TriticeaeSSRdb: a comprehensive database of simple sequence repeats in Triticeae.

Microsatellites, known as simple sequence repeats (SSRs), are short tandem repeats of 1 to 6 nucleotide motifs found in all genomes, particularly eukaryotes. They are widely used as co-dominant markers in genetic analyses and molecular breeding. Triticeae, a tribe of grasses, includes major cereal crops such as bread wheat, barley, and rye, as well as abundant forage and lawn grasses, playing a crucial role in global food production and agriculture. To enhance genetic work and expedite the improvement of Triticeae crops, we have developed TriticeaeSSRdb, an integrated and user-friendly database. It contains 3,891,705 SSRs from 21 species and offers browsing options based on genomic regions, chromosomes, motif types, and repeat motif sequences. Advanced search functions allow personalized searches based on chromosome location and length of SSR. Users can also explore the genes associated with SSRs, design customized primer pairs for PCR validation, and utilize practical tools for whole-genome browsing, sequence alignment, and in silico SSR prediction from local sequences. We continually update TriticeaeSSRdb with additional species and practical utilities. We anticipate that this database will greatly facilitate trait genetic analyses and enhance molecular breeding strategies for Triticeae crops. Researchers can freely access the database at http://triticeaessrdb.com/.

The complete chloroplast genome and phylogenetic analysis of Bauhinia glauca subsp. hupehana (Craib) T. C. Chen 1988.

Bauhinia glauca subsp. hupehana (Craib) T. C. Chen 1988, a member of the Leguminosae family, Cercidoideae subfamily, and Bauhinia genus, has a rich history of traditional usage in Chinese medicine. Renowned for its analgesic properties, it is commonly employed for managing inflammation and pain. This study aimed to sequence the complete chloroplast genome of B. glauca subsp. hupehana using Illumina paired-end sequencing data. The chloroplast genome spans 156,967 bp and consists of four main regions: the large single-copy (LSC) region (89,185 bp), the small single-copy (SSC) region (19,146 bp), and a pair of inverted repeats (IRs) (24,318 bp). The overall GC content of the chloroplast genome is 36.19%, with specific values of 33.99%, 29.79%, and 42.76% for the LSC, SSC, and IR regions, respectively. A total of 128 genes were annotated in the chloroplast genome, including 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis revealed that B. glauca subsp. hupehana is closely related to Bauhinia racemose, indicating a sister taxon relationship between the two species. This study significantly contributes to the chloroplast genomic resource for Bauhinia, laying the groundwork for future phylogenetic investigations within the genus.

De Novo Biosynthesis of Anisyl Alcohol and Anisyl Acetate in Engineered Escherichia coli.

Anisyl alcohol and its ester anisyl acetate are both important fragrance compounds and have a wide range of applications in the cosmetics, perfumery, and food industries. The currently commercially available anisyl alcohol and anisyl acetate are based on chemical synthesis. However, consumers increasingly prefer natural fragrance compounds. Therefore, it is of great significance to construct microbial cell factories to produce anisyl alcohol and anisyl acetate. In this study, we first established a biosynthetic pathway in engineered Escherichia coli MG1655 for the production of anisyl alcohol from simple carbon sources. We further increased the anisyl alcohol production to 355 mg/L by the increasing availability of erythrose-4-phosphate and phosphoenolpyruvate. Finally, we further demonstrated the production of anisyl acetate by overexpressing alcohol acetyltransferase ATF1 for the subsequent acetylation of anisyl alcohol to produce anisyl acetate. To our knowledge, this is the first report on the biosynthesis of anisyl alcohol and anisyl acetate directly from a renewable carbon source.

De novo biosynthesis of N-acetyltyramine in engineered Escherichia coli.

N-acetyltyramine as a tyramine alkaloid has drawn great attention because of its excellent anti-free radical, antithrombotic, and antitumour activity. Therefore, it is an attractive compound. In this study, we reported for the first time the construction a synthetic pathway of N-acetyltyramine in engineered Escherichia coli. First, the tyrosine decarboxylase tdc gene and arylalkylamine N-acyltransferase aanat gene were introduced into E. coli to generate a recombinant N-acetyltyramine producer with L-tyrosine as substrate. Subsequently, overexpressing aroGfbr and TyrAfbr enhance the availability of L-tyrosine to achieve de novo biosynthesis of N-acetyltyramine from glucose. Finally, overexpressing the transketolase I tktA and phosphoenolpyruvate synthase ppsA genes improved the N-acetyltyramine production to 854 mg/L.

De novo biosynthesis of τ-cadinol in engineered Escherichia coli.

τ-Cadinol is a sesquiterpene that is widely used in perfume, fine chemicals and medicines industry. In this study, we established a biosynthetic pathway for the first time in engineered Escherichia coli for production of τ-cadinol from simple carbon sources. Subsequently, we further improved the τ-cadinol production to 35.9 ± 4.3 mg/L by optimizing biosynthetic pathway and overproduction of rate-limiting enzyme IdI. Finally, the titer was increased to 133.5 ± 11.2 mg/L with a two-phase organic overlay-culture medium system. This study shows an efficient method for the biosynthesis of τ-cadinol in E. coli with the heterologous hybrid MVA pathway.

Structural Analysis of an l-Cysteine Desulfurase from an Ssp DNA Phosphorothioation System.

DNA phosphorothioate (PT) modification, in which the nonbridging oxygen in the sugar-phosphate backbone is substituted by sulfur, is catalyzed by DndABCDE or SspABCD in a double-stranded or single-stranded manner, respectively. In Dnd and Ssp systems, mobilization of sulfur in PT formation starts with the activation of the sulfur atom of cysteine catalyzed by the DndA and SspA cysteine desulfurases, respectively. Despite playing the same biochemical role, SspA cannot be functionally replaced by DndA, indicating its unique physiological properties. In this study, we solved the crystal structure of Vibrio cyclitrophicus SspA in complex with its natural substrate, cysteine, and cofactor, pyridoxal phosphate (PLP), at a resolution of 1.80 Å. Our solved structure revealed the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor, suggesting a common binding mode shared by cysteine desulfurases. In addition, although the distance between the catalytic Cys314 and the substrate cysteine is 8.9 Å, which is too far for direct interaction, our structural modeling and biochemical analysis revealed a conformational change in the active site region toward the cysteine substrate to move them close to each other to facilitate the nucleophilic attack. Finally, the pulldown analysis showed that SspA could form a complex with SspD, an ATP pyrophosphatase, suggesting that SspD might potentially accept the activated sulfur atom directly from SspA, providing further insights into the biochemical pathway of Ssp-mediated PT modification.IMPORTANCE Apart from its roles in Fe-S cluster assembly, tRNA thiolation, and sulfur-containing cofactor biosynthesis, cysteine desulfurase serves as a sulfur donor in the DNA PT modification, in which a sulfur atom substitutes a nonbridging oxygen in the DNA phosphodiester backbone. The initial sulfur mobilization from l-cysteine is catalyzed by the SspA cysteine desulfurase in the SspABCD-mediated DNA PT modification system. By determining the crystal structure of SspA, the study presents the molecular mechanism that SspA employs to recognize its cysteine substrate and PLP cofactor. To overcome the long distance (8.9 Å) between the catalytic Cys314 and the cysteine substrate, a conformational change occurs to bring Cys314 to the vicinity of the substrate, allowing for nucleophilic attack.

LI1035, a putative effector secreted by Lawsonia intracellularis, targets the MAPK pathway and regulates actin organizationin yeast and mammalian cells.

Lawsonia intracellularis is an obligate intracellular Gram-negative bacterium that has been identified as the etiological agent of the contagious disease proliferative enteropathy (PE) in a wide range of animals, mainly pigs. The genome sequence of L. intracellularis indicates that this bacterium possess a type III secretion system (T3SS), which may assist the bacterium during cell invasion and host innate immune system evasion and could be a mechanism for inducing cellular proliferation. However, the effectors secreted by the T3SS (T3Es) of L. intracellularis have not been reported. T3Es often target conserved eukaryotic cellular processes, and yeast is an established and robust model system in which to reveal their function. By screening the growth inhibition of an ordered array of Saccharomyces cerevisiae strains expressing the hypothetical genes of L. intracellularis, LI1035 was identified as the first putative effector that inhibits yeast growth. The LI1035-induced growth inhibition was rescued in two of the 14 mitogen-activated protein kinase (MAPK) yeast haploid deletion strains, suggesting that LI1035 interacts with the components of the MAPK pathway in yeast. Phosphorylation assays confirmed that LI1035 inhibits MAPK signaling cascades in yeast and mammalian cells. Actin staining assays revealed that LI1035 regulates actin organization in yeast and mammalian cells. Taken together, these results indicate that LI1035 alters MAPK pathway activity and regulates actin organization in the host. These findings may contribute to the understanding the pathogenesis of L. intracellularis and support the use of yeast as a heterologous system for the functional analysis of pathogen-specific gene products in the laboratory.

Disinfection effect of povidone-iodine in aquaculture water of swamp eel (Monopterus albus).

The swamp eel (Monopterus albus) is an important commercial farmed fish species in China. However, it is susceptible to Aeromonas hydrophila infections, resulting in high mortality and considerable economic loss. Povidone-iodine (PVP-I) is a widely used chemical disinfectant in aquaculture, which can decrease the occurrence of diseases and improve the survival. However, environmental organic matter could affect the bactericidal effectiveness of PVP-I, and the efficacy of PVP-I in aquaculture water is still unknown. In this paper, disinfection assays were conducted to evaluate the effectiveness of PVP-I against the A. hydrophila in different types of water. We found that the effective germicidal concentration of PVP-I in outdoor aquaculture water was 25 ppm for 12 h. In indoor aquaculture water with 105 CFU/mL bacteria, 10 ppm and 20 ppm of PVP-I could kill 99% and 100% of the bacteria, respectively. The minimal germicidal concentration of PVP-I in Luria-Bertani broth was 4,000 ppm. Available iodine content assay in LB solutions confirmed that the organic substance had negative impact on the effectiveness of PVP-I, which was consistent with the different efficacy of PVP-I in different water samples. Acute toxicity tests showed that the 24 h-LC50 of PVP-I to swamp eel was 173.82 ppm, which was much higher than the germicidal concentrations in outdoor and indoor aquaculture water, indicating its safety and effectivity to control the A. hydrophila. The results indicated PVP-I can be helpful for preventing the transmission of A. hydrophila in swamp eel aquaculture.

Expression and biochemical characterization of α-ketoglutarate decarboxylase from cyanobacterium Synechococcus sp. PCC7002.

α-Ketoglutarate decarboxylase (α-KGD), one member of α-keto acid decarboxylases, catalyzing non-oxidative decarboxylation of α-ketoglutarate to form succinic semialdehyde, was proposed to play critical role in completing tricarboxylic acid (TCA) cycle of cyanobacteria. Although the catalytic function of α-KGD from Synechococcus sp. PCC7002 was demonstrated previously, there was no detailed biochemical characterization of α-KGD from Synechococcus sp. PCC7002 yet. In this study, the gene encoding α-KGD from Synechococcus sp. PCC7002 was amplified and soluble expression of recombinant α-KGD was achieved by coexpressing with pTf16 chaperone plasmid in E. coli BL21 (DE3). Kinetic analysis showed that the activity of α-KGD was dependent on cofactors of thiamine pyrophosphate and divalent cation. Meanwhile this α-KGD was specific for α-ketoglutarate with respect to the decarboxylation activity despite of the pretty low activity of acetolactate synthase. The catalytic efficiency of α-KGD (the values of kcat and kcat/Km for α-ketoglutarate were 1.2s-1 and 6.3×103M-1s-1, respectively) might provide evidence for its physiological role in TCA cycle of Synechococcus sp. PCC7002.

Kinetic characterization and structural modeling of an NADP+-dependent succinic semialdehyde dehydrogenase from Anabaena sp. PCC7120.

Succinic semialdehyde dehydrogenases (SSADH) of cyanobacteria played a pivotal role in completing the cyanobacterial tricarboxylic acid cycle. The structural information of cofactor preference and catalysis for SSADH from cyanobacteria is currently available. However, the detailed kinetics of SSADH from cyanobacteria were not characterized yet. In this study, an all3556 gene encoding SSADH from Anabaena sp. PCC7120 (ApSSADH) was amplified and the recombinant ApSSADH was purified homogenously. Kinetic analysis showed that ApSSADH was an NADP+-dependent SSADH, which utilized NADP+ and succinic semialdehyde (SSA) as its preferred substrates and the activity of ApSSADH was inhibited by its substrate of SSA. At the same time, the Ser157 residue was found to function as the determinant of cofactor preference. Further study demonstrated that activity and substrate inhibition of ApSSADH would be greatly reduced by the mutation of the residues at the active site. Bioinformatic analysis indicated that those residues were highly conserved throughout the SSADHs. To our knowledge this is the first report exploring the detailed kinetics of SSADH from cyanobacteria.

Expression, purification and characterization of sll1981 protein from cyanobacterium Synechocystis sp. PCC6803.

The sll1981 protein from cyanobacterium Synechocystis sp. PCC6803 had been reported to exhibit acetolactate synthase (ALS) and L-myo-inositol-1-phosphate synthase (MIPS) activities previously. Based on amino acids sequences alignment, sll1981 protein was postulated to function as α-ketoglutarate decarboxylase (α-KGD), which played important role in completing cyanobacterial tricarboxylic acid (TCA) cycle. However the detailed enzymatic kinetics of sll1981 as ALS, MIPS and α-KGD were not determined yet. In this study, the recombinant sll1981 protein was purified from supernatant of E. coli cell and the substrate specificity of sll1981 towards pyruvate, d-glucose-6-phosphate and α-ketoglutarate was examined using homogenous recombinant sll1981. Steady-state kinetics results showed that sll1981 was a dual functional enzyme, which displayed much higher activity as α-KGD than as ALS. At the same time the MIPS activity of sll1981 was not detectable, although it was reported to be as MIPS previously. These findings not only confirmed the previous statement of the function of sll1981 as ALS and disputed the claimed function of sll1981 as MIPS, but also affirmed the new function of sll1981 as α-KGD. Therefore sll1981 was probably a key enzyme in completing the TCA cycle of Synechocystis sp. PCC6803.

In vivo mutational characterization of DndE involved in DNA phosphorothioate modification.

DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and RP stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.