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

Abstract

HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoidassociated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUa (PG1258) and the HUb (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUb using comparative expression profiling of the PG0121 (HUb) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUb has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake. INTRODUCTIONPorphyromonas gingivalis is a Gram-negative obligate anaerobe belonging to the family Bacteroidaceae that persists as a natural member of the human oral microbiota. A shift in the microbial community leading to outgrowth of this anaerobe is directly linked to periodontitis, a chronic inflammatory disease that leads to destruction of the tissues supporting the gums and ultimately, exfoliation of the teeth (Choil et al., 1990;Dzink et al., 1988;Grossi et al., 1994;Lamont & Jenkinson, 2000;Moore et al., 1991). This commensal can colonize, invade and multiply within gingival epithelial cells, as well as penetrate into deeper epithelial cell layers, potentially releasing the whole organism and/or virulence factors into the bloodstream (reviewed by Yilmaz, 2008). In addition to its ability to cause disease in the oral cavity, there are data indicating a role in systemic disease, including its ability to invade vascular endothelial cells (Dorn et al., 2000(Dorn et al., , 2002Jandik et al., 2008) and to cause aggregation of platelets (Pham et al., 2002). For many pathogenic bacteria, surface polysaccharides play a key role in immune modulation et al., 1996). HU typically acts as an accessory protein in virtually all types of nucleoprotein-mediated processes (reviewed by...

Deletion of a 77-Base-Pair Inverted Repeat Element Alters the Synthesis of Surface Polysaccharides in Porphyromonas gingivalis

“…The complete genome sequence of P. gingivalis strain W83 has been reported previously (18). The annotation in the Comprehensive Microbial Resource (CMR) database predicted that genes PG0106 through PG0120 were part of an operon, and our previous studies confirmed this annotation (20,26). As previously reported (25), a large (77-bp) inverted repeat (77bpIR) region is located 5= to PG0106.…”

“…The serotype-specific rabbit antiserum to P. gingivalis W83 and a rabbit polyclonal antibody to PG0121 have been described previously (25,26). The A-LPS-specific monoclonal antibody 1B5 was obtained from Michael Curtis, Queen Mary University of London, London, England, and the O-LPS-specific monoclonal antibody 7F12 was obtained from Richard Darveau, University of Washington, Seattle, WA.…”

“…Bacterial cell wall structure was examined by electron microscopy after cells were stained with ruthenium red, as previously described (26). Strains were streaked to BAPHK agar plates and harvested after 24 h of growth.…”

“…Previously, we showed by Northern analysis that the K-antigen locus encodes a large polycistronic message (PG0104 to PG0121) encompassing a 77-bp inverted repeat (77bpIR) region (25,26), and we determined that the histone-like protein HU (PG0121), which is encoded at the 3= end of the transcript, binds to the 77-bp sequence (27). Our working model has been that this interaction is involved in regulating transcription and/or RNA stability.…”

“…Just recently, Ltp1 was shown to inactivate (dephosphorylate) the tyrosine kinase Ptk1, which in turn was shown to control levels of exopolysaccharide production in both strain 33277 and W83 (24), showing that this tyrosine kinase modulates production and export of extracellular polysaccharide in P. gingivalis. The second regulatory mechanism that has been identified is the DNA-BII protein HU ␤-subunit (PG0121); specifically, genes in both the K-antigen synthesis locus and the A-LPS locus are downregulated in a PG0121 mutant (25,26).…”