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Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE, a Homolog of the pulE (gspE) Type II Protein Secretion Gene. Thomas J. DiChristina, 2002.Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)] . Previous studies demonstrated that a 23.3-kb S . putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T . DiChristina and E . DeLong, J . Bacteriol . 176:1468–1474, 1994) . In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems . Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors . Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems . A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S . putrefaciens) MR-1 . A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer+) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer-) B31 transconjugates . Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction . These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S . putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV) . Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids. Caio M. M. Cordova, 2002.Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis . The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools . In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene . The predicted M . pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 x 10-6 transformants/CFU/µg of plasmid DNA . However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region . Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M . pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them . An internal fragment of the M . pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M . pulmonis . Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented . These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis . Heat Shock Proteome of Agrobacterium tumefaciens: Evidence for New Control Systems. Ran Rosen, 2002.The regulation of Agrobacterium tumefaciens heat shock genes involves a transcriptional activator (RpoH) and repressor elements (HrcA-CIRCE) . Using proteome analysis and mutants in these control elements, we show that the heat shock induction of 32 (out of 56) heat shock proteins is independent of RpoH and HrcA . These results indicate the existence of additional regulatory factors in the A . tumefaciens heat shock response . Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae. Jung-Kul Lee, 2003.Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138) . In this study, NAD(P)H-dependent MDH was purified to homogeneity from C . magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography . The relative molecular masses of C . magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer . This enzyme catalyzed both fructose reduction and mannitol oxidation . The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively . C . magnoliae MDH showed high substrate specificity and high catalytic efficiency (kcat = 823 s-1, Km = 28.0 mM, and kcat/Km = 29.4 mM-1 s-1) for fructose, which may explain the high mannitol production observed in this strain . Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C . magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR) . The internal amino acid sequences of C . magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C . magnoliae MDH is an NAD(P)H-dependent tetrameric SDR . Although MDHs have been purified and characterized from several other sources, C . magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C . magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants .
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