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Participation of fad and mbt Genes in Synthesis of Mycobactin in Mycobacterium smegmatis. B. Babbette D. LaMarca, 2004.Colonies of Mycobacterium smegmatis LR222 on iron-limiting (0.1 µM Fe) minimal medium agar fluoresce under UV light due to the accumulation in the cells of the deferri form of the siderophore mycobactin . Two mutants with little or no fluorescence, designated LUN8 and LUN9, were isolated by screening colonies of transposon (Tn611)-mutagenized M . smegmatis . Ferrimycobactin prepared from iron-restricted cells of the wild type had an Rf of 0.62 on high-performance thin-layer chromatography (HPTLC) and a characteristic visible absorption spectrum with a peak near 450 nm . Similar extracts from LUN8 cells contained a small amount of ferrimycobactin with an Rf of 0.58 on HPTLC and an absorption spectrum with the peak shifted to a wavelength lower than that of the wild-type ferrimycobactin . Nuclear magnetic resonance spectroscopy studies suggested that the LUN8 mycobactin may have an altered fatty acid side chain . Mutant strain LUN9 produced no detectable mycobactin . Neither mutant strain produced measurable amounts of excreted mycobactin, although both excreted exochelin (the mycobacterial peptido-hydroxamate siderophore), and both mutants were more sensitive than the wild-type strain to growth inhibition by the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) . The transposon insertion sites were identified, and sequence analyses of the cloned flanking chromosome regions showed that the mutated gene in LUN9 was an orthologue of the Mycobacterium tuberculosis mycobactin biosynthetic gene mbtE . The mutated gene in LUN8 had homology with M . tuberculosis fadD33 (Rv1345), a gene that may encode an acyl-coenzyme A synthase and which previously was not known to participate in synthesis of mycobactin . Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export. Antonius J. A. van Maris, 2004.Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing . The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents . Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27) . Like all pyruvate decarboxylase-negative S . cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A . Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product . Ethanol formation was absent . However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen . In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type . Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast . This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export . The Active Partition Gene incC of IncP Plasmids Is Required for Stable Maintenance in a Broad Range of Hosts. Azeem Siddique, 2002.Plasmids of incompatibility group P (IncP) are capable of replication and stable inheritance in a wide variety of gram-negative bacteria . Three determinants of IncP plasmids are components of an active partition locus that is predicted to function in the segregation of plasmid copies to daughter cells . These determinants are incC, which codes for a member of the ParA family of partition ATPases; korB, which specifies a DNA-binding protein that also functions as a global transcriptional repressor; and OB, the DNA target for KorB, which occurs at multiple locations on IncP plasmids . To determine the importance and host range of the IncC/KorB partition system in the maintenance of IncP plasmids, we constructed an in-frame deletion of incC in the otherwise intact 60-kb IncP  plasmid R995 . R995 incC was found to be highly unstable in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, and Acinetobacter calcoaceticus, whereas wild-type R995 is stable in all these hosts . In addition, R995incC could not be established in Actinobacillus actinomycetemcomitans . trans-Complementation analysis showed that the coding region for IncC2 polypeptide, which is expressed from an internal translational start within the incC gene, was sufficient to restore stable maintenance to wild-type levels . The results show that the IncC/KorB active partition system of IncP plasmids is remarkably proficient for stable maintenance in diverse bacteria .
Diversity and Seasonal Variability of ß-Proteobacteria in Biofilms of Polluted Rivers: Analysis by Temperature Gradient Gel Electrophoresis and Cloning. I. H. M. Brümmer, 2003.The ß-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River . The bacterial 16S rRNA genes were cloned from each site, screened for ß-proteobacterial clones and sequenced . River biofilm clones from both rivers grouped into 9 clusters (RBFs) . RBFs 1, 2, and 3 fell into the recently described ßI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga. RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively . The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea . Seasonal changes in the relative intensity of the ß-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE) . Bands were identified by comparison of clones to community fingerprints by TGGE . Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants . The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally . They confirm the importance of certain known freshwater genera in river biofilms . The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters .
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