The microbial synthesis of sulfonolipids within the human body is likely involved in maintaining human health or causing diseases. However, the enzymes responsible for their biosynthesis remain largely unknown. In this study, we identified and verified the role of 3-ketocapnine reductase, the third-step enzyme, in the four-step conversion of L-phosphoserine into sulfobacin B both in vivo and in vitro. This finding builds upon our previous research into sulfonolipid biosynthesis, which focused on the vaginal bacterium Chryseobacterium gleum DSM 16776 and the gut bacterium Alistipes finegoldii DSM 17242. Through comprehensive gene mapping, we demonstrate the widespread presence of potential sulfonolipid biosynthetic genes across diverse bacterial species inhabiting various regions of the human body. These findings shed light on the prevalence of sulfonolipid-like metabolites within the human microbiota, suggesting a potential role for these lipid molecules in influencing the intricate biointeractions within the complex microbial ecosystem of the human body.

Sulfate-reducing microorganisms extensively contribute to the corrosion of ferrous metal infrastructure. There is substantial debate over their corrosion mechanisms. We investigated Fe⁰ corrosion with Desulfovibrio vulgaris, the sulfate reducer most often employed in corrosion studies. Cultures were grown with both lactate and Fe⁰ as potential electron donors to replicate the common environmental condition in which organic substrates help fuel the growth of corrosive microbes. Fe⁰ was corroded in cultures of a D. vulgaris hydrogenase-deficient mutant with the 1:1 correspondence between Fe⁰ loss and H₂ accumulation expected for Fe⁰ oxidation coupled to H⁺ reduction to H₂. This result and the extent of sulfate reduction indicated that D. vulgaris was not capable of direct Fe⁰-to-microbe electron transfer even though it was provided with a supplementary energy source in the presence of abundant ferrous sulfide. Corrosion in the hydrogenase-deficient mutant cultures was greater than in sterile controls, demonstrating that H₂ removal was not necessary for the enhanced corrosion observed in the presence of microbes. The parental H₂-consuming strain corroded more Fe⁰ than the mutant strain, which could be attributed to H₂ oxidation coupled to sulfate reduction, producing sulfide that further stimulated Fe⁰ oxidation. The results suggest that H₂ consumption is not necessary for microbially enhanced corrosion, but H₂ oxidation can indirectly promote corrosion by increasing sulfide generation from sulfate reduction. The finding that D. vulgaris was incapable of direct electron uptake from Fe⁰ reaffirms that direct metal-to-microbe electron transfer has yet to be rigorously described in sulfate-reducing microbes.

Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. In this study, we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome. To evaluate the robustness of fitness category assignment, Tn-seq data for three independent mutant libraries per strain were analyzed by three methods, which indicates that the Hidden Markov Model (HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size, outperforming the Bayesian and Monte Carlo simulation-based methods. Consequently, the HMM method was used to classify the fitness category. Fitness genes, categorized as essential (ES), advantage (GA), and disadvantage (GD) genes for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes. Connectivity degrees in the cofitness network decrease in the order of ES, GD, and GA/NE. In addition to accessory genes, 1599 out of 3284 core genes display differential essentiality across test strains. Within the pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.

Hyperthermus butylicus is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product. A thermostable alcohol dehydrogenase (ADH) must be present in H. butylicus to act as the key enzyme responsible for this production; however, the gene that encodes the ADH has not yet been identified. A novel ADH, HbADH2, was purified from a cell-free extract of H. butylicus, and its characteristics were determined. The gene that encodes HbADH2 was demonstrated to be HBUT_RS04850 and annotated as a hypothetical protein in H. butylicus. HbADH2 was found to be a primary–secondary ADH capable of using a wide range of substrates, including butyraldehyde and butanol. Butyraldehyde had the highest specificity constant, calculated as k_cat/K_m, with kcat and apparent K_m values of 8.00 ± 0.22 s⁻¹ and 0.59 ± 0.07 mM, respectively. The apparent K_m values for other substrates, including ethanol, 1-propanol, 2-propanol, butanol, acetaldehyde, propanal, and acetone, were 4.36 ± 0.42, 4.69 ± 0.41, 3.74 ± 0.46, 2.44 ± 0.30, 1.27 ± 0.18, 1.55 ± 0.20, and 0.68 ± 0.04 mM, respectively. The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0, respectively, while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60°C to 90°C. Based on its substrate specificity, enzyme kinetics, and thermostability, HbADH2 may be the ADH that catalyzes the production of 1-butanol in H. butylicus. The putative conserved motif sites for NAD(P)⁺ and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.

Broad-spectrum antibacterial drugs often lack specificity, leading to indiscriminate bactericidal activity, which can disrupt the normal microbial balance of the host flora and cause unnecessary cytotoxicity during systemic administration. In this study, we constructed a specifically targeted antimicrobial peptide against Staphylococcus aureus by introducing a phage-displayed peptide onto a broad-spectrum antimicrobial peptide and explored its structure–function relationship through one-factor modification. SFK2 obtained by screening based on the selectivity index and the targeting index showed specific killing ability against S. aureus. Moreover, SFK2 showed excellent biocompatibility in mice and piglet, and demonstrated significant therapeutic efficacy against S. aureus infection. In conclusion, our screening of phage-derived heptapeptides effectively enhances the specific bactericidal ability of the antimicrobial peptides against S. aureus, providing a theoretical basis for developing targeted antimicrobial peptides.

Methicillin-resistant Staphylococcus aureus (MRSA) is a current global public health problem due to its increasing resistance to the most recent antibiotic therapies. One critical approach is to develop ways to revitalize existing antibiotics. Here, we show that the phytogenic compound cinnamaldehyde (CIN) and β-lactam antibiotic combinations can functionally synergize and resensitize clinical MRSA isolates to β-lactam therapy and inhibit MRSA biofilm formation. Mechanistic studies indicated that the CIN potentiation effect on β-lactams was primarily the result of inhibition of the mecA expression by targeting the staphylococcal accessory regulator sarA. CIN alone or in combination with β-lactams decreased sarA gene expression and increased SarA protein phosphorylation that impaired SarA binding to the mecA promoter element and downregulated virulence genes such as those encoding biofilm, α-hemolysin, and adhesin. Perturbation of SarA-mecA binding thus interfered with PBP2a biosynthesis and this decreased MRSA resistance to β-lactams. Furthermore, CIN fully restored the anti-MRSA activities of β-lactam antibiotics in vivo in murine models of bacteremia and biofilm infections. Together, our results indicated that CIN acts as a β-lactam adjuvant and can be applied as an alternative therapy to combat multidrug-resistant MRSA infections.

Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo-electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6-diazo-5-oxo-L-norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

Human microbiomes, considered as a new emerging and enabling cancer hallmark, are increasingly recognized as critical effectors in cancer development and progression. Manipulation of microbiome revitalizing anticancer therapy from natural products shows promise toward improving cancer outcomes. Herein, we summarize our current understanding of the human microbiome-driven molecular mechanisms impacting cancer progression and anticancer therapy. We highlight the potential translational and clinical implications of natural products for cancer prevention and treatment by developing targeted therapeutic strategies as adjuvants for chemotherapy and immunotherapy against tumorigenesis. The challenges and opportunities for future investigations using modulation of the microbiome for cancer treatment are further discussed in this review.

The incredibly complex soil microbial communities at small scales make their analysis and identification of reasons for the observed structures challenging. Microbial community structure is mainly a result of the inoculum (dispersal), the selective advantages of those organisms under the habitat-based environmental attributes, and the ability of those colonizers to sustain themselves over time. Since soil is protective, and its microbial inhabitants have long adapted to varied soil conditions, significant portions of the soil microbial community structure are likely stable. Hence, a substantial portion of the community will not correlate to often measured soil attributes. We suggest that the drivers be ranked on the basis of their importance to the fundamental needs of the microbes: (i) those that supply energy, i.e., organic carbon and electron acceptors; (ii) environmental effectors or stressors, i.e., pH, salt, drought, and toxic chemicals; (iii) macro-organism associations, i.e., plants and their seasonality, animals and their fecal matter, and soil fauna; and (iv) nutrients, in order, N, P, and probably of lesser importance, other micronutrients, and metals. The relevance of drivers also varies with spatial and time scales, for example, aggregate to field to regional, and persistent to dynamic populations to transcripts, and with the extent of phylogenetic difference, hence phenotypic differences in organismal groups. We present a summary matrix to provide guidance on which drivers are important for particular studies, with special emphasis on a wide range of spatial and temporal scales, and illustrate this with genomic and population (rRNA gene) data from selected studies.

Kluyveromyces marxianus is a food-safe yeast with great potential for producing heterologous proteins. Improving the yield in K. marxianus remains a challenge and incorporating large-scale functional modules poses a technical obstacle in engineering. To address these issues, linear and circular yeast artificial chromosomes of K. marxianus (KmYACs) were constructed and loaded with disulfide bond formation modules from Pichia pastoris or K. marxianus. These modules contained up to seven genes with a maximum size of 15 kb. KmYACs carried telomeres either from K. marxianus or Tetrahymena. KmYACs were transferred successfully into K. marxianus and stably propagated without affecting the normal growth of the host, regardless of the type of telomeres and configurations of KmYACs. KmYACs increased the overall expression levels of disulfide bond formation genes and significantly enhanced the yield of various heterologous proteins. In high-density fermentation, the use of KmYACs resulted in a glucoamylase yield of 16.8 g/l, the highest reported level to date in K. marxianus. Transcriptomic and metabolomic analysis of cells containing KmYACs suggested increased flavin adenine dinucleotide biosynthesis, enhanced flux entering the tricarboxylic acid cycle, and a preferred demand for lysine and arginine as features of cells overexpressing heterologous proteins. Consistently, supplementing lysine or arginine further improved the yield. Therefore, KmYAC provides a powerful platform for manipulating large modules with enormous potential for industrial applications and fundamental research. Transferring the disulfide bond formation module via YACs proves to be an efficient strategy for improving the yield of heterologous proteins, and this strategy may be applied to optimize other microbial cell factories.

Insertion sequences (ISs) promote the transmission of antimicrobial resistance genes (ARGs) across bacterial populations. However, their contributions and dynamics during the transmission of resistance remain unclear. In this study, we selected IS26 as a representative transposable element to decipher the relationship between ISs and ARGs and to investigate their transfer features and transmission trends. We retrieved 2656 translocatable IS26-bounded units with ARGs (tIS26-bUs-ARGs) in complete bacterial genomes from the NCBI RefSeq database. In total, 124 ARGs spanning 12 classes of antibiotics were detected, and the average contribution rate of IS26 to these genes was 41.2%. We found that IS26-bounded units (IS26-bUs) mediated extensive ARG dissemination within the bacteria of the Gammaproteobacteria class, showing strong transfer potential between strains, species, and even phyla. The IS26-bUs expanded in bacterial populations over time, and their temporal expansion trend was significantly correlated with antibiotic usage. This wide dissemination could be due to the nonspecific target site preference of IS26. Finally, we experimentally confirmed that the introduction of a single copy of IS26 could lead to the formation of a composite transposon mediating the transmission of “passenger” genes. These observations extend our knowledge of the IS26 and provide new insights into the mediating role of ISs in the dissemination of antibiotic resistance.

Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water. It is also an opportunistic pathogen in plants, insects, animals, and humans. In response to increasing cell density, P. aeruginosa uses two acylhomoserine lactone (AHL) quorum-sensing (QS) signals (i.e., N-3-oxo-dodecanoyl homoserine lactone [3-oxo-C12-HSL] and N-butanoyl-homoserine lactone [C4-HSL]), which regulate the expression of hundreds of genes. However, how the biosynthesis of these two QS signals is coordinated remains unknown. We studied the regulation of these two QS signals in the rhizosphere strain PA1201. PA1201 sequentially produced 3-oxo-C12-HSL and C4-HSL at the early and late growth stages, respectively. The highest 3-oxo-C12-HSL-dependent elastase activity was observed at the early stage, while the highest C4-HSL-dependent rhamnolipid production was observed at the late stage. The atypical regulator RsaL played a pivotal role in coordinating 3-oxo-C12-HSL and C4-HSL biosynthesis and QS-associated virulence. RsaL repressed lasI transcription by binding the -10 and -35 boxes of the lasI promoter. In contrast, RsaL activated rhlI transcription by binding the region encoding the 5’-untranslated region of the rhlI mRNA. Further, RsaL repressed its own expression by binding a nucleotide motif located in the -35 box of the rsaL promoter. Thus, RsaL acts as a molecular switch that coordinates the sequential biosynthesis of AHL QS signals and differential virulence in PA1201. Finally, C4-HSL activation by RsaL was independent of the Las and Pseudomonas quinolone signal (PQS) QS signaling systems. Therefore, we propose a new model of the QS regulatory network in PA1201, in which RsaL represents a superior player acting at the top of the hierarchy.

Mycotoxins, which are secondary metabolites produced by toxicogenic fungi, are natural food toxins that cause acute and chronic adverse reactions in humans and animals. The genus Fusarium is one of three major genera of mycotoxin-producing fungi. Trichothecenes, fumonisins, and zearalenone are the major Fusarium mycotoxins that occur worldwide. Fusarium mycotoxins have the potential to infiltrate the human food chain via contamination during crop production and food processing, eventually threatening human health. The occurrence and development of Fusarium mycotoxin contamination will change with climate change, especially with variations in temperature, precipitation, and carbon dioxide concentration. To address these challenges, researchers have built a series of effective models to forecast the occurrence of Fusarium mycotoxins and provide guidance for crop production. Fusarium mycotoxins frequently exist in food products at extremely low levels, thus necessitating the development of highly sensitive and reliable detection techniques. Numerous successful detection methods have been developed to meet the requirements of various situations, and an increasing number of methods are moving toward highthroughput features. Although Fusarium mycotoxins cannot be completely eliminated, numerous agronomic, chemical, physical, and biological methods can lower Fusarium mycotoxin contamination to safe levels during the preharvest and postharvest stages. These theoretical innovations and technological advances have the potential to facilitate the development of comprehensive strategies for effectively managing Fusarium mycotoxin contamination in the future.