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Relevance of Peptide Uptake Systems to the Physiology and Virulence of Streptococcus agalactiae. Ulrike Samen, 2004.Streptococcus agalactiae is a major cause of invasive infections in human newborns . To satisfy its growth requirements, S . agalactiae takes up 9 of the 20 proteinogenic amino acids from the environment. Defined S . agalactiae mutants in one or several of four putative peptide permease systems were constructed and tested for peptide uptake, growth in various media, and expression of virulence traits . Oligopeptide uptake by S . agalactiae was shown to be mediated by the ABC transporter OppA1-F, which possesses two substrate-binding proteins [OppA1 and OppA2] with overlapping substrate specificities . Dipeptides were found to be taken upin parallel by the oligopeptide permease OppA1-F, by the dipeptideABC transporter DppA-E, and by the dipeptide symporter DpsA.Reverse transcription-PCR analysis revealed a polycistronicorganization of the genes oppA1-F and dppA-E and a monocistronic organization of dpsA in S . agalactiae . The results of quantitativereal-time PCR revealed a medium-dependent expression of theoperons dppA-E and oppA1-F in S . agalactiae . Growth of S . agalactiaein human amniotic fluid was shown to require an intact dpsAgene, indicating an important role of DpsA during the infectionof the amniotic cavity by S . agalactiae . Deletion of the oppBgene reduced the adherence of S . agalactiae to epithelial cellsby 26%, impaired its adherence to fibrinogen and fibronectinby 42 and 33%, respectively, and caused a 35% reduction in expressionof the fbsA gene, which encodes a fibrinogen-binding proteinin S . agalactiae . These data indicate that the oligopeptidepermease is involved in modulating virulence traits and virulencegene expression in S . agalactiae. Altering Toluene 4-Monooxygenase by Active-Site Engineering for the Synthesis of 3-Methoxycatechol, Methoxyhydroquinone, and Methylhydroquinone. Ying Tao, 2004.Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene . In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%) . Apparent Vmax values of 6.6 ± 0.9 to 10.7 ± 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 ± 0.8 nmol/min/mg of protein) . After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K . A . Canada, S . Iwashita, H . Shim, and T . K . Wood, J . Bacteriol . 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K . H . Mitchell, J . M . Studts, and B . G . Fox, Biochemistry 41:3176-3188, 2002) . By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 ± 0.2 versus 0.21 ± 0.01 nmol/min/mg of protein) . Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product . Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and produced methylhydroquinone (80%) from o-cresol . Hence, the regiospecific oxidation of o-methoxyphenol and o-cresol was changed for significant synthesis of 3-methoxycatechol, methoxyhydroquinone, 3-methylcatechol, and methylhydroquinone . The enzyme variants also demonstrated altered monohydroxylation regiospecificity for toluene; for example, G103S/A107G formed 82% o-cresol, so saturation mutagenesis converted T4MO into an ortho-hydroxylating enzyme . Furthermore, G103S/A107T formed 100% p-cresol from toluene; hence, a better para-hydroxylating enzyme than wild-type T4MO was formed . Structure homology modeling suggested that hydrogen bonding interactions of the hydroxyl groups of altered residues S103, S107, and T107 influence the regiospecificity of the oxygenase reaction . Rusty, Jammed, and Well-Oiled Hinges: Mutations Affecting the Interdomain Region of FliG, a Rotor Element of the Escherichia coli Flagellar Motor. Susan M. Van Way, 2004.The FliG protein is a central component of the bacterial flagellar motor . It is one of the first proteins added during assembly of the flagellar basal body, and there are 26 copies per motor . FliG interacts directly with the Mot protein complex of the stator to generate torque, and it is a crucial player in switching the direction of flagellar rotation from clockwise (CW) to counterclockwise and vice versa . A primarily helical linker joins the N-terminal assembly domain of FliG, which is firmly attached to the FliF protein of the MS ring of the basal body, to the motility domain that interacts with MotA/MotB . We report here the results of a mutagenic analysis focused on what has been called the hinge region of the linker . Residue substitutions in this region generate a diversity of phenotypes, including motors that are strongly CW biased, infrequent switchers, rapid switchers, and transiently or permanently paused . Isolation of these mutants was facilitated by a "sensitizing" mutation (E232G) outside of the hinge region that was accidentally introduced during cloning of the chromosomal fliG gene into our vector plasmid . This mutation partially interferes with flagellar assembly and accentuates the defects associated with mutations that by themselves have little phenotypic consequence . The effects of these mutations are analyzed in the context of a conformational-coupling model for motor switching and with respect to the structure of the C-terminal 70% of FliG from Thermotoga maritima .
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