Use of operon fusions in Mannheimia haemolytica to identify environmental and cis-acting regulators of leukotoxin transcription

Abstract

The leukotoxin of Mannheimia haemolytica is an important virulence factor that contributes to much of the pathology observed in the lungs of animals with bovine shipping fever pneumonia. We believe that identification of factors that regulate leukotoxin expression may provide insight into M. haemolytica pathogenicity. The DNA sequence upstream of the leukotoxin operon is divergently shared by P(lapT), which transcribes an arginine permease gene. The intergenic region contains several elements that are potential sites for transcriptional modulation of the promoters. We have developed plasmid-borne chloramphenicol acetyltransferase (cat) operon fusions, as well as lktC::cat chromosomal fusions, to study transcription initiation in M. haemolytica. Using these genetic tools, we have identified cis-acting sequences and environmental conditions that modulate transcription of the leukotoxin and lapT promoters. By deletion analysis, promoters were shown to rely on sequences upstream of their -10 and -35 regions for full activity. Direct repeats of the sequence TGT-N(11)-ACA and a static bend region caused by phased adenine tracts were necessary for full activation of P(lkt). A computer-generated model of the promoter's structure shows how DNA bending brings the repeat sequences within close proximity to the P(lkt) RNA polymerase, and we hypothesize that these repeats are a binding site for an activator of leukotoxin transcription. The lktC::cat operon fusion was also used to demonstrate that, like that of other RTX toxins, leukotoxin transcription is environmentally regulated. Roles for iron deprivation and temperature change were identified.

FIG. 1

(a) Nucleotide sequence of the lapT-lktC intergenic regulatory region of M. haemolytica SH1217. Bolded cytosine residues indicate the transcriptional start sites of P_lapT and P_lkt. The sequence is numbered in reference to the P_lkt transcriptional start site at +1. Underlined nucleotides indicate the predicted −10 promoter regions of P_lapT and P_lkt. Shaded nucleotides correspond to the four phased adenine tracts. Putative upstream activator sites A1, A2, and A3 are boxed. The near-consensus IHF binding site is indicated with a dotted line above the sequence, and a potential Fur binding site is marked above the nucleotides (∨∨∨∨∨). Unique enzyme restriction sites are shown. (b) Computer-predicted DNA structure of the 0.4-kb lapT/lktC promoter region that was generated by the CURVATURE program (42). Two different views were created by rotating the image along its x axis by using RasMac v2.6 (37). The image on the right has been reduced in size. Sequence elements are noted on the model. IHF and FUR are potential IHF and FUR binding sites, and A-tracts are adenine tracts. Solid arrows show the direction of transcription.

FIG. 2

Plasmid maps of cat promoter probe vector pAM2355 and operon fusion plasmids pAM2364 (lkt::cat) and pAM2365 (lapT::cat). Plasmid pAM2355 contains transcriptional terminators on either side of the cat reporter gene to prevent readthrough from other sites on the plasmid. A PCR-amplified fragment containing the lapT/lktC promoter region was ligated upstream of the promoterless cat gene in both orientations to create pAM2364 and pAM2365. All of the plasmids contain the M. haemolytica origin of replication from pYFCI. Only relevant restriction sites are shown.

FIG. 3

Primer extension analysis of pAM2364. Total RNA from M. haemolytica strain SH1217 harboring plasmid pAM2364 was obtained, and the transcriptional start site of the promoter was identified by primer extension. The asterisk indicates the mapped transcriptional start site of lkt::cat (lane 1). The DNA sequence on the right was generated by using the same primer as that used for the primer extension analysis. The −10 region of the lkt promoter is underlined for reference to the start site location.

FIG. 4

Transcription of lkt::cat and lapT::cat operon fusions in M. haemolytica. The CAT activity of SH1217 harboring pAM2364 (open circles) or pAM2365 (closed squares) was measured throughout all phases of growth in BHI at 37°C. Each point represents the average of two samples. Standard deviations are indicated with error bars. The growth curve of SH1217 harboring pAM2364 is shown as a dotted line. OD₆₀₀, optical density at 600 nm.

FIG. 5

(a) CAT activities of fusion plasmids with promoter fragments deleted. The diagram shows the lapT/lktC promoter region with particular sequence elements marked. Symbols: closed and open ovals, −10 region; dotted line, near-consensus IHF binding site; open squares, direct repeats A1, A2, and A3; closed rectangles, adenine tracts. By using the restriction enzyme listed next to the fragment, sequential deletions were made in the promoter to remove the elements indicated and then the promoter was ligated to the promoterless cat gene. The CAT activity reported is relative to that of the full-length promoter (wild type) in either the P_lkt or the P_lapT orientation, as indicated. CAT activity is the average of duplicate samples ± 1 standard deviation. Strains containing each plasmid were grown under identical conditions in BHI at 37°C for 18 h. (b) CAT activity of chromosomal operon fusions. Operon fusions equivalent to plasmids pAM2496 and pAM2506 were recombined onto the M. haemolytica chromosome to create strains SH2410 and SH2405, respectively. CAT activities were determined for cultures grown in BHI at 37°C for 18 h. Results are presented as percentages of the CAT activity of SH2020, which was set at 100%. Results are the averages of duplicate samples, and error bars show 1 standard deviation.

FIG. 6

Effect of iron depletion on leukotoxin transcription. SH2020 cells were grown for 18 h in the presence of 0, 10, 100, or 250 μM 2,2-dipyridyl (filled bars), and transcription was analyzed by CAT assay. Iron repletion was carried out by adding 200 μM FeCl₃ plus 100 or 250 μM 2,2-dipyridyl (open bars) to the culture medium. The data represent duplicate samples, and error bars represent 1 standard deviation. OD₆₀₀, optical density at 600 nm.

FIG. 7

Influence of temperature on leukotoxin transcription. SH2020 was grown at 42°C (filled squares) or at 30°C (open circles) until mid-log phase and then shifted to 42°C. CAT assays were performed on samples taken post temperature upshift. The data points represent averages of two samples, and error bars indicate 1 standard deviation. OD₆₀₀, optical density at 600 nm.

FIG. 8

Model of P_lapT and P_lkt transcription. The black line represents lapT-lktC intergenic DNA. Arrows indicate the direction of transcription, and asterisks denote transcriptional start sites of P_lapT and P_lkt. We have tried to maintain appropriate scale between RNA polymerase (RNAP) and IHF and the predicted DNA-protein interactions for open-complex formation, as well as approximate angles induced by IHF or the four adenine tracts. As shown in the model, we predict that DNA bending is an important part of stable RNA polymerase open-complex formation and transcriptional activation for both P_lapT and P_lkt. We hypothesize that transcription of P_lkt is further enhanced in the presence of an activator protein (shown here as a dimer) bound to upstream activator sites.