BackgroundExperimental designs that take advantage of high-throughput sequencing to generate datasets include RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), sequencing of 16S rRNA gene fragments, metagenomic analysis and selective growth experiments. In each case the underlying data are similar and are composed of counts of sequencing reads mapped to a large number of features in each sample. Despite this underlying similarity, the data analysis methods used for these experimental designs are all different, and do not translate across experiments. Alternative methods have been developed in the physical and geological sciences that treat similar data as compositions. Compositional data analysis methods transform the data to relative abundances with the result that the analyses are more robust and reproducible.ResultsData from an in vitro selective growth experiment, an RNA-seq experiment and the Human Microbiome Project 16S rRNA gene abundance dataset were examined by ALDEx2, a compositional data analysis tool that uses Bayesian methods to infer technical and statistical error. The ALDEx2 approach is shown to be suitable for all three types of data: it correctly identifies both the direction and differential abundance of features in the differential growth experiment, it identifies a substantially similar set of differentially expressed genes in the RNA-seq dataset as the leading tools and it identifies as differential the taxa that distinguish the tongue dorsum and buccal mucosa in the Human Microbiome Project dataset. The design of ALDEx2 reduces the number of false positive identifications that result from datasets composed of many features in few samples.ConclusionStatistical analysis of high-throughput sequencing datasets composed of per feature counts showed that the ALDEx2 R package is a simple and robust tool, which can be applied to RNA-seq, 16S rRNA gene sequencing and differential growth datasets, and by extension to other techniques that use a similar approach.

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With the completion of the genome sequence, and development of an efficient conjugation-based transformation system allowing the introduction of stable episomes, Phaeodactylum tricornutum has become an ideal platform for the study of diatom biology and synthetic biology applications. The development of plasmid-based genetic tools is the next step to improve manipulation of this species. Here, we report the identification of endogenous P. tricornutum promoters and terminators allowing selective expression of antibiotic resistance markers from stably replicating plasmids in P. tricornutum. Significantly, we developed a protocol for sequential conjugation of plasmids from Escherichia coli to P. tricornutum and demonstrated simultaneous replication of two plasmids in P. tricornutum. We developed a simple and robust conjugative system for Cas9 editing that yielded up to 60% editing efficiency of the urease gene. Finally, we constructed a plasmid encoding eight genes involved in vanillin biosynthesis that was propagated in P. tricornutum over four months with no evidence of rearrangements, with whole-plasmid sequencing indicating that the majority of mutations occurred after plasmid assembly and initial conjugation rather than during long-term propagation. The plasmid-based tools described here will facilitate investigation of the basic biology of P. tricornutum and enable synthetic biology applications.

2"The field of bacterial viruses is a fine playground for serious children who ask ambitious questions."Max DelbruckThe first bacterial intron, a self-splicing group I intron, was found to interrupt the thymidylate synthase (td) gene of the Escherichia coli phage T4 (11). The second and third bacterial group I introns were found to interrupt the aerobic (nrdB) and anaerobic (nrdD [initially named sunY]) ribonucleotide reductases of phage T4 (29,90), and another group I intron was soon discovered in the DNA polymerase gene of SPO1, a Bacillus phage (25). From this (admittedly) small sampling of phage genomes, one might have naively expected that group I introns would be abundant in phage or bacterial genomes, especially since subsequent laboratory experiments demonstrated that group I introns could propagate themselves (by a process called homing) throughout populations of intron-minus alleles with near 100% efficiency (5, 68). That a similar homing phenomenon had also been previously demonstrated for a group I intron in the large rRNA gene of yeast mitochondria (34) gave additional support to the notion that group I introns should be able to spread efficiently throughout populations. However, this expected outcome has never been realized in natural phage populations; some phage populations harbor many introns, while other related phage populations are strangely lacking in any introns whatsoever (Table 1). Why do group I introns have an unusual distribution in phage and bacterial genomes, and what potential barriers might exist to prevent their spread?A similar question might be asked of group II introns in bacteria. Much was made of the initial finding of group II introns in bacteria, as their discovery added fuel to the debate concerning the evolutionary origins of eukaryotic spliceosomal introns (22, 70) which have both structural and functional similarities to group II introns (53,79,84). Yet, the number of group II introns in bacteria is small, many of which are inferred only from database matches to reverse transcriptases or maturases encoded within known introns (13,41,73,89), and only two have been shown to splice or be mobile in vivo (48,49,55,80) (Table 2). While group II intron homing is mechanistically distinct from group I intron homing, the principle is similar; group II introns home from intron-containing to intronless alleles. Many elegant biochemical and genetic experiments have unraveled the complexities of bacterial group II intron homing (14,48,49), and based on these results, there seems no a priori reason why group II introns should not be able to spread efficiently through populations of intron-minus alleles. The paucity of bacterial group II introns becomes even more perplexing given the recent demonstration of group II intron transposition to novel chromosomal sites (15). That group II introns are abundant in mitochondrial and chloroplast genomes (52) and present in bacterial genomes but at lower levels (and absent in phages) only adds to the mystery surrounding the lack of group II intro...

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for “genome editing” in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.

The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease. Notably, a cis-acting plasmid that encodes the conjugation and CRISPR machinery conjugates from E. coli to S. enterica with high frequency compared to a trans system that separates conjugation and CRISPR machinery. In culture conditions that enhance cell-to-cell contact, conjugation rates approach 100% with the cis-acting plasmid. Targeting of single or multiplexed sgRNAs to non-essential genes results in high S. enterica killing efficiencies. Our data highlight the potential of cis-acting conjugative plasmids as a delivery system for CRISPR nucleases or other microbial-altering agents for targeted bacterial killing.

Group I introns are intervening sequences that have invaded tRNA, rRNA and protein coding genes in bacteria and their phages. The ability of group I introns to self-splice from their host transcripts, by acting as ribozymes, potentially renders their insertion into genes phenotypically neutral. Some group I introns are mobile genetic elements due to encoded homing endonuclease genes that function in DNA-based mobility pathways to promote spread to intronless alleles. Group I introns have a limited distribution among bacteria and the current assumption is that they are benign selfish elements, although some introns and homing endonucleases are a source of genetic novelty as they have been co-opted by host genomes to provide regulatory functions. Questions regarding the origin and maintenance of group I introns among the bacteria and phages are also addressed.

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.