VirB, a key transcriptional regulator of virulence plasmid genes in Shigella flexneri, forms DNA-binding site dependent foci in the bacterial cytoplasm

Abstract

VirB is a key regulator of genes located on the large virulence plasmid (pINV) in the bacterial pathogen Shigella flexneri VirB is unusual; it is not related to other transcriptional regulators, instead, it belongs to a family of proteins that primarily function in plasmid and chromosome partitioning; exemplified by ParB. Despite this, VirB does not function to segregate DNA, but rather counters transcriptional silencing mediated by the nucleoid structuring protein, H-NS. Since ParB localizes subcellularly as discrete foci in the bacterial cytoplasm, we chose to investigate the subcellular localization of VirB to gain novel insight into how VirB functions as a transcriptional anti-silencer. To do this, a GFP-VirB fusion that retains the regulatory activity of VirB and yet, does not undergo significant protein degradation in S. flexneri, was used. Surprisingly, discrete fluorescent foci were observed in live wild-type S. flexneri cells and an isogenic virB mutant using fluorescence microscopy. In contrast, foci were rarely observed (<10%) in pINV-cured cells or in cells expressing a GFP-VirB fusion carrying amino acid substitutions in the VirB DNA binding domain. Finally, the 25 bp VirB-binding site was demonstrated to be sufficient and necessary for GFP-VirB focus formation using a set of small surrogate plasmids. Combined, these data demonstrate that the VirB:DNA interactions required for the transcriptional anti-silencing activity of VirB on pINV are a prerequisite for the subcellular localization of VirB in the bacterial cytoplasm. The significance of these findings, in light of the anti-silencing activity of VirB, is discussed.ImportanceThis study reveals the subcellular localization of VirB, a key transcriptional regulator of virulence genes found on the large virulence plasmid (pINV) in Shigella. Fluorescent signals generated by an active GFP-VirB fusion form 2, 3, or 4 discrete foci in the bacterial cytoplasm, predominantly at the quarter cell position. These signals are completely dependent upon VirB interacting with its DNA binding site found either on the virulence plasmid or an engineered surrogate. Our findings: 1) provide novel insight into VirB:pINV interactions, 2) suggest that VirB may have utility as a DNA marker, and 3) raise questions about how and why this anti-silencing protein that controls virulence gene expression on pINV of Shigella spp. forms discrete foci/hubs within the bacterial cytoplasm.

FIG 1

Constructs and proof of principle experiments to assess the activity and stability of VirB fusion proteins. (A) Constructs producing VirB fused to superfolder GFP (sfGFP) at the N terminus using one of two linkers. (B) β-Galactosidase assay used to assess the regulatory activity of GFP-VirB fusions with either a SGGGG (49) or “12AA” linker (48) at the VirB-dependent icsP promoter (PicsP-lacZ; pAFW04a). Student’s t tests were used to measure statistical significance, *, P < 0.05. Note that β-galactosidase activities generated in the presence of VirB (pJNS04) and GFP-VirB (pJNS12) were equivalent to those achieved with native VirB levels in previous work (35). (C) Western blots to assess GFP-VirB protein stability, probed with an anti-VirB antibody (i) or anti-GFP antibody (ii). For these analyses, phenylmethylsulfonyl fluoride (PMSF) was added during sample preparation. (D) Densitometry of Western blots shown in panel C (subpanels i and ii, respectively). Lanes are labeled accordingly.

FIG 2

Live-cell imaging of GFP-VirB in a virB mutant strain of Shigella flexneri. (A) Quantification of foci observed during live-cell imaging of GFP-VirB in virB mutant S. flexneri using MicrobeJ. Representative cells are shown. PC, phase contrast; GFP, green fluorescent protein fluorescence (for GFP row 67.1-ms exposure; for GFP-VirB and Empty rows, 289.2-ms exposure; Empty, empty plasmid control. Bars, 1 μm in all images. Within tables, a hyphen indicates that no cells fell into this category in any of the images captured; *, maxima detected by MicrobeJ. (B) A representative field of view with magnified cell images below showing the numbers of foci commonly observed. Five fields of view were captured and analyzed across at least three independent replicates. (C) Scatterplot of mean cell length per number of foci observed in cells producing GFP-VirB, showing strong positive correlation between cell length and number of foci.

FIG 3

Live-cell imaging of GFP-VirB in a wild-type strain of S. flexneri and quantification of foci observed during live-cell imaging of GFP-VirB in wild-type S. flexneri using MicrobeJ. Representative cells are shown. Phase contrast (left column), fluorescence (middle column) (GFP row, 44.5-ms exposure; GFP-VirB and empty, 219-ms exposure), and merged (right column) images of GFP-VirB, GFP, and an empty plasmid control . Bars, 1 μm. Within tables, a hyphen indicates that no cells fell into this category in any of the images captured; *, maxima detected by MicrobeJ.

FIG 4

Live-cell imaging of GFP-VirB in a pINV-cured strain of S. flexneri and quantification of fluorescent signals observed during live-cell imaging of GFP-VirB in pINV-cured S. flexneri using MicrobeJ. Representative cells are shown. Note that only diffuse signals were observed (see also Fig. S3 in the supplemental material). Phase-contrast (left column), fluorescence (middle column) (GFP row, 40.6-ms exposure; GFP-VirB and empty rows, 223.1-ms exposure), and merged (right column) images of GFP-VirB, GFP, and an empty plasmid control. Bars, 1 μm. Within tables, a hyphen indicates that no cells fell into this category in any of the images captured; *, maxima detected by MicrobeJ.

FIG 5

Construction of a GFP-VirB DNA-binding mutant and live-cell imaging of this fusion in a virB mutant strain of S. flexneri. (A) Construct producing GFP-VirB with two amino acid substitutions, K152E and R167E, in the helix-turn-helix DNA-binding domain (denoted with blue arrows; adapted from reference 52). (B) Quantification of fluorescent signals observed during live-cell imaging of GFP-VirBK152E/R167E in virB mutant S. flexneri using MicrobeJ. Note that diffuse signals observed for this fusion were detected as maxima by MicrobeJ. Representative cells are shown. Phase-contrast (left column), fluorescence (middle column) (GFP row, 77-ms exposure; GFP-VirB, GFP-VirB K152E/R167E, and empty rows, 348.8-ms exposure), and merged (right column). Bars, 1 μm. Within the table, a hyphen indicates that no cells fell into this category in any of the images captured; *, maxima detected by MicrobeJ.

FIG 6

Live-cell imaging of GFP-VirB and controls in a pINV-cured strain of S. flexneri carrying a plasmid bearing a wild-type or mutated VirB-binding site and quantification of fluorescent foci of GFP-VirB (A) or maxima (*) (B, C, and E to G) associated with diffuse signals observed during live-cell imaging using MicrobeJ. Representative merged cell images of pINV-cured S. flexneri cells containing pHJW20 (WT PicsP) (A to D) or pMIC18 (Mutated PicsP) (E to H) are shown. No fluorescent signal was detected for the empty controls (D and H) (GFP, 157.6-ms exposure; GFP-VirB and empty, 485.3-ms exposure). Bars, 1 μm. Within tables, a hyphen indicates that no cells fell into this category in any of the images captured.

FIG 7

Live-cell imaging of GFP-VirB and controls in a pINV-cured strain of S. flexneri carrying a plasmid bearing a 25-bp VirB binding site or empty (“no-site”) binding tool control. Representative merged cell images of pINV-cured S. flexneri carrying pJNS22 (25-bp VirB-binding site) or pJNS24 (“no site” control) with either GFP-VirB, GFP-VirB K152E/R167E, GFP, or an empty plasmid control are shown (GFP, 45.1-ms exposure; GFP-VirB and empty, 297.6-ms exposure). Bars, 1 μm.