Detection of Vancomycin Resistant Genes in Intrinsically Antibiotic Resistant Bacteria from the Gut Microbiota of Indonesian Individuals.

Background: Antibiotic resistance is a global public health concern that has been exacerbated by the overuse and misuse of antibiotics, leading to the emergence of resistant bacteria. The gut microbiota, often influenced by antibiotic usage, plays a crucial role in overall health. Therefore, this study aimed to investigate the prevalence of antibiotic resistant genes in the gut microbiota of Indonesian coastal and highland populations, as well as to identify vancomycin-resistant bacteria and their resistant genes. Methods: Stool samples were collected from 22 individuals residing in Pacet, Mojokerto, and Kenjeran, Surabaya Indonesia in 2022. The read count of antibiotic resistant genes was analyzed in the collected samples, and the bacterium concentration was counted by plating on the antibiotic-containing agar plate. Vancomycin-resistant strains were further isolated, and the presence of vancomycin-resistant genes was detected using a multiplex polymerase chain reaction (PCR). Results: The antibiotic resistant genes for tetracycline, aminoglycosides, macrolides, beta-lactams, and vancomycin were found in high frequency in all stool samples (100%) of the gut microbiota. Meanwhile, those meant for chloramphenicol and sulfonamides were found in 86% and 16% of the samples, respectively. Notably, vancomycin-resistant genes were found in 16 intrinsically resistant Gram-negative bacterial strains. Among the detected vancomycin-resistant genes, vanG was the most prevalent (27.3%), while vanA was the least prevalent (4.5%). Conclusion: The presence of multiple vancomycin resistance genes in intrinsically resistant Gram-negative bacterial strains demonstrated the importance of the gut microbiota as a reservoir and hub for the horizontal transfer of antibiotic resistant genes.

Predictable and stable epimutations induced during clonal plant propagation with embryonic transcription factor.

Clonal propagation is frequently used in commercial plant breeding and biotechnology programs because it minimizes genetic variation, yet it is not uncommon to observe clonal plants with stable phenotypic changes, a phenomenon known as somaclonal variation. Several studies have linked epigenetic modifications induced during regeneration with this newly acquired phenotypic variation. However, the factors that determine the extent of somaclonal variation and the molecular changes underpinning this process remain poorly understood. To address this gap in our knowledge, we compared clonally propagated Arabidopsis thaliana plants derived from somatic embryogenesis using two different embryonic transcription factors- RWP-RK DOMAIN-CONTAINING 4 (RKD4) or LEAFY COTYLEDON2 (LEC2) and from two epigenetically distinct founder tissues. We found that both the epi(genetic) status of the explant and the regeneration protocol employed play critical roles in shaping the molecular and phenotypic landscape of clonal plants. Phenotypic variation in regenerated plants can be largely explained by the inheritance of tissue-specific DNA methylation imprints, which are associated with specific transcriptional and metabolic changes in sexual progeny of clonal plants. For instance, regenerants were particularly affected by the inheritance of root-specific epigenetic imprints, which were associated with an increased accumulation of salicylic acid in leaves and accelerated plant senescence. Collectively, our data reveal specific pathways underpinning the phenotypic and molecular variation that arise and accumulate in clonal plant populations.

Soil Mineral Composition and Salinity Are the Main Factors Regulating the Bacterial Community Associated with the Roots of Coastal Sand Dune Halophytes.

Soil salinity and mineral deficiency are major problems in agriculture. Many studies have reported that plant-associated microbiota, particularly rhizosphere and root microbiota, play a crucial role in tolerance against salinity and mineral deficiency. Nevertheless, there are still many unknown parts of plant-microbe interaction, especially regarding their role in halophyte adaptation to coastal ecosystems. Here, we report the bacterial community associated with the roots of coastal sand dune halophytes Spinifex littoreus and Calotropis gigantea, and the soil properties that affect their composition. Strong correlations were observed between root bacterial diversity and soil mineral composition, especially with soil Calcium (Ca), Titanium (Ti), Cuprum (Cu), and Zinc (Zn) content. Soil Ti and Zn content showed a positive correlation with bacterial diversity, while soil Ca and Cu had a negative effect on bacterial diversity. A strong correlation was also found between the abundance of several bacterial species with soil salinity and mineral content, suggesting that some bacteria are responsive to changes in soil salinity and mineral content. Some of the identified bacteria, such as Bacillus idriensis and Kibdelosporangium aridum, are known to have growth-promoting effects on plants. Together, the findings of this work provided valuable information regarding bacterial communities associated with the roots of sand dune halophytes and their interactions with soil properties. Furthermore, we also identified several bacterial species that might be involved in tolerance against stresses. Further work will be focused on isolation and transplantation of these potential microbes, to validate their role in plant tolerance against stresses, not only in their native hosts but also in crops.

The Underlying Nature of Epigenetic Variation: Origin, Establishment, and Regulatory Function of Plant Epialleles.

In plants, the gene expression and associated phenotypes can be modulated by dynamic changes in DNA methylation, occasionally being fixed in certain genomic loci and inherited stably as epialleles. Epiallelic variations in a population can occur as methylation changes at an individual cytosine position, methylation changes within a stretch of genomic regions, and chromatin changes in certain loci. Here, we focus on methylated regions, since it is unclear whether variations at individual methylated cytosines can serve any regulatory function, and the evidence for heritable chromatin changes independent of genetic changes is limited. While DNA methylation is known to affect and regulate wide arrays of plant phenotypes, most epialleles in the form of methylated regions have not been assigned any biological function. Here, we review how epialleles can be established in plants, serve a regulatory function, and are involved in adaptive processes. Recent studies suggest that most epialleles occur as byproducts of genetic variations, mainly from structural variants and Transposable Element (TE) activation. Nevertheless, epialleles that occur spontaneously independent of any genetic variations have also been described across different plant species. Here, we discuss how epialleles that are dependent and independent of genetic architecture are stabilized in the plant genome and how methylation can regulate a transcription relative to its genomic location.

Fermentation of Jamaican Cherries Juice Using Lactobacillus plantarum Elevates Antioxidant Potential and Inhibitory Activity against Type II Diabetes-Related Enzymes.

Jamaican cherry (Muntinga calabura Linn.) is tropical tree that is known to produce edible fruit with high nutritional and antioxidant properties. However, its use as functional food is still limited. Previous studies suggest that fermentation with probiotic bacteria could enhance the functional properties of non-dairy products, such as juices. In this study, we analyze the metabolite composition and activity of Jamaican cherry juice following fermentation with Lactobacillus plantarum FNCC 0027 in various substrate compositions. The metabolite profile after fermentation was analyzed using UPLC-HRMS-MS and several bioactive compounds were detected in the substrate following fermentation, including gallic acid, dihydrokaempferol, and 5,7-dihydroxyflavone. We also found that total phenolic content, antioxidant activities, and inhibition of diabetic-related enzymes were enhanced after fermentation using L. plantarum. The significance of its elevation depends on the substrate composition. Overall, our findings suggest that fermentation with L. plantarum FNCC 0027 can improve the functional activities of Jamaican cherry juice.

A new role for histone demethylases in the maintenance of plant genome integrity.

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development, and adaptation to environmental fluctuations of most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis, EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the transgenerational inheritance of histone marks, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases play a dual role in plants by helping to maintain transcriptional states through development and safeguard genome integrity during sexual reproduction.

Partial maintenance of organ-specific epigenetic marks during plant asexual reproduction leads to heritable phenotypic variation.

Plants differ from animals in their capability to easily regenerate fertile adult individuals from terminally differentiated cells. This unique developmental plasticity is commonly observed in nature, where many species can reproduce asexually through the ectopic initiation of organogenic or embryogenic developmental programs. While organ-specific epigenetic marks are not passed on during sexual reproduction, the fate of epigenetic marks during asexual reproduction and the implications for clonal progeny remain unclear. Here we report that organ-specific epigenetic imprints in Arabidopsis thaliana can be partially maintained during asexual propagation from somatic cells in which a zygotic program is artificially induced. The altered marks are inherited even over multiple rounds of sexual reproduction, becoming fixed in hybrids and resulting in heritable molecular and physiological phenotypes that depend on the identity of the founder tissue. Consequently, clonal plants display distinct interactions with beneficial and pathogenic microorganisms. Our results demonstrate how novel phenotypic variation in plants can be unlocked through altered inheritance of epigenetic marks upon asexual propagation.

Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity.

Inducible epigenetic changes in eukaryotes are believed to enable rapid adaptation to environmental fluctuations. We have found distinct regions of the Arabidopsis genome that are susceptible to DNA (de)methylation in response to hyperosmotic stress. The stress-induced epigenetic changes are associated with conditionally heritable adaptive phenotypic stress responses. However, these stress responses are primarily transmitted to the next generation through the female lineage due to widespread DNA glycosylase activity in the male germline, and extensively reset in the absence of stress. Using the CNI1/ATL31 locus as an example, we demonstrate that epigenetically targeted sequences function as distantly-acting control elements of antisense long non-coding RNAs, which in turn regulate targeted gene expression in response to stress. Collectively, our findings reveal that plants use a highly dynamic maternal 'short-term stress memory' with which to respond to adverse external conditions. This transient memory relies on the DNA methylation machinery and associated transcriptional changes to extend the phenotypic plasticity accessible to the immediate offspring.

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana.

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)-induced single base substitutions differed substantially from those of "background" mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations.

Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.

De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks.

Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.