@article {3129, title = {The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu.}, journal = {Nucleic Acids Res}, volume = {42}, year = {2014}, month = {2014 Jun}, pages = {6487-96}, abstract = {

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.

}, keywords = {Animals, Bacterial Proteins, Geobacter, Humans, Methylation, Mice, Nucleic Acid Conformation, RNA, Transfer, Asp, RNA, Transfer, Glu, Substrate Specificity, tRNA Methyltransferases}, issn = {1362-4962}, doi = {10.1093/nar/gku256}, author = {Shanmugam, Raghuvaran and Aklujkar, Muktak and Sch{\"a}fer, Matthias and Reinhardt, Richard and Nickel, Olaf and Reuter, Gunter and Lovley, Derek R and Ehrenhofer-Murray, Ann and Nellen, Wolfgang and Ankri, Serge and Helm, Mark and Jurkowski, Tomasz P and Jeltsch, Albert} } @article {1412, title = {Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase from Pyrococcus sp. ST04.}, journal = {Acta Crystallogr D Biol Crystallogr}, volume = {70}, year = {2014}, month = {2014 Jun}, pages = {1659-68}, abstract = {

A novel maltose-forming \α-amylase (PSMA) was recently found in the hyperthermophilic archaeon Pyrococcus sp. ST04. This enzyme shows \<13\% amino-acid sequence identity to other known \α-amylases and displays a unique enzymatic property in that it hydrolyzes both \α-1,4-glucosidic and \α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 \Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1-341) with a typical GH57 family (\β/\α)7-barrel fold and a C-domain (amino acids 342-597) composed of \α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or \β-amylases. The structural features of this novel exo-type maltose-forming \α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

}, keywords = {Amylases, Catalytic Domain, Maltose, Models, Molecular, Mutagenesis, Site-Directed, Protein Conformation, Proteolysis, Pyrococcus, Substrate Specificity}, issn = {1399-0047}, doi = {10.1107/S1399004714006567}, author = {Park, Kwang-Hyun and Jung, Jong-Hyun and Park, Sung-Goo and Lee, Myeong-Eun and Holden, James F and Park, Cheon-Seok and Woo, Eui-Jeon} } @article {808, title = {Suppression of constitutive SOS expression by recA4162 (I298V) and recA4164 (L126V) requires UvrD and RecX in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {73}, year = {2009}, month = {2009 Jul}, pages = {226-39}, abstract = {Sensing DNA damage and initiation of genetic responses to repair DNA damage are critical to cell survival. In Escherichia coli, RecA polymerizes on ssDNA produced by DNA damage creating a RecA-DNA filament that interacts with the LexA repressor inducing the SOS response. RecA filament stability is negatively modulated by RecX and UvrD. recA730 (E38K) and recA4142 (F217Y) constitutively express the SOS response. recA4162 (I298V) and recA4164 (L126V) are intragenic suppressors of the constitutive SOS phenotype of recA730. Herein, it is shown that these suppressors are not allele specific and can suppress SOS(C) expression of recA730 and recA4142 in cis and in trans. recA4162 and recA4164 single mutants (and the recA730 and recA4142 derivatives) are Rec(+), UV(R) and are able to induce the SOS response after UV treatment like wild-type. UvrD and RecX are required for the suppression in two (recA730,4164 and recA4142,4162) of the four double mutants tested. To explain the data, one model suggests that recA(C) alleles promote SOS(C) expression by mimicking RecA filament structures that induce SOS and the suppressor alleles mimic RecA filament at end of SOS. UvrD and RecX are attracted to these latter structures to help dismantle or destabilize the RecA filament.}, keywords = {Alleles, DNA Helicases, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Mutation, Missense, Rec A Recombinases, SOS Response (Genetics), Substrate Specificity}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2009.06765.x}, author = {Long, Jarukit E and Renzette, Nicholas and Sandler, Steven J} } @article {481, title = {Genome-wide gene expression patterns and growth requirements suggest that Pelobacter carbinolicus reduces Fe(III) indirectly via sulfide production.}, journal = {Appl Environ Microbiol}, volume = {74}, year = {2008}, month = {2008 Jul}, pages = {4277-84}, abstract = {Although Pelobacter species are closely related to Geobacter species, recent studies suggested that Pelobacter carbinolicus may reduce Fe(III) via a different mechanism because it lacks the outer-surface c-type cytochromes that are required for Fe(III) reduction by Geobacter sulfurreducens. Investigation into the mechanisms for Fe(III) reduction demonstrated that P. carbinolicus had growth yields on both soluble and insoluble Fe(III) consistent with those of other Fe(III)-reducing bacteria. Comparison of whole-genome transcript levels during growth on Fe(III) versus fermentative growth demonstrated that the greatest apparent change in gene expression was an increase in transcript levels for four contiguous genes. These genes encode two putative periplasmic thioredoxins; a putative outer-membrane transport protein; and a putative NAD(FAD)-dependent dehydrogenase with homology to disulfide oxidoreductases in the N terminus, rhodanese (sulfurtransferase) in the center, and uncharacterized conserved proteins in the C terminus. Unlike G. sulfurreducens, transcript levels for cytochrome genes did not increase in P. carbinolicus during growth on Fe(III). P. carbinolicus could use sulfate as the sole source of sulfur during fermentative growth, but required elemental sulfur or sulfide for growth on Fe(III). The increased expression of genes potentially involved in sulfur reduction, coupled with the requirement for sulfur or sulfide during growth on Fe(III), suggests that P. carbinolicus reduces Fe(III) via an indirect mechanism in which (i) elemental sulfur is reduced to sulfide and (ii) the sulfide reduces Fe(III) with the regeneration of elemental sulfur. This contrasts with the direct reduction of Fe(III) that has been proposed for Geobacter species.}, keywords = {Acetoin, Cytochrome c Group, Deltaproteobacteria, Ethanol, Fermentation, Ferric Compounds, Gene Expression Profiling, Genome, Bacterial, Iron, Nitrilotriacetic Acid, Oligonucleotide Array Sequence Analysis, Oxidation-Reduction, Reverse Transcriptase Polymerase Chain Reaction, RNA, Bacterial, Substrate Specificity, Sulfides, Sulfur, Sulfur-Reducing Bacteria, Thioredoxins}, issn = {1098-5336}, doi = {10.1128/AEM.02901-07}, author = {Haveman, Shelley A and Didonato, Raymond J and Villanueva, Laura and Shelobolina, Evgenya S and Postier, Bradley L and Xu, Bo and Liu, Anna and Lovley, Derek R} } @article {394, title = {Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP.}, journal = {Mol Microbiol}, volume = {70}, year = {2008}, month = {2008 Nov}, pages = {1012-25}, abstract = {The ability to manipulate protein levels is useful for dissecting regulatory pathways, elucidating gene function and constructing synthetic biological circuits. We engineered an inducible protein degradation system for use in Bacillus subtilis based on Escherichia coli and Caulobacter crescentusssrA tags and SspB adaptors that deliver proteins to ClpXP for proteolysis. In this system, modified ssrA degradation tags are fused onto the 3{\textquoteright} end of the genes of interest. Unlike wild-type ssrA, these modified tags require the adaptor protein SspB to target tagged proteins for proteolysis. In the absence of SspB, the tagged proteins accumulate to near physiological levels. By inducing SspB expression from a regulated promoter, the tagged substrates are rapidly delivered to the B. subtilis ClpXP protease for degradation. We used this system to degrade the reporter GFP and several native B. subtilis proteins, including, the transcription factor ComA, two sporulation kinases (KinA, KinB) and the sporulation and chromosome partitioning protein Spo0J. We also used modified E. coli and C. crescentus ssrA tags to independently control the degradation of two different proteins in the same cell. These tools will be useful for studying biological processes in B. subtilis and can potentially be modified for use in other bacteria.}, keywords = {Alleles, Bacillus subtilis, Bacterial Proteins, Carrier Proteins, Caulobacter crescentus, DNA-Binding Proteins, Endopeptidase Clp, Escherichia coli, Escherichia coli Proteins, Gene Expression Regulation, Bacterial, Genetic Vectors, Green Fluorescent Proteins, Plasmids, Promoter Regions, Genetic, Protein Engineering, RNA, Bacterial, Substrate Specificity}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2008.06467.x}, author = {Griffith, Kevin L and Grossman, Alan D} } @article {1212, title = {Characterization of malate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum.}, journal = {Extremophiles}, volume = {11}, year = {2007}, month = {2007 Sep}, pages = {741-6}, abstract = {

Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K ( m ) values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80 degrees C and pH 8.0) were 15 and 86 microM, respectively, with specific activity as high as 470 U mg(-1). Activity decreased more than 90\% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90\% when malate and NAD(+) were used as substrates and was not detected with NADP(+). A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.

}, keywords = {Citric Acid Cycle, Hydrogen-Ion Concentration, Kinetics, Malate Dehydrogenase, Malates, Molecular Weight, NAD, NADP, Oxaloacetic Acid, Phylogeny, Protein Conformation, Protein Subunits, Pyrobaculum, Recombinant Proteins, Sequence Analysis, Protein, Substrate Specificity, Temperature}, issn = {1431-0651}, doi = {10.1007/s00792-007-0081-2}, author = {Yennaco, Lynda J and Hu, Yajing and Holden, James F} } @article {1213, title = {Citric acid cycle in the hyperthermophilic archaeon Pyrobaculum islandicum grown autotrophically, heterotrophically, and mixotrophically with acetate.}, journal = {J Bacteriol}, volume = {188}, year = {2006}, month = {2006 Jun}, pages = {4350-5}, abstract = {

The hyperthermophilic archaeon Pyrobaculum islandicum uses the citric acid cycle in the oxidative and reductive directions for heterotrophic and autotrophic growth, respectively, but the control of carbon flow is poorly understood. P. islandicum was grown at 95 degrees C autotrophically, heterotrophically, and mixotrophically with acetate, H2, and small amounts of yeast extract and with thiosulfate as the terminal electron acceptor. The autotrophic growth rates and maximum concentrations of cells were significantly lower than those in other media. The growth rates on H2 and 0.001\% yeast extract with and without 0.05\% acetate were the same, but the maximum concentration of cells was fourfold higher with acetate. There was no growth with acetate if 0.001\% yeast extract was not present, and addition of H2 to acetate-containing medium greatly increased the growth rates and maximum concentrations of cells. P. islandicum cultures assimilated 14C-labeled acetate in the presence of H2 and yeast extract with an efficiency of 55\%. The activities of 11 of 19 enzymes involved in the central metabolism of P. islandicum were regulated under the three different growth conditions. Pyruvate synthase and acetate:coenzyme A (CoA) ligase (ADP-forming) activities were detected only in heterotrophically grown cultures. Citrate synthase activity decreased in autotrophic and acetate-containing cultures compared to the activity in heterotrophic cultures. Acetylated citrate lyase, acetate:CoA ligase (AMP forming), and phosphoenolpyruvate carboxylase activities increased in autotrophic and acetate-containing cultures. Citrate lyase activity was higher than ATP citrate synthase activity in autotrophic cultures. These data suggest that citrate lyase and AMP-forming acetate:CoA ligase, but not ATP citrate synthase, work opposite citrate synthase to control the direction of carbon flow in the citric acid cycle.

}, keywords = {Acetates, Carbon Dioxide, Citric Acid, Citric Acid Cycle, Culture Media, Deuterium, Ligases, Lyases, Pyrobaculum, Pyruvate Synthase, Substrate Specificity}, issn = {0021-9193}, doi = {10.1128/JB.00138-06}, author = {Hu, Yajing and Holden, James F} } @article {525, title = {Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens.}, journal = {J Bacteriol}, volume = {188}, year = {2006}, month = {2006 Jan}, pages = {450-5}, abstract = {The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.}, keywords = {Culture Media, Dicarboxylic Acids, Fumarates, Geobacter, Molecular Sequence Data, Operon, Oxidation-Reduction, Recombinant Proteins, Substrate Specificity, Succinate Dehydrogenase, Succinic Acid}, issn = {0021-9193}, doi = {10.1128/JB.188.2.450-455.2006}, author = {Butler, Jessica E and Glaven, Richard H and Esteve-N{\'u}{\~n}ez, Abraham and N{\'u}{\~n}ez, Cinthia and Shelobolina, Evgenya S and Bond, Daniel R and Lovley, Derek R} } @article {866, title = {Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases.}, journal = {Appl Microbiol Biotechnol}, volume = {56}, year = {2001}, month = {2001 Jul}, pages = {131-6}, abstract = {Using organically synthesized hydroxyalkanoate coenzyme A thioesters, the activities of two short-chain polyhydroxalkanoate (PHA) synthases were investigated--Ralstonia eutropha PHA synthase (a type I PHA synthase) and Ectothiorhodospira shaposhnikovii PHA synthase (a type III synthase). The results indicate that the two synthases have similar activities towards most of the monomers tested. 3-Hydroxybutyryl CoA was found to be the most efficient substrate for both synthases. Changes in the side-chain length of the monomers affect monomer reactivity, with shortening of the side-chain length having the more severe effect. Hydrophobicity in the side chain appears to play an important role in the catalytic reaction. The configuration and the position of the hydroxyl group also affect the reactivity of a monomer. Monomers with the [S] configuration can not be recognized by either synthase. Moving the hydroxyl group from the beta carbon to the alpha carbon has a much more severe effect on the reactivity of the monomer than moving the hydroxyl group to the gamma carbon. The results demonstrate that the in vitro system can be used to prepare entirely novel polymers that may not be obtainable from living cells because of metabolic restrictions.}, keywords = {Acyl Coenzyme A, Acyltransferases, Structure-Activity Relationship, Substrate Specificity}, issn = {0175-7598}, author = {Zhang, S and Kamachi, M and Takagi, Y and Lenz, R W and Goodwin, S} } @article {1199, title = {Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosus.}, journal = {J Bacteriol}, volume = {183}, year = {2001}, month = {2001 Jan}, pages = {709-15}, abstract = {

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophilic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of pyruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thought to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The active form has a molecular mass of 690+/-20 kDa and is assumed to be octomeric, while approximately 30\% of the protein is purified as a large ( approximately 1.6 MDa) complex that is not active. The apparent K(m) values and catalytic efficiencies for the substrates pyruvate and ATP (at 80 degrees C, pH 8.4) were 0.11 mM and 1.43 x 10(4) mM(-1). s(-1) and 0.39 mM and 3.40 x 10(3) mM(-1) x s(-1), respectively. Maximal activity was measured at pH 9.0 (at 80 degrees C) and at 90 degrees C (at pH 8.4). The enzyme also catalyzed the reverse reaction, but the catalytic efficiency with PEP was very low [k(cat)/K(m) = 32 (mM. s(-1)]. In contrast to several other nucleotide-dependent enzymes from P. furiosus, PpsA has an absolute specificity for ATP as the phosphate-donating substrate. This is the first PpsA from a nonmethanogenic archaeon to be biochemically characterized. Its kinetic properties are consistent with a role in gluconeogenesis, although its relatively high cellular concentration ( approximately 5\% of the cytoplasmic protein) suggests an additional function possibly related to energy spilling. It is not known whether interconversion between the smaller, active and larger, inactive forms of the enzyme has any functional role.

}, keywords = {Adenosine Monophosphate, Adenosine Triphosphate, Gluconeogenesis, Hydrogen-Ion Concentration, Phosphates, Phosphoenolpyruvate, Phosphotransferases (Paired Acceptors), Pyrococcus furiosus, Pyruvic Acid, Substrate Specificity}, issn = {0021-9193}, doi = {10.1128/JB.183.2.709-715.2001}, author = {Hutchins, A M and Holden, J F and Adams, M W} } @article {830, title = {dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12.}, journal = {Mol Microbiol}, volume = {34}, year = {1999}, month = {1999 Oct}, pages = {91-101}, abstract = {PriA, PriB and PriC were originally discovered as proteins essential for the PhiX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec-), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA{\textquoteright}s role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the PhiX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2:kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the PhiX174 model, del(priB)302 and priC303:kan mutants have almost wild-type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2:kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2:kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2:kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2:kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild-type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.}, keywords = {Bacterial Proteins, Bacteriophage mu, Cell Division, DNA Replication, DNA, Bacterial, DNA-Binding Proteins, Escherichia coli, Escherichia coli Proteins, Mutation, Phenotype, Recombination, Genetic, Replication Protein A, SOS Response (Genetics), Substrate Specificity, Suppression, Genetic}, issn = {0950-382X}, author = {Sandler, S J and Marians, K J and Zavitz, K H and Coutu, J and Parent, M A and Clark, A J} } @article {869, title = {Extracellular degradation of medium chain length poly(beta-hydroxyalkanoates) by Comamonas sp.}, journal = {Int J Biol Macromol}, volume = {25}, year = {1999}, month = {1999 Jun-Jul}, pages = {135-43}, abstract = {The PHA-degrading isolate, strain P37C, was enriched from residential compost for its ability to hydrolyze the medium chain length PHA, poly(beta-hydroxyoctanoate) (PHO). It was subsequently found to grow on a wide range of PHAs, including both short chain length and medium chain length PHAs. The isolate was identified as belonging to the genus Comamonas. Strain P37C formed clear zones on poly(beta-hydroxybutyrate) (PHB), (PHO) and poly(beta-hydroxyphenylvalerate) (PHPV) overlay plates. PHA clear zone tubes were prepared using seven different kinds of PHAs, ranging from PHB with four-carbon repeating units, to poly(beta-hydroxyoctanoate-co-beta-hydroxyundecanoate) (PHOU) with 8- and 11-carbon repeating units. There was a direct correlation between PHA side chain length and rate of hydrolysis of the PHAs. A series of PHOUs containing varying percentages of unsaturated bonds were used to make a series of epoxidized PHOUs (PHOEs) with varying percentages of epoxy functions. Results of clear zone tube assays showed that these functionalized PHAs were all biodegradable by strain P37C, and there was no apparent correlation between rate of biodegradation and the proportion of functional groups in the PHAs. Biodegradability of these PHAs was verified using respirometry and enzyme assays. Cell-free supernatants containing activity toward PHAs were prepared, and strain P37C was shown to synthesize at least two distinct PHA depolymerases for the hydrolysis of SCL and MCL PHAs.}, keywords = {Carboxylic Ester Hydrolases, Gram-Negative Aerobic Rods and Cocci, Kinetics, Polyesters, Structure-Activity Relationship, Substrate Specificity}, issn = {0141-8130}, author = {Quinteros, R and Goodwin, S and Lenz, R W and Park, W H} } @article {870, title = {Extracellular polymerization of 3-hydroxyalkanoate monomers with the polymerase of Alcaligenes eutrophus.}, journal = {Int J Biol Macromol}, volume = {25}, year = {1999}, month = {1999 Jun-Jul}, pages = {55-60}, abstract = {Previous investigations on the role of the polymerase in the synthesis of poly-3-hydroxybutyrate (PHB) are reviewed, and the results from earlier in vitro studies on the activity and selectivity of the polymerase of Alcaligenes eutrophus are discussed. In the present study the effect of glycerol on stabilizing the polymerase after purification and on eliminating the lag phase in in vitro polymerization reactions of 3-hydroxybutyl CoA (HBCoA), and 3-hydroxyvaleryl CoA (HVCoA) are described. K(M) values were determined for the activity of the polymerase with both HBCoA and HVCoA, and the rates of propagation for both monomers were estimated. With a racemic mixture of HBCoA, the enzyme polymerized only the [R] monomer.}, keywords = {Acyl Coenzyme A, Acyltransferases, Alcaligenes, Hydroxybutyrates, Kinetics, Polyesters, Substrate Specificity}, issn = {0141-8130}, author = {Lenz, R W and Farcet, C and Dijkstra, P J and Goodwin, S and Zhang, S} } @article {627, title = {Dissimilatory arsenate and sulfate reduction in Desulfotomaculum auripigmentum sp. nov.}, journal = {Arch Microbiol}, volume = {168}, year = {1997}, month = {1997 Nov}, pages = {380-8}, abstract = {A newly discovered arsenate-reducing bacterium, strain OREX-4, differed significantly from strains MIT-13 and SES-3, the previously described arsenate-reducing isolates, which grew on nitrate but not on sulfate. In contrast, strain OREX-4 did not respire nitrate but grew on lactate, with either arsenate or sulfate serving as the electron acceptor, and even preferred arsenate. Both arsenate and sulfate reduction were inhibited by molybdate. Strain OREX-4, a gram-positive bacterium with a hexagonal S-layer on its cell wall, metabolized compounds commonly used by sulfate reducers. Scorodite (FeAsO42. H2O) an arsenate-containing mineral, provided micromolar concentrations of arsenate that supported cell growth. Physiologically and phylogenetically, strain OREX-4 was far-removed from strains MIT-13 and SES-3: strain OREX-4 grew on different electron donors and electron acceptors, and fell within the gram-positive group of the Bacteria, whereas MIT-13 and SES-3 fell together in the epsilon-subdivision of the Proteobacteria. Together, these results suggest that organisms spread among diverse bacterial phyla can use arsenate as a terminal electron acceptor, and that dissimilatory arsenate reduction might occur in the sulfidogenic zone at arsenate concentrations of environmental interest. 16S rRNA sequence analysis indicated that strain OREX-4 is a new species of the genus Desulfotomaculum, and accordingly, the name Desulfotomaculum auripigmentum is proposed.}, keywords = {Arsenates, Bacteria, Anaerobic, Biotransformation, Geologic Sediments, Gram-Positive Endospore-Forming Rods, Molecular Sequence Data, Oxidation-Reduction, Phylogeny, RNA, Ribosomal, 16S, Substrate Specificity, Sulfates, Sulfides, Sulfur-Reducing Bacteria, Water Microbiology}, issn = {0302-8933}, author = {Newman, D K and Kennedy, E K and Coates, J D and Ahmann, D and Ellis, D J and Lovley, D R and Morel, F M} } @article {670, title = {Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate.}, journal = {Appl Environ Microbiol}, volume = {48}, year = {1984}, month = {1984 Jul}, pages = {81-7}, abstract = {Methanogenic bacteria with a coccobacillus morphology similar to Methanobrevibacter ruminantium were isolated from the bovine rumen. One isolate, 10-16B, represented a previously undescribed rumen population that, unlike M. ruminantium, synthesized coenzyme M, grew rapidly (mu = 0.24 h-1) on H2-CO2 in a complex medium, had simple nutritional requirements, and metabolized formate at reported rumen concentrations. H2 was metabolized to partial pressures 10-fold lower than those reported for the rumen. After H2 starvation for 26 h, strain 10-16B rapidly resumed growth when H2 was made available. The minimum concentrations of acetate (6 mM) and ammonia (less than 7 mM) that were required for optimal growth were lower than the reported acetate and ammonia concentrations in the rumen. Isoleucine and leucine stimulated growth, but only at concentrations (greater than 50 microM) higher than those reported for the rumen. Another coccobacillary methanogenic organism that synthesized coenzyme M was isolated from a different animal as were organisms that required an exogenous supply of coenzyme M. In general, methanogenic bacteria that required an exogenous supply of coenzyme M had lower maximum growth rates and more complex nutritional requirements than organisms that synthesized the cofactor. The ability of all isolates to metabolize formate below the detection limit of 10 microM indicated that, in contrast to previous reports, methanogenic bacteria have the potential to directly metabolize formate in the rumen. This study demonstrated that there are physiologically diverse populations of coccobacillary methanogenic bacteria in the rumen that can interact competitively and cooperatively.}, keywords = {Animals, Cattle, Culture Media, Enzyme Induction, Euryarchaeota, Formates, Hydrogen, Male, Mercaptoethanol, Mesna, Rumen, Substrate Specificity}, issn = {0099-2240}, author = {Lovley, D R and Greening, R C and Ferry, J G} }