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Regulation of the Intracellular Free Iron Pool by Dpr Provides Oxygen Tolerance to Streptococcus mutans. Yuji Yamamoto, 2004.Dpr is an iron-binding protein required for oxygen tolerancein Streptococcus mutans . We previously proposed that Dpr could confer oxygen tolerance to the bacterium by sequestering intracellular free iron ions that catalyze generation of highly toxic radicals [Y . Yamamoto, M . Higuchi, L . B . Poole, and Y . Kamio, J . Bacteriol. 182:3740-3747, 2000; Y . Yamamoto, L . B . Poole, R . R . Hantgan, and Y . Kamio, J . Bacteriol . 184:2931-2939, 2002] . Here, we examined the intracellular free iron status of wild-type [WT] and dpr mutant strains of S . mutans, before and after exposure to air, by using electron spin resonance spectrometry . Under anaerobic conditions, free iron ion concentrations of WT and dpr strains were 225.9 ± 2.6 and 333.0 ± 61.3 µM, respectively.Exposure of WT cells to air for 1 h induced Dpr expression andreduced intracellular free iron ion concentrations to 22.5 ±5.3 µM; under these conditions, dpr mutant cells maintainedintracellular iron concentration at 230.3 ± 28.8 µM.A decrease in cell viability and genomic DNA degradation wasobserved in the dpr mutant exposed to air . These data indicatethat regulation of the intracellular free iron pool by Dpr isrequired for oxygen tolerance in S . mutans. VanD-Type Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis. Florence Depardieu, 2004.Enterococcus faecium clinical isolates A902 and BM4538, which were resistant to relatively high levels of vancomycin (128 and 64 µg/ml, respectively) and to low levels of teicoplanin (4 µg/ml), and Enterococcus faecalis clinical isolates BM4539 and BM4540, which were resistant to moderate levels of vancomycin (16 µg/ml) and susceptible to teicoplanin (0.25 µg/ml), were studied . They were constitutively resistant by synthesis of peptidoglycan precursors ending with D-alanyl-D-lactate and harbored a chromosomal vanD gene cluster which was not transferable by conjugation to other enterococci . VanXD activity, which is not required in the absence of D-Ala-D-Ala, was low in the four strains, although none of the conserved residues was mutated; and the constitutive VanYD activity in the membrane fractions was inhibited by penicillin G . The mutations E13G in the region of D-alanine:D-alanine ligase (which is implicated in D-Ala1 binding in A902) and S319N of the serine involved in ATP binding in BM4538 and a 7-bp insertion at different locations in BM4539 and BM4540 (which led to putative truncated proteins) led to the production of an impaired enzyme and accounted for the lack of D-Ala-D-Ala-containing peptidoglycan precursors . The same 7-bp insertion in vanSD of BM4539 and BM4540 and a 1-bp deletion in vanSD of A902, which in each case led to a putative truncated and presumably nonfunctional protein, could account for the constitutive resistance . Strain BM4538, with a functional VanSD, had a G140E mutation in VanRD that could be responsible for constitutive glycopeptide resistance . This would represent the first example of constitutive van gene expression due to a mutation in the structural gene for a VanR transcriptional activator . Study of these four additional strains that could be distinguished on the basis of their various assortments of mutations confirmed that all VanD-type strains isolated so far have mutations in the ddl housekeeping gene and in the acquired vanSD or vanRD gene that lead to constitutive resistance to vancomycin . PII T-Loop Mutations Affecting Signal Transduction to NtrB Also Abolish Yeast Two-Hybrid Interactions. Isabel Martínez-Argudo, 2002.Mutations A49P and  47-53 at the T loop of the Escherichia coli GlnB (PII) protein impair regulatory interactions with the two-component sensor regulator NtrB (P . Jiang, P . Zucker, M . R . Atkinson, E . S . Kamberov, W . Tirasophon, P . Chandran, B . R . Schepke, and A . J . Ninfa, J . Bacteriol . 179: 4342-4353, 1997) . We show here that these mutations also impair interactions between PII and NtrB in the yeast two-hybrid system, indicating that defects in NtrB regulation closely reflect binding impairment . The reported results underline the strength of two-hybrid assays for analysis of interactions involving the T loop of PII proteins .
Contribution of Structural Genomics to Understanding the Biology of Escherichia coli. Allan Matte, 2003. Enterolysin A, a Cell Wall-Degrading Bacteriocin from Enterococcus faecalis LMG 2333. Trine Nilsen, 2003.A novel antimicrobial protein, designated enterolysin A, was purified from an Enterococcus faecalis LMG 2333 culture . Enterolysin A inhibits growth of selected enterococci, pediococci, lactococci, and lactobacilli . Antimicrobial activity was initially detected only on solid media, but by growing the bacteria in a fermentor under optimized production conditions (MRS broth with 4% [wt/vol] glucose, pH 6.5, and a temperature between 25 and 35°C), the bacteriocin activity was increased to 5,120 bacteriocin units ml-1 . Enterolysin A production was regulated by pH, and activity was first detected in the transition between the logarithmic and stationary growth phases . Killing of sensitive bacteria by enterolysin A showed a dose-response behavior, and the bacteriocin has a bacteriolytic mode of action . Enterolysin A was purified, and the primary structure was determined by combined amino acid and DNA sequencing . This bacteriocin is translated as a 343-amino-acid preprotein with an sec-dependent signal peptide of 27 amino acids, which is followed by a sequence corresponding to the N-terminal part of the purified protein . Mature enterolysin A consists of 316 amino acids and has a calculated molecular weight of 34,501, and the theoretical pI is 9.24 . The N terminus of enterolysin A is homologous to the catalytic domains of different cell wall-degrading proteins with modular structures . These include lysostaphin, ALE-1, zoocin A, and LytM, which are all endopeptidases belonging to the M37 protease family . The N-terminal part of enterolysin A is linked by a threonine-proline-rich region to a putative C-terminal recognition domain, which shows significant sequence identity to two bacteriophage lysins .
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