Challenges and opportunities in understanding microbial communities with metagenome assembly (accompanied by IPython Notebook tutorial)

Metagenomic investigations hold great promise for informing the genetics, physiology, and ecology of environmental microorganisms. Current challenges for metagenomic analysis are related to our ability to connect the dots between sequencing reads, their population of origin, and their encoding functions. Assembly-based methods reduce dataset size by extending overlapping reads into larger contiguous sequences (contigs), providing contextual information for genetic sequences that does not rely on existing references. These methods, however, tend to be computationally intensive and are again challenged by sequencing errors as well as by genomic repeats While numerous tools have been developed based on these methodological concepts, they present confounding choices and training requirements to metagenomic investigators. To help with accessibility to assembly tools, this review also includes an IPython Notebook metagenomic assembly tutorial. This tutorial has instructions for execution any operating system using Amazon Elastic Cloud Compute and guides users through downloading, assembly, and mapping reads to contigs of a mock microbiome metagenome. Despite its challenges, metagenomic analysis has already revealed novel insights into many environments on Earth. As software, training, and data continue to emerge, metagenomic data access and its discoveries will to grow

Development of an in vivo DNA Cloning Procedure

In this thesis, we describe the development of a method to delete and to clone specific large regions from the 1700 kilobase pExo megaplasmid of Rhizobium meliloti. In principal, the region to be cloned is flanked by FRT sites, which direct site specific recombination by the Flp recombinase. Targeting constructs were designed to include part of the IS50 from Tn5, the FRT site, an origin of transfer (oriT), and a replication origin from RK2 or the F plasmid. These constructs were directed to known Tn5-derivative insertion sites in the pExo megaplasmid. A plasmid which expresses the Flp recombinase constitutively in R. meliloti was made, and the transfer of this plasmid to FRT-flanked megaplasmid regions was shown to result in the deletion of the intervening DNA. We demonstrated that the pExo megaplasmid DNA regions could be captured in Escherichia coli, however in this case, the megaplasmid excision event appears to be directed by the oriT sites rather than the FRT sites. We present strong evidence that a specific 50 kb region contains the oriV of the pExo megaplasmid; R. meliloti strains deleted for this region could not be isolated, and this region was found to replicate autonomously in Agrobacterium tumefaciens. Preliminary sequence analysis has revealed strong homology within this region to genes encoding the RepABC replication proteins of several Rhizobium and Agrobacterium plasmids.ThesisMaster of Science (MS

Comparative genome analysis of Burkholderia phytofirmans PsJN reveals a wide spectrum of endophytic lifestyles based on interaction strategies with host plants

Burkholderia phytofirmans PsJN is a naturally occurring plant-associated bacterial endophyte that effectively colonizes a wide range of plants and stimulates their growth and vitality. Here we analyze whole genomes, of PsJN and of eight other endophytic bacteria. This study illustrates that a wide spectrum of endophytic life styles exists. Although we postulate the existence of typical endophytic traits, no unique gene cluster could be exclusively linked to the endophytic lifestyle. Furthermore, our study revealed a high genetic diversity among bacterial endophytes as reflected in their genotypic and phenotypic features. B. phytofirmans PsJN is in many aspects outstanding among the selected endophytes. It has the biggest genome consisting of two chromosomes and one plasmid, well-equipped with genes for the degradation of complex organic compounds and detoxification, e.g., 24 glutathione-S-transferase (GST) genes. Furthermore, strain PsJN has a high number of cell surface signaling and secretion systems and harbors the 3-OH-PAME quorum-sensing system that coordinates the switch of free-living to the symbiotic lifestyle in the plant-pathogen R. solanacearum. The ability of B. phytofirmans PsJN to successfully colonize such a wide variety of plant species might be based on its large genome harboring a broad range of physiological functions

Draft Genome Sequence of Frankia sp. Strain QA3, a Nitrogen-Fixing Actinobacterium Isolated from the Root Nodule of Alnus nitida

Members of the actinomycete genus Frankia form a nitrogen-fixing symbiosis with 8 different families of actinorhizal plants. We report a high-quality draft genome sequence for Frankia sp. strain QA3, a nitrogen-fixing actinobacterium isolated from root nodules of Alnus nitida