Identification, sequencing and mutagenesis of the gene for a d-carbamoylase from Agrobacterium radiobacter

Abstract A clone positive for d-carbamoylase activity (2.7 kb Hin dIII- Bam H1 DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da. The d-carbamoylase gene. named cauA , was placed under the control of T7 RNA-dependent promoter and expressed in E. coli BL21 (DE3). After induction with isopropyl-thio-β-d-galactopyranoside, the synthesis of d-carbamoylase in E. coli reached about 40% of the total protein. The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced allowed us to establish that a Pro¹⁴→Leu¹⁴ exchange leads to an inactive enzyme species, while a Cys²⁷⁹→Ser²⁷⁹ exchange did not impair the functional properties of the enxyme.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Short-chain fatty acids and polyamines in the pathogenesis of necrotizing enterocolitis: Kinetics aspects in gnotobiotic quails

Despite years of investigation, pathogenesis of necrotizing enterocolitis (NEC) remains elusive. Bacterial metabolites were implicated by several authors but their roles remain controversial. The aim of our study was to investigate the role of SCFAs and polyamines through a kinetic study of histological and macroscopical digestive lesions in monobiotic quails. Germ-free quails, inoculated with a Clostridium butyricum strain involved in a NEC case, were fed or not with a diet including lactose (7%). Quails were sacrificed at various times between D7 and D24 after bacterial inoculation. NEC-like lesions, i.e. thickening, pneumatosis, and hemorrhages, occurred only in lactose-fed quails and increased with time. The main histological characteristics were infiltrates of mononuclear cells, then heterophilic cells, then gas cyst and necrosis. The first event observed, before histological and macroscopical lesions, is a high production of butyric acid, which precedes an increase of iNOS gene expression. No difference in polyamines contents depending on the diet was observed. These results show the major role of butyric acid produced by commensal bacteria in the onset of the digestive lesions.     

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.     

A Cysteine Endopeptidase (“Dionain”) Is Involved in the Digestive Fluid of Dionaea muscipula (Venus’s Fly-trap)

The carnivorous plant Dionaea muscipula (Venus’s flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS–PAGE band protein. The name “dionain” is proposed for the enzyme.     

Human Cytidine Triphosphate Synthetase 1 Interacting Proteins

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.     

The relationship between Pseudo-nitzschia (Peragallo) and domoic acid in Scottish shellfish

The diatom genus Pseudo-nitzschia (Peragallo) associated with the production of domoic acid (DA), the toxin reposnsible for amnesic shellfish poisoning, is abundant in Scottish waters. A two year study examined the relationship between Pseudo-nitzschia cells in the water column and DA concentration in blue mussels (Mytilus edulis) at two sites, and king scallops (Pecten maximus) at one site. The rate of DA uptake and depuration differed greatly between the two species with M. edulis whole tissue accumulating and depurating 7 μg g⁻¹ (now expressed as mg kg⁻¹) per week. In contrast, it took 12 weeks for DA to depurate from P. maximus gonad tissue from a concentration of 68 μg g⁻¹ (now mg kg⁻¹) to <20 μg g⁻¹ (now mg kg^‐1). The DA depuration rate from P. maximus whole tissue was <5% per week during both years of the study. Correlations between the Pseudo-nitzschia cell densities and toxin concentrations were weak to moderate for M. edulis and weak for P. maximus. Seasonal diversity on a species level was observed within the Pseudo-nitzschia genus at both sites with more DA toxicity associated with summer/autumn Pseudo-nitzschia blooms when P. australis was observed in phytoplankton samples. This study reveals the marked difference in DA uptake and depuration in two shellfish species of commercial importance in Scotland. The use of these shellfish species to act as a proxy for DA in the environment still requires investigation.     

New pyranonaphthazarin and 2-naphthoic acid derivatives from the mangrove endophytic fungus Leptosphaerulina sp. SKS032

Two new compounds, named leptospyranonaphthazarin A (1) and leptosnaphthoic acid A (2), together with four known compounds (3–6) were isolated from an endophytic fungus Leptosphaerulina sp. SKS032. Their structures were assigned using spectroscopic methods, computational methods, and single-crystal X-ray diffraction analysis. In the antibiotic assay, compounds 1, 2, and 6 exhibited antibacterial activities against Staphylococcus aureus with minimal inhibitory concentration (MIC) values of 25.0, 50.0, and 50.0 μg/mL, respectively.