Identity gene expression in Proteus mirabilis

Abstract

Swarming colonies of independent Proteus mirabilis isolates recognize each other as foreign and do not merge together, whereas apposing swarms of clonal isolates merge with each other. Swarms of mutants with deletions in the ids gene cluster do not merge with their parent. Thus, ids genes are involved in the ability of P. mirabilis to distinguish self from nonself. Here we have characterized expression of the ids genes. We show that idsABCDEF genes are transcribed as an operon, and we define the promoter region upstream of idsA by deletion analysis. Expression of the ids operon increased in late logarithmic and early stationary phases and appeared to be bistable. Approaching swarms of nonself populations led to increased ids expression and increased the abundance of ids-expressing cells in the bimodal population. This information on ids gene expression provides a foundation for further understanding the molecular details of self-nonself discrimination in P. mirabilis.

Fig. 1.

Proteus mirabilis territorial boundaries, the ids locus, and transcription from the idsA promoter. (A) Swarm plate of the wild-type strain BB2000, BB2000 idsABCDEF deletion mutant (Δids), and BB2000 with the idsA-gfp vector pKG100 (labeled pPr-ids-gfp) or with an empty vector. A visible boundary formed between swarms of the wild type and the deletion mutant. Swarms of the wild type merged regardless of the presence of pKG100. The bar shown is 1 cm. (B) Organization of the P. mirabilis idsABCDEF locus. The arrows indicate open reading frames. The region between idsA and the diverging open reading frame (ORF) is 1,899 bp. The distance between the idsA and idsB ORFs is 73 bp. The distance between idsF and the downstream ORF is 180 bp. (C) Fluorescence of P. mirabilis cells containing the idsA promoter-gfp expression vector pKG100 or pKG104, which contains the idsAB intergenic region with gfp fused to idsB. Cells were harvested at a culture density of 4 to 5. (D) P. mirabilis BB2000 containing the idsA-gfp vector pKG102, which contains 788 bp upstream of idsA, or pKG104, which contains only 435 bp upstream of idsA, were grown either in broth or on swarm-agar plates. Broth-grown cells were obtained at a culture density of 4 to 5. Swarm cells were obtained from agar plates after 24 h (at this time active swarming had ceased). Cells were suspended in PBS, fluorescence and optical density were measured, and fluorescence units were normalized to the optical density. Bars indicate the means with standard deviations. GFP fluorescence units were normalized to optical density and measured as described in the Materials and Methods.

Fig. 2.

Migrating fronts of P. mirabilis swarms. (A) Bimodal expression of the pKG100 idsA-gfp fusion in cells of P. mirabilis at the outer edge of a swarm. The phase image is shown on the left, the fluorescence image is in the center, and the false-colored image is on the right and shows the agar background in red, fluorescence bright cells in green, and dark cells in black. Asterisks mark examples of dark cells, and arrowheads indicate swarm tracks. (B) Control. Outer swarm edge of BB2000 cells carrying a chromosomally integrated constitutive gfp (10). Asterisks mark dim cells (dark cells were not evident), and arrowheads indicate swarm tracks. The marker bars shown are 50 μm.

Fig. 3.

The idsA promoter shows a bimodal and culture density-dependent pattern of expression. (A) Histograms of individual cell fluorescence intensity in an LB broth culture at different cell densities (OD at 600 nm). We measured fluorescence of 135 individual cells at an OD of 0.13, 255 cells at an OD of 0.60, 419 cells at an OD of 1.63, and 728 cells at an OD of 4.10. Cells with fluorescence levels below 100 are dark or very dim. (B) Percentage of LB broth-grown P. mirabilis BB2000 (pKG100) cells expressing idsA-gfp in relation to culture density. (C) Expression of idsA as a function of culture density in LB broth. Fluorescence units are normalized to the optical density.

Fig. 4.

Effects of social recognition on ids expression. (A) Maximum fluorescence intensity averaged across swarm boundaries. Boundaries are between P. mirabilis BB2000 containing the idsA-gfp vector pKG102 and either BB2000 or the ids deletion mutant containing the vector control pKG101 (to allow antibiotic maintenance of the plasmids in all bacteria). We measured fluorescence in 0.65-μm by 13-μm intervals. The “0” position is in the swarm of BB2000 (pKG102) and the “1500” position is in the swarm of the vector-containing bacteria. The black box indicates the location where swarms merged or where the boundary formed. Background fluorescence in dark colonies is about 200 units. (B) Total fluorescence of P. mirabilis BB2000 (pKG102). The swarms were approaching either BB2000 containing the vector control pKG101 or the ids deletion strain carrying pKG101. Where indicated, a 0.22-μm filter divided the approaching swarms. The data are normalized to the optical density. The boxes show ranges, and the horizontal lines denote the means. There was a significant difference without the filter and no significant difference (ns) with the filter. (C) Fluorescence of P. mirabilis BB2000 (pKG102) calculated as described for panel B. Fluorescence, normalized to the optical density, was measured at discrete points along the swarm and was plotted such that the x axis measures the distance from one edge of a well to the other. The swarms met between 1 cm and 2.2 cm from the edge of the well. Markers denote means, and bars show standard errors of the means. (D) Ratios (Δids/BB2000) of the data shown in panel C, plotted as a function of the distance from the edge of the well.