Comparative transcriptomics in Yersinia pestis: a global view of environmental modulation of gene expression

Abstract

Background: Environmental modulation of gene expression in Yersinia pestis is critical for its life style and pathogenesis. Using cDNA microarray technology, we have analyzed the global gene expression of this deadly pathogen when grown under different stress conditions in vitro.

Results: To provide us with a comprehensive view of environmental modulation of global gene expression in Y. pestis, we have analyzed the gene expression profiles of 25 different stress conditions. Almost all known virulence genes of Y. pestis were differentially regulated under multiple environmental perturbations. Clustering enabled us to functionally classify co-expressed genes, including some uncharacterized genes. Collections of operons were predicted from the microarray data, and some of these were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Several regulatory DNA motifs, probably recognized by the regulatory protein Fur, PurR, or Fnr, were predicted from the clustered genes, and a Fur binding site in the corresponding promoter regions was verified by electrophoretic mobility shift assay (EMSA).

Conclusion: The comparative transcriptomics analysis we present here not only benefits our understanding of the molecular determinants of pathogenesis and cellular regulatory circuits in Y. pestis, it also serves as a basis for integrating increasing volumes of microarray data using existing methods.

Figure 1

Environmental modulation of expression of virulence genes. Shown in the squares are the putative stages of transmission/infection of Y. pestis. The TreeView charts show the transcriptional changes of the virulence genes, where columns represent different microarray experiments, and rows represent genes. Color intensities denote log₂ ratios as follows: green, negative; black, zero; red, positive; gray, missing data.

Figure 2

RT-PCR analysis of potential operons. Shown is the electrophoresis image of an RT-PCR product with the relative location of the expected size. Total RNA was used to synthesize cDNA in the presence or absence of reverse transcriptase, and the resulting cDNA samples subsequently used for RT-PCR templates, are indicated as "cDNA" or "RNA", respectively. Genomic DNA was used as a template, and is indicated as "DNA" for control PCR. "Marker" represents a DNA size marker (900, 700, 500, 300 and 100 bp from top to bottom).

Figure 3

Schematic representation of the clustered microarray data. Columns from left to right represent the different microarray experiments from up to down shown in Table 4, while rows from up to down represent genes and their corresponding gene names were listed in the order (left to right and up to down). The black vertical lines are used to define the range of clusters of co-expressed genes. Red represents up-regulation and green represents down-regulation of the corresponding genes.

Figure 4

Graphical representation of the consensus patterns by motif search. The strict consensus string, sequence logo, and PSSM are included in (a) Fur-like box; (b) PurR-like box; and (c) Fnr-like box. The underlined number is the maximum possible score with PSSM. For the sequence logo, the height of each letter indicates the relative frequency of that base at that position, while the height of each stack of letters corresponds to the sequence conservation at that position.

Figure 5

EMSA analysis of the binding of Fur protein to promoter DNA probes. Lane 1 contains the rabbit anti-F1 IgG of Y. pestis, lane 2 the specific DNA competitor, and lanes 3–7 contain 1.0, 0.7, 0.4, 0.1 and 0 μg of the recombinant Fur protein. In (A) – (I), an arrow and an asterisk indicate the probe (free) and the Fur-probe complex (bound), respectively.