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Diverse soil-resident bacteria can contribute to plant growth and health, but the molecular mechanisms enabling them to effectively colonize their plant hosts remain poorly understood. We used randomly barcoded transposon mutagenesis sequencing (RB-TnSeq) in Pseudomonas simiae, a model root-colonizing bacterium, to establish a genome-wide map of bacterial genes required for colonization of the Arabidopsis thaliana root system. We identified 115 genes (2% of all P. simiae genes) with functions that are required for maximal competitive colonization of the root system. Among the genes we identified were some with obvious colonization-related roles in motility and carbon metabolism, as well as 44 other genes that had no or vague functional predictions. Independent validation assays of individual genes confirmed colonization functions for 20 of 22 (91%) cases tested. To further characterize genes identified by our screen, we compared the functional contributions of P. simiae genes to growth in 90 distinct in vitro conditions by RB-TnSeq, highlighting specific metabolic functions associated with root colonization genes. Our analysis of bacterial genes by sequence-driven saturation mutagenesis revealed a genome-wide map of the genetic determinants of plant root colonization and offers a starting point for targeted improvement of the colonization capabilities of plant-beneficial microbes.

UnlabelledTransposon mutagenesis with next-generation sequencing (TnSeq) is a powerful approach to annotate gene function in bacteria, but existing protocols for TnSeq require laborious preparation of every sample before sequencing. Thus, the existing protocols are not amenable to the throughput necessary to identify phenotypes and functions for the majority of genes in diverse bacteria. Here, we present a method, random bar code transposon-site sequencing (RB-TnSeq), which increases the throughput of mutant fitness profiling by incorporating random DNA bar codes into Tn5 and mariner transposons and by using bar code sequencing (BarSeq) to assay mutant fitness. RB-TnSeq can be used with any transposon, and TnSeq is performed once per organism instead of once per sample. Each BarSeq assay requires only a simple PCR, and 48 to 96 samples can be sequenced on one lane of an Illumina HiSeq system. We demonstrate the reproducibility and biological significance of RB-TnSeq with Escherichia coli, Phaeobacter inhibens, Pseudomonas stutzeri, Shewanella amazonensis, and Shewanella oneidensis. To demonstrate the increased throughput of RB-TnSeq, we performed 387 successful genome-wide mutant fitness assays representing 130 different bacterium-carbon source combinations and identified 5,196 genes with significant phenotypes across the five bacteria. In P. inhibens, we used our mutant fitness data to identify genes important for the utilization of diverse carbon substrates, including a putative d-mannose isomerase that is required for mannitol catabolism. RB-TnSeq will enable the cost-effective functional annotation of diverse bacteria using mutant fitness profiling.ImportanceA large challenge in microbiology is the functional assessment of the millions of uncharacterized genes identified by genome sequencing. Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach to assign phenotypes and functions to genes. However, the current strategies for TnSeq are too laborious to be applied to hundreds of experimental conditions across multiple bacteria. Here, we describe an approach, random bar code transposon-site sequencing (RB-TnSeq), which greatly simplifies the measurement of gene fitness by using bar code sequencing (BarSeq) to monitor the abundance of mutants. We performed 387 genome-wide fitness assays across five bacteria and identified phenotypes for over 5,000 genes. RB-TnSeq can be applied to diverse bacteria and is a powerful tool to annotate uncharacterized genes using phenotype data.

Early communication was examined for 3 children (ages 20 to 27 months) who had been tracheostomized during their first year and were still cannulated. Communication means and intentions and rate of communication were examined. Potential contributions of social, cognitive, and language skills to the onset of early words were assessed. (Author/SW)

Culture-dependent and culture-independent methods were used to sample the viral metagenome within an activated sludge (AS) microbial assemblage and compare them to the diversity of activated sludge bacterial taxa. The taxonomic heterogeneity of sequences obtained in the viral metagenomic library underscores the diversity of the AS system which receives an influx of microbiota from both human and environmental sources.

Telehealth services show great promise to expand access to care while improving patient safety and reducing costs of care for all Americans. The expansion of telehealth has been slowed by a host of factors, including limited reimbursement, legal and regulatory barriers, limited provider capacity, and a lack of general public knowledge and/or acceptance of telehealth technologies and services. To hasten the expansion of telehealth requires a multifaceted and coordinated approach that will include healthcare professionals, regulators, payers, lawmakers, and patients. This paper will outline the steps necessary to scale telehealth services to a level that will help to address issues such as access to care, patient safety, quality, provider shortages, licensure and preparedness, all important elements of healthcare reform.

Neurological disorders, such as Alzheimer's disease, Parkinson's disease and stroke recovery, strike an estimated 50 million Americans each year, costing billions of dollars in medical expenses each year, not to mention the countless personal losses and the immeasurable emotional impact on families and patients that such diseases inflict. Due to the complex nature of neurological diseases, the treatment of these diseases often requires two types of care: neurological care and mental health care.

The expansion of technology into the health care field brings exciting potential for better service and quality care for an ever growing market. This is particularly an exciting time and a superb opportunity for providers serving patients in rural areas. Proper education and advice of counsel regarding the legal ramifications of establishing a telehealth program in your business is vital, as Mr. Waters points out in this article, and provider the reader key caveats to consider.