Applied and Environmental Microbiology

Microbial engineering offers potential for improving the sustainability of agriculture by providing greater control of desired microbial functions. However, successful control of engineered functions requires greater understanding of their robustness under diverse conditions including those used for plant hydroponics. Here, we studied biomass accumulation and surfactin biosynthesis by an engineered derivative of Bacillus subtilis PY79 in common plant culture media as a model system for interrogating metabolic robustness. We report the observation that PY79 and all other B. subtilis strains that we tested, including natural isolates, exhibited difficulty growing under shaking incubation in defined media where the only nitrogen sources were inorganic. In contrast, assimilation of inorganic nitrogen sources functioned relatively robustly under static incubation in these same media. Our findings may offer some guidance for use of B. subtilis in controlled environment agriculture and could aid future efforts to identify the molecular basis for the agitation-dependent effect on nitrogen assimilation.

The plant-beneficial bacterium Pseudomonas protegens CHA0 (CHA0) is widely studied for the biological control of soil-borne plant diseases. Beyond its root-colonising capabilities, CHA0 can also infect and kill insect larvae and thus exhibits a multi-host lifestyle shared with other plant- and insect-colonising bacteria. To better understand the robustness of this multi-host lifestyle, we subjected CHA0 to ten consecutive passages through larvae of the pest insect Plutella xylostella via repeated cycles of insect colonisation and killing forcing it into an insect-only lifestyle. Overall, serial passaging did not result in consistent changes in insect killing speed, larval or root colonisation, plant protection efficiency, microbial antagonism or in vitro growth. This suggests that its multi-host lifestyle was conserved following serial passage. Nonetheless, a few independently passaged lines showed an increase in larval killing speed, which in one case might be linked to choline uptake. To disentangle changes specific to the insect host from those arising due to the experimental system itself, we conducted parallel serial passages through the same system while omitting the insect host. In some of these lines, exposure to the background of the system led to changes in microbial antagonism and in in vitro growth, which likely are associated with mutations in regions encoding for regulatory systems. Our findings indicate that P. protegens CHA0 remains phenotypically stable in complex environments such as an insect host, suggesting that the multi-host lifestyle might also be conserved when applied in the field and supporting CHA0s potential for reliable biocontrol performance against both plant diseases and insect pests. Author summaryControlling insect pests with living organisms, known as biological control, offers an environmentally friendly alternative to chemical pesticides. The plant-beneficial bacterium Pseudomonas protegens CHA0 is a promising biocontrol candidate that not only colonizes plant roots but also infects and kills certain insect larvae. This ability to colonize different hosts appears to be a conserved trait also observed in other bacteria. To better understand the robustness of this multi-host lifestyle, we repeatedly exposed CHA0 to larvae of the insect pest Plutella xylostella and assessed the resulting physiological and genetic changes. Surprisingly, after ten cycles, CHA0 largely retained its insect-killing and plant-protective traits. Although a few populations showed minor changes, including slightly faster insect killing and traits associated with aspects of the experimental system, these changes were limited in scope. Overall, our findings suggest that P. protegens CHA0 does not change rapidly in complex environments such as an insect host, supporting its potential for reliable biocontrol performance in the field.

The emergence of multidrug-resistant Vibrio parahaemolyticus poses a severe threat to mariculture sustainability, highlighting the urgent need for eco-friendly antimicrobial agents. In this study, we demonstrated that L-Cysteine (L-Cys) functions as an inhibitor of V. parahaemolyticus, with a minimum inhibitory concentration of 7.5 mM. Microscopic observation and viability assays revealed that L-Cys compromises bacterial membrane integrity, ultimately leading to cell death. Further investigation indicated that the antibacterial effect is primarily attributed to the intracellular production of hydrogen sulfide (H2S) generated by L-Cys metabolism. Transcriptomic and biochemical analyses showed that L-Cys induced metabolic reprogramming by suppressing fatty acid {beta}-oxidation, one-carbon metabolism, and antioxidant enzymes. This disruption of redox homeostasis results in accelerated accumulation of reactive oxygen species (ROS). In addition to its antibacterial effect, L-Cys also effectively reduced the virulence factor of bacterial motility. Finally, L-Cys demonstrated broad-spectrum antimicrobial activity against other pathogenic Vibrio species, including V. alginolyticus and V. anguillarum. Our findings suggest that L-Cys is a promising antimicrobial agent inducing ROS to mediate membrane disruption, with the advantages of cost-effectiveness and environmental safety for controlling vibriosis. ImportanceVibrio parahaemolyticus is a significant pathogen in aquaculture and a common cause of seafood-borne gastroenteritis worldwide. The increasing prevalence of antibiotic-resistant strains of this bacterium highlights the need for alternative control agents. This study shows that L-cysteine (L-Cys) inhibits the growth of V. parahaemolyticus. Our data indicate that L-Cys is metabolized to produce hydrogen sulfide, which contributes to the accumulation of ROS and disrupts bacterial membrane integrity. Additionally, L-Cys reduces bacterial motility and shows inhibitory effects against other Vibrio species. These findings suggest that L-Cys may represent a useful agent for managing Vibrio infections in aquaculture settings.

Sink P-traps harbor microbial communities derived from environmental and human sources, yet longitudinal studies examining their stability and assembly dynamics remain limited. Here, we present, to our knowledge, the longest continuous characterization of bacteria collected from residential sink P-traps, with daily sampling over two months (n = 61 days). Samples were collected from paired sinks in a shared bathroom to identify dominant taxa, quantify temporal stability, and determine how occupant usage patterns influence community assembly. Using full-length 16S rRNA gene sequencing, we identified 3,865 unique taxa, with both sinks dominated by common sink-associated genera, including Pseudomonas, Citrobacter, Klebsiella, and Arcobacter. Despite sharing identical plumbing, environmental conditions, and cleaning regimes, the two sinks maintained distinct communities (p < 0.001). Temporal stability analyses revealed notable differences: Sink A (male; hand washing, toothbrushing, shaving) exhibited deterministic dynamics with low variability (CV = 4.9%), significant temporal autocorrelation (p = 0.001), and predictable trajectories, with time explaining 49.9% of community variation. In contrast, Sink B (female; hand washing, toothbrushing, face washing, mouthwash use) displayed stochastic dynamics with high volatility (CV = 26.5%), no significant autocorrelation (p = 0.53), and minimal temporal predictability. Differential abundance analysis revealed that Sink B was enriched in anaerobes, biofilm-forming taxa, oral microbiome associates, and preservative-resistant and lipid-degrading bacteria, while Sink A harbored a more aerobic, skin-associated community. These findings demonstrate that individual usage patterns (particularly exposure to biocidal agents) can alter P-trap community structure and temporal dynamics, with implications for microbial community prediction in residential and healthcare settings. ImportanceSink drains are increasingly recognized as reservoirs for antimicrobial-resistant pathogens, yet we lack fundamental knowledge about what drives bacterial community dynamics in these environments. By sampling paired residential sink P-traps daily for two months, we show that individual-specific behaviors, such as using products with a biocidal effect, can alter community composition from a stable, predictable state to one characterized by stochastic fluctuations. The sink exposed to mouthwash and face wash harbored more anaerobes, biofilm formers, and oral bacteria, suggesting that repeated exposure promotes disturbance-tolerant taxa rather than reducing bacterial colonization. These results provide a baseline ecological framework for understanding P-trap microbiomes and suggest that predictive monitoring of sink-associated pathogens might be feasible in stable environments but more difficult when there is variable antimicrobial exposure -- a finding directly relevant to hospital infection control.

Leptospirosis, caused by pathogenic Leptospira spp. such as L. interrogans, is a bacterial zoonosis of increasing prevalence with no consistently effective treatments in severe cases. We sought to characterize metabolic mechanisms that support L. interrogans infection in the host setting, with the ultimate goal of revealing unexplored therapeutic opportunities. We first established and validated a culture medium, which we refer to as supplemented Human Plasma-Like Medium (sHPLM). sHPLM more closely resembles the physiological environment of the human host than standard culture media, such as the EMJH (Ellinghausen-McCullough-Johnson-Harris) medium typically used for Leptospira culture. To better understand bacterial metabolism, we pioneered metabolomics in sHPLM-cultured Leptospira. Specifically, we developed a liquid chromatography mass spectrometry (LC/MS) metabolomics-based workflow for both medium analysis and stable isotope tracing with L. interrogans cultures. The application of these innovations revealed that the amino acid glutamine is a major nitrogen source for L. interrogans. A small-molecule inhibitor blocking glutamine utilization, JHU-083, effectively impaired the proliferation of sHPLM cultures. Further, adding glutamine to non-physiological EMJH medium rapidly induced a short-term proliferative boost in L. interrogans and increased biofilm formation. RNA-sequencing after glutamine exposure revealed transcriptional trends for increases in biosynthesis to support these phenotypes. Although ammonium has long been thought to be the sole nitrogen source for L. interrogans, our results demonstrate that glutamine provides a second source of nitrogen for biosynthesis and may act as a metabolite signal to alter L. interrogans physiology in ways that could influence infection. This work highlights that studying L. interrogans under physiological conditions is key to understanding mechanisms supporting infection and points to nitrogen assimilation as a potential target for therapies. Author SummaryLeptospirosis is a potentially fatal disease transmitted through water and soil contaminated with pathogenic Leptospira bacteria. Much research is currently focused on the idea that an improved understanding of how Leptospira infects hosts and causes disease may inspire the development of improved therapeutics, which are urgently needed. Focusing on Leptospira interrogans, a clinically important pathogenic species, we determined that conventional growth media are inadequate for understanding how the bacterium behaves when inside hosts. Instead, we designed an optimized formulation to mimic human blood, and we applied an underutilized technique for measuring the biochemical reactions that enable pathogen survival. These two innovations revealed that L. interrogans uses glutamine, an abundant nutrient in host blood and tissues, as a source of nitrogen for the production of biomolecules that are required for replication and infection. This discovery is notable as nitrogen demands were previously thought to be met using ammonium. Treating L. interrogans with inhibitors of both glutamine and ammonium metabolism blocked bacterial replication. We also discovered that L. interrogans increases its growth rate, upregulates its expression of biosynthetic pathways when exposed to glutamine, and increases its formation of biofilm. Our results reveal the importance of glutamine in supporting the lifecycle of leptospirosis-causing bacteria.

Yeasts ability to invade surfaces has important implications for infections and food contamination. Invasive growth in yeast is influenced by genetic and environmental factors. In this exploratory study, we investigated the effects of sodium sulfide, gene deletions, and environmental conditions on the invasive behaviour of the wine yeast strain AWRI 796. Sodium sulfide enhanced invasion in the (parent) AWRI 796 strain under nitrogen-limiting conditions, although its effect was obscured by experimental variability and pre-culture conditions. Genetic factors had a major effect on the overall invasive phenotype, with deletion of key genes suppressing invasion. Most gene-deletion mutants did not significantly affect how the colony responded to sulfide. In addition to sulfide and genotype, environmental conditions also influenced invasive behaviour. The pre-2xSLAD pre-culture condition was best for detecting sulfide-induced growth, and later plate washing time and decreased nutrient levels enhanced invasiveness. Our experimental design and findings provide a framework for understanding the determinants of yeast invasiveness, which may inform future studies on filamentous yeast behaviour.

Bacteriophages are promising alternatives for antibiotics. One challenge of developing bacteriophage-based preparation for practical application is to understand expected host range of such products. We evaluated the host range and lytic activity of BAFASAL(R), a four-phage cocktail targeting Salmonella in poultry production. For that purpose, we developed a new composite metric, the Combined Lytic Score (CLS), integrating results from two in vitro assays: serial dilutions spot test on semisolid medium and spectrophotometric growth inhibition in liquid culture. Using this approach phage cocktail was tested against collection of 72 Salmonella strains, including 55 S. Enteritidis isolates representing diverse geographic origins and genomic backgrounds. Spot test patterns were transformed into a continuous scale using the Most Probable Number (MPN) approach to estimate the number of phages required for visible lysis. In parallel, growth inhibition was quantified as the area-under-curve-based inhibition score (ANS). Both metrics were normalized and combined into CLS as a projection onto the regression line describing their correlation (R{superscript 2} {approx} 0.82). More than 65% of S. Enteritidis strains, reached normalized CLS values higher 75%, indicating high susceptibility to BAFASAL(R) in vitro. Phage susceptibility did not correlate with either phenotypic antibiotic resistance or the number of resistance and virulence genes. CLS provides a quantitative method to integrate different experimental methods of determination of bacterial susceptibility to bacteriophages and to rank bacterial strains by phage susceptibility. This approach supports robust host range determination and may facilitate regulatory evaluation and rational design of phage-based interventions in food safety and animal production. IMPORTANCEAssessment of bacteriophage host range is an important step in characterization of bacteriophage strains both in basic and translational research, yet it is still commonly based on qualitative or poorly standardized assays. This lack of harmonization limits reproducibility and complicates comparisons across studies, laboratories, and application contexts. In this work, we propose the Combined Lytic Score (CLS) as a quantitative framework that integrates outcomes from two widely used experimental approaches: serial-dilution spot assays and microtiter-based growth inhibition kinetics. By converting spot-test results into a continuous, concentration-dependent metric and combining them with normalized kinetic inhibition data, CLS enables more consistent interpretation of phage-host interaction outcomes. Application of CLS to a diverse collection of Salmonella enterica strains demonstrates how this approach can support systematic, scalable host range analyses. The CLS framework provides a practical step toward improved standardization of phage susceptibility testing, facilitating clearer data interpretation and comparison in both environmental and applied microbiology research.

Quorum sensing (QS) is a bacterial cell-cell communication system that coordinates group behaviors via self-produced signaling molecules. Serratia liquefaciens, a common mutton spoilage bacterium, precisely regulates its spoilage capacity through the AHL-mediated QS system. The results demonstrated that 20 mol/mL C6-HSL concentration-dependently enhanced AHL activity 1.26-fold, increased biofilm formation by 51.55%, and elevated protease activity and siderophore production by 37% and 26.78%, respectively. In contrast, 80 g/mL chlorogenic acid significantly inhibited AHL activity (by 26.19%), biofilm formation (by 42.54%), protease activity (by 28.92%), and motility (by 38.34%). In stored mutton, chlorogenic acid treatment reduced total plate counts by 6.1% and pH by 5.26%. Transcriptomic analysis revealed that C6-HSL treatment altered metabolic pathways such as flagellar assembly, ABC transporters, two-component systems, and secondary metabolite synthesis, in which spoilage-related genes (slyB, fimA, fliJ, iucD, cheW) were significantly up-regulated. In contrast, chlorogenic acid treatment affected pathways including amino acid metabolism, sulfur metabolism, and carbohydrate metabolism, with spoilage-related genes (fimA, tuf, ibpA, clpS, metQ) significantly down-regulated. These findings demonstrate that AHL activity plays a key role in regulating the spoilage capacity of Serratia liquefaciens, and suggest chlorogenic acid as a potential QS inhibitor with promising application in mutton preservation. ImportanceSerratia liquefaciensis a common spoilage bacterium in refrigerated foods and proliferates extensively in meat, making it one of the primary spoilage organisms. The significance of our research lies in investigating how changes in the activity of the signaling molecule AHL affect both spoilage capacity and mutton quality. Additionally, transcriptomic analysis was employed to elucidate the regulatory mechanisms by which altered AHL activity influences the spoilage potential of S. liquefaciens. The results demonstrated that variations in AHL activity significantly impacted key spoilage-related traits of S. liquefaciens, including biofilm formation, protease activity, and motility, while also contributing to improved meat quality during storage. Furthermore, the study revealed that AHL activity regulates metabolic pathways associated with spoilage as well as the expression of spoilage-related genes. These findings provide a theoretical basis for developing preservation strategies for mutton.

Controlled environment agriculture (CEA), including soilless farming systems, is rapidly expanding as a strategy to improve food security and resource efficiency. However, limited information is available on how different soilless farming system designs influence microbial populations relevant to plant health and food safety. This study investigated the effects of soilless growing systems and growing season on aerobic plate counts (APC) and bacterial community composition in nutrient solution and on bok choy (Brassica rapa subsp. chinensis) leaves. Five soilless systems, deep water culture (DWC), Kratky (KR), nutrient film technique (NFT), ebb and flow (EF), and drip irrigation (DI), were evaluated across fall and spring growing seasons. Soilless system type significantly influenced APC in nutrient solution, with the DI system consistently exhibiting the highest counts across both seasons. Increased nutrient solution pH was negatively associated with APC, whereas temperature did not significantly affect bacterial concentrations. In contrast, APC on bok choy leaves were not significantly influenced by system type, season, pH, or temperature. Bacterial community composition in nutrient solution was strongly shaped by season, soilless system type, sampling day, and temperature, as determined by 16S rRNA V4 amplicon sequencing. Microbial diversity varied primarily by system type, with limited influence of pH or temperature. Core microbiota analysis identified a small subset of taxa that persisted across systems and seasons, with Acidovorax detected in all samples. We found that soilless system design and seasonal conditions strongly influence microbial load and community structure in nutrient solution, providing a foundation for developing system-specific microbial management strategies. ImportanceUnderstanding factors that shape microbial community composition in soilless farming systems is critical for optimizing plant health, system productivity, and food safety. Microbial communities influence nutrient cycling, biofilm formation, and pathogen survival, all of which affect the ecological stability and performance of these systems. By identifying how system design, seasonal variation, and environmental conditions influence shifts in microbial populations, targeted strategies can be developed to promote beneficial microorganisms and mitigate risks associated with pathogens. This knowledge contributes to advancing safe and sustainable soilless farming practices that can meet the growing demand for fresh produce grown in controlled environments.

The genus Legionella comprises opportunistic pathogenic bacteria commonly found in natural and engineered water systems, where they interact with environmental microbes and protozoa, primarily in biofilms. Legionella pneumophila is the main causative agent of Legionnaires disease and is transmitted through inhalation of contaminated aerosols. Iron availability is a critical factor for L. pneumophila growth, persistence, and virulence, yet iron is often limited in aquatic environments. To overcome iron scarcity, many bacteria produce siderophores, secondary metabolites that scavenge ferric iron. Because siderophores are chemically diverse and species specific, they play a key role in inter-species competition and can withhold iron from competitors. Here, we investigated the effects of iron depletion and siderophore-mediated competition on L. pneumophila using commercial pyoverdines and extracellular metabolites from environmental Pseudomonas strains. Growth assays showed that L. pneumophila can grow under iron-limited conditions but with lag phases extended by more than 20 hours. Pyoverdines inhibited growth in a concentration-dependent manner, primarily increasing the time to mid-log phase (t_mid). Supernatants and crude pyoverdine extracts from siderophore-producing Pseudomonas strains caused the strongest inhibition, including lag-phase extensions of up to 55 hours or complete growth arrest. These results demonstrate that siderophore-producing bacteria can suppress L. pneumophila by limiting iron availability.

Sourdough starters contain simple microbial communities typically consisting of a few bacterial species and one or two yeast species. The yeast Maudiozyma humilis and the lactic acid bacterium Fructilactobacillus sanfranciscensis often co-occur in sourdough starters, and have been presumed to exist in a trophic relationship supported by glucose cross-feeding. However, previous research has highlighted a lack of evidence showing that yeast strains consume the glucose that F. sanfranciscensis produces. We have investigated the interaction between sourdough isolates of M. humilis and F. sanfranciscensis in a synthetic wheat sourdough medium, allowing us to control substrate composition and use flow cytometry to enumerate living and dead cells. M. humilis fitness was found to be lower in co-culture with F. sanfranciscensis than when grown alone. Analysis of spent medium composition highlighted the reliance of M. humilis on glucose rather than maltose for growth. Comparisons of predicted and measured co-culture metabolite content also revealed that F. sanfranciscensis consumed less maltose in co-culture than when grown alone. For the first time, we examined potential amino acid cross-feeding between M. humilis and F. sanfranciscensis, and found that within the pairing, F. sanfranciscensis was the main producer of amino acids. Our findings suggest that the M. humilis-F. sanfranciscensis interaction is likely to be neutral, or even competitive, with the strain identity of F. sanfranciscensis playing a defining role in the observed dominance of the bacteria and spent medium metabolite composition. ImportanceThe association of the yeast Maudiozyma humilis and the bacterium Fructilactobacillus sanfranciscensis in sourdough starters is well-documented, and together this pairing makes key functional and organoleptic contributions to the final bread product. Their relationship has historically been thought to be stabilised by cross-feeding of glucose to M. humilis. However, this theory has been drawn into question by recent research which found no evidence that M. humilis consumes the glucose produced by F. sanfranciscensis. Our understanding of cooperation, coexistence, and competition in microbial consortia affects approaches to ecosystem management in a broad variety of applied fields. The significance of our research is in demonstrating that this pairing does not interact mutualistically within a specified setting, providing support for neutral or competitive interactions as drivers of ecological stability. Research areas:

Consumption of sprouted seeds, such as alfalfa sprouts, has increased in recent years due to their perceived health benefits. However, these food products have repeatedly been associated with outbreaks of foodborne pathogens, including Salmonella enterica serovars. An S. enterica serovar Choleraesuis strain previously isolated from melon fruit internal tissues was selected as a model to explore plant-pathogen interactions on alfalfa sprouts. Using this strain, we generated a barcoded transposon mutant library comprising approximately 33,000 unique insertions. This library and a collection of individual insertion mutants derived from it were used to identify genetic mechanisms contributing to the fitness of this S. Choleraesuis strain on alfalfa sprouts. The library was screened on sprouts during cold storage at 8{degrees}C. Negative selection for mutants with insertions in eda, fabF, lpp1_2, pnp, stpA, SCHChr_03621 and two intergenic regions were identified. Competition experiments between individual insertion mutants and the wild type confirmed the phenotype of three genes: eda, coding for a keto-hydroxyglutarate-aldolase/keto-deoxy-phosphogluconate aldolase involved in the Entner-Doudoroff pathway, mnmG, encoding the glucose-inhibited division protein, and fabF, involved in fatty acid biogenesis. This study offers a genome-wide perspective on the genes enabling a plant-associated Salmonella strain to persist on alfalfa sprouts. We highlight factors that are critical not only for persistence throughout the entire cold-storage period under conditions that closely simulate real shelf-life conditions in this high-risk food matrix.

Rhamnolipids (RLs) are versatile biosurfactants produced by Pseudomonas aeruginosa with significant industrial potential. However, high production costs remain a barrier to large-scale use, necessitating genetic strategies to improve yields. While many genes are reported to influence RL production, studies often rely on qualitative phenotypic assays of questionable reliability. We systematically evaluated 29 P. aeruginosa PA14 mutants using traditional assays (Siegmund-Wagner blue plates, swarming motility) and validated the findings using Liquid Chromatography-Mass Spectrometry (LC/MS). We found that traditional phenotypic assays have a high misprediction rate ([~]35-38%), primarily due to confounding factors like variable flagellar function, colony spreading, and growth rates. Specifically, LC/MS quantification revealed that rpoN and pvdQ knockouts significantly increased total rhamnolipid titers, whereas crc, dksA, and dspI knockouts decreased production. Notably, the increased titers in rpoN and pvdQ mutants were linked to enhanced biomass accumulation rather than higher per-cell biosynthetic rates. These findings highlight the critical necessity of using quantitative analytical methods for accurate strain screening and provide a clarified set of genetic targets for metabolic engineering aimed at optimizing rhamnolipid production. ImportanceThis study addresses a critical methodological flaw in biosurfactant research: the over-reliance on qualitative phenotypic assays that too often lead to inaccurate conclusions. By systematically comparing traditional screening methods with LC/MS quantification across a collection of Pseudomonas aeruginosa mutants, we demonstrate that common assays like swarming motility and blue plates fail to accurately predict rhamnolipid production in over one-third of cases. These inaccuracies lead to the misidentification of genetic targets and may waste resources in metabolic engineering efforts. Our work provides a reliable framework for strain screening, identifies specific genes that influence rhamnolipid yields, and clarifies the biological factors--such as flagellar motility and growth dynamics--that bias traditional results. These findings are essential to optimize biosurfactant production and ensure data reproducibility.

Candida albicans is an opportunistic human fungal pathogen found in the oral cavity. Human saliva contains a 24-amino acid peptide called histatin 5 (Hst5) that has activity against C. albicans, but degradation of Hst5 by secreted aspartyl proteases (Saps) produced by C. albicans and by salivary proteases can reduce its antifungal efficacy. Building on our previous work that identified K13 and K17 as important residues for stability and activity of Hst5, we systematically investigated amino acid modification at these sites. Modifications explored the influence of hydrophobicity, charge, polarity, size, and aromaticity on Hst5s interaction with Saps and saliva. The K13R variant retained proteolytic stability and antifungal activity after incubation with Sap1, Sap2, Sap3, and Sap9, while other K13 variants generally had reduced stability and activity, emphasizing the importance of a positive charge at this position. At K17, substitutions generally enhanced proteolytic stability and improved antifungal activity after incubation with Saps. We introduced the normalized intact peptide (NIP) parameter as a tool for identifying Hst5 variants with improved stability in the presence of multiple Saps, and NIP revealed K17W as the most proteolytically stable variant overall. Additionally, we observed modest differences in peptide stability in saliva, and the K17W variant was the only variant that retained more activity than Hst5 following incubation with saliva. We further assessed the K17W variants ability to prevent biofilm formation and found it to be more effective than the parent peptide Hst5. Our findings highlight the interactions between the Hst5 K13 and K17 residues with Saps and saliva and provide a strong foundation for future Hst5 engineering efforts to improve proteolytic stability and antifungal efficacy in diverse proteolytic environments.

BackgroundLeptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., which persist in soil and water environments for extended periods of time. The mechanisms enabling this environmental survival remain elusive. Free-living amoebae (FLA) are widespread protozoa that act as reservoirs or "Trojan horses" for numerous bacterial pathogens, protecting them from stress and contributing to their persistence. Whether pathogenic Leptospira exploit similar interactions with FLA has not been resolved. Methodology/Principal FindingsUsing live confocal microscopy, flow cytometry, and gentamicin protection assays, we investigated the interactions between pathogenic (Leptospira interrogans) and saprophytic (Leptospira biflexa) leptospires with three FLA species: Acanthamoeba castellanii, Dictyostelium discoideum, and Hartmannella vermiformis. While rapid internalization was observed, entry was only partially dependent on actin-driven processes and was enhanced by the presence of live bacteria. Following internalization, bacteria persisted for at least 48h as indicated by colony-forming assays. However, no evidence of intracellular replication was detected. The number of fluorescently labeled leptospires progressively declined over time, providing further evidence of leptospires survival without multiplication. Finally, analysis of environmental soils in New Caledonia showed co-occurrence of FLA and Leptospira. Soil-derived FLA also internalized pathogenic Leptospira in vitro, showing that these interactions extend to natural isolates. Conclusions/SignificanceOur results demonstrate that free-living amoebae internalize both pathogenic and saprophytic leptospires and allow their transient persistence without replication. By providing protection and prolonging viability in soil environments, FLA may contribute to the ecological maintenance of Leptospira. These findings pinpoint FLA as potential environmental reservoirs that could play a role in shaping leptospires survival strategies relevant for transmission and host infection. Author SummaryFor bacteria living in soils and freshwater environments, survival depends on their ability to adapt to complex ecological landscapes populated by numerous predators and competitors. In such habitats, interactions with other microorganisms are unavoidable and may shape long-term survival strategies. Pathogenic Leptospira, the bacteria responsible for leptospirosis, can persist for long periods outside their hosts, yet the ecological mechanisms supporting this environmental survival remain poorly understood. In soil and freshwater ecosystems, microscopic predators known as free-living amoebae commonly feed on bacteria. However, several bacterial pathogens can survive inside these amoebae and use them as temporary shelters. Because ancestral Leptospira were soil-dwelling saprophytes, interactions with amoebae likely represent an ancient ecological relationship in which successful survival strategies may have evolved and remain conserved in present-day pathogenic species. With this perspective in mind, we used microscopy approaches and bacterial viability assays to investigate whether Leptospira interacts with amoebae. We found that several amoeba species rapidly engulf both pathogenic and non-pathogenic Leptospira. Once internalized, the bacteria remained viable for up to two days but did not multiply. We also detected both amoebae and Leptospira in the same soil samples and showed that environmental amoebae could internalize the bacteria. These findings suggest that amoebae may act as temporary shelters for Leptospira, helping them persist in soils and water and potentially contributing to the environmental stage of leptospirosis transmission.

Volatile aromatic compounds are important industrial feedstocks but also major environmental pollutants, highlighting the need for bioprocesses for their removal and valorization. Although gas-phase bioprocesses offer practical advantages for handling poorly water-soluble and highly volatile substrates, how gas-phase environments alter microbial metabolism remains poorly understood. Here, we investigated the effect of gas-phase conditions on toluene metabolism in the highly adhesive aromatic hydrocarbon-degrading bacterium Acinetobacter sp. Tol 5. A mutant lacking todC1, which encodes an essential component of the toluene dioxygenase, failed to grow on toluene in liquid culture but retained the ability to grow on solid media under a toluene atmosphere. Consistent with this phenotype, the mutant showed no detectable toluene degradation in the liquid phase, whereas it degraded toluene under gas-phase conditions after a prolonged lag phase. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the accumulation of o-cresol and p-cresol specifically in the mutant under toluene vapor, indicating that toluene metabolism had shifted to an alternative route involving cresol intermediates. In addition, transcriptome analysis identified strong induction of the mph operon encoding phenol monooxygenase (PMO), suggesting that PMO is a likely candidate enzyme mediating TDO-independent toluene oxidation under gas-phase conditions. Together, these results demonstrate that the gas-phase environment can activate an alternative catabolic route in Tol 5 that is not active during conventional liquid cultivation. Our findings highlight the importance of direct metabolic analysis under gas-phase conditions for understanding and designing bioprocesses using highly volatile substrates.

Crude glycerol is an underutilized waste stream. Viable routes for converting it to 1,3-propanediol (1,3-PDO) can conserve important resources and add value to its supply chain. Biological methods are appealing because they can circumvent expensive preprocessing steps while operating under mild conditions. Here, we show that the propanediol utilization pathway of Salmonella enterica serovar Typhimurium LT2 can be used to convert glycerol, including unprocessed crude glycerol, into 1,3-PDO under aerobic conditions in minimal media. Additionally, we demonstrate that high concentrations of expensive cofactors are not necessary to achieve optimal production titers. This study lays the groundwork for continual iteration on this pathway for bioprocess development. Key pointsO_LIS. enterica can produce 1,3-propanediol from crude glycerol alone C_LIO_LIGlycerol-to-1,3-propanediol conversion is dependent on expression of the propanediol utilization (Pdu) pathway C_LIO_LISub-saturating concentrations of exogenous vitamin B12 can boost cell growth and 1,3-propanediol yield C_LI

Phosphorus is a vital nutrient required for the functioning of living organisms. In aquatic environments, dissolved inorganic phosphate is considered its most bioavailable form. However, phosphate can be scarce, which has the potential to limit microbial metabolism and ecosystem functioning. To overcome phosphate scarcity, microbes produce alkaline phosphatase (AP) to access dissolved organic phosphorus (DOP). Here, we conducted a year-long study of alkaline phosphatase activity (APA) at the Ellen Browning Scripps Memorial Pier, a nutrient-rich coastal site. APA was observed throughout the year despite phosphate-replete conditions, suggesting that the role of APs in microbial nutrition is not completely understood. We tested the hypothesis that APA may promote acquisition of organic carbon liberated from DOP hydrolysis by growing the heterotrophic marine bacterium Ruegeria pomeroyi on three DOP compounds as sole carbon sources and assessing APA. Controlling for carbon concentration, all DOP sources supported growth, but at lower levels than glucose, with the highest growth observed on glucose-6-phosphate (G6P), followed by adenosine monophosphate (AMP) and adenosine triphosphate (ATP). Moreover, cell-specific APA was significantly enhanced in carbon-deplete conditions and during growth on G6P, relative to cultures grown on replete glucose or nucleotides. These findings suggest alkaline phosphatases (APs) are part of a generic carbon stress response and likely play a role in acquiring certain forms of organic carbon by R. pomeroyi, with implications for other taxa. Overall, this study helps advance the current state of knowledge regarding microbial phosphorus cycling and carbon utilization in aquatic environments.

Validated inactivation procedures are required for the safe handling and downstream analysis of highly pathogenic organisms, particularly those categorized as biological select agents and toxins (BSATs). TRIzol-based extraction methods are widely used for nucleic acid and protein isolation, yet their reliability for bacterial inactivation has not been comprehensively evaluated. In this study, we assessed TRIzol-based extraction methods for sample quality and inactivation reliability across a series of mock failure scenarios using five attenuated bacterial isolates: Francisella tularensis holarctica LVS, Bacillus anthracis Sterne, Yersinia enterocolitica, Mycobacterium marinum, and Burkholderia cepacia. Dilution of TRIzol to induce incomplete cell lysis for the initial extraction step, including 0% TRIzol, consistently inactivated all surrogate organisms, suggesting that downstream precipitation and sample washing reagents, including isopropanol and 70% ethanol, were sufficient to inactivate organisms in the absence of TRIzol. Several protocol failure scenarios were then evaluated to simulate human error by omitting extraction, precipitation, and washing steps individually or in combination for the most resistant organism, B. anthracis Sterne strain. Failure-scenario testing demonstrated that reliable inactivation of B. anthracis required strict adherence to the complete protocol due to the spore-forming ability of B. anthracis. Collectively, this work provides a reference with experimental evidence supporting the use of TRIzol-based extraction as a bacterial inactivation strategy for a wide range of bacterial pathogens.

Soilborne fungal pathogens pose persistent challenges to sustainable agriculture, driving demand for biological alternatives to synthetic fungicides. While actinomycetes, particularly Streptomyces have yielded numerous antifungal compounds, less-explored genera, such as Saccharomonospora represent untapped sources of novel bioactive metabolites for plant protection. Our previous work identified Saccharomonospora as strongly associated with plant disease suppression in organically-amended soils and harbored numerous uncharacterized biosynthetic gene clusters (BGCs). Here, we investigated the biosynthetic capacity and antifungal potential of Saccharomonospora using comparative genomics, untargeted metabolomics, and in vitro bioassays. Cell-free supernatants from six strains (five soil-derived type strains and one novel isolate) were evaluated against three Fusarium phytopathogens. All strains inhibited at least one pathogen, with S. xinjiangensis and S. viridis R81 exhibiting the highest and broad-spectrum activity. Metabolomic profiling of the two most bioactive strains and one moderately active strain (S. cyanea) revealed that [~]40% of detected metabolites were shared across the three strains although their relative abundances varied. S. xinjiangensis and S. viridis R81 displayed higher abundances of shared metabolite classes than S. cyanea, including alkaloids, polyketides, and peptide derivatives. Comparative genomics across the genus revealed that most BGCs, particularly those encoding non-ribosomal peptides synthetases and polyketides synthases, were strain-specific and had low sequence similarity to characterized BGCs. In contrast, BGCs encoding, indole, ectoine, arylpolyene, and terpene were ubiquitous across the genus. Collectively, these findings demonstrate that Saccharomonospora produce antifungal metabolites and harbor diverse, uncharacterized BGCs, positioning this "less-explored" actinomycete genus as a promising source of bioactive compounds for managing soilborne fungal pathogens. ImportanceActinomycetes have historically been rich sources of antifungal metabolites for agriculture and pharmaceuticals, but discovery efforts have focused largely on Streptomyces, leaving other genera underexplored. This study demonstrates that Saccharomonospora, a rare actinomycete genus, produces antifungal metabolites active against major Fusarium pathogens and harbors largely uncharacterized biosynthetic gene clusters, indicating a reservoir of novel chemistry. These findings establish Saccharomonospora as a promising yet underutilized resource for discovering new antifungal agents, expanding the toolkit for sustainable plant disease management beyond traditional actinomycete sources.