Manganese is the second most abundant transition metal found in the Earth's crust. I... more Manganese is the second most abundant transition metal found in the Earth's crust. It has a significant biological role as it is a cofactor of enzymes such as superoxide dismutase and is the key metal in the reaction center of photosystem II. In the environment, manganese is ...
The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts w... more The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) to position it for recognition of the -35 element of promoters. We report that this function depends on a hydrophobic patch on one face of the short stretch of alpha helix located at the tip of the flap domain, called the flap-tip helix. Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and sigma region 4, as determined by protease sensitivity assays, and impaired transcription from -35-dependent promoters. We suggest that contact of the flap-tip helix hydrophobic patch to the sigma region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4. This contact allowed region 4.2 recognition of the -35 promoter element and appeared to stabilize region 4 interaction with the beta' Zn(2+) binding domain. Our studies failed to detect any role for sigma region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel.
SummaryThe macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD... more SummaryThe macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma‐70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU—dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram‐negative species. Within the Gram‐negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, Ti, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy‐terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non‐coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis‐acting regulatory sequences appear to be conserved in diverse bacteria.
1.BackgroundWhile many species of bacteria have been identified that can convert soluble, reduced... more 1.BackgroundWhile many species of bacteria have been identified that can convert soluble, reduced manganese (Mn+2) into insoluble, oxidized Mn+4oxides, the mechanisms these bacteria employ and their distribution throughout the bacterial domain are less well understood. One of the best characterized MnOB is the gamma-proteobacteriumPseudomonas putidaGB-1, which uses three distinct proteins (PpMnxG, McoA and MopA) to oxidize Mn+2. The best characterized Mn oxidase enzyme is the MnxG homolog ofBacillussp. PL-12 (BaMnxG), which appears to be the only Mn oxidase in this species. MofA, found inLeptothrix discophorasp SS-1 is an additional putative Mn oxidase.ResultsBy querying publicly available databases of bacterial genome sequences for homologs to these Mn oxidase proteins, it was possible to determine the distribution of the proteins within bacteria. The overwhelming majority of homologs were found in just three phyla: proteobacteria, actinobacteria and firmicutes. These data do not p...
ABSTRACTMagnesium-dependent induction ofVibrio fischeriflagellar (Mif) biogenesis depends upon tw... more ABSTRACTMagnesium-dependent induction ofVibrio fischeriflagellar (Mif) biogenesis depends upon two diguanylate cyclases, suggesting an inhibitory role for cyclic di-GMP. Here, we report that cells defective for the sugar phosphotransferase system (PTS) exhibited a magnesium-independent phenotype similar to that of mutants of the Mif pathway. Unlike Mif mutants, PTS mutants also were hyperbioluminescent.
Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium... more Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB , respectively, for m agnesium-dependent i nduction of f lagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we intro...
Manganese is the second most abundant transition metal found in the Earth's crust. I... more Manganese is the second most abundant transition metal found in the Earth's crust. It has a significant biological role as it is a cofactor of enzymes such as superoxide dismutase and is the key metal in the reaction center of photosystem II. In the environment, manganese is ...
The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts w... more The Escherichia coli RNA polymerase beta subunit contains a flexible flap domain that interacts with region 4 of sigma(70) to position it for recognition of the -35 element of promoters. We report that this function depends on a hydrophobic patch on one face of the short stretch of alpha helix located at the tip of the flap domain, called the flap-tip helix. Disruption of the hydrophobic patch by the substitution of hydrophilic or charged amino acids resulted in a loss of the interaction between the flap and sigma region 4, as determined by protease sensitivity assays, and impaired transcription from -35-dependent promoters. We suggest that contact of the flap-tip helix hydrophobic patch to the sigma region 4 hydrophobic core is essential for stable interaction of the flap-tip helix with region 4. This contact allowed region 4.2 recognition of the -35 promoter element and appeared to stabilize region 4 interaction with the beta' Zn(2+) binding domain. Our studies failed to detect any role for sigma region 1.1 in establishing or maintaining the flap-sigma region 4 interaction, consistent with recent reports placing sigma region 1.1 in the downstream DNA channel.
SummaryThe macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD... more SummaryThe macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma‐70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU—dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram‐negative species. Within the Gram‐negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, Ti, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy‐terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non‐coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis‐acting regulatory sequences appear to be conserved in diverse bacteria.
1.BackgroundWhile many species of bacteria have been identified that can convert soluble, reduced... more 1.BackgroundWhile many species of bacteria have been identified that can convert soluble, reduced manganese (Mn+2) into insoluble, oxidized Mn+4oxides, the mechanisms these bacteria employ and their distribution throughout the bacterial domain are less well understood. One of the best characterized MnOB is the gamma-proteobacteriumPseudomonas putidaGB-1, which uses three distinct proteins (PpMnxG, McoA and MopA) to oxidize Mn+2. The best characterized Mn oxidase enzyme is the MnxG homolog ofBacillussp. PL-12 (BaMnxG), which appears to be the only Mn oxidase in this species. MofA, found inLeptothrix discophorasp SS-1 is an additional putative Mn oxidase.ResultsBy querying publicly available databases of bacterial genome sequences for homologs to these Mn oxidase proteins, it was possible to determine the distribution of the proteins within bacteria. The overwhelming majority of homologs were found in just three phyla: proteobacteria, actinobacteria and firmicutes. These data do not p...
ABSTRACTMagnesium-dependent induction ofVibrio fischeriflagellar (Mif) biogenesis depends upon tw... more ABSTRACTMagnesium-dependent induction ofVibrio fischeriflagellar (Mif) biogenesis depends upon two diguanylate cyclases, suggesting an inhibitory role for cyclic di-GMP. Here, we report that cells defective for the sugar phosphotransferase system (PTS) exhibited a magnesium-independent phenotype similar to that of mutants of the Mif pathway. Unlike Mif mutants, PTS mutants also were hyperbioluminescent.
Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium... more Flagellar biogenesis and hence motility of Vibrio fischeri depends upon the presence of magnesium. In the absence of magnesium, cells contain few or no flagella and are poorly motile or nonmotile. To dissect the mechanism by which this regulation occurs, we screened transposon insertion mutants for those that could migrate through soft agar medium lacking added magnesium. We identified mutants with insertions in two distinct genes, VF0989 and VFA0959, which we termed mifA and mifB , respectively, for m agnesium-dependent i nduction of f lagellation. Each gene encodes a predicted membrane-associated protein with diguanylate cyclase activity. Consistent with that activity, introduction into V. fischeri of medium-copy plasmids carrying these genes inhibited motility. Furthermore, multicopy expression of mifA induced other phenotypes known to be correlated with diguanylate cyclase activity, including cellulose biosynthesis and biofilm formation. To directly test their function, we intro...
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