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Regulatory Genes in the Thermoregulation of Escherichia Coli Pili Gene Transcription

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Regulatory Genes in the Thermoregulation of Escherichia Coli Pili Gene Transcription Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Regulatory genes in the thermoregulation of Escherichia coli pili gene transcription Mikael G6ransson, Kristina Forsman, and Bernt Eric Uhlin 1 Department of Microbiology, University of Ume~, S-901 87 Ume~, Sweden Expression of several different pilus adhesins by Escherichia coil is subject to thermoregulation. The surface- located fimbrial structures are present during growth at 37°C but are not produced by cells grown at lower temperatures, such as 25°C. As a step toward understanding the molecular mechanism, we have studied the role of different cistrons of a cloned pilus adhesin gene cluster (pap) from a uropathogenic E. coil isolate. By promoter cloning, mRNA analysis, and expression of subcloned genes in trans, we have identified the papl gene as the mediator of thermoregulation at the level of pilus adhesin gene transcription. Expression of the major pilus subunit gene O~apA) and several other pilus protein cistrons appeared to be dependent on stimulation by the papB and papl gene products. Constructs carrying different pap DNA regions indicated that none of the known Pap proteins acts directly as thermosensor. The chromosomal rpoH gene and RpoH ~ factor did not appear to be required for pap transcription, and the thermoregulation of pilus gene transcription must be different from that of the heat shock regulon. By overexpressing the papI gene product from an expression plasmid in trans, we could circumvent the temperature regulation and turn on production of pilus adhesin at low temperature. Our results suggest that the level of mRNA encoding the PapI activator is limiting at low growth temperatures and that thermoregulation is due to a determinant in the papl-papB intercistronic region. [Key Words: Adhesion genesl environmental regulationl thermoregulated mRNA synthesisl transcription activationl regulatory networks] Received August 8, 1988; revised version accepted October 31, 1988. Microorganisms presumably multiply as rapidly as envi- pression of adhesive properties correlating to virulence ronmental conditions permit. Studies of enteric bacteria are often temperature dependent, with optimum at 37°C such as Escherichia coli and Salmonella typhimurium and reduced at lower temperatures (De Graaf et al. 19801 show that they have evolved mechanisms that permit G6ransson and Uhlin 1984}. Other conditional factors, rapid growth under favorable conditions and aid in sur- e.g., growth substrate, may also influence the expression vival under conditions that are unfavorable for growth of pilus adhesins, and it appears that regulatory mecha- {Neidhardt 1987}. Changes in the growth conditions may nisms have evolved to allow for successful colonization therefore cause more or less complex alterations in the of a given niche under appropriate environmental condi- biochemistry and properties of the cells. Regulatory net- tions (Silverman et al. 1984). works such as the heat shock regulon, the SOS regulon, Analysis of the molecular mechanism(sl involved in and the cAMP-CRP regulon are examples of how mul- thermoregulation of adhesion should increase our tigene systems may be affected coordinately by changes knowledge about how external stimuli are transmitted in environmental conditions. In addition to the net- to the level of gene expression. Molecular genetic works and regulons that most bacteria seem to possess, studies of several determinants of pilus adhesins in E. one may find that individual isolates can express special coli have shown that there are multicistronic gene properties lacking in the majority of a given species. The clusters encoding the different major and minor pilus ability to adhere to surfaces in the surroundings is an proteins and the biogenesis of these surface organelles example of such a property, and this kind of interaction {for reviews, see Mooi and de Graaf 19851 Uhlin et al. between the microorganism and the environment is par- 1985bi Normark et al. 19861. Using gene operon fusions ticularly important and evident in the case of infectious to lacZ, we obtained evidence suggesting that the tem- disease (Ofek and Beachey 1980}. Adhesion by E. coli to perature regulation of digalactoside-binding pili {Pap human epithelial cells, as exemplified in the case of uro- pilil expression operates at the level of transcription of at pathogenic isolates, is commonly mediated by receptor- least the major pilin gene, papA [G6ransson and Uhlin specific pilus adhesins (Korhonen et al. 19821 Svenson et 1984}. Subsequent analysis established that there are al. 1983). The appearance of pili on the bacteria and ex- two cistrons in the region upstream of papA {papB and paplI, that papa is cotranscribed with papB, and that papB and papI seem to be involved in regulation of papA tCorrespondingauthor. expression {Bdida et al. 1985, 19881 see also Fig. 1}. We GENES & DEVELOPMENT3:123-130 © 1989 by Cold SpringHarbor Laboratory ISSN 0890-9369/89 $1.00 123 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press G6ransson et al. have studied the role of different pap cistrons in the tem- papC region and perhaps even farther (Baga et al. 1985, perature regulation of pilus production and expression of 1987, 1988; Uhlin et al. 1985a). Therefore we asked the adhesion properties. whether or not expression of all genes required for bio- genesis of pili and adhesin was dependent on the regula- tory genes and sequences located upstream of papA. One Results way to test this was to determine whether pilus produc- tion and expression of adhesin could be made constitu- Thermoregulation of Pap pilus adhesin is mediated by tive at botll high and low temperatures if the region up- the papI-papB region stream of papA were replaced by DNA sequences en- In addition to the major pilin subunit gene, papA, the coding a temperature-independent promoter region. Our formation of pilus adhesin involves several accessory studies with the E. coli alaS gene promoter suggested genes and minor pilus protein genes (i.e., papC-papH, that it would be suitable, considering the relative Fig. 1). Most distal from papA is the papG gene, and it strength and apparent temperature independence of that has been shown to encode the pilus protein, which is promoter upon cloning into operon fusion vectors (Sj6- responsible for the binding specificity of the Pap adhesin berg et al. 1986, and unpubl.; see also Table 2). The (Lund et al. 1987). The papA gene is part of an operon plasmid pHMG93 contains all of the pap genes required starting with papB, and although most transcripts seem for production of pili and adhesin, whereas the region to terminate between papA and papH, there is evidence including papI and part of the papB gene has been re- that some transcription continues through the papH- placed by DNA encoding the 5' end and the promoter of A ~lkb i E H H B pPAP5 t --I pa~ pa~B papa papH pap£) DapE l:~ papG (pSR322) E B H pHMG93 I Hl i I~/3/~22)/L_I P(alaS) E H pHMG1 pHMG15 E pPAP226 pPAP218 E B pHMG61 1 P(alaS) i lkb E S T A TV H T ~ ~A pHMG79 P(UV5) (,oACYC184) S BE pHMG94 J---// //~~ (pACYC184) P(UM5) Figure 1. Genetic and physical maps of plasmids: (AI Constructs expressing Pap pilus-adhesin genes or lacZ operon fusions; {B) PapB and PapI overproducing plasmids are shown below a map of the papI-papB-papA region. The parts representing cloning vector DNA are indicated by interrupted lines and are not drawn to scale. The filled parts of the bars show the coding regions of the indicated pap genes. Hatched portions show that only a part of a gene is remaining. Vertical arrowheads indicate cleavage sites for different restric- tion endonucleases: (E) EcoRI; (H) HindIII; {B) BamHI; (S) SphI; (T) TaqI; (A) ApaI; (V) EcoRV. Horizontal arrows indicate direction of transcription from promoters, as discussed in the text. (A) The insertion mutation in papB, which constitutes the difference between pHMG 15 and the wild-type derivative pHMG 1. 124 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Temperature-regulated activator of gene expression Table 1. Temperature-dependent and constitutive expression f of Pap pilus adhesin Adhesin Pilus phenotype a phenotype b Strain 37°C 26°C 37°C 26°C L HB 101/pPAPS + - + - B• HB101/pHMG93 + + + + HBlO1/pBR322 .... J96 + - + - J96/pHMG94 + + + + q i a Examined by electron microscopy. b Mannose-resistant hemagglutination assay (MRHA}. more (M. G6ransson, K. Forsman, B.E. Uhlin, unpubl.). Temperature-shift experiments {from 25-37°C) with bacteria carrying the different fusion plasmids also showed that the kinetics of increase in 13-galactosidase expression was similar (Fig. 3J. These results suggested tl that the gene products from papB and papI by them- 0 selves were not the cause of the temperature effect. The rpoH (htpR) gene product of E. coli has been E F shown to activate transcription of a set of genes (the heat shock regulon) upon shifts from low to high growth tem- Figure 2. Electron microscopic analysis of pilus expression: peratures while acting as an alternative or-factor in the (A) HB101/pPAP5 at 37°C; (B) HB101/pPAP5 at 26°C; {C) HB101/pHMG93 at 37°C; {D) HB101/pHMG93 at 26°C; (E) HBlO1/pBR322 at 37°C; (F) HBI01/pBR322 at 26°C. the alas genc. E. coli strain HB101 harboring thc dif- ferent plasmids was grown at 37°C and at 26°C. Thc bac- 114(; 1 teria were then analyzed for production of pill by clcc- tron microscopy {Fig. 2) and for expression of hemagglu- tinating ability. As summarized in Table 1, the cloned 150 wild-type pap genes (pPAP5) were temperature depen- dent in their expression, but the alaS promoter substitu- tion IpHMG93) resulted in constitutive expression.
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