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Model System for High-Throughput Screening of Novel Human Immunodeficiency Virus Protease Inhibitors in Escherichia coli. Ting-Jen Cheng, 2004.Novel human immunodeficiency virus (HIV) protease inhibitors are urgently needed for combating the drug-resistance problem in the fight against AIDS . To facilitate lead discovery of HIV protease inhibitors, we have developed a safe, convenient, and cost-effective Escherichia coli-based assay system . This E . coli-based system involves coexpression of an engineered ß-galactosidase as an HIV protease substrate and the HIV protease precursor comprising the transframe region and the protease domain . Autoprocessing of the HIV protease precursor releases the mature HIV protease . Subsequently, the HIV protease cleaves ß-galactosidase, resulting in a loss of the ß-galactosidase activity, which can be detected in high-throughput screens . Using Food and Drug Administration-approved HIV protease inhibitors, this E . coli-based system is validated as a surrogate screening system for identifying inhibitors that not only possess inhibitory activity against HIV protease but also have solubility and permeability for in vivo activity . The usefulness of the E . coli-based system was demonstrated with the identification of a novel HIV protease inhibitor from a library of compounds that were prepared by an amide-forming reaction with transition-state analog cores . A novel inhibitor with a sulfonamide core of amprenavir, E2, has shown good correlation with the in vitro enzymatic assay and in vivo E . coli-based system . This system can also be used to generate drug resistance profiles that could be used to suggest therapeutic uses of HIV protease inhibitors to treat the drug-resistant HIV strains . This simple yet efficient E . coli system not only represents a screening platform for high-throughput identification of leads targeting the HIV proteases but also can be adapted to all other classes of proteases . BetS Is a Major Glycine Betaine/Proline Betaine Transporter Required for Early Osmotic Adjustment in Sinorhizobium meliloti. Alexandre Boscari, 2002.Hybridization to a PCR product derived from conserved betaine choline carnitine transporter (BCCT) sequences led to the identification of a 3.4-kb Sinorhizobium meliloti DNA segment encoding a protein (BetS) that displays significant sequence identities to the choline transporter BetT of Escherichia coli (34%) and to the glycine betaine transporter OpuD of Bacillus subtilis (30%) . Although the BetS protein shows a common structure with BCCT systems, it possesses an unusually long hydrophilic C-terminal extension (169 amino acids) . After heterologous expression of betS in E . coli mutant strain MKH13, which lacks choline, glycine betaine, and proline transport systems, both glycine betaine and proline betaine uptake were restored, but only in cells grown at high osmolarity or subjected to a sudden osmotic upshock . Competition experiments demonstrated that choline, ectoine, carnitine, and proline were not effective competitors for BetS-mediated betaine transport . Kinetic analysis revealed that BetS has a high affinity for betaines, with Kms of 16 ± 2 µM and 56 ± 6 µM for glycine betaine and proline betaine, respectively, in cells grown in minimal medium with 0.3 M NaCl . BetS activity appears to be Na+ driven . In an S . meliloti betS mutant, glycine betaine and proline betaine uptake was reduced by about 60%, suggesting that BetS represents a major component of the overall betaine uptake activities in response to salt stress . ß-Galactosidase activities of a betS-lacZ strain grown in various conditions showed that betS is constitutively expressed . Osmotic upshock experiments performed with wild-type and betS mutant cells, treated or not with chloramphenicol, indicated that BetS-mediated betaine uptake is the consequence of immediate activation of existing proteins by high osmolarity, most likely through posttranslational activation . Growth experiments underscored the crucial role of BetS as an emerging system involved in the rapid acquisition of betaines by S . meliloti subjected to osmotic upshock . Characterization of the Campylobacter jejuni Heptosyltransferase II Gene, waaF, Provides Genetic Evidence that Extracellular Polysaccharide Is Lipid A Core Independent. Neil J. Oldfield , 2002.Campylobacter jejuni produces both lipooligosaccharide (LOS) and a higher-molecular-weight polysaccharide that is believed to form a capsule . The role of these surface polysaccharides in C . jejuni-mediated enteric disease is unclear; however, epitopes associated with the LOS are linked to the development of neurological complications . In Escherichia coli and Salmonella enterica serovar Typhimurium the waaF gene encodes a heptosyltransferase, which catalyzes the transfer of the second L-glycero-D-manno-heptose residue to the core oligosaccharide moiety of lipopolysaccharide (LPS), and mutation of waaF results in a truncated core oligosaccharide . In this report we confirm experimentally that C . jejuni gene Cj1148 encodes the heptosyltransferase II enzyme, WaaF . The Campylobacter waaF gene complements an S . enterica serovar Typhimurium waaF mutation and restores the ability to produce full-sized lipopolysaccharide . To examine the role of WaaF in C . jejuni, waaF mutants were constructed in strains NCTC 11168 and NCTC 11828 . Loss of heptosyltransferase activity resulted in the production of a truncated core oligosaccharide, failure to bind specific ligands, and loss of serum reactive GM1, asialo-GM1, and GM2 ganglioside epitopes . The mutation of waaF did not affect the higher-molecular-weight polysaccharide supporting the production of a LOS-independent capsular polysaccharide by C . jejuni . The exact structural basis for the truncation of the core oligosaccharide was verified by comparative chemical analysis . The NCTC 11168 core oligosaccharide differs from that known for HS:2 strain CCUG 10936 in possessing an extra terminal disaccharide of galactose-ß(1,3) N-acetylgalactosamine . In comparison, the waaF mutant possessed a truncated molecule consistent with that observed with waaF mutants in other bacterial species . Molecular Characterization of the Acid-Inducible asr Gene of Escherichia coli and Its Role in Acid Stress Response. Vaida , 2003.Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity . These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis . Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown . We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0) . We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0) . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E . coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa . Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids . This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively . Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene . N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor . Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock . Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide . Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E . coli under acid shock conditions . Characterization of Streptococcus mutans Strains Deficient in EIIABMan of the Sugar Phosphotransferase System. Jacqueline Abranches, 2003.The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci . The role of EIIABMan (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159 . The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIABMan is involved in sugar uptake and in carbohydrate catabolite repression . PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S . mutans EIIABMan transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS . Additionally, there appears to be a fructose PTS that is different than FruI and FruCD . To determine whether EIIABMan controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIABMan-deficient strains . In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIABMan is required for optimal expression of gtfBC . No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression . Two-dimensional gel analysis of batch-grown cells of the EIIABMan-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIABMan has a pleiotropic effect on gene expression .
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