Identification of genes essential for Moraxella catarrhalis growth under iron-limiting conditions using Genomic Array Footprinting (GAF). Moraxella catarrhalis BBH18

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. Overall design: 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.

人体宿主的铁扣留作用是对抗卡他莫拉菌（Moraxella catarrhalis）等呼吸道病原体的第一道免疫防线，该菌因此会在定植与感染过程中经历铁匮乏阶段。本研究借助高通量全基因组筛选技术——基因组阵列足迹分析（genomic array footprinting, GAF），明确了卡他莫拉菌在铁匮乏环境中生长所必需的遗传要素。为此，我们构建了大型marinerT7转座子突变体库（约28000株独立转座子突变株），分别在铁限制条件与对照生长条件下培养：铁限制条件通过30μM去铁胺（Desferal, DF30）螯合铁离子实现，对照条件则采用脑心浸液肉汤（brain heart infusion broth, DF0）。分别于指数生长期与早期静止期回收突变株，以此制备突变株特异性cDNA探针，将探针与定制化NimbleGen GAF微阵列进行杂交。本研究相关结果的进一步讨论详见下述文献：Stefan P.W. de Vries、Peter Burghout、Jeroen D. Langereis、Aldert Zomer、Peter W.M. Hermans、Hester J. Bootsma发表于《分子微生物学（Molecular Microbiology）》的论文《卡他莫拉菌在铁限制条件下生长的遗传需求》。实验设计概述：分别从胁迫条件（铁限制，DF30）与对照条件（DF0）下的指数生长期、早期静止期回收的突变株中制备cDNA探针；将探针与定制化GAF 4x72K NimbleGen GAF微阵列杂交以完成信号读取。