The rise of antibiotic-resistant bacteria has led to an urgent need for rapid detection of drug resistance in clinical samples, and improvements in global surveillance. Here we show how de Bruijn graph representation of bacterial diversity can be used to identify species and resistance profiles of clinical isolates. We implement this method for Staphylococcus aureus and Mycobacterium tuberculosis in a software package (‘Mykrobe predictor') that takes raw sequence data as input, and generates a clinician-friendly report within 3 minutes on a laptop. For S. aureus, the error rates of our method are comparable to gold-standard phenotypic methods, with sensitivity/specificity of 99.1%/99.6% across 12 antibiotics (using an independent validation set, n=470). For M. tuberculosis, our method predicts resistance with sensitivity/specificity of 82.6%/98.5% (independent validation set, n=1,609); sensitivity is lower here, probably because of limited understanding of the underlying genetic mechanisms. We give evidence that minor alleles improve detection of extremely drug-resistant strains, and demonstrate feasibility of the use of emerging single-molecule nanopore sequencing techniques for these purposes.

This study characterized genetic interactions between the maize (Zea mays) genes dull1 (du1), encoding starch synthase III (SSIII), and isa2, encoding a noncatalytic subunit of heteromeric isoamylase-type starch-debranching enzyme (ISA1/ISA2 heteromer). Mutants lacking ISA2 still possess the ISA1 homomeric enzyme. Eight du1 -mutations were characterized, and structural changes in amylopectin resulting from each were measured. In every instance, the same complex pattern of alterations in discontinuous spans of chain lengths was observed, which cannot be explained solely by a discrete range of substrates preferred by SSIII. Homozygous double mutants were constructed containing the null mutation isa2-339 and either du1-Ref, encoding a truncated SSIII protein lacking the catalytic domain, or the null allele du1-R4059. In contrast to the single mutant parents, double mutant endosperms affected in both SSIII and ISA2 were starch deficient and accumulated phytoglycogen. This phenotype was previously observed only in maize sugary1 mutants impaired for the catalytic subunit ISA1. ISA1 homomeric enzyme complexes assembled in both double mutants and were enzymatically active in vitro. Thus, SSIII is required for normal starch crystallization and the prevention of phytoglycogen accumulation when the only isoamylase-type debranching activity present is ISA1 homomer, but not in the wild-type condition, when both ISA1 homomer and ISA1/ISA2 heteromer are present. Previous genetic and biochemical analyses showed that SSIII also is required for normal glucan accumulation when the only isoamylase-type debranching enzyme activity present is ISA1/ISA heteromer. These data indicate that isoamylase-type debranching enzyme and SSIII work in a coordinated fashion to repress phytoglycogen accumulation.Semicrystalline glucan polymers that form insoluble starch granules are found in the vast majority of organisms within the Archaeplastida lineage of primary photosynthetic eukaryotes. This group, which comprises glaucophytes, rhodophytes (red algae), and Chloroplastida (green algae and land plants), in general does not contain soluble glucan polymers of substantial size. Conversely, with a few exceptions, essentially all other eukaryotes and prokaryotes utilize the soluble polymer glycogen for the storage of Glc and lack any insoluble glucans. Starch granules and their constituent polymers are capable of storing many more Glc units than chemically similar but soluble glucans, and this is likely to have provided a selective advantage during the establishment and spread of the photosynthetic eukaryotes. Support for this suggestion comes from the facts that the primary photosynthetic eukaryotes are a monophyletic group (Rodríguez-Ezpeleta et al., 2005). In light of the important role of starch metabolism in these organisms, including the land plants, it is of interest to understand the molecular mechanisms that generate semicrystalline glucans and how these processes differ from those that generate glycogen.Starch granules are made up of two t...

ADP-glucose pyrophosphorylase (AGPase) provides the nucleotide sugar ADP-glucose and thus constitutes the first step in starch biosynthesis. The majority of cereal endosperm AGPase is located in the cytosol with a minor portion in amyloplasts, in contrast to its strictly plastidial location in other species and tissues. To investigate the potential functions of plastidial AGPase in maize (Zea mays) endosperm, six genes encoding AGPase large or small subunits were characterized for gene expression as well as subcellular location and biochemical activity of the encoded proteins. Seven transcripts from these genes accumulate in endosperm, including those from shrunken2 and brittle2 that encode cytosolic AGPase and five candidates that could encode subunits of the plastidial enzyme. The amino termini of these five polypeptides directed the transport of a reporter protein into chloroplasts of leaf protoplasts. All seven proteins exhibited AGPase activity when coexpressed in Escherichia coli with partner subunits. Null mutations were identified in the genes agpsemzm and agpllzm and shown to cause reduced AGPase activity in specific tissues. The functioning of these two genes was necessary for the accumulation of normal starch levels in embryo and leaf, respectively. Remnant starch was observed in both instances, indicating that additional genes encode AGPase large and small subunits in embryo and leaf. Endosperm starch was decreased by approximately 7% in agpsemzm-or agpllzm-mutants, demonstrating that plastidial AGPase activity contributes to starch production in this tissue even when the major cytosolic activity is present.Plant ADP-glucose pyrophosphorylase (AGPase) catalyzes the production of ADP-glucose (ADPGlc) and inorganic pyrophosphate (PPi) from Glc-1-P and ATP, thus generating the nucleotide sugar used by starch synthases to incorporate glucosyl units into starch. ADPGlc formation is an important metabolic control point and thus has been a genetic engineering target for crop improvement. Altering AGPase activity by transgenic means resulted in elevated yields in several starch-producing crops, including rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), and potato (Solanum tuberosum), although the precise nature of the metabolic and developmental changes that result remains to be elucidated (Stark et al., 1992;Greene and Hannah, 1998;Smidansky et al., 2002Smidansky et al., , 2003Smidansky et al., , 2007Hannah et al., 2012).AGPase localization appears to be strictly plastidial in most plant tissues, whereas in cereal endosperms, the majority of the enzyme is cytosolic and a minor form resides within amyloplasts (for review, see ComparotMoss and Denyer, 2009;Hannah and Greene, 2009;Geigenberger, 2011). Such an arrangement necessitates distinct modes of regulation and metabolic control of starch biosynthesis compared with other plants, including transport of ADPGlc and Glc phosphate (Glc-1-P and/or Glc-6-P) from the cytosol into the amyloplast. Subcellular fractionation revealed that 85% to 95% ...