Project description:In this study, the intergeneric hybrids F1, F2, BC1F1, BC1F2, and BC2F1 from Elytrigia elongata and Triticum aestivum crosses were produced to study their chromosome pairing behavior. The average E. elongata chromosome configuration of the two F1 hybrids agreed with the theoretical chromosome configuration of 21I+7II, indicating that the genomic constitution of this F1 hybrid was ABDStStEeEbEx. Compared with the BC1F1 generation, the BC2F1 generation showed a rapid decrease in the number of E. elongata chromosomes and the BC1F2 generation showed a more extensive distribution of E. elongata chromosomes. In addition, pairing between wheat and E. elongata chromosomes was detected in each of the wheat-E. elongata hybrid progenies, albeit rarely. Our results demonstrated that genomic in situ hybridization (GISH) using an E. elongata genomic DNA probe offers a reliable approach for characterizing chromosome pairing in wheat and E. elongata hybrid progenies.
Project description:BACKGROUND:Trematodes have a complex life cycle with animal host changes and alternation of parthenogenetic and hermaphrodite generations. The parthenogenetic generation of the worm (rediae) from the first intermediate host Littorina littorea was used for chromosome spreads production. Karyotype description of parasitic flatworm Himasthla elongata Mehlis, 1831 (Digenea: Himasthlidae) based on fluorochrome banding and 18S rDNA mapping. RESULTS:Chromosome spreads were obtained from cercariae embryos and redial tissue suspensions with high pressure squash method.74.4 % of the analysed spreads contained 12 chromosome pairs (2n?=?24). Chromosome classification was performed according to the morphometry and nomenclature published. H. elongata spread chromosomes had a rather bead-like structure. Ideograms of DAPI-banded chromosomes contained 130 individual bands. According to flow cytometry data, the H. elongata genome contains 1.25 pg of DNA, so one band contains, on average, 9.4 Mb of DNA. Image bank captures of individual high-resolution DAPI-banded chromosomes were provided. Differential DAPI- and CMA3-staining revealed the chromatin areas that differed in AT- or GC-content. Both dyes stained chromosomes all along but with varying intensities in different areas. FISH revealed that vast majority (95.0 %) of interphase nuclei contained one signal for 18S rDNA. This corresponded to the number of nucleoli per cell detected by observations in vivo. The rDNA signal was observed on one or two homologs of chromosome 10 in 72.2 % of analysed chromosome spreads, therefore chromosome 10 possessed the main rDNA cluster and minor ones on chromosomes 3 and 6, that corresponds with AgNOR results. CONCLUSIONS:Himasthla elongata chromosomes variations presented as image bank. Differential chromosome staining with fluorochromes and FISH used for 18S rDNA mapping let us to conclude: (1) Himasthla elongata karyotype is 2n?=?24; (2) chromosome number deviates from the previously studied echinostomatids (2n?=?14-22); (3). Chromosome 10 possesses the main rDNA cluster with the minor ones existing on chromosomes 3 and 6.
Project description:For osmoadaptation the halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. H. elongata does not rely entirely on synthesis but can accumulate ectoine by uptake from the surrounding environment with the help of the osmoregulated transporter TeaABC. Disruption of the TeaABC-mediated ectoine uptake creates a strain that is constantly losing ectoine to the medium. However, the efflux mechanism of ectoine in H. elongata is not yet understood. H. elongata possesses four genes encoding mechanosensitive channels all of which belong to the small conductance type (MscS). Analysis by qRT-PCR revealed a reduction in transcription of the mscS genes with increasing salinity. The response of H. elongata to hypo- and hyperosmotic shock never resulted in up-regulation but rather in down-regulation of mscS transcription. Deletion of all four mscS genes created a mutant that was unable to cope with hypoosmotic shock. However, the knockout mutant grew significantly faster than the wildtype at high salinity of 2 M NaCl, and most importantly, still exported 80% of the ectoine compared to the wildtype. We thus conclude that a yet unknown system, which is independent of mechanosensitive channels, is the major export route for ectoine in H. elongata.
Project description:Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species.
Project description:The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.
Project description:Himanthalia elongata is a brown oceanic seaweed rich in bioactive compounds. It could play an important role in food production because of its antimicrobial and antioxidant properties. Three strains belonging to the Lactobacillus casei group (Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus rhamnosus) and a Bacillus subtilis strain were used for the solid-state fermentation of commercial seaweeds, and bacterial growth was monitored using the plate count method. High-pressure processing (HPP) was also employed (6000 bar, 5 min, 5 °C) before extraction. The antimicrobial activity of the extracts was tested in terms of the main food pathogenic bacteria (Salmonella spp., Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Bacillus cereus), and the phenolic content was estimated using the Folin-Ciocalteau method. In addition, targeted UHPLC-MS2 methods were used to unravel the profile of phlorotannins. H. elongata allowed the growth of the L. casei group strains and B. subtilis, showing the fermentability of this substrate. Significant antimicrobial activity toward L. monocytogenes was observed in the extracts obtained from unfermented samples, but neither fermentation nor HPP enhanced the natural antimicrobial activity of this seaweed species. The content in the phenolic compounds decreased because of the fermentation process, and the amount of phenolics in both the unfermented and fermented H. elongata extracts was very low. Despite phlorotannins being related to the natural antimicrobial activity of this brown seaweed, these results did not support this association. Even if fermentation and HPP were not proven to be effective tools for enhancing the useful compounds of H. elongata, the seaweed was shown to be a suitable substrate for L. casei group strains as well as for B. subtilis growth, and its extracts exhibited antimicrobial activity toward foodborne pathogens.
Project description:Salt tolerance in the ?-proteobacterium Halomonas elongata is linked to its ability to produce the compatible solute ectoine. The metabolism of ectoine production is of great interest since it can shed light on the biochemical basis of halotolerance as well as pave the way for the improvement of the biotechnological production of such compatible solute. Ectoine belongs to the biosynthetic family of aspartate-derived amino-acids. Aspartate is formed from oxaloacetate, thereby connecting ectoine production to the anaplerotic reactions that refill carbon into the tricarboxylic acid cycle (TCA cycle). This places a high demand on these reactions and creates the need to regulate them not only in response to growth but also in response to extracellular salt concentration. In this work, we combine modeling and experiments to analyze how these different needs shape the anaplerotic reactions in H. elongata. First, the stoichiometric and thermodynamic factors that condition the flux distributions are analyzed, then the optimal patterns of operation for oxaloacetate production are calculated. Finally, the phenotype of two deletion mutants lacking potentially relevant anaplerotic enzymes: phosphoenolpyruvate carboxylase (Ppc) and oxaloacetate decarboxylase (Oad) are experimentally characterized. The results show that the anaplerotic reactions in H. elongata are indeed subject to evolutionary pressures that differ from those faced by other gram-negative bacteria. Ectoine producing halophiles must meet a higher metabolic demand for oxaloacetate and the reliance of many marine bacteria on the Entner-Doudoroff pathway compromises the anaplerotic efficiency of Ppc, which is usually one of the main enzymes fulfilling this role. The anaplerotic flux in H. elongata is contributed not only by Ppc but also by Oad, an enzyme that has not yet been shown to play this role in vivo. Ppc is necessary for H. elongata to grow normally at low salt concentrations but it is not required to achieve near maximal growth rates as long as there is a steep sodium gradient. On the other hand, the lack of Oad presents serious difficulties to grow at high salt concentrations. This points to a shared role of these two enzymes in guaranteeing the supply of oxaloacetate for biosynthetic reactions.
Project description:Neisseria elongata, a common oral bacterium, has been recognized as a cause of infections such as infective endocarditis, septicemia, and osteomyelitis. Neisseria-induced infective endocarditis, although infrequently reported, typically arises after dental procedures. Without antibiotic therapy, its complications can be severe. We report the case of a 27-year-old man who presented with fever, severe dyspnea, and a leg abscess from cellulitis. An echocardiogram showed a vegetation-like echogenic structure on the septal leaflet of the patient's native tricuspid valve, and an insignificant Gerbode defect. Three blood cultures grew gram-negative, antibiotic-susceptible coccobacilli that were confirmed to be N. elongata. Subsequent DNA sequencing conclusively isolated N. elongata subsp nitroreducens as the organism responsible for the infective endocarditis. The patient recovered after 21 days of antibiotic therapy. In addition to the patient's unusual case, we discuss the nature and isolation of N. elongata and its subspecies.