Identification of an Enterococcus faecium strain isolated from raw bovine milk co-harbouring the oxazolidinone resistance genes optrA and poxtA in China.

Oxazolidinones are potent antimicrobial agents used to treat human infections caused by multidrug-resistant Gram-positive bacteria. The growing resistance to oxazolidinones poses a significant threat to public health. In August 2021, a linezolid-resistant Enterococcus faecium BN83 was isolated from a raw milk sample of cow in Inner Mongolia, China. This isolate exhibited a multidrug resistance phenotype and was resistant to most of drugs tested including linezolid and tedizolid. PCR detection showed that two mobile oxazolidinones resistance genes, optrA and poxtA, were present in this isolate. Whole genome sequencing analysis revealed that the genes optrA and poxtA were located on two different plasmids, designated as pBN83-1 and pBN83-2, belonging to RepA_N and Inc18 families respectively. Genetic context analysis suggested that optrA gene on plasmid pBN83-1 was located in transposon Tn6261 initially found in E. faecalis. Comprehensive analysis revealed that Tn6261 act as an important horizontal transmission vector for the spread of optrA in E. faecium. Additionally, poxtA-bearing pBN83-2 displayed high similarity to numerous plasmids from Enterococcus of different origin and pBN83-2-like plasmid represented a key mobile genetic element involved in movement of poxtA in enterococcal species. The presence of optrA- and poxtA-carrying E. faecium in raw bovine milk represents a public health concern and active surveillance is urgently warranted to investigate the prevalence of oxazolidinone resistance genes in animal-derived food products.

Preparation of Ni based mesoporous Al2O3 catalyst with enhanced CO2 methanation performance.

A Ni based mesoporous γ-Al2O3 (MA) catalyst was prepared via partial hydrolysis without organic surfactants and employed in the carbon dioxide methanation reaction. The obtained catalysts were characterized by N2 adsorption-desorption, H2-TPR, XRD, XPS, TG, SEM and TEM-EDS techniques. CO2 methanation was performed in a fixed-bed reactor. A high surface area of MA with excellent hydrothermal stability was obtained, which promoted the dispersion of nickel species, producing a better catalytic performance. Incorporation of more NiO species into the Ni/MA catalyst increased the amount of active metallic Ni sties, further improving the catalytic activity and CH4 selectivity. Moreover, the monolithic skeleton of MA with fabric-like walls suppressed the aggregation of active metallic Ni sites and carbon deposition, enhancing the catalyst's stability, which provides a new insight for potential industrial applications.

FLOWERING LOCUS T duplication coordinates reproductive and vegetative growth in perennial poplar.

Annual plants grow vegetatively at early developmental stages and then transition to the reproductive stage, followed by senescence in the same year. In contrast, after successive years of vegetative growth at early ages, woody perennial shoot meristems begin repeated transitions between vegetative and reproductive growth at sexual maturity. However, it is unknown how these repeated transitions occur without a developmental conflict between vegetative and reproductive growth. We report that functionally diverged paralogs FLOWERING LOCUS T1 (FT1) and FLOWERING LOCUS T2 (FT2), products of whole-genome duplication and homologs of Arabidopsis thaliana gene FLOWERING LOCUS T (FT), coordinate the repeated cycles of vegetative and reproductive growth in woody perennial poplar (Populus spp.). Our manipulative physiological and genetic experiments coupled with field studies, expression profiling, and network analysis reveal that reproductive onset is determined by FT1 in response to winter temperatures, whereas vegetative growth and inhibition of bud set are promoted by FT2 in response to warm temperatures and long days in the growing season. The basis for functional differentiation between FT1 and FT2 appears to be expression pattern shifts, changes in proteins, and divergence in gene regulatory networks. Thus, temporal separation of reproductive onset and vegetative growth into different seasons via FT1 and FT2 provides seasonality and demonstrates the evolution of a complex perennial adaptive trait after genome duplication.

Enhanced phytoremediation of volatile environmental pollutants with transgenic trees.

Small, volatile hydrocarbons, including trichloroethylene, vinyl chloride, carbon tetrachloride, benzene, and chloroform, are common environmental pollutants that pose serious health effects. We have developed transgenic poplar (Populus tremula x Populus alba) plants with greatly increased rates of metabolism and removal of these pollutants through the overexpression of cytochrome P450 2E1, a key enzyme in the metabolism of a variety of halogenated compounds. The transgenic poplar plants exhibited increased removal rates of these pollutants from hydroponic solution. When the plants were exposed to gaseous trichloroethylene, chloroform, and benzene, they also demonstrated superior removal of the pollutants from the air. In view of their large size and extensive root systems, these transgenic poplars may provide the means to effectively remediate sites contaminated with a variety of pollutants at much faster rates and at lower costs than can be achieved with current conventional techniques.

Poplar (Populus spp.).

Although species within the genus Populus are, in general, easier to transform and regenerate in vitro than most other trees, many poplar species are very recalcitrant. Many protocols that previously have been reported were developed for a specific genotype or species. Thus, it has often been necessary to re-optimize a protocol each time research is initiated with a new genotype. The method presented in this chapter has been effective for a wide variety of poplar genotypes.

Transgenic modification of gai or rgl1 causes dwarfing and alters gibberellins, root growth, and metabolite profiles in Populus.

In Arabidopsis and other plants, gibberellin (GA)-regulated responses are mediated by proteins including GAI, RGA and RGL1-3 that contain a functional DELLA domain. Through transgenic modification, we found that DELLA-less versions of GAI (gai) and RGL1 (rgl1) in a Populus tree have profound, dominant effects on phenotype, producing pleiotropic changes in morphology and metabolic profiles. Shoots were dwarfed, likely via constitutive repression of GA-induced elongation, whereas root growth was promoted two- to threefold in vitro. Applied GA(3 )inhibited adventitious root production in wild-type poplar, but gai/rgl1 poplars were unaffected by the inhibition. The concentrations of bioactive GA(1) and GA(4) in leaves of gai- and rgl1-expressing plants increased 12- to 64-fold, while the C(19) precursors of GA(1) (GA(53), GA(44) and GA(19)) decreased three- to ninefold, consistent with feedback regulation of GA 20-oxidase in the transgenic plants. The transgenic modifications elicited significant metabolic changes. In roots, metabolic profiling suggested increased respiration as a possible mechanism of the increased root growth. In leaves, we found metabolite changes suggesting reduced carbon flux through the lignin biosynthetic pathway and a shift towards allocation of secondary storage and defense metabolites, including various phenols, phenolic glucosides, and phenolic acid conjugates.

Transgenic sterility in Populus: expression properties of the poplar PTLF, Agrobacterium NOS and two minimal 35S promoters in vegetative tissues.

Transgenic sterility is a desirable trait for containment of many kinds of transgenes and exotic species. Genetically engineered floral sterility can be imparted by expression of a cytotoxin under the control of a predominantly floral-tissue-specific promoter. However, many otherwise desirable floral promoters impart substantial non-floral expression, which can impair plant health or make it impossible to regenerate transgenic plants. We are therefore developing a floral sterility system that is capable of attenuating undesired background vegetative expression. As a first step towards this goal, we compared the vegetative expression properties of the promoter of the poplar (Populus trichocarpa Torr. & Gray) homolog of the floral homeotic gene LEAFY (PTLF), which could be used to impart male and female flower sterility, to that of three candidate attenuator-gene promoters: the cauliflower mosaic virus (CaMV) 35S basal promoter, the CaMV 35S basal promoter fused to the TMV omega element and the nopaline synthase (NOS) promoter. The promoters were evaluated via promoter::GUS gene fusions in a transgenic poplar hybrid (Populus tremula L. x P. alba L.) by both histochemical and fluorometric GUS assays. In leaves, the NOS promoter conveyed the highest activity and had a mean expression level 5-fold higher than PTLF, whereas the CaMV 35S basal promoter fused to the omega element and the CaMV 35S basal promoter alone directed mean expression levels that were 0.5x and 0.35x that of PTLF, respectively. Differential expression in shoots, leaves, stems and roots was observed only for the NOS and PTLF promoters. Strongest expression was observed in roots for the NOS promoter, whereas the PTLF promoter directed highest expression in shoots. The NOS promoter appears best suited to counteract vegetative expression of a cytotoxin driven by the PTLF promoter where 1:1 toxin:attenuator expression is required.

Gene and enhancer trap tagging of vascular-expressed genes in poplar trees.

We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the beta-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS expression phenotypes are dominant and are scored in primary transformants, this system does not require rounds of sexual recombination, a typical barrier to developmental genetic studies in trees. Gene and enhancer trap lines defining genes expressed during primary and secondary vascular development were identified and characterized. Collectively, the vascular gene expression patterns revealed that approximately 40% of genes expressed in leaves were expressed exclusively in the veins, indicating that a large set of genes is required for vascular development and function. Also, significant overlap was found between the sets of genes responsible for development and function of secondary vascular tissues of stems and primary vascular tissues in other organs of the plant, likely reflecting the common evolutionary origin of these tissues. Chromosomal DNA flanking insertion sites was amplified by thermal asymmetric interlaced PCR and sequenced and used to identify insertion sites by reference to the nascent Populus trichocarpa genome sequence. Extension of the system was demonstrated through isolation of full-length cDNAs for five genes of interest, including a new class of vascular-expressed gene tagged by enhancer trap line cET-1-pop1-145. Poplar gene and enhancer traps provide a new resource that allows plant biologists to directly reference the poplar genome sequence and identify novel genes of interest in forest biology.

Activation tagging of a dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature.

We identified a dwarf transgenic hybrid poplar (Populus tremula x Populus alba) after screening of 627 independent activation-tagged transgenic lines in tissue culture, greenhouse, and field environments. The cause of the phenotype was a hyperactivated gene encoding GA 2-oxidase (GA2ox), the major gibberellin (GA) catabolic enzyme in plants. The mutation resulted from insertion of a strong transcriptional enhancer near the transcription start site. Overexpression of the poplar GA2ox gene (PtaGA2ox1) caused hyperaccumulation of mRNA transcripts, quantitative shifts in the spectrum of GAs, and similarity in phenotype to transgenic poplars that overexpress a bean (Phaseolus coccineus) GA2ox gene. The poplar PtaGA2ox1 sequence was most closely related to PsGA2ox2 from pea (Pisum sativum) and two poorly known GA2oxs from Arabidopsis (AtGA2ox4 and AtGA2ox5). The dwarf phenotype was reversible through gibberellic acid application to the shoot apex. Transgenic approaches to producing semidwarf trees for use in arboriculture, horticulture, and forestry could have significant economic and environmental benefits, including altered fiber and fruit production, greater ease of management, and reduced risk of spread in wild populations.