Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro

Abstract

Vibrio cholerae is the etiologic bacterial agent of cholera, a severe diarrheal disease endemic in much of the developing world. The V. cholerae genome contains 3,890 genes distributed between a large and a small chromosome. Although the large chromosome encodes the majority of recognizable gene products and virulence determinants, the small chromosome carries a disproportionate number of hypothetical genes. Thus, little is known about the role of the small chromosome in the biology of this organism or other Vibrio species. We have used the rabbit ileal loop model of V. cholerae infection to obtain in vivo-grown cells under near midexponential conditions in the small-intestinal environment. We compared the global transcriptional pattern of these in vivo-grown cells to those grown to midexponential phase in rich medium under aerobic conditions. Under both conditions, the genes showing the highest levels of expression reside primarily on the large chromosome. However, a shift occurs in vivo that results in many more small chromosomal genes being expressed during growth in the intestine. Our analysis further suggests that nutrient limitation (particularly iron) and anaerobiosis are major stresses experienced by V. cholerae during growth in the rabbit upper intestine. Finally, relative to in vitro growth, the intestinal environment significantly enhanced expression of several virulence genes, including those involved in phenotypes such as motility, chemotaxis, intestinal colonization, and toxin production.

Figure 1

Graphical representation of V. cholerae gene expression in LB. (Upper) Distribution of small-chromosome genes. (Lower) Distribution of large-chromosome genes. Three thousand eight hundred-ninety genes were analyzed by using GENESPRING, and the expression levels of these genes are represented by normalized intensities. On the basis of expression levels, dashed lines divide the total genes into three equal areas: top one-third, middle one-third, and bottom one-third. One hundred seven genes, whose expression levels were >3.5, are not listed.

Figure 2

Functional classes of differentially expressed V. cholerae genes. (A) Functional categories of the 300 genes with the highest expression levels in vitro (aerobic growth in LB) and in vivo (growth in rabbit ileal loops). (B) Functional categories of genes showing 2-fold or greater changes under in vivo compared with in vitro growth conditions. The percentage of genes in each category appears above each bar.

Figure 3

RT-PCR of total RNA isolated from rabbit ileal loops. PCR was performed as described in Materials and Methods by using primers specific for selected V. cholerae genes and either reverse-transcribed RNA (cDNA) or chromosomal DNA as template.