From the Cover: PNAS Plus: The essential gene set of a photosynthetic organism

Synechococcus elongatus PCC 7942 is a model organism used for studying photosynthesis and the circadian clock, and it is being developed for the production of fuel, industrial chemicals, and pharmaceuticals. To identify a comprehensive set of genes and intergenic regions that impacts fitness in S. elongatus, we created a pooled library of ∼250,000 transposon mutants and used sequencing to identify the insertion locations. By analyzing the distribution and survival of these mutants, we identified 718 of the organism’s 2,723 genes as essential for survival under laboratory conditions. The validity of the essential gene set is supported by its tight overlap with well-conserved genes and its enrichment for core biological processes. The differences noted between our dataset and these predictors of essentiality, however, have led to surprising biological insights. One such finding is that genes in a large portion of the TCA cycle are dispensable, suggesting that S. elongatus does not require a cyclic TCA process. Furthermore, the density of the transposon mutant library enabled individual and global statements about the essentiality of noncoding RNAs, regulatory elements, and other intergenic regions. In this way, a group I intron located in tRNA^Leu, which has been used extensively for phylogenetic studies, was shown here to be essential for the survival of S. elongatus. Our survey of essentiality for every locus in the S. elongatus genome serves as a powerful resource for understanding the organism’s physiology and defines the essential gene set required for the growth of a photosynthetic organism.Determining the sets of genes necessary for survival of diverse organisms has helped to identify the fundamental processes that sustain life across an array of environments (1). This research has also served as the starting point for efforts by synthetic biologists to design organisms from scratch (2, 3). Despite the importance of essential gene sets, they have traditionally been challenging to gather because of the difficulty of observing mutations that result in lethal phenotypes. More recently, the pairing of transposon mutagenesis with next generation sequencing, referred to collectively as transposon sequencing (Tn-seq), has resulted in a dramatic advance in the identification of essential gene sets (4–7). The key characteristic of Tn-seq is the use of high-throughput sequencing to screen for the fitness of every transposon mutant in a pooled population to measure each mutation’s impact on survival. These data can be used to quantitatively ascertain the effect of loss-of-function mutations at any given locus, intragenic or intergenic, in the conditions under which the library is grown (8). Essential gene sets for 42 diverse organisms distributed across all three domains have now been defined, largely through the use of Tn-seq (9). A recently developed variation on Tn-seq, random barcode transposon site sequencing (RB-TnSeq) (10), further minimizes the library preparation and sequencing costs of whole-genome mutant screens.Despite the proliferation of genome-wide essentiality screens, a complete essential gene set has yet to be defined for a photosynthetic organism. A collection of phenotyped Arabidopsis thaliana mutants has been created but extends to only one-tenth of Arabidopsis genes (11). In algae, efforts are underway to produce a Tn-seq–like system in Chlamydomonas reinhardtii; however, the mutant library currently lacks sufficient saturation to determine gene essentiality (12). To date, the essential genes for photoautotrophs have only been estimated by indirect means, such as by comparative genomics (13). The absence of experimentally determined essential gene sets in photosynthetic organisms, despite their importance to the environment and industrial production, is largely because of the difficulty and time required for genetic modification of these organisms.Cyanobacteria comprise an extensively studied and ecologically important photosynthetic phylum. They are responsible for a large portion of marine primary production and have played a foundational role in research to decipher the molecular components of photosynthesis (14, 15). Synechococcus elongatus PCC 7942 is a particularly well-studied member of this phylum because of its genetic tractability and streamlined genome (16). As a result, it has been developed as a model photosynthetic organism and a production platform for a number of fuel products and high-value chemicals (17). Despite the importance of S. elongatus for understanding photosynthesis and industrial production, 40% of its genes have no functional annotation, and only a small portion of those that do have been studied experimentally.Here, we use RB-TnSeq, a method that pairs high-density transposon mutagenesis and pooled mutant screens, to probe the S. elongatus genome for essential genes and noncoding regions. We categorized 96% of 2,723 genes in S. elongatus as either essential (lethal when mutated), beneficial (growth defect when mutated), or nonessential (no phenotype when mutated) under standard laboratory conditions. Furthermore, we determined the genome-wide essentiality of noncoding RNAs (ncRNAs), regulatory regions, and intergenic regions. Our investigation has produced an extensive analysis of the loci essential for the growth of a photosynthetic organism and developed a powerful genomic tool that can be used for additional screens under a wide array of ecologically and industrially relevant growth conditions.     

Anterior chiasmal optic nerve avulsion.

In a case of traumatic avulsion of the optic nerve at the anterior chiasm, MR imaging provided highly specific images of the injury site, including the absence of the optic nerve within the optic canal and the point of transection at the anterior portion of the chiasm. This was confirmed clinically and histopathologically. MR imaging should be considered in cases of suspected chiasmal injury.     

The hypercoagulability states.

Although interest in coagulation has usually centered about the various hemorraghic disorders, it is hyperactivity of the hemostatic system with its thrombotic complications which has a far greater clinical impact. The hypercoagulability states are a vague group of disorders not well defined by the laboratory. They fall into two distinct groups: (1) conditions promoting venous thrombosis by activating the coagulation mechanism and (2) conditions promoting arterial thrombosis by platelet plug formation. Dealing with both of these groups separately the various disorders associated with a hypercoagulable state are discussed as well as their pathophysiologic basis; Special emphasis is placed on the laboratory evaluation of these disorders.     

Factors affecting the performance of factor VIII coagulant activity assays. Results of proficiency surveys of the College of American Pathologists.

To evaluate sources of interlaboratory variation in factor VIII coagulant assays and the effects of uniform calibration on it, data were analyzed from Survey Set H2-C, 1987, of the College of American Pathologists, Northfield, Ill, into which the College of American Pathologists Reference Preparation for Factor VIII was incorporated. Peer group performance and its improvement by uniform calibration were evaluated by comparing means, precisions, and numbers of acceptable survey scores. Results showed a poorer performance by less experience laboratories and by those that used undesirable calibrators. Substantial improvement was seen with uniform calibration, especially among laboratories that had employed poorer calibrators. Although variance component analysis showed that the type of calibrator was the most important contributor to interlaboratory variation, it accounted for only 10% of total variability. Participant summaries and questionnaires that spanned several years revealed steady improvement in methodology but no striking changes in interlaboratory variation.