Search Results (97)

Large-scale bioprocesses often suffer from spatial heterogeneities, which impact microbial performance and often lead to phenotypic population heterogeneity. To better understand these effects at the single-cell level, this study applied, for the first time, automated real-time flow cytometry (ART-FCM) to monitor L-phenylalanine production with an Escherichia coli triple reporter strain in a fed-batch process with glycerol as the carbon source. The strain was cultivated in both a well-mixed stirred-tank bioreactor (STR) and a scale-down two-compartment bioreactor (TCB), consisting of an STR and a coiled flow inverter (CFI) in bypass, to simulate spatial heterogeneities. ART-FCM enabled autonomous, high-frequency sampling every 20 min, allowing for real-time tracking of fluorescence signals linked to growth (rrnB-mEmerald), oxygen availability (narGHIJ-CyOFP1), and product formation (aroFBL-mCardinal2). The STR exhibited uniform reporter expression and higher biomass accumulation, while the TCB showed delayed product formation and pronounced phenotypic diversification depending on the set mean residence time in the CFI. Single-cell fluorescence distributions revealed that the shorter mean residence time in the CFI resulted in pronounced subpopulation formation, whereas longer exposure attenuated heterogeneity, indicating transcriptional adaptation. This finding highlights a critical aspect of scale-down studies: increased exposure duration to perturbations can enhance population robustness. Overall, this study demonstrates the relevance of ART-FCM, in combination with a multi-reporter strain, as a pioneering tool for capturing dynamic cellular behavior and correlating it to process performance, providing deeper insights into microbial heterogeneity under fluctuating bioprocess conditions. Full article

Peroxiredoxins (Prxs; EC 1.11.1.15) are a group of thiol peroxidases that catalyze the detoxification of H₂O₂ and other organic hydroperoxides. The ripening of pepper (Capsicum annuum L.) fruit involves significant phenotypic, physiological, and biochemical changes. Based on the available pepper plant genome, eight PRX genes were identified and named CaPRX1, CaPRX1-Cys, CaPRX2B, CaPRX2E, CaPRX2F, CaPRX2-CysBAS1, CaPRX2-CysBAS2, and CaPRX Q. Among these, only CaPRX1-Cys was not detected in the transcriptome (RNA-Seq) of sweet pepper fruits reported previously. This study analyzes the modulation of these seven CaPRX genes during ripening and after treating fruits with nitric oxide (NO) gas. A time-course expression analysis of sweet pepper fruit during ripening revealed that two genes were upregulated (CaPRX1 and CaPRX2E), two were downregulated (CaPRX2B and PRX Q), and three were unaffected (CaPRX2F, CaPRX2-CysBAS1, and CaPRX2-CysBAS2). Gene expression was also studied in three hot pepper varieties with varying capsaicin contents (Piquillo < Padrón < Alegría riojana), showing a differential expression pattern during ripening. Furthermore, NO treatment of sweet pepper fruits triggered the upregulation of CaPRX2B and CaPRXQ genes and the downregulation of CaPRX1 and CaPRX2-CysBAS1 genes, while the other three remained unaffected. Among the CaPrx proteins, four (CaPrx2B, CaPrx2-CysBAS1, CaPrx2-CysBAS2, and CaPrx2E) were identified as susceptible to S-nitrosation, as determined by immunoprecipitation assays with an antibody against S-nitrocysteine and further mass spectrometry analyses. These findings indicate the diversification of PRX genes in pepper fruits and how some of them are regulated by NO, either at the level of gene expression or through protein S-nitrosation, a NO-promoting post-translational modification (PTM). Given that Prxs play a crucial role in stress tolerance, these data suggest that Prxs are vital components of the antioxidant system during pepper fruit ripening, an event that is accompanied by physiological nitro-oxidative stress. Full article

Pummelo (Citrus maxima) is an ancestral species that has given rise to several major citrus varieties, such as sweet orange (C. x aurantium var. sinensis) and grapefruit (C. x aurantium var. paradisi). This species is also cultivated and its fruit consumed, particularly in Asia. Over the course of evolution, the allogamous reproduction of pummelos and the absence of asexual multiplication have contributed to its diversification. To assess its phenotypic diversity and the chemical composition of leaf and peel essential oils, genetic analysis using DNA markers is an essential prerequisite to ensure the identity and if varieties belong to this species. Fifty-eight accessions classified as grapefruits or pummelos were analyzed using 42 SSRs, 4 Indels, and 36 SNP markers. Based on the allelic composition of these markers, 20 cultivars were detected belonging to pummelos, 18 cultivars to grapefruits, and 11 were interspecific hybrids. The grapefruit inter-cultivar SSR diversity is null. The genetic origin of five interspecific hybrids is elucidated. The level of phenotypic diversity and of essential oil composition corroborate the modes of diversification, with high levels for those resulting from crosses and very low levels for the group of grapefruit mutants. Only the characteristics of breeding selection (pulp color, acidity and aspermia) are variable in grapefruits. In the composition of leaf essential oils (LEOs), nine profiles were detected in grapefruits based on variations in six compounds (neral, geranial, β-phellandrene, γ-terpinene, (E)-β-ocimene, and β-pinene). The seven interspecific hybrids involving pummelo as one parent show particular LEO profiles but without specific compounds, with the exception of p-cymenene which is present only in Wheeny. The diversity of peel essential oils in pummelos is lower, but variations in γ-terpinene, β-pinene, limonene, and myrcene make it possible to define seven profiles. With genetic verification the chemical and phenotypic diversity of the two species, pummelo and grapefruit, revealed in this study can be used as a reference for behavior in a specific environment. Full article

Xyloglucan endotransglucosylases/hydrolases (XTHs) are key enzymes involved in cell wall remodeling by modifying xyloglucan–cellulose networks, thereby influencing plant growth, development, and secondary cell wall formation. While the roles of XTHs have been extensively studied in primary and secondary growth, their functions in the formation and thickening of lignified nut shells remain largely unknown. Pecan (Carya illinoinensis), an economically important nut crop, develops a hard, lignified shell that protects the seed during fruit maturation. In this study, we performed a comprehensive genome-wide characterization of the XTH gene family in pecan and identified 38 XTH genes, which were categorized into four distinct phylogenetic groups. Structural analyses of the deduced proteins revealed conserved catalytic residues alongside divergent loop regions, suggesting functional diversification. Expression profiling across various tissues and among pecan cultivars with contrasting shell phenotypes indicated that specific XTH genes may play critical roles in shell structure formation. Moreover, gene regulatory networks in thin- and thick-shelled pecans provided new insights into the molecular mechanisms underlying shell development and thickness regulation. These findings lay a foundation for future genetic improvement strategies targeting nut shell traits in woody perennials. Full article

The R2R3-MYB transcription factor GAMYB plays crucial roles in plant growth and development, but the biological functions of SlGAMYB1 in tomato remain poorly understood. Here, we investigated the roles of SlGAMYB1 by overexpressing a miR159-resistant version (35S:SlGAMYB1^m) in tomato. Transgenic plants exhibited a dwarf phenotype with reduced internode elongation, which was associated with decreased bioactive gibberellin (GA) levels due to transcriptional repression of SlGA3ox1 and activation of SlGA2ox1/2/4/5. Additionally, 35S:SlGAMYB1^m altered leaf morphology by inhibiting cell proliferation through downregulation of cell cycle genes, resulting in larger but fewer epidermal cells. Intriguingly, 35S:SlGAMYB1^m plants displayed increased floral organ number, a process likely mediated by the upregulation of SlWUS rather than GA signaling. These findings demonstrate that SlGAMYB1 regulates diverse aspects of tomato development through both GA-dependent and independent pathways, providing new insights into the functional diversification of GAMYB genes and potential strategies for genetic improvement of tomato architecture and yield. Full article

Environmental stresses significantly influence crop growth and productivity, acting as powerful selective pressures in plant evolution. The AP2/ERF superfamily is crucial for plant development and stress responses, orchestrating key regulatory pathways. This study explores the adaptive evolution of AP2/ERF genes across 15 key plant species, focusing on expansion and contraction patterns driven by amplification through multi-omics analyses. Across 15 plant genomes, we identified 1495 AP2/ERF genes. AP2/ERF genes demonstrated preferential retention following amplification, underscoring their importance in genomic stability and functional adaptation. Notably, the amplification-associated AP2 subfamily exhibited substantial expansion in quinoa (CqAP2/ERFs), emphasizing its role in stress adaptation. Robust regulatory networks were identified between CqAP2/ERFs, AtAP2/ERFs, and fatty acid pathways, highlighting their contributions to stress resilience. Transcriptomic analyses in Arabidopsis thaliana further validated the conserved correlation of these networks. Functional predictions based on phenotypic and RNA-seq data revealed the involvement of AP2/ERFs in key stress response and developmental processes. By integrating genomic, metabolomic, phenotypic, transcriptomic, and protein interaction data, this study uncovers novel regulators and adaptive pathways of AP2/ERFs, providing insights into their evolutionary diversification post-amplification. These findings establish a comprehensive framework for understanding the pivotal roles of AP2/ERFs in enhancing plant stress tolerance. Full article

This study examines the impact of inhaled tobramycin therapy on the within-host changes in P. aeruginosa strains isolated from Bulgarian patients with CF prior to and post treatment. Genotypic comparison by RAPD-PCR indicated that most of the pre-treatment isolates had a high similarity and were genetically comparatively close to strains from other countries with known increased morbidity or treatment requirements. Most of the post-treatment isolates were, however, genetically distant from their pre-treatment counterparts, showing genotypic diversification after the treatment. Phenotypic comparisons showed a lower ODmax reached during groswth and an increased lag-time in the post-treatment isolates. All strains were capable of invasion and intracellular reproduction within A549 cultured cells. The addition of sub-inhibitory amounts (1/4 or 1/2 MIC) of tobramycin during growth showed the higher relative fitness (as a percentage of the untreated control) of the post-treatment strains. The effects of sub-MICs on biofilm growth did not show such a pronounced trend. However, when a resazurin-based viability test was applied, the advantage of the post-treatment strains was confirmed for both broth and biofilm cultures. In spite of that, according to the determined MIC values, all isolates were tobramycin-sensitive, and the data from this study imply the development of tolerance to the antibiotic in the strains that survived the treatment. Full article

As one of the largest gene families in plants, the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) genes are involved in important biological processes, such as plant growth and development and response to bio-/abiotic stresses. The rubber tree (Hevea brasiliensis Müll. Arg.) is the primary commercial source of natural rubber globally. In this study, 274 LRR-RLK genes were comprehensively identified and classified into 21 subclades of the rubber tree genome. Members belonging to the same subclade exhibited comparable gene structures and possessed conserved protein motifs. Gene duplication analysis detected 35 tandem duplication genes and 81 segmental duplication genes. Cis-element analysis of HbLRR-RLK promoters identified light, hormone, stress, and development-related cis-elements. Tissue-specific expression profiling revealed that 73% (200/274) of HbLRR-RLKs were expressed in at least one of seven analyzed tissues. Protein–protein interaction (PPI) network identified 584 potential interactions among the HbLRR-RLKs. Additionally, subcellular localization analysis suggested that HbPSKR2 (HbLRR-RLK174) is a plasma membrane-localized receptor, and the gene could restore the short-root phenotype of the atpskr mutant in Arabidopsis. These results provide a comprehensive structure to facilitate analysis of the evolution and functional diversification of LRR-RLKs in the rubber tree. Full article

The calcareous nannoplankton comprises haptophyte eukaryotes known as coccolithophores, capable of calcifying elaborate external skeletons (coccoliths s.l.) which differ morphologically depending on the phase of the life cycle considered, and the locus (intra- or extracellular) of mineralization. No study is currently available that analyzes the impact of these differences on coccolith morphology. An analysis of the assembly of their crystals is conducted here in search of the following: (1) identical traits across life cycles; (2) fossil records diagnostic of extracellular calcification; and (3) influence of the geometry of biomineralization during the diploid phase on the long-term evolution of a clade. This study shows patterns such as correlation of characters and structural imprint that unify the haploid and diploid phases, indicating a strong cellular integrity and offering potent means to determine life cycles in living and fossil communities. It also shows that differences in diversity patterns and longevity among families and orders depend on coccolith geometry, concentric geometry being more favorable to stability, and superposition geometry facilitating morphological diversification. Extinction occurs when the potential for diversification is attained. Finally, I propose that the evolution of biomineralization in the calcareous nannoplankton may have been more complex than initially thought, with intra- and extracellular calcification evolving independently. Full article

The surface adhesion and stiffness of underlying substrates mediate the geometry, mechanics, and self-organization of expanding bacterial colonies. Recent studies have qualitatively indicted that stiffness may impact bacterial attachment and accumulation, yet the variation in the cell-to-surface adhesion with substrate stiffness remains to be quantified. Here, by developing a cell-level force–distance spectroscopy (FDS) technique based on atomic force microscopy (AFM), we simultaneously quantify the cell–surface adhesion and stiffness of the underlying substrates to reveal the stiffness-dependent adhesion of the phototrophic bacterium Chromatium okenii. As the stiffness of the soft substrate, modeled using a low-melting-point (LMP) agarose pad, was varied between 20 kPa and 120 kPa by changing the agarose concentrations, we observed a progressive increase in the mean adhesion force by over an order of magnitude, from $0.21\pm 0.10$ nN to $2.42\pm 1.16$ nN. In contrast, passive polystyrene (PS) microparticles of comparable dimensions showed no perceptible change in their surface adhesion, confirming that the stiffness-dependent adhesive interaction of C. okenii is of a biological origin. Furthermore, for Escherichia coli, the cell–surface adhesion varied between $0.29\pm 0.17$ nN and $0.39\pm 0.20$ nN, showing a weak dependence on the substrate stiffness, thus suggesting that stiffness-modulated adhesion is a species-specific trait. Finally, by quantifying the adhesion of the C. okenii population across different timescales, we reported the emergent co-existence of weak and strongly adherent sub-populations, demonstrating diversification of the adherent phenotypes over the growth stages. Taken together, these findings suggest that bacteria, depending on the species and their physiological stage, may actively modulate cell-to-surface adhesion in response to the stiffness of soft surfaces. While the surface properties, for instance, hydrophobicity (or hydrophilicity), play a key role in mediating bacterial attachment, this work introduces substrate stiffness as a biophysical parameter that could reinforce or suppress effective surface interactions. Our results suggest how bacteria could leverage stiffness-dependent adhesion and the diversity therein as functional traits to modulate their initial attachment to, colonization of, and proliferation on soft substrates during the early stages of biofilm development. Full article

In communication systems, the signal and preference for the signal have to match, limiting phenotypic variation. Yet, communication systems evolve, but the mechanisms of how phenotypic variation can come into existence while not disrupting the match are poorly understood. Geographic variation in communication can provide insights into the diversification of these systems. Females of the katydid Neoconocephalus triops use the pulse rate and call structure for call recognition. Using behavioral experiments, we determined preferences for pulse rate at two relevant ambient temperatures and preferences for call structure (continuous, versed) in females from Puerto Rico and Costa Rica. Puerto Rican females had closed preference at both tested temperatures, indicating high selectivity for pulse rate. In contrast, Costa Rican females had a closed preference only at 20 °C; at 25 °C the females were unselective toward higher than natural pulse rates. Additionally, Puerto Rican females were not selective for call structure, whereas Costa Rican females preferred versed calls. It is not clear whether the differences in pulse preference were due to neural constraints or different selective pressures, however, they may facilitate further divergence and reproductive isolation. Importantly, the reduced selectivity for call structure or pulse rate allows calls to display the necessary variation for the communication system to evolve. Full article

The mutualistic relationship between Streptococcus thermophilus (S. thermophilus) and L. delbrueckii subsp. bulgaricus (L. bulgaricus) is responsible for milk coagulation, gel formation, and the flavour of yogurt. Under set-style yogurt processing conditions, the performance of a mixed culture composed of these species depends on key technological parameters such as the capacity for acidification and proteolytic activity. This study aimed to determine the extent of phenotypic diversity by comparing the key traits of acidification and proteolytic activity among isolates found in yogurt starter cultures. Seventy-two isolates from three industrial starter cultures were ranked by either their fast or slow acidification activity (time to reach pH 4.6, 16 h), proteolytic activity, cell envelope proteinase (CEP) activity, redox potential and titratable acidity. The integration of multiple phenotype measures by hierarchical clustering and non-metric dimensional scaling (NMDS) clustered groups of isolates by multifactor similarity. A significant difference (p-value < 0.05) was observed between the clusters regarding redox potential and the proteolytic activity of both S. thermophilus and L. bulgaricus. The integration of multiple phenotypes points to the diversification that may have occurred over repeated culturing of yogurt starter bacteria. The phenotypic diversity may explain the divergence in starter performance and be used to refine the formulation of new starter cultures. Future work will investigate the correlation between the activity of specific enzymes based on the phenotype to explain the separation between the fast and slow acidification of isolates. Full article

As one of the world’s most crucial food crops, maize plays a pivotal role in ensuring food security and driving economic growth. The diversification of maize variety breeding is significantly enhancing the cumulative benefits in these areas. Precise measurement of phenotypic data is pivotal for the selection and breeding of maize varieties in cultivation and production. However, in outdoor environments, conventional phenotyping methods, including point cloud processing techniques based on region growing algorithms and clustering segmentation, encounter significant challenges due to the low density and frequent loss of point cloud data. These issues substantially compromise measurement accuracy and computational efficiency. Consequently, this paper introduces a Constrained Region Point Cloud Phenotyping (CRPCP) algorithm that proficiently detects the phenotypic traits of multiple maize plants in sparse outdoor point cloud data. The CRPCP algorithm consists primarily of three core components: (1) a constrained region growth algorithm for effective segmentation of maize stem point clouds in complex backgrounds; (2) a radial basis interpolation technique to bridge gaps in point cloud data caused by environmental factors; and (3) a multi-level parallel decomposition strategy leveraging scene blocking and plant instances to enable high-throughput real-time computation. The results demonstrate that the CRPCP algorithm achieves a segmentation accuracy of 96.2%. When assessing maize plant height, the algorithm demonstrated a strong correlation with manual measurements, evidenced by a coefficient of determination R² of 0.9534, a root mean square error (RMSE) of 0.4835 cm, and a mean absolute error (MAE) of 0.383 cm. In evaluating the diameter at breast height (DBH) of the plants, the algorithm yielded an R² of 0.9407, an RMSE of 0.0368 cm, and an MAE of 0.031 cm. Compared to the PointNet point cloud segmentation method, the CRPCP algorithm reduced segmentation time by more than 44.7%. The CRPCP algorithm proposed in this paper enables efficient segmentation and precise phenotypic measurement of low-density maize multi-plant point cloud data in outdoor environments. This algorithm offers an automated, high-precision, and highly efficient solution for large-scale field phenotypic analysis, with broad applicability in precision breeding, agronomic management, and yield prediction. Full article

Background/Objectives: Staphylococcus pseudintermedius is part of the normal microbiota in dogs. Since 2006, an increase in multidrug-resistant clones of methicillin-resistant S. pseudintermedius has been reported, as well as zoonotic transmission. Longitudinal investigations into clonal population structures, antibiotic resistance patterns, and the presence of resistance and virulence genes are important tools for gaining knowledge of the mechanisms behind the emergence of such clones. Methods: We investigated 87% of all non-repetitive MRSP isolates from dogs and cats in Sweden over a ten-year period (n = 356). All isolates were subjected to staphylococcal chromosomal cassette mec identification, whole-genome sequencing, multi-locus sequence typing, and analyses of genomic relatedness, as well as investigation of phenotypical resistance patterns and the presence of antibiotic resistance genes and virulence genes. Results: A considerable increase over time in the number of clonal lineages present was observed, indicating genomic diversification, and four clones became dominant: ST71, ST258, ST265, and ST551. In total, 96% of the isolates were multidrug-resistant. Statistically significant differences in resistance to several antibiotic classes between the four dominant clones were present. All isolates carried several virulence genes encoding factors associated with attachment, colonization, toxin synthesis, quorum sensing, antibiotic resistance, and immune evasion. Full article

Maize is a highly versatile crop holding significant importance in global food, feed and nutritional security. Grain yield is a complex trait and difficult to improve without targeting the improvement of grain yield attributing traits, which are relatively less complex in nature. Hence, considering the erosion in genetic diversity, there is an urgent need to use wild relatives for genetic diversification and unravel the genomic regions for grain yield attributing traits in maize. Thus, the current study aimed to identify quantitative trait loci (QTLs) linked with grain yield and yield attributing traits. Two BC₂F₂ populations developed from the cross of LM13 with Zea parviglumis (population 1) and LM14 with Zea parviglumis (population 2) were genotyped and phenotyped in field conditions in the kharif season. BC₂F_2:3 lines in both populations were phenotyped again for grain yield and attributing traits in the spring season. In total, three QTLs each for ear height (EH), two QTLs for flag leaf length (FLL) and one QTL each for ear diameter (ED), plant height, flag leaf length (FLL), flag leaf width and 100 kernel-weight were identified in population 1. In population 2, two QTLs for kernel row per ear (KRPE) and one QTL for FLL were detected in. QTLs for EH, FLL and KPRE showed consistency across seasons. Among the identified QTLs, six QTLs were found to be co-localized near identified genomic regions in previous studies, validating their potential in contributing to trait expression. The identified QTLs can be utilized for marker assisted selection, transferring favorable alleles from wild relatives in modern maize. Full article