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Prevention and Cure of Systemic Escherichia coli K1 Infection by Modification of the Bacterial Phenotype. Naseem Mushtaq, 2004.Escherichia coli is a common cause of meningitis and sepsis in the newborn infant, and the large majority of isolates from these infections produce a polysialic acid (PSA) capsular polysaccharide, the K1 antigen, that protects the bacterial cell from immune attack . We determined whether a capsule-depolymerizing enzyme, by removing this protective barrier, could alter the outcome of systemic infection in an animal model . Bacteriophage-derived endosialidase E (endoE) selectively degrades the PSA capsule on the surface of E . coli K1 strains . Intraperitoneal administration of small quantities of recombinant endoE (20 µg) to 3-day-old rats, colonized with a virulent strain of K1, prevented bacteremia and death from systemic infection . The enzyme had no effect on the viability of E . coli strains but sensitized strains expressing PSA to killing by the complement system . This study demonstrates the potential therapeutic efficacy of agents that cure infections by modification of the bacterial phenotype rather than by killing or inhibition of growth of the pathogen . Temporal Transcription Map of the Virulent Streptococcus thermophilus Bacteriophage Sfi19. Marco Ventura, 2004. Functional Annotation of Class I Lysyl-tRNA Synthetase Phylogeny Indicates a Limited Role for Gene Transfer. Alexandre Ambrogelly, 2002.Functional and comparative genomic studies have previously shown that the essential protein lysyl-tRNA synthetase (LysRS) exists in two unrelated forms . Most prokaryotes and all eukaryotes contain a class II LysRS, whereas most archaea and a few bacteria contain a less common class I LysRS . In bacteria the class I LysRS is only found in the  -proteobacteria and a scattering of other groups, including the spirochetes, while the class I protein is by far the most common form of LysRS in archaea . To investigate this unusual distribution we functionally annotated a representative phylogenetic sampling of LysRS proteins . Class I LysRS proteins from a variety of bacteria and archaea were characterized in vitro by their ability to recognize Escherichia coli tRNALys anticodon mutants . Class I LysRS proteins were found to fall into two distinct groups, those that preferentially recognize the third anticodon nucleotide of tRNALys (U36) and those that recognize both the second and third positions (U35 and U36) . Strong recognition of U35 and U36 was confined to the pyrococcus-spirochete grouping within the archaeal branch of the class I LysRS phylogenetic tree, while U36 recognition was seen in other archaea and an example from the
-proteobacteria . Together with the corresponding phylogenetic relationships, these results suggest that despite its comparative rarity the distribution of class I LysRS conforms to the canonical archaeal-bacterial division . The only exception, suggested from both functional and phylogenetic data, appears to be the horizontal transfer of class I LysRS from a pyrococcal progenitor to a limited number of bacteria .
Transcriptional Switch On of ssgA by A-Factor, Which Is Essential for Spore Septum Formation in Streptomyces griseus. Haruka Yamazaki, 2003.A-factor (2-isocapryloyl-3R-hydroxymethyl-  -butyrolactone) triggers morphological development and secondary metabolism in Streptomyces griseus . A transcriptional activator (AdpA) in the A-factor regulatory cascade switches on a number of genes required for both processes . AdBS11 was identified in a library of the DNA fragments that are bound by AdpA and mapped upstream of ssgA, which is essential for septum formation in aerial hyphae . Gel mobility shift assays and DNase I footprinting revealed three AdpA-binding sites at nucleotide positions about -235 (site 1), -110 (site 2), and +60 (site 3) with respect to the transcriptional start point, p1, of ssgA . ssgA had two transcriptional start points, one starting at 124 nucleotides (p1) and the other starting at 79 nucleotides (p2) upstream of the start codon of ssgA . Of the three binding sites, only sites 1 and 2 were required for transcriptional activation of p1 and p2 by AdpA . The transcriptional switch on of ssgA required the extracytoplasmic function sigma factor,
 AdsA, in addition to AdpA . However, it was unlikely that
AdsA recognized the two ssgA promoters, since their -35 and -10 sequences were not similar to the promoter sequence motifs recognized by
BldN, a
AdsA homologue of Streptomyces coelicolor A3(2) . An ssgA disruptant formed aerial hyphae, but did not form spores, irrespective of the carbon source of the medium, which indicated that ssgA is a member of the whi genes . Transcriptional analysis of ssfR, located just upstream of ssgA and encoding an IclR-type transcriptional regulator, suggested that no read-through from ssfR into ssgA occurred, and ssgA was transcribed in the absence of ssfR . ssgA was thus found to be controlled by AdpA and not by SsfR to a detectable extent . SsfR appeared to regulate spore septum formation independently of SsgA or through interaction with SsgA in some unknown way, because an ssfR disruptant also showed a whi phenotype .
Viability of and Plasmid Retention in Frozen Recombinant Escherichia coli over Time: a Ten-Year Prospective Study. Gina L. Koenig, 2003.The long-term viability and plasmid retention of recombinant Escherichia coli strains were investigated by real-time testing of master cell banks (MCBs) stored at the Roche Molecular Systems Culture Collection (RMSCC) . MCBs at the RMSCC were cryogenically frozen and stored at -80°C for long-term preservation . At regular intervals during a period of 5 to more than 10 years, representative cryovials of each MCB were tested for viability and plasmid retention . Plasmid retention and viability for all 30 MCBs were stable over time . Twenty-seven MCBs maintained high levels of plasmid retention (at or near 100%), while three MCBs showed lower plasmid retention rates (ranging from 13.9 to 96.5%) that were consistent over time . New MCBs with high plasmid retention were created from two of the MCBs with lower plasmid retention by selective pressure with high levels of antibiotics . These new MCBs have shown stable viability and high plasmid retention over the first 5 months of storage . In conclusion, this study shows that properly selected, frozen and stored MCBs retain viability and maintain plasmid retention over time . Moreover, it is possible to recover cultures with high plasmid retention from MCBs with low plasmid retention by selecting clones grown in the presence of high levels of antibiotics .
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