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Telomere Exchange between Linear Replicons of Borrelia burgdorferi. Wai Mun Huang, 2004.Spirochetes in the genus Borrelia carry a linear chromosome and numerous linear plasmids that have covalently closed hairpin telomeres . The overall organization of the large chromosome of Borrelia burgdorferi appears to have been quite stable over recent evolutionary time; however, a large fraction of natural isolates carry differing lengths of DNA that extend the right end of the chromosome between about 7 and 20 kbp relative to the shortest chromosomes . We present evidence here that a rather recent nonhomologous recombination event in the B . burgdorferi strain Sh-2-82 lineage has replaced its right chromosomal telomere with a large portion of the linear plasmid lp21, which is present in the strain B31 lineage . At least two successive rounds of addition of linear plasmid genetic material to the chromosomal right end appear to have occurred at the Sh-2-82 right telomere, suggesting that this is an evolutionary mechanism by which plasmid genetic material can become part of the chromosome . The unusual nonhomologous nature of this rearrangement suggests that, barring horizontal transfer, it can be used as a unique genetic marker for this lineage of B . burgdorferi chromosomes . Azole Resistance in Candida glabrata: Coordinate Upregulation of Multidrug Transporters and Evidence for a Pdr1-Like Transcription Factor. John-Paul Vermitsky, 2004.Candida glabrata has emerged as a common cause of fungal infection . This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented . Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target . Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C . glabrata these include CDR1 and PDH1 . By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6- to 15-fold upregulation of CDR1 compared to susceptible strains . In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three- to fivefold, while ERG11 expression was minimally changed . Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants) . Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2- to 20-fold . Effects of antifungal treatment on gene expression in susceptible C . glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4- to 12-fold . These findings suggest that these transporter genes are regulated by a common mechanism . In support of this, a mutation associated with laboratory resistance was identified in the C . glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae . The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation . Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1 . Contribution of Membrane-Binding and Enzymatic Domains of Penicillin Binding Protein 5 to Maintenance of Uniform Cellular Morphology of Escherichia coli. David E. Nelson, 2002.Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD) . However, only one, PBP 5, has a demonstrated physiological function . In its absence, certain mutants of E . coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells . To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5 . Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD . However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound . Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required . A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal ß-sheet domain of PBP 6 . In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional . The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core . Functional Characterization of Cysteine Residues in GlpT, the Glycerol 3-Phosphate Transporter of Escherichia coli. Mon-Chou Fann, 2003.In Escherichia coli, the GlpT transporter, a member of the major facilitator superfamily, moves external glycerol 3-phosphate (G3P) into the cytoplasm in exchange for cytoplasmic phosphate . Study of intact cells showed that both GlpT and HisGlpT, a variant with an N-terminal six-histidine tag, are inhibited (50% inhibitory concentration  35 µM) by the hydrophilic thiol-specific agent p-mercurichlorobenzosulfonate (PCMBS) in a substrate-protectable fashion; by contrast, two other thiol-directed probes, N-maleimidylpropionylbiocytin (MPB) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), have no effect . Use of variants in which the HisGlpT native cysteines are replaced individually by serine or glycine implicates Cys-176, on transmembrane helix 5 (TM5), as the major target for PCMBS . The inhibitor sensitivity of purified and reconstituted HisGlpT or its cysteine substitution derivatives was found to be consistent with the findings with intact cells, except that a partial response to PCMBS was found for the C176G mutant, suggesting the presence of a mixed population of both right-side-out (RSO) (resistant) and inside-out (ISO) (sensitive) orientations after reconstitution . To clarify this issue, we studied a derivative (P290C) in which the RSO molecules can be blocked independently due to an MPB-responsive cysteine in an extracellular loop . In this derivative, comparisons of variants with (P290C) and without (P290C/C176G) Cys-176 indicated that this residue shows substrate-protectable inhibition by PCMBS in the ISO orientation in proteoliposomes . Since PCMBS gains access to Cys-176 from both periplasmic and cytoplasmic surfaces of the protein (in intact cells and in a reconstituted ISO orientation, respectively) and since access is unavailable when the substrate is present, we propose that Cys-176 is located on the transport pathway and that TM5 has a role in lining this pathway .
Bifidobacterium longum Requires a Fructokinase (Frk; ATP:D-Fructose 6-Phosphotransferase, EC 2.7.1.4) for Fructose Catabolism. Cristina I. Caescu, 2004.Although the ability of Bifidobacterium spp . to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined . This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum . The B . longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag . The expressed enzyme was purified by affinity chromatography on a Co2+-based column, and the pH and temperature optima were determined . A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Kmfructose = 0.739 ± 0.18 mM and KmATP = 0.756 ± 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM) . It was also found that frk is inducible by fructose and is subject to glucose-mediated repression . Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose .
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