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Complete Sequences of Six Penicillin-Binding Protein Genes from 40 Streptococcus pneumoniae Clinical Isolates Collected in Japan. Yumiko Sanbongi, 2004.All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S . pneumoniae isolates collected in Japan, were completely sequenced . The MICs of penicillin for these strains varied between 0.015 and 8 µg/ml . In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains . These six strains were highly resistant (MICs  2 µg/ml) to cefotaxime . The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains . In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 µg/ml) was the highest imipenem MIC detected . In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were
1 µg/ml . In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 µg/ml) was the highest cefotaxime MIC detected . On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed . The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S . pneumoniae resistance to ß-lactam antibiotics . Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with ß-lactam resistance .
Construction of an Integration-Proficient Vector Based on the Site-Specific Recombination Mechanism of Enterococcal Temperate Phage  FC1.Hee-Youn Yang, 2002.The genome of temperate phage FC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination . In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage
FC1 was constructed . A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1 . E . faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1 . Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E . faecalis KBL 707 chromosome . This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage
FC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector . The transformation efficiency of pEMJ1-1 was as high as 6 x 103 transformants/µg of DNA . In addition, a vector (pATTB1) containing the 290-bp attB region was constructed . pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences . The results indicate that the integration vector system based on the site-specific recombination mechanism of phage
FC1 can be used for genetic engineering in E . faecalis and in other hosts .
Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritella abyssi. Ying Xu, 2003.The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCaseMab), a new, strictly psychrophilic and piezophilic bacterial species, was purified . OTCaseMab displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes . In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state . The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof . OTCaseMab displays higher Km values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue  -N-phosphonoacetyl-L-ornithine (PALO) . OTCaseMab differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high Km values and the lower sensitivity to PALO . The Km for ornithine, however, is substantially lower at low temperatures . A survey of the catalytic efficiencies (kcat/Km) of OTCases adapted to different temperatures showed that OTCaseMab activity remains suboptimal at low temperature despite the 4.5-fold decrease in the Km value for ornithine observed when the temperature is brought from 20 to 5°C . OTCaseMab adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits .
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