Transcriptional organization, regulation and role of the Porphyromonas gingivalis W83 hmu haemin-uptake locus

Abstract

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.

Two-component signal transduction systems in oral bacteria

“…Among the targets of HaeR identified by ChIP-seq were 5ʹ untranslated sequences upstream of PGN_0704 ( ihtA ), PGN_0687 ( htrA ) and PGN_0556 ( hmuS ). RR binding to these sequences was confirmed by EMSA, so we conclude that the three loci, consistent with their previously established roles in haemin transport, are directly regulated by PGN_0753 [55–57]. …”

Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves increased consumption of formate and reduced utilization of lactate

“…As a consequence of being a cofactor of antioxidant enzymes, such as superoxidase dismutase (SOD), this metal has antioxidant properties (Archibald & Fridovich, 1982;Hiraoka et al, 2000;Nakayama, 1990Nakayama, , 1994Yim et al, 1990). In agreement with the functional properties of these metals, the downregulation of iron transporters such as the major haemin uptake system hmu (PG1551) was observed (Dashper et al, 2005;Lewis et al, 2006) (Table 2). On the other hand, iron-storage proteins such as the chaperone like proteinencoding gene dps (PG0090) and the ferritin-encoding gene PG1286 were upregulated in mid-exponential phase cells (Table 1).…”

“…Such coordinate regulation of multiple genes having operonic organization (e.g. the feoB2 and hmu loci reported previously) is an excellent way to validate the accuracy of our microarray results (Dashper et al, 2005;Lewis et al, 2006). …”

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

“…The molecular mechanisms underlying iron uptake have been extensively studied (Olczak et al, 2005). Genes involved in iron acquisition include the hmuYRSTUV locus (PG1551-PG1556), which encodes a novel hybrid haemin-uptake system (Brunner et al, 2010;Lewis et al, 2006;Olczak et al, 2005Olczak et al, , 2008Simpson et al, 2000Simpson et al, , 2004Smalley et al, 2011); the hbp35 gene (PG0616), which encodes a haemin-binding protein that also exhibits thioredoxin activity (Hiratsuka et al, 2010;Shoji et al, 2010); the surface protein HusA (PG2227), which acts as a haemophore with very high affinity (Gao et al, 2010); the tlr (PG0644) iron transport locus Slakeski et al, 2000); the haemin-binding protein FetB (PG0669) and the surrounding genes in that locus; and PG0465, the ferric uptake regulator (fur) (Olczak et al, 2005). In addition, regulatory mechanisms controlling expression of iron acquisition genes have been identified, including the transcriptional activator encoded by PG1237, which has been shown to control expression of the hmu locus (Wu et al, 2009).…”