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Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis.

ABSTRACT: An hemR (hemin-regulated) gene from Porphyromonas gingivalis ATCC 53977 has been isolated and characterized. This gene is present downstream from the prtT gene, previously cloned in this laboratory. In addition, another putative gene, ORF1, was identified between hemR and prtT. The complete nucleotide sequences of ORF1 and hemR were determined, and the deduced amino acid sequence of ORF1 and HemR proteins corresponded to 16- and 48-kDa proteins, respectively. The amino termini of the HemR protein exhibited significant homology with iron-regulated, TonB-dependent outer membrane receptor proteins from various bacteria, while the carboxyl terminus of the HemR protein displayed almost complete identity with a P. gingivalis PrtT protease domain. PCR analyses confirmed the existence of such extensive homology between the carboxyl termini of both the prtT and hemR genes on the P. gingivalis chromosome. Northern blots indicated that ORF1 was part of a 1.0-kb mRNA and was positively regulated by hemin levels. On the other hand, the hemR gene was apparently a part of a 3.0-kb polycistronic message and was negatively regulated at the transcriptional level by hemin. Primer extension analysis of the hemR gene revealed that the transcription start site was at a C residue located within ORF1. An examination of HemR::lacZ constructs in both Escherichia coli and P. gingivalis confirmed hemin repression of hemR expression in both organisms. Moreover, the HemR protein expressed in E. coli was detected by an antiserum from a periodontitis patient heavily colonized with P. gingivalis but not by serum from a periodontally healthy patient or by antisera against hemin-grown P. gingivalis cells. Therefore, it is likely that the 48-kDa HemR protein can be expressed only under hemin-restricted conditions. These results suggest that we have isolated a hemin-regulated gene, hemR, which encodes a 48-kDa protein that may be a TonB-dependent outer membrane protein.

SUBMITTER: Karunakaran T

PROVIDER: S-EPMC178912 | biostudies-other | 1997 Mar

REPOSITORIES: biostudies-other

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Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis.

Karunakaran T T Madden T T Kuramitsu H H

Journal of bacteriology 19970301 6

An hemR (hemin-regulated) gene from Porphyromonas gingivalis ATCC 53977 has been isolated and characterized. This gene is present downstream from the prtT gene, previously cloned in this laboratory. In addition, another putative gene, ORF1, was identified between hemR and prtT. The complete nucleotide sequences of ORF1 and hemR were determined, and the deduced amino acid sequence of ORF1 and HemR proteins corresponded to 16- and 48-kDa proteins, respectively. The amino termini of the HemR protei ...[more]

PMID: 9068634

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Project description:Periodontal disease is a chronic inflammatory disease in which the oral pathogen Porphyromonas gingivalis plays an important role. Porphyromonas gingivalis expresses virulence determinants in response to higher hemin concentrations, but the underlying regulatory processes remain unclear. Bacterial DNA methylation has the potential to fulfil this mechanistic role. We characterized the methylome of P. gingivalis, and compared its variation to transcriptome changes in response to hemin availability. Porphyromonas gingivalis W50 was grown in chemostat continuous culture with excess or limited hemin, prior to whole-methylome and transcriptome profiling using Nanopore and Illumina RNA-Seq. DNA methylation was quantified for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC). Of all 1,992 genes analyzed, 161 and 268 were respectively over- and under-expressed with excess hemin. Notably, we detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify altered methylation and expression responses to hemin availability in P. gingivalis, with insights into mechanisms regulating its virulence in periodontal disease. IMPORTANCE DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis, an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the regulatory processes underlying these effects remain unknown. We profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin that reflect health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin. Joint analyses identified coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease.

Dataset Information

Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis.

Publications

Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis.

Similar Datasets

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