Expression of the Oligopeptide Permease Operon of Moraxella catarrhalis Is Regulated by Temperature and Nutrient Availability

Abstract

Moraxella catarrhalis causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Together, these two conditions contribute to enormous morbidity and mortality worldwide. The oligopeptide permease (opp) ABC transport system is a nutritional virulence factor important for the utilization of peptides. The substrate binding protein OppA, which binds peptides for uptake, is a potential vaccine antigen, but little was known about the regulation of gene expression. The five opp genes oppB, oppC, oppD, oppF, and oppA are in the same open reading frame. Sequence analysis predicted two promoters, one located upstream of oppB and one within the intergenic region between oppF and oppA. We have characterized the gene cluster as an operon with two functional promoters and show that cold shock at 26°C for ≤ 0.5 h and the presence of a peptide substrate increase gene transcript levels. Additionally, the putative promoter upstream of oppA contributes to the transcription of oppA but is not influenced by the same environmental cues as the promoter upstream of oppB. We conclude that temperature and nutrient availability contribute to the regulation of the Opp system, which is an important nutritional virulence factor in M. catarrhalis.

FIG 1

Diagram of the opp operon in the wild-type (WT) strain and deleted segments in the prm mutant (not to scale). Numbers denote base pairs, and small arrows indicate promoter regions. Black arrows denote open reading frames, and gray arrows denote the drug resistance cassette.

FIG 2

Results of reverse transcriptase PCR showing that the opp genes are transcribed as an operon. RNA was isolated from wild-type O35E cultures grown to late log phase in brain heart infusion medium. Primers were designed to span the end of one gene through the beginning of another (intergenic region) or the middle of each gene, as indicated. Lanes 1, 4, 7, 10, 13, and 16 are RNA with reverse transcriptase and polymerase. Lanes 2, 5, 8, 11, 14, and 17 are RNA with no reverse transcriptase and only polymerase. Lanes 3, 6, 9, 12, 15, and 18 are DNA with polymerase. Lane 19 has no template with reverse transcriptase and polymerase.

FIG 3

Results of quantitative real-time PCR to determine the levels of the oppB, oppC, oppD, oppF, and oppA transcripts in the prm mutant compared to wild-type levels. RNA was isolated from bacterial cultures grown from an OD₆₀₀ 0.07 to an OD₆₀₀ of 0.8 at 37°C. Transcript quantity was normalized to the level of the housekeeping gene gyrB. All results are the averages of results from 3 separate experiments performed in triplicate. Error bars represent standard errors of the means from 3 biological replicates. See the text for statistical analysis.

FIG 4

Characterization of mutants. (A) Immunoblot assay of wild-type and prm whole bacterial lysates probed with OppF antiserum (1:10,000). OppF is ∼36 kDa, as indicated by the arrow. (B) Immunoblot assay of wild-type and prm whole bacterial lysates probed with OppA antiserum (1:1,000,000). OppA is ∼75 kDa, as indicated by the arrow. A secondary anti-rabbit horseradish peroxidase conjugate was used at a 1:2,000 dilution for detection for both immunoblots. (C) Coomassie blue-stained SDS-PAGE gel of the 2 whole bacterial lysates showing equal protein loading. Molecular mass markers (in kilodaltons) are indicated on the left.

FIG 5

Growth curves of wild-type strain O35E and the prm knockout mutant in chemically defined medium broth supplemented with arginine (top) or triornithine (bottom). The x axis is time in hours, and the y axis is the optical density at 600 nm. Each point is the average of data from 5 wells.

FIG 6

Temperature alters opp gene transcription during growth. Quantitative real-time PCR was performed on 50 ng of RNA per sample isolated from WT bacterial cultures grown to an OD₆₀₀ of 0.8 at 37°C in BHI medium. Normalized transcript levels were statistically higher (*), as determined by a t test (P < 0.05), at 26°C than at 37°C for all the opp genes. Results are the averages of data from 3 experiments performed in triplicate, with standard deviations denoted with error bars.

FIG 7

Impact of timed cold shock exposure on oppB and oppA gene transcription. Quantitative real-time PCR was performed on 50 ng of RNA per sample isolated from WT bacterial cultures grown to an OD₆₀₀ of 0.3 at 37°C and subjected to growth at 26°C for set time intervals of 0.5, 1, 2, and 3 h. Transcript levels were normalized to the level of the housekeeping gene gyrB. Transcript levels of oppB and oppA at 37°C were compared with those at 26°C. Results are the averages of data from 3 experiments performed in triplicate, with standard deviations denoted with error bars, and statistical significance (*) was determined with a t test (P < 0.05).

FIG 8

Quantitative real-time PCR results showing the normalized oppA transcript levels in the prm mutant during timed cold shock intervals. RNA was isolated from bacterial cultures grown to an OD₆₀₀ of 0.3 at 37°C, followed by growth for timed intervals of 0.5, 1, and 3 h at 37°C or cold shock at 26°C. Normalized results are the averages of results from 2 separate experiments performed in triplicate, with standard deviations denoted with error bars, and statistical significance (*) was determined with a t test (P < 0.05).

FIG 9

Nutrient availability impacts opp gene transcription. Quantitative real-time PCR was performed on 50 ng of RNA per sample isolated from bacterial cultures grown to an OD₆₀₀ of 0.8 at 37°C and 26°C in chemically defined medium supplemented with free arginine (R) or chemically defined medium supplemented with a peptide as the only source of arginine (VANRP). Normalized transcript levels were statistically higher (*), as determined by a t test (P < 0.05), in medium supplemented with the peptide at 26°C but not at 37°C for all the opp genes. Results are the averages of data from 3 experiments performed in triplicate, with standard deviations denoted with error bars.