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Fimbrial Phase Variation in Bordetella Pertussis: a Novel Mechanism for Transcriptional Regulation

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Fimbrial Phase Variation in Bordetella Pertussis: a Novel Mechanism for Transcriptional Regulation The EMBO Journal vol.9 no.9 pp.2803 - 2809, 1990 Fimbrial phase variation in Bordetella pertussis: a novel mechanism for transcriptional regulation Rob Willems, Annet Paul, The expression of B.pertussis virulence genes is affected Han G.J.van der Heide, Anja R.ter Avest and by growth conditions such as temperature, and the concen- Frits R.Mooi tration of MgSO4. Under nonpermissive conditions, i.e. a temperature of 25°C or the presence of 20 mM MgSO4, Laboratory of Bacteriology, National Institute of Public Health and the genes are repressed. When the cells are subsequently Environmental Protection, PO Box 1, 3720 BA Bilthoven, The shifted to permissive conditions, production is resumed Netherlands (Lacey, 1960; Idigbe et al., 1981). Recent evidence indicates Communicated by P.H.Makela that these growth conditions act through the bvg locus, which codes for three polypeptides involved in sensory transduction Fimbriae belong to a class of extracellular filamentous (Stibitz et al., 1988; Knapp and Mekalanos, 1988; Arico proteins which are involved in the attachment of bacteria et al., 1989). Indeed most B.pertussis virulence factors are to host tissues. Bordetella pertussis, the etiological agent not produced in strains harbouring transposon insertions in of whooping cough, produces two serologically distinct the bvg locus (Weiss et al., 1983). Thus most B.pertussis fimbriae. We show that, like a number of other B.per- virulence genes are part of a single regulon, positively tussis virulence genes, transcription of the runbrial regulated by bvg. In addition to this coordinate regulation, subunit genes (fum) is positively controlled by trans-acting fimbrial genes are subject to a second type of control, which polypeptides encoded by the bvg locus. In addition to this occurs independently of other virulence genes. Thus a coordinate control, transcription of the fim genes is particular B.pertussis strain may produce both types of regulated at an individual level by phase variation. This fimbriae, only one type, or no fimbriae at all. The process is characterized by a switching between a high mechanism underlying this fimbrial phase variation probably and low level of expression of a particularfim gene. We involves changes at the DNA level, since the capacity to have identified a conserved DNA region, located close to produce a particular fimbria at a high or low level is a the start of the fim genes, which is likely to be involved relatively stable characteristic which is passed on to progeny. in both positive regulation by the bvg locus, and phase In vivo, presumably under immunological pressure, fimbrial variation. This promoter region contains a stretch of phase variation can be readily observed (Preston et al., - 15 C residues and it appears that phase transitions 1980). In this paper we identify DNA regions involved in occur by small insertions or deletions in this C-rich the regulation of transcription of B.pertussis fimbrial genes, region. We propose that these mutations affect transcrip- and discuss the mechanism underlying fimbrial phase tion of thefim genes by varying the distance between the variation. Our results suggest that bvg regulation and phase binding site for an activator and the -10 box. The fim variation are linked at the molecular level. promoter shows homology with the pertussis toxin pro- moter, which is also positively regulated by the bvg locus. Results Key words: Bordetella/fimbriae/phase variation/promoter/ regulation Effect of bvg on expression of fimbrial genes Two B.pertussis strains were used to study the effect of bvg on expression offim genes, the Wellcome 28 strain which produces high amounts of serotype 2 and 3 fimbriae, and Introduction the Tohama (BP536) strain which produces high amounts Bordetella pertussis is the causative agent of whooping cough of serotype 2 fimbriae, and low amounts of serotype 3 or pertussis, a serious respiratory disease. A large number fimbriae (Figure 1). When the Wellcome 28 strain was of virulence factors have been implicated in the pathogenesis grown in the presence of 20 mM MgSO4, a condition of pertussis including at least four toxins (pertussis toxin, known to repress bvg-controlled genes, no serotype 2 or 3 adenylate cyclase, tracheal cytotoxin and dermonecrotic fimbrial subunits were detected (Figure 1). The presence of toxin) and two types of adhesins (the filamentous hemag- MgSO4 also decreased the amount of serotype 2 fimbriae glutinin, and fimbriae) (see Weiss and Hewlett, 1986 and produced by the Tohama strain, although repression was less Mooi, 1988 for reviews). B.pertussis produces two severe than observed in the Wellcome 28 strain (Figure 1). serologically distinct fimbriae, designated serotype 2 and No fimbrial subunits were detected in a derivative of Tohama serotype 3 fimbriae, which are composed of subunits with (BP347) containing a transposon insertion in the bvg locus slightly different molecular sizes: 22 500 and 22 000 daltons, (Figure 1). These observations indicate that, like a number respectively (Ashworth et al., 1982; Irons et al., 1985; of other B.pertussis virulence genes, thefim2 andfim3 genes Zhang et al., 1985). We have shown (Mooi et al., 1987) are subject to bvg regulation. that in addition to the genes coding for the serotype 2 and 3 fimbriae (fim2 andfin3, respectively), B.pertussis strains Identification of the fim3 promoter region contain a third fimbrial gene (fimX) which codes for an as Approximately 170 bp upstream of the start codon offim3, yet unidentified product. several inverted repeats are observed which may function Oxford University Press 2803 R.Willems et al. pRIP 102 100 bp Sf, Xma I Sph l-a Sphl-b Sol I Sph I-c I-LXmaI S- s o o co I C .f ... r r p ftm3 r :- .:: I : Fig. 2. Physical and genetic map of the DNA region harbouring the .:.: :. fim3 gene. The thick line and shaded boxes represent B.pertussis and pEMBL8 DNA, respectively. Only restriction enzyme recognition sites lg relevant for this study are shown. The Sphlb-Sphlc DNA fragment s _ _ ... 7 _ __ .-. r _- was used to detect fim3 mRNA. Abbreviations: ir, inverted repeat; : ^ E ._ w w p, promoter; bp, base pair. 500 - / 7/ / 7, c r/ MgSO4 400 - / 0 / 7, 7 0 / + MgS04 -::: O C\M / a) 300 - E / 0 / E -& ._ / _ - / 0 200 - / :>r Fig. 1. Production of serotype 2 and 3 fimbrial subunits. Total cell 40 0 lysates from strains, grown at 37°C in the absence or presence of 1) / 20 mM MgSO4, were analyzed by immunoblotting. The blots were 100 incubated with a monoclonal antibody directed against serotype 2 (a-fim2 Moab), or 3 (a-fim3 Moab) subunits. Positions of fimbrial / subunits are indicated by arrows. The bands which are sometimes observed below the arrows are proteolytic fragments of the fimbrial W28 B52 B1 1 1 31 10 B1 15 Bp536 B113 subunits. Abbreviations: W28, Wellcome 28. Fig. 3. Production of serotype 3 fimbriae, as determined by a whole as (rho-independent) transcriptional terminators (Mooi et al., cell ELISA. Microtitre plates were coated with cells grown in the 1989) (Figure 2). Thus it seemed likely that transcription presence or absence of 20 mM MgSO4 and the amount of serotype 3 of thefim3 gene was initiated downstream of these putative fimbriae was determined with a monoclonal antibody raised against serotype 3 fimbriae. The amount of serotype 3 fimbriae produced by terminators, i.e. from within a region located not more than the Wellcome 28 strain was arbitrarily set at 100%. B110, BIll and 170 bp from thefim3 start codon. The following experiment B1 15 are derivatives of B52 containing pMMB-fim3w+, was performed to determine if transcription was indeed pMMBfim3w+-and pMMBfim3bJ8- -, respectively. B113 was initiated from within this region. A 904 bp SphI fragment, derived from BP536 by passage through a rabbit. Abbreviation: W28, derived from the Wellcome 28 strain (Figure 2), was inserted Wellcome 28. into the multicloning site of pMMB67. The SphI fragment contains one of the putative transcriptional terminators When the plasmids were mobilized into B.pertussis strain located upstream of fim3, and a complete copy of the B52, a derivative of Tohama in which the fim2 and fim3 fim3w+ gene (in naming the fim genes we observe the genes have been inactivated by insertions, fim3w+ was following conventions: 2, 3 or X indicate the serotype; expressed at high levels (Figure 3). Compared to the w[ellcome], t[ohama], bll, etc. indicate the strain from Wellcome 28 strain, production was approximately five times which the gene is derived, and + and - indicate whether higher in B52 carrying pMMB-fim3w'. This higher the gene was expressed at high or low levels in the original production is probably due to a gene dose effect. Production strain, respectively). Plasmid pMMB67 contains the tac pro- of fimbriae was independent of the orientation of the moter and the lacI repressor, and genes cloned into its multi- fim3w+ gene in PMMB67 (Figure 3), indicating that cloning site can be expressed at high levels in E. coli if the transcription of fim3w+ is not dependent on a vector tac promoter is derepressed by addition of IPTG (isopropyl- promoter, but initiated from within the SphI fragment. When 0l-thiogalactopyranoside). Two recombinant plasmids which B.pertussis strains harbouring pMMB-fim3w+ were contained the fim3w+ gene in the correct or inverse grown in the presence of MgSO4, a seven-fold decrease in orientation relative to the tac promoter, designated fimbriae production was observed relative to controls grown pMMB-fim3w+ and pMMBfim3w+ -, respectively, were without MgSO4 (Figure 3), suggesting that expression of selected for further study. In E. coli strains harbouring the cloned fim3w+ gene was still bvg-dependent. pMMB-film3w+, no serotype 3 fimbrial subunits were Addition of IPTG did not significantly affect produc- detected on immunoblots even when the tac promoter was tion of fimbriae by B.pertussis strains carrying derepressed by addition of IPTG (results not shown).
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