Kristina Forsman

Upstream activating sequences that are shared by two divergently transcribed operons mediate cAMP-CRP regulation of pilus-adhesin in Escherichia coli

Transcription of the genes encoding pilus‐adhesin of serotype F13 in digalactoside‐binding Escherichia coli required activation by the cAMP‐CRP complex. Analysis of protein‐DNA interaction in vitro showed that CRP bound in a cAMP‐dependent manner to a sequence located 0.2 kb upstream of the point of transcription initiation of the pilus subunit operon. The cAMP‐CRP activation included, in addition to the main pilus operon, the oppositely oriented operon encoding the Papl regulatory protein. Furthermore, the auto‐regulatory product of the promoter‐proximal gene (papB) in the pilus subunit operon was found to stimulate the papl transcriptional unit. Thus the cAMP‐CRP complex and PapB might act in concert and indirectly promote pili synthesis by stimulating expression of the Papl positive regulator. The results of trans complementation experiments and analyses using lacZ operon fusion derivatives showed that the cAMP‐CRP activation also operated directly in cis on the pilus subunit operon. The region containing the CRP binding site appeared to function as an upstream activating sequence since deletion abolished expression even when the pap regulatory proteins Papl and PapB were supplied in trans. The implications for possible mechanisms of transcriptional activation by the cAMP‐CRP complex at this novel location between the two oppositely oriented operons are discussed.

The gene for Best's macular dystrophy is located at 11q13 in a Swedish family

A large Swedish family with more than 250 cases of Bests macular dystrophy has been clinically and genetically studied. The gene was traced to a couple born in central Sweden in the 17th century. Highly significant evidence for genetic linkage to DNA markers on chromosome 11q13 was detected. A lod score of 15.12 was obtained at recombination fraction 0.01 with DNA marker INT2 (also called FGF3). The retinally expressed gene ROM1, which maps to the same chromosomal region is a candidate for this genetic disease.

Oligomeric interaction of the PapB transcriptional regulator with the upstream activating region of pili adhesin gene promoters in Escherichia coli.

Transcriptional regulation of the pap genes, which encode fimbrial adhesins in uropathogenic Escherichia coli, depends on an upstream activating region. This region contains binding sites for a transcription factor, PapB, which is a member of a growing family of putative regulatory proteins found in several virulence‐associated fimbrial gene systems. To assess the nature of the PapB binding sites, we studied different naturally occurring variants and a number of in vitro constructed mutant binding sites. DNase I footprinting analysis and visualization of the PapB–DNA complex by atomic force microscopy showed that the protein occupied a DNA region of more than 50 bp. Purified PapB protein was shown to recognize a motif including a 9 bp repeat sequence containing T/A triplets at a conserved position. PapB binding was affected by distamycin, and the results were consistent with the possibility that the binding to DNA occurred through minor groove interaction. From these analyses and estimation of the relative number of PapB proteins per binding site, we suggest that PapB binds the DNA in an oligomeric fashion and may function as an architectural factor in the transcriptional control of adhesin expression.

GENETIC HETEROGENEITY OF AUTOSOMAL DOMINANT AMELOGENESIS IMPERFECTA DEMONSTRATED BY ITS EXCLUSION FROM THE AIH2 REGION ON HUMAN CHROMOSOME 4Q

Amelogenesis imperfecta (AI) is a group of hereditary enamel defects, characterized by large clinical diversity. On the basis of differences in clinical manifestation and inheritance pattern, 14 different subtypes have been recognized. A locus for autosomal dominant AI (ADAI) of local hypoplastic type was recently mapped to the region between D4S392 and D4S395 on the long arm of chromosome 4. To test whether the chromosome 4 locus is responsible for other forms of AI as well, a linkage study was carried out with 17 families representing at least five clinical forms of ADAI. Admixture tests for heterogeneity performed with the marker D4S2456 gave statistical support for genetic heterogeneity of ADAI with the odds 78:1. Linkage to the ADAI locus on chromosome 4q (AIH2) could only be demonstrated with families expressing the local hypoplastic type, and there was no support for heterogeneity within that group of families. Furthermore, linkage could be excluded for five families with other clinical forms of ADAI. The data therefore demonstrated that ADAI is genetically heterogeneous, and that at least two loci for it exist.

Functional and structural homology among regulatory cistrons of pili-adhesin determinants in Escherichia coli

SummaryExpression of the digalactoside-binding Pap pili involves two trans-acting regulatory genes, papB and papI. Using pap-lac operon fusions and DNA hybridization probes derived from pap DNA we tested whether or not other pili-adhesin determinants from different Escherichia coli strains encode homologs to the pap regulatory genes. Digalactoside-specific clones of serotypes F72 and F11 complemented papB and papI mutants of the Pap (serotype F13) clone and DNA hybridization analysis showed that the clones are homologous in the DNA sequences encoding the two regulatory genes. Similar results were obtained with an S-pili determinant which mediates binding to sialic acid-containing receptors and the findings suggest that the regulatory regions may be more conserved than other genes in different pili-adhesin gene clusters. Determinants for type 1-pili (mannose-specific binding) and for pili associated with enterotoxigenic E. coli (K88, K99, CFAI, CFAII) did not appear to contain DNA sequences homologous to papB or papI. E. coli strain J96, which was the origin of the pap DNA, was found to carry two additional copies of papB-papI homologous sequences in the chromosome. In strains expressing more than one kind of pili the trans-active gene products thereby may allow for regulatory interaction between separate pili-adhesin gene systems.

Refined genetic localization of the Best disease gene in 11q13 and physical mapping of linked markers on radiation hybrids.

Abstract Best’s macular dystrophy, also known as vitelliform macular degeneration type 2 (VMD-2), is an autosomal dominant eye disorder that causes reduced visual acuity. It generally manifests itself in the teenage years. The gene mutated in VMD-2 patients may provide valuable insight into the biological mechanisms of the far more common disorder age-related macular degeneration. The VMD-2 gene has been localized to 11q13 between UGB and FcɛRI. In order to clone the gene positionally, a large Swedish VMD-2 family dating back to the 17th century was studied for recombinations. Since the last study, another 40 microsatellite markers have been analyzed in the family; the closest centromeric flanking marker, D11S4076, revealed two recombinations and the closest telomeric flanking marker, UGB, revealed one recombination. The recombinations have occurred in affected individuals, which eliminates the potential problem of reduced penetrance. The order and physical distance between 22 markers located at proximal 11q13 were analyzed on the G3 Stanford radiation-reduced cell hybrids. The data suggest that the VMD-2 region flanked by the microsatellite markers D11S4076 and UGB is approximately 980 kb.

Genetics of Histone-Like Protein H-NS/H1 and Regulation of Virulence Determinants in Enterobacteria

Isolates of E. coli associated with intestinal or with extraintestinal disease are often characterized by their ability to express different properties thought to contribute to bacterial virulence. Examples of such properties are the synthesis of various types of adhesins, production of different kinds of cytotoxins (e.g. enterotoxins and hemolysins), ability to invade host tissue cells, and expression of certain capsule- and O-antigens. Changes in growth conditions may profoundly affect how different virulence-associated properties are expressed. The type of growth substrate, osmolarity, and growth temperature are examples of environmental factors found to influence the phenotypic expression of some of the virulence associated properties monitored under laboratory conditions. We have studied the expression of E. coli adhesins typically produced by many isolates from urinary tract infections in man. The P-specific adhesins are fimbrial adhesins, also referred to as pili, and they mediate binding to α-D-Gal-(1–4)-β-D-Gal-containing glycolipid structures. Genetic studies of the pap gene cluster have revealed functions of several of the gene products required for biogenesis of this type of adhesin (see Tennent et al., 1990 for a recent review).

Biogenesis of Galα(1–4)Gal binding P-pili of Uropathogenic E. Coli

Escherichia coli is the most common cause of urinary tract infections (UTI). Depending on the virulence of the organism and the efficiency of the host defense system, these UTI appear either as asymptomatic bacteriuria, acute cystitis or acute pyelonephritis. A number of putative virulence properties have been recognized in E. coli isolates associated with acute pyelonephritis in otherwise uncompromised children. One such property is the ability to express mannose-resistant (MR) hemagglutinins. Most E. coli are capable of forming type 1 pili that carry a mannosespecific adhesin. Pyelonephritogenic E. coli additionally carry one or more chromosomal gene clusters that encode pili associated with MR hemagglutination. Of the several MR hemagglutinins that are known today, the best characterized is the Galα(1–4)Gal-specific adhesin associated with P-pili. The name P-pili refers to the observation that this adhesin acts as a hemagglutinin for erythrocytes which express any of the P blood group antigens.