Transcriptional response of Pasteurella multocida to defined iron sources

Abstract

Pasteurella multocida was grown in iron-free chemically defined medium supplemented with hemoglobin, transferrin, ferritin, and ferric citrate as iron sources. Whole-genome DNA microarrays were used to monitor global gene expression over seven time points after the addition of the defined iron source to the medium. This resulted in a set of data containing over 338,000 gene expression observations. On average, 12% of P. multocida genes were differentially expressed under any single condition. A majority of these genes encoded P. multocida proteins that were involved in either transport and binding or were annotated as hypothetical proteins. Several trends are evident when the data from different iron sources are compared. In general, only two genes (ptsN and sapD) were expressed at elevated levels under all of the conditions tested. The results also show that genes with increased expression in the presence of hemoglobin did not respond to transferrin or ferritin as an iron source. Correspondingly, genes with increased expression in the transferrin and ferritin experiments were expressed at reduced levels when hemoglobin was supplied as the sole iron source. Finally, the data show that genes that were most responsive to the presence of ferric citrate did not follow a trend similar to that of the other iron sources, suggesting that different pathways respond to inorganic or organic sources of iron in P. multocida. Taken together, our results demonstrate that unique subsets of P. multocida genes are expressed in response to different iron sources and that many of these genes have yet to be functionally characterized.

FIG. 1.

Hierarchical clustering of microarray expression data. The ratio of fluorescence intensities between control and experimental populations across four experiments (hemoglobin, transferrin, ferritin, and ferric citrate) was log transformed and normalized between experiments by subtracting the mean change (n-fold). Red and green represent the increase (n-fold) and the decrease (n-fold), respectively, in gene expression. Data from an experiment using BHI medium as the iron source are presented for reference only and were not used for clustering. Unweighted hierarchical clustering using correlation as a similarity measure was performed by using Spotfire DecisionSite 6.3 software. Tables display selected genes from the clusters highlighted by the corresponding colors. Genes encoding hypothetical proteins are designated by “hyp.” The cluster tables contain genes that were generally expressed at elevated levels in response to transferrin and ferritin (A), expressed at elevated levels in response to all conditions except hemoglobin (B), expressed at reduced levels in all experiments (C), expressed at elevated levels in response to hemoglobin only (D), and expressed at elevated levels in all experiments (E).

FIG. 2.

Numbers of P. multocida genes that were significantly altered in expression. Significance was determined by using SAM software as described in Materials and Methods. The numbers enclosed by overlapping circles represent the numbers of genes whose expression was elevated (A) or lowered (B) under the conditions indicated.

FIG. 3.

Percentages of P. multocida genes whose expression was significantly altered upon exposure to defined iron sources. The total numbers of genes that were expressed at significant levels throughout the experiments are displayed below the supplemented iron sources. Genes are grouped by functional category, and the values shown are the percentages of the total number of genes that were transcriptionally altered.