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Four Variants of the Citrobacter freundii AmpC-Type Cephalosporinases, Including Novel Enzymes CMY-14 and CMY-15, in a Proteus mirabilis Clone Widespread in Poland. El, 2004.Twenty-nine Proteus mirabilis isolates from 17 Polish hospitals were analyzed . The isolates were resistant to a variety of antimicrobials, and their patterns of resistance to ß-lactams resembled those of the constitutive class C cephalosporinase (AmpC) producers . Indeed, ß-lactamases with a pI of  9.0 were found in all of the isolates, and they were subsequently identified as four AmpC-type cephalosporinases, CMY-4, -12, -14, and -15, of which the two last ones were novel enzyme variants . The enzymes were of Citrobacter freundii origin and were closely related to each other, with CMY-4 likely being the evolutionary precursor of the remaining ones . The blaCMY genes were located exclusively in chromosomal DNA, within EcoRI restriction fragments of the same size of
10 kb . In the CMY-12- and -15-producing isolates, an additional fragment of
4.5 kb hybridized with the blaCMY probe as well, which could have arisen from a duplication event during the evolution of the genes . In all of the isolates, the ISEcp1 mobile element, which most probably is involved in mobilization of the C . freundii ampC gene, was placed at the same distance from the 5' ends of the blaCMY genes, and sequences located between them were identical in isolates carrying each of the four genes . These data suggested that a single chromosome-to-chromosome transfer of the ampC gene from C . freundii to P . mirabilis could have initiated the spread and evolution of the AmpC-producing P . mirabilis in Poland . The hypothesis seems to be confirmed by pulsed-field gel electrophoresis typing, which revealed several cases of close relatedness between the P . mirabilis isolates from distant centers and showed an overall similarity between the majority of the multiresistant isolates .
An Internationally Spread Clone of Streptococcus pneumoniae Evolves from Low-Level to Higher-Level Penicillin Resistance by Uptake of Penicillin-Binding Protein Gene Fragments from Nonencapsulated Pneumococci. Christoph Hauser, 2004.Low-level penicillin resistance in an international Streptococcus pneumoniae serotype 19F clone emerging in Switzerland was characterized by mutations in the penicillin-binding protein PBP2x . Some isolates of this clone had evolved to higher resistance levels (penicillin MICs of 0.094 and 1 µg/ml), probably by acquisition of pbp2x fragments from local nonencapsulated pneumococci . Signal Sequence Mutations as Tools for the Characterization of LamB Folding Intermediates. Amy Rizzitello Duguay, 2002.lamBA23DA25Y and lamBA23YA25Y tether LamB to the inner membrane by blocking signal sequence processing . We isolated suppressors of lamBA23DA25Y and lamBA23YA25Y, all of which mapped within the LamB signal sequence . Most interesting were mutations that changed an amino acid with a strong positive charge to an amino acid with no charge . Further characterization of two such suppressors revealed that they produce functional LamB that is localized to the outer membrane with its entire signal sequence still attached . Biochemical analysis shows that mutant LamB monomer chases into an oligomeric species with properties different from those of wild-type LamB trimer . Because assembly of mutant LamB is slowed, these mutations provide useful tools for the characterization of LamB folding intermediates . TraG-Like Proteins of Type IV Secretion Systems: Functional Dissection of the Multiple Activities of TraG (RP4) and TrwB (R388). Gunnar Schröder, 2003.TraG-like proteins are essential components of type IV secretion systems . During secretion, TraG is thought to translocate defined substrates through the inner cell membrane . The energy for this transport is presumably delivered by its potential nucleotide hydrolase (NTPase) activity . TraG of conjugative plasmid RP4 is a membrane-anchored oligomer that binds RP4 relaxase and DNA . TrwB (R388) is a hexameric TraG-like protein that binds ATP . Both proteins, however, lack NTPase activity under in vitro conditions . We characterized derivatives of TraG and TrwB truncated by the N-terminal membrane anchor (TraG  2 and TrwB1) and/or containing a point mutation at the putative nucleotide-binding site (TraG2K187T and TraGK187T) . Unlike TraG and TrwB, truncated derivatives behaved as monomers without the tendency to form oligomers or aggregates . Surface plasmon resonance analysis with immobilized relaxase showed that mutant TraGK187T was as good a binding partner as the wild-type protein, whereas truncated TraG monomers were unable to bind relaxase . TraG2 and TrwB1 bound ATP and, with similar affinity, ADP . Binding of ATP and ADP was strongly inhibited by the presence of Mg2+ or single-stranded DNA and was competed for by other nucleotides . Compared to the activity of TraG2, the ATP- and ADP-binding activity of the point mutation derivative TraG2K187T was significantly reduced . Each TraG derivative bound DNA with an affinity similar to that of the native protein . DNA binding was inhibited or competed for by ATP, ADP, and, most prominently, Mg2+ . Thus, both nucleotide binding and DNA binding were sensitive to Mg2+ and were competitive with respect to each other .
Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid. Ryoji Mitsui, 2003.During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase . When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced . These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde . To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B . cepacia TM1 . The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain . Incorporation of [14C]formaldehyde into the cell constituents was increased by overexpression of the genes . Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain . These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde . This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria .
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