Copper Efflux System Required in Murine Lung Infection by Haemophilus influenzae Composed of a Canonical ATPase Gene and Tandem Chaperone Gene Copies.

Copper is an essential micronutrient but is toxic at high concentrations. In Haemophilus influenzae mechanisms of copper resistance and its role in pathogenesis are unknown; however, our previous genetic screen by transposon insertion-site sequencing implicated a putative cation transporting ATPase () in survival in a mouse lung infection model. Here, we demonstrate that H.

High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism.

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis.

A novel zinc binding system, ZevAB, is critical for survival of nontypeable Haemophilus influenzae in a murine lung infection model.

Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative bacterial pathogen that causes upper and lower respiratory infections. Factors required for pulmonary infection by NTHI are not well understood. Previously, using high-throughput insertion tracking by deep sequencing (HITS), putative lung colonization factors were identified.

High-throughput insertion tracking by deep sequencing for the analysis of bacterial pathogens.

Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis.

Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model.

Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection.

A microtiter assay was developed for the improved detection of inorganic phosphate released from adenosine 5'-triphosphate (ATP) in the glutamine synthetase biosynthetic assay. In this assay, ascorbic acid replaces the traditionally used ferrous sulfate to reduce the phosphomolybdate complex. As a result, increased color development, linearity, and sensitivity are achieved.

Intracellular symbionts and other bacteria associated with deer ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts.

The diversity of bacteria associated with the deer tick (Ixodes scapularis) was assessed using PCR amplification, cloning, and sequencing of 16S rRNA genes originating from seven ticks collected from Nantucket Island and Wellfleet, Cape Cod, Mass. The majority of sequences obtained originated from gram-negative proteobacteria. Four intracellular bacteria were detected including strains of Ehrlichia, Rickettsia, and Wolbachia and an organism related to intracellular insect symbionts from the Cytophaga-Flavobacterium-Bacteroides group.