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Impaired Growth Rates in Milk of Lactobacillus helveticus Peptidase Mutants Can Be Overcome by Use of Amino Acid Supplements. Jeffrey E. Christensen, 2003.To evaluate the contribution of intracellular peptidases to the growth of the 14-amino-acid (aa) auxotroph Lactobacillus helveticus CNRZ32, single- and multiple-peptidase-deletion mutants were constructed . Two broad-specificity aminopeptidases (PepC and PepN) and X-prolyl dipeptidyl aminopeptidase (PepX) were inactivated through successive cycles of chromosomal gene replacement mutagenesis . The inactivation of all three peptidases in JLS247 (  pepC
pepN
pepX)
did not affect the growth rate in amino acid-defined medium . However,
the peptidase mutants generally had decreased specific growth rates
when acquisition of amino acids required hydrolysis of the proteins
in milk, the most significant result being a 73% increase in
generation time for JLS247 . The growth rate deficiencies in milk were
overcome by amino acid supplements with some specificity to each of
the peptidase mutants . For example, milk supplementation with Pro
resulted in the most significant growth rate increase for
pepX
strains and a 7-aa supplement (Asn, Cys, Ile, Pro, Ser, Thr, and Val)
resulted in a JLS247 growth rate indistinguishable from that of the
wild type . Our results show that characterization of the activities
of the broad-specificity aminopeptidases had little predictive
value regarding the amino acid supplements found to enhance the milk
growth rates of the peptidase mutant strains . These results represent
the first determination of the physiological roles with respect to
specific amino acid requirements for peptidase mutants grown in milk .
Multidrug Resistance of a Porin Deletion Mutant of Mycobacterium smegmatis. Joachim Stephan, 2004.Mycobacteria contain an outer membrane of unusually low permeability which contributes to their intrinsic resistance to many agents . It is assumed that small and hydrophilic antibiotics cross the outer membrane via porins, whereas hydrophobic antibiotics may diffuse through the membrane directly . A mutant of Mycobacterium smegmatis lacking the major porin MspA was used to examine the role of the porin pathway in antibiotic sensitivity . Deletion of the mspA gene caused high-level resistance of M . smegmatis to 256 µg of ampicillin/ml by increasing the MIC 16-fold . The permeation of cephaloridine in the mspA mutant was reduced ninefold, and the resistance increased eightfold . This established a clear relationship between the activity and the outer membrane permeation of cephaloridine . Surprisingly, the MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the mspA mutant were increased 2- to 10-fold . This is in contrast to those for Escherichia coli, whose sensitivity to these agents was not affected by deletion of porin genes . Uptake of the very hydrophobic steroid chenodeoxycholate by the mspA mutant was retarded threefold, which supports the hypothesis that loss of MspA indirectly reduces the permeability by the lipid pathway . The multidrug resistance of the mspA mutant highlights the prominent role of outer membrane permeability for the sensitivity of M . smegmatis to antibiotics . An understanding of the pathways across the outer membrane is essential to the successful design of chemotherapeutic agents with activities against mycobacteria . Effects of Phosphate and Light on Growth of and Bioactive Peptide Production by the Cyanobacterium Anabaena Strain 90 and Its Anabaenopeptilide Mutant. Sari Repka, 2004.Cyanobacteria synthesize several types of bioactive secondary metabolites . Anabaena strain 90 produces three types of bioactive peptides, microcystins (inhibitors of protein phosphatases 1 and 2A), anabaenopeptilides, and anabaenopeptins (serine protease inhibitors) . To investigate the role of the anabaenopeptilides in Anabaena, wild-type strain 90 (WT) and its anabaenopeptilide deficient mutant (MU) were cultured with various light and phosphate levels to evaluate the effects and coeffects of these growth factors on the concentrations of the three classes of peptides and the growth characteristics . WT and MU grew in comparable ways under the different growth conditions . The total peptide concentration in WT was significantly higher than that in MU (2.5 and 1.4 µg/mg [dry weight], respectively) . Interestingly, the average concentration of anabaenopeptins was significantly higher in MU than in WT (0.59 and 0.24 µg/mg [dry weight], respectively) . The concentration of microcystins was slightly but not statistically significantly higher in MU than in WT (1.0 and 0.86 µg/mg [dry weight], respectively) . In WT, the highest peptide concentrations were usually found after 13 days in cultures grown at medium light intensities (23 µmol m–2 s–1) and with the highest phosphate concentrations (2,600 µg liter–1) . In MU, the highest peptide concentrations were found in 13-day-old cultures grown at medium light intensities (23 µmol m–2 s–1) and with phosphate concentrations greater than 100 µg liter–1 . The higher concentrations of anabaenopeptins in MU may compensate for the absence of anabaenopeptilides . These findings clearly indicate that these compounds may have some linked function in the producer organism, the nature of which remains to be discovered . Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen. Yong Ge, 2002.Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates . The two components of this enzyme were hyperexpressed and anaerobically purified . Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe2S2 centers . Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism . TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 µM, kcat = 3.9 ± 0.2 s-1, and KmO2 = 16 ± 2 µM (100 mM sodium phosphate, pH 7.0; 25°C), where KmO2 represents the Km for O2 and KmA represents the Km for the aromatic substrate . The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate  3-chlorobenzoate > p-toluate
4-chlorobenzoate >> o-toluate
2-chlorobenzoate . The transformation of each of the first five compounds was well coupled to O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol . In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate . However, the apparent Km of TADO for these benzoates was >100 µM, indicating that they do not effectively inhibit the turnover of good substrates .
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