Tiegang Xu

A novel DNA modification by sulphur

Streptomyces lividans has a novel DNA modification, which sensitises its DNA to degradation during electrophoresis (the Dnd phenotype). The entire gene cluster (dnd) involved in this modification was localized on an 8 kb DNA fragment and was expressed in a S. lividans deletion mutant (dnd) and in several heterologous hosts. Disruption of the dnd locus abolishes the Dnd phenotype, and gain of the dnd locus conferred the Dnd phenotype respectively. Extensive analysis of the dnd gene cluster revealed five open reading frames, whose hypothetic functions suggested an incorporation of sulphur or a sulphur‐containing substance into S. lividans genome, yet in an unknown manner. The Dnd phenotype was also discovered to exist in DNA of widespread bacterial species of variable origin and diverse habitat. Similarly organized gene clusters were found in several bacterial genomes representing different genera and in eDNA of marine organisms, suggesting such modification as a widespread phenomenon. A coincidence between the Dnd phenotype and DNA modification by sulphur was demonstrated to occur in several representative bacterial genomes by the in vivo35S‐labelling experiments.

A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella.

A novel, site-specific, DNA backbone S-modification (phosphorothioation) has been discovered, but its in vivo function(s) have remained obscure. Here, we report that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolated from Escherichia coli, while protecting its own DNA by site-specific phosphorothioation. A cloned 15-kb gene cluster from S. enterica conferred both host-specific restriction and DNA S-modification on E. coli. Mutational analysis of the gene cluster proved unambiguously that the S-modification prevented host-specific restriction specified by the same gene cluster. Restriction activity required three genes in addition to at least four contiguous genes necessary for DNA S-modification. This functional overlap ensures that restriction of heterologous DNA occurs only when the host DNA is protected by phosphorothioation. Meanwhile, this novel type of host-specific restriction and modification system was identified in many diverse bacteria. As in the case of methylation-specific restriction systems, targeted inactivation of this gene cluster should facilitate genetic manipulation of these bacteria, as we demonstrate in Salmonella.

Functional analysis of spfD gene involved in DNA phosphorothioation in Pseudomonas fluorescens Pf0-1.

DNA phosphorothioation is widespread in many bacterial species. By homology analysis of the dnd gene cluster in Pseudomonas fluorescens Pf0‐1, a spfBCDE gene cluster involved in DNA phosphorothioation was localized. Disruption of the spfD gene, a dndD homolog, caused the loss of the Dnd phenotype and demonstrated the involvement of spfD in DNA phosphorothioation in P. fluorescens Pf0‐1. The ATPase activity of SpfD suggests that SpfD could hydrolyze ATP to provide the energy required in the DNA phosphorothioate modification process.

DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus

BackgroundA novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system.ResultsA 6,665-bp DNA region carrying just five ORFs (dndA-E) was defined as the sole determinant for modification of the DNA backbone in S. lividans to form phosphorothioate. This provides a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. While dndA is clearly transcribed independently, dndB-E constitute an operon, as revealed by RT-PCR analysis. An efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these dnd genes. The Dnd- phenotype caused by specific in-frame deletion of the dndA, C, D, and E genes or the enhanced Dnd phenotype resulting from in-frame deletion of dndB could be restored by expression vectors carrying the corresponding dnd genes. Interestingly, overdosage of DndC or DndD, but not other Dnd proteins, in vivo was found to be detrimental to cell viability.ConclusionDNA phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. Overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host.

Advanced Nanoporous Materials for Micro-Gravimetric Sensing to Trace-Level Bio/Chemical Molecules

Functionalized nanoporous materials have been developed recently as bio/chemical sensing materials. Due to the huge specific surface of the nano-materials for molecular adsorption, high hopes have been placed on gravimetric detection with micro/nano resonant cantilevers for ultra-sensitive sensing of low-concentration bio/chemical substances. In order to enhance selectivity of the gravimetric resonant sensors to the target molecules, it is crucial to modify specific groups onto the pore-surface of the nano-materials. By loading the nanoporous sensing material onto the desired region of the mass-type transducers like resonant cantilevers, the micro-gravimetric bio/chemical sensors can be formed. Recently, such micro-gravimetric bio/chemical sensors have been successfully applied for rapid or on-the-spot detection of various bio/chemical molecules at the trace-concentration level. The applicable nanoporous sensing materials include mesoporous silica, zeolite, nanoporous graphene oxide (GO) and so on. This review article focuses on the recent achievements in design, preparation, functionalization and characterization of advanced nanoporous sensing materials for micro-gravimetric bio/chemical sensing.

Direct evidence that ThiI is an ATP pyrophosphatase for the adenylation of uridine in 4-thiouridine biosynthesis.

Over a hundred post-transcriptional modifications are made to nucleosides in RNA, including the best characterized 4-thiouridine (s4U) at position 8 in bacterial tRNA. The s4U serves as a near-UV photosensor, undergoing a photoinduced cross-linking reaction with cytidine-13 when exposed to near-UV light. The cross-linked tRNAs are inefficient aminoacylation substrates, and therefore protein synthesis stops, triggering entry into a controlled growth arrest. Two enzymes, ThiI and IscS, are proposed to be involved in the modification of uridine to 4-thiouridine. IscS removes a sulfur atom from free cysteine, and the terminal sulfur is transferred to ThiI, where a persulfide is likely formed at cysteine-456 prior to ThiI’s effect on tRNA modification. Two chemical mechanisms were proposed to account for the generation of s4U from the persulfide group and activated uridine residue, but the activation of uridine by adenylation has not been proven in either of the two mechanisms. In this paper, we provide direct evidence for the formation of an adenylation intermediate in the modification process using electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS), ATP pyrophosphatase activity analysis, and an isotopic tracer method. ThiI was shown previously as a sulfurtransferase, which was responsible for the transfer of a persulfide to tRNA. It shares an adenylation-specific P-loop motif (SGGFDS) within the PPi synthetase family, seemingly required for the activation of uridine by adenylation (Scheme 1). In order to examine and prove the potential adenylation role of the ThiI protein in 4-thiouridine biosynthesis, a recombinant protein was expressed (see the Supporting Information) and assayed in vitro for its proposed ATP pyrophosphatase activity by monitoring the production of PPi using the EnzCheck pyrophosphate assay kit (Producer). The kinetic constants observed for this reaction are summarized in Table 1. The specific activity of ThiI toward

High-Q in-plane resonance-mode cantilever bio/chemical sensor for real-time detection in liquids

Reported is an lateral-mode resonant cantilever sensor that features a much depressed liquid drag force, thereby, achieving a much higher quality factor of 249 in water and higher specific mass sensitivity in liquids, compared with traditional out-of-plane resonant cantilevers. With 8.8Hz/pg sensitivity calibrated by specific binding streptavidin-labled Dynabeads with the biotin-immobilized cantilever in Phosphate Buffered Saline (PBS) solution, specific-antibody immobilized cantilevers have real-time detected E. coli in PBS solution. Real-time detection of Hg2+ ions in aqueous Hg(NO3)2 is also carried out by using the sensing-group-modified cantilevers, resulting in a finer noise-limited resolution than 100ppb.

Functionalized mesoporous thin-film directly self-assembled on resonant-cantilevers for batch-producible chemical sensors

The paper reports a top-down/bottom-up combined resonant micro-cantilever chemical sensor, where functionalized mesoporous thin-film (MTF) is directly self-assembled on the sensing region of the integrated cantilever. By using the batch-producible nano construction technique, a large number of such sensors can be volume fabricated with uniform performance among individual sensors or different runs. More importantly, sensing groups can be simultaneously constructed at the pore inner surface when the MTF is directly constructed on the cantilever. With -NH2 groups modified, the functionalized MTF is grown onto the surface of the cantilever free-end, the micro-gravimetric sensor exhibits quick response and high-sensitive detection to gaseous CO2.