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Nitrosomonas europaea Expresses a Nitric Oxide Reductase during Nitrification. Hubertus J. E. Beaumont, 2004.In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea . Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans . NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells . NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene . The findings demonstrate the presence of an alternative site of N2O production in N . europaea . Mechanisms of Resistance in Multiple-Antibiotic-Resistant Escherichia coli Strains of Human, Animal, and Food Origins. Yolanda Sáenz, 2004.Seventeen multiple-antibiotic-resistant nonpathogenic Escherichia coli strains of human, animal, and food origins showed a wide variety of antibiotic resistance genes, many of them carried by class 1 and class 2 integrons . Amino acid changes in MarR and mutations in marO were identified for 15 and 14 E . coli strains, respectively . Bactericidal Effects of a Fusion Protein of Llama Heavy-Chain Antibodies Coupled to Glucose Oxidase on Oral Bacteria. A. Szynol, 2004.Enzymes such as lactoperoxidase and glucose oxidase (GOx) are used as antimicrobial agents in oral care products . Their low specificities and substantiveness can be reduced by covalent coupling of antimicrobial molecules to antibodies . Variable domains (VHH) derived from llama heavy-chain antibodies are particularly suited for such an approach . The antibodies are composed solely of heavy-chain dimers; therefore, production of active fusion proteins by using molecular biology-based techniques is less complicated than production by use of conventional antibodies . In this study, a fusion protein consisting of VHH and GOx was constructed and expressed by Saccharomyces cerevisiae . A llama was immunized with Streptococcus mutans strain HG982 . Subsequently, B lymphocytes were isolated and cDNA fragments encoding the VHH fragments were obtained by reverse transcription-PCR . After construction of a VHH library in Escherichia coli and screening of the library against mutans group streptococci and Streptococcus sanguinis strains, we found two VHH fragments with high specificities for S . mutans strains . A GOx gene was linked to the two VHH genes and cloned into S . cerevisiae yeasts . The yeasts expressed and secreted the recombinant proteins into the growth medium . The test of binding of fusion proteins to oral bacteria through their VHH fragments showed that S . mutans had been specifically targeted by GOx-S120, one of the fusion protein constructs . A low concentration of the fusion protein was also able to selectively kill S . mutans within 20 min in the presence of lactoperoxidase and potassium iodide . These findings demonstrate that the fusion protein GOx-VHH is potentially valuable in the selective killing of target bacteria such as S . mutans . Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:L-Alanine Ligase (MurC) from Haemophilus influenzae. Clifford D. Mol, 2003.UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan . The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg2+ and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn2+ have been determined to 1.85- and 1.7-Å resolution, respectively . These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine . An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound . The MurC active site clearly shows that the  -phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group . These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates .
Transcriptional Regulation and Posttranslational Activity of the Betaine Transporter BetL in Listeria monocytogenes Are Controlled by Environmental Salinity. Roy D. Sleator, 2003.While the genetic elements contributing to the salinity tolerance of Listeria monocytogenes have been well characterized, the regulatory signals and responses (genetic and/or biochemical) that govern these mechanisms have yet to be elucidated . Encoded by betL, the first genetic element to be linked to listerial osmotolerance, the secondary betaine uptake system BetL is a member of the betaine-carnitine-choline transporter family . Preceded by consensus  A- and
B-dependent promoter
sites, betL is constitutively expressed and transcriptionally
up-regulated in response to salt stress . The nisin-controlled
expression system was used to achieve salinity-independent, controlled
betL expression in Listeria . In the absence of
NaCl-activated transcriptional control, BetL activity was found to be a
function of environmental salinity, showing optimal activity in buffer
supplemented with 1 to 2% NaCl (osmolality, 417 to 719
mosmol/kg) . In addition, BetL was activated rapidly (half-life, 2 min)
in response to an osmotic upshift imposed by adding 2% NaCl to
50 mM potassium phosphate
buffer .
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