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Motility of Urease-Deficient Derivatives of Helicobacter pylori. Shumin Tan, 2004.Early studies of a ureB mutant derivative of Helicobacter pylori had suggested that urease is needed for motility and that urease action helps energize flagellar rotation . Here we report experiments showing that motility is unaffected by deletion of ureA and ureB (urease genes) or by inactivation of ureB alone, especially if H . pylori strains used as recipients for transformation with mutant alleles are preselected for motility . This result was obtained with the strain used in the early studies (CPY3401) and also with 15 other strains, 3 of which can colonize mice . We conclude that urease is not needed for H . pylori motility . The Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo. Roger S. Smith, 2002.Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl . These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI . LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone . There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells . In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P . aeruginosa had on colonization of the lung . We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P . aeruginosa to colonize the lung . To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice . In this model, 3O-C12-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1  (IL-1) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1ß, inducible protein 10, and T-cell activation gene 3 . Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression . The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain . We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment . Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections . Therefore, the quorum-sensing systems of P . aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation .
The Small Subunit of M · AquI Is Responsible for Sequence-Specific DNA Recognition and Binding in the Absence of the Catalytic Domain. Hatice Pinarbasi, 2003.AquI DNA methyltransferase (M · AquI) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'-CCCGGG-3' . M · AquI is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M · HhaI . Recently, we reported the subcloning, overexpression, and purification of the subunits (  and ß) of M · AquI separately . Here we describe the DNA binding properties of M · AquI . The results presented here indicate that the ß subunit alone contains all of the information for sequence-specific DNA recognition and binding . The first step in the sequence-specific recognition of DNA by M · AquI involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs IX and X, found in all known C5 DNA methyltransferases, contained in the ß subunit . The
subunit enhances the binding of the ß subunit to DNA specifically and nonspecifically . It is likely that the addition of the
subunit to the ß subunit stabilizes the conformation of the ß subunit and thereby enhances its affinity for DNA indirectly . Addition of S-adenosyl-L-methionine and its analogues S-adenosyl-L-homocysteine and sinefungin enhances binding, but only in the presence of the
subunit . These compounds did not have any effect on DNA binding by the ß subunit alone . Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the ß subunit alone did not form a covalent complex with its specific sequence in the absence or presence of S-adenosyl-L-methionine . However, the addition of the
subunit to the ß subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC .
Incidence of Enteric Viruses in Groundwater from Household Wells in Wisconsin. Mark A. Borchardt, 2003.Recent studies on the contamination of groundwater with human enteric viruses have focused on public water systems, whereas little is known about the occurrence of viruses in private household wells . The objective of the present study was to estimate the incidence of viruses in Wisconsin household wells located near septage land application sites or in rural subdivisions served by septic systems . Fifty wells in seven hydrogeologic districts were sampled four times over a year, once each season . Reverse transcriptase PCR (RT-PCR), followed by Southern hybridization, was used to detect enteroviruses, rotavirus, hepatitis A virus (HAV), and Norwalk-like viruses (NLVs) . In addition, cell culture was used to detect culturable enteroviruses . Companion water samples were collected for total coliforms, Escherichia coli, fecal enterococci, F-specific RNA coliphages, nitrate, and chloride analyses . Among the 50 wells, four (8%) were positive for viruses by RT-PCR . Three wells were positive for HAV, and the fourth well was positive for both rotavirus and NLV in one sample and an enterovirus in another sample . Contamination was transient, since none of the wells was virus positive for two sequential samples . Culturable enteroviruses were not detected in any of the wells . Water quality indicators were not statistically associated with virus occurrence, although some concordance was noted for chloride . The present study is the first in the United States to systematically monitor private household wells for virus contamination and, combined with data for public wells, provides further insight on the extent of groundwater contamination with human enteric viruses . Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin. Christiane Szekat, 2003.The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp . strain HIL Y-85,54728 . Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II . The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster . For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection . In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed . To test the expression system, three mutants were constructed . In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine . S16I mersacidin was produced only in small amounts . The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin . Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity .
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