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Applied and Environmental Microbiology, June 2005, p. 2949-2954, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2949-2954.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Real-Time Quantitative Reverse Transcription-PCR Analysis of Expression Stability of Actinobacillus pleuropneumoniae Housekeeping Genes during In Vitro Growth under Iron-Depleted Conditions

K. Klitgaard Nielsen* and M. Boye

Danish Institute for Food and Veterinary Research, Copenhagen, Denmark

Received 20 September 2004/ Accepted 13 December 2004

The aims of the present investigation were to develop and test a sensitive and reproducible method for the study of gene expression in the porcine lung pathogen Actinobacillus pleuropneumoniae by real-time quantitative reverse transcription (RT)-PCR and to evaluate a number of suitable internal controls, as such controls have not been defined yet for this bacterium. Bacterial gene expression was studied during in vitro exponential and early stationary growth in medium with and without sufficient iron, respectively. First, the stability of expression of five genes, the glyA, tpiA, pykA, recF, and rhoAP genes involved in basic housekeeping, was evaluated on the basis of the mean pairwise variation. All the housekeeping genes included were stably expressed under the conditions investigated and consequently were included in the normalization procedure. Next, the geometric mean of the internal control genes was used to correct five genes of interest. These genes were three genes involved in iron acquisition (tbpA, exbB, and fhuD), the heat shock protein gene groEL, and a putative quorum-sensing gene (luxS). The level of tbpA, exbB, and fhuD expression in A. pleuropneumoniae showed significant up-regulation under iron-restricted conditions compared to bacteria grown in medium with sufficient iron. The observed expression patterns of the genes of interest were consistent with previous observations. This study therefore lends further support to the use of real-time quantitative RT-PCR, with the glyA, tpiA, pykA, recF, and rhoAP genes as internal controls, for future similar gene expression studies in A. pleuropneumoniae.

* Corresponding author. Mailing address: Danish Institute for Food and Veterinary Research, Bülowsvej 27, DK-1790 Copenhagen, Denmark. Phone: 45 72 34 62 65. Fax: 45 72 34 60 01. E-mail: kni{at}dfvf.dk.

Applied and Environmental Microbiology, June 2005, p. 2949-2954, Vol. 71, No. 6
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.6.2949-2954.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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