Applied Microbiology and Biotechnology最新文献

Campylobacter jejuni, a major cause of foodborne zoonotic infections worldwide, shows a paradoxical ability to survive despite its susceptibility to environmental and food-processing stressors. This resilience is likely due to the bacterium entering a viable but non-culturable state, often within biofilms, or even initiating biofilm formation as a survival strategy. This study presents an innovative application of NanoLuc bioluminescence to accurately monitor the development of C. jejuni biofilms on various substrates, such as polystyrene plates, mucin-coated surfaces, and chicken juice matrices. Introduction of NanoLuc luciferase in a pathogenic C. jejuni strain enables rapid non-invasive holistic observation, capturing a spectrum of cell states that may comprise live, damaged, and viable but non-culturable populations. Our comparative analysis with established biofilm quantification methods highlights the specificity, sensitivity, and simplicity of the NanoLuc assay. The assay is efficient and offers precise cell quantification and thus represents an important complementary or alternative method to conventional biofilm monitoring methods. The findings of this study highlight the need for a versatile approach and suggest combining the NanoLuc assay with other methods to gain comprehensive insight into biofilm dynamics.

• Innovative NanoLuc bioluminescence assay for sophisticated biofilm quantification.

• Holistic monitoring of C. jejuni biofilm by capturing live, damaged and VBNC cells.

• Potential for improving understanding of biofilm development and structure.

Azospirillum argentinense Az19 is an osmotolerant plant growth-promoting bacterium that protects maize plants from drought. In this work, we explored the role of trehalose in the superior performance of Az19 under stress. The trehalase-coding gene treF was constitutively expressed in Az19 through a miniTn7 system. The resulting recombinant strain, Az19F, did not accumulate trehalose, was affected in its capacity to cope with salt-, osmotic-, and UV-stress, and showed higher reactive oxygen species levels. Physiological alterations were also observed under normal conditions, such as increased growth in biofilms, higher motility, and decreased auxin secretion. Even so, the capacity of Az19F to colonize maize roots was not affected, either under normal or drought conditions. When inoculated in maize, both Az19 and Az19F strains promoted plant growth similarly under normal irrigation. However, unlike Az19, the trehalose-deficient strain Az19F could not improve the height, aerial fresh weight, or relative water content of maize plants under drought. Notably, Az19F triggered an exacerbated oxidative response in the plants, resulting in higher levels of antioxidant and phenolic compounds. We conclude that the role of trehalose metabolism in A. argentinense Az19 transcends stress tolerance, being also important for normal bacterial physiology and its plant growth-promoting activity under drought.

• Trehalose is required by Az19 for full tolerance to salt-, osmotic-, and UV-stress.

• A restriction in trehalose accumulation alters Az19 normal cell physiology.

• Trehalose contributes to Az19-induced maize growth promotion under drought.

Malaria, a parasitic disease caused by Plasmodium spp. and transmitted by Anopheles mosquitoes, remains a major global health issue, with an estimated 249 million cases and 608,000 deaths in 2022. Rapid and accurate diagnosis and treatment are crucial for malaria control and elimination. However, limited access to sensitive molecular tests means that microscopic examination and rapid diagnostic tests (RDT) are the most used methods in endemic areas, despite their lower diagnostic accuracy. Therefore, there is a need for developing sensitive, simple, accurate, and rapid diagnostic tools suitable for field conditions. Herein, we aimed to explore the potential of the enzymatic recombinase amplification assay (ERA® Technology) as a remote laboratory test by evaluating and validating the GENEYE® ERA Plasmodium detection kit in Brazilian endemic areas. A cross-sectional cohort study was conducted between June and August of 2023 in the Brazilian Amazon. The study enrolled 323 participants residing in three malaria-affected regions: Cruzeiro do Sul and Mâncio Lima (Acre State) and Guajará (Amazonas State). The participants were tested for malaria by microscopy, rapid diagnostic tests (RDT), nested PCR (nPCR), quantitative real-time PCR (qPCR), and ERA. The sensitivity, specificity, and predictive values were assessed using nPCR as a gold standard. Plasmodium prevalence was 21.7%, 18.8%, 19.2%, 21.7%, and 21.7% by nPCR, microscopy, RDT, qPCR, and ERA respectively. Using nPCR as the standard, qPCR, and ERA showed a sensitivity of 100%. In comparison, microscopy and RDT showed a sensitivity of 87.1% and 88.6%, a negative predictive value (NPV) of 96.56 and 96.93, and kappa values of 0.91 and 0.92, respectively. For Plasmodium falciparum, the sensitivity of qPCR and ERA was 100% while the sensitivity of microscopy and RDT was 96.9% and 93.7%, and the NPV was 99.66 and 99.32, respectively. For Plasmodium vivax, only ERA showed the same sensitivity of nPCR. The sensitivity, NPV, and kappa values were 78.85%, 97.27, and 0.87 for qPCR and microscopy, and 84.21%, 97.94, and 0.9 for RDT. The data presented here show that the GENEYE® ERA Plasmodium detection kit offers a promising alternative to traditional malaria diagnostic methods. Its high sensitivity, specificity, fast processing time, and operational simplicity position it as a valuable point-of-care diagnostic tool, particularly in resource-limited and remote malaria-endemic areas.

• GENEYE® ERA kit detects Plasmodium in under 25 min, no DNA purification needed.

• The kit matches or exceeds the compared methods in sensitivity and specificity.

• The kit is suitable for accurate testing in low-infrastructure, point-of-care settings.

Chiral diaryl alcohols, such as (4-chlorophenyl)(pyridin-2-yl)methanol, are important intermediates for pharmaceutical synthesis. However, using alcohol dehydrogenases (ADHs) in the asymmetric reduction of diaryl ketones to produce the corresponding alcohols is challenging due to steric hindrance in the substrate binding pockets of the enzymes. In this study, the steric hindrance of the ADH from Geotrichum candidum NBRC 4597 (G. candidum acetophenone reductase, GcAPRD) was eliminated by simultaneous site-directed mutagenesis of Phe56 (in the large pocket) and Trp288 (in the small pocket). As a result, two double mutants, Phe56Ile/Trp288Ala, and Phe56Ala/Trp288Ala, exhibited much higher specific activities towards 2-(4′-chlorobenzoyl)pyridine (4.5 μmol/min/mg and 3.4 μmol/min/mg, respectively) than the wild type (< 0.2 μmol/min/mg). In whole-cell-catalyzed asymmetric reductions of diaryl ketones, Phe56Ile/Trp288Ala significantly increased the isolated yields, which were over 90% for the reactions of most of the tested substrates. Regarding enantioselectivity, Phe56Ile/Trp288Ala and Phe56Ala/Trp288Ala, and Trp288Ala generally exhibited similar selectivity to produce (R)-alcohols with up to 97% ee.

• Phe56 in Geotrichum reductase (GcAPRD) was mutated to eliminate steric hindrance.

• Mutation at Phe56 increased enzymatic activity and expanded substrate specificity.

• Phe56Ile/Trp288Ala showed high activity and (R)-selectivity towards diaryl ketones.

In the present investigation, 13% ± 0.84 of the extracted and purified phycocyanin from Phormidium versicolor was obtained, with a purity of 0.69 following dialysis. FT-IR analysis of purified phycocyanin revealed stretching vibration peaks in the profiles of the functional groups of N–H, O–H, C = O, N–H, C = O, and C = NH⁺. The phycocyanin had a significant DPPH radical scavenging ability (IC₅₀ = 0.6 ± 0.02 mg mL⁻¹) confirmed with FRAP assay, and it exhibited microbiological activity between 1.25 and 2.5 mg mL⁻¹ against Candida albicans, Klebsiella pneumoniae, and Enterococcus faecalis. Phycocyanin showed no cytotoxic and improved the viability of HEK-293. It was added to skin cream at a rate of 6 mg g⁻¹ because of its significant yield extraction and biological activity. At 10 mg mL⁻¹, a bactericidal activity has been noted, inhibiting the growth of bacteria responsible for inflammatory skin conditions. For 60 days, the emulsion’s stability was monitored at room temperature, 25 °C, and 45 °C. The appearance of the batch kept at 45 °C was changed to beige after 7 days, while the others were kept for 15 days. Skin creams enhanced with phycocyanin were found to be stable over the course of storage at both room temperature and 25 °C, based on centrifugation stability analysis. But starting on the fifteenth day, the items kept at 45 °C were unstable. Thus, the current study’s findings are in favor of using phycocyanin as an antioxidant in cosmetic products. However, further investigation is required before using it in clinical trials.

• Phycocyanin extraction field (13%) is particularly significant compared to other cyanobacteria.

• Phycocyanin at 0.6 μg g−1 in skin cream fights bacteria in skin inflammation.

• Phycocyanin-enriched cream was stable at room temp, 25 °C, and unstable at 45 °C after day 15.

Nonstructural protein 3C, a master protease of Picornaviridae, plays a critical role in viral replication by directly cleaving the viral precursor polyprotein to form the viral capsid protein and antagonizing the host antiviral response. Additionally, 3C protease, as a tool enzyme, is involved in regulating polyprotein expression. Here, the 3C mutant gene (3Cm), fused with a small ubiquitin-like modifier (SUMO) tag at the N-terminal and featuring a mutation at position 127, was inserted into the cold-shock plasmid pCold of Escherichia coli for expression. Meanwhile, the P1-∆2A plasmid was constructed for expression in Pichia pastoris. The expressions of 3C protein and P1 precursor protein were confirmed by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (SDS-PAGE), and western blot (WB) analysis. The results showed that the wild-type 3C protease is toxic to the host, not only inhibiting protein expression but also inducing the degradation of the host. Moreover, mutation of the 127th amino acid from leucine (L) to proline (P) on the β-ribbon of 3C enhanced the overexpression capacity of 3C in E. coli while maintaining enzymatic activity. Subsequently, 100 µg P1 protein was utilized as a substrate to investigate the cleavage efficiency of 3C protease at various concentrations, temperatures, durations, and pH levels. The results showed that the target protein was cleaved when the protease reached 8 μg. We also found that the presence of the N-terminal SUMO tag did not affect the cleavage activity of 3Cm. The optimal cleavage activity was observed between 25 and 37 °C, with the peak cleavage efficiency of 89% at 30 °C for 2 h. More than 50% of the substrate was degraded within 1 h at 30 °C. Its optimal pH range is between 7 and 8. Remarkably, the P1 protein, cleaved by 3Cm protease, can further form virus-like particles (VLPs) in vitro.

• Expression and purification of toxic protein 3C protease in E. coli

• Cleavage efficiency assessment of 3C protease at various temperatures, durations, and pH

• Assembly of virus-like particles of FMDV by cleaving the precursor polyprotein in vitro

Syngas fermentation to ethanol has reached industrial production. Further improvement of this process would be aided by quantitative understanding of the influence of imposed reaction conditions on the fermentation performance. That requires a reliable model of the microbial kinetics. Data were collected from 37 steady states in chemostats and from many batch experiments that use Clostridium authoethanogenum. Biomass-specific rates from CO conversion experiments were related to each other according to simple reaction stoichiometries and the Pirt equation, with only the ratio of ethanol to acetate production remaining as degree of freedom. No clear dependency of this ratio on dissolved concentrations, such as CO or acetic acid concentration, was found. This is largely caused by the lack of knowledge about the dependency of the CO uptake rate (and hence all other rates) on the CO concentration. This knowledge gap is caused by a lack of dissolved CO measurements. For dissolved H₂, a similar gap applies. Modelling H₂ consumption adds more degrees of freedom to the system, so that more structured experiments with H₂ is needed. The inhibition of gas consumption by acetate and ethanol is partly known but needs further study.

• Set of Clostridium autoethanogenum syngas fermentation data from chemostats.

• Unstructured kinetic models can relate most biomass-specific rates to dilution rates.

• Lack of dissolved gas measurements limits deeper understanding.

The halotolerant yeast Scheffersomyces spartinae, commonly found in marine environments, holds significant potential for various industrial applications. Despite this, its genetic characteristics have been relatively underexplored. In this study, we isolated a strain of S. spartinae named YMxiao from seawater in Zhoushan City, China. Through scanning electron microscopy and flow cytometry, we characterized S. spartinae YMxiao cells as urn-shaped, demonstrating asymmetric division via budding, and possessing a diploid genome. Compared to the model yeast Saccharomyces cerevisiae, S. spartinae YMxiao exhibited greater tolerance to various stressful conditions. Furthermore, S. spartinae YMxiao was capable of utilizing xylose, mannitol, sorbitol, and arabinose as sole carbon sources for growth. We conducted whole-genome sequencing of S. spartinae YMxiao using a combination of Nanopore and Illumina technologies, resulting in a telomere-to-telomere complete genome assembly of 12 Mb. Genome annotation identified 5311 protein-coding genes, 214 tRNA genes, and 236 transposable elements distributed across 8 chromosomes. Comparative genomics between S. spartinae strains YMxiao and ARV011 revealed genomic variations and evolutionary patterns within this species. Notably, certain genes in S. spartinae strains were found to be under strong positive selection. Additionally, we developed a genetic manipulation protocol that successfully enabled gene knockouts in S. spartinae. Our findings not only enhance our understanding of the S. spartinae genome but also provide a foundation for future research into its potential biotechnological applications.

• The unique phenotypes and genetic characteristics of S. spartinae were disclosed.

• Comparative genomics showed vast genetic variations between S. spartinae strains.

• Genetic manipulation protocol was established for S. spartinae strain.

Metabolomics is a cutting-edge omics technology that identifies metabolites in organisms and their environments and tracks their fluctuations. This field has been extensively utilized to elucidate previously unknown metabolic pathways and to identify the underlying causes of metabolic changes, given its direct association with phenotypic alterations. However, metabolomics inherently has limitations that can lead to false positives and false negatives. First, most metabolites function as intermediates in multiple biochemical reactions, making it challenging to pinpoint which specific reaction is responsible for the observed changes in metabolite levels. Consequently, metabolic processes that are anticipated to vary with metabolite concentrations may not exhibit significant changes, generating false positives. Second, the range of metabolites identified is contingent upon the analytical conditions employed. Until now, no analytical instrument or protocol has been developed that can capture all metabolites simultaneously. Therefore, some metabolites are changed but are not detected, generating false negatives. In this review, we offer a novel and systematic assessment of the limitations of omics technologies and propose-specific strategies to minimize false positives and false negatives through multi-omics approaches. Additionally, we provide examples of multi-omics applications in microbial metabolic engineering and host-microbiome interactions, helping other researchers gain a better understanding of these strategies.

• Metabolomics identifies metabolic shifts but has inherent false positive/negatives.

• Multi-omics approaches help overcome metabolomics’ inherent limitations.

Type VI secretion system 2 (T6SS2) of Vibrio parahaemolyticus is required for cell adhesion and autophagy in macrophages; however, other phenotypes conferred by this T6SS have not been thoroughly investigated. We deleted TssL2, a key component of T6SS2 assembly, to explore the role of the T6SS2 in environmental adaptation and virulence. TssL2 deletion reduced Hcp2 secretion, suggesting that TssL2 played an important role in activity of functional T6SS2. We found that TssL2 was necessary for cell aggregation, wrinkly phenotype formation, and participates in motility and biofilm formation by regulating related genes, suggesting that TssL2 was essential for V. parahaemolyticus to adapt changing environments. In addition, this study demonstrated TssL2 significantly affected adhesion, cytotoxicity, bacterial colonization ability, and mortality in mice, even the levels of the proinflammatory cytokines IL-6 and IL-8, suggesting that TssL2 was involved in bacterial virulence and immunity. Proteome analysis revealed that TssL2 significantly affected the expression of 163 proteins related to ABC transporter systems, flagellar assembly, biofilm formation, and multiple microbial metabolism pathways, some of which supported the effect of TssL2 on the different phenotypes of V. parahaemolyticus. Among them, the decreased expression of the T3SS1 and T2SS proteins was confirmed by the results of gene transcription, which may be the main reason for the decrease in cytotoxicity. Altogether, these findings further our understanding of T6SS2 components on environmental adaption and virulence during bacterial infection.

• The role of T6SS2 in V. parahaemolyticus was far from clear.

• TssL2 participates in cell aggregation, wrinkly phenotype formation, motility, and biofilm formation.

• TssL2 is essential for cell bacterial colonization, cytotoxicity, virulence, and proinflammatory cytokine production.