bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic and biochemical analyses of Escherichia coli strains having a mutation in the structural gene (poxB) for pyruvate oxidase
Authors: Chang Y Y , Cronan J E
Published: January 2020

Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated from an aceEF (pyruvate dehydrogenase-deficient) strain by selection for a complete absence of growth on medium lacking acetate. Extracts of two of the mutants were shown to contain normal levels of pyruvate oxidase antigen, although the enzymatic activities of the extracts were reduced or absent. The poxB locus was mapped by using closely linked transposon insertions to min 18.7 of the E. coli linkage map between the cmlA and aroA loci, a location far removed from that of the regulatory gene, poxA.
Chemotaxis of Pseudomonas aeruginosa: involvement of methylation
Authors: Craven R C , Montie T C
Published: January 2020

The involvement of a protein methyl transfer system in the chemotaxis of Pseudomonas aeruginosa was investigated. When a methionine auxotroph of P. aeruginosa was starved for methionine, chemotaxis toward serine, measured by a quantitative capillary assay, was reduced 80%, whereas background motility was unaffected or increased. When unstarved bacteria were labeled with L-[methyl-3H]methionine, a labeled species of 73,000 molecular weight which was methylated in response to stimulation by L-serine was identified. Under appropriate electrophoretic conditions, the 73,000 molecular weight species was resolved into two bands, both of which responded to stimulation by L-serine, L-arginine, and alpha-aminoisobutyrate (AIB) with an increased incorporation of methyl label. Arginine, which elicited the strongest chemotactic response in the capillary assay, also stimulated the greatest methylation response. Methylation of the 73,000 molecular weight species reached a maximum 10 min after stimulation by AIB and returned to the unstimulated level upon removal of the AIB. In vitro labeling of cell extracts with S-adenosyl[methyl-3H]methionine indicated that the 73,000 molecular weight species are methylated by an S-adenosylmethionine-mediated reaction. These results indicate that chemotaxis of P. aeruginosa toward amino acids is mediated by dynamic methylation and demethylation of methyl-accepting chemotaxis proteins analogous to those of the enteric bacteria.
Characterization of the folic acid C9-N10-cleaving enzyme of Dictyostelium minutum V3
Authors: De Wit R J , van der Velden R J , Konijn T M
Published: January 2020

Folic acid is a chemoattractant for the slime mold Dictyostelium minutum V3. The activity of extracellular folic acid is regulated by a folic acid C9-N10 splitting enzyme (FAS). The products were identified as pterin-6-aldehyde and p-amino-benzoylglutamic acid. The enzyme was stabilized by EDTA. For the extracellular enzyme, the Km was 10(-7) M, and the optimal pH was 4.0. During starvation, FAS activity was mainly secreted into the medium; after 3 h, a plateau was reached. The membrane-bound activity was constant, but only 12% of the extracellular activity at 3 h. Intracellular activity also increased up to 3 h to a level of 23% of the extracellular FAS. The substrate recognition of FAS was found to be based on 4-O or N3 or both, N5 or N8 or both, N10, and the p-aminobenzoic acid moiety, whereas 2-NH2, N1, and the glutamic acid moiety were not recognized. Other slime mold species were found to secrete FAS with 20-fold or more reduced activity than D. minutum V3.
Genetic analysis of a pleiotropic deletion mutation (delta igf) in Bacillus subtilis
Authors: Fujita Y , Fujita T
Published: January 2020

A delta igf mutation of Bacillus subtilis (formerly called fdpAl) is a large deletion causing pleiotropic defects. The mapping of the delta igf deletion by phage PBS1 transduction revealed the following map order: sacA, thiC, hsrE, delta igf, ts199, purA. To analyze the pleiotropic nature of the delta igf mutation, mutants affected in each property of the pleiotropic mutation were isolated, and the mutations were mapped. iol and gnt mutants could not grow on inositol and gluconate, respectively, and fdp mutants were affected only in fructose-bisphosphatase. The map order from sacA to purA was as follows: sacA, thiC, hsrE, iol-6, gnt-4, fdp-74, hsrB, ts199, purA. The delta igf deletion covered loci from iol-6 to hsrB.
Regulation of ribonucleoside diphosphate reductase mRNA synthesis in Escherichia coli
Authors: Hanke P D , Fuchs J A
Published: January 2020

A RNA-DNA hybridization assay for ribonucleoside diphosphate reductase (RDP reductase) mRNA was used to determine whether control of RDP reductase synthesis in Escherichia coli is at the level of RNA transcription. The correlation observed between the level of RDP reductase enzymatic activity and the rate of RDP reductase mRNA synthesis suggested that the control is at the level of RNA transcription. No increase in RDP reductase enzymatic activity or RDP reductase mRNA was observed during the first 15 min after removal of thymine from a thymine-requiring culture. Thereafter, the rate of RDP reductase mRNA synthesis increased linearly for approximately 75 min before beginning to level off. The addition of thymine to a culture starved for thymine resulted in a decreasing rate of RDP reductase mRNA synthesis. However, 30 min of growth in the presence of thymine was required before the rate of RDP reductase mRNA synthesis dropped to the rate observed in an exponentially growing culture. Inhibition of DNA synthesis caused by shifting a culture of a polC mutant or a dnaB mutant to nonpermissive growth conditions resulted in an increase in the rate of RDP reductase mRNA synthesis similar to that observed for a thymine-starved culture.
Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources
Authors: Tesh M J , Morse S A , Miller R D
Published: January 2020

The utilization of amino acids and other compounds as carbon and energy sources by Legionella pneumophila was examined. Based on the stimulation of oxygen consumption in washed-cell suspensions, glutamate, serine, threonine, and tyrosine were the only amino acids which were utilized as energy sources. Other stimulators of oxygen uptake were lactate, pyruvate, acetate, fumarate, and succinate. Citrate was a good stimulator only when the bacteria were grown in the presence of the substrate. Radiolabeling studies showed that [14C]glutamate was rapidly metabolized, with the label distributed evenly in all cell fractions. [14C]pyruvate and [14C]acetate were incorporated into the lipid-containing cell fraction, whereas glucose and glycerol were found in both the lipid- and polysaccharide-containing cell fractions. Radiorespirometry of differentially labeled [14C]glucose indicated that this compound was metabolized primarily by the pentose phosphate and Entner-Doudoroff pathways rather than by the glycolytic pathway.
Characterization of lactose transport in Kluyveromyces lactis
Authors: Dickson R C , Barr K
Published: January 2020

We have determined that lactose uptake in Kluyveromyces lactis is mediated by an inducible transport system. Induction, elicited by lactose or galactose, of the transporter required protein synthesis. Transport of lactose required an energy-generating system and occurred by an active process, since an intracellular lactose concentration 175 times greater than the extracellular concentration could be obtained. The Km for lactose transport was about 2.8 mM in uninduced and lactose- or galactose-induced cells. The lactose transporters in K. lactis and Escherichia coli appear to be different since they respond uniquely to inhibition by substrate analogs.
Suppressed nonsense mutations in the araC gene of Escherichia coli provide three novel variant proteins
Authors: Schecter J B
Published: January 2020

A total of 400 suppressible mutations have been isolated in the araC gene of Escherichia coli. Based on deletion mapping, growth patterns when suppressed, and intragenic recombination, 37 mutants have been determined to contain unique mutations. Rapid plate assays were developed to test for each of the three AraC protein functions: inducing araBAD, repressing araBAD, and araC self-repression. The 185 mutant proteins, resulting from 37 mutants each suppressed by five different suppressors, were assayed for each of the three AraC functions. These plate assays showed that: (i) for each function, some areas of the gene map are more sensitive to mutation than other areas, and (ii) three of the mutant AraC proteins were unlike previously characterized AraC mutants. Enzyme assays on the mutant proteins confirmed their novel character. The first mutant cannot induce araBAD but retains the capacity to perform both repression functions; and the second and third can each perform one of the two repression functions better than it can perform the other. These characteristics suggest that previously proposed models of ara regulation are incomplete.
Stability of plasmids R1-19 and R100 in hyper-recombinant Escherichia coli strains and in Salmonella typhimurium strains
Authors: Gómez-Eichelmann M C , Torres H K
Published: January 2020

Plasmids R1-19 and R100 dissociate in hyper-recominant Escherichia coli strains in a way that is similar to but slower than dissociation in Salmonella typhimurium. The results presented suggest that the molecular mechanism for plasmid dissociation in hyper-recombinant E. coli strains is different than that in S. typhimurium strains.
Neurospora crassa mutant impaired in glutamine regulation
Authors: González A , Tenorio M , Vaca G , Mora J
Published: January 2020

The final products of the catabolism of arginine that can be utilized as nitrogen sources by Neurospora crassa are ammonium, glutamic acid, and glutamine. Of these compounds, only glutamine represses arginase and glutamine synthetase. We report here the isolation and characterization of a mutant of N. crassa whose arginase, glutamine synthetase, and amino acid accumulations are resistant to glutamine repression (glnI). This mutant has a greater capacity than the wild type (glns) to accumulate most of the arginine and some of the glutamine in osmotically sensitive compartments while growing exponentially. Nonetheless, the major part of the glutamine remains soluble and metabolically available for repression. We propose that the lower repression of glutamine synthetase by glutamine in this mutant could be a necessary condition for sustaining the higher flow of nitrogen for the accumulation of amino acids observed in ammonium excess and that, if glutamine is the nitrogen signal that regulates the arginine accumulation of the vesicle, the glnr mutant has also escaped this control. Finally, in the glnr mutant, some glutamine resynthesis is necessary for arginine biosynthesis and accumulation.
Temperature-sensitive autolysis-defective mutants of Escherichia coli
Authors: Harkness R E , Ishiguro E E
Published: January 2020

Two independently isolated temperature-sensitive autolysis-defective mutants of Escherichia coli LD5 (thi lysA dapD) were characterized. The mutants were isolated by screening the survivors of a three-step enrichment process involving sequential treatments with bactericidal concentrations of D-cycloserine, benzyl-penicillin, and D-cycloserine at 42 degrees C. Cultures of the mutants underwent autolysis during beta-lactam treatment, D-cycloserine treatment, or diaminopimelic acid deprivation at 30 degrees C. The same treatments at 42 degrees C inhibited growth but did not induce lysis of the mutants. The minimum inhibitory concentrations of selected beta-lactam antibiotics and D-cycloserine were identical for the parent and mutant strains at both 30 and 42 degrees C. Both mutants failed to form colonies at 42 degrees C, and both gave rise to spontaneous temperature-resistant revertants. The revertants exhibited the normal lytic response when treated with D-cycloserine and beta-lactams or when deprived of diaminopimelic acid at 42 degrees C. The basis for the autolysis-defective phenotype of these mutants could not be determined. However, a nonspecific in vitro assay for peptidoglycan hydrolase activity in cell-free extracts indicated that both mutants were deficient in a peptidoglycan hydrolase. Both mutations were localized to the 56- to 61-min region of the E. coli chromosome by F' complementation.
The wild-type gene for glutamine synthetase restores ammonia control of nitrogen fixation to Gln- (glnA) mutants of Rhodopseudomonas capsulata
Authors: Scolnik P A , Virosco J , Haselkorn R
Published: January 2020

The wild-type glnA gene, coding for glutamine synthetase, was cloned from the photosynthetic bacterium Rhodopseudomonas capsulata by using a cosmid library to complement the Gln- phenotype of an Escherichia coli glnA deletion strain. The original cosmid plasmid contained 37 kilobase pairs (kbp) of R. capsulata DNA, of which only 2 kbp was necessary for Gln complementation in E. coli. A plasmid containing this 2-kbp insert was mobilized into G29, a Gln- mutant of R. capsulata which is also unable to repress nitrogenase in ammonia-containing media (Nifc phenotype). The 2-kbp fragment restored glutamine-independent growth and ammonia repression of nitrogenase, indicating that in R. capsulata, production of the signal for nitrogen repression of nif depends on the activity of the glnA gene. Repression of nitrogenase was shown, by hybridization of RNA to cloned nif DNA, to occur at the level of transcription in the wild-type and the complemented G29 strains.
Teichoicase from Bacillus subtilis Marburg
Authors: Kusser W , Fiedler F
Published: January 2020

The properties of a teichoic acid degrading enzyme (teichoicase) isolated from Bacillus subtilis Marburg are described. The purified enzyme showed phosphodiesterase activity but not phosphomonoesterase activity, and it had an absolute substrate specificity for alpha-glucosylated glycerol teichoic acid, the endogenous cell wall teichoic acid of the enzyme-producing cell. The substrate was degraded by an exo-mechanism yielding the monomer alpha-D-glucose 1 leads to 2 (sn)glycero-3-phosphate. When B. subtilis Marburg was grown in a rich medium, enzyme activity was detected in extracts from sporulating cells. Teichoicase activity was present in a mutant blocked in stage II of the sporulation process but was absent in a mutant blocked in stage O. It was concluded that teichoicase is active on enzyme-producing cells since the reaction product could be detected in their culture supernatant. Attempts to demonstrate analogous enzyme activity in other Bacillus strains failed. The enzyme could be used for the rapid detection of alpha-glucosylated glycerol teichoic acid and for the controlled alteration of native bacterial cell surfaces exhibiting the appropriate structure.
Uric acid utilization by Mycobacterium intracellulare and Mycobacterium scrofulaceum isolates
Authors: Falkinham J O , George K L , Parker B C , Gruft H
Published: January 2020

Forty-nine human and environmental isolates of Mycobacterium intracellulare and Mycobacterium scrofulaceum were tested for their ability to grow on uric acid and a number of its degradation products. Nearly all (88 to 90%) strains used uric acid or allantoin as a sole nitrogen source; fewer (47 to 69%) used allantoate, urea, or possibly ureidoglycollate. Enzymatic activities of one representative isolate demonstrated the existence of a uric acid degradation pathway resembling that in other aerobic microorganisms.
Expression of the Rickettsia prowazekii citrate synthase gene in Escherichia coli
Authors: Wood D O , Atkinson W H , Sikorski R S , Winkler H H
Published: January 2020

Recombinant DNA techniques were used to isolate the Rickettsia prowazekii citrate synthase gene on the plasmid vector pBR322 by functional complementation of a gltA mutation of Escherichia coli K-12. Analysis of citrate synthase activity in crude extracts revealed that the enzyme expressed in E. coli retains the regulatory control mechanisms characteristic of the rickettsial enzyme.
Tn3 labeling of a cryptic plasmid found in the plant pathogenic bacterium Pseudomonas tabaci and mobilization of RSF1010 by donation
Authors: Obukowicz M , Shaw P D
Published: January 2020

pBPW1, a conjugative cryptic plasmid isolated from the plant pathogenic bacterium Pseudomonas tabaci BR2, was labeled with Tn3. pBPW1::Tn3 and RSF1010 mobilization into Pseudomonas mellea recipients were separate events, not involving recombination of the two plasmids during conjugation.
SOS induction by P1 Km miniplasmids
Authors: Capage M A , Scott J R
Published: January 2020

We have constructed (in vitro) a set of P1 miniplasmids. The smallest of these that could function as an independent replicon contained the right side of EcoRI-5 plus all of EcoRI-8. Those miniplasmids that lack EcoRI-6 induce the SOS pathway of the cell as shown by (i) increased expression of the recA operon, (ii) excision of the cryptic genetic element e14, (iii) spontaneous induction of lambda, and (iv) dependence of e14 excision on recA+ function. This induction was contingent upon the replication of the P1 Km miniplasmids from their P1 origin and, thus, was apparently caused by an aberrant initiation of DNA replication. When P1 EcoRI-6 was present in cis or trans with a P1 Km miniplasmid, neither e14 nor lambda was excised, but the expression of the recA operon was still induced. These results suggest that P1 EcoRI fragments 5 and 8 are insufficient for normal replication, and thus our P1 Km miniplasmids induced SOS functions. A product of EcoRI-6 may partially restore normal replication.
Synthesis of biodegradative threonine dehydratase in Escherichia coli: role of amino acids, electron acceptors, and certain intermediary metabolites
Authors: Hobert E H , Datta P
Published: January 2020

The specific activity of inducible biodegradative threonine dehydratase (EC 4.2.1.16) in Escherichia coli K-12 increased significantly when the standard tryptone-yeast extract medium or a synthetic mixture of 18 L-amino acids was supplemented with 10 mM KNO3 or 50 mM fumarate and with 4 mM cyclic AMP. In absolute terms, almost four times as much enzyme was produced in the amino acid medium as in the tryptone-yeast extract medium. Enzyme induction in the amino acid medium was sensitive to catabolite repression by glucose, gluconate, glycerol, and pyruvate. An analysis of amino acid requirements for enzyme induction showed that a combination of only four amino acids, threonine, serine, valine, and isoleucine, produced high levels of threonine dehydratase provided that both fumarate and cyclic AMP were present. Immunochemical data revealed that the enzyme synthesized in the presence of these four amino acids was indistinguishable from that produced in the tryptone-yeast extract or the medium with 18 amino acids. We interpret these results to mean that not the amino acids themselves but some metabolites derived anaerobically in reactions involving an electron acceptor may function as putative regulatory molecule(s) in the anaerobic induction of this enzyme.
Organization and subcloning of the dacA-rodA-pbpA cluster of cell shape genes in Escherichia coli
Authors: Stoker N G , Broome-Smith J K , Edelman A , Spratt B G
Published: January 2020

The transducing bacteriophage lambda pBS10 carries a small cluster of Escherichia coli penicillin-binding protein/cell shape genes, including pbpA, rodA, and dacA. Deletion mapping and subcloning showed that these genes, and the gene for a cytoplasmic membrane protein of molecular weight 54,000, are located within a 5.6-kilobase region and are probably contiguous. The dacA gene, which codes for penicillin-binding protein 5, was cloned on a 1.5-kilobase fragment into a low-copy-number plasmid vector, but insertion into high-copy-number plasmids produced deleterious effects on bacterial growth, and the plasmids could not be stably maintained. The direction of transcription of dacA was determined. The rodA gene was cloned on a 1.6-kilobase fragment into both low- and high-copy-number plasmids, and the identification of its gene product is described in the accompanying paper (Stoker et al., J. Bacteriol. 155:854-859). The pbpA gene, which codes for penicillin-binding protein 2, was cloned on a 3.7-kilobase fragment in low-copy-number plasmids, but insertion of the fragment into high-copy-number plasmids resulted in deleterious effects on bacterial growth, and the plasmids could not be stably maintained.
Type 3 fimbriae of Klebsiella sp.: molecular characterization and role in bacterial adhesion to plant roots
Authors: Korhonen T K , Tarkka E , Ranta H , Haahtela K
Published: January 2020

Type 3 fimbriae of Klebsiella were purified and characterized. The fimbriae were 4 to 5 nm in diameter and 0.5 to 2 microns long. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the fimbrillin had an apparent molecular weight of 23,500, and it differed from enterobacterial type 1 fimbrillins in its amino acid composition. Hydrophobic amino acids comprised 33.6% of all amino acids in the fimbrillin, which lacked cystine, phenylalanine, and arginine. Serologically, the type 3 fimbriae were also distinct from the type 1 fimbriae. Purified type 3 fimbriae agglutinated tannin-treated human blood group O erythrocytes; this confirms the role of type 3 fimbriae as hemagglutinins. Purified 125I-labeled type 3 fimbriae bound to the roots of Poa pratensis, and this binding could be inhibited by Fab fragments to the purified fimbriae. Anti-type 3 fimbriae Fab fragments also inhibited bacterial adhesion to plant roots. These results demonstrate that type 3 fimbriae mediate adhesion of klebsiellas to plant roots. Eight nitrogen-fixing strains of Klebsiella also produced type 3 fimbriae when grown under anaerobic nitrogen fixation conditions. It is proposed that type 3 fimbriae are involved in the establishment of the plant-bacterium association concerning nitrogen-fixing Klebsiella strains.
Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae
Authors: Chan R K , Melnick L M , Blair L C , Thorner J
Published: January 2020

MAT alpha haploids with mutations in the STE13 or KEX2 gene, and MATa haploids with mutations in the STE6 or STE14 gene, do not mate with wild-type cells of the opposite mating type. We found that such mutants were able to mate with partners that carry mutations (sst1 and sst2) that cause cells to be supersensitive to yeast mating pheromone action. Mating ability of MAT alpha ste13 and MAT alpha kex2 mutants could also be restored by adding normal MAT alpha cells to mating mixtures or by adding just the appropriate purified pheromone (alpha-factor). Therefore, the mating deficiencies caused by the ste13 and kex2 lesions, and by inference, the ste6 and ste14 mutations, appear to result only from secretion of an insufficient amount of pheromone or a nonfunctional pheromone.
Alternative explanation for excision repair deficiency caused by the polAex1 mutation
Authors: Wahl A F , Hockensmith J W , Kowalski S , Bambara R A
Published: January 2020

An investigation of the mechanism of the polAex1 mutation in vitro suggested that the excision repair deficiency observed in vivo does not result from an inability of the enzyme to nick translate. The defect appears to reside in the inability of the enzyme to effectively generate a nick structure to serve as a substrate for DNA ligase.
DNA repair in competent cells of Bacillus subtilis
Authors: Mita I , Sadaie Y , Kada T
Published: January 2020

A series of isogenic transformable strains of Bacillus subtilis carrying the uvr-19 or rec-43 mutation or both were constructed. Both mutations made competent cells defective in repairing UV-irradiated cellular or transforming DNA, and their effects were additive in a doubly deficient strain, suggesting that two repair processes, requiring uvr-19+ and rec-43+ gene products, are independently functional in competent cells of B. subtilis.
Genetic control of the hexose phosphate transport system of Escherichia coli: mapping of deletion and insertion mutations in the uhp region
Authors: Kadner R J , Shattuck-Eidens D M
Published: January 2020

The Escherichia coli transport system responsible for the accumulation of a number of sugar phosphates is encoded by the uhp region and is induced by external, but not intracellular, glucose 6-phosphate. To delineate the genetic organization of the uhp region, a total of 225 independent point, deletion, and transposon Tn10 insertion mutations were collected. Mutations conferring the Uhp-phenotype were obtained on the basis of their resistance to fosfomycin and their inability to use sugar phosphates as carbon source. Deletions of uhp sequences were obtained as a consequence of imprecise excision of Tn10 insertions located on either side of uhp. Conjugal crosses between these deletions and the point of insertion mutations allowed determination of the relative order of the uhp alleles and of the deletion endpoints. Specialized lambda transducing phages carrying a uhpT-lac operon fusion and various amounts of adjacent uhp material were isolated and used as genetic donors. Results from these crosses corroborated those obtained in the conjugal crosses. The locations of the mutant alleles were compared with the regulatory properties of Uhp+ revertants of these alleles. This comparison suggested the existence of at least three genes in which mutation yields the Uhp-phenotype. Mapping experiments were consistent with the gene order pyrE-gltS-uhpTRA-ilvB, where uhpT encodes the transport system and uhpR and uhpA are regulatory genes whose products are necessary for proper uhp regulation.
Molecular Cloning of the
uhp
Region and Evidence for a Positive Activator for Expression of the Hexose Phosphate Transport System of
Escherichia coli
Authors: Shattuck-Eidens Donna M. , Kadner Robert J.
Published: January 2020

The
uhp
locus of
Escherichia coli
contains genes for the sugar phosphate transport system (
uhpT
) and the regulatory system which allows its induction by external glucose 6-phosphate (
uhpRA
). The
uhp
region was cloned onto high-copy-number plasmids, both from Uhp
+
plasmids of the Clarke-Carbon collection and from genetically characterized specialized transducing phages carrying
uhpT-lac
operon fusions. Two Clarke-Carbon plasmids and their Uhp
+
subclones in pBR322 shared restriction sites defining the
uhp
region, but exhibited different regulation of Uhp expression and dependence on chromosomal
uhp
genotype. Plasmid pLC17-47 and derivatives conferred constitutive glucose 6-phosphate uptake activity in all strains, even those with complete deletions of
uhp
. These plasmids also rendered constitutive the expression of a chromosomal
uhpT-lac
operon fusion. Plasmid pLC40-33 conferred inducible Uhp expression, which required the presence of the
uhpA
+
gene on the chromosome. The induced transport levels in all strains carrying these plasmids were not appreciably amplified over haploid levels. Similar behavior was seen with the cloned operon fusions. A fusion-bearing plasmid that carried an intact regulatory system (
uhpR
+
A
+
) exhibited
trans
-dominant constitutive expression of β-galactosidase, regardless of the chromosomal
uhp
genotype. In contrast, the cloned fusion carrying only
uhpR
+
gave glucose 6-phosphate-inducible production of β-galactosidase that was dependent on the presence of chromosomal
uhpA
+
. Expression of both fusions in the haploid state was inducible. From these results, it was concluded that the
uhpA
product is necessary for
uhpT
transcription and that elevated dosage of
uhpA
results in at least partially constitutive expression of
uhpT
. A tentative model for
uhp
regulation is presented.
Expression of a cloned K88ac adhesion antigen determinant: identification of a new adhesion cistron and role of a vector-encoded promoter
Authors: Kehoe M , Winther M , Dougan G
Published: January 2020

The determinant for the K88ac adherence antigen of porcine enterotoxigenic Escherichia coli has been cloned previously onto the vector plasmid pBR322 to form the K88ac-pBR322 hybrid plasmid pMK005 (M. Kehoe et al., Nature [London] 291:122-126). Further studies on the expression of the K88ac antigen from pMK005 are presented in this paper. Expression was found to be dependent mainly on the P1 promoter of the pBR322 vector. The natural K88ac promoter was apparently not cloned from the original parental K88ac plasmid. The P1 promoter was deleted and replaced by a DNA sequence encoding the promoter-operator region of the E. coli tryptophan (Trp) operon. Cells harboring the Trp-pMK005 hybrid plasmid expressed high levels of K88ac antigen when the Trp promoter was repressed. If the promoter was derepressed either by growing the cells in low concentrations of tryptophan or in the presence of indole acrylic acid, growth of the cells harboring the Trp-pMK005 hybrid plasmid was inhibited. A quantitative assay was used to measure the levels of K88ac antigen expressed by cells harboring different pMK005::Tn5 plasmids. All cells were found to express a reduced level of K88ac antigen, providing evidence that a single transcription unit, initiating at promoter P1 of pBR322, may be involved in the expression of the K88ac antigen. By constructing specific deletion and insertion mutants of pMK005, a fifth adhesion cistron, tentatively named adhE, was identified and mapped at the proximal end of the K88ac determinant. Although the cistron is required for high-level expression of K88ac surface-associated fimbriae, as yet no gene product has been assigned to adhE.
Mutation causing overproduction of outer membrane protein OmpF and suppression of OmpC synthesis in Escherichia coli
Authors: Koga-Ban Y , Mutoh N , Inokuchi K , Mizushima S
Published: January 2020

A novel mutation affecting the synthesis of major outer membrane proteins OmpF and OmpC in Escherichia coli K-12 is described. The mutation resulted in overproduction of the OmpF protein with concomitant suppression of OmpC synthesis. This mutation, designated as ompFp100, was mapped at 21 min on the E. coli chromosome map with the gene order aroA-aspC-ompF4-ompFp100-asnS-pyrD. This mutation was cis-dominant to the expression of the ompF gene. In addition, the direction of the mRNA transcription of the ompF gene was from asnS to aspC. These results strongly indicate that ompFp100 is a promoter mutation of the ompF gene. Introduction of an ompF mutation, which causes the disappearance of the OmpF protein, into strains carrying the ompFp100 mutation resulted in the reappearance of the OmpC protein in the outer membrane. Addition of a high concentration of sucrose to the medium, which suppresses the OmpF synthesis and stimulates the OmpC synthesis in the wild-type strain, resulted in the reappearance of the OmpC protein in the ompFp100 mutant with concomitant suppression of the overproduction of the OmpF protein. These results suggest that suppression of OmpC synthesis in the ompFp100 mutant is due to overproduction of the OmpF protein.
Isolation and preliminary characterization of Escherichia coli mutants deficient in exonuclease VII
Authors: Vales L D , Rabin B A , Chase J W
Published: January 2020

Strains of Escherichia coli containing reduced levels of exonuclease VII activity due to mutations in the xseB gene have been isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Seven mutants of independent origin deficient in exonuclease VII activity were obtained. Four of these contained defects in xseA, a locus which has been previously identified, and three others contained mutations in a gene distinct from xseA, which we have designated xseB. Genetic mapping studies place the xseB locus between proC and dnaZ. Exonuclease VII purified from KLC835 (xseA+ xseB3) is more heat labile than enzyme purified from the parent strain PA610 (xse+), showing that xseB is a structural gene for exonuclease VII. The isolation of lambda transducing phage carrying xseA is also described.
DNA double-strand breakage and removal of cross-links in Deinococcus radiodurans
Authors: Kitayama S , Asaka S , Totsuka K
Published: January 2020

Mitomycin C-sensitive mutants of Deinococcus radiodurans were isolated which were either resistant to or only moderately sensitive to far UV (254 nm) or gamma rays. They were also sensitive to irradiation at 365 nm in the presence of 4,5',8-trimethylpsoralen. They were classified into seven complementary groups (mtcA through mtcG) by transformation experiments. Interstrand cross-links in the DNA duplex induced by mitomycin C were removed in the cells of two mutants (mtcD and mtcE) as in the wild type, whereas the other mutants were deficient in this repair. After a sublethal dosage of mitomycin C, single- and double-stranded cuts of cross-linked DNA were observed in the wild-type cells during postincubation. This removal of cross-links in DNA seems to be indispensable for the cells since their colony-forming ability was markedly reduced if they were postincubated under an inhibitory condition for repair of these lesions.
Isolation of rel mutants of Escherichia coli B/r
Authors: Little R , Mou S W , Bremer H
Published: January 2020

A method that relies on the biological effect of near-UV (340-nm) irradiation is described by which large numbers of independent rel mutants of Escherichia coli B/r may be rapidly isolated.
Plasmid visualization and nif gene location in nitrogen-fixing Azospirillum strains
Authors: Plazinski J , Dart P J , Rolfe B G
Published: January 2020

A modified gel electrophoresis technique provided a reproducible way of detecting and isolating plasmids with molecular weights ranging from 12 X 10(6) to 370 X 10(6) for Azospirillum species. Analysis with the nifHD region of Rhizobium trifolii showed that the Azospirillum nif genes were chromosomally located in all eight strains investigated and not on endogenous plasmids.
Metabolism of carbon monoxide by Rhodopseudomonas gelatinosa: cell growth and properties of the oxidation system
Authors: Uffen R L
Published: January 2020

Rhodopseudomonas gelatinosa 1 grew as an anaerobic facultative methylotroph with carbon monoxide as the sole carbon and energy source. Carbon from CO was assimilated into cell material via the ribulose 1,5-bisphosphate carboxylase cycle. The CO oxidation system in R. gelatinosa was induced during growth with the gas substrate. Light-grown cells did not oxidize CO. Surprisingly, when strain 1 cells grown in the dark with CO were transferred to growth with both CO and light, they continued to use CO and then photometabolized after the CO gas flow was stopped. This change in the energy-yielding substrate resulted in a diauxic growth response. The use of CO in preference to light energy forms the basis of a system in the cells that controls photosynthetic differentiation. CO oxidation was assayed as CO-methyl viologen oxidoreductase. Methyl viologen reduction only occurred with CO; the dye was not reduced with other C1 compounds. In vitro methyl viologen was reduced best at 24 degrees C and at pH values above 8.5. Whole cells exhibited a Km of 12.5 microM for CO and a Vmax of 3,800 nmol of CO oxidized per mg of protein per min. This was a low-potential oxidation reaction that readily reduced the viologen dye triquat (1,1'-trimethylene-2,2'-dipyridilium dibromide) (E degrees' = -548 mV).
Repair and mutagenesis in Escherichia coli K-12 after exposure to various alkyl-nitrosoguanidines
Authors: Todd M L , Schendel P F
Published: January 2020

The mutagenic and toxic effects of a series of N-alkyl-N'-nitro-N-nitrosoguanidines were examined in Escherichia coli K-12. The role of nucleotide excision repair, the SOS response, and the adaptive response in both the reduction and the production of the biological effects of these chemicals was tested. The effects of ethyl-nitrosoguanidine are similar in nucleotide excision repair-proficient and -deficient strains, but both the mutagenicity and the toxicity of alkyl groups larger than two carbons are significantly reduced by the presence of this repair system. Similarly, when alkyl groups are larger than two carbons, the umuC gene product is essential for the production of a fraction of the mutations that these lesions produce. The induction of the adaptive response had a significant effect on the toxicity of all of the chemicals tested, but its effect on mutagenicity was less uniform, having a larger effect on ethylating and propylating agents than on butylating and amylating agents.
The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number
Authors: Erhart E , Hollenberg C P
Published: January 2020

The copy number of 2 mu DNA-derived plasmids in CIR+ Saccharomyces cerevisiae transformants is determined by its selective marker and is usually much lower than that of the endogenous plasmid. Only plasmids containing the leu2 allele of pJDB219, designated as leu2-d, under selective conditions displayed a higher copy number than did endogenous 2 mu DNA and by displacement generated cured cells. Spontaneous loss of 2 mu DNA occurred with a frequency of about 0.02% per generation. Curing plasmids, like pMP78, have copy numbers of 35; noncuring plasmids, like pDB248 or YEp6, have copy numbers of 4 to 8. The 2 mu DNA copy number in strains AH22 and YNN27 were determined to be 40 and 100, respectively. The high copy number of leu2-d-containing plasmids can be explained by its weak expression of less than 5% that of the wild-type LEU2 gene. The leu2-d allele has a deletion of the 5'-end sequence starting from 29 base pairs before the ATG initiation codon, but surprisingly, its expression is still regulated. On YRp7, which contains the chromosomal autonomic replication sequence ARS1, the defective leu2-d allele could not complement a leu2 host strain. This suggests a more stringent control of replication of ARS1-containing plasmids than of 2 mu-containing plasmids.
D-(-)-poly-beta-hydroxybutyrate in membranes of genetically competent bacteria
Authors: Reusch R N , Sadoff H L
Published: January 2020

D-(-)-Poly-beta-hydroxybutyrate is a constituent of the membranes and the cytoplasms of genetically competent Azotobacter vinelandii, Bacillus subtilis, and Haemophilus influenzae cells. Within each species the concentration of D-(-)-poly-beta-hydroxybutyrate in the membranes and cytoplasm correlates with transformability. Fluorescence analysis of the thermotropic lipid phase transitions in A. vinelandii and B. subtilis cells indicates that D-(-)-poly-beta-hydroxybutyrate forms an organized gel structure in the membranes which is very labile. The concentration of organized D-(-)-poly-beta-hydroxybutyrate in the membranes, which can be estimated from the intensity of its phase transition, can be used to assess the competence of a culture.
Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum
Authors: Lamed R , Setter E , Bayer E A
Published: January 2020

The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Outer membrane protein K of Escherichia coli: purification and pore-forming properties in lipid bilayer membranes
Authors: Whitfield C , Hancock R E , Costerton J W
Published: January 2020

Protein K, a recently described outer membrane protein correlated with encapsulation in Escherichia coli (Paakkanen et al., J. Bacteriol. 139:835-841, 1979), has been purified to apparent homogeneity. Purification was based upon the noncovalent association of protein K with peptidoglycan, and the purified protein was shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. Incorporation of small amounts (10(-10) to 10(-11) M) of purified protein K into artificial lipid bilayers resulted in an increase, by many orders of magnitude, in membrane conductance. The increased conductance resulted from the formation of large, water-filled, ion-permeable channels exhibiting single-channel conductance in 1.0 M KCl of 1.83 nS. The membrane conductance showed a linear relationship between current and applied voltage and was not voltage induced or regulated. The channel was permeable to large organic ions (e.g., Tris+ Cl-) and, based upon a pore length of 7.5 nm, a minimum channel diameter of 1.2 nm was estimated; these properties resemble values for other enteric porins. The possible biological role of the pores produced by protein K is discussed.
Sulfite oxidase activity in Thiobacillus novellus
Authors: Southerland W M , Toghrol F
Published: January 2020

Thiobacillus novellus shows a maximum induction of sulfite oxidase activity and a maximum growth rate as a result of supplementing the autotrophic growth medium with 4.0 microM ammonium molybdate. Cells grown in the presence of molybdate showed approximately 10-fold increases in the amount of enzyme-associated molybdenum and in the sulfite-to-cytochrome c and sulfite-to-ferricyanide reductase activities. The effect of exogenous molybdate was not discernible with cells grown in the absence of thiosulfate. Tungsten inhibited the growth of T. novellus and the expression of sulfite oxidase activity.
Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida
Authors: Darveau R P , MacIntyre S , Buckley J T , Hancock R E
Published: January 2020

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.
Effect of heme on Bacteroides distasonis catalase and aerotolerance
Authors: Gregory E M , Fanning D D
Published: January 2020

Parallel increases in intracellular catalase activity and resistance to extracellular H2O2 and to hyperbaric O2 toxicity were observed when Bacteroides distasonis VPI 4243 (ATCC 8503, type strain) was grown in either complex or defined medium containing graded amounts of hemin. Virtually all of the cells with high catalase activity (greater than 200 U/mg) remained viable upon exposure at 37 degrees C to 100-lb/in2 O2 on agar surfaces for 1 h, whereas low-catalase cells (less than 10 U/mg) lost 1.2 log units of viable cells during that treatment. Upon exposure to 500 microM H2O2, high-catalase cells lost 0.4 log units of the initial viable colonies during the same period in which low-catalase cells lost 3 log units of viable cells. The superoxide dismutase activity was the same in each test culture. These data support the role of intracellular catalase in protecting B. distasonis from oxidative damage resulting from hyperbaric oxygenation or H2O2 exposure. Catalase activity elicited by adding hemin to cells grown previously in medium lacking hemin was inhibited only 40% by prior incubation of the cells with chloramphenicol (30 micrograms/ml) and only 22% with rifampin (5 micrograms/ml). A model which is consistent with these data involves the production of an apocatalase in cells grown in low-hemin medium. Addition of hemin to the cells would result in a rapid chloramphenicolor rifampin-insensitive stimulation of catalase activity followed by further de novo biosynthesis of catalase.
Use of lambda unc transducing bacteriophages in genetic and biochemical characterization of H+-ATPase mutants of Escherichia coli
Authors: Mosher M E , Peters L K , Fillingame R H
Published: January 2020

The eight subunits of the H+-ATPase of Escherichia coli are coded by the genes of the unc operon, which maps between bglB and asnA. A collection of unc mutations were transferred via P1 transduction into a strain in which lambda cI857 S7 was inserted into bglB. The lambda phage was induced, and asnA+ transducing phage that carried unc were selected. Transducing phage carrying mutations in the uncA, B, D, E, and F genes were used for complementation analysis with a collection of unc mutants, including mutants which had been reported previously but not genetically characterized. Some mutations gave a simple complementation pattern, indicating a single defective gene, whereas other mutations gave more complex patterns. Two mutants (uncE105 and uncE107) altered in the proteolipid (omega) subunit of F0 were not complemented by any of the lambda unc phage, even though both mutants had a fully functional F1 ATPase and therefore normal A and D genes. Hence, only limited conclusions can be drawn from genetic complementation alone, since it cannot distinguish normal from abnormal genes in certain classes of unc mutants. The lambda unc phage proved to be essential in characterizing several mutants defective in F0-mediated H+ translocation. The unc gene products were overproduced by heat induction of the lysogenized lambda unc phage to determine whether all the F0 subunits were in the membrane. Two mutants that gave a simple complementation pattern, indicative of one defective gene, did not assemble a three-subunit F0. The uncB108 mutant was shown to lack the chi subunit of F0 but to retain psi and omega. Trace amounts of an altered omega subunit and normal amounts of chi and psi were found in the uncE106 mutant. A substitution of aspartate for glycine at residue 58 of the protein was determined by DNA sequence analysis of the uncE gene cloned from the lambda uncE106 phage DNA. One of the omega-defective, noncomplementing mutants (uncE107) was shown to retain all three F0 subunits. The uncE gene from this mutant was also sequenced to confirm an asparagine-for-aspartate substitution at position 61 (the dicyclohexylcarbodiimide-binding site) of the omega subunit.