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A Second Lysine-Specific Serine Protease from Lysobacter sp . Strain IB-9374. Shigeru Chohnan, 2004.A second lysyl endopeptidase gene (lepB) was found immediately upstream of the previously isolated lepA gene encoding a highly active lysyl endopeptidase in Lysobacter genomic DNA . The lepB gene consists of 2,034 nucleotides coding for a protein of 678 amino acids . Amino acid sequence alignment between the lepA and lepB gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa) . The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity . The lepB gene encoding the propeptide and mature-enzyme portions was overexpressed in Escherichia coli, and the inclusion body produced generated active LepB through appropriate refolding and processing . The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA . LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides . The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it . Efficacy of Cidofovir in a Murine Model of Disseminated Progressive Vaccinia. Johan Neyts, 2004.An animal model that mimics progressive disseminated vaccinia was elaborated . To this end nude (athymic) mice were inoculated intracutaneously with vaccinia virus in the lumbosacral area . Viral replication (DNA) in the skin was detected as early as day 2 postinfection (p.i.) . Mice developed typical vaccinia lesions at the site of inoculation by day 4 to 6 p.i . By about 2 weeks p.i., the infection had spread all over the body, a situation reminiscent of disseminated vaccinia in humans . The infection resulted in viremia and spread of the virus to visceral organs, as well as to the brain . Topical treatment with cidofovir, initiated at the day of infection or at day 1 p.i., completely protected against virus-induced cutaneous lesions and against associated mortality . When treatment was initiated at a later time (day 2 to 5 p.i.), a partial but marked protective effect was noted, which can be explained by the fact that by that time, the virus had spread from the skin to the visceral organs . Next, infected animals were left untreated until the time (  2 weeks p.i.) at which disseminated vaccinia had developed . When systemic treatment with cidofovir was initiated at that time, it caused lesions to heal and regress . In most of these animals, lesions had completely (or almost completely) disappeared by day 10 to 15 after the start of therapy . The observation that cidofovir is able to cause healing of disseminated vaccinia lesions in animals should have implications for the therapy of complications of vaccination against smallpox .
Mosaic Structure and Molecular Evolution of the Leukotoxin Operon (lktCABD) in Mannheimia (Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi. Robert L. Davies, 2002.The mosaic structure and molecular evolution of the leukotoxin operon (lktCABD) was investigated by nucleotide sequence comparison of the lktC, lktB, and lktD genes in 23 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains . Sequence variation in the lktA gene has been described previously (R . L . Davies et al., J . Bacteriol . 183:1394–1404, 2001) . The leukotoxin operon of M . haemolytica has a complex mosaic structure and has been derived by extensive inter- and intraspecies horizontal DNA transfer and intragenic recombination events . However, the pattern of recombination varies throughout the operon and among the different evolutionary lineages of M . haemolytica . The lktA and lktB genes have the most complex mosaic structures with segments derived from up to four different sources, including M . glucosida and P . trehalosi . In contrast, the lktD gene is highly conserved in M . haemolytica . The lktC, lktA, and lktB genes of strains representing the major ovine lineages contain recombinant segments derived from bovine or bovine-like serotype A2 strains . These findings support the previous conclusion that host switching of bovine A2 strains from cattle to sheep has played a major role in the evolution of the leukotoxin operon in ovine strains of M . haemolytica . Homologous segments of donor and recipient alleles are identical, or nearly identical, indicating that the recombinational exchanges occurred relatively recent in evolutionary terms . The 5' and 3' ends of the operon are highly conserved in M . haemolytica, which suggests that multiple horizontal exchanges of the complete operon have occurred by a common mechanism such as transduction . Although the lktA and lktB genes both have complex mosaic structures and high nucleotide substitution rates, the amino acid diversity of LktB is significantly lower than that of LktA due to a higher degree of evolutionary constraint against amino acid replacement . The recombinational exchanges within the leukotoxin operon have had greatest effect on LktA and probably provide an adaptive advantage against the host antibody response by generating novel antigenic variation at surface-exposed sites . Strains of Escherichia coli O157:H7 Differ Primarily by Insertions or Deletions, Not Single-Nucleotide Polymorphisms. Indira T. Kudva, 2002.Escherichia coli O157:H7 (O157) strains demonstrate varied pulsed-field gel electrophoresis patterns following XbaI digestion, which enable epidemiological surveillance of this important human pathogen . The genetic events underlying PFGE differences between strains, however, are not defined . We investigated the mechanisms for strain variation in O157 by recovering and examining nucleotide sequences flanking each of the XbaI restriction enzyme sites in the genome . Our analysis demonstrated that differences between O157 strains were due to discrete insertions or deletions that contained the XbaI sites polymorphic between strains rather than single-nucleotide polymorphisms in the XbaI sites themselves . These insertions and deletions were found to be uniquely localized within the regions of the genome that are specific to O157 compared to E . coli K-12 (O islands), suggesting that strain-to-strain variation occurs in these O islands . These results may be utilized to devise novel strain-typing tools for this pathogen . The IntP C-Terminal Segment Is Not Required for Excision of Bacteriophage Mx8 from the Myxococcus xanthus Chromosome. Nobuki Tojo, 2003.During lysogenization of myxophage Mx8, phage DNA can be integrated into the attB site of the Myxococcus xanthus chromosome through site-specific recombination . We previously demonstrated that the Mx8 attP site is located within the coding sequence of the Mx8 intP gene . Hence, the integration of Mx8 into the M . xanthus chromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR . To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of the intP-attP or intR-attR regions as well as the lacZ gene . The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision . For Mx8 excision, a gene designated xis was shown to be required in addition to intR . Natural Genetic Transformation in Monoculture Acinetobacter sp . Strain BD413 Biofilms. Larissa Hendrickx, 2003.Horizontal gene transfer by natural genetic transformation in Acinetobacter sp . strain BD413 was investigated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm . Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation . The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction . Biofilms were highly amenable to natural transformation . They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells . An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies . The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure . Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of Acinetobacter sp . strain BD413 .
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