The Lipoprotein LpqW Is Essential for the Mannosylation of Periplasmic Glycolipids in Corynebacteria

Phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) are essential components of the cell wall and plasma membrane of mycobacteria, including the human pathogen Mycobacterium tuberculosis, as well as the related Corynebacterineae. We have previously shown that the lipoprotein, LpqW, regulates PIM and LM/LAM biosynthesis in mycobacteria. Here, we provide direct evidence that LpqW regulates the activity of key mannosyltransferases in the periplasmic leaflet of the cell membrane. Inactivation of the Corynebacterium glutamicum lpqW ortholog, NCgl1054, resulted in a slow growth phenotype and a global defect in lipoglycan biosynthesis. The NCgl1054 mutant lacked LAMs and was defective in the elongation of the major PIM species, AcPIM2, as well as a second glycolipid, termed Gl-X (mannose-α1–4-glucuronic acid-α1-diacylglycerol), which function as membrane anchors for LM-A and LM-B, respectively. Elongation of AcPIM2 and Gl-X was found to be dependent on expression of polyprenol phosphomannose (ppMan) synthase. However, the ΔNCgl1054 mutant synthesized normal levels of ppMan, indicating that LpqW is not required for synthesis of this donor. A spontaneous suppressor strain was isolated in which lipoglycan synthesis in the ΔNCgl1054 mutant was partially restored. Genome-wide sequencing indicated that a single amino acid substitution within the ppMan-dependent mannosyltransferase MptB could bypass the need for LpqW. Further evidence of an interaction is provided by the observation that MptB activity in cell-free extracts was significantly reduced in the absence of LpqW. Collectively, our results suggest that LpqW may directly activate MptB, highlighting the role of lipoproteins in regulating key cell wall biosynthetic pathways in these bacteria.     

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

BioMart and Bioconductor: a powerful link between biological databases and microarray data analysis

biomaRt is a new Bioconductor package that integrates BioMart data resources with data analysis software in Bioconductor. It can annotate a wide range of gene or gene product identifiers (e.g. Entrez-Gene and Affymetrix probe identifiers) with information such as gene symbol, chromosomal coordinates, Gene Ontology and OMIM annotation. Furthermore biomaRt enables retrieval of genomic sequences and single nucleotide polymorphism information, which can be used in data analysis. Fast and up-to-date data retrieval is possible as the package executes direct SQL queries to the BioMart databases (e.g. Ensembl). The biomaRt package provides a tight integration of large, public or locally installed BioMart databases with data analysis in Bioconductor creating a powerful environment for biological data mining.     

Long,Processive Enzymatic DNA Synthesis Using 100% Dye-Labeled Terminal Phosphate-Linked Nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by φ29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

Cleavage of neuronal synaptosomal-associated protein of 25 kDa by exogenous matrix metalloproteinase-7

Matrix metalloproteinases (MMPs) belong to a family of zinc dependent enzymes best studied for their role in cancer and inflammation. Though MMPs typically target extracellular proteins, here we show that MMP-7, an MMP family member which lacks a C-terminal hemopexin-like domain, can cleave an intraneuronal protein that is critical to vesicular fusion and neurotransmitter release, synaptosomal-associated protein of 25 kDa (SNAP-25). Western blot analysis using an N-terminal specific antibody on extracts from cultured neurons suggests that cleavage occurs towards the C-terminal portion of SNAP 25. Additional studies with recombinant SNAP-25 demonstrate that cleavage occurs at amino acid 129. The ability of MMP-7 to cleave SNAP-25 is diminished by chlorpromazine and phenylarsine oxide, inhibitors of clathrin dependent endocytosis. Together, these results imply that exogenous MMP-7 can access an intraneuronal substrate and suggest that additional studies may be warranted to determine if SNAP function is impaired with brain inflammation.     

The Nonenzymatic Hydrolysis of Oligoribonucleotides VII. Structural Elements Affecting Hydrolysis

Abstract

Several elements of oligoribonucleotide structure are important for efficient hydrolysis. We have found that the following factors influence oligoribonucleotide hydrolysis: (i) single-stranded structure of RNA flanking the scissile phosphodiester bond, (ii) the substituent on atom C-5 of the uridine adjacent to the cleaved internucleotide bond, (iii) the position of the scissile UA phosphodiester bond within a hairpin loop, (iv) the concentration of formamide, urea, ethanol and sodium chloride.