Transferase Family Research Articles

Regulation of wheat yield by soil multifunctionality and metagenomic-based microbial degradation potentials under crop rotations

Crop rotation benefits soil fertility and crop yield by providing organic components including cellulose, lignin, chitin, and glucans that are mainly degraded by soil microbial carbohydrate-active enzymes (CAZymes). However, the impacts of crop rotation on soil microbial CAZyme genes are not well understood. Hence, CAZyme genes and families involved in the degradation of differentially originated organic components were investigated using metagenomics among distinct crop rotations. Crop rotation had a more significant effect on soil nitrogen than on carbon fractions with higher content in the complex rotation referring to alfalfa (Medicago sativa L.; 4 year)-potato (Solanum tuberosum L.; 1 year)-winter wheat (3 year; A4PoW3). The composition of soil microbial CAZyme genes related to the degradation of fungi-derived components was more affected by crop rotation compared with the degradation of plant- and bacteria-derived components. The total abundance of CAZyme genes and families was significantly higher in the complex rotation. Notably, CAZyme genes belonging to glycoside hydrolase and glycosyl transferase families had more connections in their network. Moreover, key genes including CE4, GH20, and GH23 assembled toward the middle of the network, and were significantly regulated by dominant soil nitrogen fractions including soil potential nitrogen mineralization and microbial biomass nitrogen. Soil multifunctionality was mostly explained by the composition and total abundance of CAZyme genes, but wheat grain yield was profoundly regulated by fungi-derived components degradation genes under effects of dominant nitrogen fractions. Overall, the findings provide deep insight into the degradation potentials of soil microbial CAZyme genes for developing sustainable crop rotational agroecosystems.

UGT1A7 altered HER2-positive breast cancer response to trastuzumab by affecting epithelial-to-mesenchymal transition: A potential biomarker to identify patients resistant to trastuzumab treatment.

HER2-positive (HER2+) breast cancer accounts for 20-30% of all breast cancers. Although trastuzumab has significantly improved the survival of patients with HER2+ breast cancer, more than 70% of patients develop drug resistance within one year of treatment. Differential-gene-expression analysis of trastuzumab-sensitive and resistant HER2+ breast cancer cell lines from GSE15043 was performed to identify the biomarkers associated with trastuzumab resistance. Differential biomarker expression was confirmed in FFPE tissues collected from clinical HER2+ breast cancer tumor samples that were sensitive or resistant to trastuzumab treatment. UGT1A7, a member of the uronic acid transferase family, was associated with trastuzumab resistance. UGT1A7 expression was downregulated in trastuzumab-resistant tumor tissues and in a cell line that developed trastuzumab resistance (BT474TR). Overexpressing UGT1A7 in BT474TR restored their sensitivity to trastuzumab treatment, whereas downregulating UGT1A7 expression in parental cells led to trastuzumab resistance. Importantly, UGT1A7 localized to the endoplasmic reticulum and altered stress responses. Furthermore, downregulating UGT1A7 expression promoted epithelial-to-mesenchymal transition (EMT) by affecting TWIST, SNAIL, and GRP78 expression and the AMP-activated protein kinase signaling pathway, thus contributing to trastuzumab resistance. This study demonstrated the important role and novel mechanisms of UGT1A7 in tumor responses to trastuzumab. Low UGT1A7 expression plays an important role in EMT and contributes to trastuzumab resistance. UGT1A7 has the potential to be developed as a biomarker for identifying patients who are resistant to trastuzumab treatment.

O-glycosylation of SARS-CoV-2 spike protein by host O-glycosyltransferase strengthens its trimeric structure.

Protein O-glycosylation, also known as mucin-type O-glycosylation, is one of the most abundant glycosylation in mammalian cells. It is initially catalyzed by a family of polypeptide GalNAc transferases (ppGalNAc-Ts). The trimeric spike protein (S) of SARS-CoV-2 is highly glycosylated and facilitates the virus's entry into host cells and membrane fusion of the virus. However, the functions and relationship between host ppGalNAc-Ts and O-glycosylation on the S protein remain unclear. Herein, we identify 15 O-glycosites and 10 distinct O-glycan structures on the S protein using an HCD-product-dependent triggered ETD mass spectrometric analysis. We observe that the isoenzyme T6 of ppGalNAc-Ts (ppGalNAc-T6) exhibits high O-glycosylation activity for the S protein, as demonstrated by an on-chip catalytic assay. Overexpression of ppGalNAc-T6 in HEK293 cells significantly enhances the O-glycosylation level of the S protein, not only by adding new O-glycosites but also by increasing O-glycan heterogeneity. Molecular dynamics simulations reveal that O-glycosylation on the protomer-interface regions, modified by ppGalNAc-T6, potentially stabilizes the trimeric S protein structure by establishing hydrogen bonds and non-polar interactions between adjacent protomers. Furthermore, mutation frequency analysis indicates that most O-glycosites of the S protein are conserved during the evolution of SARS-CoV-2 variants. Taken together, our finding demonstrate that host O-glycosyltransferases dynamically regulate the O-glycosylation of the S protein, which may influence the trimeric structural stability of the protein. This work provides structural insights into the functional role of specific host O-glycosyltransferases in regulating the O-glycosylation of viral envelope proteins.

Transcriptomic Association Analysis of the Metabolic Mechanism of Sulfamethoxazole in Channel Catfish (Ictalurus punctatus).

Sulfamethoxazole is a widely used antimicrobial drug used to treat bacterial diseases in aquaculture. To understand the gene expression in channel catfish liver after treatment with sulfamethoxazole, in this study, the treatment group received sulfamethoxazole (100 mg/kg bw), which was administered orally once, and samples were taken at 5 h, 12 h, and 6 d after the administration of sulfamethoxazole, while the control group was orally administered sterile water. To further identify potentially significant genes, a transcriptome analysis using RNA-seq was carried out. More than 50 million high-quality reads were found. After filtering and quality analysis, these reads were identified as 54,169,682, 51,313,865, 51,608,845, and 49,333,491. After counting 23,707 of these transcripts for gene expression, it was discovered that 14,732 of them had genes with differential expression. Moreover, we found that the annotation with the most GO variation was "cellular process" (1616 genes), "metabolic process" (1268 genes), "binding" (1889 genes), and "catalytic activity" (1129 genes). KEGG pathways showed that the "metabolic pathway" was the pathway that was significantly enriched in both experimental groups when comparing the experimental groups: 5 h and 12 h (128 genes); 5 h and 6 d (332 genes); and 12 h and 6 d (348 genes). Also, UDP- glucuronosyltransferase (ugt), which is associated with glucuronidation, and UDP-glucuronosyltransferase 2C1-like (ugt2a1) showed significant upregulation. Carboxylesterase 5A-like (ces3), which promotes fatty acyl and cholesteryl ester metabolism, and the glutathione transferase family were upregulated in the expression of sulfamethoxazole metabolism in the liver, which significantly affected the metabolic effects of the drug. Meanwhile, dypd, uck2b, and rrm2, which are related to nucleotide synthesis and metabolism, were upregulated. Our study extends the knowledge of gene expression in drug metabolism in channel catfish and further provides insight into the molecular mechanism of sulfamethoxazole metabolism.

Association of maternal phthalates exposure and metabolic gene polymorphisms with congenital heart diseases: a multicenter case-control study

BackgroundThe majority of congenital heart diseases (CHDs) are thought to result from the interactions of genetics and the environment factors. This study aimed to assess the association of maternal non-occupational phthalates exposure, metabolic gene polymorphisms and their interactions with risk of CHDs in offspring.MethodsA multicenter case-control study of 245 mothers with CHDs infants and 268 control mothers of health infant was conducted from six hospitals. Maternal urinary concentrations of eight phthalate metabolites were measured by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Twenty single nucleotide polymorphisms (SNPs) in cytochrome P450 family 2 subfamily C member 9 (CYP2C9) and 19 (CYP2C19), uridine diphosphate (UDP) glucuronosyl transferase family 1 member A7 (UGT1A7), family 2 member B7 (UGT2B7) and B15(UGT2B15) genes were genotyped. The multivariate logistic regressions were used to estimate the association between maternal phthalates exposure or gene polymorphisms and risk of CHDs. Generalized multifactor dimensionality reduction (GMDR) was used to analyze the gene–gene and gene–phthalates exposure interactions.ResultsThere was no significant difference in phthalate metabolites concentrations between the cases and controls. No significant positive associations were observed between maternal exposure to phthalates and CHDs. The SNPs of UGT1A7 gene at rs4124874 (under three models, log-additive: aOR = 1.74, 95% CI:1.28–2.37; dominant: aOR = 1.86, 95% CI:1.25–2.78; recessive: aOR = 2.50, 95% CI: 1.26–4.94) and rs887829 (under the recessive model: aOR = 13.66, 95% CI: 1.54–121) were significantly associated with an increased risk of CHDs. Furthermore, the associations between rs4124874 (under log-additive and dominant models) of UGT1A7 were statistically significant after the false discovery rate correction. No significant gene-gene or gene-phthalate metabolites interactions were observed.ConclusionsThe polymorphisms of maternal UGT1A7 gene at rs4124874 and rs887829 were significantly associated with an increased risk of CHDs. More large-scale studies or prospective study designs are needed to confirm or refute our findings in the future.

Disruption of a glycosyl transferase family 17 protein alters cadmium accumulation and resistance in rice (Oryza sativa)

Disruption of a glycosyl transferase family 17 protein alters cadmium accumulation and resistance in rice (Oryza sativa)

OR16-02 Histone ADP-Ribosylation Site Mutations As Oncogenic Drivers In Breast And Ovarian Cancers

Abstract Disclosure: D. Huang: None. C.V. Camacho: None. A. Nagari: None. W.L. Kraus: Other; Self; - Founder and stockholder for Ribon Therapeutics, Inc. - Founder, stockholder, scientific advisory board member, member of the board of directors for ARase Therapeutics, Inc. - Coholder of U.S. Patent. Recent studies have identified oncohistones, which are encoded by histone genes that harbor missense mutations, as a new class of cancer drivers. We found that many of the oncohistone mutations identified with the highest frequencies in human cancers occur on glutamate (Glu, E) and aspartate (Asp, D) residues, which are potential sites of ADP-ribosylation (ADPRylation). ADPRylation is a post-translational modification, which is catalyzed by the PARP family of ADP-ribosyl transferases and results in the covalent attachment of ADP-ribose on protein substrates. Unlike other well characterized histone modifications, the functions of histone ADPRylation are poorly characterized. Mass spectrometry analyses confirmed that 20 Glu or Asp residues subjected to oncohistone mutation are bona fide sites of ADPRylation by PARP-1. Ectopic expression of each of six different oncohistone mutants from three different core histones (H2B, H3, and H4) altered the proliferation of at least 2 out of 3 cancer cell lines, including MCF-7 and MDA-MB-231 breast cancer cells, as well as OVCAR-3 ovarian cancer cells. We focused on H2B-D51, whose mutation resulted in enhanced cell proliferation in both of the breast cancer cell lines for further characterization of its potential transcription regulatory effects. Proteomic (histone PTMs) and genomic (ATAC-Seq, ChIP-Seq and RNA-Seq) analyses revealed that loss of ADPRylation on H2B-D51 by mutation to alanine (Ala, A) or asparagine (Asn, N) dramatically enhanced p300-mediated acetylation of H2B, altered chromatin accessibility, and changed the pattern of gene expression in MDA-MB-231 cells. To determine the effects of H2B-D51 ADPRylation on cancer cell growth in vivo and their responses to the FDA-approved PARP inhibitor Niraparib, we performed xenograft tumor growth assays in immunocompromised mice using MDA-MB-231 cells ectopically expressing wild-type or D51A mutant histone H2B. Ectopic expression of the H2B-D51A mutant led to a significant increase in tumor growth versus wild-type H2B. Interestingly, these effects were inhibited by treatment with Niraparib, suggesting that the H2B-D51A mutant alone does not confer resistance to PARP inhibitor. Taken together, this study demonstrates that functional sites of Glu/Asp ADPRylation on histones are mutated in cancers, allowing cancer cells to escape the growth-regulating effects of ADPRylation. (This work was supported by grants from the NIH/NIDDK and CPRIT, and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K.) Presentation: Friday, June 16, 2023

Screening of wild species and transcriptome profiling to identify differentially regulated genes in response to late blight resistance in potato.

Late blight (Phytophthora infestans) is a serious disease of potatoes. The aim of this study was to screen wild potato species and identify differentially expressed genes (DEGs) associated with late blight resistance. Wild potato species such as PIN45 (Solanum pinnatisectum), CPH62 (Solanum cardiophyllum), JAM07 (Solanum jamesii), MCD24 (Solanum microdontum), PLD47 (Solanum polyadenium), and cv. Kufri Bahar (control) were tested by artificial inoculation of P. infestans under controlled conditions. Transcriptomes of the leaf tissues (96h post-inoculation) were sequenced using the Illumina platform. Statistically significant (p < 0.05) DEGs were analyzed in wild species by comparison with the control, and upregulated (>2 log2 fold change, FC) and downregulated (<-2 log2 FC) genes were identified. DEGs were functionally characterized with Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Selected genes were validated by real-time PCR analysis to confirm RNA-seq results. We identified some upregulated genes associated with late blight resistance in wild species such as cytochrome P450, proline-rich protein, MYB transcription factor MYB139, ankyrin repeat-containing protein, and LRR receptor-like serine/threonine-protein kinase in PIN45; glucosyltransferase, fructose-bisphosphate aldolase, and phytophthora-inhibited protease 1 in CPH62; steroid binding protein and cysteine proteinase 3 in JAM07; glycine-rich cell wall structural protein 1 and RING finger protein in MCD24; and cysteine proteinase 3 and major latex protein in PLD47. On the other hand, downregulated genes in these species were snakin-2 and WRKY transcription factor 3 in PIN45; lichenase and phenylalanine ammonia-lyase 1 in CPH62; metallothionein and LRR receptor-like serine/threonine-protein kinase in JAM07; UDP-glucoronosyl/UDP-glucosyl transferase family protein and steroid binding protein in MCD24; and cytoplasmic small heat shock protein class I and phosphatase PLD47. Our study identified highly resistant wild potato species and underlying genes such as disease resistance, stress response, phytohormones, and transcription factors (e.g., MYB, WRKY, AP2/ERF, and AN1) associated with late blight resistance in wild potato species.

Autonomous action and cooperativity between the ONECUT2 transcription factor and its 3' untranslated region.

The transcription factor ONECUT2 (OC2) is a master transcriptional regulator operating in metastatic castration-resistant prostate cancer that suppresses androgen receptor activity and promotes neural differentiation and tumor cell survival. OC2 mRNA possesses an unusually long (14,575nt), evolutionarily conserved 3' untranslated region (3' UTR) with many microRNA binding sites, including up to 26 miR-9 sites. This is notable because miR-9 targets many of the same genes regulated by the OC2 protein. Paradoxically, OC2 expression is high in tissues with high miR-9 expression. The length and complex secondary structure of OC2 mRNA suggests that it is a potent master competing endogenous RNA (ceRNA) capable of sequestering miRNAs. Here, we describe a novel role for OC2 3' UTR in lethal prostate cancer consistent with a function as a ceRNA. A plausible ceRNA network in OC2-driven tumors was constructed computationally and then confirmed in prostate cancer cell lines. Genes regulated by OC2 3' UTR exhibited high overlap (up to 45%) with genes driven by the overexpression of the OC2 protein in the absence of 3' UTR, indicating a cooperative functional relationship between the OC2 protein and its 3' UTR. These overlapping networks suggest an evolutionarily conserved mechanism to reinforce OC2 transcription by protection of OC2-regulated mRNAs from miRNA suppression. Both the protein and 3' UTR showed increased polycomb-repressive complex activity. The expression of OC2 3' UTR mRNA alone (without protein) dramatically increased the metastatic potential by in vitro assays. Additionally, OC2 3' UTR increased the expression of Aldo-Keto reductase and UDP-glucuronyl transferase family genes responsible for altering the androgen synthesis pathway. ONECUT2 represents the first-described dual-modality transcript that operates as both a key transcription factor driving castration-resistant prostate cancer and a master ceRNA that promotes and protects the same transcriptional network.

QSAR study of benzofuran and indole derivatives to predict new compounds as histone lysine methyl transferase inhibitors

Initiation and progression of several diseases by post-translational histone modifications are considered a worldwide problem. Enhancer of Zeste Homologue 2 (EZH2), which belongs to the histone-lysine N-methyl transferase (HKMT) family, has been emphasised as a promising target for cancer therapy. It is a major challenge for the scientific community to find novel approaches to treating this disease. In this study, a series of 51 derivatives of the benzofuran and indole families, previously experimentally evaluated against HKMT, was used to develop the best model with promising anticancer activity. The multiple linear regression (MLR) method, implemented in QSARINS software, was used with a genetic algorithm for variable selection. According to QSARINS, the model with two descriptors (minHBint4 and Wlambdal.unity) was found to be the best and its parameters fit well, and its validation was well established. The applicability domain was also validated for this model. Furthermore, its robustness (R2 = 0.9328), stability (Q2LOO = 0.9212, Q2LMO = 0.9187), and good predictive power (R2ext = 0.929) were also verified. Hence, this model was assumed to have predictive HKMT anticancer activity for designing active compounds. Molecular docking was also performed to identify binding interactions, and new molecules with better predicted biological activity (pIC50) were designed. The binding energy of the three designed compounds demonstrated higher binding activity at the target receptor, followed by complex stability, determined by a 100 ns molecular dynamics simulation and binding free energy calculation. Density functional theory (DFT) and pharmacokinetic analyses also confirmed their drug-like properties. Finally, it can be declared that the proposed tools allow rapid and economical identification of potential anti-HKMT drugs (anticancer drugs) for further development.

Co-conserved sequence motifs are predictive of substrate specificity in a family of monotopic phosphoglycosyl transferases.

Monotopic phosphoglycosyl transferases (monoPGTs) are an expansive superfamily of enzymes that catalyze the first membrane-committed step in the biosynthesis of bacterial glycoconjugates. MonoPGTs show a strong preference for their cognate nucleotide diphospho-sugar (NDP-sugar) substrates. However, despite extensive characterization of the monoPGT superfamily through previous development of a sequence similarity network comprising >38,000 nonredundant sequences, the connection between monoPGT sequence and NDP-sugar substrate specificity has remained elusive. In this work, we structurally characterize the C-terminus of a prototypic monoPGT for the first time and show that 19 C-terminal residues play a significant structural role in a subset of monoPGTs. This new structural information facilitated the identification of co-conserved sequence "fingerprints" that predict NDP-sugar substrate specificity for this subset of monoPGTs. A Hidden Markov model was generated that correctly assigned the substrate of previously unannotated monoPGTs. Together, these structural, sequence, and biochemical analyses have delivered new insight into the determinants guiding substrate specificity of monoPGTs and have provided a strategy for assigning the NDP-sugar substrate of a subset of enzymes in the superfamily that use UDP-di-N-acetyl bacillosamine. Moving forward, this approach may be applied to identify additional sequence motifs that serve as fingerprints for monoPGTs of differing UDP-sugar substrate specificity.

Activity-dependent post-translational regulation of palmitoylating and depalmitoylating enzymes in the hippocampus.

Activity-induced changes in protein palmitoylation can regulate the plasticity of synaptic connections, critically impacting learning and memory. Palmitoylation is a reversible post-translational modification regulated by both palmitoyl-acyl transferases that mediate palmitoylation and palmitoyl thioesterases that depalmitoylate proteins. However, it is not clear how fluctuations in synaptic activity can mediate the dynamic palmitoylation of neuronal proteins. Using primary hippocampal cultures, we demonstrate that synaptic activity does not impact the transcription of palmitoylating and depalmitoylating enzymes, changes in thioesterase activity, or post-translational modification of the depalmitoylating enzymes of the ABHD17 family and APT2 (also known as LYPLA2). In contrast, synaptic activity does mediate post-translational modification of the palmitoylating enzymes ZDHHC2, ZDHHC5 and ZDHHC9 (but not ZDHHC8) to influence protein-protein interactions, enzyme stability and enzyme function. Post-translational modifications of the ZDHHC enzymes were also observed in the hippocampus following fear conditioning. Taken together, our findings demonstrate that signaling events activated by synaptic activity largely impact activity of the ZDHHC family of palmitoyl-acyl transferases with less influence on the activity of palmitoyl thioesterases.

GmGSTU23 Encoding a Tau Class Glutathione S-Transferase Protein Enhances the Salt Tolerance of Soybean (Glycine max L.).

Salt stress has a detrimental impact on crop yield, quality, and profitability. The tau-like glutathione transferases (GSTs) represent a significant group of enzymes that play a crucial role in plant stress responses, including salt stress. In this study, we identified a tau-like glutathione transferase family gene from soybean named GmGSTU23. Expression pattern analysis revealed that GmGSTU23 was predominantly expressed in the roots and flowers and exhibited a concentration-time-specific pattern in response to salt stress. Transgenic lines were generated and subjected to phenotypic characterization under salt stress. The transgenic lines exhibited increased salt tolerance, root length, and fresh weight compared to the wild type. Antioxidant enzyme activity and malondialdehyde content were subsequently measured, and the data revealed no significant differences between the transgenic and wild-type plants in the absence of salt stress. However, under salt stress, the wild-type plants exhibited significantly lower activities of SOD, POD, and CAT than the three transgenic lines, whereas the activity of APX and the content of MDA showed the opposite trend. We identified changes in glutathione pools and associated enzyme activity to gain insights into the underlying mechanisms of the observed phenotypic differences. Notably, under salt stress, the transgenic Arabidopsis's GST activity, GR activity, and GSH content were significantly higher than those of the wild type. In summary, our findings suggest that GmGSTU23 mediates the scavenging of reactive oxygen species and glutathione by enhancing the activity of glutathione transferase, thereby conferring enhanced tolerance to salt stress in plants.

Streptomyces lividans 66 produces a protease inhibitor via a tRNA-utilizing enzyme interacting with a C-minus NRPS.

Small peptide aldehydes (SPAs) with protease inhibitory activity are naturally occurring compounds shown to be synthesized by nonribosomal peptide synthetases (NRPS). SPAs are widely used in biotechnology and have been utilized as therapeutic agents. They are also physiologically relevant and have been postulated to regulate the development of their producing microorganisms. Previously, we identified an NRPS-like biosynthetic gene cluster in Streptomyces lividans 66 that lacked a condensation (C) domain but included a tRNA-Utilizing Enzyme (tRUE) belonging to the leucyl/phenylalanyl (L/F) transferase family. This system was predicted to direct the synthesis of a novel SPA, which we named livipeptin. Using evolutionary genome mining approaches, here we confirm the presence of L/F transferase tRUEs within the genomes of diverse Streptomyces and related organisms, including fusions with the anticipated C-minus NRPS-like protein. We then demonstrate genetic functional cooperation between the identified L/F-transferase divergent tRUE homolog with the C-minus NRPS, leading to the synthesis of a metabolic fraction with protease inhibitory activity. Semisynthetic assays in the presence of RNAse revealed that the productive interaction between the tRUE and the C-minus NRPS enzymes is indeed tRNA-dependent. We expect our findings to boost the discovery of SPAs, as well as the development of protease-mediated biotechnologies, by exploiting the uncovered genetic basis for synthesizing putative acetyl-leu/phe-arginine protease inhibitors. Furthermore, these results will facilitate the purification and structural elucidation of livipeptin, which has proven difficult to chemically characterize. Significance The discovery of natural products biosynthetic genes marks a significant advancement in our understanding of these metabolites, e.g. of their evolution, activity and biosynthesis, but also opens biotechnological opportunities and knowledge to advance genome mining approaches. We made this possible by uncovering a new biosynthetic pathway in Streptomyces lividans 66 shown to direct the synthesis of a strong protease inhibitor, termed livipeptin, following unprecedented biosynthetic rules and genes. Thus, by shedding light on the genetic mechanisms predicted to govern the production of acetyl-leu/phe-arginine protease inhibitors, including the elusive livipeptin, this study enables novel protease-mediated biotechnologies as well as approaches for discovering protease inhibitors from genome data.

Modification of plant cell walls with hydroxycinnamic acids by BAHD acyltransferases.

In the last decade it has become clear that enzymes in the "BAHD" family of acyl-CoA transferases play important roles in the addition of phenolic acids to form ester-linked moieties on cell wall polymers. We focus here on the addition of two such phenolics-the hydroxycinnamates, ferulate and p-coumarate-to two cell wall polymers, glucuronoarabinoxylan and to lignin. The resulting ester-linked feruloyl and p-coumaroyl moities are key features of the cell walls of grasses and other commelinid monocots. The capacity of ferulate to participate in radical oxidative coupling means that its addition to glucuronoarabinoxylan or to lignin has profound implications for the properties of the cell wall - allowing respectively oxidative crosslinking to glucuronoarabinoxylan chains or introducing ester bonds into lignin polymers. A subclade of ~10 BAHD genes in grasses is now known to (1) contain genes strongly implicated in addition of p-coumarate or ferulate to glucuronoarabinoxylan (2) encode enzymes that add p-coumarate or ferulate to lignin precursors. Here, we review the evidence for functions of these genes and the biotechnological applications of manipulating them, discuss our understanding of mechanisms involved, and highlight outstanding questions for future research.

A New Face of the Old Gene: Deletion of the PssA, Encoding Monotopic Inner Membrane Phosphoglycosyl Transferase in Rhizobium leguminosarum, Leads to Diverse Phenotypes That Could Be Attributable to Downstream Effects of the Lack of Exopolysaccharide.

The biosynthesis of subunits of rhizobial exopolysaccharides is dependent on glycosyltransferases, which are usually encoded by large gene clusters. PssA is a member of a large family of phosphoglycosyl transferases catalyzing the transfer of a phosphosugar moiety to polyprenol phosphate; thus, it can be considered as priming glycosyltransferase commencing synthesis of the EPS repeating units in Rhizobium leguminosarum. The comprehensive analysis of PssA protein features performed in this work confirmed its specificity for UDP-glucose and provided evidence that PssA is a monotopic inner membrane protein with a reentrant membrane helix rather than a transmembrane segment. The bacterial two-hybrid system screening revealed interactions of PssA with some GTs involved in the EPS octasaccharide synthesis. The distribution of differentially expressed genes in the transcriptome of the ΔpssA mutant into various functional categories indicated complexity of cell response to the deletion, which can mostly be attributed to the lack of exopolysaccharide and downstream effects caused by such deficiency. The block in the EPS biosynthesis at the pssA step, potentially leading to an increased pool of UDP-glucose, is likely to be filtered through to other pathways, and thus the absence of EPS may indirectly affect the expression of proteins involved in these pathways.

Disruption of MenT2 toxin impairs the growth of Mycobacterium tuberculosis in guinea pigs.

Toxin-antitoxin (TA) systems are abundantly present in the genomes of various bacterial pathogens. TA systems have been implicated in either plasmid maintenance or protection against phage infection, stress adaptation or disease pathogenesis. The genome of Mycobacterium tuberculosis encodes for more than 90 TA systems and 4 of these belong to the type IV subfamily (MenAT family). The toxins and antitoxins belonging to type IV TA systems share sequence homology with the AbiEii family of nucleotidyl transferases and the AbiEi family of putative transcriptional regulators, respectively. Here, we have performed experiments to understand the role of MenT2, a toxin from the type IV TA system, in mycobacterial physiology and disease pathogenesis. The ectopic expression of MenT2 using inducible vectors does not inhibit bacterial growth in liquid cultures. Bioinformatic and molecular modelling analysis suggested that the M. tuberculosis genome has an alternative start site upstream of the annotated menT2 gene. The overexpression of the reannotated MenT2 resulted in moderate growth inhibition of Mycobacterium smegmatis. We show that both menT2 and menA2 transcript levels are increased when M. tuberculosis is exposed to nitrosative stress, in vitro. When compared to the survival of the wild-type and the complemented strain, the ΔmenT2 mutant strain of M. tuberculosis was more resistant to being killed by nitrosative stress. However, the survival of both the ΔmenT2 mutant and the wild-type strain was similar in macrophages and when exposed to other stress conditions. Here, we show that MenT2 is required for the establishment of disease in guinea pigs. Gross pathology and histopathology analysis of lung tissues from guinea pigs infected with the ∆menT2 strain revealed significantly reduced tissue damage and inflammation. In summary, these results provide new insights into the role of MenT2 in mycobacterial pathogenesis.

Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I.

Rhamnogalacturonan I (RG-I) is a major plant cell wall pectic polysaccharide defined by its repeating disaccharide backbone structure of [4)-α-D-GalA-(1,2)-α-L-Rha-(1,]. A family of RG-I:Rhamnosyltransferases (RRT) has previously been identified, but synthesis of the RG-I backbone has not been demonstrated in vitro because the identity of Rhamnogalacturonan I:Galaturonosyltransferase (RG-I:GalAT) was unknown. Here a putative glycosyltransferase, At1g28240/MUCI70, is shown to be an RG-I:GalAT. The name RGGAT1 is proposed to reflect the catalytic activity of this enzyme. When incubated together with the rhamnosyltransferase RRT4, the combined activities of RGGAT1 and RRT4 result in elongation of RG-I acceptors in vitro into a polymeric product. RGGAT1 is a member of a new GT family categorized as GT116, which does not group into existing GT-A clades and is phylogenetically distinct from the GALACTURONOSYLTRANSFERASE (GAUT) family of GalA transferases that synthesize the backbone of the pectin homogalacturonan. RGGAT1 has a predicted GT-A fold structure but employs a metal-independent catalytic mechanism that is rare among glycosyltransferases with this fold type. The identification of RGGAT1 and the 8-member Arabidopsis GT116 family provides a new avenue for studying the mechanism of RG-I synthesis and the function of RG-I in plants.

Molecular characteristics of glutathione transferase gene family in a neglect medical Spirometra tapeworm.

The Spirometra mansoni is a neglect medical tapeworm, its plerocercoid larvae can parasitize in humans and animals, causing sparganosis. In this study, 17 new members of the glutathione transferase (GST) family were sequenced and characterized in S. mansoni. Clustering analysis displayed the categorization of SmGSTs into two main clades. RT-qPCR illustrated that 7 GST genes were highly expressed in the plerocercoid stage while 8 GSTs were highly expressed in the adult. rSmGST has the typical C- and N-terminal double domains of glutathione transferase. Immunolocalization revealed that natural SmGST is mainly located in the epidermis and parenchyma of plerocercoid, and in the epidermis, parenchyma, uterus and egg shell of adult worm. The optimum activity for rSmGST was found to be pH 6.5 and 25°C. The evolutionary tree showed a high level of diversity of cestodes GSTs. SmGSTs contained both conserved family members and members in the process of further diversification. The findings in this study will lay a foundation to better explore the underlying mechanisms of GSTs involved in Spirometra tapeworms.

Genome-Wide Identification and Expression Analysis of Isopentenyl transferase Family Genes during Development and Resistance to Abiotic Stresses in Tea Plant (Camellia sinensis).

The tea plant is an important economic crop and is widely cultivated. Isopentenyl transferase (IPT) is the first and rate-limiting enzyme of cytokinin (CK) signaling, which plays key roles in plant development and abiotic stress. However, the IPT gene family in tea plants has not been systematically investigated until now. The phylogenetic analyses, gene structures, and conserved domains were predicted here. The results showed that a total of 13 CsIPT members were identified from a tea plant genome database and phylogenetically classified into four groups. Furthermore, 10 CsIPT members belonged to plant ADP/ATP-IPT genes, and 3 CsIPTs were tRNA-IPT genes. There is a conserved putative ATP/GTP-binding site (P-loop motif) in all the CsIPT sequences. Based on publicly available transcriptome data as well as through RNA-seq and qRT-PCR analysis, the CsIPT genes which play key roles in the development of different tissues were identified, respectively. Furthermore, CsIPT6.2 may be involved in the response to different light treatments. CsIPT6.4 may play a key role during the dormancy and flush of the lateral buds. CsIPT5.1 may play important regulatory roles during the development of the lateral bud, leaf, and flower. CsIPT5.2 and CsIPT6.2 may both play key roles for increased resistance to cold-stress, whereas CsIPT3.2 may play a key role in improving resistance to high-temperature stress as well as drought-stress and rewatering. This study could provide a reference for further studies of CsIPT family’s functions and could contribute to tea molecular breeding.