bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme
Authors: Grabau C , Cronan J E
Published: January 2020

The pyruvate oxidase structural gene (poxB) of Escherichia coli was cloned into derivatives of plasmid pBR322. The gene was first cloned into a cosmid vector by selection for the tetracycline resistance determinant of a closely linked Tn10 insertion (no direct selection for the gene was available). Subsequent subcloning resulted in localization of the gene to a 3.1-kilobase-pair DNA segment. Two of the smaller poxB plasmids were shown to cause the overproduction of oxidase activity (by six- to eightfold), and one of these plasmids was shown to encode a protein having the size and antigenic determinants of pyruvate oxidase. Introduction of poxB plasmids into strains (aceEF) lacking pyruvate dehydrogenase activity relieved the aerobic growth requirement of the strains for exogenous acetate.
Genetic analysis of autolysin-deficient and flagellaless mutants of Bacillus subtilis
Authors: Pooley H M , Karamata D
Published: January 2020

Three mutants with an autolysin-deficient and flagellaless phenotype (lyt) were genetically analyzed and compared with three thermosensitive flagellaless mutants. In view of the near indistinguishability of their phenotypes, all six mutations were assigned to fla loci. They were distributed into four linkage groups, designated flaA through flaD. flaA and flaB map between pyrD and thyA, flaD maps between aroD and lys, and, in agreement with a previous report, flaC maps near hisA. A locus associated with hypermotility, ifm-3, maps near the latter marker. Introduction of ifm-3 into lyt-1- and flaA4-containing strains led to partial suppression of the nonmotile phenotype. We discuss the possibility that the cellular concentration of autolysins is regulated by the expression of fla genes. Discrepancies with respect to previous mapping of flaA and flaB are accounted for.
Genetic analysis of the low calcium response in Yersinia pestis mu d1(Ap lac) insertion mutants
Authors: Goguen J D , Yother J , Straley S C
Published: January 2020

Yersinia pestis strain KIM requires plasmid pCD1 for expression of the low calcium response, plague virulence antigen V, and virulence. We constructed Mu d1(Ap lac) insertion mutants of this plasmid which were unable to express the low calcium response. The insertions mapped to a 17-kilobase region of the plasmid. By determining the orientation of the insertions and examining beta-galactosidase production from the Mu d1 lac genes, we determined that this region contains three units of transcription, one of which is transcribed in a direction opposite the direction of transcription of the other two. Transcription of at least two of these units was induced significantly at 37 degrees C compared with 26 degrees C. Ca2+ (2.5 mM) and ATP (18 mM) had no significant effect on the level of expression of the Mu d1 lac genes of these mutants. All insertions in the region strongly reduced production of the V antigen. Insertions from each unit of transcription also reduced virulence in mice.
Defective plasma membrane assembly in yeast secretory mutants
Authors: Tschopp J , Esmon P C , Schekman R
Published: January 2020

Yeast mutants that are conditionally blocked at distinctive steps in secretion and export of cell surface proteins have been used to monitor assembly of integral plasma membrane proteins. Mutants blocked in transport from the endoplasmic reticulum (sec18), from the Golgi body (sec7 and sec14), and in transport of secretory vesicles (sec1) show dramatically reduced assembly of galactose and arginine permease activities. Simultaneous induction of galactose permease and alpha-galactosidase (a secreted glycoprotein) in sec mutant cells at the nonpermissive temperature (37 degrees C) shows that both activities accumulate and can be exported coordinately when cells are returned to the permissive temperature (24 degrees C) in the presence or absence of cycloheximide. Plasma membrane fractions isolated from sec mutant cells radiolabeled at 37 degrees C have been analyzed by two-dimensional sodium dodecyl sulfate-gel electrophoresis. Although most of the major protein species seen in plasma membranes from wild-type cells are not efficiently localized in sec18 or sec7, several of these proteins appear in plasma membranes from sec1 cells. These results may be explained by contamination of plasma membrane fractions with precursor vesicles that accumulate in sec1 cells. Alternatively, some proteins may branch off during transport along the secretory pathway and be inserted into the plasma membrane by a different mechanism.
Release of respiratory control in Escherichia coli after bacteriophage adsorption: process independent of DNA injection
Authors: Letellier L , Labedan B
Published: January 2020

Adsorption of phages T4, T5, and BF23 to previously starved Escherichia coli cells triggered the immediate release of respiratory control. A similar stimulation of respiration was induced after T4 ghost attachment, showing that this process was independent of the mechanism of DNA injection. Rather, this change in the respiratory rate was related to the transient depolarization of the cytoplasmic membrane also induced after phage and ghost adsorption. Both processes were suppressed by addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the case of T4 (phage and ghosts) but not of T5 and BF23. The increase in respiratory rate observed after phage adsorption was of a magnitude similar to that induced by protonophores. Since other treatments that depolarize the membrane without a massive proton influx did not increase the rate of respiration of starved bacteria with the same efficiency, these results suggest that phage adsorption induced an entry of protons into the cell cytoplasm.
Structural modifications in the peptidoglycan of Escherichia coli associated with changes in the state of growth of the culture
Authors: Pisabarro A G , de Pedro M A , Vázquez D
Published: January 2020

By determining the composition in muropeptides of the murein of a number of strains of Escherichia coli, purified from cells at various states of growth, the sacculus was found to be considerably modified when cells stop active growth. Murein from resting cells becomes hypercross-linked and richer in covalently bound lipoprotein, whereas the mean length of the glycan chains is considerably reduced. The alteration of the sacculus occurs progressively during the transitions from active growth to stationary phase and vice versa.
Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger
Authors: Meixner-Monori B , Kubicek C P , Habison A , Kubicek-Pranz E M , Röhr M
Published: January 2020

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.
Cloning of rfaG, B, I, and J genes for glycosyltransferase enzymes for synthesis of the lipopolysaccharide core of Salmonella typhimurium
Authors: Kadam S K , Rehemtulla A , Sanderson K E
Published: January 2020

R-prime plasmids carrying the pyrE-rfa-cysE region of the chromosome of Salmonella typhimurium were isolated by using the vector pULB113 (RP4::mini-Mu). One of the R-prime plasmids was used as a source of DNA to clone the rfa genes for lipopolysaccharide synthesis to pBR322. The following three hybrid plasmids were constructed: pKZ15, with a 4.0-kilobase EcoRI fragment of S. typhimurium DNA, containing the rfaG gene; pKZ27, a 9-kilobase BglII fragment with the rfaG, rfaB, and rfaI genes; and pKZ26, a 7.7-kilobase HindIII fragment with the rfaG, rfaB, rfaI, and rfaJ genes. We propose that these cloned genes code for four glycosyltransferases used for synthesis of the lipopolysaccharide core region (rfaG for glucosyltransferase I; rfaI for galactosyltransferase I; rfaB for galactosyltransferase II; and rfaJ for glucosyltransferase II). For all four genes, mutants which lacked the appropriate enzyme activity were complemented by the plasmids to give completed core lipopolysaccharide with O (somatic) side chains; for rfaG, rfaB, and rfaI, mutants gave restored or even amplified levels of the appropriate glycosyltransferase in in vitro assays. We show that the order of genes in the region is pyrE-rfaG-(rfaB-rfaI)-rfaJ-rfaL-rfaF -cysE.
Transport of AMP by Rickettsia prowazekii
Authors: Atkinson W H , Winkler H H
Published: January 2020

Rickettsia prowazekii possesses an exchange transport system for AMP. Chromatographic analysis of the rickettsiae demonstrated that transported AMP appeared intracellularly as AMP, ADP, and ATP, and no hydrolytic products appeared in either the intracellular or extracellular compartments. The phosphorylation of AMP to ADP and ATP was prevented by pretreatment of the cells with 1 mM N-ethylmaleimide without inhibiting the transport of AMP. Although no efflux was demonstrable in the absence of nucleotide in the medium, the intracellular adenine nucleotide pool could be exchanged with external unlabeled adenine nucleotides. Both ADP and ATP were as effective as AMP at inhibiting the uptake of [3H]AMP. Although this transport system was inhibited by low temperature (0 degrees C) and partially inhibited by the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (1 mM), it was relatively insensitive to KCN (1 mM). The uptake of AMP at 34 degrees C had an apparent Kt for influx of 0.4 mM and a Vmax of 354 pmol min-1 per mg. At 0 degrees C there was a very rapid and unsaturable association of AMP with these organisms. Correction of the uptake data at 34 degrees C for the 0 degrees C component lowered the apparent Kt to 0.15 mM. Both magnesium and phosphate ions are required for optimal transport activity. Chemical measurements of the total intracellular nucleotide pools demonstrated that this system was not a net adenine nucleotide transport system, but that uptake of AMP was the result of an exchange with internal adenine nucleotides.
Identification of pKM101-encoded loci specifying potentially lethal gene products
Authors: Winans S C , Walker G C
Published: January 2020

Two pKM101-encoded loci (designated kilA and kilB) have been identified which elaborate products that are potentially lethal to the bacterial cell. The lethal effects of each of these products is inhibited by two other plasmid-encoded loci, designated korA and korB (for kil override). Both korA and korB are required to control the lethality of either kil gene. In the presence of korA and korB both kil genes have other phenotypes: kilB is necessary for conjugal transfer, whereas kilA is responsible for the small-colony morphology on defined media that is characteristic of pKM101-containing strains (the Slo phenotype).
Isolation and properties of beta-glucosidase from Ruminococcus albus
Authors: Ohmiya K , Shirai M , Kurachi Y , Shimizu S
Published: January 2020

An enzyme active against p-nitrophenyl-beta-D-glucoside was purified from logarithmic-phase cells of Ruminococcus albus cultivated in a medium containing ball-milled cellulose. The purification yielded homogeneous enzyme after an approximately 520-fold increase in specific activity and a 9% yield. The enzyme was identified as a beta-glucosidase because it can hydrolyze cellobiose and cellooligosaccharides to glucose from the nonreducing ends.
Cloning, sequence analysis, and expression of alteration of the mRNA stability gene (ams+) of Escherichia coli
Authors: Chanda P K , Ono M , Kuwano M , Kung H
Published: January 2020

The ams+ gene, which influences the stability of mRNA in Escherichia coli was cloned in pBR322. The product of the gene, which is a 17,000-dalton protein, was expressed in expression vector pRC23, a derivative of pBR322. The molecular weight is consistent with sequencing analysis which shows that the gene contains 595 nucleotides and has an open reading frame of 149 amino acids. We discussed the possible role(s) of the ams+ gene product in affecting mRNA stability.
Polymorphic extracellular glucoamylase genes and their evolutionary origin in the yeast Saccharomyces diastaticus
Authors: Yamashita I , Maemura T , Hatano T , Fukui S
Published: January 2020

DNA coding for extracellular glucoamylase genes STA1 and STA3 was isolated from DNA libraries of two Saccharomyces diastaticus strains, each carrying STA1 or STA3. Cells transformed with a plasmid carrying either the STA1 or STA3 gene secreted glucoamylases having the same enzymatic and immunological properties and the same electrophoretic mobilities in acrylamide gel electrophoresis as those of authentic glucoamylases. Southern blot analysis of genomic DNA from S. diastaticus and a glucoamylase-non-secreting yeast, Saccharomyces cerevisiae, revealed that the STA1 and STA3 loci of S. diastaticus showed a high degree of homology, and that both yeast species (S. diastaticus and S. cerevisiae) contained DNA segments highly homologous to those of the extracellular glucoamylase genes. Restriction maps of the homologous DNA segments suggested that the extracellular glucoamylase genes of S. diastaticus may have arisen from recombination among the resident DNA segments in S. cerevisiae.
Amplification of a major membrane-bound DNA sequence of Bacillus subtilis
Authors: Sargent M G , Bennett M F
Published: January 2020

A membrane-bound DNA sequence from Bacillus subtilis was subcloned into a plasmid which can replicate in Escherichia coli but not in B. subtilis. This plasmid hybridized with an 11-kilobase HindIII fragment which is the major particle-bound fragment in lysates treated with HindIII. The plasmid integrated into the B. subtilis chromosome at the region of homology, conferring chloramphenicol resistance on the recipient. The inserted resistance was mapped close to purA by using the generalized transducing phage AR9. In one chloramphenicol-resistant strain, the pMS31 region was repeated at least 20 times. A large proportion of the copies of the cloned region were present in the particle fraction, indicating that the capacity to bind this region of the chromosome was substantially in excess of the normal dose of the region. The structure of the particle-bound region was sensitive to ionic detergents and high salt concentrations but was not greatly affected by RNase or ethidium bromide. The basis of a specific DNA-membrane interaction can now be studied by using the amplified region, without the complications of sequences required for autonomous plasmid replication.
Uptake of phenol by Trichosporon cutaneum
Authors: Mörtberg M , Neujahr H Y
Published: January 2020

The soil yeast Trichosporon cutaneum, which is distinguished by having a strictly oxidative metabolism, can be induced to utilize phenol as a sole carbon source. The present paper shows that such phenol-induced cells contain a specific, energy-dependent uptake system for phenol. Phenol uptake is not directly linked to its o-hydroxylation inside the cell, the first step of phenol metabolism. The Km for uptake is 235 +/- 30 microM, that for hydroxylation only 4.5 +/- 0.5 microM. Further, the phenol analog 2,6-dimethylphenol, which can not be hydroxylated, competes with phenol for the uptake system. The pH dependence of uptake indicates that phenolate is an essential form during the uptake process. The energy requirement for phenol uptake is indicated by effects of various inhibitors of energy generation, including proton-conducting uncouplers. Direct monitoring of proton movements in a pH-stat during phenol uptake indicates a phenol-proton symport. One proton is cotransported with every phenol molecule. Phenol competes with the uptake of sucrose and glycerol by cells grown on these substrates. Under such conditions the uptake of phenol seems to proceed through a different system, with lower affinity for phenol than in phenol-grown cells.
Enzymatic detection of phospholipid biosynthetic enzymes after electroblotting
Authors: Poole M A , Fischl A S , Carman G M
Published: January 2020

The membrane-associated enzymes phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase; EC 2.7.8.8) from Saccharomyces cerevisiae were detected enzymatically after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting. Enzyme activities were measured on nitrocellulose blots by using pure enzyme preparations as well as Triton X-100-solubilized membranes. Phosphatidylinositol synthase activity migrated to Mr 34,000, and phosphatidylserine synthase activity migrated to Mr 23,000.
Interaction of UAG suppressors and omnipotent suppressors in Saccharomyces cerevisiae
Authors: Song J M , Liebman S W
Published: January 2020

Haploids bearing the dominant UAG suppressor, SUP7-a, and various alleles of the omnipotent suppressor sup35 were examined. The presence of the UAG suppressor reduced the efficiency of some alleles of sup35, and caused other sup35 alleles to be lethal. A nonclassical interaction of the dominant suppressor tRNA and the ribosome is proposed to explain these observations.
Efficiency of induction of prophage lambda mutants as a function of recA alleles
Authors: Dutreix M , Bailone A , Devoret R
Published: January 2020

Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.
Exocellular esterase and emulsan release from the cell surface of Acinetobacter calcoaceticus
Authors: Shabtai Y , Gutnick D L
Published: January 2020

An esterase activity has been found, both in the cell-free growth medium and on the cell surface of the hydrocarbon-degrading Acinetobacter calcoaceticus RAG-1. The enzyme catalyzed the hydrolysis of acetyl and other acyl groups from triglycerides and aryl and alkyl esters. Emulsan, the extracellular heteropolysaccharide bioemulsifier produced by strain RAG-1, was also a substrate for the enzyme. Gel filtration showed that the cell-free enzyme was released from the cell surface either emulsan free or associated with the bioemulsifier. The partially purified enzyme was found to interact specifically with the esterified fully active emulsan, but not with the deesterified polymer. A role for esterase in emulsan release from the cell surface was indicated when the enzyme was preferentially depleted from the cell surface under conditions in which emulsan was not released. Such cells lost the capacity to release the biopolymer.
Stimulation of beta(1----3)glucan synthetase of various fungi by nucleoside triphosphates: generalized regulatory mechanism for cell wall biosynthesis
Authors: Szaniszlo P J , Kang M S , Cabib E
Published: January 2020

Particulate fractions from the taxonomically diverse fungi Achlya ambisexualis, Hansenula anomala, Neurospora crassa, Cryptococcus laurentii, Schizophyllum commune, and Wangiella dermatitidis were found to catalyze the time-dependent incorporation of glucose from UDP-[14C]glucose into a water-insoluble material. The reaction was stimulated by bovine serum albumin. The product was characterized as beta(1----3)glucan on the basis of its resistance to alpha- and beta-amylase and susceptibility to beta(1----3)glucanase. With the exception of the preparation from A. ambisexualis, all others were stimulated by nucleoside triphosphates and their analogs. The best activators were GTP and guanosine 5'-(gamma-thio)triphosphate. It is concluded that the stimulation by nucleotides, previously found with the glucan synthetase of Saccharomyces cerevisiae, is a regulatory mechanism that was well conserved during fungal evolution, presumably because of its importance in controlling cell wall biosynthesis and cell growth.
Structure of the core oligosaccharide from lipopolysaccharide of Erwinia carotovora
Authors: Sandulache R , Prehm P
Published: January 2020

The lipopolysaccharide of Erwinia carotovora was analyzed by quantitative sugar analysis, methylation analysis, and chromic oxide oxidation. This led to the following structure of the core oligosaccharide: (Formula; see text).
Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis
Authors: Nicholson W L , Chambliss G H
Published: January 2020

Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.
Mutations in the rpoH (htpR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma 70 subunit of RNA polymerase
Authors: Grossman A D , Zhou Y N , Gross C , Heilig J , Christie G E , Calendar R
Published: January 2020

Escherichia coli K-12 strain 285c contains a mutation in rpoD, the gene encoding the sigma subunit of RNA polymerase. The 70-kilodalton sigma polypeptide encoded by this allele is unstable, and this instability leads to temperature-sensitive growth. We describe the isolation and characterization of four temperature-resistant pseudorevertants of 285c that can grow at high temperature. Each of these revertants increased the stability of the sigma 70 mutant protein. The map position of the suppressor mutations was close to that of the rpoH (htpR) gene. A multicopy plasmid containing the intact rpoH gene restored the temperature-sensitive phenotype. Marker rescue experiments established the positions of three of the alleles within the rpoH gene. One mutation has been sequenced and causes a leucine-to-tryptophan change 7 amino acids from the carboxyl terminus of the rpoH gene product.
Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli
Authors: Chen L , Rhoads D , Tai P C
Published: January 2020

We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was the case not only with the OmpA protein, which is synthesized by free polysomes and hence is presumably exported posttranslationally in the cell, but also with alkaline phosphatase, which is synthesized only by membrane-bound polysomes and has been shown to be secreted cotranslationally in the cells. Prolonged incubation rendered the precursors inactive for subsequent translocation. Posttranslational translocation was impaired, like cotranslational translocation, by inhibitors of the proton motive force and by treatment of the vesicles with protease. Since it appears that E. coli can translocate the same proteins either cotranslationally or posttranslationally, the cotranslational mode may perhaps be more efficient, but not obligatory, for the secretion of bacterial proteins.
Locations of hook-associated proteins in flagellar structures of Salmonella typhimurium
Authors: Homma M , Iino T
Published: January 2020

Hooks of the flagella of Salmonella typhimurium were purified from an flaL mutant. Hook-associated proteins, namely HAP1, HAP2, and HAP3, were separated from them, and the antibody against each HAP was prepared. By immunoelectron microscopic observation, these three kinds of antiHAP antibodies were found to bind on the distal ends of hooks of filamentless mutants consistently with their composition of HAPs. The antiHAP2 antibody bound to the very tops of the claw-shaped ends of the hooks which contain all three HAPS. The antibodies against HAP1 and HAP3 bound to the basal areas and the middle areas, respectively, of the claw-shaped ends. The order of disassembly of the component proteins by heat treatment of the hook structure from the filamentless mutants was (HAP2, HAP3) greater than HAP1 greater than hook protein. These observations were consistent with our layered structure model: HAP1, HAP3, and HAP2 are assembled at the distal end of the hook in this sequence. All three HAPs were detected in the hook-filament complexes prepared from a flagellate strain. When the hook-filament structure was treated with antibody against HAP1 and with the anti-rabbit immunoglobulin G antibody, the antibody aggregate was observed in the region corresponding to the boundary between filament and hook. This observation strongly suggests that HAP1 is the protein connecting filament with hook. The locations of HAP2 and HAP3 in the hook-filament structure were not clarified with the same procedure.
Involvement of protein sulfhydryls in the trigger reaction of rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides
Authors: Miyakawa T , Kaji M , Yasutake T , Jeong Y K , Tsuchiya E , Fukui S
Published: January 2020

The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.
Duplication of Escherichia coli during inhibition of net phospholipid synthesis
Authors: Pierucci O , Rickert M
Published: January 2020

In Escherichia coli BB26-36, the inhibition of net phospholipid synthesis during glycerol starvation affected cell duplication in a manner that was similar in some respects to that observed during the inhibition of protein synthesis. Ongoing rounds of chromosome replication continued, and cells in the D period divided. The initiation of new rounds of chromosome replication and division of cells in the C period were inhibited. Unlike the inhibition of protein synthesis, however, the accumulation of initiation potential in dnaA and dnaC mutants at the nonpermissive temperature was not affected by the inhibition of phospholipid synthesis. Furthermore, proteins synthesized during the inhibition of phospholipid synthesis can be utilized later for division. The results are consistent with a dual requirement for protein and phospholipid synthesis for both the inauguration of new rounds of chromosome replication and the initiation of septum formation. Once initiated, both processes progress to completion independent of continuous phospholipid and protein synthesis.
Release of cell wall peptides into culture medium by exponentially growing Escherichia coli
Authors: Goodell E W , Schwarz U
Published: January 2020

Escherichia coli W7 cells were found to release three different muropeptides into the culture medium: tetrapeptide (L-Ala-D-Glu-meso-diaminopimelic acid-D-Ala), tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and a previously undescribed dipeptide (meso-diaminopimelic acid-D-Ala). From the rate of release of these three peptides, it was calculated that 6 to 8% of the murein in the sacculus was lost per generation.
High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA
Authors: Santamaria R I , Gil J A , Martin J F
Published: January 2020

An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained.
Cloning and expression in Escherichia coli of Vibrio parahaemolyticus thermostable direct hemolysin and thermolabile hemolysin genes
Authors: Taniguchi H , Ohta H , Ogawa M , Mizuguchi Y
Published: January 2020

Two hemolysin genes of Vibrio parahaemolyticus WP1, a thermostable direct (TSD) hemolysin gene and a thermolabile hemolysin gene, were cloned into the pBR322 vector in Escherichia coli K-12 C600. A large amount of the TSD hemolysin produced in E. coli K-12 accumulated in the periplasmic space. The TSD hemolysin gene was localized on a 0.9-kilobase HindIII-BamHI fragment by identifying qualitatively the production of the TSD hemolysin by a reverse passive hemagglutination assay in the osmotic shock fluid. The thermolabile hemolysin gene was isolated on a 1.3-kilobase HindIII-PstI fragment by selection with the hemolysin on blood agar. Southern blot hybridization and colony hybridization experiments indicated that the TSD hemolysin gene was present in the chromosomal DNA of 15 Kanagawa phenomenon-positive strains but not in 14 negative strains, whereas the thermolabile hemolysin gene was detected in all strains. No homologous DNA sequences to TSD and thermolabile hemolysin genes were detected in the chromosomes of Vibrio cholerae, Vibrio vulnificus, non-O1 V. cholerae, and Vibrio anguillarum.
H2, N2, and O2 metabolism by isolated heterocysts from Anabaena sp. strain CA
Authors: Smith R L , Kumar D , Zhang X K , Tabita F R , Van Baalen C
Published: January 2020

Metabolically active heterocysts isolated from wild-type Anabaena sp. strain CA showed high rates of light-dependent acetylene reduction and hydrogen evolution. These rates were similar to those previously reported in heterocysts isolated from the mutant Anabaena sp. strain CA-V possessing fragile vegetative cell walls. Hydrogen production was observed with isolated heterocysts. The ratio of C2H4 to H2 produced ranged from 0.9 to 1.2, and H2 production exhibited unique biphasic kinetics consisting of a 1 to 2-min burst of hydrogen evolution followed by a lower, steady-state rate of hydrogen production. This burst was found to be dependent upon the length of the dark period immediately preceding illumination and may be related to dark-to-light ATP transients. The presence of 100 nM NiCl2 in the growth medium exerted an effect on both acetylene reduction and hydrogen evolution in the isolated heterocysts from strain CA. H2-stimulated acetylene reduction was increased from 2.0 to 3.2 mumol of C2H4 per mg (dry weight) per h, and net hydrogen production was abolished. A phenotypic Hup- mutant (N9AR) of Anabaena sp. strain CA was isolated which did not respond to nickel. In isolated heterocysts from N9AR, ethylene production rates were the same under both 10% C2H2-90% Ar and 10% C2H2-90% H2 with or without added nickel, and net hydrogen evolution was not affected by the presence of 100 nM Ni2+. Isolated heterocysts from strain CA were shown to have a persistent oxygen uptake of 0.7 mumol of O2 per mg (dry weight) per h, 35% of the rate of whole filaments, at air saturating O2 levels, indicating that O2 impermeability is not a requirement for active heterocysts.
Irreversible binding to the receptor of bacteriophages T5 and BF23 does not occur with the tip of the tail
Authors: Heller K J , Schwarz H
Published: January 2020

Treatment of purified tails of bacteriophage T5 with 0.05% sodium dodecyl sulfate specifically removed pb2, a protein of 108,000 molecular weight (108K), from the tail. Although these tails were devoid of the single straight tail fiber, they still inhibited adsorption of T5 to Escherichia coli cells. Reconstitution of these tails with pb2 increased the efficiency of inhibition of T5 adsorption. Treatment of tails with 0.1% sodium dodecyl sulfate removed, in addition to pb2, a protein of 67K from phage T5 and one of 60K from phage BF23. These tails failed to inhibit phage adsorption, and no reconstitution was achieved. Reconstitution of T5 tails with pb2 from BF23, and of BF23 tails with pb2 from T5, did not alter the host receptor specificity of the tails. Binding of untreated T5 tails to small FhuA receptor particles revealed that binding occurred with the conical part of the tail and that pb2 was most likely released from the tail upon binding. From these results and from recent observations with T5-BF23 hybrid phages (K.J. Heller, Virology 139:11-21, 1984), we conclude that the receptor-binding proteins of T5 and BF23 are the 67K and 60K proteins, respectively, and that they are not located at the tip of the tail but rather at or near the site where the straight tail fiber is attached to the conical part of the tail.
Nucleotide sequence of ermA, a macrolide-lincosamide-streptogramin B determinant in Staphylococcus aureus
Authors: Murphy E
Published: January 2020

The complete nucleotide sequence of ermA, the prototype macrolide-lincosamide-streptogramin B resistance gene from Staphylococcus aureus, has been determined. The sequence predicts a 243-amino-acid protein that is homologous to those specified by ermC, ermAM, and ermD, resistance determinants from Staphylococcus aureus, Streptococcus sanguis, and Bacillus licheniformis, respectively. The ermA transcript, identified by Northern analysis and S1 mapping, contains a 5' leader sequence of 211 bases which has the potential to encode two short peptides of 15 and 19 amino acids; the second, longer peptide has 13 amino acids in common with the putative regulatory leader peptide of ermC. The coding sequence for this peptide is deleted in several mutants in which macrolide-lincosamide-streptogramin B resistance is constitutively expressed. Potential secondary structures available to the leader sequence of the wild-type (inducible) transcript and to constitutive deletion, insertion, and point mutations provide additional support for the translational attenuation model for induction of macrolide-lincosamide-streptogramin B resistance.
Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli
Authors: Freitag C S , Abraham J M , Clements J R , Eisenstein B I
Published: January 2020

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.
Guidance of Cytophaga sp. strain U67 gliding on the sheaths of Oscillatoria princeps
Authors: McGrath C F , Burchard R P
Published: January 2020

Individual cells of Cytophaga sp. strain U67 glided in helical patterns on the surface of sheaths deposited by the cyanobacterium Oscillatoria princeps. Possible bases for the helical substructure of the sheath are discussed.
Preferential inhibition of plasmid replication in vivo by altered DNA gyrase activity in Escherichia coli
Authors: Uhlin B E , Nordström K
Published: January 2020

The thermosensitive growth phenotype exerted by runaway-mutant plasmids was suppressed by sublethal doses of the DNA gyrase inhibitors novobiocin or nalidixic acid, although the latter drug was less efficient. A novobiocin-resistant gyrB mutant Escherichia coli strain prevented expression of the runaway phenotype at 37 to 42 degrees C in the absence of any drug.
Analysis of plasmid deletional instability in Bacillus subtilis
Authors: Hahn J , Dubnau D
Published: January 2020

Using a model system, we have studied deletion formation in Bacillus subtilis. When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B. subtilis, an intramolecular recombination event usually occurred. Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate. Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry. Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination. The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B. subtilis and does not require that the competent state be established.
Expression of Actinomyces viscosus antigens in Escherichia coli: cloning of a structural gene (fimA) for type 2 fimbriae
Authors: Donkersloot J A , Cisar J O , Wax M E , Harr R J , Chassy B M
Published: January 2020

A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression of A. viscosus antigens and to gain insight into the structure of A. viscosus type 1 and type 2 fimbriae. Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A. viscosus cells. The proteins responsible for these reactions were identified in three clones. Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively. Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that AV1402 expresses a gene (fimA) for a subunit of A. viscosus type 2 fimbriae.
In vitro and in vivo activation of L-serine deaminase in Escherichia coli K-12
Authors: Newman E B , Dumont D , Walker C
Published: January 2020

Escherichia coli L-serine deaminase (L-SD) in crude extracts made in glycylglycine could be activated by incubation with iron sulfate and dithiothreitol. This activation could also be demonstrated in vitro in two mutants which were physiologically deficient in L-SD activity in vivo. This suggests that these mutants were deficient not in L-SD but in an enzyme(s) activating L-SD. The suggestion is made that production of a functional L-SD in vivo requires activation of the structural gene product by an enzyme or enzymes that reduce the protein to an active form.
Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3
Authors: Tomás J M , Jofre J T
Published: January 2020

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.
Globoside-specific adhesins of uropathogenic Escherichia coli are encoded by similar trans-complementable gene clusters
Authors: Lund B , Lindberg F P , Båga M , Normark S
Published: January 2020

Uropathogenic Escherichia coli frequently express globoside-specific adhesins, shown to mediate binding to uroepithelial cells. For one gene cluster pap, it recently has been demonstrated that globoside binding is not dependent on expression of the pilus subunit gene papA. Instead, two other pap genes papF and papG are specifically required for globoside binding (F. P. Lindberg et al., EMBO J. 3:1167-1173, 1984). By restriction enzyme mapping, DNA hybridization, DNA sequencing, and protein expression in minicells, we show that three gene clusters encoding globoside binding have a very similar structure and gene organization, although they were cloned from different E. coli isolates. Major differences between the adhesin clones were restricted to the central part of the pilin gene (papA) and to one of the two adhesin gene (papG). The three functional units required for biogenesis of globoside-binding pili, i.e., pilin synthesis, pilin export, and pilin assembly, as well as expression of adhesion function, were all trans complementable among the gene clusters.
Transient increase of Ca2+ uptake as a signal for mating pheromone-induced differentiation in the heterobasidiomycetous yeast Rhodosporidium toruloides
Authors: Miyakawa T , Tachikawa T , Jeong Y K , Tsuchiya E , Fukui S
Published: January 2020

The role of Ca2+ for the signaling of rhodotorucine A, a mating pheromone of Rhodosporidium toruloides, was investigated. The efficiency with which the target cells responded to the mating pheromone was dependent on the Ca2+ concentration in the medium. The pheromone induced a very rapid and transient increase of Ca2+ uptake in the recipient cell. We concluded that the transient increase in the intracellular Ca2+ concentration could play an essential role in the control of differentiation by the pheromone.
Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements
Authors: Niaudet B , Jannière L , Ehrlich S D
Published: January 2020

Linear DNA molecules composed of a central region nonhomologous with the Bacillus subtilis chromosome and two flanking regions homologous with the chromosome can integrate into the chromosome, provided that the homologous regions have the same relative orientation. The resulting chromosome can be maintained in a haploid or in a merodiploid cell together with a parental chromosome. This can most easily be explained by supposing that the integration occurs by crossing over at each homologous region and that a part of the chromosome between these regions is deleted and replaced by the central nonhomologous region of the integrating molecule. If no essential genes were replaced during that process a haploid cell would be obtained; if essential genes were replaced a merodiploid cell would be obtained. The use of appropriate linear molecules therefore should allow the induction of deletions, extending from a given chromosomal site in a predetermined direction, and defined duplications in the B. subtilis chromosome.