bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of an incompatibility region of mini-Rts1 that contains five direct repeats
Authors: Kamio Y , Terawaki Y
Published: January 2020

The plasmid mini-Rts1, consisting of an EcoRI/HindIII fragment of about 1.8 kilobases (kb), contains an incompatibility determinant in its EcoRI/AccI region (0.5 kb). The nucleotide sequence of this incompatibility fragment was determined. A most striking feature of the sequence is the presence of five 24-base pair direct repeats. Four out of the five repeating units, which are contained in a 0.2-kb EcoRI/HincII fragment, were cloned en bloc in pACYC184 and found to express Rts1-specific incompatibility. In addition, the copy number of the mini-Rts1 plasmid appeared to be increased threefold upon removal of the 0.2-kb incompatibility region (incI) from the plasmid. This deletion derivative of mini-Rts1, as well as mini-Rts1, was maintained stably at 37 degrees C, but was cured at a high frequency at 42 degrees C. A possible role of the repeated nucleotide sequence was discussed. By subcloning the mini-Rts1 DNA, a second inc determinant (incII) was found on the AccI fragment, which is contiguous to the 0.5-kb EcoRI/AccI fragment.
Cloning of a pleiotropic gene that positively controls biosynthesis of A-factor, actinorhodin, and prodigiosin in Streptomyces coelicolor A3(2) and Streptomyces lividans
Authors: Horinouchi S , Hara O , Beppu T
Published: January 2020

A-factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone), an autoregulating factor originally found in Streptomyces griseus, is involved in streptomycin biosynthesis and cell differentiation in this organism. A-factor production is widely distributed among actinomycetes, including Streptomyces coelicolor A3(2) and Streptomyces lividans. A chromosomal pleiotropic regulatory gene of S. coelicolor A3(2) controlling biosynthesis of A-factor and red pigments was cloned with a spontaneous A-factor-deficient strain of S. lividans HH21 and plasmid pIJ41 as a host-vector system. The restriction endonuclease KpnI-digested chromosomal fragments were ligated into the plasmid vector and introduced by transformation into the protoplasts of strain HH21. Three red transformants thus selected were found to produce A-factor and to carry a plasmid with the same molecular weight, and a 6.4-megadalton fragment was inserted in the KpnI site of pIJ41. By restriction endonuclease mapping and subcloning, a restriction fragment (1.2 megadaltons, approximately 2,000 base pairs) bearing the gene which causes concomitant production of A-factor and red pigments was determined. The red pigments were identified by thin-layer chromatography and spectroscopy to be actinorhodin and prodigiosin, both of which are the antibiotics produced by S. coelicolor A3(2). The cloned fragment was introduced into the A-factor-negative mutants (afs) of S. coelicolor A3(2) by using pIJ702 as the vector, where it complemented one of these mutations, afsB, characterized by simultaneous loss of A-factor and red pigment production. We conclude that the cloned gene pleiotropically and positively controls the biosynthesis of A-factor, actinorhodin, and prodigiosin.
The muc genes of pKM101 are induced by DNA damage
Authors: Elledge S J , Walker G C
Published: January 2020

A gene fusion was constructed in vitro that resulted in the synthesis of a hybrid protein consisting of the amino-terminal segment of the MucB protein of the mutagenesis-enhancing plasmid pKM101 joined to an enzymatically active carboxy-terminal segment of the beta-galactosidase protein. In strains bearing this fusion, beta-galactosidase activity was induced by UV radiation and other DNA-damaging agents. A genetic analysis of the regulation of expression of the phi (mucB'-lacZ') fusion was consistent with the LexA protein acting as the direct repressor of the mucB gene. Examination of the expression of the mucA and phi (mucB'-lacZ') gene products in maxicells in the presence and absence of a high-copy-number plasmid carrying the lexA+ gene demonstrated that lexA regulated both the mucA and mucB genes, thus supporting our conclusion that the two genes are organized in an operon with the mucA gene transcribed first. An analysis of the effects of the recA430(lexB30) mutation on muc expression led to the discovery of the differential ability of the recA430 gene product to induce expression of a dinB::Mu d1(Ap lac) fusion located on the chromosome and the same phi (dinB'-lacZ+) fusion cloned into plasmid pBR322. Models to account for the role of the recA430 allele on the expression of damage-inducible genes and on mutagenesis are discussed.
Regulation of Nostoc sp. phycobilisome structure by light and temperature
Authors: Anderson L K , Rayner M C , Sweet R M , Eiserling F A
Published: January 2020

Nostoc sp. strain MAC cyanobacteria were green in color when grown in white light at 30 degrees C and contained phycobilisomes that had phycoerythrin and phycocyanin in a molar ratio of 1:1. Cells grown for 4 to 5 days in green light at 30 degrees C or white light at 39 degrees C turned brown and contained phycoerythrin and phycocyanin in a molar ratio of greater than 2:1. In addition to the change in pigment composition, phycobilisomes from brown cells were missing a 34.5-kilodalton, rod-associated peptide that was present in green cells. The green light-induced changes were typical of the chromatic adaptation response in cyanobacteria, but the induction of a similar response by growth at 39 degrees C was a new observation. Phycobilisomes isolated in 0.65 M phosphate buffer (pH 7) dissociate when the ionic strength or pH is decreased. Analysis of the dissociation products from Nostoc sp. phycobilisomes suggested that the cells contained two types of rod structures: a phycocyanin-rich structure that contained the 34.5-kilodalton peptide and a larger phycoerythrin-rich complex. Brown Nostoc sp. cells that lacked the 34.5-kilodalton peptide also lacked the phycocyanin-rich rod structures in their phycobilisomes. These changes in phycobilisome structure were indistinguishable between cells cultured at 39 degrees C in white light and those cultured at 30 degrees C in green light. A potential role is discussed for rod heterogeneity in the chromatic adaptation response.
Expression of hydroxamate and phenolate siderophores by Shigella flexneri
Authors: Payne S M , Niesel D W , Peixotto S S , Lawlor K M
Published: January 2020

Shigella flexneri strains were assayed for the ability to synthesize and utilize phenolate and hydroxamate siderophores. The hydroxamate aerobactin was synthesized by all isolates tested, whereas phenolates were only rarely produced. Expression of aerobactin was accompanied by production of a single iron-regulated outer membrane protein (Mr = 74,000). This protein was not produced by a mutant defective in aerobactin utilization and may serve as the aerobactin receptor. Phenolate (enterobactin)-producing strains synthesized three additional outer membrane proteins (Mr = 74,000, 81,000, and 83,000) in response to iron starvation. These proteins are the same apparent size as those produced by Escherichia coli K-12 strains. Ent sequences are apparently present in strains which do not synthesize this compound. Although normally silent, ent genes can be activated in Ent- strains to produce Ent+ variants. These laboratory variants are phenotypically indistinguishable from clinical Ent+ isolates.
Inducible reactivation of bacteriophage T7 damaged by methyl methanesulfonate or UV light
Authors: Dodson L A , Masker W E
Published: January 2020

We examined the effects of host mutations affecting "SOS"-mediated UV light reactivation on the survival of bacteriophage T7 damaged by UV light or methyl methanesulfonate (MMS). Survival of T7 alkylated with MMS was not affected by the presence of plasmid pKM101 or by a umuC mutation in the host. The survival of UV light-irradiated T7 was similar in umuC+ and umuC strains but was slightly enhanced by the presence of pKM101. When phage survival was determined on host cells preirradiated with a single inducing dose of UV light, these same strains permitted higher survival than that seen with noninduced cells for both UV light- and MMS-damaged phage. The extent of T7 reactivation was approximately proportional to the UV light inducing dose inflicted upon each bacterial strain and was dependent upon phage DNA damage. Enhanced survival of T7 after exposure to UV light or MMS was also observed after thermal induction of a dnaB mutant. Thus, lethal lesions introduced by UV light or MMS are apparently repaired more efficiently when host cells are induced for the SOS cascade, and this inducible reactivation of T7 is umuC+ independent.
Identification of the pifC gene and its role in negative control of F factor pif gene expression
Authors: Miller J F , Malamy M H
Published: January 2020

The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection. We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene. A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis. This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map. Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB.
Control of triglyceride synthesis by the intracellular level of long-chain acyl coenzyme A for lipid synthesis
Authors: Kamiryo T
Published: January 2020

Candida lipolytica mutants defective in acyl coenzyme A synthetase I synthesized triglyceride to a markedly less extent than did the wild-type yeast, when grown on oleic acid. The synthesis of triglyceride was controlled by the level of long-chain acyl coenzyme A available for lipid synthesis, whereas the synthesis of phospholipids was hardly affected.
Isolation and characterization of chain-terminating nonsense mutations in a porin regulator gene, envZ
Authors: Garrett S , Taylor R K , Silhavy T J
Published: January 2020

We isolated four independent amber mutations in gene envZ, whose product is involved in the regulation of porin protein genes ompF and ompC. The envZ amber strains exhibit an OmpF-/+ OmpC- porin phenotype and express other envelope protein genes at wild-type levels. This phenotype is clearly different from that of the previously isolated class of envZ mutants that exhibit an OmpF- OmpC+ phenotype and a pleiotropic decrease in the expression of several exported protein genes, including lamB and phoA. The addition of the local anesthetic procaine to wild-type strains also causes a pleiotropic decrease in the expression of genes ompF, lamB, and phoA. However, procaine has no effect on the synthesis of LamB or PhoA protein in the envZ amber strains. Thus, although EnvZ protein is required for the full expression of ompF and ompC, it apparently is not normally involved in the expression of other envelope protein genes. One interpretation of these results is that the EnvZ protein can be altered either by mutation or by procaine to a form that interferes with the expression of several envelope protein genes other than ompF and ompC. Finally, complementation analysis with ompR insertion mutations supports the physical data of Mizuno et al. (J. Biol. Chem 257:13692-13698, 1982) that suggest that envZ is cotranscribed with ompR from a single promoter in the order ompR envZ.
Postincision steps of photoproduct removal in a mutant of Bacillus cereus 569 that produces UV-sensitive spores
Authors: Weinberger S , Evenchick Z , Hertman I
Published: January 2020

An excision-defective mutant of Bacillus cereus 569 is normal in incision and repair synthesis, but rejoining of incision breaks is defective, resulting in accumulation of low-molecular-weight DNA after UV irradiation. The defect in removal of photoproducts by exonuclease after incision renders both vegetative cells and dormant spores of the mutant sensitive to UV. A similarity is indicated to the uvrD mutation described recently in Escherichia coli.
Mapping the xyl, mtl, and lct loci in Escherichia coli K-12
Authors: Lin R J , Hill C W
Published: January 2020

We report a correction to the Escherichia coli K-12 genetic map. The order of genes near minute 80 was found to be xyl-mtl-lct, not xyl-lct-mtl. The mapping utilized two different Tn10 insertion mutations, zia-138::Tn10 between xyl and mtl and zib-137::Tn10 distal to lct.
Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum, and R. meliloti
Authors: Djordjevic M A , Zurkowski W , Shine J , Rolfe B G
Published: January 2020

Two self-transmissible Sym(biosis) plasmids, one encoding pea-specific nodulation and nitrogen-fixation functions (plasmid pJB5JI) and the other encoding clover-specific nodulation and nitrogen-fixation functions (plasmid pBR1AN) were used to determine whether the symbiotic genes encoded on these plasmids are expressed in various members of the Rhizobiaceae. The host specificity of Rhizobium trifolii and R. leguminosarum Sym plasmid-cured strains could be directly determined by the transfer to these strains of the appropriate Sym plasmid. The nodulation of white clovers was restored by either plasmid pJB5JI or pBR1AN when these plasmids were transferred to two transposon Tn5-induced hair-curling (Hac-) R. trifolii mutants. In addition, lucerne nodulation was restored to a Hac- R. meliloti mutant when either plasmid pBR1AN or pJB5JI was transferred to this strain. The phenotype of nonmucoid (Muc-) Rhizobium mutants, which had altered cell surfaces, was not influenced by the transfer to these strains of plasmid pBR1AN or plasmid pJB5JI.
Rates of peptidoglycan turnover and cell growth of Bacillus subtilis are correlated
Authors: Cheung H Y , Vitković L , Freese E
Published: January 2020

Peptidoglycan turnover was measured by the decrease of trichloroacetic acid-precipitable label in cells labeled with N-acetyl-D-[14C]glucosamine. The rate of turnover was reduced strongly by the inhibition of RNA or protein synthesis and weakly by the inhibition of lipid, peptidoglycan, or DNA synthesis. It increased with the growth rate (which was controlled by the concentration of oxomethylvalerate limiting the intracellular isoleucine supply) to the same degree in stringent (rel+) and isogenic relaxed (relA) strains. In these and all other strains tested, the turnover rate (k) increased with the growth rate (g) according to the equation, k = 0.70 X g1.38, even when the growth rate was systematically altered by changes in the temperature or in the composition of the medium.
Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions
Authors: Fleming T P , Nahlik M S , McIntosh M A
Published: January 2020

The vector Mu d(Apr lac) was utilized to construct operon fusions in the Escherichia coli enterobactin (ent) biosynthetic and transport genes. Enzyme assays indicated a 5- to 15-fold increase in the expression of beta-galactosidase when the fusion strains were grown under iron-deficient conditions. The polarity effects seen by Mu d insertions into entA, entC, and entE were consistent with a single operon, entA(CGB)E. The direction of transcription from iron-regulated promoters was determined by directional transfer of selected genetic markers after the insertion of F'ts114 lac+. Regulatory mutants were isolated in the fusion strains by the selection for constitutive expression of beta-galactosidase and the iron-regulated outer membrane proteins.
Protein turnover in Azotobacter vinelandii during encystment and germination
Authors: Ruppen M E , Garner G , Sadoff H L
Published: January 2020

Protein turnover occurs during differentiation of Azotobacter vinelandii 12837 to the extent of 50% during encystment and 7% during germination. The addition of rifampin at the initiation of encystment prevents encystment and inhibits turnover. In germinating cysts, protein turnover is essential owing to an apparent lack of certain amino acid biosynthetic enzymes. The capacity to synthesize sulfur-containing amino acids from inorganic precursors is regained about halfway through the germination process.
Effect of cerulenin on macromolecule synthesis in chemoheterotrophically and photoheterotrophically grown Rhodopseudomonas sphaeroides
Authors: Shepherd W D , Kaplan S
Published: January 2020

The antibiotic cerulenin causes the immediate cessation of phospholipid biosynthesis in both chemoheterotrophic and photoheterotrophic cultures of Rhodopseudomonas sphaeroides. Macromolecule biosynthesis in photoheterotrophic cells was unaffected by cerulenin for the first 2 h after antibiotic addition and then continued at a reduced rate for an additional 8 h. In contrast, macromolecule biosynthesis in chemoheterotrophic cells was severely affected by cerulenin within the first 2 h of treatment. Pulse-labeling of protein after cerulenin addition revealed that all subcellular fractions were equally affected by the action of cerulenin with chemoheterotrophic cell fractions more profoundly affected than those derived from photoheterotrophic cells. Protein insertion into the intracytoplasmic membrane of photoheterotrophic cells continued for up to 6 h after the onset of cerulenin treatment. Residual macromolecule synthesis was correlated with the presence of the photosynthetic membrane system under all conditions of growth.
L-Sorbose metabolism in Klebsiella pneumoniae and Sor+ derivatives of Escherichia coli K-12 and chemotaxis toward sorbose
Authors: Sprenger G A , Lengeler J W
Published: January 2020

L-Sorbose degradation in Klebsiella pneumoniae was shown to follow the pathway L-sorbose leads to L-sorbose-1-phosphate leads to D-glucitol-6-phosphate leads to D-fructose-6-phosphate. Transport and phosphorylation of L-sorbose was catalyzed by membrane-bound enzyme IIsor of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system, specific for and regulated by this ketose and different from all other enzymes II described thus far. Two soluble enzymes, an L-sorbose-1-phosphate reductase and a D-glucitol-6-phosphate dehydrogenase, were involved in the conversion of L-sorbose-1-phosphate to D-fructose-6-phosphate. This dehydrogenase was temperature sensitive, preventing growth of wild-type strains of K. pneumoniae at temperatures above 35 degrees C in the presence of L-sorbose. The enzyme was distinct from a second D-glucitol-6-phosphate dehydrogenase involved in the metabolism of D-glucitol. The sor genes were transferred from the chromosome of nonmotile strains of K. pneumoniae by means of a new R'sor+ plasmid to motile strains of Escherichia coli K-12. Such derivatives not only showed the temperature-sensitive Sor+ phenotype characteristic for K. pneumoniae or Sor+ wild-type strains of E. coli, but also reacted positively to sorbose in chemotaxis tests.
Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature
Authors: Brooks S J , Collins J J , Brill W J
Published: January 2020

The effect of elevated growth temperature on nif mRNA synthesis, nif protein synthesis, and nitrogenase activity was investigated in wild-type and NifL- strains of Klebsiella pneumoniae. Nitrogenase activity is not affected at 41 degrees C; however, nitrogenase is not synthesized at that temperature. Transcription of three nif operons studied is repressed at 41 degrees C. We show that the nifL protein is not required for repression by high temperature and propose that repression of nif at 41 degrees C results from a reversible inactivation of the nifA gene product.
Purification and characteristics of a gamma-glutamyl kinase involved in Escherichia coli proline biosynthesis
Authors: Smith C J , Deutch A H , Rushlow K E
Published: January 2020

gamma-Glutamyl kinase, the first enzyme of the proline biosynthetic pathway, was purified to homogeneity from an Escherichia coli strain resistant to the proline analog 3,4-dehydroproline. The enzyme had a native molecular weight of 236,000 and was apparently comprised of six identical 40,000-dalton subunits. Enzymatic activity of the protein was detectable only in assays containing highly purified gamma-glutamyl phosphate reductase, the second enzyme of the proline pathway. Plots of gamma-glutamyl kinase activity as a function of glutamate concentration were sigmoidal, with a half-saturation value for glutamate of 33 mM, whereas plots of enzyme activity as a function of ATP concentration displayed typical Michaelis-Menten kinetics with a Km for ATP of 4 X 10(-4) M. Enzyme activity was insensitive to the glutamate analog L-methionine-DL-sulfoximine, but ADP was a potent competitive inhibitor. Characteristics of the enzyme were compared with those of a gamma-glutamyl kinase partially purified from a 3,4-dehydroproline-sensitive E. coli. These results indicated that the only major difference was that the enzyme from the 3,4-dehydroproline-resistant strain was 100-fold less sensitive to feedback inhibition by proline.
Genetic and molecular analyses of Escherichia coli K1 antigen genes
Authors: Silver R P , Vann W F , Aaronson W
Published: January 2020

The plasmid pSR23, composed of a 34-kilobase E. coli chromosomal fragment inserted into the BamHI site of the pHC79 cosmid cloning vector, contains genes encoding biosynthesis of the K1 capsular polysaccharide. Deletions, subclones, and Tn5 insertion mutants were used to localize the K1 genes on pSR23. The only deletion derivative of pSR23 that retained the K1 phenotype lacked a 2.7-kilobase EcoRI fragment. Subclones containing HindIII and EcoRI fragments of pSR23 did not produce K1. Cells harboring pSR27, a subclone containing a 23-kilobase BamHI fragment, synthesized K1 that was not detectable extracellularly. Six acapsular Tn5 insertion mutants of three phenotypic classes were observed. Class I mutants synthesized K1 only when N-acetylneuraminic acid (NANA) was provided in the medium. Reduced amounts of K1 were detectable in cell extracts of class II mutants. Class III mutants did not produce detectable K1 in either extracts or when cells were provided exogenous NANA. All mutants had sialyltransferase activity. Analysis in the E. coli minicell system of proteins expressed by derivatives of pSR23 identified a minimum of 12 polypeptides, ranging in size from 18,000 to 80,000 daltons, involved in K1 biosynthesis. The 16-kilobase coding capacity required for the proteins was located in three gene clusters designated A, B, and C. We propose that the A cluster contains a NANA operon of two genes that code for proteins with apparent molecular weights of 45,000 and 50,000. The A region also includes a 2-kilobase segment involved in regulation of K1 synthesis. The B region encoding five protein species appears responsible for the translocation of the polymer from its site of synthesis on the cytoplasmic membrane to the cell surface. The C region encodes four protein species. Since the three gene clusters appear to be coordinately regulated. we propose that they constitute a kps regulon.
Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol
Authors: Oosawa K , Imae Y
Published: January 2020

The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli. Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III. The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell. The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other. Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol. These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs. Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.
Components of the regular surface array of Aquaspirillum serpens MW5 and their assembly in vitro
Authors: Kist M L , Murray R G
Published: January 2020

The two-layered regular surface array of Aquaspirillum serpens MW5 was removed from cell envelopes and dissociated into subunits by treatment with 6 M urea. The surface components reassembled onto an outer membrane surface and self-assembled into planar sheets in vitro in the presence of Ca2+ or Sr2+. The two layers were removed sequentially from cell envelopes by a two-step extraction procedure involving initial treatment with a high-pH buffer to remove the outermost surface layer and subsequent treatment with 6 M urea to remove the innermost layer. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the outer and inner layers of the array were composed of two proteins with molecular weights of 125,000 and 150,000, respectively. The two layers assembled sequentially; the 150,000-molecular-weight protein formed an array on an outer membrane surface, and the 125,000-molecular-weight protein required that array as a template for its in vitro assembly.
Physical map of Salmonella typhimurium LT2 DNA in the vicinity of the proA gene
Authors: Riley M , O'Reilly C , McConnell D
Published: January 2020

More than 55 kilobases of chromosomal DNA of Salmonella typhimurium LT2, including the gpt, proA, ataA, and newD genes, were cloned in plasmid vector pULB113. The locations of the genes and selected restriction endonuclease cleavage sites were established, and some of the restriction enzyme fragments were subcloned in plasmid vector pBR322.
Recombination between short direct repeats in Streptomyces lavendulae plasmid DNA
Authors: Nakano M M , Ogawara H , Sekiya T
Published: January 2020

Streptomyces lavendulae S985 carried two plasmids, pSL1 and pSL2. pSL2 contained all of the pSL1 sequences plus a tandem duplication of 900 base pairs from a region of pSL1. Sequence analysis of the duplication junction suggested that the duplication occurred by recombination between short direct repeats of as little as 5 base pairs.
Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum
Authors: Meissner P S , Falkinham J O
Published: January 2020

A Chesapeake Bay water isolate of Mycobacterium scrofulaceum containing a 115-megadalton plasmid (pVT1) grew in the presence of 100 microM HgCl2 and converted soluble 203Hg2+ to volatile mercury at a rate of 50 pmol/10(8) cells per min. Cell extracts contained a soluble mercuric reductase whose activity was not dependent on exogenously supplied thiol compounds. The enzyme displayed nearly identical activity when either NADH or NADPH served as the electron donor. A spontaneously cured derivative lacking pVT1 failed to grow in the presence of 100 microM HgCl2 and possessed no detectable mercuric reductase activity.
Enzyme III stimulation of cyclic AMP synthesis in an Escherichia coli crp mutant
Authors: Daniel J
Published: January 2020

Cyclic AMP (cAMP) synthesis in Escherichia coli is altered in cAMP receptor protein mutants and in phosphoenolpyruvate:sugar phosphotransferase transport system mutants. The stimulation of cAMP synthesis observed in cAMP receptor protein-deficient mutants is largely dependent upon enzyme III of the phosphoenolpyruvate:sugar phosphotransferase transport system. The phosphoenolpyruvate:sugar phosphotransferase transport system enzyme I is not required for elevated cAMP synthesis. These results suggest that enzyme III plays an important role in regulating adenylate cyclase activity.
Functional limits of the araIc promoter suggest an additional regulatory site for araBAD expression
Authors: Horwitz A H , Miyada C G , Wilcox G
Published: January 2020

The araBAD promoter is defined, in part, by two types of cis-acting constitutive mutations, araIc at position -35 and araXc at position -10. Subcloning experiments demonstrated that the araIc and araIcXc promoters require DNA sequence information out to position -53 to -56 for maximum constitutive expression. This is 8 to 10 base pairs more DNA than is generally thought to be necessary for RNA polymerase interaction. The -53 to -56 region is required for glucose repression, suggesting that an additional factor interacts in this region and is necessary for maximum expression.
Effect of growth temperature on the acquisition of iron by Salmonella typhimurium and Escherichia coli
Authors: Worsham P L , Konisky J
Published: January 2020

We have examined the effect of growth temperature on three systems normally induced under conditions of iron limitation: synthesis of the siderophore enterochelin (enterobactin), transport of ferric enterochelin, and production of the outer membrane protein which serves as the colicin I receptor. We found that although Salmonella typhimurium produces less enterochelin when grown at 42 degrees C, synthesis of this siderophore was not diminished in Escherichia coli grown under the same conditions. Growth at 42 degrees C under a condition of iron stress led to a reduction in the ability of cells to transport ferric enterochelin in both organisms. A two- to threefold decrease in the number of colicin I receptors was observed in cells of E. coli or S. typhimurium grown at 42 degrees C as compared with the number of receptors observed in cells grown at 37 degrees C. The colicin I receptor was shown not to be inherently unstable at 42 degrees C. By using a cir-lacZ operon fusion, it was shown that at least part of the decrease in receptor levels found in cells grown at high temperature was the result of decreased transcription of cir, the receptor structural gene. The effect of growth temperature on these systems was shown to be independent of fur, a regulatory element which mediates their enhanced production in response to iron stress. We suggest that a second regulatory element common to gene products involved in iron sequestration may be responsible for temperature regulation of these systems.
Physical map of the chromosomal region of Corynebacterium diphtheriae containing corynephage attachment sites attB1 and attB2
Authors: Rappuoli R , Ratti G
Published: January 2020

The chromosome of Corynebacterium diphtheriae C7 was recently shown to contain two equivalent attachment sites (attB1 and attB2) for lysogenization by corynephages (R. Rappuoli, J.L. Michel, and J.R. Murphy, J. Bacteriol. 153:1202-1210, 1983). Portions of bacterial chromosome containing each attB site, as well as a 3.5-kilobase (kb) EcoRI fragment containing both attB1 and attB2 sites, were cloned in the pUC8 plasmid vector. Restriction endonuclease mapping and Southern blot hybridization analysis of restriction endonuclease fragments showed that attB1 and attB2 are 2.25 kb apart on the chromosome. Furthermore, a 0.85-kb HincII-EcoRI restriction endonuclease fragment containing attB1, a 0.77-kb HincII-BamHI fragment containing attB2, and a 1.2-kb EcoRI-BamHI fragment containing attP share short homologous regions. No homology was detected between the sequences flanking the two attB sites. The isolation of a segregant which had lost the entire chromosomal segment contained between attB1 and attB2 suggests that this region is not essential for growth.
Physiology of a temperature-sensitive mutant of Saccharomyces cerevisiae defective in phosphofructokinase activity
Authors: Banerjee S , Getz G S , Garg M
Published: January 2020

In this paper, we describe a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae (P5-9) which at a restrictive temperature (36 degrees C) shows a pleiotropic defect for transport of many different metabolites. The temperature sensitivity of the mutant is closely related to a reduction in phosphofructokinase activity. This conclusion is based on the following criteria. (i) Both the primary isolate, designated P5-9 (ts [rho-] Ino-), which is an inositol auxotroph and respiration deficient, and a purified derivative, SB4 (ts [rho+] Ino+ ), which is respiration competent and capable of growing in the absence of inositol, are temperature sensitive for growth and ethanol production in media containing glucose or fructose as the sole carbon source. (ii) The respiration-competent derivative SB4 is not temperature sensitive in media containing glycerol or glycerol-pyruvate; glucose inhibits its growth at 36 degrees C in these media. (iii) Assays of glycolytic enzymes in P5-9 and SB4 extracts, prepared from cells incubated for 1 to 2 h at 36 degrees C before harvesting, show selective reduction in phosphofructokinase activity. Analysis of tetrads derived from the cross of mutant and nonmutant haploids indicates that temperature sensitivity for growth is due to a single gene or to two closely linked genes. The biochemical analysis of spores from seven such tetrads revealed a uniform cosegregation of temperature sensitivity for growth and phosphofructokinase activity. Transport and ATP levels were drastically reduced in SB4 cells incubated at 36 degrees C for 1 to 2 h with glucose as the carbon source, but not when glycerol-pyruvate or lactate was the energy source. Therefore, depletion of energy as a result of phosphofructokinase inactivation appears to be the cause of the pleiotropic transport defect observed in the mutant.
Elemental composition of bacterial metachromatic inclusions determined by electron microprobe X-ray analysis
Authors: Webster J A , Fay D D , Costa J L , Jones P M , Hugh R
Published: January 2020

Electron microscopy and microprobe X-ray analysis were used to study metachromatic inclusions of Spirillum itersonii , Corynebacterium diphtheriae, and Micrococcus luteus. In situ metachromatic inclusions were electron dense and contained phosphorus and divalent cations. Metachromatic inclusions isolated by anion-exchange column chromatography and by isoosmolar Metrizamide density gradient centrifugation were similar in composition to in situ inclusions.
A Mu gin complementing function and an invertible DNA region in Escherichia coli K-12 are situated on the genetic element e14
Authors: van de Putte P , Plasterk R , Kuijpers A
Published: January 2020

The Gin product catalyzes an inversion of 3,000 base pairs of DNA in the genome of bacteriophage Mu. The orientation of the invertible of G-region determines the host range of the phage. Gin- mutants are complemented by a host function in strain HB101 and several other Escherichia coli K-12 strains. At least three clones in the E. coli gene bank described previously (L. Clarke and J. Carbon, Cell 9:91-99, 1976) contained the gin complementing function. This function, which we named pin, catalyzes an inversion of 1,800 base pairs in the adjacent DNA. The invertible region, named the P-region, together with pin, was further subcloned on pBR322. Conjugation and transduction experiments mapped the pin gene between the genes purB and fabD near position 25 on the E. coli chromosome. Also situated in this region is e14, a cryptic, UV- excisable , genetic element (A. Greener and C.W. Hill, J. Bacteriol . 144:312-321, 1980). We demonstrated that pin and the P-region are part of e 14. The e 14 element was cloned on pBR322 by genetic manipulation techniques in vivo. It has the properties of a defective prophage containing integration and excision functions and a SOS-sensitive repressor.
Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis
Authors: Riley M I , Dickson R C
Published: January 2020

We isolated and identified mutant strains of Kluyveromyces lactis that are defective for the Leloir pathway enzymes galactokinase, transferase, and epimerase, and we termed these loci GAL1 , GAL7 , and GAL10 , respectively. Genetic data indicate that these three genes are tightly linked, having an apparent order of GAL7 - GAL10 - GAL1 . This same gene order has been observed in Saccharomyces cerevisiae. Strains harboring gal7 mutations have elevated levels of beta-galactosidase, coded by an unlinked gene, galactokinase, and epimerase activities under uninduced conditions. We investigated the genetic basis of this constitutive gene expression and found no recombinants between the constitutive and Gal- phenotypes among 76 tetrads, suggesting that either GAL7 or a tightly linked gene codes for a regulatory function. This is the second gene that has been shown to specifically coregulate expression of the genes coding for beta-galactosidase and the Leloir pathway enzymes.
Genetic mapping of pheU, an Escherichia coli gene for phenylalanine tRNA
Authors: Gallagher P J , Schwartz I , Elseviers D
Published: January 2020

We report the genetic mapping of pheU , an Escherichia coli gene for phenylalanine tRNA. This gene was located near 94.5 min on the E. coli map. There are no other known tRNA or ribosomal genes in its immediate vicinity.
Rhizobium japonicum nitrogenase Fe protein gene (nifH)
Authors: Fuhrmann M , Hennecke H
Published: January 2020

A 12.1-kilobase PstI fragment from Rhizobium japonicum, which contains homology to both the Klebsiella pneumoniae and the Rhizobium meliloti nifH genes, was cloned into vector pHE3 . The nifH -homologous region was localized on the restriction enzyme cleavage map by Southern blot hybridization experiments. DNA fragments overlapping the R. japonicum nifH gene were subcloned into plasmid vectors to allow the expression of this region in Escherichia coli minicells. The nifH gene product (the polypeptide of the nitrogenase Fe protein) was thus found to have a molecular weight of 33,000. The complete nucleotide sequence of the nifH gene was established, and the amino acid sequence of its gene product was deduced. The reading frame is 882 nucleotides long, corresponding to 294 amino acids which add up to a polypeptide with a molecular weight of 31,525. There was extensive sequence homology with nifH genes or gene products from other nitrogen-fixing bacteria. The transcription initiation site of the R. japonicum nifH gene was found to lie 153 nucleotides upstream from the coding region and was preceded by a characteristic promoter sequence. A potential terminator region was located 13 nucleotides downstream from the coding region.
Heterogeneity of Rhizobium lipopolysaccharides
Authors: Carlson R W
Published: January 2020

The lipopolysaccharides ( LPSs ) from strains of Rhizobium leguminosarum, Rhizobium trifolii, and Rhizobium phaseoli were isolated and partially characterized by mild acid hydrolysis and by polyacrylamide gel electrophoresis. Mild acid hydrolysis results in a precipitate which can be removed by centrifugation or extraction with chloroform. The supernatant contains polysaccharides which, in general, are separated into two fractions ( LPS1 and LPS2 ) by Sephadex G-50 gel filtration chromatography. The higher-molecular-weight LPS1 fractions among the various Rhizobium strains are highly variable in composition and reflect the variability reported in the intact LPSs (R. W. Carlson and R. Lee, Plant Physiol. 71:223-228, 1983; Carlson et al., Plant Physiol. 62:912-917, 1978; Zevenhuizen et al., Arch. Microbiol. 125:1-8, 1980). The LPS1 fraction of R. leguminosarum 128C53 has a higher molecular weight than all other LPS1 fractions examined. All LPS2 fractions examined are oligosaccharides with a molecular weight of ca. 600. The major sugar component of all LPS2 oligosaccharides is uronic acid. The LPS2 compositions are similar for strains of R. leguminosarum and R. trifolii, but the LPS2 from R. phaseoli was different in that it contained glucose, a sugar not found in the other LPS2 fractions or found only in trace amounts. Polyacrylamide gel electrophoretic analysis shows that each LPS contains two banding regions, a higher-molecular-weight heterogeneous region often containing many bands and a lower-molecular-weight band. The lower-molecular-weight bands of all LPSs have the same electrophoretic mobility, which is greater than that of lysozyme. The banding pattern of the heterogeneous regions varies among the different Rhizobium strains. In the case of R. leguminosarum 128C53 LPS, the heterogeneous region of a higher molecular weight than is this region from all other Rhizobium strains examined and consists of many bands separated from one another by a small and apparently constant molecular weight interval. When the heterogeneous region of R. Leguminosarum 128C53 LPS was cut from the gel and analyzed, its composition was found to be that of the intact LPS, whereas the lower-molecular-weight band contains only sugars found in the LPS2 oligosaccharide. In the case of R. leguminosarum 128C63 and R. trifolii 0403 LPSs, the heterogeneous regions are similar and consist of several band s separated by a large-molecular-weight interval with a the major band of these heterogeneous regions having the lowest molecular weight with an electrophoretic mobility near that of beta-lactoglobulin. The heterogeneous region from R. phaseoli 127K14 consists of several bands with electrophoretic mobilities near that of beta-lactoglobulin, whereas this region from R. trifolii 162S7 shows a continuous staining region, indicating a great deal of heterogeneity. The results described in this paper are discussed with regard to the reported properties of Escherichia coli and Salmonella LPSs.
Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13
Authors: Lehrbach P R , Zeyer J , Reineke W , Knackmuss H J , Timmis K N
Published: January 2020

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .
Virulence plasmid-associated autoagglutination in Yersinia spp
Authors: Skurnik M , Bölin I , Heikkinen H , Piha S , Wolf-Watz H
Published: January 2020

The autoagglutination of Yersinia enterocolitica was dependent on the presence of the virulence plasmid and on the active growth of bacteria in tissue culture media at 37 degrees C. Cultures with a high initial concentration of bacteria failed to autoagglutinate , indicating that synthesis of new virulence plasmid-associated surface factors was essential for autoagglutination. The synthesis of a plasmid-encoded polypeptide (molecular weight, 240,000), designated P1, that could be dissociated under strongly reducing conditions into subunits of 52,500 daltons was found to be correlated with autoagglutination. Further, a strain of Yersinia pseudotuberculosis [ YPIII ( PIB102 )], which has Tn5 inserted within the structural gene of P1 that prevents the synthesis of P1, failed to autoagglutinate , in contrast to the wild-type strain, strongly indicating that P1 is involved in this phenomenon. It was also found by immunoblotting that in addition to the common response to temperature, the P1 proteins of Y. enterocolitica and Y. pseudotuberculosis were immunologically related.
Streptococcus faecalis R plasmid pJH1 contains an erythromycin resistance transposon (Tn3871) similar to transposon Tn917
Authors: Banai M , LeBlanc D J
Published: January 2020

The R plasmid pJH1 contains a 5.1-kilobase transposon ( Tn3871 ) that mediates inducible resistance to erythromycin. Three AvaI digestion fragments from this transposon are identical in size to and homologous with three AvaI-derived fragments from the previously described erythromycin resistance transposon Tn917 . These three DNA fragments account for greater than 90% of both transposons.
Detection and characterization of Tn2501, a transposon included within the lactose transposon Tn951
Authors: Michiels T , Cornelis G
Published: January 2020

The DNA sequence spanning coordinates 9.9 to 16.4 kilobases of the lactose transposon Tn951 ( Cornelis et al., Mol. Gen. Genet. 160:215-224, 1978) constitutes a transposable element by itself. Unlike Tn951 ( Cornelis et al., Mol. Gen. Genet. 184:241-248, 1981), this element, called Tn2501 , transposes in the absence of any other transposon. Transposition of Tn2501 proceeds through transient cointegration and duplicates 5 base pairs of host DNA. Tn2501 is flanked by nearly perfect inverted repeats (44 of 48), related to the inverted repeats of Tn21 ( Zheng et al., Nucleic Acids Res. 9:6265-6278, 1982). Unlike Tn21 , Tn2501 does not confer mercury resistance.
Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function
Authors: Sutton B C , Stanley J , Zelechowska M G , Verma D P
Published: January 2020

A first visible step in the nodulation of legumes by Rhizobium spp. is the deformation and curling of root hairs. We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti. Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera. Pseudomonas putida gave unambiguous expression of the root hair curling genes. This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain. The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R. japonicum strains. Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P. putida. We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans.
New mutation affecting the synthesis of some membrane proteins and sporulation in Bacillus subtilis
Authors: Matsuzaki S , Kobayashi Y
Published: January 2020

A new mutation, mpo, which affects the synthesis of some membrane proteins and sporulation in Bacillus subtilis was identified. The mpo mutation was tightly linked to the overproduction of membrane proteins MP32 and MP18 (molecular weights of 32,000 and 18,000, respectively) and the temperature-sensitive sporulation phenotype. Genetic analysis showed that the mpo mutation maps between the spoIIIB and lys loci.