bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic evidence that outer membrane binding of starch is required for starch utilization by Bacteroides thetaiotaomicron
Authors: Anderson K L , Salyers A A
Published: November 2016

Mutagenesis of Bacteroides thetaiotaomicron with the transposon Tn4351 produced five classes of mutants that were not able to grow on amylose or amylopectin. These classes of mutants differed in their ability to grow on maltoheptaose (G7) and in the level of starch-degrading enzymes produced when bacteria were grown on maltose. All of the mutants were deficient in starch binding. Since one class of mutants retained normal levels of starch-degrading enzymes, this indicates that binding of the starch molecule by a cell surface receptor is necessary for starch utilization by B. thetaiotaomicron. Analysis of a starch-negative mutant that grew on G7 indicated that B. thetaiotaomicron possessed two starch-binding components or sites. One component (site A), apparently missing in this mutant, had an absolute preference for larger starch oligomers, whereas the other component (site M) also had a high affinity for maltodextrins (G4 through G7). Mutants not able to grow on maltodextrins (greater than G4) probably lacked both of these binding components. Only one class of mutants did not grow normally on maltose, but instead had a 4- to 5-h lag on maltose and a slower growth rate than the wild type. This class of mutants did not produce any of the starch-degrading enzymes or bind starch, even when growing on maltose. Such a phenotype probably resulted from transposon inactivation of a central regulatory gene or a gene encoding an enzyme that produces the inducer. The fact that both the degradative enzymes and the starch-binding activity were affected in this mutant indicates that genes encoding the cell surface starch-binding site are under the same regulatory control as genes encoding the enzymes.
Genetic and physiological relationships among the miaA gene, 2-methylthio-N6-(delta 2-isopentenyl)-adenosine tRNA modification, and spontaneous mutagenesis in Escherichia coli K-12
Authors: Connolly D M , Winkler M E
Published: November 2016

The miaA tRNA modification gene was cloned and located by insertion mutagenesis and DNA sequence analysis. The miaA gene product, tRNA delta 2-isopentenylpyrophosphate (IPP) transferase, catalyzes the first step in the biosynthesis of 2-methylthio-N6-(delta 2-isopentenyl)-adenosine (ms2i6A) adjacent to the anticodon of several tRNA species. The translation start of miaA was deduced by comparison with mod5, which encodes a homologous enzyme in yeasts. Minicell experiments showed that Escherichia coli IPP transferase has a molecular mass of 33.5 kilodaltons (kDa). Transcriptional fusions, plasmid and chromosomal cassette insertion mutations, and RNase T2 mapping of in vivo miaA transcription were used to examine the relationship between miaA and mutL, which encodes a polypeptide necessary for methyl-directed mismatch repair. The combined results showed that miaA, mutL, and a gene that encodes a 47-kDa polypeptide occur very close together, are transcribed in the same direction in the order 47-kDa polypeptide gene-mutL-miaA, and likely form a complex operon containing a weak internal promoter. Three additional relationships were demonstrated between mutagenesis and the miaA gene or ms2i6A tRNA modification. First, miaA transcription was induced by 2-aminopurine. Second, chromosomal miaA insertion mutations increased the spontaneous mutation frequency with a spectrum distinct from mutL mutations. Third, limitation of miaA+ bacteria for iron, which causes tRNA undermodification from ms2i6A to i6A, also increased spontaneous mutation frequency. These results support the notion that complex operons organize metabolically related genes whose primary functions appear to be completely different. In addition, the results are consistent with the idea that mechanisms exist to increase spontaneous mutation frequency when cells need to adapt to environmental stress.
Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli
Authors: Urbanowski M L , Stauffer G V
Published: November 2016

The metR-mediated activation of the Salmonella typhimurium metE and metH genes was shown to be modulated by homocysteine, an intermediate in the methionine biosynthetic pathway. Homocysteine stimulates expression of a metE-lacZ gene fusion four- to fivefold by increasing transcription from the metE promoter. In contrast, homocysteine plays an inhibitory role in the metR-mediated activation of the metH gene, decreasing expression of a metH-lacZ gene fusion threefold.
Cyanide inactivation of hydrogenase from Azotobacter vinelandii
Authors: Seefeldt L C , Arp D J
Published: November 2016

The effects of cyanide on membrane-associated and purified hydrogenase from Azotobacter vinelandii were characterized. Inactivation of hydrogenase by cyanide was dependent on the activity (oxidation) state of the enzyme. Active (reduced) hydrogenase showed no inactivation when treated with cyanide over several hours. Treatment of reversibly inactive (oxidized) states of both membrane-associated and purified hydrogenase, however, resulted in a time-dependent, irreversible loss of hydrogenase activity. The rate of cyanide inactivation was dependent on the cyanide concentration and was an apparent first-order process for purified enzyme (bimolecular rate constant, 23.1 M-1 min-1 for CN-). The rate of inactivation decreased with decreasing pH. [14C]cyanide remained associated with cyanide-inactivated hydrogenase after gel filtration chromatography, with a stoichiometry of 1.7 mol of cyanide bound per mol of inactive enzyme. The presence of saturating concentrations of CO had no effect on the rate or extent of cyanide inactivation of hydrogenases. The results indicate that cyanide can cause a time-dependent, irreversible inactivation of hydrogenase in the oxidized, activatable state but has no effect when hydrogenase is in the reduced, active state.
Catalytic and substrate-binding domains of endoglucanase 2 from Bacteroides succinogenes
Authors: McGavin M , Forsberg C W
Published: November 2016

Endoglucanase 2 (EG2) of the cellulolytic ruminal anaerobe Bacteroides succinogenes is a 118-kilodalton (kDa) enzyme which binds to cellulose and produces cellotetraose as the end product of hydrolysis. The purified enzyme was treated with the protease trypsin in an attempt to isolate peptides which retained the ability to either hydrolyze soluble carboxymethyl cellulose or bind to insoluble cellulose. There was no loss in endoglucanase activity (carboxymethylcellulase) over a period of 2 h following the addition of trypsin. In comparison, there was a greater than eightfold reduction in the binding of carboxymethylcellulase activity to crystalline cellulose. A Lineweaver-Burk plot with amorphous cellulose as the substrate revealed that the trypsin-digested enzyme had an identical Vmax but a 1.9-fold-lower Km in comparison with the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the trypsin-digested enzyme revealed two major peptides of 43 and 51 kDa (p43 and p51). The 43-kDa peptide was able to bind to both amorphous and crystalline cellulose, whereas p51 did not. Purified p51 had a molar activity toward carboxymethyl cellulose which was identical to that of the intact enzyme, but activity toward both amorphous and crystalline cellulose was reduced approximately twofold. Two high-titer monoclonal antibodies from mice immunized with the intact protein recognized p43 but not p51. The results are consistent with a bifunctional organization of EG2, in which the 118-kDa enzyme is composed of a 51-kDa catalytic domain and a highly antigenic 43-kDa substrate-binding domain. In terms of its domain structure and activity toward cellulose, EG2 is very similar to cellobiohydrolase II of Trichoderma reesei.
Isolation and characterization of the rRNA gene clusters of Halobacterium marismortui
Authors: Mevarech M , Hirsch-Twizer S , Goldman S , Yakobson E , Eisenberg H , Dennis P P
Published: November 2016

Two rRNA operons of Halobacterium marismortui were identified and cloned into plasmid pBR322 as 10- and 20-kilobase-pair (kbp) HindIII fragments, respectively. Restriction maps of the 10-kbp clone (pHH10) and an 8-kbp HindIII-ClaI subclone (pHC8) of the other operon were established. Southern hybridization of 16S, 23S, and 5S rRNA probes to the clones demonstrated that both operons code for the three rRNA species. By S1 nuclease analysis, the transcription initiation sites, some of the processing sites within the primary transcripts, and the boundaries of the mature 16S and 23S rRNA molecules were determined. Both operons are transcribed in vivo. Comparison of the two operons indicated that they are not identical. The most striking difference between the operons is the existence of three putative transcription initiation sites in one operon (HC8) and only one such site in the other operon (HH10). The regions surrounding these 5' transcript end sites share a high level of sequence similarity to each other and to the rRNA promoter regions of other halophilic archaebacteria. Analysis of the proximal 130 nucleotides of the two 16S rRNA genes indicated greater-than-expected sequence heterogeneity. There are a 2-base-pair insertion in the HC8 16S gene and 10 additional sites of nucleotide sequence heterogeneity.
DNA sequence and regulation of the gene (cbpA) encoding the 42-kilodalton cytoplasmic membrane carotenoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942
Authors: Reddy K J , Masamoto K , Sherman D M , Sherman L A
Published: November 2016

The gene (cbpA) coding for a carotenoid-binding protein of the cyanobacterium Synechococcus sp. strain PCC 7942 (Anacystis nidulans R2) has been cloned and sequenced. A polyclonal antibody against the protein was used to identify immunoreactive clones from a lambda gt11 expression library of Synechococcus strain PCC 7942. The initial positive clone (lambda gtAN42) contained a 0.9-kilobase (kb) chromosomal fragment, which was used to detect a larger chromosomal fragment from a lambda EMBL3 library. The lambda EMBL3 recombinant, lambda EM109, contained an 18-kb portion of the Synechococcus strain PCC 7942 chromosome. The open reading frame of cbpA encoded 450 amino acids which give rise to a protein of 49,113 daltons. The hydrophobicity plot indicates that the protein may have a 49-residue signal sequence which is cleaved to yield a mature protein of 43,709 daltons. The protein has been localized in the cytoplasmic membrane by biochemical procedures as well as by electron microscopic immunocytochemistry. Northern (RNA) blot analysis indicates that transcription of cbpA is tightly regulated by DNA topology, light intensity, and iron concentration. Transcription is greatly induced by growth under high light intensities and repressed during growth under iron-deficient conditions. The DNA gyrase inhibitor novobiocin specifically inhibited the light-induced transcription. In Northern blots, the gene-specific probe hybridized to two size classes of RNA, with lengths of 2.0 and 6.2 kb. Since cbpA appears to be a component of the 6.2-kb transcript, it is likely part of a larger operon.
Mapping of the Escherichia coli acid glucose-1-phosphatase gene agp and analysis of its expression in vivo by use of an agp-phoA protein fusion
Authors: Pradel E , Boquet P L
Published: November 2016

The agp gene of Escherichia coli encodes an acid glucose-1-phosphatase, one of the numerous phosphatases optimally active between pH 4 and 6 found in the periplasmic space of this bacterium. An agp-phoA protein fusion linked to a gene conferring kanamycin resistance was inserted into the chromosome in place of agp by homologous recombination and was mapped to minute 22.6. Because the activity of glucose-1-phosphatase cannot be measured accurately in whole cells, the alkaline phosphatase activity of the agp-phoA hybrid protein was used to monitor the expression of the chromosomal agp gene. The expression of agp was subject to catabolite repression but was unaffected by the concentration of inorganic phosphate in the growth medium. The product of the agp gene was required for growth on glucose-1-phosphate as the sole carbon source, a function for which alkaline phosphatase or other acid phosphatases cannot substitute.
Translational coupling in the threonine operon of Escherichia coli K-12
Authors: Little S , Hyde S , Campbell C J , Lilley R J , Robinson M K
Published: November 2016

In an attempt to express the two distal genes of the Escherichia coli threonine operon, the majority of the first gene in the operon, thrA, was removed and a series of transcriptional fusions were constructed placing the thrB and thrC genes downstream of either the trp or hybrid tac promoter. Analysis of the proteins produced by cells containing these fusions revealed that although the distal gene, thrC, was efficiently expressed, the proximal gene, thrB, was not expressed at a detectable level. A translational fusion was constructed which fused the cat gene in phase to the last 800 base pairs of thrA followed by thrB and thrC. Cells containing this fusion produced high levels of both the thrB and thrC gene products, showing that translation of thrB requires translation through thrA; thus, thrA and thrB are translationally coupled. In addition, it was found that a sequence between 220 and 57 base pairs before the start of thrB was necessary to allow translational coupling to occur.
Formation of crystals of the insecticidal proteins of Bacillus thuringiensis subsp. aizawai IPL7 in Escherichia coli
Authors: Oeda K , Inouye K , Ibuchi Y , Oshie K , Shimizu M , Nakamura K , Nishioka R , Takada Y , Ohkawa H
Published: November 2016

Escherichia coli JM103 cells harboring expression plasmid pTB1 or pKC6 synthesized the 130- and 135-kilodalton insecticidal proteins, respectively, of Bacillus thuringiensis subsp. aizawai IPL7, and both products accumulated as cytoplasmic inclusion bodies. Amorphous inclusions which contained contaminating proteins, together with the corresponding insecticidal proteins, were formed in cultures at 37 degrees C, but bipyramidal crystals practically free of contaminants were observed at 30 degrees C. Although 9.8% of the amino acids were substituted between these two proteins, both protein crystals had the same shape as those of the parental B. thuringiensis strain, which produced both proteins.
Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis
Authors: Hecht D W , Malamy M H
Published: November 2016

Conjugal transposons play an important role in the dissemination of antibiotic resistance determinants in the streptococci and have been postulated to exist in Bacteroides fragilis. To investigate the presence of conjugal transposons in B. fragilis, we employed a Tra- derivative of the transfer factor pBFTM10 contained in the chimeric plasmid pGAT400 delta BglII. We attempted to restore transferability to this plasmid from a series of transconjugants generated by crossing B. fragilis TMP230 containing the TET transfer factor with B. fragilis TM4000, a standard recipient. Transconjugant TM4.2321 transferred pGAT400 delta BglII to Escherichia coli HB101 at almost the same frequency as did the Tra+ parental plasmid, pGAT400. Analysis of the transferred plasmids revealed the presence of 9.6 kilobases of additional DNA in every case but at different positions in independent isolates. The presence of this DNA, designated Tn4399, allowed the pGAT400 delta BglII derivatives to retransfer from the TM4000 background to B. fragilis or E. coli recipients. DNA hybridization studies demonstrated the presence of one copy of Tn4399 in TMP230 and three copies at new sites in TM4.2321. Tn4399 is a new B. fragilis transposon with unique transfer properties that may play a role in the dissemination of drug resistance genes. It differs from previously described conjugal transposons by its ability to mobilize nonconjugal plasmids in cis.
Control of transducer methylation levels in Escherichia coli: investigation of components essential for modulation of methylation and demethylation reactions
Authors: Russell C B , Stewart R C , Dahlquist F W
Published: November 2016

During bacterial chemotaxis in Escherichia coli, adaptation is accomplished by reversible methylation of the transmembrane signal transducers. Methyl groups are added by the CheR protein in a slow response to attractants and removed by the CheB protein in response to repellents. The methylesterase activity of the CheB protein is modulated by a factor that is controlled in a global fashion throughout the cell. By controlling the level of expression of the cheR, cheB, and transducer genes with exogenous promoters on multicopy plasmids, we demonstrate that the modulating factor exists in stoichiometric concentrations relative to CheB protein and that the generation or efficacy of this factor requires the cheA and/or cheW gene products, suggesting that phosphorylation of the methylesterase by CheA may be involved in its global activation. We show that in the absence of any modulation of the CheB activity, the CheR methyltransferase activity is modulated in a local fashion at the transducers, most likely as a result of a conformational change in the transducer protein brought about by the binding of ligand, and does not require CheA or CheW.
Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation
Authors: Gupta S , Clark D P
Published: November 2016

Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance.
Klebsiella pneumoniae pulS gene encodes an outer membrane lipoprotein required for pullulanase secretion
Authors: D'Enfert C , Pugsley A P
Published: November 2016

The product of the Klebsiella pneumoniae gene pulS, which is located downstream from the pullulanase structural gene (pulA), is essential for the cell surface localization and extracellular release of pullulanase in Escherichia coli K-12. pulS is transcribed in the opposite direction to pulA, from which it is separated by a region of 624 nucleotides. Although this latter region contains a new component of the maltose regulon, pulB, which is transcribed from the pulA promoter, it is not required for pullulanase synthesis or secretion. Unlike pulA and all other pullulanase secretion genes characterized so far, the expression of pulS is not induced by growth in the presence of maltose and is unaffected by mutations in the maltose regulator gene malT. The pulS gene product was identified as a ca. 12-kilodalton outer membrane lipoprotein. The characterization of PulS brings to three the number of identified proteins which are known to be required for pullulanase secretion in addition to the components of the signal sequence-dependent general protein export pathway.
DnaA protein overproduction abolishes cell cycle specificity of DNA replication from oriC in Escherichia coli
Authors: Pierucci O , Rickert M , Helmstetter C E
Published: November 2016

Initiation of DNA replication from oriC in Escherichia coli takes place at a specific time in the cell division cycle, whether the origin is located on a chromosome or a minichromosome, and requires participation of the product of the dnaA gene. The effects of overproduction of DnaA protein on the cell cycle specificity of the initiation event were determined by using minichromosome replication as the assay system. DnaA protein was overproduced by inducing the expression of plasmid-encoded dnaA genes under control of either the ptac or lambda pL promoter. Induction of DnaA protein synthesis caused a burst of minichromosome replication in cells at all ages in the division cycle. The magnitude of the burst was consistent with the initiation of one round of replication per minichromosome in all cells. The replication burst was followed by a period of reduced minichromosome replication, with the reduction being greater at 30 than at 41 degrees C. The results support the idea that the DnaA protein participates in oriC replication at a stage that is limiting for initiation. Excess DnaA protein enabled all cells to achieve the state required for initiation of DNA polymerization by either effecting or overriding the normal limiting process.
Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus
Authors: Speelmans G , de Vrij W , Konings W N
Published: November 2016

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.
Identification and sequence analysis of the Bacillus subtilis W23 xylR gene and xyl operator
Authors: Kreuzer P , Gärtner D , Allmansberger R , Hillen W
Published: November 2016

The xyl operator of Bacillus subtilis W23 was identified by deletion analysis of the xyl regulatory region as a 25-base-pair (bp) sequence located 10 bp downstream from the xyl promoter. The outer 10 bp of the xyl operator exhibit perfect palindromic symmetry, while 5 central bp are nonpalindromic. It was demonstrated that the penultimate base pair near the end of this sequence is important for repressor binding. The location of the xylR gene encoding the repressor was determined by its ability to mediate xylose-dependent repression of a xyl-cat fusion on a multicopy plasmid. The nucleotide sequence of 1,355 bp from this DNA was analyzed and contains an open reading frame with a coding capacity for 384 amino acids leading to a protein with a calculated molecular weight of 42,270. A mutant with a deletion in this reading frame showed no repression of the xyl-cat fusion. The coding sequence is preceded by a suitable ribosome-binding sequence and uses GTG as a start codon and TAA as a stop codon. The relationship of these results to corresponding data obtained from B. subtilis 168 is discussed.
Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum
Authors: Baron S F , Ferry J G
Published: November 2016

The membrane-associated coenzyme F420-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained alpha, beta, and gamma subunits (molecular weights of 43,000, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F420-active alpha 1 beta 1 gamma 1 trimer (molecular weight, 109,000). The hydrogenase contained 1 mol of flavin adenine dinucleotide (FAD), 1 mol of nickel, 12 to 14 mol of iron, and 11 mol of acid-labile sulfide per mol of the 109,000-molecular-weight species, but no selenium. The isoelectric point was 5.6. The amino acid sequence I-N3-P-N2-R-N1-EGH-N6-V (where N is any amino acid) was conserved in the N-termini of the alpha subunits of the F420-hydrogenases from M. formicicum and Methanobacterium thermoautotrophicum and of the largest subunits of nickel-containing hydrogenases from Desulfovibrio baculatus, Desulfovibrio gigas, and Rhodobacter capsulatus. The purified F420-hydrogenase required reductive reactivation before assay. FAD dissociated from the enzyme during reactivation unless potassium salts were present, yielding deflavoenzyme that was unable to reduce coenzyme F420. Maximal coenzyme F420-reducing activity was obtained at 55 degrees C and pH 7.0 to 7.5, and with 0.2 to 0.8 M KCl in the reaction mixture. The enzyme catalyzed H2 production at a rate threefold lower than that for H2 uptake and reduced coenzyme F420, methyl viologen, flavins, and 7,8-didemethyl-8-hydroxy-5-deazariboflavin. Specific antiserum inhibited the coenzyme F420-dependent but not the methyl viologen-dependent activity of the purified enzyme.
L-, P-, and M-ring proteins of the flagellar basal body of Salmonella typhimurium: gene sequences and deduced protein sequences
Authors: Jones C J , Homma M , Macnab R M
Published: November 2016

The flgH, flgI, and fliF genes of Salmonella typhimurium encode the major proteins for the L, P, and M rings of the flagellar basal body. We have determined the sequences of these genes and the flgJ gene and examined the deduced amino acid sequences of their products. FlgH and FlgI, which are exported across the cell membrane to their destinations in the outer membrane and periplasmic space, respectively, both had typical N-terminal cleaved signal-peptide sequences. FlgH is predicted to have a considerable amount of beta-sheet structure, as has been noted for other outer membrane proteins. FlgI is predicted to have an even greater amount of beta-structure. FliF, as is usual for a cytoplasmic membrane protein of a procaryote, lacked a signal peptide; it is predicted to have considerable alpha-helical structure, including an N-terminal sequence that is likely to be membrane-spanning. However, it had overall a quite hydrophilic sequence with a high charge density, especially towards its C terminus. The flgJ gene, immediately adjacent to flgI and the last gene of the flgB operon, encodes a flagellar protein of unknown function whose deduced sequence was hydrophilic and may correspond to a cytoplasmic protein. Several aspects of the DNA sequence of these genes and their surrounds suggest complex regulation of the flagellar gene system. A notable example occurs within the flgB operon, where between the end of flgG (encoding the distal rod protein of the basal body) and the start of flgH (encoding the L-ring protein) there was an unusually long noncoding region containing a potential stem-loop sequence, which could attenuate termination of transcription or stabilize part of the transcript against degradation. Another example is the interface between the flgB and flgK operons, where transcription termination of the former may occur within the coding region of the latter.
A global response induced in Escherichia coli by redox-cycling agents overlaps with that induced by peroxide stress
Authors: Greenberg J T , Demple B
Published: November 2016

Escherichia coli treated with nontoxic levels of the superoxide-generating redox-cycling agents menadione and paraquat showed dramatic changes in protein composition as monitored by two-dimensional gel analysis. The distribution of proteins synthesized after treatment with these agents overlapped significantly with that seen after hydrogen peroxide treatment, and it included all the proteins in the oxyR regulon. The redox-cycling agents also elicited the synthesis of at least 33 other proteins that were not seen with hydrogen peroxide, including three heat shock proteins, the Mn-containing superoxide dismutase, the DNA repair protein endonuclease IV, and glucose-6-phosphate dehydrogenase. At least some of these redox-inducible proteins appear to be part of a specific response to intracellular superoxide. E. coli is thus equipped with a network of inducible responses against oxidative damage, controlled in multiple regulatory pathways.
Subcellular localization of the nodD gene product in Rhizobium leguminosarum
Authors: Schlaman H R , Spaink H P , Okker R J , Lugtenberg B J
Published: November 2016

In Rhizobium strains the transcription of symbiosis plasmid-localized nod genes, except nodD, is induced by plant flavonoids and requires the nodD gene product. In order to localize NodD protein in R. leguminosarum, a NodD protein-specific antiserum was raised against a lacZ'-'nodD gene fusion product. Using these antibodies, we determined that the NodD protein is located exclusively in the cytoplasmic membrane of wild-type R. leguminosarum biovar viciae cells. This localization is independent of the presence of inducers. In a Rhizobium strain that overproduced the NodD protein, the protein was present both in the cytoplasmic membrane and the cytosol, indicating an influence of the protein abundance on its ultimate subcellular localization. It was estimated that 20 to 80 molecules of NodD protein were present per wild-type Rhizobium cell. A model which combines the localization and the DNA-binding properties of the NodD protein as well as the observed association of flavonoids with the cytoplasmic membrane is discussed.
Isolation, nucleotide sequence, and preliminary characterization of the Escherichia coli K-12 hemA gene
Authors: Verkamp E , Chelm B K
Published: November 2016

The Escherichia coli hemA gene, essential for the synthesis of 5-aminolevulinic acid (ALA), was isolated and sequenced. The following criteria identified the cloned gene as hemA. (i) The gene complemented a hemA mutation of E. coli. (ii) The gene was localized to approximately 26.7 min on the E. coli chromosomal linkage map, consistent with the location of the mapped hemA locus. Furthermore, DNA sequence analysis established that the cloned gene lay directly upstream of prfA, which encodes polypeptide chain release factor 1. (iii) Deletion of the gene resulted in a concomitant requirement for ALA. The hemA gene directed the synthesis of a 46-kilodalton polypeptide in maxicell experiments, as predicted by the coding sequence. The DNA and deduced amino acid sequences of the E. coli hemA gene displayed no detectable similarity to the ALA synthase sequences which have been characterized from a variety of organisms, but are very similar to the cloned Salmonella typhimurium hemA sequences (T. Elliott, personal communication). Results of S1 nuclease protection experiments showed that the hemA mRNA appeared to have two different 5' ends and that a nonoverlapping divergent transcript was present upstream of the putative distal hemA transcriptional start site.
Region of the streptococcal plasmid pMV158 required for conjugative mobilization
Authors: Priebe S D , Lacks S A
Published: November 2016

The nonconjugative streptococcal plasmid pMV158 can be mobilized by the conjugative streptococcal plasmid pIP501. We determined the sequence of the 1.1-kilobase EcoRI fragment of pMV158 to complete the DNA sequence of the plasmid. We showed that an open reading frame, mob (able to encode a polypeptide of 58,020 daltons), is required for mobilization of pMV158. An intergenic region present in the EcoRI fragment contains four lengthy palindromes that are found also in one or more of the staphylococcal plasmids pT181, pE194, and pUB110. One palindromic sequence, palD, which is common to all four plasmids, also appeared to be necessary for mobilization. Circumstantial evidence indicates that this sequence contains both an oriT site and the mob promoter. The Mob protein is homologous in its amino-terminal half to Pre proteins encoded by pT181 and pE194 that were shown by others to be essential for site-specific cointegrative plasmid recombination; their main biological function may be plasmid mobilization.
Participation of the lytic replicon in bacteriophage P1 plasmid maintenance
Authors: Yarmolinsky M B , Hansen E B , Jafri S , Chattoraj D K
Published: November 2016

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.
An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis
Authors: Collis C M , Grigg G W
Published: November 2016

A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain.
Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA)
Authors: Loprasert S , Negoro S , Okada H
Published: November 2016

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.
Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA
Authors: Defais M , Lesca C , Monsarrat B , Hanawalt P
Published: November 2016

Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M: Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as lambda, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage lambda in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of lambda phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, lambda phage replication seems to switch prematurely from the theta mode to the rolling circle mode.
Cloning, nucleotide sequence, and hybridization studies of the type IIb heat-labile enterotoxin gene of Escherichia coli
Authors: Pickett C L , Twiddy E M , Coker C , Holmes R K
Published: November 2016

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.
Dual mechanism for stimulation of glutamate transport by potassium ions in Streptococcus mutans
Authors: Sato Y , Noji S , Suzuki R , Taniguchi S
Published: November 2016

An ATP-driven primary transport system operative for L-glutamate or L-aspartate in Streptococcus mutans is, through the entire pH range from 5.5 to 8.5, specifically stimulated by extracellular potassium ions. The stimulation by potassium ions observed in the low pH range between 5.5 and 7 has been interpreted to be due to potassium ion-dependent regulation of the intracellular pH (the first mechanism). In the high pH range from 7 to 8.5, on the other hand, the present study demonstrates that potassium stimulation is essentially not associated with such intracellular pH regulation. This conclusion is based on our observation that potassium stimulation in the high pH range is insensitive to a proton conductor, carbonyl cyanide-p-trifluoromethoxy-phenyl-hydrazone. Since none of the other monovalent cations, including sodium, rubidium, ammonium, and Tris ions, could replace potassium ions in significantly stimulating glutamate transport, it is most likely that the influx of potassium ions specifically cancels the membrane potential derived by movement of glutamate with the net negative charges across a membrane and thus facilitates transport (the second mechanism). The second mechanism appears to be operative even in a low pH range, in addition to the first mechanism.
Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis
Authors: Mueller J P , Taber H W
Published: November 2016

Cytochrome aa3 is one of two terminal oxidase complexes in the Bacillus subtilis electron transport chain. A novel genetic strategy was devised which permitted the isolation of B. subtilis mutants lacking cytochrome aa3 by selection for streptomycin-resistant clones which failed to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Two mutations were studied intensively. Spectroscopic examination showed that each mutant lacked cytochrome aa3; they were also asporogenous and unable to grow on lactate as the sole carbon and energy source. These mutations were mapped to a locus designated ctaA, located at 127 degrees between pyrD and metC on the B. subtilis chromosome. Both ctaA mutations were closely linked by transformation to the pycA locus. The ctaA locus and a portion of the pycA locus were cloned from a B. subtilis integration library constructed in Escherichia coli. A recombinant plasmid containing a 4.0-kilobase insert of B. subtilis DNA could transform both ctaA mutants to CtaA+. Gene disruption and complementation experiments with subcloned fragments revealed that the ctaA locus consisted of a single transcriptional unit about 1.35 kilobase pairs in size. The nucleotide sequence of the ctaA transcriptional unit contains a single open reading frame capable of coding for a protein with a predicted molecular weight of 34,065. The predicted protein is extremely hydrophobic, with several probable membrane-spanning domains. No sequence similiarity was found between ctaA and the highly conserved procaryotic and mitochondrial oxidase polypeptides. Cloning and sequence analysis of two ctaA mutations revealed that one allele is a nonsense mutation in the carboxy terminus and the other is a missense mutation in the amino terminus; this indicates that the pleiotropic phenotype conferred by each mutation was caused by loss of CtaA or of its activity. Genetic evidence suggests that the ctaA gene product is required as an accessory protein in the genetic expression, posttranslational biogenesis, or both, of the cytochrome aa3 complex and during an early stage of sporogenesis.
Variation in buoyant density of whole cells and isolated cell walls of Streptococcus faecium (ATCC 9790)
Authors: Glaser D , Haines M , Bylund J , Higgins M
Published: November 2016

The buoyant density of whole cells of Streptococcus faecium varies with growth rate and during the cell cycle. Two possible explanations for this were explored: (i) the density of cell walls may vary, and (ii) the proportions of wall and cytoplasm may vary. We tested the first possibility by isolating walls from chilled, unfixed populations of S. faecium cells and fractionating them on Percoll density gradients. Mean cell wall density averaged 4% less than whole-cell density and did not vary significantly with growth rate. In addition, walls isolated from heavy and light fractions of a population of cells did not differ significantly in density. Thus, variation in the density of isolated cell walls could not account for the observed variation in whole-cell density within or between populations. Using previously published measurements of the physical dimensions of S. faecium cells, we also found that the relative proportions of wall and cytoplasm (see the second possibility above) could not account for the observed changes in whole-cell buoyant density.
Analysis of the transcriptional unit encoding the genes for rubredoxin (rub) and a putative rubredoxin oxidoreductase (rbo) in Desulfovibrio vulgaris Hildenborough
Authors: Brumlik M J , Voordouw G
Published: November 2016

The nucleotide sequence of a 2.0-kilobase-pair EcoRI restriction fragment upstream from the gene (rub, 162 base pairs) encoding rubredoxin from Desulfovibrio vulgaris Hildenborough indicates that it is part of a larger transcriptional unit, containing an additional 378-base-pair open reading frame which terminates 16 nucleotides from the translational start of the rub gene and could encode a polypeptide of 14 kilodaltons (kDa). Northern (RNA) blotting of RNA isolated from both D. vulgaris Hildenborough and Escherichia coli TG2 transformed with plasmid pJK29, which contains both genes on a 1.1-kilobase-pair SalI insert, confirms that the genes for this 14-kDa polypeptide and rubredoxin are present on a single transcript of 680 nucleotides. Strong evidence that the 14-kDa polypeptide is also a redox protein is provided by the fact that its NH2 terminus is homologous to desulforedoxin, which has been isolated from D. gigas as a small dimeric redox protein (36 amino acids per monomer), coordinating two iron atoms. Since rubredoxin is a potential redox partner for the 14-kDa protein, it has been tentatively named rubredoxin oxidoreductase, produced by the rbo gene. Southern blotting indicates that the rbo-rub operon is present in several species and strains of sulfate-reducing bacteria.
Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b
Authors: Chen Y P , Yoch D C
Published: November 2016

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.