bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Rhizobium japonicum hydrogen uptake genes
Authors: Haugland R A , Cantrell M A , Beaty J S , Hanus F J , Russell S A , Evans H J
Published: January 2020

Recombinant cosmids from a gene library of the DNA from Hup+ Rhizobium japonicum 122DES previously have been shown to restore hydrogenase activity when transferred by conjugation into certain Hup- mutants of R. japonicum. We generated a restriction map covering 32.2 kilobases of this cosmid DNA. At least 25.3 kilobases of the cosmid pHU1 were shown to have the same arrangement as those in the genome of strain 122DES. Analysis of Tn5 insertions into the 122DES genome indicates that hup-specific sequences occur in a region spanning about 15 kilobases of insert DNA within pHU1. Introduction of pHU1 into five out of six R. japonicum Hup- mutants resulted in a Hup+ phenotype in some transconjugants. Three of the mutations appear to be in transcriptional units completely contained within pHU1, whereas the other two must be in genes that are at least partially contained within pHU1. pBR235 derivatives containing fragments of hup DNA can be transferred into the R. japonicum Hup- mutant PJ18nal if the derivatives contain a region of homology with the R. japonicum genome. The hup mutation in strain PJ18nal appears to be dominant. The hup genes in R. japonicum strain 122DES appear to be organized in at least two, and probably three, transcriptional units.
Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid
Authors: Russel M , Model P
Published: January 2020

To determine whether the fip gene of Escherichia coli, which is required for filamentous phage assembly, is required for cell viability, we replaced the chromosomal copy of the gene with an inactive copy introduced on a plasmid. We found that the fip gene is dispensable. The method we devised, which should be generally useful, was also tested with an inactivated rho gene. As expected, the rho gene is essential.
Mapping of trxB, a mutation responsible for reduced thioredoxin reductase activity
Authors: Haller B L , Fuchs J A
Published: January 2020

The mutation (trxB) responsible for reduced thioredoxin reductase activity has been mapped on the Escherichia coli K-12 chromosome clockwise from aroA between 20 and 21 min. The gene order in this region of the E. coli chromosome was found to probably be serC-aroA-trxB. The location of gshA, the structural gene for gamma-glutamylcystein synthetase, relative to srl and recA also was determined.
Bacillus cereus electron transport and proton motive force during aerotaxis
Authors: Laszlo D J , Niwano M , Goral W W , Taylor B L
Published: January 2020

Aerotaxis (migration towards oxygen) of Bacillus cereus M63, a motile strain, was inhibited by potassium cyanide and 2-heptyl-4-hydroxyquinoline N-oxide, indicating a requirement for both the terminal oxidase (cytochrome aa3) and the cytochrome b segment of the electron transport system. The concentration of oxygen that gave a half-maximal aerotactic response (K0.5) was 0.31 microM, which was similar to the Km for respiration (0.80 microM). The proton motive force increased from -135 to -177 mV when anaerobic cells were aerated, and it is proposed that the signal for aerotaxis is the increase in proton motive force that results from increased respiration. A strain of B. cereus T initially used in this study was immotile, grew as long chains of cells, and was deficient in autolytic enzyme. B. cereus M63 is a spontaneous derivative of B. cereus T that has normal motility.
Heterogeneity of lipid A: comparison of lipid A types from different gram-negative bacteria
Authors: Mattsby-Baltzer I , Gemski P , Alving C R
Published: January 2020

Chloroform-soluble purified lipid A preparations from 10 sources, including five Escherichia coli strains (EH100, K-12, O127, O111, RCDC), two Salmonella strains (Salmonella typhimurium, Salmonella minnesota R595), Shigella sonnei II, and a hybrid of Shigella flexneri and E. coli K-12, were compared with lipid A from S. flexneri. Purified lipid A from S. flexneri was earlier found to be composed of eight fractions. The various lipid A preparations were assayed by thin-layer chromatography. Chromatograms were stained for phosphate or carbohydrate by molybdenum blue or orcinol, respectively. The number of major bands found for each lipid A preparation varied between 7 and 10, depending on the source. Comparable bands, based on Rf, were found among all of the different lipid A preparations, but the quantity of each band varied between the sources of lipid A. Four bands (designated 2, 3, 7, and 8) were abundant in every preparation. Variations of conditions used for preparing lipid A, such as changing of hydrolysis time, did not affect the appearance of lipid A on thin-layer chromatography. Change in the type of acid used for hydrolysis also did not affect the band pattern, but it did change the quantitative amounts of the various bands to some degree.
Protein on the cell surface of the moderately halophilic phototrophic bacterium Rhodospirillum salexigens
Authors: Evers D , Weckesser J , Drews G
Published: January 2020

A cell surface protein (Mr 68,000) of the moderately but obligately halophilic phototrophic bacterium Rhodospirillum salexigens was identified by two independent methods: first, by labeling the cell surface with radioactive iodine and lactoperoxidase, and second, by washing cells in 30% sucrose to remove proteins attached to the cell surface by ionic bonds. The identified protein very likely represents the outermost layer of the cell envelope of R. salexigens as observed by electron microscopy. The protein was isolated. Its isoelectric point was determined to be 4.4; the excess of acidic over basic amino acids was found to be 18.3 mol%; and its average hydrophobicity was 2.26 kJ per residue.
Study of regulation and transport of hemolysin by using fusion of the beta-galactosidase gene (lacZ) to hemolysin genes
Authors: Juarez A , Härtlein M , Goebel W
Published: January 2020

Operon and gene fusions between lacZ and the hemolysin genes, hlyC and hlyA, were performed. These two genes are essential for the synthesis of active hemolysin and are transcribed from a common promoter (p1). Whereas the amount of hemolysin produced in Escherichia coli is not changed by altering the hly gene dose, beta-galactosidase activity follows the gene dosage in both types of fusions when lacZ comes under the control of p1. This indicates that hemolysin is not negatively regulated on the transcription or translation level. The products of the gene fusions hlyC::lacZ and hlyA::lacZ were identified in maxicells as stable proteins of 146,000 and 220,000 daltons, respectively. Both fusion proteins possess beta-galactosidase activity indicating that the performed fusions of lacZ to the hly genes do not destroy the reading frame of hlyC and hlyA. The fusion proteins HlyC-beta-gal and HlyA-beta-gal were predominantly detected in the cytoplasm, confirming previous data which suggested that the primary gene products of hlyC and hlyA are not transported across the cytoplasmic membrane.
Methylammonium transport in Anacystis nidulans R-2
Authors: Boussiba S , Dilling W , Gibson J
Published: January 2020

Methylammonium was taken up rapidly by illuminated cells of Anacystis nidulans R-2, leading to internal concentrations of 1.3 +/- 0.1 mM within 1 min, and a gradient of up to 200 between the cells and medium. Accumulation of 14CH3NH3+ required at least 5 mM NaCl, but the uptake rate was independent of medium pH between 6.5 and 9. The kinetics of uptake could be resolved into an initial fast phase lasting less than 1 min (approximate Km, 7.2 microM; Vmax, 12.5 nmol min-1 mg of protein-1 at 15 degrees C). A second, slower phase associated with product formation was eliminated by preincubation with methionine sulfoximine, a specific inhibitor of glutamine synthetase; the rapid phase was unaffected by this treatment. Ammonium ions competed with 14CH3NH3+ for entry, and addition of 5 microM NH4+ or 100 microM CH3NH3+ released 14CH3NH3+ accumulated during the rapid phase of entry. Small additions of NH4+ made at the same time as additions of 14CH3NH3+ delayed the start of radioactivity uptake by a time which corresponded accurately with the period needed for the complete removal of the added NH4+. The effects of inhibitors on accumulation and carbocyanine dye fluorescence suggest that ATP-dependent membrane potential was needed to drive 14CH3NH3+ transport. Spheroplasts were as active as whole cells in accumulating NH4+ and 14CH3NH3+, indicating that soluble periplasmic components are not involved in the translocation. Some significant differences between the translocation of 14CH3NH3 and that of NH4+ were observed: growth with NH4+ in place of NO3- repressed 14CH3NH3+ accumulation ability without affecting the NH4+ uptake rate Na+ was not required for NH4+ uptake, and concentration of KCl inhibitory with 14C3NH3+ did not reduce NH4+ uptake.
H+/ATP stoichiometry of cowpea Rhizobium sp. strain 32H1 cells grown under nitrogen-fixing and nitrogen-nonfixing conditions
Authors: Gober J W , Kashket E R
Published: January 2020

The obligate aerobe Cowpea Rhizobium sp. strain 32H1 in axenic culture is able to fix N2 when grown under 0.2% O2 but not when grown under 21% O2. It was, therefore, of interest to investigate ATP synthesis in these cells grown under the two conditions. When respiring in buffers having pHs ranging from 6 to 8.5, cells grown under either O2 tension maintained an intracellular pH more alkaline than the exterior. The transmembrane chemical gradient of H+ (delta pH) was essentially the same under both conditions of growth, decreasing from ca. 90 mV at medium pH 6 to ca. 30 mV at pH 8.5. However, the transmembrane electrical gradient (delta psi) was significantly higher in cells grown under 21% O2 (150 to 166 mV) than in cells grown under 0.2% O2, the latter being 16 mV at pH 6 and increasing to 88 mV at pH 8.5. Therefore, the proton motive force of 21% O2-grown cells ranged from 237 mV at external pH 6 to 185 mV at pH 8.5, compared with a proton motive force of 114 to 121 mV in the 0.2% O2-grown cells. The cells grown in 0.2% O2 had the same proton motive force whether tested at 21 or at 0.2% O2. The phosphorylation potential, calculated from the intracellular ATP, ADP, and Pi concentrations, was 424 mV in the 21% O2-grown cells and 436 mV in the 0.2% O2-grown cells. Thus, the 21% O2-grown cells translocated 1.8 to 2.3 H+/ATP synthesized by the H+-ATPase, whereas the H+/ATP ratio for 0.2% O2-grown cells was 3.7 to 3.8.
Aerobactin iron uptake sequences in plasmid ColV-K30 are flanked by inverted IS1-like elements and replication regions
Authors: Perez-Casal J F , Crosa J H
Published: January 2020

By using Southern blot hybridization procedures, we found that a specific sequence within a 16.3-kilobase HindIII restriction fragment of pColV-K30 was also present in at least three other pColV-K30 HindIII fragments. Restriction endonuclease mapping of these HindIII fragments indicated that two of these repeated sequences, identified as IS1-like, occur in reverse orientation adjacent to the ends of the aerobactin iron uptake region, also included in the 16.3-kilobase HindIII fragment. There are two distinct replication regions enclosing the IS1-flanked aerobactin genes. A pColV-K30 BamHI restriction endonuclease fragment, carrying one of these replication regions, showed homology with the F plasmid EcoRI fragment f5, which carries the F replication sequences.
Specificity and mechanism of ferrioxamine-mediated iron transport in Streptomyces pilosus
Authors: Müller G , Raymond K N
Published: January 2020

Although the ferrioxamines are an important and well-characterized class of siderophores produced by several species of Nocardia, Streptomyces, Micromonospora, Arthrobacter, Chromobacterium, and Pseudomonas, no studies of the mechanism of ferrioxamine-mediated iron uptake have been performed for an organism which produces the siderophore. This is the first report of metal transport in Streptomyces pilosus mediated by the native ferrioxamines B, D1, D2, and E. 55Fe accumulation in these ferrioxamines was dependent on metabolic energy and was a saturable process with increasing complex concentration. The apparent Km for [55Fe]ferrioxamine B uptake was approximately 0.2 microM. Both chromic desferriferrioxamine B and [67Ga]desferriFerrioxamine B were transported at rates similar to those of the 55Fe complexes: this implies that no decomplexation or reduction of the metal ion is required for transport, since the chromic complexes are kinetically inert and the gallium complexes have no stable divalent state as a possible reduction product. In addition, isomers of inert chromic desferriferrioxamine B complexes were used to probe the stereospecificity of the ferrioxamine uptake system. The chromic complexes were separated into three fractions by cationic exchange chromatography and assigned as two cis and a (mixture of) trans geometrical isomer(s) by their visible spectra. [55Fe]ferrioxamine B uptake was equally inhibited by each isomer, suggesting that no differentiation between cis and trans geometrical isomers occurs. In the presence of chromic desferriferrioxamine B isomers, the uptake rates for 55Fe-labeled ferrioxamines E, D1, and D2 were even more strongly reduced than was that for [55Fe]ferrioxamine B itself. From these results we conclude that all the ferrioxamines tested are transported into the cells by the same uptake system.
DNA sequence of the F traALE region that includes the gene for F pilin
Authors: Frost L S , Paranchych W , Willetts N S
Published: January 2020

The complete sequence of a 1.4-kilobase PstI fragment containing the F transfer genes traA, -L, and -E is presented. The traA reading frame has been located both genetically and by comparing the primary structure of F pilin (the traA product) predicted by the DNA sequence to the amino acid composition and sequence of N- and C-terminal peptides isolated from purified F pilin. Taken together, these data show that there is a leader peptide of 51 amino acids and that F pilin contains 70 amino acids, giving molecular weights of 13,200 for F propilin and 7,200 for mature F pilin. Secondary structure predictions for F pilin revealed a reverse turn that precedes the sequence Ala-Met-Ala51, a classic signal peptidase cleavage site. The N-terminal alanine residue is blocked by an acetyl group as determined by 1H-nuclear magnetic resonance spectroscopy. The traL and traE genes encode proteins of molecular weights 10,350 and 21,200, respectively. According to DNA sequence predictions, these proteins do not contain signal peptide leader sequences. Secondary structure predictions for these proteins are in accord with traLp and traEp being membrane proteins in which hydrophobic regions capable of spanning the membrane are linked by sequences that form turns and carry positively charged residues capable of interacting with the membrane surface.
Vitamin requirements of three spiroplasmas
Authors: Chang C J
Published: January 2020

A chemically defined medium (CC-494M) was used to study the vitamin requirements of three spiroplasmas representing three distinct serogroups: flower spiroplasmas [Spiroplasma floricola and FS (SR-3)] and honeybee spiroplasma [HBS (AS-576)]. Nicotinic acid and riboflavin were essential to spiroplasma growth. Nicotinamide could substitute for nicotinic acid. Populations of S. floricola, FS (SR-3), and HBS (AS-576) reached 3.2 X 10(9), 1.96 X 10(10), and 6.1 X 10(9) CFU/ml, respectively, when nicotinic acid (0.036 mg/liter) and riboflavin (0.014 mg/liter) were supplied.
Regulatory role of phosphate and other anions in transport of ADP and ATP by Rickettsia prowazekii
Authors: Winkler H H , Daugherty R M
Published: January 2020

ADP and ATP were transported in Rickettsia prowazekii by an obligate exchange system without prior hydrolysis. The uptake of ATP and ADP by the obligate exchange system in R. prowazekii was dependent upon the anionic composition of the medium. The rate of transport of ATP was about three times greater than that of ADP in the absence of anions, and the rates of transport of both were about doubled by a variety of anions. However, phosphate anions were able to stimulate greatly the uptake of ADP so that in the presence of these anions, the uptake of ATP and that of ADP were about equal. Millimolar concentrations of anions were required to elicit the stimulation of ADP and ATP transport. The ADP-dependent efflux of ADP and ATP was also greatly stimulated by phosphate anions. The stimulation of ADP and ATP transport required that the anions be present in the external medium, as preincubation of the rickettsiae with phosphate anions was neither necessary nor sufficient. The competitive inhibition of ATP uptake by ADP required phosphate anions, indicating that phosphate anions increased the affinity of ADP for the transport system. The role of phosphate in the regulation of ATP and ADP exchange and its significance are discussed.
Transposon Tn10-like tetracycline resistance determinants in Haemophilus parainfluenzae
Authors: Levy S B , Buu-Hoi A , Marshall B
Published: January 2020

Tetracycline resistance (Tcr) determinants from three different strains of Haemophilus parainfluenzae expressed 10-fold higher levels of resistance when mated into Escherichia coli. No plasmid was found in any of the E. coli recipients, even in matings in which a plasmid was identified in the donor Haemophilus sp. The Tcr determinant from Haemophilus sp. caused instability of resident plasmids in the recipient E. coli: all plasmids were lost within 30 generations in antibiotic-free media. However, by serial subculture in antibiotics, stable resident plasmids were obtained which carried the Tcr determinant from Haemophilus sp. and were transferable by conjugation and transformation among E. coli strains. All Haemophilus determinants hybridized with a probe for the Tcr determinant on Tn10, which bears inducible Tcr. However, Haemophilus determinants were constitutively resistant to tetracycline in the Haemophilus donors and in the E. coli recipients. This constitutive expression was recessive to wild-type Tn10 in the same cell, indicating that the constitutive phenotype resulted from the absence of an active repressor. Restrictive enzyme analysis of various E. coli plasmid derivatives bearing a Tcr determinant from Haemophilus sp. demonstrated that the inserted DNA was of similar size (8.95 to 9.35 kilobases), close to that of Tn10. Heteroduplex analysis and DNA:DNA hybridization confirmed that the Tcr determinant from Haemophilus sp. had greater than 90% homology with the Tn10 determinant, including the DNA sequence for the repressor.
Identification of additional genes on transposon Tn10: tetC and tetD
Authors: Braus G , Argast M , Beck C F
Published: January 2020

Two genes (tetC and tetD) were identified and located on transposon Tn10 between gene tetA and insertion sequence IS10R. Genes tetC and tetD encode proteins of apparent subunit molecular weights of 23,000 and 18,000, respectively. The TetD protein was found to be membrane associated. Tetracycline resistance levels promoted by transposon Tn10 were found to be unaffected in Escherichia coli K-12 when mutants lacking tetC or tetC and tetD were tested. The nucleotide sequence of genes tetC and tetD is reported in the accompanying article (K. Schollmeier and W. Hillen, J. Bacteriol. 160:499-503, 1984).
Identification of Escherichia coli region III flagellar gene products and description of two new flagellar genes
Authors: Bartlett D H , Matsumura P
Published: January 2020

Region III flagellar genes in Escherichia coli are involved with the assembly and rotation of the flagella, as well as taxis. We subcloned the flaB operon from a lambda fla transducing phage onto plasmid pMK2004. Two additional genes were found at the flaB locus, and we subdivided the flaB gene into flaB1, flaBII, and flaBIII. The cheY suppressor mutations which have previously been mapped to flaB were further localized to flaB11 (Parkinson et al., J. Bacteriol. 155:265-274, 1983). Until now, gene product identification has not been possible for these genes because of their low levels of gene expression. Overexpression of the flagellar genes was accomplished by placing the flaB operon under the control of the lacUV5 or tac promoters. Plasmid-encoded proteins were examined in a minicell expression system. By correlating various deletions and insertions in the flaB operon with the ability to complement specific flagellar mutants and code for polypeptides, we made the following gene product assignments: flaB 1, 60 kilodaltons; flaB 11, 38 kilodaltons; flaB111, 28 kilodaltons; flaC, 56 kilodaltons; fla0, 16 kilodaltons; and flaE, 54 kilodaltons.
Regulation of phenylalanine biosynthesis in Rhodotorula glutinis
Authors: Fiske M J , Kane J F
Published: January 2020

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.
Two-component suppression of recF143 by recA441 in Escherichia coli K-12
Authors: Volkert M R , Margossian L J , Clark A J
Published: January 2020

Sensitivity to UV irradiation conferred by recF143 was partially suppressed by recA441 (also known as tif-1). A temperature-conditional component depended on uvrA function and is thought to involve thermal induction of excision repair enzymes. In a uvrA6 mutant, a temperature-independent component of suppression was seen. This is thought to indicate that recA441 also caused temperature-independent changes in recA activity. Two hypotheses are offered to explain how recA441 produced both thermosensitive and thermoindependent effects.
Transcriptional organization within an Escherichia coli cell division gene cluster: direction of transcription of the cell separation gene envA
Authors: Sullivan N F , Donachie W D
Published: January 2020

A cluster of at least 14 genes, each concerned with some aspect of cell envelope growth, morphogenesis, or function, is located at 2 min on the genetic map of Escherichia coli. We located the envA cell division gene and its promoter within the cluster and determined the direction of transcription of the gene by constructing fusions between its promoter and the galK coding sequence. In addition, we identified the promoter of a possible new gene lying between envA and the secA gene. We also present evidence from gene fusion studies which shows the direction of transcription of the ftsZ(sulB) division gene. The direction of transcription is the same for all three promoters and is the same as that of all other cluster genes for which this is known. We discuss the significance of this observation, together with the fact that every gene examined in sufficient detail within the cluster appears to have its own promoter and to be able to be expressed from isolated cloned fragments. Using a novel variable-copy plasmid vector, we demonstrate that the DNA fragment containing the envA gene is not stably maintained in multiple copies. The construction of two independent, nonoverlapping deletions allows us to conclude that the envA product itself is responsible for this effect.
An acidic or basic amino acid at position 26 of the b subunit of Escherichia coli F1F0-ATPase impairs membrane proton permeability: suppression of the uncF469 nonsense mutation
Authors: Jans D A , Hatch L , Fimmel A L , Gibson F , Cox G B
Published: January 2020

The uncF469 allele differed from normal in that a G----A base change occurred at nucleotide 77 of the uncF gene, resulting in a TAG stop codon rather than the tryptophan codon TGG. Two partial revertant strains were isolated which retained the uncF469 allele but formed a partially functional b-subunit, due to suppression of the uncF469 nonsense mutation. From the altered isoelectric points of the b-subunits from these strains, it was concluded that the suppressor gene of partial revertant strain AN1956 inserts an acidic amino acid for the TAG codon, and that the suppressor gene of partial revertant strain AN1958 inserts a basic amino acid. The membranes of both partial revertant strains showed impaired permeability to protons on removal of F1-ATPase. The membranes of both strains, however, were able to carry out oxidative phosphorylation, and the ATPase activities of both were resistant to the inhibitor dicyclohexylcarbodiimide.
Dissimilation of aromatic compounds in Rhodotorula graminis: biochemical characterization of pleiotropically negative mutants
Authors: Durham D R , McNamee C G , Stewart D B
Published: January 2020

Microorganisms oxidize many aromatic compounds through the dihydroxylated intermediates catechol and protocatechuate and through the beta-ketoadipate pathway. The catabolic sequences used by the yeast Rhodotorula graminis for the dissimilation of aromatic compounds were elucidated after biochemical analysis of pleiotropically negative mutant strains. Growth properties of one mutant strain revealed that benzoate-4-hydroxylase was required for the utilization of phenylalanine, mandelate, and benzoate. Analysis of benzoate-4-hydroxylase- and p-hydroxybenzoate hydroxylase-deficient mutants provided genetic evidence that benzoate was hydroxylated in the para position forming p-hydroxybenzoate. Enzyme assays and growth studies with wild-type and mutant strains of R. graminis indicated that separate and highly specific hydroxylases oxidized p-hydroxybenzoate and m-hydroxybenzoate to protocatechuate. Examination of a protocatechuate 3,4-dioxygenase-deficient mutant demonstrated the role of the protocatechuate branch of the eucaryotic beta-ketoadipate pathway for the utilization of phenylalanine, mandelate, benzoate, and m-hydroxybenzoate. Salicylate, on the other hand, was shown to be metabolized through catechol. Thus, R. graminis differs from other yeasts such as Trichosporon cutaneum and Rhodotorula mucilaginosa in that it contains both branches of the beta-ketodipate pathway.
Escherichia coli recBC deletion mutants
Authors: Chaudhury A M , Smith G R
Published: January 2020

Mutants of Escherichia coli with deletions of the recB and recC genes were obtained by two methods using transposable DNA elements. The phenotypes of these mutants are similar to those of mutants with recBC point mutations. These results indicate that the RecBC gene products, exonuclease V, is not essential for the growth of E. coli but is important for DNA repair and recombination.
Genetic system of Schwanniomyces alluvius determined by diad analysis of fusion products
Authors: James A P , Zahab D M
Published: January 2020

The genetics of Schwanniomyces alluvius, a yeast that secretes alpha-amylase, were investigated. No mating types have been detected in this haploid organism. Hybrids were produced by protoplast fusion, and these were subjected to diad analysis by using two-spored asci. Results showed that the diploidy introduced by cell fusion persists through successive spore generations. It was concluded that in this organism, sporulation is preceded by the fusion of mitotic products, regardless of the ploidy of the latter. Routine procedures for constructing novel strains would, no doubt, be hampered by this failure of the sporulation process to restore haploidy. Nevertheless, chromosomal instability of hybrids, as indicated both by heterogeneity of fusion products and by a high frequency of morbidity among their segregants, may permit the use of classical genetic techniques for strain construction.
Protein and nucleic acid synthesis during synchronized growth of Plasmodium falciparum
Authors: Gritzmacher C A , Reese R T
Published: January 2020

Plasmodium falciparum, the human malarial parasite, was synchronized for asexual growth and pulse labeled to determine when RNA, protein, and DNA synthesis occurred. RNA was synthesized during two periods in the 48-h developmental cycle, protein synthesis occurred throughout the cycle, and most DNA was synthesized just before nuclear division (schizogony).
Spectrophotometric determination of affinities of peptides for their transport systems in Escherichia coli
Authors: Perry D , Gilvarg C
Published: January 2020

The use of novel synthetic peptides to measure peptide transport by spectrophotometric means is described. These peptides contain glycine residues alpha-substituted with thiophenol and are recognized as substrates by both peptide transport systems and intracellular peptidases of Escherichia coli (Kingsbury et al., Gilvarg, C., Proc. Natl. Acad. Sci. U.S.A. 81:4573-4576, 1984). Transport and peptidase cleavage results in the intracellular release of thiophenol, which exits rapidly from the cell. The release of thiophenol from these peptides by cell suspensions can be measured with Ellman sulfhydryl reagent [5,5'-dithiobis(2-nitrobenzoic acid)] and provides a direct determination of the rate of peptide transport. The reductions in thiophenol release from these peptides resulting from the addition of peptide competitors enable the affinities of the competitors for their transport systems to be determined. By this method, it is shown that the dipeptide transport system is more restrictive with respect to changes in the amino acid sidechains of its substrates than those of the oligopeptide transport system.
Isolation of a versatile Serratia marcescens mutant as a host and molecular cloning of the aspartase gene
Authors: Takagi T , Kisumi M
Published: January 2020

An extracellular nuclease-deficient, antibiotic-sensitive, and restrictionless mutant was isolated from the wild-type strain of Serratia marcescens Sr41 by four rounds of mutagenesis. The mutant was transformed efficiently with plasmid DNAs prepared from Escherichia coli and S. marcescens, and was used as a host for the cloning of the aspartase gene (aspA+) of S. marcescens. Cells carrying the cloned aspA+ gene on a multicopy plasmid produced ca. 39-fold more aspartase than did control cells, and the level of enzyme overproduction was in proportion to the copy number of the aspA+ recombinant plasmid. Aspartase was identified as a polypeptide of molecular weight 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships
Authors: Surin B P , Rosenberg H , Cox G B
Published: January 2020

The DNA nucleotide sequence of four genes for the phosphate-specific transport system of Escherichia coli is reported. Along with the DNA sequence for the phoS gene reported previously (Surin et al., J. Bacteriol. 157:772-778, 1984; Magota et al., J. Bacteriol. 157:909-917, 1984), this study completes the nucleotide sequence of the phosphate-specific transport region. The complete sequence (including phoS) contains five open reading frames oriented in the same direction, each preceded by a putative ribosome-binding site near the presumed translation initiation codon ATG. The complete sequence is transcribed counterclockwise, in the order phoS pstC pstA pstB phoU. Genetic complementation shows that of the four open reading frames in the new sequence, three correspond to known mutant alleles; the fourth, which was designated pstC, has not been described before and could not be related to any known mutant allele. We have confirmed that pstA was allelic to phoT32. The pstC, pstB, and phoU gene products were identified as peripheral membrane proteins. The pstA gene product appears to be an integral membrane protein.
Two control systems modulate the level of glutaminyl-tRNA synthetase in Escherichia coli
Authors: Cheung A Y , Watson L , Söll D
Published: January 2020

We studied the regulation of in vivo expression of Escherichia coli glutaminyl-tRNA synthetase at the transcriptional and translational level by analysis of glnS mRNA and glutaminyl-tRNA synthetase levels under a variety of growth conditions. In addition, strains carrying fusions of the beta-galactosidase structural gene and the glnS promoter were constructed and subsequently used for glnS regulatory studies. The level of glutaminyl-tRNA synthetase increases with the increasing growth rate, with a concomitant though much larger increase in glnS mRNA levels. Thus, transcriptional control appears to mediate metabolic regulation. It is known that glnR5, a regulatory mutation unlinked to glnS, causes overproduction of glutaminyl-tRNA synthetase. Here we showed that the glnR5 product enhances transcription of glnS 10- to 15-fold. The glnR5 mutation does not affect metabolic control. Thus, glnS appears to be regulated by two different control systems affecting transcription. Furthermore, our results suggest post-transcriptional regulation of glutaminyl-tRNA synthetase.
Nodules are induced on alfalfa roots by Agrobacterium tumefaciens and Rhizobium trifolii containing small segments of the Rhizobium meliloti nodulation region
Authors: Hirsch A M , Drake D , Jacobs T W , Long S R
Published: January 2020

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.
Location of some proteins involved in peptidoglycan synthesis and cell division in the inner and outer membranes of Escherichia coli
Authors: Rodríguez-Tébar A , Barbas J A , Vázquez D
Published: January 2020

Inner and outer membranes of Escherichia coli were separated by isopycnic centrifugation in sucrose gradients and analyzed for the presence of penicillin-binding proteins. All penicillin-binding proteins--except penicillin-binding protein 3, which is found almost exclusively in the cytoplasmic membrane and is involved in septum formation--are also found in gradient fractions corresponding to the outer membrane. Our results support the hypothesis that approximately half of the total amount of penicillin-binding proteins may be sacculus-located proteins linked to the outer membrane, probably through peptidoglycan bridges.
A mutation in Escherichia coli K-12 results in a requirement for thiamine and a decrease in L-serine deaminase activity
Authors: Newman E B , Miller B , Colebrook L D , Walker C
Published: January 2020

Mutants of Escherichia coli K-12 deficient in L-serine deaminase (L-SD) activity have been isolated. These strains required thiamine and grew normally when it was provided. The decrease in L-SD activity caused no obvious metabolic deficiency. A study of revertants and transductants showed that a single mutation was responsible for the thiamine requirement and for the decrease in L-SD activity.
Stable inheritance of plasmid R1 requires two different loci
Authors: Gerdes K , Larsen J E , Molin S
Published: January 2020

The largest EcoRI fragment from plasmid R1 mediates a stability phenotype which is required to ensure the stable inheritance of this low-copy-number plasmid. When covalently linked to small, unstable R1 derivatives, this fragment makes the plasmids as stable as the wild-type R1 plasmid. A genetic analysis showed that two independently acting stabilization functions are encoded by this EcoRI fragment, both of which have the potential of partial stabilization of mini-R1 plasmids. The two loci are located at opposite ends of the fragment. Stabilization was also obtained by inserting these regions in unrelated, unstable plasmids from the p15 group. One of the two functions was very efficient in stabilizing such foreign replicons. Besides the stability phenotype, these genes exert incompatibility in an allele-specific manner. The stability functions do not seem to interfere seriously with the copy number of the plasmid.
Mapping of a mutation affecting regulation of iron uptake systems in Escherichia coli K-12
Authors: Bagg A , Neilands J B
Published: January 2020

The site of a mutation resulting in constitutive derepression of iron uptake systems has been localized at 15.7 min on the genetic map of Escherichia coli K-12.
Evidence for an internal promoter in the Escherichia coli threonine operon
Authors: Saint Girons I , Margarita D
Published: January 2020

We constructed plasmids carrying the two first genes of the threonine operon from which the major promoter was deleted in vitro by digestion with BAL 31 nuclease. These plasmids continued to express the second gene (thrB) of the operon as judged by their ability to complement a threonine auxotroph. These data indicate that, in addition to the major promoter thrP, there was an internal promoter, thrBp, which could be used for the transcription of the thrB, the second gene of the operon. Additional evidence was given by subcloning a 230-base-pair segment of the operon in a plasmid suitable for detection of translation initiation signals and promoters. The thrBp promoter was thus shown to lie within a 61-base-pair fragment at the 3' end of the first gene, thrA, of the threonine operon.
Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides
Authors: Muller E D , Chory J , Kaplan S
Published: January 2020

We report the cloning and characterization of the gene product of the gene for the form II ribulose bisphosphate carboxylase from Rhodopseudomonas sphaeroides. We present evidence that the form II enzyme is encoded by a single gene in R. sphaeroides; however, this gene does hybridize to a second chromosomal locus.
Conjugation-mediated genetic exchange in Legionella pneumophila
Authors: Dreyfus L A , Iglewski B H
Published: January 2020

Genetic exchange mechanisms, to our knowledge, have not been reported for Legionella pneumophila, and consequently, studies on the genetic organization of L. pneumophila have not appeared in the literature. Here, we describe gene transfer mediated by broad host range conjugative plasmids in Legionella spp. Escherichia coli strains carrying plasmids RP1 and R68.45 (IncP1), S-a (IncW), and R40a (IncC), but not plasmids of incompatibility groups FI, FII, and FV, served as donors in matings with L. pneumophila Knoxville 1 (LPK-1). Transconjugants selected by resistance to kanamycin (RP1, R68.45, and S-a) and carbenicillin (R40a) were observed at frequencies of 6.6 X 10(-3), 4.7 X 10(-3), 2.2 X 10(-4), and 5.4 X 10(-5), respectively. Plasmid transfer was not affected by DNase added to the mating medium. After plasmid transfer, LPK-1 stably maintained RP1, R68.45, and S-a, but not R40a. Plasmid-containing LPK-1 isolates also served as donors in agar plate matings with E. coli W1485-1 and naladixic acid-resistant mutants of LPK-1, Legionella micdadei, and Legionella longbeachii. Recombinational exchange of a chromosomal trait was demonstrated when a thymidine auxotroph of L. pneumophila was repaired by R68.45-mediated chromosomal mobilization of a prototrophic donor strain.
Analysis of the individual regulatory components of the IncFII plasmid replication control system
Authors: Womble D D , Dong X , Luckow V A , Wu R P , Rownd R H
Published: January 2020

Replication of the IncFII plasmid NR1 is controlled by regulating the amount of synthesis of the repA1 initiator protein at both the transcriptional and translational levels. We have examined mutations which have altered each of these levels of regulation, resulting in different plasmid copy numbers. The genes which encode each of the individual wild-type or mutant regulatory components from the replication control region of NR1 have been cloned independently into pBR322 vectors, and their effects in trans, either individually or in various combinations, on plasmid incompatibility, stability, copy number, and repA1 gene expression have been defined.
Alpha mating type-specific expression of mutations leading to constitutive agglutinability in Saccharomyces cerevisiae
Authors: Doi S , Yoshimura M
Published: January 2020

Two mutants of Saccharomyces cerevisiae have been isolated and characterized. The mutants were constitutively agglutinable at 36 degrees C, the temperature at which wild-type cells agglutinate only after induction by mating pheromone. The mutant cells had other properties specific for the normal alpha cell type, i.e., conjugation with a cells, response to a mating pheromone, and production of alpha mating pheromone. The two mutations, cag1 and cag2, were recessive and expressed only in alpha cells. cag1 is linked very closely to the MAT locus, but cag2 is unlinked to the MAT locus. These cag mutations complemented ste3-1. These results indicate that CAG genes are novel alpha-specific genes involved in the regulation of sex agglutinin synthesis.
Molecular analysis of transferable tetracycline resistance plasmids from Clostridium perfringens
Authors: Abraham L J , Rood J I
Published: January 2020

Conjugative tetracycline resistance plasmids from 15 Clostridium perfringens isolates from piggeries were analyzed by restriction endonuclease digestion and agarose gel electrophoresis. Seven isolates from one farm were found to carry a 47-kilobase pair (kb) plasmid, pJIR5, which had EcoRI, XbaI, and ClaI profiles that were identical to those of a previously characterized plasmid, pCW3. An isolate from a second farm was found to carry a plasmid, pJIR6, which also was indistinguishable from pCW3. Five additional isolates from a third farm carried a 67-kb plasmid, pJIR2, which had at least 29 kb of DNA in common with pCW3. Finally, two isolates from a fourth farm were found to carry a 50-kb plasmid pJIR4, which appeared to consist of an entire pCW3 molecule with a 3-kb insertion. Comparative restriction maps of pCW3, pJIR2, and pJIR4 that identified the regions of homology among these plasmids were constructed. We suggest that many conjugative tetracycline resistance plasmids in C. perfringens may contain a pCW3-like core.
Regulation of a cya-lac fusion by cyclic AMP in Salmonella typhimurium
Authors: Jovanovich S B
Published: January 2020

cya-lac and crp-lac operon fusions were isolated in Salmonella typhimurium by using the phage Mu d1(lac cts Apr). Both transduction and reversion analyses have indicated that lac expression is controlled by the appropriate promoter, e.g., either crpp or cyap. By using chromosomal mobilization techniques, we found that cya had a clockwise direction of transcription on the standard S. typhimurium map. The cya-lac fusions could be complemented by Escherichia coli F'133, which covers cya, with a resultant 17 to 38% decrease in cya expression. Cyclic AMP was found to be able to repress the expression of the cya-lac fusion ninefold when present at 25 mM. This repression was not seen in crp backgrounds, and hence is mediated by the cAMP receptor protein. Repression of cya was also found upon growth on carbon sources known to elicit high cyclic AMP levels.
Three-dimensional architecture of the cell sheath and septa of Methanospirillum hungatei
Authors: Shaw P J , Hills G J , Henwood J A , Harris J E , Archer D B
Published: January 2020

The methanogenic bacterium Methanospirillum hungatei exists as filaments which have a very unusual cell wall architecture, comprising a long cylindrical sheath within which there may be many individual cells arranged in a line. The sheath has a two-dimensional crystalline structure, and the cells are separated within the tube by septa which also have a crystalline structure. We have used computer image processing of tilted-view electron micrographs to analyze the structure in negative stains of both of these components in three dimensions. The repeating unit of the sheath consists of four approximately spherical domains ca. 2.5 nm in diameter arranged in a row. Based on observations of the type of lattice imperfections that occur, we suggest that each of the domains represents a separate polypeptide subunit and that the subunits are incorporated into the wall one by one. The septa are circular plates of remarkably constant size. They are normally found as double layers. They are hexagonally symmetrical and consist of trimerically associated subunits which interact about dimer axes to form an open network containing large pores ca. 15 nm in diameter.
Isolation of a DNA Fragment Containing Replication Functions from IncP2 Megaplasmid pMG2
Authors: Prince Alice S. , Barlam Tamar
Published: January 2020

Replication functions with IncP2 specificity were identified on a 3-kilobase DNA fragment isolated from the 400-kilobase
Pseudomonas
megaplasmid pMG2.
Autogenous posttranscriptional regulation of RNA polymerase beta and beta' subunit synthesis in Escherichia coli
Authors: Dennis P P , Nene V , Glass R E
Published: January 2020

Bacterial strains carrying poorly suppressed amber mutations in the RNA polymerase beta subunit gene (rpoB) exhibit regulatory compensation. This compensation allows these strains to produce an adequate content of RNA polymerase to support a near normal rate of growth despite the poorly suppressed amber mutation. The primary compensatory mechanism permitting the elevated expression functions by permitting a much more efficient (up to threefold) loading of ribosomes at the beta cistron translation initiation site on the mRNA. This result supports the concept that the production of beta and beta' RNA polymerase subunits are autogenously regulated at the level of mRNA translation; this translational mechanism is clearly distinct from the transcriptional mechanism regulating beta and beta' expression described previously (P. P. Dennis, Proc. Natl. Acad. Sci. U.S.A. 74:5416-5420, 1977).