Promoter architecture of the Porphyromonas gingivalis fimbrillin gene

Abstract

Porphyromonas gingivalis fimbriae can mediate adherence to many of the available substrates in the oral cavity. Expression of P. gingivalis fimbriae is regulated at the transcriptional level by environmental signals, such as temperature and hemin concentration. The arrangement of the upstream promoter and regulatory sequences required for transcription and control of the fimbrial structural gene (fimA) was investigated. Primer extension analysis demonstrated that the transcriptional start site of the fimA gene is located 41 bp upstream from the translational start codon. A region (upf) spanning 648 bp upstream of the start codon to 44 bp downstream of the translational start site was cloned upstream of a promoterless lacZ reporter gene. A series of deletion and base substitution mutations were then generated in the upf region. The constructs were introduced into the chromosome of P. gingivalis, and promoter activity measured by assaying levels of beta-galactosidase. The results showed that fimA contains sequences resembling sigma70 promoter consensus sequences, consisting of a -10 region (TATGAC) located at -18 to -23 and a -35 region (TTGTTG) located at -41 to -46 from the transcriptional start point. The AT-rich upstream sequences spanning bases -48 to -85 and bases -90 to -240 were required for full expression of the fimA gene, indicating the existence of positive regulation regions. Moreover, the -48 to -64 region may constitute an UP element, contributing to promoter activity in P. gingivalis. Thus, our data suggest that the P. gingivalis fimA gene has a transcription complex consisting of -10 and -35 sequences, an UP element, and additional AT-rich upstream regulatory sequences.

FIG. 1

Transcriptional start site mapping of P. gingivalis fimA, using primers PE1 (a) and PE3 (b) Lanes: 1, primer extension with P. gingivalis 33277 (a) and P. gingivalis UPF (b) RNAs as templates; G, T, C, and A, nucleotide-specific sequencing reactions.

FIG. 2

Core promoter region of the fimA gene. (A) Schematic map of the fimA promoter region fused with the lacZ reporter gene. (B) Sequences of the core promoter region of fimA with and without mutations. UPF represents wild-type promoter sequence; the sequences subsequently mutated are underlined and in boldface. The sequences of eight mutated promoters are also shown. Base substitutions are represented by underlined and bolded letters and deletion mutations are shown by the symbol ▵, with the number representing deleted base pairs.

FIG. 3

Homologous recombination between pUPF5 or its derivatives (pMP) and the P. gingivalis chromosome. The thicker lines represent the DNA fragment containing the fimA gene, fimA promoter region, lacZ gene, and erythromycin resistance gene (Ery). (A) The homologous recombination occurs upstream of the mutation. (B) The recombination occurs downstream of the mutation. Pmut. and Pwild, mutant and wild-type promoters, respectively.

FIG. 4

PCR and Southern blot analyses of P. gingivalis MP150. (A) Chromosomal DNAs from three isolates of P. gingivalis MP150 analyzed by PCR with forward primer FP1 and reverse primer lacZ2. Lanes: 1, DNA standard; 2 to 4, isolates of P. gingivalis MP150. (B) DNA samples analyzed by Southern blotting. DNA was digested with SspI and HindIII and probed with a 1.4-bp fimA fragment. Lanes: 1, DNA standard; 2, UPF; 3 to 5, isolates of MP150.

FIG. 5

Effects of fimA mutations in the promoter region on transcriptional activity. See Fig. 2 for depiction of mutations. β-Galactosidase level is presented in Miller units as described in the text. Data represent the means and standard errors obtained from at least three independent experiments.

FIG. 6

RNA polymerase (ς⁷⁰)-fimA interaction. (A) DNA used in the mobility shift DNA binding assay. The region 5′ of the DNA fragment was tagged with an EcoRI site, and +1 corresponds to the transcriptional start site of the fimA gene. (B) Mobility shift DNA binding assay. Lanes: 1, fimA promoter fragment only; 2, fimA promoter fragment and E. coli RNA holoenzyme; 3, fimA promoter, RNA holoenzyme, and calf thymus DNA.

FIG. 7

Nucleotide sequence of the fimA promoter region. The underlined and boldface sequences represent −10, −35, UP element, and transcriptional start site regions. The −71 to −240 region contains positive regulatory sequences. RBS, ribosome binding site.