Swarming-coupled expression of the Proteus mirabilis hpmBA haemolysin operon

Abstract

The HpmA haemolysin toxin of Proteus mirabilis is encoded by the hpmBA locus and its production is upregulated co-ordinately with the synthesis and assembly of flagella during differentiation into hyperflagellated swarm cells. Primer extension identified a sigma(70) promoter upstream of hpmB that was upregulated during swarming. Northern blotting indicated that this promoter region was also required for concomitant transcription of the immediately distal hpmA gene, and that the unstable hpmBA transcript generated a stable hpmA mRNA and an unstable hpmB mRNA. Transcriptional luxAB fusions to the DNA regions 5' of the hpmB and hpmA genes confirmed that hpmB sigma(70) promoter activity increased in swarm cells, and that there was no independent hpmA promoter. Increased transcription of the hpmBA operon in swarm cells was dependent upon a 125 bp sequence 5' of the sigma(70) promoter -35 hexamer. This sequence spans multiple putative binding sites for the leucine-responsive regulatory protein (Lrp), and band-shift assays with purified Lrp confirmed the presence of at least two such sites. The influence on hpmBA expression of the key swarming positive regulators FlhD(2)C(2) (encoded by the flagellar master operon), Lrp, and the membrane-located upregulator of the master operon, UmoB, was examined. Overexpression of each of these regulators moderately increased hpmBA transcription in wild-type P. mirabilis, and the hpmBA operon was not expressed in any of the flhDC, lrp or umoB mutants. Expression in the mutants was not recovered by cross-complementation, i.e. by overexpression of FlhD(2)C(2), Lrp or UmoB. Expression of the zapA protease virulence gene, which like hpmBA is also upregulated in swarm cells, did not require Lrp, but like flhDC it was upregulated by UmoB. The results indicate intersecting pathways of control linking virulence gene expression and swarm cell differentiation.

Fig. 1

Primer extension mapping of mRNA termini 5′ of (a) hpmB and (b) hpmA. Wild-type vegetative (v) cells and differentiated swarm (s) cells were isolated from LB agar plates at 1·5 and 4 h after seeding, respectively. T, G, C and A indicate the DNA sequence lanes. (a) The hpmB transcript start site (+1) lies 8 bp 3′ of a putative promoter which is compared with the consensus σ⁷⁰ promoter sequence. (b) Several transcript termini (*) were detected in the 284-base region 5′ of hpmA. The previously suggested (Uphoff & Welch, 1990) −35 and −10 hexamers 104 bp 5′ of the putative hpmA σ⁷⁰ promoter are boxed.

Fig. 2

Northern hybridization of a hpmA probe to total cellular RNA isolated from wild-type differentiated swarm cells carrying pBluescript (−), pGF74 (hpmBA) or pGF72 (ΔphpmBA). A 281- to 6583-base RNA ladder (Promega) was used to determine transcript size. Fluorographs were photographed using a Kodak DC-40 camera and transcripts were quantified using Kodak Digital Science 1D software.

Fig. 3

Northern hybridization of hpmA and hpmB probes to total cellular RNA isolated from differentiating wild-type cells collected 2, 3, and 4 h after seeding onto LB agar. The positions of the hpmA and hpmBA transcripts are indicated and hpmB degradation products are bracketed. Fluorography was carried out for 4 h (left panel) and 240 h (right panel). Transcript size was determined using a 281- to 6583-base RNA ladder (Promega).

Fig. 4

DNA sequence of the mutT-hpmB intergenic region showing the hpmB transcription start site, the putative σ⁷⁰ and σ²⁸ promoter sequences, and four putative Lrp-binding sites (Lrp 1-4).

Fig. 5

(a) Transcriptional fusions of the non-coding region 5′ of hpmBA to luxAB. A 712 bp region 5′ of hpmB encompassing putative promoter sequences (black boxes, σ⁷⁰ and σ²⁸) and four putative Lrp-binding sites (arrows, 1-4) was inserted 5′ of luxAB in the reporter plasmid pQF120 (Ronald et al., 1990), creating pLUX712. Deletion derivatives were constructed by PCR (PCR1, PCR2 and PCR3) and/or digestion with restriction endonucleases: D, DraI; P, PstI; X, XhoI. Peak luciferase activities in relative light units (RLU) of wild-type P. mirabilis (WT) and the flhDC null mutant (flhDCΩ) carrying the transcriptional fusions (pLUX) are shown on the right. (b) Luciferase activity over the swarm-cell differentiation cycle of wild-type cells carrying pLUX712. Cells were isolated from seeded agar plates at hourly intervals between 2 and 7 h post-inoculation. Peak swarm cell differentiation was at 5 h.

Fig. 6

Band-shift analysis of Lrp binding to the regulatory region 5′ of hpmBA. End-labelled DNA fragments of a 183 bp and a 239 bp region 5′ of hpmB encompassing Lrp sites 3 and 4, and 2, 3 and 4, respectively (see Fig. 3, nucleotides 529 to 711 and 473 to 711, respectively) were incubated with 0-30 ng purified His-Lrp protein. A control was performed with a labelled DNA fragment containing the 157 bp 5′ of the P. mirabilis flhB flagella gene (Claret & Hughes, 2000b). Samples were electrophoresed through a 6% polyacrylamide gel and DNA bands were visualized by fluorography.

Fig. 7

Northern hybridization of hpmA (left column), zapA (middle column) and flhDC (right column) probes to total cellular RNA isolated from differentiated wild-type swarm cells and the lrp::Tn5, umoBΩ, and flhDCΩ mutants carrying pBAD (−), pBADflhDC (flhDC), pBADlrp (lrp) or pBADumoB (umoB). Cells were collected 4 h after seeding onto LB agar. Transcript size was determined using a 281- to 6583-base RNA ladder (Promega). Fluorographs were photographed using a Kodak DC-40 camera and transcripts were quantified using Kodak Digital Science 1D software.