Circadian Clock and Hypoxia.

The timing of life on Earth is remarkable: between individuals of the same species, a highly similar temporal pattern is observed, with shared periods of activity and inactivity each day. At the individual level, this means that over the course of a single day, a person alternates between two states. They are either upright, active, and communicative or they lie down in a state of (un)consciousness called sleep where even the characteristic of neuronal signals in the brain shows distinctive properties. The circadian clock governs both of these time stamps-activity and (apparent) inactivity-making them come and go consistently at the same approximate time each day. This behavior thus represents the meeting of two pervasive systems: the circadian clock and metabolism. In this article, we will describe what is known about how the circadian clock anticipates daily changes in oxygen usage, how circadian clock regulation may relate to normal physiology, and to hypoxia and ischemia that can result from pathologies such as myocardial infarction and stroke.

From the outer space to the inner cell: deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox state and absolute abundance quantification of extracellular, membrane, and cytosolic proteins.

Understanding cellular mechanisms of stress management relies on omics data as a valuable resource. However, the lack of absolute quantitative data on protein abundances remains a significant limitation, particularly when comparing protein abundances across different cell compartments. In this study, we aimed to gain deeper insights into the proteomic responses of the Gram-positive model bacterium Bacillus subtilis to disulfide stress. We determined proteome-wide absolute abundances, focusing on different sub-cellular locations (cytosol and membrane) as well as the extracellular medium, and combined these data with redox state determination. To quantify secreted proteins in the culture medium, we developed a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria. We concentrated extracellular proteins, which are highly diluted in the medium, using StrataClean beads along with a set of standard proteins to determine the extent of the concentration step. The resulting data set provides new insights into protein abundances in different sub-cellular compartments and the extracellular medium, along with a comprehensive proteome-wide redox state determination. Our study offers a quantitative understanding of disulfide stress management, protein production, and secretion in B. subtilis. IMPORTANCE: Stress responses play a crucial role in bacterial survival and adaptation. The ability to quantitatively measure protein abundances and redox states in different cellular compartments and the extracellular environment is essential for understanding stress management mechanisms. In this study, we addressed the knowledge gap regarding absolute quantification of extracellular proteins and compared protein concentrations in various sub-cellular locations and in the extracellular medium under disulfide stress conditions. Our findings provide valuable insights into the protein production and secretion dynamics of B. subtilis, shedding light on its stress response strategies. Furthermore, the developed protocol for absolute quantification of extracellular proteins in bacteria presents a practical and efficient approach for future studies in the field. Overall, this research contributes to the quantitative understanding of stress management mechanisms and protein dynamics in B. subtilis, which can be used to enhance bacterial stress tolerance and protein-based biotechnological applications.

Alpha-glucans from bacterial necromass indicate an intra-population loop within the marine carbon cycle.

Phytoplankton blooms provoke bacterioplankton blooms, from which bacterial biomass (necromass) is released via increased zooplankton grazing and viral lysis. While bacterial consumption of algal biomass during blooms is well-studied, little is known about the concurrent recycling of these substantial amounts of bacterial necromass. We demonstrate that bacterial biomass, such as bacterial alpha-glucan storage polysaccharides, generated from the consumption of algal organic matter, is reused and thus itself a major bacterial carbon source in vitro and during a diatom-dominated bloom. We highlight conserved enzymes and binding proteins of dominant bloom-responder clades that are presumably involved in the recycling of bacterial alpha-glucan by members of the bacterial community. We furthermore demonstrate that the corresponding protein machineries can be specifically induced by extracted alpha-glucan-rich bacterial polysaccharide extracts. This recycling of bacterial necromass likely constitutes a large-scale intra-population energy conservation mechanism that keeps substantial amounts of carbon in a dedicated part of the microbial loop.

Let There Be Light: Genome Reduction Enables Bacillus subtilis to Produce Disulfide-Bonded Gaussia Luciferase.

Bacillus subtilis is a major workhorse for enzyme production in industrially relevant quantities. Compared to mammalian-based expression systems, B. subtilis presents intrinsic advantages, such as high growth rates, high space-time yield, unique protein secretion capabilities, and low maintenance costs. However, B. subtilis shows clear limitations in the production of biopharmaceuticals, especially proteins from eukaryotic origin that contain multiple disulfide bonds. In the present study, we deployed genome minimization, signal peptide screening, and coexpression of recombinant thiol oxidases as strategies to improve the ability of B. subtilis to secrete proteins with multiple disulfide bonds. Different genome-reduced strains served as the chassis for expressing the model protein Gaussia Luciferase (GLuc), which contains five disulfide bonds. These chassis lack extracellular proteases, prophages, and key sporulation genes. Importantly, compared to the reference strain with a full-size genome, the best-performing genome-minimized strain achieved over 3000-fold increased secretion of active GLuc while growing to lower cell densities. Our results show that high-level GLuc secretion relates, at least in part, to the absence of major extracellular proteases. In addition, we show that the thiol-disulfide oxidoreductase requirements for disulfide bonding have changed upon genome reduction. Altogether, our results highlight genome-engineered Bacillus strains as promising expression platforms for proteins with multiple disulfide bonds.

Diamide-based screening method for the isolation of improved oxidative stress tolerance phenotypes in Bacillus mutant libraries.

IMPORTANCE: During their life cycle, bacteria are exposed to a range of different stresses that need to be managed appropriately in order to ensure their growth and viability. This applies not only to bacteria in their natural habitats but also to bacteria employed in biotechnological production processes. Oxidative stress is one of these stresses that may originate either from bacterial metabolism or external factors. In biotechnological settings, it is of critical importance that production strains are resistant to oxidative stresses. Accordingly, this also applies to the major industrial cell factory Bacillus subtilis. In the present study, we, therefore, developed a screen for B. subtilis strains with enhanced oxidative stress tolerance. The results show that our approach is feasible and time-, space-, and resource-efficient. We, therefore, anticipate that it will enhance the development of more robust industrial production strains with improved robustness under conditions of oxidative stress.

Enhancing bacterial fitness and recombinant enzyme yield by engineering the quality control protease HtrA of Bacillus subtilis.

IMPORTANCE: In the expanding market of recombinant proteins, microbial cell factories such as Bacillus subtilis are key players. Microbial cell factories experience secretion stress during high-level production of secreted proteins, which can negatively impact product yield and cell viability. The CssRS two-component system and CssRS-regulated quality control proteases HtrA and HtrB play critical roles in the secretion stress response. HtrA has a presumptive dual function in protein quality control by exerting both chaperone-like and protease activities. However, its potential role as a chaperone has not been explored in B. subtilis. Here, we describe for the first time the beneficial effects of proteolytically inactive HtrA on α-amylase yields and overall bacterial fitness.

Impact of Pneumococcal and Viral Pneumonia on the Respiratory and Intestinal Tract Microbiomes of Mice.

With 2.56 million deaths worldwide annually, pneumonia is one of the leading causes of death. The most frequent causative pathogens are Streptococcus pneumoniae and influenza A virus. Lately, the interaction between the pathogens, the host, and its microbiome have gained more attention. The microbiome is known to promote the immune response toward pathogens; however, our knowledge on how infections affect the microbiome is still scarce. Here, the impact of colonization and infection with S. pneumoniae and influenza A virus on the structure and function of the respiratory and gastrointestinal microbiomes of mice was investigated. Using a meta-omics approach, we identified specific differences between the bacterial and viral infection. Pneumococcal colonization had minor effects on the taxonomic composition of the respiratory microbiome, while acute infections caused decreased microbial complexity. In contrast, richness was unaffected following H1N1 infection. Within the gastrointestinal microbiome, we found exclusive changes in structure and function, depending on the pathogen. While pneumococcal colonization had no effects on taxonomic composition of the gastrointestinal microbiome, increased abundance of Akkermansiaceae and Spirochaetaceae as well as decreased amounts of Clostridiaceae were exclusively found during invasive S. pneumoniae infection. The presence of Staphylococcaceae was specific for viral pneumonia. Investigation of the intestinal microbiomés functional composition revealed reduced expression of flagellin and rubrerythrin and increased levels of ATPase during pneumococcal infection, while increased amounts of acetyl coenzyme A (acetyl-CoA) acetyltransferase and enoyl-CoA transferase were unique after H1N1 infection. In conclusion, identification of specific taxonomic and functional profiles of the respiratory and gastrointestinal microbiome allowed the discrimination between bacterial and viral pneumonia. IMPORTANCE Pneumonia is one of the leading causes of death worldwide. Here, we compared the impact of bacterial- and viral-induced pneumonia on the respiratory and gastrointestinal microbiome. Using a meta-omics approach, we identified specific profiles that allow discrimination between bacterial and viral causative.