TEXT

Top Abstract Text References

Pasteurella multocida is a gram-negative, nonmotile, rod-shaped, facultative anaerobe that has been isolated from a wide range of mammals and birds throughout the world. This organism is the etiologic agent of a variety of economically significant diseases, including fowl cholera in poultry, hemorrhagic septicemia in cattle and buffalo, atrophic rhinitis in swine, and snuffles in rabbits (9, 12). The global distribution, severity of disease caused, and the wide variety of livestock affected by P. multocida account for considerable economic losses due to this pathogen worldwide (12).

Iron is an essential nutrient for most organisms due to its important role in metabolic electron transport chains. Due to the presence of specialized protein carriers such as transferrin and lactoferrin in body fluids, the concentration of free iron normally present in mammalian and avian hosts is not enough to support the in vivo growth of bacteria (1, 2). Successful pathogens must therefore possess an effective response to the limited iron conditions encountered upon entry into a host. Previous studies illustrate the fact that the identities of genes and pathways involved in the acquisition, transport, and utilization of iron in P. multocida are poorly understood (5, 6, 10, 11, 13, 16). Here we have utilized whole-genome microarray analysis to identify genes with altered expression patterns when P. multocida is grown under iron-limiting conditions.

Bacterial growth and RNA isolation. P. multocida PM70 was grown to log phase in a flask of brain-heart infusion (BHI) medium (Becton Dickinson) at 37°C. The culture was split into two 180-ml volumes, briefly centrifuged at 4°C, washed with 1× phosphate-buffered saline (pH 7.0), and centrifuged again. One pellet was resuspended in 180 ml of BHI medium, and the other was resuspended in 180 ml of BHI medium containing the iron chelator 2,2'-dipyridyl (200 µM) (Sigma, St. Louis, Mo.). The resuspended cultures were incubated on a rotary shaker at 37°C, and 30-ml volumes were removed 15, 30, 60, and 120 min after resuspension. These samples were briefly centrifuged at 4°C, and the pellets were flash frozen in dry ice and ethanol. Total RNA extractions were performed with RNeasy Maxi columns (Qiagen, Chatsworth, Calif.), with DNase digestions done on the column by adding 82 Kunitz units of enzyme (Qiagen) and incubating at room temperature for 15 min.

Microarray analysis. Gene expression analysis with DNA microarrays was performed as described elsewhere (http: //www.cbc.umn.edu/ResearchProjects/AGAC/Pm/Pmarraydata.html) (8a). In brief, a library of targets representing all 2,014 open reading frames (ORFs) from P. multocida PM70 (AE004439) was constructed with primers designed to amplify fragments of 500 bp from each ORF from genomic DNA. Two successive rounds of PCR were performed to minimize genomic DNA contamination in the products of amplification, and the final 100-µl reactions were checked for quality on agarose gels and purified with MultiScreen PCR plates (Millipore, Bedford, Mass.). The 1,936 (96%) ORF segments that were successfully amplified were printed using a Total Array System robot (BioRobotics, Boston, Mass.). RNA from P. multocida grown in BHI medium alone or BHI medium containing 2,2'-dipyridyl were labeled with Cy3 and Cy5, respectively, and competitively hybridized with the printed microarrays. Images of the hybridized arrays were obtained with a Scanarray 5000 microarray scanner (GSI Lumonics, Watertown, Mass.). Two independent hybridizations using independent RNA extractions were performed for each time point. Fluorescent intensities for individual spots were normalized based on the total intensity of fluorescence in the Cy3 and Cy5 channels. Hierarchical clustering and analysis were performed using the publicly available programs Cluster and Treeview (M. Eisen; http://www.microarrays.org/software).

P. multocida genes with altered expression profiles. A complete set of all results is presented on our website at http: //www.cbc.umn.edu/ResearchProjects/AGAC/Pm/Pmarraydata.html. Of the 1,936 ORFs represented on the array, 135 had changes in expression of at least twofold over the course of the experiment (Table 1 and Fig. 1). The remaining ORFs either were not well measured or did not have detectably altered expression levels. A twofold change in the level of expression was used as an indication of significance based on the reproducibility of results obtained in our lab. These genes can be functionally classified based on homology with previously described proteins in public databases (Fig. 2). The classification strategy we used was based on the system developed for the Haemophilus influenzae genome (4). Several genes involved in energy metabolism had altered expression levels. For example, three genes encoding glycolysis enzymes (gapdh, pgk, and eno) had, on average, 2.8-fold decreases in expression in response to low iron conditions, while lactate dehydrogenase expression increased nearly 8-fold. The gene encoding Fnr, a transcriptional activator of genes involved in anaerobic metabolism, was expressed at 3.6-fold higher levels. Interestingly, some genes that are normally transcriptionally activated by Fnr, such as fumarate reductase and formate dehydrogenase (frdABCD and fdxGHI), had an over fourfold decrease in average expression. Additionally, the hypothetical protein encoded by ORF 0064 decreased 3.8-fold and is homologous to YfiD, a pyruvate formate lyase also regulated by Fnr. These discrepancies in Fnr regulation may be due to the disruption of FeS cluster cofactors that Fnr has been proposed to utilize for oxygen sensing (8). The expression of arcA, a transcriptional repressor of aerobic metabolism genes, decreased nearly threefold. In contrast, four genes involved in amino acid biosynthesis (aroA, trpG, hisH, and ilvM) had an average increase in expression of 2.6-fold. Together, these data show that in general the expression levels of genes involved in energy metabolism decreased 2.1- to 6-fold while those of genes involved in DNA and central intermediary metabolism, as well as those involved in amino acid biosynthesis, generally increased (Fig. 2).

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Functional classification of P. multocida genes that had altered expression levels, of twofold or more, in response to low iron conditions. Open and closed bars represent the numbers of genes that decreased and increased in expression, respectively.

View larger version (37K): [in this window] [in a new window]   FIG. 1.   Hierarchical clustering of 135 P. multocida ORFs that had significantly altered expression levels under low iron conditions. Clustering and visualization were performed using the software programs Cluster and Treeview. Red and green colors represent fold increase and decrease, respectively, in gene expression in response to low iron conditions. The gene expression profiles for the 15-, 30-, 60-, and 120-min time points are shown in the panel on the left, and corresponding ORF numbers in the P. multocida genome are also shown.

Utility of the microarray-based approach for profiling of gene expression in bacteria. A major advantage of the microarray approach is that it enables simultaneous profiling of the transcriptional activity of the entire bacterial genome in a time- and cost-efficient manner. This is especially important in ascribing possible functions to the many hypothetical genes discovered through the whole genome sequencing approach. Furthermore, knowledge of the transcriptional profile of the entire genome enables a holistic approach to the understanding of the metabolic state of the organism under various conditions. Apart from being relatively inexpensive to set up and perform, the use of two-color hybridizations permits comparative analyses of bacterial gene expression by obviating many of the sources of variation inherent in other methods, including single color- or radiolabel-based hybridization methods and many of the PCR-based approaches.