thermophilus is a key dairy starter culture widely used in the production of yogurt and cheese, where its robustness in stress adaptation and growth performance is critical for industrial fermentation efficiency. Cyclic di-AMP (c-di-AMP) is a key second messenger involved in regulating osmotic homeostasis and stress adaptation in bacteria. However, the role of c-di-AMP in S. thermophilus remains unexplored. Here, cdaA, encoding a diadenylate cyclase containing a conserved DisA_N domain responsible for c-di-AMP synthesis, was identified in S. thermophilus S-3 through bioinformatic analysis and validated by LC-MS/MS. The cdaA gene knockout strain, S-3ΔcdaA, was unable to synthesize c-di-AMP and exhibited a markedly shortened lag phase. Stress response assays revealed that S-3ΔcdaA was sensitive to osmotic and ethanol stress, indicating that cdaA is essential for stress tolerance. Specifically, S-3ΔcdaA showed almost no growth in chemically defined medium (CDM) supplemented with 300 mM potassium or sodium chloride, whereas the wild-type S-3 reached OD600 values of 0.72 and 0.52, respectively. Under 5% ethanol stress, the specific growth rate of S-3ΔcdaA dropped significantly to 0.35 relative to S-3 compared to 1.10 in CDM medium alone. Interestingly, S-3ΔcdaA retained the ability to grow under 0.01% oxgall stress, reaching an OD600 of 0.83, while growth of S-3 was completely abolished. It suggested that absence of cdaA confers a survival advantage under bile salt stress. Taken together, these results showed that cdaA is critical for osmotic and ethanol tolerance but negatively modulates bile salt resistance in S. thermophilus.

Transcriptomic Analysis Reveals the Time-Dependent Mechanism of Antifungal Activity in Bacillus velezensis GHZJ-1

The results suggest that facing continuously increased environmental stress over time, GHZJ-1 undergoes global transcriptional reprogramming and resource reallocation, downregulating basal metabolism to construct a synergistic antagonistic system coupling chemical defense with nutritional competition.

Trehalose-6-phosphate synthase promotes thermotolerance by governing glycolytic flux in Cryptococcus deneoformans

Growth at physiologically relevant temperatures is essential for fungal pathogenesis and is controlled by several cellular factors. The evolution of fungal thermotolerance is concerning as warming environments may promote the emergence of new pathogens. Trehalose, a disaccharide absent in mammals, plays a central role in thermotolerance by stabilizing proteins and membranes during heat stress. Trehalose is synthesized from glucose-6-phosphate (G6P) and uridine-diphosphate-glucose (UDPG) in two steps catalyzed by trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). Here, we investigated genetic suppression of Tps1 function in Cryptococcus deneoformans, a species in the Cryptococcus pathogenic species complex. Tps1 is essential for growth at 37°C in C. deneoformans and spontaneous suppressor mutations restored the growth of tps1Δ mutants at 37°C. Whole-genome sequencing followed by variant calling analysis primarily identified loss-of-function mutations in the gene encoding hexokinase 1 (Hxk1). Targeted gene deletion mutants further showed that loss of either HXK1 or HXK2 can bypass tps1Δ in a carbon source-dependent manner. The tps1Δ mutant exhibited elevated hexokinase activity, accumulation of G6P and glycogen, and ATP depletion after heat shock. Deletion of HXK1 or HXK2 restored hexokinase activity and partially restored G6P and ATP levels in the tps1Δ mutant, while glycogen remained elevated, indicating that excess glycolytic flux underlies the tps1Δ high-temperature growth defect. Overall, our study uncovers a previously unappreciated mechanism of Tps1-mediated heat adaptation in C. deneoformans, by revealing that Tps1 functions as a critical metabolic gatekeeper that safeguards glycolytic flux to sustain growth at elevated temperatures. Article summary Trehalose is crucial for fungal thermal adaptation and mutants lacking trehalose are inviable at 37°C. This study examined how genetic suppressors restore viability at 37°C in mutants lacking TPS1, which encodes trehalose-6-phosphate synthase. Through whole-genome sequencing of spontaneous suppressor isolates and variant calling analysis, mutations were identified in HXK1. Gene deletion mutants and biochemical assays show these mutations alter glycolytic flux. We show that tps1Δ mutants exhibit unbridled glycolysis, and their growth at 37°C is restored by hxk1Δ mutations that reduce glycolytic flux. This study highlights the interdependence between Tps1 and Hxk1, which may have broader relevance across organisms.

Stage V sporulation protein G as a global regulator of biofilm formation and virulence in Staphylococcus species: molecular mechanisms and therapeutic potential

A Bacillus thuringiensis ΔcdsR-based expression system for insecticidal proteins identified using AlphaFold3.

Environmentally friendly biopesticides, such as those based on the bacterium Bacillus thuringiensis (Bt), have an important role to play in Integrated Pest Management systems. An increasingly diverse range of pesticidal proteins is being discovered, led by advances in genome sequencing and the bioinformatic identification of candidate genes. In this work, we utilized an AlphaFold3-based structure-prediction approach to identify candidate genes and, in parallel, developed a Bt chassis strain that can be universally used to express new pesticidal proteins. This chassis strain is based on a cdsR deletion strain with a controllable cell death pathway that decouples toxin production from sporulation, enabling cytoplasmic hyperexpression via strong promoters and encapsulation of recombinant proteins. These characteristics provide superior efficacy, UV stability, and environmental safety compared with conventional Bt formulations. Several of the identified proteins displayed insecticidal activity when expressed in this strain, providing a versatile framework for next-generation biocontrol and sustainable pest-management innovation.