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 nucleoid-associated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUα (PG1258) and the HUβ (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 HUβ using comparative expression profiling of the PG0121 (HUβ) 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 HUβ 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.

Fig. 1.

Western analysis of PG0121-HU expression in E. coli MG1655 hupAB double mutant and complementation of low pH sensitivity. (a) Western analysis of PG0121-HU (9 kDa) expression without addition of the transcriptional inducer IPTG using PG0121 antiserum. Lanes: (1) Molecular mass ladder; (2) SG 790, E. coli MG1655 hupAB double mutant; (3) SG 886, E. coli MG1655 hupAB double mutant + emptypTrcHis2A ; (4) SG 903, E. coli MG1655 hupAB double mutant + plasmid pTrcHis2A expressing E. coli HUβ coding region; (5) SG 918, E. coli MG1655 hupAB double mutant + plasmid pTrcHis2C expressing PG0121 HU coding region; (6) SG 919, E. coli MG1655 hupAB double mutant + empty pTrcHis2C; (7) SG 632, E. coli MG1655 wild-type; (8) SG 883, E. coli MG1655 WT + empty pTrcHis2A ; (9) 250 ng pure HU0121 control. (b) E. coli strains were incubated in LB broth at pH 2.5 for the time indicated. The cells were then diluted and plated and c.f.u. were determined. The data represent the mean of four biological replicates with sem.

Fig. 2.

Complementation of cell-division defect of E. coli MG1655 hupAB double mutant with PG0121 HU. E. coli strains were grown to mid-exponential growth phase in LB broth and then imaged at 100× magnification with dark field microscopy. (a) E. coli MG1655 parent strain. (b) SG 790; E. coli MG1655 hupAB double mutant. (c) SG 886; E. coli MG1655 hupAB double mutant + empty pTrcHis2A. (d) SG 903; E. coli MG1655 hupAB double mutant + pTrcHis2A expressing E. coli HU-β coding region. (e) SG 918; E. coli MG1655 hupAB double mutant + pTrcHis2C expressing PG0121 HU coding region. The images shown are representative of three independent experiments. Bars, 10 µm.

Fig. 3.

Northern blot analysis of PG0106 and PG121 expression during different phases of growth. Total RNA was isolated from the wild-type strain W83 during early (OD₆₀₀ 0.3), mid- (OD₆₀₀ 0.6) and late- (OD₆₀₀ 1.2) exponential growth. The blot on the left shows the transcripts that hybridize to PG0106 during the three different phases of growth. The blot on the right shows the transcripts that hybridized to a probe for PG0121 (HU) during the three different phases of growth. See text for description.

Fig. 4.

The PG0121 HU mutant demonstrates an altered cell-division phenotype under iron-limiting growth conditions. P. gingivalis strains were grown to mid-exponential growth phase in TSB broth that was treated with, 2′-dipyridyl to chelate the iron and then imaged at 100× magnification with dark field microscopy. (a) Parent strain P. gingivalis W83. (b) PG0121 HU mutant strain. (c) Knock-in complemented P. gingivalis strain PgTcKI_1. The images shown are representative of three independent experiments. Bar, 10 µm.