Porphyromonas gingivalis PgFur Is a Member of a Novel Fur Subfamily With Non-canonical Function

Abstract

Porphyromonas gingivalis , a keystone pathogen of chronic periodontitis, uses ferric uptake regulator homolog (PgFur) to regulate production of virulence factors. This study aimed to characterize PgFur protein in regard to its structure-function relationship. We experimentally identified the 5′ mRNA sequence encoding the 171-amino-acid-long PgFur protein in the A7436 strain and examined this PgFur version as a full-length protein. PgFur protein did not bind to the canonical Escherichia coli Fur box, but the wild-type phenotype of the mutant Δ pgfur strain was restored partially when expression of the ecfur gene was induced from the native pgfur promoter. The full-length PgFur protein contained one zinc atom per protein monomer, but did not bind iron, manganese, or heme. Single cysteine substitutions of CXXC motifs resulted in phenotypes similar to the mutant Δ pgfur strain. The modified proteins were produced in E. coli at significantly lower levels, were highly unstable, and did not bind zinc. The pgfur gene was expressed at the highest levels in bacteria cultured for 24 h in the absence of iron and heme or at higher levels in bacteria cultured for 10 h in the presence of protoporphyrin IX source. No influence of high availability of Fe 2+ , Zn 2+ , or Mn 2+ on pgfur gene expression was observed. Two chromosomal mutant strains producing protein lacking 4 ( pgfur Δ 4aa ) or 13 ( pgfur Δ 13aa ) C-terminal amino acid residues were examined in regard to importance of the C-terminal lysine-rich region. The pgfur Δ 13aa strain showed a phenotype typical for the mutant Δ pgfur strain, but both the wild-type PgFur protein and its truncated version bound zinc with similar ability. The Δ pgfur mutant strain produced higher amounts of HmuY protein compared with the wild-type strain, suggesting compromised regulation of its expression. Potential PgFur ligands, Fe 2+ , Mn 2+ , Zn 2+ , PPIX, or serum components, did not influence HmuY production in the Δ pgfur mutant strain. The mutant pgfur Δ 4aa and pgfur Δ 13aa strains exhibited affected HmuY protein production. PgFur, regardless of the presence of the C-terminal lysine-rich region, bound to the hmu operon promoter. Our data suggest that cooperation of PgFur with partners/cofactors and/or protein/DNA modifications would be required to accomplish its role played in an in vivo multilayer regulatory net...

Porphyromonas gingivalis HmuY and Bacteroides vulgatus Bvu—A Novel Competitive Heme Acquisition Strategy

“…The gene-encoding Bvu protein was amplified using PCR (primers are listed in Table 2 ) and inserted into the pMAL-c5x_His plasmid as described previously [ 50 ]. B. vulgatus Bvu protein (GenBank ID: ABR39853), lacking the predicted signal peptide sequence (MKRYLSIITILGMMLLPFSAC), was overexpressed in E. coli Rosetta (DE3)RIL cells.…”

PgRsp Is a Novel Redox-Sensing Transcription Regulator Essential for Porphyromonas gingivalis Virulence

“…Linear DNA was used for electroporation of the wild-type A7436 strain [39]. To generate the complemented mutant strain (TO11 + pgrsp), the pgrsp gene together with the promoter region was amplified using PCR and subsequently cloned into XhoI and BamHI restriction sites of a pTIO-tetQ plasmid [40] and introduced into the TO11 mutant strain by electroporation. Homologous recombination was confirmed by PCR and DNA sequencing (Microsynth, Balgach, Switzerland).…”

“…Gene expression was initially examined in bacteria cultured for 20 h in iron/heme rich (Hm) or depleted (DIP) medium using microarray analysis as reported previously [13,16]. Additionally, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used as reported previously [16,40]. Total RNA was isolated from 5 × 10 7 to 4 × 10 8 P. gingivalis cells grown for 4, 10, and 24 h in iron/heme rich or iron/heme depleted medium, or one hour after exposure to oxidative stress using the Total RNA Mini Kit (A&A Biotechnology, Gdynia, Poland) and the Clean-Up RNA Concentrator Kit (A&A Biotechnology).…”

“…For overexpression of PgRsp protein in fusion with N-terminally attached His-tag and maltose-binding protein (MBP), pMAL_c5x plasmid (New England Biolabs), modified as described previously (pMAL c5x_His plasmid), was used [40]. The pgrsp gene was amplified by PCR using primers listed in Table S1 and cloned into the pMAL c5x_His plasmid using restriction-free cloning [42].…”

Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling

“…Due to the ability of free iron to form toxic hydroxyl radicals through the Fenton reaction (Guerinot 1994 ), the essential high-affinity uptake systems of iron and iron homeostasis in bacteria must be tightly regulated, and in most bacteria, these processes are under control of the global metalloregulator Ferric uptake regulator (Fur) (Hantke 2001 ). Fur was first described in Escherichia coli (Hantke 1981 ), where it controls the expression of more than 90 genes, and its chemical properties and role in homeostasis has since been studied in homologs from multiple bacteria, including Mycobacterium smegmatis (Gao et al 2019 ), Acidovorax citrulli (Liu et al 2019 ), Campylobacter jejuni (Sarvan et al 2019 ), Porphyromonas gingivalis (Smiga et al 2019 ) and Salmonella enterica serovar Typhimurium (Wang et al 2019 ). Although Fur was originally described as a repressor of genes coding for components of the ferric uptake systems found in the cell membrane, it is now understood to control the expression of toxins such as hemolysin and exotoxins, as well as proteins involved in iron-scavenging and uptake systems (Prince et al 1991 ; Vasil and Ochsner 1999 ).…”