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. 2023 Apr;119(4):439-455.
doi: 10.1111/mmi.15033. Epub 2023 Feb 20.

Non-specific and specific DNA binding modes of bacterial histone, HU, separately regulate distinct physiological processes through different mechanisms

Affiliations

Non-specific and specific DNA binding modes of bacterial histone, HU, separately regulate distinct physiological processes through different mechanisms

Subhash C Verma et al. Mol Microbiol. 2023 Apr.

Abstract

The histone-like protein HU plays a diverse role in bacterial physiology from the maintenance of chromosome structure to the regulation of gene transcription. HU binds DNA in a sequence-non-specific manner via two distinct binding modes: (i) random binding to any DNA through ionic bonds between surface-exposed lysine residues (K3, K18, and K83) and phosphate backbone (non-specific); (ii) preferential binding to contorted DNA of given structures containing a pair of kinks (structure-specific) through conserved proline residues (P63) that induce and/or stabilize the kinks. First, we show here that the P63-mediated structure-specific binding also requires the three lysine residues, which are needed for a non-specific binding. Second, we demonstrate that substituting P63 to alanine in HU had no impact on non-specific binding but caused differential transcription of diverse genes previously shown to be regulated by HU, such as those associated with the organonitrogen compound biosynthetic process, galactose metabolism, ribosome biogenesis, and cell adhesion. The structure-specific binding also helps create DNA supercoiling, which, in turn, may influence directly or indirectly the transcription of other genes. Our previous and current studies show that non-specific and structure-specific HU binding appear to have separate functions- nucleoid architecture and transcription regulation- which may be true in other DNA-binding proteins.

Keywords: HU; non-specific binding; nucleoid architecture; structure-specific binding; transcription regulation.

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Conflict of interest statement

Conflict of Interest Statement: The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Crystal structures of HU-DNA complexes. (A) Crystal structure of E. coli HUα2 (α subunits in blue and cornflower blue) in a complex with linear DNA (PDB 6O8Q). (B) Crystal structure of Anabaena HU (two subunits in plum and purple) in a complex with a distorted DNA (PDB 1P51), with E. coli HUα2 (shown in A) superimposed on it. The lysine residues, K3, K18, and K83, and the proline residue P63 of both HU subunits are shown in red. Any contacts between these residues and DNA are shown as black lines.
Fig. 2.
Fig. 2.
Effect of substituting lysine and P63 residues of HUα2 on DNA binding. Binding of purified wild-type HUα2 or its variants harboring indicated amino acid substitutions to a 6-carboxy fluorescein labeled linear DNA or cruciform DNA. Each data point on Y-axis represents a change in milipolarization (mP) units at indicated protein concentrations in fluorescence polarizations assays. Kd (nM) estimated by fitting binding curves to a Hill equation are given in the table. ND = not determined.
Fig. 3.
Fig. 3.
Effect of substituting lysine and P63 residues of HUα2 on supercoiling inducing ability. The left panel outlines the assay carried out to determine the ability of HUα2 to induce negative supercoils in a nicked plasmid DNA. The middle panel shows the electrophoresis of the DNA products from the assay in the presence of the indicated concentrations of wild-type HUα2 protein or its variants. The right panel shows densitometric lane traces of the gel shown in the middle panel.
Fig. 4.
Fig. 4.
Effect of substituting lysine and P63 residues of HUα2 on gal transcription (A) Amount of galP2 transcript originating from the galP2 promoter of pSA850 plasmid in the presence of 200 nM GalR and the indicated concentrations of HUα2 protein or its variants. The amount of galP2 transcript was normalized with that of RNAI and RNAII transcripts originating from the same plasmid. Each data point represents the galP2/(RNAI + RNAII) transcript ratio relative to that in the presence of GalR alone which was set to 100. The gel image used to quantify transcript amount is shown in Fig. S3). (B) Levels of β-galactosidase activity of the chromosomal galE-lacZ transcriptional reporter in the E. coli strains of indicated genotypes: Graphical and error bars represent averages and standard deviation of triplicate biological replicates, respectively. Experiment was performed twice, with similar results. Statistically significant differences were determined by 1-way ANOVA with Tukey’s multiple comparisons test. ns not significant; ** adjusted p-value 0.001; *** adjusted p-value <0.001.
Fig. 5.
Fig. 5.
Effect of substituting the P63 residue of HUα2 on global gene expression. Volcano plots showing differential expression of genes between E. coli strains of the indicated genotypes. X-axis represents log2 of the fold-change (Log2FC) in RNA levels and y-axis represents the −log10 of false discovery rate (FDR) of each gene. Vertical dotted lines are positioned at a log2 fold-change of 0.5 or −0.5 and horizontal dotted lines are positioned at the −log10 of 0.05 FDR. Genes in red are identified as differentially expressed genes (DEGs), determined using glmTreat function at FDR<0.05 and log2 fold-change >0.5 (plus or minus). Venn diagram shows DEGs common between two the comparison groups.
Fig. 6.
Fig. 6.
Role of HU in transcription regulation of type 1 fimbriae genes (A) Representative fluorescence and differential interference contrast images of WT cells, or WT cells harboring fimA::gfpmut2 transcriptional reporter. (B) Percentage of cells expressing GFP fluorescence above background in E. coli strains harboring the indicated deletion mutations in hupA and hupB genes and mutations in the hupA gene to introduce P63A amino acid substitution in the HUα subunit. Graphical and error bars represent averages and standard deviation of at least four fluorescence images each containing more than 200 cells. Experiment was performed twice, with similar results. Statistically significant differences as determined by 1-way ANOVA with Dunnett’s multiple comparisons test. ns not significant; *** adjusted p-value 0.0001; **** adjusted p-value <0.0001.
Fig. 7.
Fig. 7.
Role of HU in the formation of type I fimbriae on the surface of E. coli cells Scanning electron micrographs of representative cells of E. coli strains harboring the indicated deletion mutations in hupA, hupB, and type I fimbriae genes. Scale bar 1 μm.
Fig. 8.
Fig. 8.
Role of HU in regulating the orientation of the fimS switch (A) The position of Hinfl restriction site when fimS switch is in ON or OFF phase. Red arrows represent the binding sites of the primers fimE-u1 and fimA-l1 used for polymerase chain reaction of fimS region. Solid red lines represent the PCR products and sizes of Hinfl restriction fragments in ON or OFF phase. IR inverted repeats (B) Agarose geel after electrophoresis of Hinfl digestion reactions of the PCR products of chromosomal DNA of E. coli strains harboring the indicated deletion mutations in hupA and hupB genes and mutations in the hupA gene to introduce P63A amino acid substitution in the HUα subunit. Numbers correspond to genotypes in panel B. M DNA marker. (C) Percentage amount of 520 bp fragment from total amount of 520 bp and 423 bp fragments. Graphical and error bars represent averages and standard deviation of three biological replicates respectively. Experiment was performed twice, with similar results. Statistically significant differences as determined by 1-way ANOVA with Dunnett’s multiple comparisons test. ns not significant; ** adjusted p-value 0.003; *** adjusted p-value <0.001.
Figure 9.
Figure 9.
A model for mechanism, prevalence, and function of non-specific and structure-specific DNA binding modes of HU in E. coli Two subunits of HU are depicted as blue and purple with DNA binding amino acid residues shown as red circles. DNA is depicted in gold. The bound DNA is in the straight conformation in non-specific mode and sharpy bent in structure-specific mode. While non-specific binding mode is widespread in the chromosome and primarily involved in chromosome organization, structure-specific binding mode is limited and involved in transcription regulation. TF Transcription factor; RNAP RNA polymerase.

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