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. 2016 Jun;31(3):270-83.
doi: 10.1111/omi.12122. Epub 2015 Sep 15.

Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis

Y Dou  1 , W Aruni  1 , A Muthiah  1 , F Roy  1 , C Wang  1 , H M Fletcher  1   2
Affiliations

Studies of the extracytoplasmic function sigma factor PG0162 in Porphyromonas gingivalis

Y Dou et al. Mol Oral Microbiol. 2016 Jun.

Abstract

PG0162, annotated as an extracytoplasmic function (ECF) sigma factor in Porphyromonas gingivalis, is composed of 193 amino acids. As previously reported, the PG0162-deficient mutant, P. gingivalis FLL350 showed significant reduction in gingipain activity compared with the parental strain. Because this ECF sigma factor could be involved in the virulence regulation in P. gingivalis, its genetic properties were further characterized. A 5'-RACE analysis showed that the start of transcription of the PG0162 gene occurred from a guanine (G) residue 69 nucleotides upstream of the ATG translation initiation codon. The function of PG0162 as a sigma factor was confirmed in a run-off in vitro transcription assay using the purified rPG0162 and RNAP core enzyme from Escherichia coli with the PG0162 promoter as template. As an appropriate PG0162 inducing environmental signal is unknown, a strain overexpressing the PG0162 gene designated P. gingivalis FLL391 was created. Compared with the wild-type strain, transcriptome analysis of P. gingivalis FLL391 showed that approximately 24% of the genome displayed altered gene expression (260 upregulated genes; 286 downregulated genes). Two other ECF sigma factors (PG0985 and PG1660) were upregulated more than two-fold. The autoregulation of PG0162 was confirmed with the binding of the rPG0162 protein to the PG0162 promoter in electrophoretic mobility shift assay. In addition, the rPG0162 protein also showed the ability to bind to the promoter region of two genes (PG0521 and PG1167) that were most upregulated in P. gingivalis FLL391. Taken together, our data suggest that PG0162 is a sigma factor that may play an important role in the virulence regulatory network in P. gingivalis.

Keywords: extracytoplasmic function sigma factor; periodontitis; sigma 24; virulence.

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Figures

Fig. 1
Fig. 1
The secondary and tertiary structure prediction of PG0162. (A) Model of PG0162. Blue – N terminal end loop and helix, Red – C terminal end helix, Sky Blue and Green – most conserved sigma −70 domain 2, Brown & orange - sigma −70 promoter interacting SR-4 domain. (B) Sigma 70 domain identified in PG0162 (Position 35–93; 132–187, tan color). (C) Surface model of PG0162. (D) Surface model of PG0162, 90° rotation. (E) Secondary structure of sigma 70 subdomain-2. (F) Secondary structure of sigma 70 subdomain-4.
Fig. 2
Fig. 2
5′ RLM-RACE showed that the transcription of PG0162 start from a guanine which is located 69 nt upstream of the translation start codon. (A) PCR amplification of PG0162 promoter region was showed about 120 bp. (B) Promoter region of PG0162. The transcription start site G is bold shadowed; the translation start codon ATG is bold italic; the −35 and −10 elements were bolded, italic and underlined; the ribosome binding site GAGGA was underlined.
Fig. 3
Fig. 3
In vitro transcription analysis of PG0162. (A) Western blot of rPG0162. (B) In vitro transcription product analysis. L: φX174 DNA/HinfI Dephosphorylated Marker; Lane 1: no rPG0162; Lane 2: with rPG0162; Lane 3: RNaseA was added; Lane 4: DNaseI was added.
Fig. 4
Fig. 4
Electrophoretic mobility shift assay showed that rPG0162 could bind to the 10 fmol of promoter of PG0162, PG0521, and PG1167. (A) rPG0162 bind to PG0162 promoter. Lane 1, no protein; Lane 2, 0.5 pmol of rPG0162; Lane 3, 1 pmol of rPG0162; Lane 4, 2 pmol of rPG0162; Lane 5, 2 pmol of rPG0162 plus unlabeled PG0162. (B) Promoter regions of PG0521 and PG1167, the −35 and −10 elements were underlined; the putative transcription start site were bolded. (C) rPG0162 could bind to promoter of PG0521. Lane 1, no protein; lane 2, 0.5 pmol of rPG0162; lane 3, 1 pmol of rPG0162; lane 4, 1 pmol of rPG0162 and non-labeled PG0521. (D) rPG0162 could bind to promoter of PG1167. Lane 1, no protein; lane 2, 0.5 pmol of rPG0162; lane 3, 1 pmol of rPG0162; lane 4, 5 pmol of rPG0162; lane 5, 5 pmol of rPG0162 and non-labeled PG1167 promoter DNA. (E) Logo of −35 and −10 consensus motifs recognized by PG0162. (F) rPG0162 did not bind to promoter of PG0985. (G) rPG0162 did not bind to promoter of PG1660. F and G: Lane 1, no protein; lane 2, 0.5 pmol of rPG0162; lane 3, 1 pmol of rPG0162; lane 4, 5 pmol of rPG0162. (H) rPG0162 did not bind to promoter of PG1661. Lane 1, no protein; lane 2, 0.5 pmol of rPG0162; lane 3, 1 pmol of rPG0162.
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