The Yersinia pestis NlpD Lipoprotein is important for iron assimilation and is functionally linked to the twin-arginine translocation system

Cultures of Y. pestis Kimberley53 (Kim53) virulent strain and Kim53∆nlpD were grown in heart infusion broth supplemented with 0.2% xylose and 2.5 mM CaCl2 for 5 h at 37°C, 200rpm. Total RNA samples were extructed and used for cRNA synthesis and labelling (using Cy3-CTP and Cy5-CTP). cRNA of two independent samples from each strain were labeled and hybridized to custom made Agilent 8x15K slide. Goal: Identifing alterations in gene expression profile between the wild-type Kim53 virulent strain and Kim53∆nlpD. Those altered mRNA transcrips will be used for better understanding of the cause for Kim53∆nlpD attenuation. Y. perstis two independent biological replicates were performed. Extracted total RNA were used for cRNA synthesis and labeling with Cy3/Cy5-CTP, hybridized to Agilent custom-made 8x15K slide format. For bacterial total RNA preparation, bacterial colonies were grown on BHIA plats for 48 h at 28°C , harvested and diluted in heart infusion broth (HIB) supplemented with 0.2% xylose and 2.5 mM CaCl2 to an OD660 of 0.01 and grown over night at 28°C in a shaker (200 rpm). The resulting cultures were diluted in fresh broth to an OD660 of 0.05 and allowed to grow for 5 h at 37°C. Aliquots of ~5×108 cfu were collected by centrifugation and cells were used for RNA preperation from Y. pestis Kimberley53 (Kim53) virulent strain and Kim53∆nlpD (fully attenuated) strain. Differential expression was evaluate using linear models for designed microarray experiments. Genes with differences corresponding to P<0.05 in either the high or the low photomultiplicator scans and that had signal to control or control to signal ratios ³2.0 were considered to be differentially regulated.