Clostridium aquiflavi sp. nov., isolated from yellow water of Nongxiangxing baijiu in the Yibin region of China.

A Gram-stain-positive, strictly anaerobic, endospore-forming and rod-shaped (0.6-0.8×2.7-13.1 µm) bacterium, designated as 5 N-1T, was isolated from a yellow water sample collected from the manufacturing process of Nongxiangxing baijiu in the Yibin region of Sichuan, PR China. Growth occurred at 15-40 °C (optimum growth at 37 °C), at pH 6.0-9.0 (optimum growth at pH 7.0) and in NaCl concentrations of 0-1 % (w/v) and ethanol concentrations of 0-2 % (v/v). The major fatty acids in strain 5 N-1T were C16 : 0, C18 : 0 and C14 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unidentified aminophospholipids and one unidentified lipid. Phylogenetic analysis of its 16S rRNA gene sequence indicated that strain 5 N-1T was most closely related to Clostridium weizhouense YB-6T (97.70 %) and Clostridium uliginosum DSM 12992T (97.56 %). The average nucleotide identity and digital DNA‒DNA hybridization values between strain 5 N-1T and the above two type strains were 80.89 and 80.05 % and 25.80 and 25.30 %, respectively, which were all below the species thresholds. The genome size of strain 5 N-1T was 3.5 Mbp and the DNA G+C content was 27.5 mol%. Based on the results of phenotypic and genotypic analyses, strain 5 N-1T represents a novel species of the genus Clostridium, for which the name Clostridium aquiflavi sp. nov. is proposed. The type strain is Clostridium aquiflavi 5 N-1T (=CICC 24886T=JCM 35355T).

Fermentation characteristics and postacidification of yogurt by Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 at optimal inoculum ratio.

This study aimed to investigate the symbiosis between Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047. In addition, the effect of their different inoculum ratios was determined, and comparison experiments of fermentation characteristics and storage stability of milk fermented by their monocultures and cocultures at optimal inoculum ratio were performed. We found the time to obtain pH 4.6 and ΔpH during storage varied among 6 inoculum ratios (1:1, 2:1, 10:1, 19:1, 50:1, 100:1). By the statistical model to evaluate the optimal ratio, the ratio of 19:1 was selected, which exhibited high acidification rate and low postacidification with pH values remaining between 4.2 and 4.4 after a 50-d storage. Among the 3 groups included in our analyses (i.e., the monocultures of S. thermophilus CICC 6038 [St] and Lb. bulgaricus CICC 6047 [Lb] and their cocultures [St+Lb] at 19:1), the coculture group showed higher acidification activity, improved rheological properties, richer typical volatile compounds, more desirable sensor quality after the fermentation process than the other 2 groups. However, the continuous accumulation of acetic acid during storage showed that acetic acid was more highly correlated with postacidification than d-lactic acid for the Lb group and St+Lb group. Our study emphasized the importance of selecting an appropriate bacterial consortium at the optimal inoculum ratio to achieve favorable fermentation performance and enhanced postacidification stability during storage.

Carbon export from leaves is controlled via ubiquitination and phosphorylation of sucrose transporter SUC2.

All multicellular organisms keep a balance between sink and source activities by controlling nutrient transport at strategic positions. In most plants, photosynthetically produced sucrose is the predominant carbon and energy source, whose transport from leaves to carbon sink organs depends on sucrose transporters. In the model plant Arabidopsis thaliana, transport of sucrose into the phloem vascular tissue by SUCROSE TRANSPORTER 2 (SUC2) sets the rate of carbon export from source leaves, just like the SUC2 homologs of most crop plants. Despite their importance, little is known about the proteins that regulate these sucrose transporters. Here, identification and characterization of SUC2-interaction partners revealed that SUC2 activity is regulated via its protein turnover rate and phosphorylation state. UBIQUITIN-CONJUGATING ENZYME 34 (UBC34) was found to trigger turnover of SUC2 in a light-dependent manner. The E2 enzyme UBC34 could ubiquitinate SUC2 in vitro, a function generally associated with E3 ubiquitin ligases. ubc34 mutants showed increased phloem loading, as well as increased biomass and yield. In contrast, mutants of another SUC2-interaction partner, WALL-ASSOCIATED KINASE LIKE 8 (WAKL8), showed decreased phloem loading and growth. An in vivo assay based on a fluorescent sucrose analog confirmed that SUC2 phosphorylation by WAKL8 can increase transport activity. Both proteins are required for the up-regulation of phloem loading in response to increased light intensity. The molecular mechanism of SUC2 regulation elucidated here provides promising targets for the biotechnological enhancement of source strength.

Development and validation of a PMA-qPCR method for accurate quantification of viable Lacticaseibacillus paracasei in probiotics.

The effectiveness of probiotic products hinges on the viability and precise quantification of probiotic strains. This study addresses this crucial requirement by developing and validating a precise propidium monoazide combination with quantitative polymerase chain reaction (PMA-qPCR) method for quantifying viable Lacticaseibacillus paracasei in probiotic formulations. Initially, species-specific primers were meticulously designed based on core genes from the whole-genome sequence (WGS) of L. paracasei, and they underwent rigorous validation against 462 WGSs, 25 target strains, and 37 non-target strains across various taxonomic levels, ensuring extensive inclusivity and exclusivity. Subsequently, optimal PMA treatment conditions were established using 25 different L. paracasei strains to effectively inhibit dead cell DNA amplification while preserving viable cells. The developed method exhibited a robust linear relationship (R 2 = 0.994) between cycle threshold (Cq) values and viable cell numbers ranging from 103 to 108 CFU/mL, with an impressive amplification efficiency of 104.48% and a quantification limit of 7.30 × 103 CFU/mL. Accuracy assessments revealed biases within ±0.5 Log10 units, while Bland-Altman analysis demonstrated a mean bias of 0.058 Log10, with 95% confidence limits of -0.366 to 0.482 Log10. Furthermore, statistical analysis (p = 0.76) indicated no significant differences between theoretical and measured values. This validated PMA-qPCR method serves as a robust and accurate tool for quantifying viable L. paracasei in various sample matrices, including pure cultures, probiotics as food ingredients, and composite probiotic products, thereby enhancing probiotic product quality assurance and contributing to consumer safety and regulatory compliance.

Identification and quantification of viable Lacticaseibacillus rhamnosus in probiotics using validated PMA-qPCR method.

The identification and quantification of viable bacteria at the species/strain level in compound probiotic products is challenging now. Molecular biology methods, e.g., propidium monoazide (PMA) combination with qPCR, have gained prominence for targeted viable cell counts. This study endeavors to establish a robust PMA-qPCR method for viable Lacticaseibacillus rhamnosus detection and systematically validated key metrics encompassing relative trueness, accuracy, limit of quantification, linear, and range. The inclusivity and exclusivity notably underscored high specificity of the primers for L. rhamnosus, which allowed accurate identification of the target bacteria. Furthermore, the conditions employed for PMA treatment were fully verified by 24 different L. rhamnosus including type strain, commercial strains, etc., confirming its effective discrimination between live and dead bacteria. A standard curve constructed by type strain could apply to commercial strains to convert qPCR Cq values to viable cell numbers. The established PMA-qPCR method was applied to 46 samples including pure cultures, probiotics as food ingredients, and compound probiotic products. Noteworthy is the congruity observed between measured and theoretical values within a 95% confidence interval of the upper and lower limits of agreement, demonstrating the relative trueness of this method. Moreover, accurate results were obtained when viable L. rhamnosus ranging from 103 to 108 CFU/mL. The comprehensive appraisal of PMA-qPCR performances provides potential industrial applications of this new technology in quality control and supervision of probiotic products.

Asymptotic tests for Hardy-Weinberg equilibrium in hexaploids.

Hexaploids, a group of organisms containing three complete sets of chromosomes in a single nucleus, are of utmost importance to evolutionary studies and breeding programs. Many studies have focused on hexaploid linkage analysis and QTL mapping in controlled crosses, but little methodology has been developed to reveal how hexaploids diversify and evolve in natural populations. We formulate a general framework for studying the pattern of genetic variation in autohexaploid populations through testing deviation from Hardy-Weinberg equilibrium (HWE) at individual molecular markers. We confirm that hexaploids cannot reach exact HWE but can approach asymptotic HWE at 8-9 generations of random mating. We derive a statistical algorithm for testing HWE and the occurrence of double reduction for autopolyploids, a phenomenon that affects population variation during long evolutionary processes. We perform computer simulation to validate the statistical behavior of our test procedure and demonstrate its usefulness by analyzing a real data set for autohexaploid chrysanthemum. When extended to allohexaploids, our test procedure will provide a generic tool for illustrating the genome structure of hexaploids in the quest to infer their evolutionary status and design association studies of complex traits.

Heterophylly Quantitative Trait Loci Respond to Salt Stress in the Desert Tree Populus euphratica.

Heterophylly, or leaf morphological changes along plant shoot axes, is an important indicator of plant eco-adaptation to heterogeneous microenvironments. Despite extensive studies on the genetic control of leaf shape, the genetic architecture of heterophylly remains elusive. To identify genes related to heterophylly and their associations with plant saline tolerance, we conducted a leaf shape mapping experiment using leaves from a natural population of Populus euphratica. We included 106 genotypes grown under salt stress and salt-free (control) conditions using clonal seedling replicates. We developed a shape tracking method to monitor and analyze the leaf shape using principal component (PC) analysis. PC1 explained 42.18% of the shape variation, indicating that shape variation is mainly determined by the leaf length. Using leaf length along shoot axes as a dynamic trait, we implemented a functional mapping-assisted genome-wide association study (GWAS) for heterophylly. We identified 171 and 134 significant quantitative trait loci (QTLs) in control and stressed plants, respectively, which were annotated as candidate genes for stress resistance, auxin, shape, and disease resistance. Functions of the stress resistance genes ABSCISIC ACIS-INSENSITIVE 5-like (ABI5), WRKY72, and MAPK3 were found to be related to many tolerance responses. The detection of AUXIN RESPONSE FACTOR17-LIKE (ARF17) suggests a balance between auxin-regulated leaf growth and stress resistance within the genome, which led to the development of heterophylly via evolution. Differentially expressed genes between control and stressed plants included several factors with similar functions affecting stress-mediated heterophylly, such as the stress-related genes ABC transporter C family member 2 (ABCC2) and ABC transporter F family member (ABCF), and the stomata-regulating and reactive oxygen species (ROS) signaling gene RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). A comparison of the genetic architecture of control and salt-stressed plants revealed a potential link between heterophylly and saline tolerance in P. euphratica, which will provide new avenues for research on saline resistance-related genetic mechanisms.