J Bacteriol 183(6): 1983-1989

Identification of an <em>Actinobacillus pleuropneumoniae</em> Consensus Promoter Structure

Department of Microbiology, Michigan State University, East Lansing, Michigan 48824-1101

^{Corresponding author. Mailing address: Department of Microbiology, Michigan State University, East Lansing, MI 48824-1101. Phone: (517) 355-6515. Fax: (517) 353-8957. E-mail:}ude.usm.tolip@sklum.

Received 2000 Nov 15; Accepted 2000 Dec 20.

Abstract

Actinobacillus pleuropneumoniae promoter-containing clones were isolated from a genomic DNA library constructed in our lVET promoter trap vector pTF86. The promoter-containing clones were identified by their ability to drive expression of the promoterless luxAB genes of Vibrio harveyi. The degree of expression was quantifiable, and only high-expression or “hot” promoters were used for this study. Nine clones were sequenced, and their transcriptional start sites were determined by primer extension. The sequences upstream of the start site were aligned, and a consensus promoter structure for A. pleuropneumoniae was identified. The consensus promoter sequence for A. pleuropneumoniae was found to be TATAAT and TTG/AAA, centered approximately 10 and 35 bp upstream of the transcriptional start site, respectively. A comparison of the A. pleuropneumoniae consensus with other prokaryotic consensus promoters showed that the A. pleuropneumoniae consensus promoter is similar to that found in other eubacteria in terms of sequence, with an identical −10 element and a similar but truncated −35 element. However, the A. pleuropneumoniae consensus promoter is unique in the spacing between the −10 and −35 elements. The promoter spacing was analyzed by site-directed mutagenesis, which demonstrated that optimal spacing for an A. pleuropneumoniae promoter is shorter than the spacing identified for Escherichia coli and Bacillus subtilis promoters.

Abstract

Actinobacillus pleuropneumoniae is the causative agent of an acute necrotizing hemorrhagic pleuropneumonia in swine (13, 19, 22). Unfortunately, there is not an abundance of knowledge about what gene products play an important role in A. pleuropneumoniae disease. Our laboratory has developed an in vivo expression technology (IVET) system to identify gene products that have a role in the pathogenesis of swine pleuropneumonia (6). This IVET system has been used to identify gene promoters that are specifically induced during infection.

This IVET approach is based on the complementation of a defined attenuated riboflavin-requiring auxotroph (Rib^{) by a promoter trap vector that contains promoterless copies of the genes necessary to complement the genetic lesion in riboflavin synthesis. If the fragment of}A. pleuropneumoniae genomic DNA ligated into the vector contains a functional promoter, the rib genes are expressed and the auxotroph is able to survive and cause disease in experimentally infected pigs. The goal of our IVET studies is to recover clones from infected pigs, to characterize the promoters that are selected, and to determine what gene(s) lies downstream of each promoter to identify its role in A. pleuropneumoniae pathogenesis. A part of this work is to characterize these in vivo-expressed promoters and compare their structure to that of housekeeping gene promoters. However, the housekeeping or sigma-70 promoter structure in A. pleuropneumoniae is unknown.

The sigma-70 promoters in Escherichia coli are characterized by two nucleotide sequences that are centered at positions −35 and −10 relative to the transcriptional start site. The accepted consensus sequences are TTGACA and TATAAT for the −35 and −10 regions, respectively. These sequences are separated by 17 ± 1 nucleotides (10, 11, 16). There is a similar structure for other well-studied organisms such as Bacillus subtilis (12), but the research on promoter structures in pathogens such as A. pleuropneumoniae is limited.

Only a few attempts have been made to identify promoter elements in A. pleuropneumoniae, and none of the sigma factors have been identified or characterized (5, 9, 14). The results from these experiments show no clear similarity to E. coli promoters or to promoters from other eubacteria. This raises the question as to the structure of a sigma-70-like promoter in A. pleuropneumoniae and how it compares with other eubacterial promoters. Since several genes encoding antibiotic resistance markers that are readily expressed in E. coli are not expressed in A. pleuropneumoniae (25), it is likely that the A. pleuropneumoniae consensus promoter does differ in some way from that found in E. coli.

The goal of this study was to identify and characterize promoter sequences active in A. pleuropneumoniae under standard laboratory growth conditions. We identified DNA fragments with promoter activity by their ability to express promoterless lux genes and identified their transcriptional start sites by primer extension analysis. We have compared the DNA sequences of these active promoters and propose a consensus promoter sequence for A. pleuropneumoniae.

ACKNOWLEDGMENTS

This work was supported by USDA CSREES grant 98-02202.

We thank Robin Shea and Troy Fuller for their contributions to this work. We also thank Lee Kroos for his expert guidance and critical review of the work presented.

ACKNOWLEDGMENTS

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