Search Results (143)

The genus Aegilops comprises important wild relatives of cultivated wheat and represents a valuable genetic resource for wheat improvement. In this study, the complete chloroplast genomes of five Aegilops species (Ae. crassa, Ae. cylindrica, Ae. juvenalis, Ae. tauschii, and Ae. triuncialis) collected from Kazakhstan and Uzbekistan were sequenced, assembled, and comparatively analyzed. The chloroplast genomes exhibited a conserved quadripartite structure consisting of a large single-copy (LSC), a small single-copy (SSC), and two inverted repeat (IR) regions. Genome sizes ranged from 135,612 to 136,840 bp, with an identical GC content of 38% across all species. Comparative analyses revealed high structural conservation among chloroplast genomes, particularly within IR regions, whereas greater sequence divergence was observed in the non-coding regions of the LSC and SSC. Sliding-window analysis identified several highly polymorphic regions, including rpl32-trnL(UAG), ndhF-rpl32, trnC(GCA)-rpoA, psbA, and ndhD, which may serve as potential DNA barcodes and informative markers for phylogenetic studies. A total of 850 chloroplast simple sequence repeats (SSRs) were detected, predominantly A/T-rich mononucleotide repeats. Codon usage analysis demonstrated a conserved preference for A/U-ending codons across all species. Ka/Ks analysis indicated that most chloroplast protein-coding genes are under strong purifying selection, although relatively elevated evolutionary rates were detected in rpoA and ycf4. Phylogenetic analyses based on complete chloroplast genomes strongly supported sectional relationships within Aegilops and confirmed close maternal relationships among several species. Overall, this study provides chloroplast genome resources for Aegilops and contributes to understanding chloroplast genome evolution, phylogeny, and molecular marker development. Full article

The misuse of antibiotics is causing widespread antibiotic resistance, creating an urgent need for new treatment options such as nanoparticle-based therapies. This study aimed to compare silver nanoparticles (AgNPs) produced via green synthesis methods with those made through traditional chemical processes. Furthermore, the study investigated and contrasted the bacterial responses to these two types of AgNPs over a 21-day period of selection pressure using experimental evolution techniques. Analysis using scanning electron microscopy and transmission electron microscopy revealed a consistent, uniform morphology among the AgNPs produced via chemical methods. In contrast, AgNPs synthesized through green methods displayed an irregular morphology. Despite these morphological differences, all nanoparticles from both synthesis approaches were under 100 nm in diameter. These findings were further supported by the absorption spectrum data, which showed a maximum absorption peak between the 400 and 500 nm wavelength range. E. coli exposed to green synthesized AgNPs for 21 days adapted to their presence, exhibiting both enhanced resistance to the green synthesized AgNPs themselves and the development of cross-resistance to ionic silver, a pattern not observed in chemically synthesized AgNP-selected populations. Populations selected using chemical synthesized AgNPs did not develop increased resistance to either chemically or green synthesized AgNPs; however, they showed a slight increase in resistance to ionic silver. Genomics analysis identified polymorphism in genes in a green synthesized AgNP-resistant line including but not limited to the multidrug efflux transporter system (EmrAB), DUF4756 family protein (D1792_RS05680), putative zinc-binding protein YnfU/cold shock-like protein (ynfU/cspB) and imcF-related family protein (D1792_RS10035). Bacterial resistance to chemical AgNPs involves specific polymorphisms in key bacterial components like the RNA polymerase sigma factor (RpoE) and the EmrAB efflux pump. Collectively, the method used to synthesize the AgNPs influences their antibacterial efficacy and the likelihood of bacteria developing resistance. Understanding this interaction is vital for developing effective and resistance-controlled applications of AgNPs across medicine, environmental science, and industry. Full article

Vibrio splendidus is an important opportunistic pathogen that causes diseases in aquatic animals, and its persisters increase the difficulty of aquaculture disease control. The stringent response is a central pathway in bacteria for coping with environmental stress, and the signaling molecule (p)ppGpp, synthesized under the regulation of RelA/SpoT homologs, is closely associated with persister formation and virulence modulation. However, the regulatory mechanisms linking the stringent response to persister formation and virulence in V. splendidus remain unclear. In this study, the core gene deletion strains ΔrelA and ΔrelAΔspoT were constructed via homologous recombination. Combined with D₂O single-cell Raman spectroscopy, transcriptomics, and phenotypic assays, we systematically characterized the biological effects of stringent response inactivation. The results showed that the loss of relA and spoT significantly reduced persister formation and key virulence traits while enhancing biofilm formation. Single-cell Raman spectroscopy analysis indicated that persisters remained metabolically active, accompanied by changes in different cellular components. Transcriptome analysis revealed that the absence of stringent response affected multiple pathways, including ribosomal function, energy metabolism, two-component systems, and quorum sensing. Additionally, the sigma factor RpoS may potentially exert a compensatory function in ΔrelAΔspoT strain, but this requires further validation. In conclusion, the stringent response positively regulates persister formation and virulence in V. splendidus, despite the existence of complex regulatory mechanisms. This study provides a theoretical basis for the development of anti-infective strategies targeting stringent response in aquatic pathogens. Full article

Background/Objectives: The σ factor of bacterial RNA polymerase (RNAP) directs promoter recognition, recruits RNAP to initiate transcription, and is released from the elongation complex to participate in subsequent rounds of initiation. However, the dynamic recycling mechanism of the primary σ factor, σ⁷⁰ (RpoD), during transcription in Escherichia coli remains poorly understood. Methods: We employed in vivo and in vitro interaction assays to screen for σ⁷⁰-interacting partners under different growth conditions. Protein localization studies were performed using fluorescence microscopy. The transcriptomic profile of ΔclpB, ΔdnaK, ΔhtpG, or ΔyhgF mutant was assessed by RNA-seq. Results: The molecular chaperones ClpB, DnaK, HtpG, and the RNA-binding protein YhgF interacts with RpoD both in vivo and in vitro, and the interaction in vivo is growth medium-dependent (LB vs. ABTGcasa). During exponential growth, each of these proteins co-localizes with the nucleoid. The transcriptome profile in ΔclpB, ΔhtpG or ΔyhgF mutant is mutant-specific to some extent; differentially expressed genes (DEGs) associated with amino acid metabolism and lipopolysaccharide biosynthesis are down-regulated in ΔclpB, ΔhtpG or ΔyhgF mutant in a manner that is growth medium-dependent, in agreement with the medium-dependent interaction of RpoD with the chaperones and YhgF. In contrast, the absence of DnaK resulted in delays to initiation of replication with a slow growth, and decreases cell motility, accompanied by down-regulated flagellar assembly and up-regulated amino acid metabolism genes. In summary, ClpB, DnaK, HtpG, and YhgF may regulate transcription by directly interacting with σ⁷⁰. The σ factor recycling guides global transcription to select genes for transcription and subsequently allows cells to cope with the changing environments by responding to the nutrient level as a signal. Full article

Background: The global burden of drug-resistant Mycobacterium tuberculosis continues to threaten tuberculosis control efforts, largely due to the emergence and transmission of resistance-associated genetic mutations. Molecular epidemiology provides critical insights into mutation profiles and resistance associations, yet the interplay among key mutations and their contributions to complex resistance patterns remains poorly understood, particularly in high-burden settings. Methods: A retrospective, cross-sectional, laboratory-based design was used to analyze 111 phenotypically confirmed drug-resistant isolates. Molecular drug susceptibility testing (DST) for first- and second-line anti-tuberculosis drugs was performed at the National Health Laboratory Service (NHLS) TB reference laboratory. Drug-resistance profiles were classified according to World Health Organization (WHO) definitions. Descriptive and inferential statistical analyses were conducted to determine mutation frequencies, co-occurrence patterns, and associations with resistance profiles. Results: rpoB (D435V 38.7%; S450L 36.0%) and katG (S315T 80.2%) mutations predominated, forming the core molecular basis of MDR-TB, while 15% harbored inhA promoter mutations associated with low-level isoniazid resistance. The most frequent combinations included rpoB S450L with katG S315T and rpoB D435V with katG S315T, consistent with multidrug-resistant tuberculosis (MDR-TB) profiles. Nearly 48% showed dual resistance to fluoroquinolones and second-line injectables. Conclusion: This study highlights the predominance of resistance-associated mutations and their co-occurrence patterns in shaping MDR-TB profiles in the study setting. The observed burden of second-line drug resistance underscores the importance of comprehensive resistance testing. These findings support the use of mutation profiling for rapid diagnosis and informed treatment decisions, while emphasizing the need for ongoing local surveillance to guide TB control efforts. Full article

Acidithiobacillus caldus is perpetually exposed to multiple extreme environmental stresses. CsrA, functioning as a post-transcriptional regulator of physiological metabolism, acts as a differential modulator, facilitating more economical and efficient adaptation to extreme environments. The csrA expression recombinant strain was constructed in A. caldus MTH-04 by conjugative transfer technology pJD215. Physiological characterization revealed enhanced acid tolerance, significantly elongated flagella, elevated extracellular secretion, and altered biofilm composition. Notably, intracellular concentrations of free glutamate and aspartate increased to 24.18 mg/L and 16.07 mg/L, respectively. The secondary structure of CsrA protein was determined in vitro through circular dichroism spectroscopy and size-exclusion chromatography. Electrophoretic Mobility Shift Assay (EMSA) successfully demonstrated in vitro binding activity of CsrA to the rpoS leader mRNA. CsrA suppresses rpoS mRNA translation by competing with ribosomes for binding sites, thereby negatively regulating rpoS expression. Critical binding sites were further validated through site-directed mutagenesis. Through EMSA, RT-qPCR and the translation reporter system, it was also found that CsrA has a dual regulatory function for nearby flagella- and motility-related gene clusters (flgC, 07035, motD, 15040), which also implies the global regulatory role of CsrA. In summary, a potential overall post-transcriptional regulatory mechanism based on CsrA and rpoS by extremophile A. caldus was proposed. Finally, the efficiency of bioleaching application by csrA overexpression strain was improved by 20.81%. Full article

Background/Objectives: Salmonella Heidelberg (SH) is a globally distributed pathogen associated with gastrointestinal disease in humans and animals and frequently affects poultry. Among the classic strategies used in vaccine development, evolutionary engineering enables the generation of attenuated bacterial strains through exposure to selective pressures such as antibiotics. In this study, spontaneous antibiotic-resistant mutant strains of SH were generated by exposure to high concentrations of streptomycin and rifampicin, after which their phenotypic and genotypic characteristics were evaluated. Methods: The wild-type strain SA628 wt was subjected to continuous and discontinuous selection under antibiotic pressure. Phenotypic characterization included biochemical profiling and antibiotic susceptibility testing. Whole-genome sequencing was performed to identify genetic changes affecting virulence- and resistance-associated genes, plasmid content, and point mutations using variant calling approaches. The potential functional relationships of the mutated genes were further analyzed through genetic network analysis. Results: The mutant strains SA628 mut1 and SA628 mut3 were obtained through discontinuous selection, whereas strain SA628 mut2 was generated under continuous selection. Phenotypically, all the mutant strains exhibited resistance to streptomycin, whereas SA628 mut2 and SA628 mut3 also exhibited resistance to rifampicin. Genomic analyses revealed mutations in rpoS, ascD, ynfE, rpoB, and cyaA associated with discontinuous selection and in iscU, ybiO, rpoB, and rsmG associated with continuous selection. Network analysis indicated that these genes are functionally connected within regulatory and metabolic interaction networks, including global transcriptional regulation, anaerobic metabolism, cAMP-mediated signaling, translation, and iron–sulfur cluster biogenesis. Conclusions: Collectively, these findings suggest that antibiotic-driven selection promotes coordinated genetic changes affecting stress responses and metabolism, which may contribute to reduced virulence. This work provides insights into bacterial adaptation under antibiotic stress and supports the potential use of evolutionary engineering for the development of attenuated strains. Full article

Background: The subtribe Pleurothallidinae is a diverse group within Orchidaceae with a complex taxonomic history. Comparative plastome analysis can provide insights into genome evolution and facilitate phylogenetic reconstruction. Methods: Here we analyzed 25 complete chloroplast genomes representing 15 genera, including 14 newly assembled genomes, to investigate plastome evolution in this subtribe. Results: All genomes exhibited the typical quadripartite structure (148, 246–158, 138 bp) with conserved gene content (128–134 genes). While most protein-coding genes were under purifying selection, we detected signatures of positive selection in specific lineages. Notably, ndhF in Lepanthes tachirensis showed a markedly elevated Ka/Ks ratio (3.65), which may be associated with adaptation to an extensive distributional range. ENC-plot analysis indicated that natural selection, rather than mutation pressure alone, shapes codon usage bias, with patterns varying among species from different geographic regions. Nucleotide diversity analysis identified eight hypervariable intergenic regions (psbK-psbI, atpI-rps2, petN-psbM, psbB-psbT, petD-rpoA, rpoA-rps11, rps3-rpl22, ccsA-ndhD) suitable as candidate molecular markers. Phylogenetic analysis confirmed that Lepanthes and Pleurothallis are non-monophyletic as traditionally defined. Conclusions: These findings expand plastome resources for Pleurothallidinae, reveal lineage-specific patterns of selection, and provide molecular markers for future taxonomic and evolutionary studies. Full article

Rifampicin-resistant tuberculosis (RR-TB) remains a major threat to global TB control, primarily driven by mutations in the rpoB gene of Mycobacterium tuberculosis (Mtb). Most resistance-conferring mutations occur within the 81-base pair RIF resistance determining region (RRDR), particularly at codons S450L, H445Y/D, and D435V, which are strongly associated with high level resistance. However, increasing evidence of low-frequency and disputed variants both within and beyond the RRDR reveals a broader genetic spectrum that contributes to diagnostic uncertainty and variable phenotypic outcomes. This review summarizes current knowledge of high frequency, low frequency, and disputed rpoB mutations and their implications for molecular detection of RIF resistance. Structural analyses show that specific amino acid substitutions alter key hydrogen bonds or create steric hindrance in the RIF-binding pocket, leading to diverse resistance levels. Despite the success of molecular platforms such as Xpert MTB/RIF and line probe assays, their hotspot-based detection limits sensitivity to noncanonical variants. Lowering the minimum inhibitory concentration (MIC) breakpoint and integrating sequencing-based approaches, such as targeted and whole-genome sequencing, can enhance detection accuracy. A combined genomic and phenotypic framework will be essential to close existing diagnostic gaps and advance precision guided management of RIF-resistant and multidrug-resistant tuberculosis. Full article

The endophytic Streptomyces sp. PRh3 (PRh3), isolated from Dongxiang wild rice (DXWR), exhibited impaired biosynthetic capacity in the laboratory. To address this defect, rifampicin-based ribosome engineering was first applied to PRh3 to generate PRh3-r55, which acquired a characteristic H473Y rifampicin-resistant mutation in rpoB to activate the production of two β-carboline alkaloids JBIR-133 and JBIR-134. Then the biosynthetic gene cluster (BGC) ksl was introduced into PRh3-r55 for heterologous expression, generating PRh3-r55K. This combined approach achieved a synergistic effect, enabling the strain to produce not only the expected JBIR-133, JBIR-134, and kitasetaline, but also a novel sulfur-containing molecule, novkitasetaline. Structural elucidation identified novkitasetaline as a unique tryptamine-substituted kitasetaline derivative at the C-3 position of its pyridine ring. Notably, this structural modification conferred significant antimalarial activity to novkitasetaline, rendering it active against drug-sensitive Plasmodium falciparum 3D7 (IC₅₀ = 32.65 ± 2.93 μM) and three other drug-resistant P. falciparum strains: K13^C580Y, Dd2, and HB3 (IC₅₀ = 45.98 ± 4.17~59.67 ± 3.15 μM), primarily by disrupting late-stage parasite development. These efforts not only identified a promising antimalarial lead compound but also demonstrated that combining ribosome engineering with heterologous expression is an effective strategy for discovering bioactive natural products from Streptomyces. Full article

The aim of this study was to identify the causative agent of bacterial blight and basal glume rot of winter wheat that appeared in Serbia in 2023. To characterize the isolated bacteria (eight isolates in total), their cultural, biochemical, pathogenic, and genetic characteristics were examined. Based on the results of the LOPAT test, the isolates were classified into Pseudomonas Group Ia. The syrB and syrD genes were simultaneously detected in six wheat isolates—P0123, P0223, P0323, P0423, P0523, and P0823—while two isolates, P1123 and P1323, lacked both genes. Multilocus sequence typing (MLST) of the gapA, gltA, gyrB, and rpoD genes identified six isolates (P0123, P0223, P0323, P0423, P0523, and P0823) as Pseudomonas syringae pv. atrofaciens, whereas the remaining two isolates (P1123 and P1323) were most closely related to P. poae. Phylogenetic analysis revealed three genetically heterogeneous subgroups of P. syringae pv. atrofaciens among the wheat isolates from Serbia. Pathogenicity tests demonstrated that wheat isolates are able cause disease on wheat seedlings using three different inoculation methods: spraying the entire seedling, trimming the leaves before spraying, and wounding the leaves with multiple needles followed by spraying. Overall, isolates P0123 and P0423 were identified as the most virulent, inducing pronounced blight symptoms on wheat seedlings. In contrast, isolates P1123 and P1323 were weakly virulent and are therefore considered to be secondary or accompanying factors in plants already infected with more aggressive isolates, rather than primary pathogens responsible for disease development. This study contributes to a deeper understanding of the ecology, distribution, and pathogenic potential of bacterial communities associated with wheat blight disease in Serbia. Full article

Tomato is one of the most important vegetable crops in Costa Rica, where favorable environmental conditions enabled year-round production but also promote bacterial diseases. In recent years, pith necrosis has been frequently observed; nevertheless, the causal agents remain unidentified in the country. This study evaluated bacteria associated with symptomatic plants collected in the Central Valley of Costa Rica. From 32 plants, 61 bacterial isolates were obtained, described morphologically, and characterized through basic biochemical tests. Partial sequencing of the 16S rRNA gene revealed diverse bacterial taxa, predominantly belonging to the genus Pseudomonas. Thirteen isolates were selected for pathogenicity assays, which confirmed variable virulence levels. Multilocus sequence analysis based on concatenated sequences of the 16S rRNA, gyrB, rpoD, and rpoB genes identified Pseudomonas alliivorans LTM 13.1.2, P. flavescens LTM 14.2.2, and P. capsici LTM 78.3.2 as causal agents of pith necrosis. Additionally, P. straminea LTM 78.2.1 and Cedecea sp. LTM 72.2.1 caused tissue discoloration. Whole-genome sequencing of the two most virulent isolates (LTM 13.1.2 and LTM 78.3.2) supported their taxonomic classification and revealed virulence-associated genes and biosynthetic clusters. This study represents the first report of these Pseudomonas species as tomato pathogens in Costa Rica and expands their known distribution and host ranges. Full article

In recent years, the cultivated area of hazelnuts in Chile has increased significantly. Along with this rapid expansion, biotic constraints that affect the optimal development of the crop have been identified. Among these, bacterial blight disease caused by Xanthomonas arboricola pv. corylina has been particularly relevant. This pathogen has a global distribution and is present in all hazelnut-producing countries. In the spring of 2023, hazelnut orchards were sampled from the Maule to Biobío Regions of Chile. The Chilean isolates recovered from hazelnut tissues showing symptoms of bacterial blight were characterized by their ability to grow on different semi-selective media, their carbohydrate utilization profiles, hypersensitivity response in tobacco plants, and biochemical tests. Additionally, the isolates were identified based on the 16S rRNA gene and multilocus sequence analysis (MLSA) on the rpoD, gyrB, and atpD genes. The results showed that the X. arboricola pv. corylina Chilean isolates differed from previously reported isolates in other geographic areas as they are capable of metabolizing sorbitol and mannitol. Using MLSA and average nucleotide identity (ANI) comparison, these isolates were grouped into four and five phylogenetic clades, respectively, representing a significant difference from what has been reported in similar international studies. Full article

Indocalamus Nakai, a genus within the tribe Arundinarieae, has significant horticultural and economic value. However, its classification has long been challenging, and due to limited sampling, both intra- and intergeneric phylogenetic relationships, as well as its evolutionary history, remain unclear. In this study, extensive field surveys and comprehensive sample collection were conducted to address these challenges. A total of 31 complete plastomes of Indocalamus species were assembled. All plastomes exhibit a typical quadripartite structure, ranging in length from 139,555 bp to 139,791 bp, and contain 137 genes, including 90 protein-coding genes, 39 tRNA genes, and 8 rRNA genes. Phylogenetic analyses indicate that Indocalamus is polyphyletic and divided into three distinct clades (IV, V, and X). Based on integrated phylogenomic and morphological evidence, we propose a revised classification of Indocalamus into three major sections. Fossil-calibrated divergence estimates reveal that the major clades of Indocalamus are not monophyletic, highlighting a complex reticulate evolutionary history exemplifying the widespread rapid radiation observed in temperate woody bamboos. The intensification of the East Asian monsoon is likely to have played a key role in driving the rapid radiation of these lineages. Additionally, several clade-specific DNA barcodes (trnT-trnE, petN-trnC, petA-psbJ, and petD-rpoA) were identified, which will enhance the identification of Indocalamus and its closely related genera. This study, through extensive sampling and integration of morphological and molecular phylogenetic evidence, provides a preliminary delimitation of the genus Indocalamus, elucidates its complex evolutionary history, and lays a solid foundation for future systematic research and horticultural applications. Full article

The molecular identification of valuable genotypes is an important problem of germplasm management. In this study, we evaluated the potential of 11 universal primer pairs for the DNA barcoding of locally derived cultivars of subtropical crops (actinidia, feijoa, citrus, and tea). A total of 47 accessions (elite cultivars, forms, and breeding lines) of these four genera were included in the study. The efficiency of the following universal primers was assessed using Sanger sequencing: ITS-p5/ITS-u4, ITS-p5/ITS-u2, ITS-p3/ITS-u4, 23S,4.5S&5S, 16S, petB/petD, rpl23/rpl2.l, rpl2 intron, rpoC1 intron, trnK intron, and trnE-UUC/trnT-GUU. Among these primers, trnE-UUC/trnT-GUU showed greater intraspecific polymorphisms, while rpl2 intron and 16S displayed the lowest polymorphism levels in all crops. In addition, the 23S,4.5S & 5S, and rpoC1 intron were efficient for intraspecific analysis of tea and actinidia species. Using five efficient chloroplast primers, a total of 22/6 SNPs/InDels were observed in tea accessions, 45/17 SNPs/InDels in actinidia, 23/3 SNPs/InDels in mandarins, and 5/4 SNPs/InDels in feijoa. These results will be useful for the further development of DNA barcodes of related accessions. Full article