Construction and analysis of hemin binding protein mutants in the oral pathogen Treponema denticola.
Treponema denticola, a periodontal pathogen, can use hemin as its sole iron source. The organism synthesizes two low-iron-induced outer-membrane hemin-binding proteins, HbpA and HbpB. To characterize genetically the function of these two novel proteins, standard recombinant DNA procedures and electroporation were used to construct T. denticola strains in which the genomic copies of either hbpA or both hbpA and hbpB were interrupted with an erythromycin resistance cassette. Northern blot and RT-PCR analyses verified that the normal hbpA transcripts were missing in both mutants. The hbpA mutation also had a polar effect on the transcription of hbpB and thus neither mutant strain transcribes the downstream hbpB gene. The parental and hbp mutant strains had similar growth properties in normal media, but the mutants reached a lower cell density than parental cells in iron-restricted media. The results indicate that HbpA and/or HbpB are required for efficient iron utilization but that there is an additional system that can help T. denticola acquire iron. The growth defect of the mutants was totally restored by lactoferrin but only partially restored by adding exogenous hemin or inorganic iron. Thus, hbpA and/or hbpB specifically facilitate hemin and iron utilization under low iron conditions and are presumably important for T. denticola virulence in the host environment.
Xu
Xiaoping
X
Kolodrubetz
David
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D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D006427:Hemin / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014208:Treponema / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D006427:Hemin; D007501:Iron