High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model–based analyses of transposon-insertion sequencing data

The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data

ARTIST: High-Resolution Genome-Wide Assessment of Fitness Using Transposon-Insertion Sequencing

Transposon-insertion sequencing (TIS) is a powerful approach for deciphering genetic requirements for bacterial growth in different conditions, as it enables simultaneous genome-wide analysis of the fitness of thousands of mutants. However, current methods for comparative analysis of TIS data do not adjust for stochastic experimental variation between datasets and are limited to interrogation of annotated genomic elements. Here, we present ARTIST, an accessible TIS analysis pipeline for identifying essential regions that are required for growth under optimal conditions as well as conditionally essential loci that participate in survival only under specific conditions. ARTIST uses simulation-based normalization to model and compensate for experimental noise, and thereby enhances the statistical power in conditional TIS analyses. ARTIST also employs a novel adaptation of the hidden Markov model to generate statistically robust, high-resolution, annotation-independent maps of fitness-linked loci across the entire genome. Using ARTIST, we sensitively and comprehensively define Mycobacterium tuberculosis and Vibrio cholerae loci required for host infection while limiting inclusion of false positive loci. ARTIST is applicable to a broad range of organisms and will facilitate TIS-based dissection of pathways required for microbial growth and survival under a multitude of conditions

High-Throughput Sequencing Enhanced Phage Display Identifies Peptides That Bind Mycobacteria

Bacterial cell wall components have been previously used as infection biomarkers detectable by antibodies. However, it is possible that the surface of the Mycobacterium tuberculosis (M. tb), the causative agent of tuberculosis (TB), also possesses molecules which might be non-antigenic. This makes the probing of biomarkers on the surface of M. tb cell wall difficult using antibodies. Here we demonstrate the use of phage display technology to identify peptides that bind to mycobacteria. We identified these clones using both random clone picking and high throughput sequencing. We demonstrate that random clone picking does not necessarily identify highly enriched clones. We further showed that the clone displaying the CPLHARLPC peptide which was identified by Illumina sequencing as the most enriched, binds better to mycobacteria than three clones selected by random picking. Using surface plasmon resonance, we showed that chemically synthesised CPLHARLPC peptide binds to a 15 KDa peptide from M.tb H37Rv whole cell lysates. These observations demonstrate that phage display technology combined with high-throughput sequencing is a powerful tool to identify peptides that can be used for investigating potential non-antigenic biomarkers for TB and other bacterial infections

El mobbing como proceso psicosocial. Consecuencias en el deterioro de la subjetividad

En el presente trabajo se intentará precisar, desde una perspectiva pluridisciplinar, los alcances terminológicos del fenómeno "mobbing" con el objeto de construir una herramienta metodológica, útil en investigación psicosocial, como en el esclarecimiento de esta forma de violencia que perjudica los procesos saludables en el desarrollo de la subjetividad. El "acoso laboral-mobbing" es una problemática que solo puede ser comprendida desde un enfoque multidisciplinar que favorezca la identificación de aquellas condiciones limitantes del sujeto laboral en la construcción de su identidad. Teniendo en cuenta aquellos factores derivados de la cultura organizacional y de la contextualización del mercado de trabajo en la Argentina, que operan como facilitadores de dicha problemática. Donde la interacción que se construye entre el sujeto y la cultura organizacional pueda ser propiciadora de mobbing, fenómeno que al ser identificado crea las condiciones de posibilidad de su propia prevención.Fil: Martín, Elsa Inés. Universidad Nacional de Mar del Plata. Facultad de Ciencias Económicas y Sociales; Argentina.Fil: Alfonso, Néstor Manuel. Universidad Nacional de Mar del Plata. Facultad de Ciencias Económicas y Sociales; Argentina

Mycobacterium tuberculosis FtsX extracellular domain activates the peptidoglycan hydrolase, RipC

Bacterial growth and cell division are coordinated with hydrolysis of the peptidoglycan (PG) layer of the cell wall, but the mechanisms of regulation of extracellular PG hydrolases are not well understood. Here we report the biochemical, structural, and genetic analysis of the Mycobacterium tuberculosis homolog of the transmembrane PG-hydrolase regulator, FtsX. The purified FtsX extracellular domain binds the PG peptidase Rv2190c/RipC N-terminal segment, causing a conformational change that activates the enzyme. Deletion of ftsEX and ripC caused similar phenotypes in Mycobacterium smegmatis, as expected for genes in a single pathway. The crystal structure of the FtsX extracellular domain reveals an unprecedented fold containing two lobes connected by a flexible hinge. Mutations in the hydrophobic cleft between the lobes reduce RipC binding in vitro and inhibit FtsX function in M. smegmatis. These studies suggest how FtsX recognizes RipC and support a model in which a conformational change in FtsX links the cell division apparatus with PG hydrolysis

Genome-wide Phenotypic Profiling Identifies and Categorizes Genes Required for Mycobacterial Low Iron Fitness

Iron is vital for nearly all living organisms, but during infection, not readily available to pathogens. Infectious bacteria therefore depend on specialized mechanisms to survive when iron is limited. These mechanisms make attractive targets for new drugs. Here, by genome-wide phenotypic profiling, we identify and categorize mycobacterial genes required for low iron fitness. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), can scavenge host-sequestered iron by high-affinity iron chelators called siderophores. We take advantage of siderophore redundancy within the non-pathogenic mycobacterial model organism M. smegmatis (Msmeg), to identify genes required for siderophore dependent and independent fitness when iron is low. In addition to genes with a potential function in recognition, transport or utilization of mycobacterial siderophores, we identify novel putative low iron survival strategies that are separate from siderophore systems. We also identify the Msmeg in vitro essential gene set, and find that 96% of all growth-required Msmeg genes have a mutual ortholog in Mtb. Of these again, nearly 90% are defined as required for growth in Mtb as well. Finally, we show that a novel, putative ferric iron ABC transporter contributes to low iron fitness in Msmeg, in a siderophore independent manner

Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids

Staphylococcus aureus contains two distinct teichoic acid (TA) polymers, lipoteichoic acid (LTA) and wall teichoic acid (WTA), which are proposed to play redundant roles in regulating cell division. To gain insight into the underlying biology of S. aureus TAs, we used a small molecule inhibitor to screen a highly saturated transposon library for cellular factors that become essential when WTA is depleted. We constructed an interaction network connecting WTAs with genes involved in LTA synthesis, peptidoglycan synthesis, surface protein display, and D-alanine cell envelope modifications. Although LTAs and WTAs are synthetically lethal, we report that they do not have the same synthetic interactions with other cell envelope genes. For example, D-alanylation, a tailoring modification of both WTAs and LTAs, becomes essential when the former, but not the latter, are removed. Therefore, D-alanine–tailored LTAs are required for survival when WTAs are absent. Examination of terminal phenotoypes led to the unexpected discovery that cells lacking both LTAs and WTAs lose their ability to form Z rings and can no longer divide. We have concluded that the presence of either LTAs or WTAs on the cell surface is required for initiation of S. aureus cell division, but these polymers act as part of distinct cellular networks

Transposon junction sequencing accurately reflects true library content.

<p>A. A Mtb mutant library is created by phage-delivery of transposons, disrupting each genome with a single insertion. Shown is a schematic of 6 mutant chromosomes spanning three genes (A–C), with transposons—red arrows—disrupting one of the three genes. After growing the library on 7H10 media, we pooled surviving mutants. In this schematic, gene C is required for optimal growth and thus mutants with transposons in gene C are lost. We isolated genomic DNA from the survivors for transposon site mapping. B. We sheared the genomic DNA by sonication, and repaired frayed ends to create blunt ends. We then used Taq polymerase to generate A-tails, allowing the ligation of T-tailed adapters. Finally, we selectively amplified transposon junctions using primers recognizing the transposon end and the adapter. Primers used for amplification contain all requisite sequences to permit direct sequencing of amplicons on an Illumina Genome Analyzer 2. C. We created a library of identified transposon insertion mutants in known relative quantities. DNA from the library was prepared for transposon junction sequencing. Insertion counts were plotted against the known relative quantity of the mutant in the library. D. To further confirm that read counts were a representation of the number of genomes in the library, we estimated the number of PCR template molecules. For each gene, we plotted the estimate of template molecule count against the read counts.</p

RNAs required for growth <i>in vitro</i>.

<p>A. IGV plot for genomic region containing the rnpB, the RNA component of RNaseL. Tracks, from top to bottom: 1. Histogram of insertion counts, 2. Comprehensive heat-map of requirement of 500-bp windows, 3. Position of annotated genes, 4. Position of TA dinucleotide sites, 5. Position of rnpB. B. IGV plot for genomic region containing the tmRNA. Tracks, from top to bottom: 1. Histogram of insertion counts, 2. Comprehensive heat-map of requirement of 500-bp windows, 3. Position of annotated genes, 4. Position of TA dinucleotide sites, 5. Position of the tmRNA.</p