Contact sites of AraC protein to the regulatory site aral of the Escherichia coli araBAD operon have been determined by the chemical-interference technique. DNA AraC protein, which plays an inducing and repressing role in regulating the araBAD operon (9-16), does not possess any region of amino acid sequence with close homology to the consensus of the DNA-contacting domains of the abovementioned proteins (8, 17). Also, AraC protein appears to associate with a more extensive DNA region, protecting about 40 base pairs from nuclease digestion (9, 10). Therefore, we have used the binding-interference technique developed by Siebenlist and Gilbert (18) to examine the DNA contact sites that this protein makes with DNA to look for different possible modes of protein-DNA interaction. These studies reveal that AraC protein binds one face of the DNA in three adjacent major groove regions of araI site. AraC protein binds this site to induce or repress the araBAD promoter (15, 16). Consistent with the apparent contacts, the consensus of AraC binding sites contains three regions of conserved bases, and these lie in the three regions of contact.In view of the unusual binding pattern shown by AraC protein, we wished to determine if the protein binding unit is comprised of two, three, or some other number of subunits of AraC. We used the gel electrophoresis assay of DNA binding to accurately quantitate specifically bound protein and DNA. The results of these experiments show that AraC protein binds to araI as a dimer and to araO,, the site to which AraC binds to repress the araC promoter, also as a dimer. MATERIALS AND METHODSStrains, Plasmids, and General Methods. Strains, plasmids, and general methods were as described (15,(19)(20)(21). Plasmid pTD383 contains the araI site on a 170-base-pair fragment that is released by EcoRI and HindIll digestion. The araO, site is on a 300-base-pair fragment that is released by EcoRI and HindIII digestion of plasmid pTD242. AraC protein was purified as described (21).Binding Interference Assay. The assay is based on the method of Siebenlist and Gilbert (18). Phosphates of a 32P-end-labeled DNA fragment were ethylated by adding 100 ,ul of saturated ethylnitrosourea (Sigma) in 95% ethanol to a 100-,ul DNA sample in 50 mM sodium cacodylate, pH 8.0/0.1 mM EDTA and incubating at 50'C for 30-60 min. Purines were methylated by adding 1 ul of dimethyl sulfate to the DNA in 200 ,4 of 50 mM sodium cacodylate, pH 8.0/10 mM MgCI2/0.1 mM EDTA and incubating for 1 min at 20°C. This reaction was stopped by adding 50 ,4 of 1 M Tris HCl, pH 7.5/1 M 2-mercaptoethanol/1.5 M sodium acetate/0.05 M magnesium acetate/1 mM EDTA/0.1 mg of tRNA per ml. All reactions were timed to yield approximately one modification per DNA molecule. Modified DNA samples were precipitated with ethanol, washed, dried under vacuum, and resuspended in 10 mM Tris HCI, pH 8.0/1 mM EDTA (TE buffer).DNA recovered from the gel electrophoresis binding assay (see below) was cleaved at the positions of the modifications. To display ...

No abstract

The exopolysaccharide alginate is a maJor virulence factor ofPseudomonas aeruginosa strains that infect the lungs of cystic fibrosis patients. The synthesis of alginate is almost uniquely associated with the pathogenicity ofP. aeruginosa within the environment of the cystic fibrosis lung. The gene a4gC is one of the essential alginate biosynthetic genes and codes for the enzyme phosphomannomutase. In this report, we present data on the transcriptional regulation of algC expression. The activity of the algC promoter is modulated by the response regulator, AlgR1, a member of the two-component signal transduction protein family, which also regulates other alginate-specific promoters. In both mucoid (alginate-positive) and nonmucoid (alginate-negative) P. aeruginosa strains, transcriptional activation ofalgC increased with the osmolarity of the culture medium. This osmolarity-induced activation was found to be dependent on AlgRl. AlgRl was found to interact directly with the algC promoter. Deletion mapping, in conjunction with mobility shift assays, showed that AlgRl specifically bound with two regions ofalgC upstream DNA. A fragment spanning nucleotide positions -378 to -73 showed strong specific binding, while a fragment located between positions -73 and +187 interacted relatively weakly with AlgR1. Phosphorylation of the AlgRl protein resulted in the stimulation of its in vitro ability to bind to the algC promoter region (a fragment spanning nucleotides -378 to -73). Transcription from the aIgC promoter, which has significant homology with the RNA polymerase o-54 (RpoN) recognition sequence, decreased in an rpoN mutant of P. aeruginosa.

No abstract

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5′‐nucleotidase activity, the level of secretion of the 5′‐nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as α2‐macroglobulin. As macrophage‐ or mast cell surface‐associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP‐using enzymes generate various phosphorylated or non‐phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.

No abstract

Emerging and reemerging human viral pathogens pose great public health concerns since therapeutics against these viruses are limited. Thus, there is an urgent need to develop novel drugs that can block infection of either a specific virus or a number of viruses. Viral entry is thought to be an ideal target for potential therapeutic prevention. One of the challenges of developing antivirals is that most of these viruses are highly pathogenic and therefore require high biosafety-level containment. In this study, we have adopted a comparative high-throughput screening protocol to identify entry inhibitors for three enveloped viruses (Marburg virus, influenza virus H5N1, and Lassa virus) using a human immunodeficiency virus–based pseudotyping platform. We demonstrate the utility of this approach by screening a small compound library and identifying putative entry inhibitors for these viruses. One major advantage of this protocol is to reduce the number of false positives in hit selection, and we believe that the protocol is useful for inhibitor screening for many enveloped viruses.

SnmrD.a.ryThe 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-l, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2D b cells with the sitespecific Db-restricted CTL dones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitopecoding regions from variant cell DNAs. Cells selected for resistance to CTL done Y-1 (K-l; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (I(-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wildtype and mutant cell-transforming proteins.T umors induced by DNA viruses acquire spedfic antigens that provide targets for T lymphocyte-mediated immune responses modulating tumor cell growth in vivo and abrogating cellular transformation in vitro (1-4). Specific CTL recognition of tumor cells requires the presentation by MHC class I molecules of processed tumor antigen to the T lymphocyte antigen receptor. Tumor cell variants that escape CTL immunosurveiUance have been described (5-18). In most instances, the emergence of CTL escape variant cells is associated with the decrease or absence of MHC class I antigens required for antigen recognition by . In other cases, a complete loss of tumor antigen from the variants has been reported (6,10,(17)(18)(19).