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pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates in Portugal. Isabel Portugal, 2004.The nucleotide sequences of the pncA genes within 55 multidrug-resistant pyrazinamide-resistant Mycobacterium tuberculosis clinical isolates were determined . Fifty-three out of the 55 isolates were pyrazinamidase (PZase) negative . Four strains contained a wild-type pncA gene, and PZase activity was undetectable in two of these strains . Seven of the 18 identified pncA mutations found have not been described in previous studies . Molecular Evolution of the dotA Gene in Legionella pneumophila. Kwan Soo Ko, 2003.The molecular evolution of dotA, which is related to the virulence of Legionella pneumophila, was investigated by comparing the sequences of 15 reference strains (serogroups 1 to 15) . It was found that dotA has a complex mosaic structure . The whole dotA gene of Legionella pneumophila subsp . pneumophila serogroups 2, 6, and 12 has been transferred from Legionella pneumophila subsp . fraseri . A discrepancy was found between the trees inferred from the nucleotide and deduced amino acid sequences of dotA, which suggests that multiple hits, resulting in synonymous substitutions, have occurred . Gene phylogenies inferred from three different segments (the 5'-end region, the central, large periplasmic domain, and the 3'-end region) showed impressively dissimilar topologies . This was concordant with the sequence polymorphisms, indicating that each region has experienced an independent evolutionary history, and was evident even within the same domain of each strain . For example, the PP2 domain was found to have a heterogeneous structure, which led us hypothesize that the dotA gene of L . pneumophila may have originated from two or more different sources . Comparisons of synonymous and nonsynonymous substitutions demonstrated that the PP2 domain has been under strong selective pressure with respect to amino acid change . Split decomposition analysis also supported the intragenic recombination of dotA . Multiple recombinational exchange within the dotA gene, encoding an integral cytoplasmic membrane protein that is secreted, probably provided increased fitness in certain environmental niches, such as within a particular biofilm community or species of amoebae . The YSIRK-G/S Motif of Staphylococcal Protein A and Its Role in Efficiency of Signal Peptide Processing. Taeok Bae, 2003.Many surface proteins of pathogenic gram-positive bacteria are linked to the cell wall envelope by a mechanism requiring a C-terminal sorting signal with an LPXTG motif . Surface proteins of Streptococcus pneumoniae harbor another motif, YSIRK-G/S, which is positioned within signal peptides . The signal peptides of some, but not all, of the 20 surface proteins of Staphylococcus aureus carry a YSIRK-G/S motif, whereas those of surface proteins of Listeria monocytogenes and Bacillus anthracis do not . To determine whether the YSIRK-G/S motif is required for the secretion or cell wall anchoring of surface proteins, we analyzed variants of staphylococcal protein A, an immunoglobulin binding protein with an LPXTG sorting signal . Deletion of the YSIR sequence or replacement of G or S significantly reduced the rate of signal peptide processing of protein A precursors . In contrast, cell wall anchoring or the functional display of protein A was not affected . The fusion of cell wall sorting signals to reporter proteins bearing N-terminal signal peptides with or without the YSIRK-G/S motif resulted in hybrid proteins that were anchored in a manner similar to that of wild-type protein A . The requirement of the YSIRK-G/S motif for efficient secretion implies the existence of a specialized mode of substrate recognition by the secretion pathway of gram-positive cocci . It seems, however, that this mechanism is not essential for surface protein anchoring to the cell wall envelope . Rope-Producing Strains of Bacillus spp . from Wheat Bread and Strategy for Their Control by Lactic Acid Bacteria. Olimpia Pepe, 2003.Two types of white wheat bread (high- and low-type loaves) were investigated for rope spoilage . Thirty of the 56 breads tested developed rope spoilage within 5 days; the high-type loaves were affected by rope spoilage more than the low-type loaves . Sixty-one Bacillus strains were isolated from ropy breads and were characterized on the basis of their phenotypic and genotypic traits . All of the isolates were identified as Bacillus subtilis by biochemical tests, but molecular assays (randomly amplified polymorphic DNA PCR assay, denaturing gradient gel electrophoresis analysis, and sequencing of the V3 region of 16S ribosomal DNA) revealed greater Bacillus species variety in ropy breads . In fact, besides strains of B . subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus were also identified . All of the ropy Bacillus isolates exhibited amylase activity, whereas only 32.4% of these isolates were able to produce ropiness in bread slices after treatment at 96°C for 10 min . Strains of lactic acid bacteria previously isolated from sourdough were first selected for antirope activity on bread slices and then used as starters for bread-making experiments . Prevention of growth of approximately 104 rope-producing B . subtilis G1 spores per cm2 on bread slices for more than 15 days was observed when heat-treated cultures of Lactobacillus plantarum E5 and Leuconostoc mesenteroides A27 were added . Growth of B . subtilis G1 occurred after 7 days in breads started with Saccharomyces cerevisiae T22, L . plantarum E5, and L . mesenteroides A27 . Sequence Diversity and Functional Conservation of the Origin of Replication in Lactococcal Prolate Phages. Jasna Rakonjac, 2003.Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis . In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found . The nonconserved sequence was confined to the template for an untranslated transcript, PE1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved . All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant PE1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins . Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the PE1-T transcript is required for replication . This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages .
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