Assessing characteristics of RNA amplification methods for single cell RNA sequencing.

BACKGROUND: Recently, measurement of RNA at single cell resolution has yielded surprising insights. Methods for single-cell RNA sequencing (scRNA-seq) have received considerable attention, but the broad reliability of single cell methods and the factors governing their performance are still poorly known. RESULTS: Here, we conducted a large-scale control experiment to assess the transfer function of three scRNA-seq methods and factors modulating the function. All three methods detected greater than 70% of the expected number of genes and had a 50% probability of detecting genes with abundance greater than 2 to 4 molecules. Despite the small number of molecules, sequencing depth significantly affected gene detection. While biases in detection and quantification were qualitatively similar across methods, the degree of bias differed, consistent with differences in molecular protocol. Measurement reliability increased with expression level for all methods and we conservatively estimate measurements to be quantitative at an expression level greater than ~5-10 molecules. CONCLUSIONS: Based on these extensive control studies, we propose that RNA-seq of single cells has come of age, yielding quantitative biological information.

Biochemical characterization of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: meso-2,6-diaminopimelate ligase (MurE) from Verrucomicrobium spinosum DSM 4136(T.).

Verrucomicrobium spinosum is a Gram-negative bacterium that is related to bacteria from the genus Chlamydia. The bacterium is pathogenic towards Drosophila melanogaster and Caenorhabditis elegans, using a type III secretion system to facilitate pathogenicity. V. spinosum employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the V. spinosum genome provides evidence that the bacterium should be able to synthesize peptidoglycan de novo, since all of the necessary genes are present. The enzyme UDP-N-acetylmuramoyl-l-alanyl-d-glutamate: meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The murE ortholog from V. spinosum (murE Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:meso-2,6-diaminopimelate ligase activity in vivo using functional complementation. In vitro analysis using the purified recombinant enzyme demonstrated that MurEVs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. meso-Diaminopimelate was the preferred substrate with a K m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurEVs. Our kinetic analysis and structural model of MurEVs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that V. spinosum incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a V. spinosum culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.

Whole-genome sequence of Enterobacter sp. strain SST3, an endophyte isolated from Jamaican sugarcane (Saccharum sp.) stalk tissue.

Enterobacter sp. strain SST3 is an endophytic bacterium isolated from Saccharum spp. Here we present its annotated draft genome that may shed light on its role as a bacterial endophyte of sugarcane. To our knowledge, this is the first genome announcement of a sugarcane-associated bacterium from the genus Enterobacter.

Genomic and Biochemical Analysis of the Diaminopimelate and Lysine Biosynthesis Pathway in Verrucomicrobium spinosum: Identification and Partial Characterization of L,L-Diaminopimelate Aminotransferase and UDP-N-Acetylmuramoylalanyl-D-glutamyl-2,6-meso-Diaminopimelate Ligase.

The Gram-negative bacterium Verrucomicrobium spinosum has attracted interest in recent years following the sequencing and annotation of its genome. Comparative genomic analysis of V. spinosum using diaminopimelate/lysine metabolic genes from Chlamydia trachomatis suggests that V. spinosum employs the L,L-diaminopimelate aminotransferase (DapL) pathway for diaminopimelate/lysine biosynthesis. The open reading frame corresponding to the putative dapL ortholog was cloned and the recombinant enzyme was shown to possess L,L-diaminopimelate aminotransferase activity in vitro. In vivo analysis using functional complementation confirmed that the dapL ortholog was able to functionally complement an E. coli mutant that confers auxotrophy for diaminopimelate and lysine. In addition to its role in lysine biosynthesis, the intermediate diaminopimelate has an integral role in peptidoglycan biosynthesis. To this end, the UDP-N-acetylmuramoylalanyl-d-glutamyl-2,6-meso-diaminopimelate ligase ortholog was also identified, cloned, and was shown to possess meso-diaminopimelate ligase activity in vivo. The L,L-diaminopimelate aminotransferase pathway has been experimentally confirmed in several bacteria, some of which are deemed pathogenic to animals. Since animals, and particularly humans, lack the genetic machinery for the synthesis of diaminopimelate/lysine de novo, the enzymes involved in this pathway are attractive targets for development of antibiotics. Whether dapL is an essential gene in any bacteria is currently not known. V. spinosum is an excellent candidate to investigate the essentiality of dapL, since the bacterium employs the DapL pathway for lysine and cell wall biosynthesis, is non-pathogenic to humans, facile to grow, and can be genetically manipulated.