Outer space is an extreme environment and the survival of many microorganisms after spaceflight is well established. However, adaptations of Bacillus subtilis to space stress, particularly metabolism, are largely unknown. Here, we first performed a spaceflight mission of the B. subtilis TD7 strain and compared the spaceflight-exposed strain with the wild-type in terms of their phenotype, biofilm formation and secondary metabolism. The spaceflight-exposed strain exhibited slower growth, different morphology and decreased biofilm formation. Importantly, a declinein lipopeptide production was observed after spaceflight. Multi-omics approaches were used to uncover the molecular mechanisms underlying secondary metabolism and 997 differentially expressed genes (DEGs) were found, involving the TCA cycle, fatty acid degradation, amino acid biosynthesis and quorum sensing systems. Further analysis of 26 lipopeptide-related DEGs further elucidated the relationship between the space environment and secondary metabolism regulation. Our findings could contribute to a better understanding of the relationship between the space environment and microbial adaptation mechanisms.

Advances in neuromicrobiology and related omics technologies have reinforced the idea that unseen microbes play critical roles in human cognition and behaviour. Included in this research is evidence indicating that gut microbes, through direct and indirect pathways, can influence aggression, anger, irritability and antisocial behaviour. Moreover, gut microbes can manufacture chemicals that are known to compromise cognition. For example, recent court decisions in the United States and Europe acknowledge that gut microbes can produce high levels of ethanol, without consumption of alcohol by the defendants. The dismissal of driving while intoxicated charges in these cases-so-called auto-brewery syndrome-highlights the way in which microbiome knowledge will enhance the precision, objectivity and fairness of our legal systems. Here in this opinion essay, we introduce the concept of the 'legalome'-the application of microbiome and omics science to forensic psychiatry and criminal law. We argue that the rapid pace of microbial discoveries, including those that challenge ideas of free will and moral responsibility, will necessitate a reconsideration of traditional legal doctrines and justifications of retributive punishment. The implications extend beyond the courtroom, challenging us to reconsider how environmental factors-from diet to socioeconomic conditions-might shape preventative and rehabilitative efforts through their effects on the microbiome.

Daptomycin (DAP) is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, and its biosynthesis is precisely regulated by a complex regulatory network. Although the biosynthetic pathway of DAP has been elucidated, the regulatory mechanism governing its biosynthesis at the transcriptional level is not yet fully understood. In the present study, a new transcriptional regulator, DhyR, was identified. A deletion mutant of dhyR was constructed using the CRISPR-Cas9 tool to elucidate the biological role of DhyR thanks to functional and transcriptomic analyses. The results demonstrated that DhyR positively regulates DAP biosynthesis in S. roseosporus. The in-frame deletion of the dhyR gene resulted in a significant downregulation of the transcription levels of all structural genes within the DAP biosynthetic gene cluster and a significant decrease in DAP yield. In contrast, overexpression of dhyR enhanced the transcription levels of the DAP biosynthetic gene cluster, leading to a 23% increase in DAP yield. Deletion of dhyR caused significant changes in the expression of multiple genes involved in carbohydrate metabolism, energy metabolism and amino acid metabolic pathways through transcriptome analysis. Especially, deletion of dhyR led to a significant downregulation of transcription levels of three DAP biosynthesis-associated genes, including atrA, depR1 and ssig-05090. In summary, DhyR positively regulates DAP biosynthesis in S. roseosporus by influencing the expression of the DAP gene cluster and modulating precursor flux. It functions as a pleiotropic regulator of primary and secondary metabolism in S. roseosporus.

The DnaK (Hsp70) protein is an essential ATP-dependent chaperone foldase and holdase found in most organisms. In this study, combining multiple experimental approaches we determined FliC as major interaction partner of DnaK in the opportunistic bacterial pathogen Pseudomonas aeruginosa. Implementing immunofluorescence microscopy and electron microscopy techniques DnaK was found extracellularly associated to the assembled filament in a regular pattern. dnaK repression led to intracellular FliC accumulation and motility impairment, highlighting DnaK essentiality for FliC export and flagellum assembly. SPOT-membrane peptide arrays coupled with artificial intelligence analyses suggested a highly dynamic DnaK-FliC interaction landscape involving multiple domains and transient complexes formation. Remarkably, invitro fast relaxation imaging (FReI) experiments mimicking ATP-deprived extracellular environment conditions exhibited DnaK ATP-independent holdase activity, regardless of its co-chaperone DnaJ and its nucleotide exchange factor GrpE. We present a model for the DnaK-FliC interactions involving dynamic states throughout the flagellum assembly stages. These results expand the classical view of DnaK chaperone functioning and introduce a new participant in the Pseudomonas flagellar system, an important trait for bacterial colonisation and virulence.

Immunoassays represent sensitive, easy-to-use, and cost-effective tests useful for the detection of the SARS-CoV-2 virus. In this manuscript, we report on the binding specificity of a pair of novel monoclonal antibodies (MAbs) generated against the SARS-CoV-2 nucleocapsid protein (NP) and their development into sensitive sandwich enzyme-linked immunosorbent assays (sELISA) and a lateral flow immunoassay (LFIA). Binding of these MAbs to hCoVs is limited to variants of SARS-CoV-2 and SARS-CoV NP. Chemiluminescent and absorbance spectroscopy sELISAs report a limit of detection (LOD) for the SARS-CoV-2 B.1.1.529 NP variant at 15 pg/mL, and the LFIA using a red-dyed 200 nm particle at 10 ng/mL. The sELISA exhibits broad SARS-CoV-2 viral variant detection with assay LOD for SARS-CoV-2 B.1.1.529 virus at 1.4 × 105 genome copies per mL (p ≤ 0.001). The availability of these MAbs should facilitate continued investment in the commercial development of immunoassays to increase global SARS-CoV-2 detection technologies.

The discovery at the end of the 20th century of genes that induce cell death revolutionised the biocontaintment of genetically manipulated bacteria for environmental or agricultural applications. These bacterial 'killer' genes were then assayed for their potential to target and control malignant cells in human cancers. The identification of the bacteriomes in different human organs and tissues, coupled with the observation that bacteria tend to accumulate near tumours, has opened new avenues for anti-cancer strategies. This progress, along with recent insights into how cancer cells evade immune response, has prompted innovative therapeutic approaches. Tumour microenvironments are typically nutrient-rich, characterised by low oxygen tensions and very resistant to immune responses. Two recent studies in MBT highlight the promise of using Salmonella typhimurium and Escherichia coli as vectors in novel cancer treatments. Engineered S. typhimurium strains can generate adjuvant flagellin-antigen complexes that function as insitu vaccines, hence increasing the immunogenic responses within tumour environment. Similarly, gut E. coli can be used as vectors to targert tumour cells in colon cancer, enabling both diagnostic applications and localised treatments. Both approaches hold significant potential to improve patient survival outcomes.

Advancing microbiome-gut-brain axis science requires systematic, rational and translational approaches to bridge the critical knowledge gaps currently preventing full exploitation of the gut microbiome as a tractable therapeutic target for gastrointestinal, mental and brain health. Current research is still marked by many open questions that undermine widespread application to humans. For example, the lack of mechanistic understanding of probiotic effects means it remains unclear why even apparently closely related strains exhibit different effects invivo. For the therapeutic application of live microbial psychobiotics, consensus on their application as adjunct treatments to conventional neuromodulators, use in unmedicated populations or in at-risk cohorts with sub-clinical symptomatology is warranted. This missing information on both sides of the therapeutic equation when treating central nervous system (CNS) conditions makes psychobiotic research challenging, especially when compared to other pharmaceutical or functional food approaches. Expediting the transition from positive preclinical data to proven benefits in humans includes interpreting the promises and pitfalls of animal behavioural assays, as well as navigating mechanism-informed decision making to select the right microbe(s) for the job. In this review, we consider how these decisions can be supported in light of information accrued from a range of clinical studies across healthy, at-risk and pathological study populations, where specific strains have been evaluated in the context of gastrointestinal physiology, brain function and behaviour. Examples of successful, partial and unsuccessful translation from bench to bedside are considered. We also discuss the developments in in silico analyses that have enhanced our understanding of the gut microbiome and that have moved research towards pinpointing the host-microbe interactions most important for optimal gut-brain axis function. Combining this information with knowledge from functional assays across invitro and exvivo domains and incorporating model organisms can prime the discovery pipelines with the most promising and rationally selected psychobiotic candidates.

Pseudomonas putida is widely used in industrial applications, including the recombinant proteins production, because of its natural advantageous properties. In this study, the gene encoding FleQ, the primary regulator of flagellar synthesis, was deleted to construct a new non-motile P. putida KT2440-derived strain (ΔfleQ). The non-motile cells showed reduced biofilm formation and enhanced expression of a heterologous gene in nutrient-rich media compared with the wild-type (WT) strain, attributed to the reallocation of cellular resources from flagellar synthesis and cellular motility. Additionally, the ΔfleQ strain exhibited enhanced tolerance to chloramphenicol, indicating higher ribosome production, confirmed by a higher RNA/protein ratio relative to the WT. While the WT strain showed decreased growth and a three-fold increase in reporter gene activity in minimal media, the ΔfleQ strain maintained consistent reporter gene expression and exhibited a relatively higher growth rate. This suggests that the FleQ is involved in modulating proteome allocation based on nutrient quality. The removal of FleQ allows for more flexible resource allocation, creating a chassis strain with nutrient quality-independent gene expression capacity, which could be valuable in industrial applications where consistent output is essential.

ABSTRACTLive biotherapeutic products (LBPs), including engineered bacteria, are rapidly emerging as potential therapeutic interventions. These innovative therapies can serve as live in situ drug delivery platforms for the direct deposition of therapeutic payloads, including complex biologics, at sites of disease. This approach offers a platform likely to enhance therapeutic efficacy and decrease off‐target side effects. LBPs also can likely be distributed at a relatively low price point, as their production can be economically scaled up. LBPs represent an exciting new means for ensuring healthy lives and promoting well‐being for all ages, aligning with the World Health Organization's sustainable development goal 3.