Porphyromonas gingivalis, an oral bacterium associated with periodontal disease, requires haemin for growth. Although several multigenic clusters encoding haemin-uptake systems are present on the genome of P. gingivalis, little is known regarding their transcriptional organization and expression. This study identified a 23 kDa iron-regulated haemin-binding protein encoded by a larger than previously reported variant of hmuY. It was shown that the hmu locus is larger than previously reported and is composed of six genes, hmuYRSTUV, encoding a novel hybrid haemin-uptake system. The locus has an operonic organization and the transcriptional start site is located 292 bp upstream of hmuY. The data indicate that the regulation of the operon is iron-dependent. Interestingly, differential regulation within the operon was demonstrated, resulting in excess of the hmuYR message encoding the outer-membrane proteins when compared to the full-length transcript. In addition, the hmuY transcript is more prevalent than the hmuR transcript. Secondary structure analysis of the hmuYRSTUV mRNA predicted the formation of several potential stem-loops in the 59 ends of hmuR-and hmuS-specific mRNAs, consistent with the differential regulation observed. Finally, it was demonstrated that haemin binding and uptake are elevated in iron-depleted conditions and are reduced 45 % and 70 %, respectively, in an hmu-deficient strain when compared to the parental strain, indicating that the hmu locus plays a major role in haemin acquisition in P. gingivalis. Since homologues of the hmu locus were also found in Bacteroides fragilis, Bacteroides thetaiotaomicron and Prevotella intermedia, these findings may have implications for a better understanding of haemin acquisition in those organisms as well.

Low Molecular Weight Tyrosine Phosphatases (LMWTP) are widespread in prokaryotes; however, understanding of the signaling cascades controlled by these enzymes is still emerging. P. gingivalis, an opportunistic oral pathogen, expresses a LMWTP, Ltp1, that is differentially regulated in biofilm communities. Here we characterize the enzymatic activity of Ltp1 and, through the use of mutants that lack Ltp1 or expresses catalytically defective Ltp1, show that tyrosine phosphatase activity constrains both monospecies biofilm development and community development with the antecedent oral biofilm constituent S. gordonii. Exopolysaccharide production is downregulated by Ltp1 through transcriptional regulation of multiple genes involved in biosynthesis and transport. Furthermore, Ltp1 regulates transcriptional activity of luxS and thus impacts AI-2 dependent signaling in biofilm communities. In the absence of Ltp1 transcription across the hmu hemin uptake locus is reduced, and consequently uptake of hemin is impaired in the Ltp1 mutant. The gingipain proteinases Kgp and RgpA/B remain phosphorylated in the Ltp1 mutant. Phosphorylated Rgps are poorly secreted, whereas cell surface activity of phosphorylated Kgp is enhanced. By controlling the activity of several virulence-associated properties, Ltp1 may restrain the pathogenic potential of P. gingivalis and maintain a commensal interaction with the host.

Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is a recognized periodontopathogen. It exhibits a high degree of aerotolerance and is able to survive in host cells, indicating that efficient oxidative stress protection mechanisms must be present in this organism. Manganese homeostasis plays a major role in oxidative stress protection in a variety of organisms; however, the transport and role of this metal in P. gingivalis is not well understood. Analysis of the genome of P. gingivalis W83 revealed the presence of two genes encoding homologs of a ferrous iron transport protein, FeoB1 and FeoB2. FeoB2 has been implicated in manganese accumulation in P. gingivalis. We sought to determine the role of the FeoB2 protein in metal transport as well as its contribution to resistance to oxygen radicals. Quantitative reverse transcriptase PCR analyses demonstrated that expression of feoB2 is induced in the presence of oxygen. The role of FeoB2 was investigated using an isogenic mutant strain deficient in the putative transporter. We characterized the FeoB2-mediated metal transport using 55 Fe 2؉ and 54 Mn 2؉ . The FeoB2-deficient mutant had dramatically reduced rates of manganese uptake (0.028 pmol/min/10 7 bacteria) compared with the parental strain (0.33 pmol/min/10 7 bacteria) (after 20 min of uptake using 50 nM of 54 Mn 2؉ ). The iron uptake rates, however, were higher in the mutant strain (0.75 pmol/min/10 7 bacteria) than in the wild type (0.39 pmol/min/10 7 bacteria). Interestingly, reduced survival rates were also noted for the mutant strain after exposure to H 2 O 2 and to atmospheric oxygen compared to the parental strain cultured under the same conditions. In addition, in vitro infection of host cells with the wild type, the FeoB2-deficient mutant, and the same-site revertant revealed that the mutant had a significantly decreased capability for intracellular survival in the host cells compared to the wild-type strain. Our results demonstrate that feoB2 encodes a major manganese transporter required for protection of the bacterium from oxidative stress generated by atmospheric oxygen and H 2 O 2 . Furthermore, we show that FeoB2 and acquisition of manganese are required for intracellular survival of P. gingivalis in host cells.

Although hemin is an indispensable nutrient for the oral pathogen Prevotella intermedia, not much is known regarding the molecular mechanisms of hemin acquisition. The availability of the genomic sequence of the bacterium allowed us to apply proteomic approaches to identify proteins that may be mediating the hemin acquisition process. As hemin acquisition mechanisms have been shown to be induced in iron-depleted conditions, we applied proteomic approaches to detect those proteins whose expressions were affected by iron. We analyzed 40 protein spots and identified 19 such proteins. Interestingly, two proteins drastically upregulated in iron-depleted conditions, PIN0009 and PINA0611, are homologs of hemin uptake receptors in other bacteria. PIN0009 is predicted to be an outer membrane lipoprotein. It is encoded by a gene that is the first of a seven-gene genomic locus encoding proteins of a novel hemin acquisition system. The second protein, PINA0611, is a homolog of numerous TonB-dependent outer membrane receptors including outer membrane iron uptake receptors of various Gram-negative bacteria. There was also another protein, regulated by iron, that was previously demonstrated to bind hemoglobin in P. intermedia. Finally, we identified a thioredoxin-like protein that has a novel outer membrane location.