Anaerobic regulation of Actinobacillus actinomycetemcomitans leukotoxin transcription is ArcA/FnrA-independent and requires a novel promoter element.
The periodontal pathogen, Actinobacillus actinomycetemcomitans, produces a 116-kDa leukotoxin that appears to help the bacterium evade the innate host immune response. The expression of leukotoxin is induced when cells are grown anaerobically, a condition found in the subgingival crevice. This regulation most likely occurs at the transcriptional stage since the levels of leukotoxin RNA are induced by hypoxic growth. In order to map the leukotoxin promoter element(s) responsible for oxygen regulation, deletion and linker-scanning mutations were cloned into a transcriptional reporter gene plasmid and then tested in A. actinomycetemcomitans grown aerobically or anaerobically. A 35-bp DNA element, at position -36 to -70, was found to be responsible for the repression of leukotoxin synthesis in aerobically grown A. actinomycetemcomitans. The sequence of this oxygen response element (ORE) does not match the consensus binding sites for known DNA binding proteins, not even Fnr or ArcA which play major roles in oxygen regulation in other bacteria. However, since sequence analysis alone cannot disprove a role for the Fnr or ArcAB pathways in leukotoxin regulation, the genes for the Fnr and ArcA homologues in A. actinomycetemcomitans were identified, mutated by targeted insertional mutagenesis and assessed for loss of oxygen regulation. Deletion of either fnr or arcA altered the expression of numerous A. actinomycetemcomitans proteins, but leukotoxin expression was still repressed by oxygen. These results, coupled with the promoter mutation analyses, lead to the conclusion that A. actinomycetemcomitans employs a novel pathway in the aerobic/anaerobic regulation of leukotoxin synthesis.
Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900, USA. Kolodrubetz@uthscsa.edu
David
D
Kolodrubetz
Linda
L
Phillips
Chris
C
Jacobs
Alex
A
Burgum
Ellen
E
Kraig
D016976:Aggregatibacter actinomycetemcomitans / Q000235:genetics*; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D005098:Exotoxins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D005098:Exotoxins; C018579:leukotoxin