ABSTRACT: Identification of genes essential for Moraxella catarrhalis growth under iron-limiting conditions using Genomic Array Footprinting (GAF): Expression analysis of directed gene deletion mutants.
Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To further characterize the functional consequences of the gene deletions (GAF identified genes), we determined transcriptome profiles of the wild-type and directed mutant strains during exponential growth in BHI medium. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology.
Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To this end, a large marinerT7 transposon mutant library (~28,000 independent transposon mutants) was grown under iron-limiting conditions, achieved by sequestration of iron by 30 M-BM-5M Desferal (DF30), and under control growth conditions (brain heart infusion broth, DF0). Mutants were recovered at exponential- and the early-stationary growth phase and used for the generation of mutant-specific cDNA probes that were hybridized to custom-designed NimbleGen GAF microarrays. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology. cDNA probes generated from mutants recovered during the exponential growth phase, or early-stationary phase under challenge (iron-limiting, DF30) or control conditions (DF0). Hybridization on GAF 4x72K custom design Nimblegen GAF arrays for read-out.
Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To further characterize the functional consequences of the gene deletions (GAF identified genes), we determined transcriptome profiles of the wild-type and directed mutant strains during exponential growth in BHI medium. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology. M. catarrhalis BBH18 directed mutants (n = 3) and their parental wild-type BBH18 strain (n = 4) were expanded to mid-log phase (OD620nm, 1.2 to 1.4) and RNA was isolated as previously described (de Vries et al., 2010 J Bacteriol 192: 3574-3583). Total RNA was labeled according to standard Nimblegen gene expression array protocols and hybridized to 4x72K custom design Nimblegen arrays for read-out.
Project description:Iron-sequestration by the human host is a first line defense against respiratory pathogens like Moraxella catarrhalis, which consequently experiences a period of iron-starvation during colonization and infection. We determined the genetic requirements for M. catarrhalis growth during iron-starvation using the high-throughput genome-wide screening technology genomic array footprinting (GAF). To this end, a large marinerT7 transposon mutant library (~28,000 independent transposon mutants) was grown under iron-limiting conditions, achieved by sequestration of iron by 30 µM Desferal (DF30), and under control growth conditions (brain heart infusion broth, DF0). Mutants were recovered at exponential- and the early-stationary growth phase and used for the generation of mutant-specific cDNA probes that were hybridized to custom-designed NimbleGen GAF microarrays. The results described in this study are further discussed in Stefan P.W. de Vries, Peter Burghout, Jeroen D. Langereis, Aldert Zomer, Peter W.M. Hermans, Hester J. Bootsma: Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions, Molecular Microbiology.
Project description:Identification of genes essential for Moraxella catarrhalis growth under iron-limiting conditions using Genomic Array Footprinting (GAF)
Project description:Total RNA samples were isolated from M. catarrhalis grown in BHI (iron-replete condition, designated DF0) and in BHI containing 30 mM Desferal (iron-limiting condition, designated DF30). Total RNA and M. catarrhalis genome-directed primers (GDPs) were used for synthesis of Cy3- or Cy5-labeled cDNA samples. Three independent M. catarrhalis growth experiments and total RNA isolations were used for synthesis of cDNA samples, and a total of six DNA microarray hybridizations were performed in this study. Keywords: Effect of Growth Environment on Global Gene Expression by Moraxella catarrhalis
Project description:Moraxella catarrhalis is a human-restricted bacterial pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Iron is a co-factor required for the function of proteins involved in respiration and DNA replication, and thus is essential to life for most bacterial species. As iron is sequestered by host tissues, all host-adapted pathogens encounter periods of iron starvation. Previous studies have defined the repertoire of genes required for growth of M. catarrhalis under iron restricted conditions, and described differential expression at the transcriptional level. However, global changes in protein expression in response to iron starvation have not been investigated. Here we provide a SWATH-MS dataset describing differential expression of the M. catarrhalis CCRI-195ME proteome under iron restricted versus iron replete conditions.
Project description:Comparison of the gene expression profile of Moraxella catarrhalis grown in the presence of 20% pooled human sputum in chemically-defined medium relative to Moraxella catarrhalis grown in chemically-defined medium alone.
