Applied and Environmental Microbiology最新文献

Bacillus cereus is a foodborne pathogen of growing concern due to its persistence and antimicrobial resistance. To identify host determinants influencing susceptibility to phage BCP8-2, a mini-Tn10 transposon mutant library of B. cereus ATCC 14579 was constructed and screened for altered phage susceptibility. A mutant (BC2012) showing partial resistance carried an insertion in BC_RS27090 (previously BC_5432), encoding a putative GtrB-like bactoprenol glycosyltransferase. Complementation restored phage sensitivity, confirming its functional involvement. The mutant showed a slight reduction in efficiency of plating, whereas, adsorption assay revealed significantly reduced phage binding and infectivity, particularly under cation-rich conditions. Furthermore, adsorption kinetics and one-step growth curve in mutant supported that gtrB mutation affects initial adsorption. Microscopy showed altered surface morphology with thinner, smoother cell walls. These data indicate that gtrB affects surface properties essential for cation-dependent phage adsorption and infection and provide a foundation for future studies on the role of surface glycosylation and ionic interactions in Gram-positive phage biology.IMPORTANCEPathogenic Bacillus cereus is resilient across diverse environments and produces diverse toxins linked to foodborne outbreaks. Bacteriophages provide an effective strategy to control B. cereus; however, molecular targets of phage-host interaction are poorly understood, limiting the effective phage-based control. The current study addresses this gap by identifying a putative bactoprenol glycosyltransferase (GtrB), a viral factor in phage BCP8-2 susceptibility. Data obtained highlight a partial reduction of phage infection in the gtrB mutant, demonstrating that GtrB-like glycosyltransferase significantly contributes to BCP8-2 phage infection and adsorption. Our findings provide significant insights into advancing phage therapy, antibiotic resistance, and B. cereus biocontrol in food and clinical settings.

Chili pepper (Capsicum annuum L.) is a vital vegetable crop susceptible to yield loss from Alternaria alternata (Fr.), Keissl., which causes leaf spot/blight and fruit rot. Harnessing beneficial rhizobacteria offers an eco-friendly strategy to enhance plant resilience to biotic stressors. In vitro screening identified Pseudomonas chlororaphis strains viz., P. chlororaphis subsp. aurantiaca (ARS-38) and P. chlororaphis subsp. chlororaphis (RP-4) as potent antifungal strains, inhibiting A. alternata growth by 62% and 64%, respectively. In a zinc (Zn)-amended medium, their antifungal activity increased further, reducing fungal growth by 70% with individual strains and 93% when combined. Microscopy revealed fungal growth inhibition linked to conidia and conidiophore disintegration. In planta under open-field pot conditions, A. alternata infection resulted in 100% disease incidence (DI) and altered histochemical, photosynthetic, and enzyme responses. Treatment with ARS-38 + RP-4 in Zn-amended soil enhanced plant resistance by increasing lignin and phenolic accumulation and reducing enzyme activities, resulting in a 96% reduction in DI. Zinc supplementation improved plant health and enhanced disease suppression when combined with the bacterial strains. Combining rhizobacteria with Zn (2 mg/kg) emerges as a promising strategy to mitigate Alternaria leaf spot disease and enhance biostress tolerance in chili plants.

Importance: Biological control offers a sustainable approach for reducing fungal diseases while limiting reliance on chemical fungicides. This study shows that two Pseudomonas chlororaphis strains, ARS-38 and RP-4, combined with low-level zinc supplementation, strongly suppress Alternaria alternata, an important chili pepper pathogen. The combined treatment reduced fungal growth in vitro and substantially decreased disease development in plants while improving structural, physiological, and biochemical responses associated with plant health. The findings demonstrate the potential value of integrating beneficial rhizobacteria with micronutrient supplementation for sustainable disease management and provide a basis for further development of microbial-micronutrient strategies for crop protection.

Azospirillum brasilense is a non-photosynthetic bacterium that possesses the ability to produce carotenoids, in which squalene is a critical intermediate of the carotenoid biosynthetic pathway. Farnesyl pyrophosphate (FPP) serves as a precursor for the production of sesquiterpenoids; hence, its availability is a major bottleneck in the microbial biosynthesis of several major plant sesquiterpenes. We improved the utility of A. brasilense Cd for the heterologous production of sesquiterpenes by enhancing intracellular availability of FPP and disrupted the chromosomal hpnC gene to block the squalene biosynthetic pathway to decrease a competing sink for FPP, along with the introduction of the heterologous Escherichia coli ispA gene, together with additional copies of the native A. brasilense dxps and crtE genes. These genetic manipulations, along with the expression of codon-harmonized santalene synthase from Santalum album, enabled an increase in the production of α-santalene by 50-fold, α-exo-bergamotene by 20-fold, and β-santalene by 10-fold in A. brasilense Cd. Additionally, we developed a fructose-inducible santalene synthase expression system, which may be more efficient and economical than the isopropyl β-D-1-thiogalactopyranoside-inducible system. Finally, by cloning and expressing genes encoding cytochrome P450 monooxygenase and its cognate NADPH-cytochrome P450 reductase, we have shown the potential of A. brasilense to produce santalols, the most desired ingredient of sandalwood oil.IMPORTANCESandalwood oil is among the most valuable natural fragrances and is also known for its diverse biological activities. The biosynthesis of santalene, a major constituent of sandalwood oil, originates from the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) via the MEP pathway. This pathway is naturally present in Azospirillum brasilense, a plant growth-promoting rhizobacterium, making it a suitable host for the heterologous production of sesquiterpenes. This study has shown the possibility of using A. brasilense as a promising host-vector system for enhancing the production of santalene isomers by several fold. It has also shown the superiority of a fructose-inducible system over the isopropyl β-D-1-thiogalactopyranoside-inducible system for the industrial production of high-value secondary metabolites in this bacterium.

The aspA gene of Yersinia pestis exhibits a pronounced polymorphism at codon 363, with the dominant TTG allele encoding an inactive aspartate ammonia-lyase (AspA) while the GTG allele encoding an active enzyme. We previously demonstrated that this polymorphism drives an environment-dependent fitness trade-off, yet the underlying mechanism remains unclear. To address this, we first characterized the phenotypic differences between the two strains and revealed a clear growth-stress survival trade-off: the GTG (AspA-active) strain grew faster and produced more ATP, whereas the TTG (AspA-inactive) strain, despite slower growth, exhibited superior survival under nutrient limitation with oxidative, anaerobic, or hyperosmotic stress. Exogenous aspartate reversed the GTG strain's disadvantages under anaerobic or cold stress. To dissect the metabolic basis, we performed ¹³C-labeled metabolic flux analysis on these two strains. The GTG strain rapidly converted aspartate to fumarate via AspA, fueling the TCA cycle and boosting ATP production. In contrast, the TTG strain displayed stalled aspartate utilization and accumulated amino acids and lipids. Supplementation with TCA cycle intermediates (fumarate, succinate, or malate) partially rescued TTG growth, confirming that reduced TCA flux limits its proliferation. Collectively, AspA activity orchestrates a growth-stress survival trade-off through metabolic reprogramming, underlying the antagonistic pleiotropy of Y. pestis in aspartate-available or stressful environments. The "metabolic congestion" strategy of the TTG strain enhances survival under multiple stresses relevant to the flea gut, and it is plausible that this metabolic reprogramming may contribute to transmission and provide a selective advantage under flea-borne conditions.IMPORTANCEThe plague bacterium Yersinia pestis encounters oxidative, anaerobic, and hyperosmotic stresses-conditions relevant to its flea gut environment during transmission. This study reveals that a single, naturally occurring polymorphism in the aspA gene shifts the bacterium's metabolism from a growth-focused mode to a "metabolic congestion" state that enhances survival under these stresses in vitro. Our work provides a mechanistic framework for understanding how metabolic reprogramming could influence adaptation to vector-like conditions, offering a plausible basis for the environment-dependent fitness trade-offs that may shape transmission dynamics.

Establishing biocontrol agents (BCAs) within grapevine tissues could provide a means of protecting internal plant compartments, but little is known about their spatial and temporal fate after vascular delivery or whether the graft union constrains their distribution. We compared the internal recovery of two biologically contrasting BCAs, the bacterium Pseudomonas chlororaphis M71 and the filamentous fungus Trichoderma atroviride SC1, following controlled stem injection into rooted and grafted grapevine plants. A xylem-mobile dye was used as a reference for immediate passive fluid transport, while viable microbial recovery was assessed over time and complemented by quantitative isolation and microscopy. At 48 h, the dye generally showed broader longitudinal distribution than either of the microorganisms. The two microorganisms displayed markedly different patterns. M71 persisted for up to 56 days, but remained predominantly associated with tissues close to the injection site. In contrast, SC1 was recovered from a broader range of tissues, including roots, and remained detectable for up to 56 days. In grafted plants, both microorganisms were recovered on both sides of the graft union, indicating that the graft interface was not an absolute barrier, although recovery depended on microbial identity and inoculation position. Green fluorescent protein-labeled M71 cells and SC1 hyphae were observed within xylem vessels, confirming internal localization and, for SC1, vegetative development after injection. These results show that, and how, both microbial biology and grafted-plant architecture influence the internal fate of injected BCAs in grapevine.IMPORTANCEBeneficial microorganisms established inside grapevine tissues could potentially protect from infections regarding internal tissues, but little is known about how such microorganisms distribute and persist inside grapevine after injection into the plant tissues. This study shows that two biocontrol agents (BCAs) with contrasting biological characteristics follow markedly different spatial and temporal patterns. Pseudomonas chlororaphis M71 persisted mainly near the site of introduction, whereas Trichoderma atroviride SC1 was recovered from a broader range of tissues and developed hyphae within xylem vessels. Importantly, the graft union was not an absolute barrier to microbial recovery. These findings provide a framework for selecting where and when BCAs should be introduced during grapevine propagation and highlight that vascular access, persistence, and broad within-plant establishment are distinct properties that should be considered when developing endophytic biocontrol strategies.

Atmospheric and room-temperature plasma (ARTP) is a potent tool for microbial strain engineering; however, the mechanistic basis of its genome-wide mutagenic effects remains poorly understood. Utilizing Saccharomyces cerevisiae as a model, we performed a comprehensive characterization of the mutational landscape and DNA repair pathways elicited by ARTP. Under high-lethality conditions (~99% mortality), ARTP treatment induced a substantial mutational burden, with SNVs and InDels rates increasing 25.2- and 36.7-fold relative to untreated controls, respectively. These mutations exhibited a pronounced bias toward C-T/G-A and A-T/T-A substitutions and InDels within AT-rich regions. Remarkably, the frequency of loss of heterozygosity (LOH) surged 57.4-fold, suggesting a potent capacity for inducing DNA breaks. Through sectored colony assays, we demonstrate that ARTP primarily inflicts pre-replicative double-strand breaks (DSBs), with reciprocal crossovers serving as the predominant driver of terminal LOH. The distribution and pattern of LOH were also altered in ARTP-treated cells. Mechanistically, our study establishes that the translesion synthesis polymerases Rev1 and Pol ζ are indispensable for converting ARTP-induced lesions-likely pyrimidine dimers-into fixed point mutations. In contrast, Rad30 facilitates high-fidelity bypass across these dimers. The base excision repair pathway appears largely dispensable for the processing of these lesions. In contrast, the nucleotide excision repair pathway plays a predominant role in mitigating ARTP-induced mutations. Finally, the strategic application of iterative ARTP mutagenesis facilitated the rapid evolution of acetate-tolerant strains. Our findings provide fundamental insights into the molecular signatures of ARTP mutagenesis, offering a framework for its optimized application in directed evolution.

Importance: While ARTP is a cornerstone of non-thermal mutagenesis in industrial biotechnology, its genetic impact has long been treated as a "black box." This study provides a high-resolution molecular dissection of ARTP-induced genomic alterations using a diploid yeast model. We demonstrate that ARTP is not merely a source of point mutations but a potent catalyst for pre-replicative double-strand breaks and extensive mitotic recombination. By elucidating the pivotal roles of specific DNA repair pathways in mutation fixation, we transition ARTP from a purely empirical screening tool to a mechanistically grounded platform for directed evolution. Furthermore, we demonstrate the practical utility of this platform by evolving acetate-tolerant strains and identifying the underlying genetic drivers of their resilience.