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The RpoS Sigma Factor in the Dissimilatory Fe(III)-Reducing Bacterium Geobacter sulfurreducens. Cinthia Núñez, 2004.Geobacter sulfurreducens RpoS sigma factor was shown to contribute to survival in stationary phase and upon oxygen exposure . Furthermore, a mutation in rpoS decreased the rate of reduction of insoluble Fe(III) but not of soluble forms of iron . This study suggests that RpoS plays a role in regulating metabolism of Geobacter under suboptimal conditions in subsurface environments . Carbonic Anhydrase Is Essential for Growth of Ralstonia eutropha at Ambient CO2 Concentrations. Bernhard Kusian, 2002.Mutant strain 25-1 of the facultative chemoautotroph Ralstonia eutropha H16 had previously been shown to exhibit an obligately high-CO2-requiring (HCR) phenotype . Although the requirement varied with the carbon and energy sources utilized, none of these conditions allowed growth at the air concentration of CO2 . In the present study, a gene designated can and encoding a ß-carbonic anhydrase (CA) was identified as the site altered in strain 25-1 . The mutation caused a replacement of the highly conserved glycine residue 98 by aspartate in Can . A can deletion introduced into wild-type strain H16 generated mutant HB1, which showed the same HCR phenotype as mutant 25-1 . Overexpression of can in Escherichia coli and mass spectrometric determination of CA activity demonstrated that can encodes a functional CA . The enzyme is inhibited by ethoxyzolamide and requires 40 mM MgSO4 for maximal activity . Low but significant CA activities were detected in wild-type H16 but not in mutant HB1, strongly suggesting that the CA activity of Can is essential for growth of the wild type in the presence of low CO2 concentrations . The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO2 concentrations requires sufficient CA activity rather than the specific function of Can . The metabolic function(s) depending on CA activity remains to be identified .
 Integrase Complementation at the Level of DNA Binding and Complex Formation.Simone E. Nunes-Düby, 2002.Site-specific recombinases of the Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites . Each protomer may cleave its own site as a monomer in cis (as for Cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for Flp recombinase) . The crystal structure of the
Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on
integrase (Int) generated data that were not in agreement . Support for cis and trans cleavage came from assays with bispecific DNA substrates for
and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively . The data presented here do not provide new evidence for cis cleavage, but they strongly suggest that the previously described complementation results cannot be used in support of a trans-cleavage mechanism . We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-order attL intasome structures . The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions . Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present . This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis . Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (in cis) by mutant
Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the
Int mutants .
The Escherichia coli Methyl-Directed Mismatch Repair System Repairs Base Pairs Containing Oxidative Lesions. Jennifer Wyrzykowski, 2003.
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