bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and nucleotide sequence of a DNA fragment from Bacillus natto that enhances production of extracellular proteases and levansucrase in Bacillus subtilis
Authors: Nagami Y , Tanaka T
Published: November 2016

A DNA fragment from Bacillus natto IFO3936 has been cloned which enhances the production of both extracellular alkaline and neutral proteases in Bacillus subtilis. The DNA sequence analysis around the gene responsible for the hyperproduction, prtR, revealed one open reading frame (comprising 60 amino acid residues) which was bounded by potential transcriptional and translational regulatory signals in its preceding and following regions. This open reading frame was not homologous to the published sequences of the structural genes of the two proteases. The calculated molecular weight (7,109) of the polypeptide predicted from the DNA sequence is much smaller than those of the two proteases, indicating that the gene product is distinct from those enzymes. In-frame fusion between the N-terminal region of the coding sequence and the lacZ gene of Escherichia coli demonstrated that the coding region was indeed translated in vivo. By deletion analysis it was suggested that prtR was the structural gene for the 60-amino-acid polypeptide. Cells carrying a prtR plasmid secreted both proteases 40 to 400 times more than the cells carrying the vector alone. Furthermore, it was found that prtR also enhanced the production of levansucrase by 1 or 2 orders of magnitude. There was no difference, however, in the amount of the other extracellular enzymes such as alpha-amylase, RNase, and alkaline phosphatase. These results indicate that prtR is specific for the hyperproduction of the proteases and levansucrase.
Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer membranes
Authors: Armstrong S K , Parr T R , Parker C D , Hancock R E
Published: November 2016

The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures. The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCl was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cl- and a maximum possible conductance through the channel of 1.28 nS.
Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene
Authors: Lampel K A , Uratani B , Chaudhry G R , Ramaley R F , Rudikoff S
Published: November 2016

The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase.
Purification and characterization of glucose oxidase from ligninolytic cultures of Phanerochaete chrysosporium
Authors: Kelley R L , Reddy C A
Published: November 2016

Glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography. The enzyme is a flavoprotein with an apparent native molecular weight of 180,000 and a denatured molecular weight of 80,000. This enzyme does not appear to be a glycoprotein. It gives optimal activity with D-glucose, which is stoichiometrically oxidized to D-gluconate. The enzyme has a relatively broad pH optimum of 4 to 5. It is inhibited by Ag+ (10 mM) and o-phthalate (100 mM), but not by Cu2+, NaF, or KCN (each 10 mM).
Nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus
Authors: Jones C L , Khan S A
Published: November 2016

The complete nucleotide sequence of the enterotoxin B gene from Staphylococcus aureus, as well as the 5' and 3' flanking regions, was determined. Starting from an ATG initiator codon, an open reading frame encoded the enterotoxin B precursor that consisted of 266 amino acids (Mr, 31,400). The 5' terminal portion of the gene encodes a signal peptide 27 amino acids long. The deduced amino acid sequence matched, with a few exceptions, the published amino acid sequence of enterotoxin B. The structural gene was flanked on the 5' side by a promoter-like sequence and on the 3' side by a palindromic structure followed by a thymine-rich region that resembled a transcription termination signal. Downstream from the entB gene were two overlapping open reading frames corresponding to 134 and 185 amino acids in the opposite orientation. The signal sequence of the enterotoxin B precursor resembled that of other secreted proteins found in other bacteria.
Escherichia coli B/r leuK mutant lacking pseudouridine synthase I activity
Authors: Searles L L , Jones J W , Fournier M J , Grambow N , Tyler B , Calvo J M
Published: November 2016

Escherichia coli B/r strain EB146 containing mutation leuK16 has elevated levels of enzymes involved in the synthesis of leucine, valine, isoleucine, histidine, and tryptophan (Brown et al., J. Bacteriol. 135:542-550, 1978). We show here that strain EB146 (leuK16) has properties that are similar to those of E. coli and Salmonella typhimurium hisT strains. In tRNA1Leu from both hisT and leuK strains, positions 39 and 41 are uridine residues rather than pseudouridine residues. Furthermore, in tRNA3Leu and tRNA4Leu from a leuK strain, uridine residues at positions 39 and 40, respectively, are unmodified. Pseudouridine synthase I activity is missing in extracts of strain EB146 (leuK16), and extracts of strain EB146 (leuK16) and of a hisT strain do not complement one another in vitro. Four phenotypes of strain EB146 (leuK16), leucine excretion, wrinkled colony morphology, and elevated levels of leu and his enzymes, are complemented by a plasmid having a 1.65-kilobase DNA fragment containing the E. coli K-12 hisT locus. These results indicate that either leuK codes for pseudouridine synthase I (and is thus a hisT locus in reality) or, less likely, it codes for a product that affects the synthesis or activity of pseudouridine synthase I.
Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae
Authors: Kong Q T , Wu Q L , Ma Z F , Shen S C
Published: November 2016

Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae has been demonstrated. Studies on the oxygen regulation of nifB-lacZ and nifH-lacZ fusions in the presence of the nifLA operon, which contains either an intact or a deleted nifL gene, indicate that possibly both the nifL promoter and the nifL product are responsible for nif repression by oxygen.
Identification of a specific membrane-particle-associated DNA sequence in Bacillus subtilis
Authors: Sargent M G , Bennett M F
Published: November 2016

After the Bacillus subtilis nucleoid was dissected with restriction endonucleases, a specific DNA sequence from the purA region was isolated in a particulate form that probably originated from the cell membrane. Precise definition of the binding region within this sequence was achieved by a novel procedure based on a previously reported observation that additional copies of the binding region, introduced into the chromosome using an integrative plasmid, were also predominantly particle bound. Subsections of the original plasmid insertion were cloned into the integrative plasmid and introduced into B. subtilis, in which they became tandemly reiterated under appropriate selective conditions. HaeIII sites in the vector, flanking each insertion, were used to excise the latter for subsequent tests of particle association. Examination of 10 strains containing subsections of the original 5.2-kilobase-pair region showed that the binding region was confined to 283 base pairs. This was confirmed by dissection in vitro of a larger, isolated, particle-bound sequence. The nucleotide sequence of a 1,300-base-pair region that contained this site was determined. The entire region had a notably high A + T content and was deficient in open reading frames for transcription.
Arginine catabolism in Agrobacterium strains: role of the Ti plasmid
Authors: Dessaux Y , Petit A , Tempé J , Demarez M , Legrain C , Wiame J M
Published: November 2016

We present a study of the enzymatic activities involved in the pathway for arginine catabolism by Agrobacterium tumefaciens. Nitrogen from arginine is recovered through the arginase-urease pathway; the genes for these two activities are probably chromosomally born. Arginase was found to be inducible during growth in the presence of arginine or ornithine. Urease was constitutively expressed. Ornithine, resulting from the action of arginase on arginine, could be used as a nitrogen source via transamination to delta 1-pyrroline-5-carboxylate and reduction of the latter compound to proline by a reductase (both enzymatic activities are probably chromosomally encoded). Ornithine could also be used as a carbon source. Thus, we identified an ornithine cyclase activity that was responsible for direct conversion of ornithine to proline. This activity was found to be Ti plasmid encoded and inducible by growth in medium containing octopine or nopaline. The same activity was also chromosomally encoded in some Agrobacterium strains. In such strains, this activity was inducible during growth in arginine-containing medium.
Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants
Authors: Wolf J B , Brey R N
Published: November 2016

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.
Critical micelle concentrations of lipoteichoic acids
Authors: Wicken A J , Evans J D , Knox K W
Published: November 2016

Purified lipoteichoic acids (LTAs) from several gram-positive organisms have been shown, by methods involving spectral changes of an added merocyanine dye probe, to have critical micelle concentrations in the range of 1 to 10 micrograms/ml, suggesting that acylated LTAs in their monomer forms may represent the major configuration of extracellular LTAs in bacterial culture fluids. The critical micelle concentrations obtained did not differ markedly with degree of carbohydrate substitution of the polymers. The significance of these findings in relation to the biological properties of LTA is discussed.
Cloning and expression of the genes specifying Shiga-like toxin production in Escherichia coli H19
Authors: Huang A , de Grandis S , Friesen J , Karmali M , Petric M , Congi R , Brunton J L
Published: November 2016

Some strains of Escherichia coli produce a protein which is cytotoxic for Vero cell and HeLa cell monolayers. This toxin is very similar to the toxin of Shigella dysenteriae 1 and has been named verotoxin or E. coli Shiga-like toxin. It has been shown that toxin conversion is due to a group of bacteriophages, one of which has been designated H-19B. In this study we report hybridization experiments showing that part of the H-19B genome is homologous to phage lambda. We have cloned a 1.7-kilobase BalI-BglII fragment from the genome of H-19B into pUC18. The recombinant plasmid confers the ability to produce high levels of Shiga-like toxin on transformed E. coli cells. We demonstrate using an in vitro transcription/translation system that the cloned fragment specifies the two verotoxin subunit peptides which have masses of 31 and 5.5 kilodaltons. The identity of peptides was confirmed by immunoprecipitation with verotoxin antiserum and protein A-Sepharose beads.
Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12
Authors: Hoffmann H , Fischer E , Kraut H , Braun V
Published: November 2016

A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed. The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried the fhuACD genes of pLC19-19 of the Clarke and Carbon collection. At low temperature (27 degrees C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA. The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA. Upon runaway replication of pHK232 at 37 degrees C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA. For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins. Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA. This procedure yielded FhuA protein free from other membrane proteins. The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively. As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and T1.
Determination of the chromosomal locations of four Bacillus subtilis genes which code for a family of small, acid-soluble spore proteins
Authors: Connors M J , Howard S , Hoch J , Setlow P
Published: November 2016

The chromosomal locations of four genes which code for small, acid-soluble spore proteins (SASP) in Bacillus subtilis have been determined. Although these four genes code for extremely homologous small, acid-soluble spore proteins (greater than 65% sequence identity), the genes are not clustered but are located at 70 degrees (adjacent to glyB [sspB gene]), 115 degrees (between metC and pyr cluster [sspD gene]), 180 degrees (between metB and kauA [sspC gene]), and 260 degrees (between ilvC and aroG [sspA gene]) on the B. subtilis genetic map.
Tn5-induced mutations affecting sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha
Authors: Chandra T S , Friedrich C G
Published: November 2016

Mutants of Thiosphaera pantotropha defective in chemolithoautotrophic growth were obtained by transpositional mutagenesis with Tn5 coding for kanamycin resistance. The suicide vehicle for introducing Tn5 to T. pantotropha was pSUP5011 harbored by Escherichia coli. Kanamycin-resistant isolates were screened for the inability to grow with reduced sulfur compounds (Sox-). Four classes of Sox- mutants were obtained. Three were of different pleiotropic phenotypes: (i) unable to grow with formate, nitrate, and xanthine; (this class strongly suggested the involvement of a molybdenum cofactor in inorganic sulfur-oxidizing ability); (ii) no growth with hydrogen; (iii) slight growth with hydrogen and formate. Two plasmids, pHG41 (about 450 kilobase pairs) and pHG42 (110 kilobases), were identified in lysates of T. pantotropha. In one Sox- mutant pHG41 could not be detected. Revertant analysis suggested that pHG41 and pHG42 were not involved in the Sox character.
Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe
Authors: Erratt J A , Nasim A
Published: November 2016

A recombinant plasmid pool of the Saccharomyces diastaticus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevisiae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had identical restriction maps, which differed from the map of the STA1 gene (I. Yamashita and S. Fukui, Agric. Biol. Chem. 47:2689-2692, 1983). The glucoamylase activity exhibited by all S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diastaticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.
Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro
Authors: Lowery R G , Saari L L , Ludden P W
Published: November 2016

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by interconversion of the Fe protein between a modified and an unmodified form. Since the discovery of the activation process in 1976, investigators have been unable to demonstrate the inactivation (modification) reaction in vitro. In this study, NAD-dependent modification and concomitant inactivation of the Fe protein were demonstrated in crude extracts of R. rubrum. Activation of the in vitro-modified Fe protein by activating enzyme and structural similarity between the in vivo and in vitro modifications are presented as evidence that the in vitro modification is the physiologically relevant ADP-ribosylation reaction. Using a partially purified preparation, we showed that the inactivating enzyme activity is stimulated by divalent metal ions and ADP, that O2-denatured Fe protein will not serve as a substrate, and that dithionite inhibits the modification reaction.
Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection
Authors: Knight C D , Rossen L , Robertson J G , Wells B , Downie J A
Published: November 2016

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.
Mevalonic acid is partially synthesized from amino acids in Halobacterium cutirubrum: a 13C nuclear magnetic resonance study
Authors: Ekiel I , Sprott G D , Smith I C
Published: November 2016

13C nuclear magnetic resonance revealed an unusual pathway for the biosynthesis of lipids in Halobacterium cutirubrum and H. halobium. Mevalonic acid was not synthesized from three acetyl-coenzyme A molecules, as has been suggested previously, and the branch-methyl and methine carbons in phytanyl chains were derived from neither acetate nor glycerol. Instead, they were supplied by the degradation of amino acids, in particular of lysine. Presumably, two different types of two-carbon fragments were used simultaneously by halobacteria for the biosynthesis of mevalonate. The labeling pattern of squalene supported the above conclusions. Based on these data, a general scheme is proposed to account for the contribution of lysine-to-lipid biosynthesis.
Cloning and characterization of livH, the structural gene encoding a component of the leucine transport system in Escherichia coli
Authors: Nazos P M , Antonucci T K , Landick R , Oxender D L
Published: November 2016

The physical location of the genetically defined livH gene was mapped in the 17-kilobase plasmid pOX1 by using transposon Tn5 inactivation mapping and further confirmed by subcloning and complementation analysis. These results indicated that the livH gene maps 3' to livK, the gene encoding the leucine-specific binding protein. Moreover, the nucleotide sequence of the livH gene and its flanking regions was determined. The livH gene is encoded starting 47 base pairs downstream from the livK gene, and it is transcribed in the same direction as the livK gene. The livK-livH intergenic region lacks promoter sequences and contains a GC-rich sequence that could lead to the formation of a stable stem loop structure. The coding sequence of the livH gene, which is 924 base pairs, specifies a very hydrophobic protein of 308 amino acid residues. Expression of livH-containing plasmids in minicells suggested that a poorly expressed protein with an Mr of 30,000 could be the livH gene product.
Cloning of the gene for indoleacetic acid-lysine synthetase from Pseudomonas syringae subsp. savastanoi
Authors: Glass N L , Kosuge T
Published: November 2016

The phytopathogen Pseudomonas syringae subsp. savastanoi incites the production of galls on olive and oleander plants. Gall formation is dependent on bacterial production of the phytohormone indoleacetic acid (IAA). The genetic determinants for IAA synthesis are located on a plasmid (pIAA) and are organized in an operon in oleander strains of the bacterium. P. syringae subsp. savastanoi further converts IAA to an amino acid conjugate, 3-indole-acetyl-epsilon-L-lysine (IAA-lysine). The gene for IAA-lysine synthetase (iaaL) was found on the IAA plasmid by screening pIAA deletion mutants for the ability to convert IAA to IAA-lysine. The iaaL locus was then cloned in the vector pUC8 from a bank of P. syringae subsp. savastanoi EW2009 plasmid DNA to construct recombinant plasmid pLG87. The specific activity of IAA-lysine synthetase in Escherichia coli transformed with pLG87 was 47 times higher than that of the enzyme extract from P. syringae subsp. savastanoi. The direction of transcription of the iaaL gene was determined to be opposite to that of the IAA operon. The location of the iaaL gene on pIAA1 was mapped by Tn5 insertion mutagenesis to a 2.5-kilobase-pair fragment 2 kilobase pairs from the IAA operon.
IS1-dependent generation of high-copy-number replicons from bacteriophage P1 Ap Cm as a mechanism of gene amplification
Authors: Froehlich B J , Watkins C , Scott J R
Published: November 2016

Mutant P1 Ap Cm lysogens were isolated in which the drug resistance genes resident on the plasmid prophage P1 Ap Cm are amplified by a novel mechanism. The first step required for amplification is IS1-mediated rearrangement of the P1 Ap Cm prophage. The drug resistance genes are amplified from the rearranged P1 Ap Cm prophage by the formation of a plasmid (P1dR) which contains the two resistance genes. The P1dR plasmid is an independent replicon about one-half the size of P1 Ap Cm that can be maintained at a copy number eightfold higher than that at which P1 Ap Cm can be maintained. It contains no previously identified replication origin and is dependent on the Rec+ function of the host.
Unrelated conjugative plasmids have sequences which are homologous to the leading region of the F factor
Authors: Golub E I , Low K B
Published: November 2016

Conjugative plasmids from various incompatibility groups which carry DNA homologous to the ssb gene of the F factor were found to have additional homology with the F factor. This region homologous with F was located on both sides of the ssb gene and occupied a considerable part of the leading region, i.e., the 12.9-kilobase portion of F transferred first during conjugation. This region was the only region of the F factor which has a homologous counterpart on many plasmids.
Effect of chloramphenicol and ethidium bromide on the level of ornithine carbamoyltransferase in Neurospora crassa
Authors: Zerez C R , Weiss R L
Published: November 2016

The specific activity of the nuclear-gene-encoded, mitochondrial arginine biosynthetic enzyme ornithine carbamoyltransferase (EC 2.1.3.3) in Neurospora crassa was elevated in mycelia treated with chloramphenicol or ethidium bromide. The increase in specific activity was caused by an increase in the number of mature enzyme molecules rather than by the activation of a preexisting enzyme. Chloramphenicol and ethidium bromide appeared to act indirectly via arginine-mediated derepression. However, derepression did not appear to result from a drug-mediated decrease in the arginine pool.
Entrainment and temperature dependence of the response oscillator in Halobacterium halobium
Authors: Schimz A , Hildebrand E
Published: November 2016

Halobacterium halobium spontaneously reverses its swimming direction with a frequency of about 0.1 s-1. The self-sustained periodicity can be entrained by rhythmic light stimuli within a limited range between 0.13 and 0.3 s-1. Increase of temperature increases the reversal frequency and shortens the time to reach maximal responsiveness to attractant stimuli during a cycle. Also, the period of absolute refractoriness to repellent stimuli is shortened. The results illustrate characteristic properties of biological oscillators.
Identification, immunochemical characterization, and purification of a major lipoprotein antigen associated with the inner (cytoplasmic) membrane of Escherichia coli
Authors: Doherty H , Yamada H , Caffrey P , Owen P
Published: November 2016

A major antigenic constituent of the inner membrane of Escherichia coli ML308-225 was identified as a 28.5-kilodalton lipoprotein containing covalently bound glycerol and palmitate. This lipoprotein corresponded to antigen 47 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H.R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978) and to new lipoprotein 4 described for E. coli B by Ichihara et al. (S. Ichihara, H. Hussain, and S. Mizushima, J. Biol. Chem. 256:3125-3129, 1980). Experiments involving isopycnic centrifugation of spheroplast envelopes indicated that antigen 47 was enriched in cytoplasmic membrane subfractions of low density. The protein did not manifest an obvious association with peptidoglycan of the types displayed by the bound form of the Braun (Lpp) lipoprotein, the 21-kilodalton peptidoglycan-associated lipoprotein, or the ompF/C gene products. Antibodies specific for antigen 47 were used to demonstrate that the molecule was immunologically distinct from both the Braun lipoprotein and the peptidoglycan-associated lipoprotein of E. coli. Antigens of similar molecular mass to and cross-reacting with antigen 47 were present in the envelopes of eight type species of the Enterobacteriaceae. A protocol for the purification of antigen 47, based upon its solubility in a chloroform-methanol-water mixture, was developed.
camR, a negative regulator locus of the cytochrome P-450cam hydroxylase operon
Authors: Koga H , Aramaki H , Yamaguchi E , Takeuchi K , Horiuchi T , Gunsalus I C
Published: November 2016

A 4.27-kilobase insert from a HindIII DNA library of Pseudomonas putida carrying the CAM plasmid allowed coordinate expression of genes camD and camC under control of camR, an upstream regulator. The camC gene specifies cytochrome P-450cam, and camD specifies the 5-exo-alcohol dehydrogenase. A 1.38-kilobase deletion from the insert results in the constitutive expression of genes camC and camD; transformation in trans restores the substrate control, indicating that camR is a negative regulator.
Incompatibility repressor in a RepA-like replicon of the IncFI plasmid ColV2-K94
Authors: Weber P C , Palchaudhuri S
Published: November 2016

The replication region Rep1 of the IncFI plasmid ColV2-K94 was cloned on self-replicating restriction fragments. Rep1 was structurally and functionally homologous to the RepA replicon of IncFII R plasmids. Despite this close relationship, these two replication systems were compatible with each other. The nucleotide sequence of the copA incompatibility-replication control gene of Rep1 was determined and compared with the copA sequence of RepA. Six base changes were found in a 24-base-pair span of the copA gene; these may result in the formation of a new, more stable, 49-base stem-loop structure in the potential CopA RNA repressor molecule. We postulate that these alterations weaken the interaction between RNA transcripts of the Rep1 and RepA replicons.
High A + T content conserved in DNA sequences upstream of leuABCD in Escherichia coli and Salmonella typhimurium
Authors: Haughn G W , Wessler S R , Gemmill R M , Calvo J M
Published: November 2016

The nucleotide sequence of over 800 base pairs of DNA upstream of leuP was determined for Escherichia coli and Salmonella typhimurium. In both of these enteric bacteria, approximately 500 base pairs of A + T-rich sequences separates leuP from an upstream open reading frame. Although these A + T-rich sequences share little homology, the distribution of A + T base pairs within the region is strikingly conserved. Deletion of the A + T-rich sequences upstream of the E. coli leu operon does not markedly affect the strength of the leu promoter in vivo.
Production of aerobactin by some species of the genus Yersinia
Authors: Stuart S J , Prpic J K , Robins-Browne R M
Published: November 2016

Seventy-seven isolates of Yersinia species were examined for evidence of hydroxamate biosynthesis. A total of 7 of 12 Y. frederiksenii, 1 of 5 Y. kristensenii, and 5 of 5 Y. intermedia isolates synthesized an hydroxamate species which was chromatographically and electrophoretically identical to aerobactin. Proof that this material was indeed aerobactin was obtained for one strain of Y. frederiksenii by using mass spectrometry. None of 50 Y. enterocolitica nor any of 5 Y. pseudotuberculosis isolates produced hydroxamates.
New role for photoreversible pyrimidine dimers in induction of prototrophic mutations in excision-deficient Escherichia coli by UV light
Authors: Ruiz-Rubio M , Woodgate R , Bridges B A , Herrera G , Blanco M
Published: November 2016

UV mutagenesis to His+ in certain recA441 lexA51 bacteria was not photoreversible, indicating that pyrimidine dimers are not target lesions. Photoreversibility was observed in recA+ lexA51 bacteria, showing that pyrimidine dimers are needed to activate the recA+ protein (unlike the recA441 protein) to perform a function in UV mutagenesis distinct from cleavage of the lexA repressor.
Contrasting mechanisms of envZ control of mal and pho regulon genes in Escherichia coli
Authors: Case C C , Bukau B , Granett S , Villarejo M R , Boos W
Published: November 2016

The envZ11 missense mutation in the regulatory gene envZ pleiotropically repressed synthesis of OmpF, alkaline phosphatase, and several proteins of the maltose regulon. Procaine treatment of wild-type cells resulted in the same phenotype through an envZ+-mediated mechanism. Here we show that envZ11-procaine act differently on the mal and pho regulons. In the mal system, the expression of the positive regulator gene malT, measured as beta-galactosidase activity of a malT-lac+ operon fusion, was drastically reduced by procaine treatment or by the envZ11 mutation. In contrast, expression of the positive regulator of the pho regulon phoB was not reduced by procaine treatment. The products of the regulatory genes phoM, phoR, and phoU were also not required for procaine action. Procaine and envZ11 inhibited expression of only two products of the pho regulon, alkaline phosphatase and the PhoE porin. The conclusion that envZ11-procaine act differently on the mal and the pho regulons is supported by our ability to isolate second-site mutations with a Mal+ PhoA- phenotype in an envZ11 strain.
Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9
Authors: Kobayashi T , Kato C , Kudo T , Horikoshi K
Published: November 2016

Most of the cloned penicillinase from alkalophilic Bacillus sp. strain 170 and alkaline phosphatase were released into the culture medium by Escherichia coli strains bearing plasmid pEAP1 or pEAP2 (T. Kudo, C. Kato, and K. Horikoshi, J. Bacteriol. 156:949-951, 1983). We analyzed the basis for excretion of periplasmic enzymes in the cells bearing these plasmids. Several experiments such as subcloning, insertion of a chloramphenicol acetyltransferase cartridge, and DNA sequencing were done. A dormant kil gene in plasmid pMB9 was expressed by a promoter of the inserted DNA fragment of alkalophilic Bacillus sp. strain 170, and as a result, the outer membrane of E. coli became permeable, allowing the proteins to be excreted without cell lysis.
Reversibility of SOS-associated division inhibition in Escherichia coli
Authors: Maguin E , Lutkenhaus J , D'Ari R
Published: November 2016

In Escherichia coli the SOS response, induced by DNA-damaging treatments, includes two systems of cell division inhibition, SfiA and SfiC, which are thought to prevent cell division by interacting with the division protein FtsZ. It is shown here that SfiA-mediated division inhibition is readily reversible, even in the absence of de novo protein synthesis, suggesting that functional FtsZ molecules can be recovered from SfiA-FtsZ complexes. The action of SfiC, on the other hand, is essentially irreversible; induction by expression of the recA (Tif) mutation for 60 min results in division inhibition that continues for at least 180 min after the end of the induction period. An excess of the presumed target molecule FtsZ, furnished by a multicopy plasmid, suppresses the action of SfiA but not SfiC. Simultaneous induction of SfiA and SfiC results in irreversible division inhibition, showing that SfiC is epistatic to SfiA. The irreversibility of SfiC action is most readily accounted for by assuming that the SfiC product, unlike SfiA, is stable. The reversibility of SfiA action is slower in a lon mutant, in which the SfiA protein is partially stabilized. From the kinetics of division resumption in the absence of protein synthesis, we estimated the in vivo half-life of the SfiA protein to be 10 min in a lon+ strain and 170 min in a lon mutant.
Overproduction of FtsZ suppresses sensitivity of lon mutants to division inhibition
Authors: Lutkenhaus J , Sanjanwala B , Lowe M
Published: November 2016

Escherichia coli lon mutants are sensitive to UV light and other DNA-damaging agents. This sensitivity is due to the loss of the lon-encoded ATP-dependent proteolytic activity which results in increased stability of the cell division inhibitor SulA. Introduction of the multicopy plasmid pZAQ containing the ftsZ gene, which is known to increase the level of FtsZ, suppressed the sensitivity of lon mutants to the DNA-damaging agents UV and nitrofurantoin. Alterations of pZAQ which reduced the expression of ftsZ reduced the ability of this plasmid to suppress the UV sensitivity. Examination of the kinetics of cell division revealed that pZAQ did not suppress the transient filamentation seen after exposure to UV, but did suppress the long-term inhibition that is normally observed. lon strains carrying pZAQ could stably maintain a multicopy plasmid carrying sulA (pBS2), which cannot otherwise be introduced into lon mutants. In addition, the increased temperature sensitivity of lexA(Ts) strains containing pBS2 was suppressed by pZAQ. These results suggest that SulA inhibits cell division by inhibiting FtsZ and that this interaction is stoichiometric.
Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine
Authors: Laten H M , Valentine P J , van Kast C A
Published: November 2016

By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation. Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase. The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation. These data can be explained by postulating the existence of two enzyme activities not previously reported in S. cerevisiae. The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-). The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).
Transcriptional analysis of beta-lactamase regulation in Bacillus licheniformis
Authors: Salerno A J , Lampen J O
Published: November 2016

The expression of the blaP gene for the beta-lactamase of Bacillus licheniformis was examined by transcriptional analyses. Radiolabeled probes containing the blaP gene or various regions 3' or 5' to it were used to analyze RNA samples prepared from induced and uninduced cultures of wild-type and mutant B. licheniformis strains. The level of blaP mRNA was low in uninduced wild-type cells. At 37 degrees C, blaP mRNA levels reached a maximum 1 h after induction while rising up to 180-fold and then declined, but remained severalfold above the uninduced level for several hours. The rate of beta-lactamase synthesis was roughly proportional to the levels of blaP mRNA in both wild-type and mutant strains, indicating that regulation of beta-lactamase formation occurs primarily at the level of transcription. Turnover of blaP mRNA in the presence of rifampin was rapid, giving a blaP mRNA half-life of about 2 min. Yet, high levels of blaP mRNA were maintained for at least 1 h after removal of free inducer. Three blaP mRNAs of 1.2, 2.9, and 3.4 kilobases were produced from the blaP promoter. The most abundant made up about 97% of all blaP transcripts and was also the smallest, ending at a transcriptional terminator located about 60 bases 3' to the blaP structural gene. Variables such as incubation temperature, cytotoxicity of inducer, and type of strain had essentially no effect on the ratio of large blaP mRNA to total blaP mRNA. The 2.9- and 3.4-kilobase blaP mRNAs identify potential locations of genetically linked regulators of beta-lactamase synthesis.
Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis"
Authors: Thorne L , Garduno F , Thompson T , Decker D , Zounes M , Wild M , Walfield A M , Pollock T J
Published: November 2016

A gene from Bacillus thuringiensis subsp. "israelensis" was cloned from the large plasmids of this subspecies and was shown to code for a mosquitocidal polypeptide. The gene could be expressed in either Escherichia coli, Bacillus subtilis, or B. thuringiensis subsp. "israelensis" to produce the larvicidal activity. Similarly, a Lepidoptera-specific toxin gene from B. thuringiensis subsp. "kurstaki" was also cloned and expressed in E. coli and B. subtilis. Both cloned genes were sequenced and subjected to computer analysis. A long open translational reading frame coded for the B. thuringiensis subsp. "kurstaki" gene product. However, the B. thuringiensis subsp. "israelensis" clone was composed of two adjacent open reading frames oriented as if they were in a transcriptional operon. The products of the cloned genes retained their specificity for either Lepidoptera or Diptera. The control regions immediately preceding the toxin genes of both B. thuringiensis subspecies showed considerable DNA homology, most likely because both toxins are expressed only during sporulation. In addition, the deduced amino acid sequences from the two contiguous B. thuringiensis subsp. "israelensis" genes bore a striking resemblance to the deduced amino acid sequence from the single larger B. thuringiensis subsp. "kurstaki" gene, as if these two arrangements were evolutionarily related.
Characterization of Escherichia coli mutants completely defective in synthesis of cyclopropane fatty acids
Authors: Grogan D W , Cronan J E
Published: November 2016

The synthesis of cyclopropane fatty acids (CFA) in bacteria represents a biochemically and physiologically unique membrane modification whose importance for the cell remains unknown, despite extensive study of a Cfa- mutant of Escherichia coli and of the cloned cfa gene. Recently we reported the isolation of new Cfa- mutants (D. W. Grogan and J. E. Cronan, Jr., Mol. Gen. Genet. 196:367-372, 1984). Molecular-genetic and biochemical analysis indicated that these were null mutants of the E. coli cfa locus which were formed by inversions of a chromosomal segment. Isogenic Cfa+ and Cfa- strains were constructed from one such mutant and subjected to various stress conditions. In nearly all cases, both strains responded equally, but certain treatments, such as repeated freezing and thawing, favored the survival of Cfa+ strains over Cfa- strains. Though not essential, CFA thus appeared to play some beneficial role (or roles) in the bacterial cell.
Osmoregulation of the maltose regulon in Escherichia coli
Authors: Bukau B , Ehrmann M , Boos W
Published: November 2016

The maltose regulon consists of four operons that direct the synthesis of proteins required for the transport and metabolism of maltose and maltodextrins. Expression of the mal genes is induced by maltose and maltodextrins and is dependent on a specific positive regulator, the MalT protein, as well as on the cyclic AMP-catabolite gene activator protein complex. In the absence of an exogenous inducer, expression of the mal regulon was greatly reduced when the osmolarity of the growth medium was high; maltose-induced expression was not affected, and malTc-dependent expression was only weakly affected. Mutants lacking MalK, a cytoplasmic membrane protein required for maltose transport, expressed the remaining mal genes at a high level, presumably because an internal inducer of the mal system accumulated; this expression was also strongly repressed at high osmolarity. The repression of mal regulon expression at high osmolarity was not caused by reduced expression of the malT, envZ, or crp gene or by changes in cellular cyclic AMP levels. In strains carrying mutations in genes encoding amylomaltase (malQ), maltodextrin phosphorylase (malP), amylase (malS), or glycogen (glg), malK mutations still led to elevated expression at low osmolarity. The repression at high osmolarity no longer occurred in malQ mutants, however, provided that glycogen was present.