bluuebunny/crossref_metadata_2025 huggingface dataset:
Overproduction of subunit a of the F0 component of proton-translocating ATPase inhibits growth of Escherichia coli cells
Authors: Kanazawa H , Kiyasu T , Noumi T , Futai M
Published: January 2020

A hybrid plasmid, pKY159, carrying the promoter and the proximal region of the gene cluster for proton-translocating ATPase caused growth inhibition of Escherichia coli cells (K. Yamaguchi and M. Yamaguchi, J. Bacteriol. 153:550-554, 1983). The mechanism of this growth inhibition was studied, especially in terms of the responsible gene(s). Insertion of IS1, IS5, or gamma delta between the promoter and the gene for a possible component of the ATPase of 14,000 daltons (14K protein) released the inhibitory effect by pKY159. Deletion of the gene for subunit a also released the effect. However, deletion in the gene for the 14K protein released the effect only with an additional insertion within the gene. These results suggested that overproduction of subunit a is closely related to growth inhibition, whereas the 14K protein is not.
Purification and properties of glutathione reductase from the cyanobacterium Anabaena sp. strain 7119
Authors: Serrano A , Rivas J , Losada M
Published: January 2020

An NADPH-glutathione reductase (EC 1.6.4.2) has been purified 6,000-fold to electrophoretic homogeneity from the filamentous cyanobacterium Anabaena sp. strain 7119. The purified enzyme exhibits a specific activity of 249 U/mg and is characterized by being a dimeric flavin adenine dinucleotide-containing protein with a ratio of absorbance at 280 nm to absorbance at 462 nm of 5.8, a native molecular weight of 104,000, a Stokes radius of 4.13 nm, and a pI of 4.02. The enzyme activity is inhibited by sulfhydryl reagents and heavy-metal ions, especially in the presence of NADPH, with oxidized glutathione behaving as a protective agent. As is the case with the same enzyme from other sources, the kinetic data are consistent with a branched mechanism. Nevertheless, the cyanobacterial enzyme presents three distinctive features with respect to that isolated from non-photosynthetic organisms: (i) absolute specificity for NADPH, (ii) an alkaline optimum pH value of ca. 9.0, and (iii) strong acidic character of the protein, as estimated by column chromatofocusing. The kinetic parameters are very similar to those found for the chloroplast enzyme, but the molecular weight is lower, being comparable to that of non-photosynthetic microorganisms. A protective function, analogous to that assigned to the chloroplast enzyme, is suggested.
Mini-Mu-duction as a test for genetic complementation in Escherichia coli
Authors: Cronan J E
Published: January 2020

In mini-Mu-duction, segments of host DNA bracketed between two copies of an internally deleted Mu phage (a mini-Mu) can be packaged within Mu phage particles. Upon infection of a second host strain, the DNA injected by these particles can insert into the chromosomal DNA in a reaction catalyzed by the phage A gene product (transposase), which is independent of homologous recombination. This results in a partially diploid host strain in which the duplicated host DNA is bracketed by two copies of the mini-Mu phage (Faelen et al., Mol. Gen. Genet. 176:191-197, 1979). The frequency of mini-Mu-duction reported previously was low (10(-8) to 10(-9) per recipient cell) thus limiting its use to rather stable mutational lesions. I have increased the frequency of mini-Mu-duction 10- to 100-fold by use of a helper phage lacking the kil gene and by UV irradiation of the phage stocks. I have also shown that mini-Mu-duction is a reliable complementation assay in rec+ as well as recA recipient strains. This genetic complementation test does not require prior gene localization and (due to the extended host range of phage Mu) should be applicable to many enterobacterial species.
New chloramphenicol resistance locus in Bacillus subtilis
Authors: Anderson L M , Henkin T M , Chambliss G H , Bott K F
Published: January 2020

A spontaneously occurring, noninducible, chloramphenicol-resistant mutant of Bacillus subtilis 168 has a mutation (cam-2) which maps in the ribosomal protein region of the chromosome near dal. Its presence does not confer dependence on chloramphenicol. Ribosomes of the cam-2 strain remained sensitive to chloramphenicol in in vitro protein synthesis. No chloramphenicol acetyltransferase activity could be detected.
Methylation of ribosomal proteins in bacteria: evidence of conserved modification of the eubacterial 50S subunit
Authors: Amaro A M , Jerez C A
Published: January 2020

Methylation of the 50S ribosomal proteins from Bacillus stearothermophilus, Bacillus subtilis, Alteromonas espejiana, and Halobacterium cutirubrum was measured after the cells were grown in the presence of [1-14C]methionine or [methyl-3H]methionine or both. Two-dimensional polyacrylamide gel electrophoretic analysis revealed, in general, similar relative electrophoretic mobilities of the methylated proteins from each eubacterium studied. Proteins known to be structurally and functionally homologous in several microorganisms were all methylated. Thus, the following group of proteins, which appear to be involved in peptidyltransferase or in polyphenylalanine-synthesizing activity in B. stearothermophilus (P.E. Auron and S. R. Fahnestock, J. Biol. Chem. 256:10105-10110, 1981), were methylated (possible Escherichia coli methylated homologs are indicated in parentheses): BTL5(EL5), BTL6(EL3), BTL8(EL10), BTL11(EL11), BTL13(EL7L12) and BTL20b(EL16). In addition, the pentameric ribosomal complex BTL13 X BTL8, analogous to the complex EL7L12 X EL10 of E. coli, contained methylated proteins. Analysis of the methylated amino acids in the most heavily methylated proteins, BSL11 from B. subtilis and BTL11 from B. stearothermophilus, showed the presence of epsilon-N-trimethyllysine as the major methylated amino acid in both proteins, in agreement with known data for E. coli. In addition, BSL11 appeared to contain trimethylalanine, a characteristic, modified amino acid previously described only in EL11 from E. coli. These results and those previously obtained from other bacteria indicate a high degree of conservation for ribosomal protein methylation and suggest an important, albeit unknown, role for the modification of these components in eubacterial ribosomes.
Pleomorphism of fusobacteria isolated from the cockroach hindgut
Authors: Foglesong M A , Cruden D L , Markovetz A J
Published: January 2020

Fusobacteria are commonly isolated from the hindgut of the cockroach Eublaberus posticus . Eleven strains isolated from E. posticus by us were keyed to four species, Fusobacterium necrophorum, F. varium , F. gonidiaformans , and F. prausnitzii , using current taxonomic criteria. With the exception of F. gonidiaformis , all species showed rods with swollen centers and large bodies. The pleomorphism of F. varium was examined by phase microscopy and scanning and transmission electron microscopy. The pleomorphic process begins with a gradual swelling at the center of the rod until a large round body is formed. Some of these round bodies then fragment, giving rise to rod-shaped cells. When 10% yeast extract was added to growth media, pleomorphism was not induced. A dialyzable factor was found to account for this observation. Fermentation of [1-14C]glutamic acid gives rise to butyrate labeled in the carboxyl carbon, indicating that butyrate is formed by the hydroxyglutarate pathway which may be characteristic for the genus Fusobacterium.
Genome complexity of methanogenic bacteria
Authors: Klein A , Schnorr M
Published: January 2020

The genome complexities of different methanogenic bacteria were investigated by using an optical method to study renaturation kinetics of single-stranded DNA. The observed genome sizes ranged from 1.0 X 10(9) to 1.8 X 10(9) daltons, which is a typical range for procaryotic cells. Melting profiles of the DNA of three methanogenic species from different families show fractions which have a higher A . T content than the average DNA of that species.
Unique developmental characteristics of the swarm and short cells of Proteus vulgaris and Proteus mirabilis
Authors: Falkinham J O , Hoffman P S
Published: January 2020

Swarming cells of Proteus mirabilis and Proteus vulgaris could be distinguished from their short-cell counterparts by virtue of their synthesis (or lack of synthesis) of certain enzymes and outer membrane proteins. Urease synthesis was constitutive in swarm cells and uninducible in short cells. In contrast, phenylalanine deaminase was inducible in both short and swarm cells, demonstrating that transcriptional and translational processes were functional. During swarm cell development, the amount of one outer membrane protein (45 kilodaltons) fell and the amounts of two others (50 and 28.3 kilodaltons) rose significantly, the level of cytochrome b decreased, and the synthesis of cytochromes a and d were repressed. Respiratory activities of swarm cells were greatly diminished, suggesting that energy for swarming came from fermentation rather than from respiration. Widespread changes in the pattern of enzyme activities, in cytochrome composition, and in the composition and type of outer membrane proteins suggest that they are due to transcriptional regulation.
Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate
Authors: Goldberg J B , Ohman D E
Published: January 2020

Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis commonly produce a capsule-like exopolysaccharide called alginate. The alginate-producing (Alg+) phenotype results in a mucoid colony morphology and is an unstable trait. A mutant of P. aeruginosa FRD (a cystic fibrosis isolate) was obtained which was temperature sensitive for alginate production ( Algts ). At elevated growth temperatures (41 degrees C), no alginate was detected in culture supernatants of the Algts mutant, but yields of alginate increased as the temperature of incubation was reduced. The mutation responsible for the Algts phenotype, alg-50(Ts), has been mapped to a region of the FRD chromosome closely linked to trp-2. The alg-50(Ts) marker did not map near the met-l-linked chromosomal mutations responsible for the instability of the Alg+ phenotype. A broad host range cosmid cloning system based upon derivatives of plasmid RK2 was used to construct a P. aeruginosa clone bank. After transfer of the clone bank to the Algts mutant, hybrid plasmids were obtained which complemented the Algts defect. Deletion mapping of the original 20.3 kilobases of P. aeruginosa DNA cloned showed that a 4.7-kilobase fragment would complement the alg-50(Ts) mutation.
Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium
Authors: Nakamura T , Iwahashi H , Eguchi Y
Published: January 2020

S-Sulfocysteine synthase was isolated from Salmonella typhimurium LT-2 to homogeneous form with polyacrylamide gel electrophoresis. The molecular weight of this enzyme was determined to be ca. 55,000. The enzyme consisted of two identically sized subunits, and it contained one pyridoxal phosphate per subunit. The enzyme catalyzed the biosynthesis of cysteine or S-methylcysteine from sulfide or methanethiol and O-acetylserine, respectively, in addition to the formation of S-sulfocysteine from thiosulfate and O-acetylserine. The enzyme is identical to cysteine synthase B. The intracellular level of this enzyme was regulated by lesser extents of the same factors as those effective for cysteine synthase A.
Helical macrofiber formation in Bacillus subtilis: inhibition by penicillin G
Authors: Zaritsky A , Mendelson N H
Published: January 2020

The folding process required for helical macrofiber formation after the outgrowth of Bacillus subtilis spores was found to be blocked by very low concentrations of penicillin G (1 to 3 ng/ml). Under such conditions, growth and septation without cell separation resulted in characteristic disorganized multicellular structures. Higher concentrations (4 and 10 ng/ml) were needed to inhibit spore outgrowth and vegetative growth, respectively.
Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica
Authors: Miyakawa T , Kadota T , Okubo Y , Hatano T , Tsuchiya E , Fukui S
Published: January 2020

Mating pheromone-induced alteration of the cell surface proteins of haploid cells, presumed to play crucial roles in the specific cell-cell interactions during sexual conjugation of Tremella mesenterica , was investigated. Exposed surface proteins were revealed by lactoperoxidase-catalyzed iodination in combination with polyacrylamide gel electrophoresis and autoradiography. From comparison of the molecular species of 125I-labeled surface proteins of the vegetative and the gamete (mating pheromone-treated) cells of the two compatible mating types (ab and AB), it was suggested that a striking change in cell surface structure occurs during the differentiation; although labeled protein species of the vegetative cells of the two mating types were indistinguishable, several new species, both mating type specific and nonspecific, appeared in the gamete cells. Turnover of the labeled proteins of the vegetative cells was negligible, whereas that of the gamete cells was rapid with release of low-molecular-weight labeled proteins in the medium. A role for the labeled surface proteins of the gamete cells in the cell-cell interactions during sexual conjugation was suggested by the following: the surface changes were induced by mating pheromone; the labeled proteins were preferentially localized on the surface of the mating tube; the labeled species appeared sequentially during the differentiation; and mating type-specific species were present in both mating types.
Escherichia coli K-12 mutation that inactivates biodegradative threonine dehydratase by transposon Tn5 insertion
Authors: Goss T J , Datta P
Published: January 2020

From a collection of kanamycin-resistant mutants of Escherichia coli K-12 isolated by transposon Tn5 mutagenesis, we have identified a mutant that lacks functional biodegradative threonine dehydratase (EC 4.2.1.16) by direct enzyme assay and by the loss of cross-reacting material with affinity-purified antibodies against the purified enzyme. Aerobic and anaerobic growth of this strain on various carbon sources failed to reveal a phenotype. Evidence for the insertional inactivation of threonine dehydratase by Tn5 was obtained by cloning the DNA segments flanking the Tn5 insertion site into pBR322 and hybridizing the cloned DNA to a synthetic oligodeoxynucleotide probe complementary to the DNA segment coding for a unique hexapeptide at the amino terminus end of the enzyme; the region of homology to the synthetic cDNA sequence appears to be located within about 500 nucleotides from one end of Tn5. Genetic analysis with the transposon element that caused insertional inactivation located the tdc gene at min 67 on the E. coli chromosome.
Effect of purine and pyrimidine limitations on RNA synthesis in Bacillus subtilis
Authors: Vasantha N , Galliers E M , Hansen J N
Published: January 2020

The effects of varying the intracellular levels of GTP or UTP on the rate of RNA synthesis in Bacillus subtilis were studied. The levels of these nucleotides were manipulated by pyrimidine limitation in a pyr auxotroph, by purine limitation in a pur auxotroph, or by the addition of decoyinine , which specifically inhibits GMP synthesis. Decreased levels of UTP and GTP were accompanied by dramatically decreased synthesis and accumulation of stable RNAs (tRNA and rRNA), but mRNA synthesis was less affected. However, sporulation was initiated only when the intracellular level of GTP decreased.
Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli
Authors: Gardner A L , Aronson A I
Published: January 2020

The structural gene for glutamine synthetase (glnA) in Bacillus subtilis ( glnAB ) cloned in the lambda vector phage Charon 4A was used to transduce a lysogenic glutamine auxotrophic Escherichia coli strain to prototrophy. The defective E. coli gene ( glnAE ) was still present in the transductant since it could be transduced. In addition, curing of the prototroph resulted in the restoration of glutamine auxotrophy. Proteins in crude extracts of the transductant were examined by a "Western blotting" procedure for the presence of B. subtilis or E. coli glutamine synthetase antigen; only the former was detected. Growth of the strain in media without glutamine was not curtailed even when the bacteriophage lambda pL and pRM promoters were hyperrepressed . The specific activities and patterns of derepression of glutamine synthetase in the transductant were similar to those of B. subtilis, with no evidence for adenylylation. The information necessary for regulation of glnAB must be closely linked to the gene and appears to function in E. coli.
Anaerobiosis, formate, nitrate, and pyrA are involved in the regulation of formate hydrogenlyase in Salmonella typhimurium
Authors: Barrett E L , Kwan H S , Macy J
Published: January 2020

Three groups of mutants defective in the fermentative production of gas were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). One group consisted of strains which lacked hydrogenase. The mutation site for this group was located in the vicinity of the known hyd gene. A second group consisted of mutants which lacked the formate dehyrogenase associated with hydrogenase. The mutation site was located in four of them. It was not in the vicinity of the previously described fhlD gene but was instead located at 93 min on the Salmonella map. The third mutant group, which consisted of strains that produced gas in triple sugar iron agar but not in nutrient agar supplemented with glucose, appeared to be pyrA mutants. The insertion site was located in the vicinity of pyrA , and they required arginine and pyrimidines for growth. Expression of the lac operon in the hyd mutants was induced by anaerobiosis. It was only slightly increased by the addition of formate under anaerobic conditions and slightly decreased by the addition of nitrate. Nitrate had no effect in an hyd ::Mu d strain that also carried a chlC::Tn10 insertion. Full expression of the lac operon in the fhl mutants required both formate and anaerobic conditions. The presence of nitrate in addition to formate resulted in activities about half those obtained in its absence, even in the fhl ::Mu d chlC::Tn10 double mutant. In the absence of formate, nitrate reduced expression only in the fhl ::Mu d single mutants. Expression of the lac operon among the pyrA mutants was repressed by arginine and cytosine and also by anaerobiosis. An explanation for the involvement of pyrA in aerobic and anaerobic energy metabolism is proposed.
Enhancement of chemotaxis in Spirochaeta aurantia grown under conditions of nutrient limitation
Authors: Terracciano J S , Canale-Parola E
Published: January 2020

Spirochaeta aurantia M1 cells were grown in a chemostat under conditions of energy and carbon source limitation. The chemotactic responses of the chemostat-grown cells were compared with those of S. aurantia cells grown in batch culture in the presence of excess energy and carbon source. Chemotactic responses were measured by determining the number of cells that entered a capillary tube containing a solution of attractant. S. aurantia cells grown in the chemostat under energy and carbon source limitation exhibited enhanced chemotactic responses and detected lower concentrations of attractant, as compared with cells grown in batch culture. The chemotactic response toward an attractant was specifically enhanced when that attractant was the growth-limiting energy and carbon source. The medium used contained either D-glucose or D-xylose as the sole energy and carbon source. Cells had the greatest chemotactic response toward glucose when grown at a dilution rate (D) of 0.045 h-1 under glucose limitation and toward xylose when grown at D = 0.06 h-1 under xylose limitation. When cells were grown under glucose limitation (D = 0.045 h-1), they sensed concentrations of attractant (glucose) ca. 1,000 times lower than those sensed by batch-grown cells. A similar enhancement of sensing ability (toward xylose) was observed in cells grown under xylose limitation. The results indicated that S. aurantia cells are able to regulate their chemosensory system in response to nutrient limitation. Maximum enhancement of chemotaxis occurs in cells growing at very low concentrations of energy and carbon source. Most likely, this property provides the spirochetes with competitive advantages when the availability of nutrients becomes severely limited in their habitats.
Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions
Authors: Jeter R M , Olivera B M , Roth J R
Published: January 2020

In this paper, we report that the enteric bacterium Salmonella typhimurium synthesized cobalamin de novo under anaerobic culture conditions. Aerobically, metE mutants of S. typhimurium need either methionine or cobalamin as a nutritional supplement for growth. The growth response to cobalamin depends upon a cobalamin-requiring enzyme, encoded by the gene metH, that catalyzes the same reaction as the metE enzyme. Anaerobically, metE mutants grew without any nutritional supplements; the metH enzyme functioned under these conditions due to the endogenous biosynthesis of cobalamin. This conclusion was confirmed by using a radiochemical assay to measure cobalamin production. Insertion mutants defective in cobalamin biosynthesis (designated cob) were isolated in the three major branches of the cobalamin biosynthetic pathway. Type I mutations blocked the synthesis of cobinamide, type II mutations blocked the synthesis of 5,6-dimethylbenzimidazole, and type III mutations blocked the synthesis of cobalamin from cobinamide and 5,6-dimethylbanzimidazole. Mutants that did not synthesize siroheme (cysG) were blocked in cobalamin synthesis. Genetic mapping experiments showed that the cob mutations are clustered in the region of the S. typhimurium chromosome between supD (40 map units) and his (42 map units). The discovery that S. typhimurium synthesizes cobalamin de novo only under anaerobic conditions raises the possibility that anaerobically grown cells possess a variety of enzymes which are dependent upon cobalamin as a cofactor.
Isolation of pigmentation mutants of the green filamentous photosynthetic bacterium Chloroflexus aurantiacus
Authors: Pierson B K , Keith L M , Leovy J G
Published: January 2020

Mutants deficient in the production of bacteriochlorophyll c (Bchl c) and one mutant lacking colored carotenoids were isolated from the filamentous gliding bacterium Chloroflexus aurantiacus. Mutagenesis was achieved by using UV radiation or N-methyl-N'-nitro-N-nitrosoguanidine. Several clones were isolated that were deficient in Bchl c synthesis. All reverted. One double mutant deficient both in Bchl c synthesis and in the synthesis of colored carotenoids under anaerobic conditions was isolated. Isolation of a revertant in Bchl c synthesis from this double mutant produced a mutant strain of Chloroflexus that grew photosynthetically under anaerobic conditions and lacked colored carotenoids. Analysis of pigment contents and growth rates of the mutants revealed a positive association between growth rate and content of Bchl c under light-limiting conditions.
Host-dependent transposon Tn5-mediated streptomycin resistance
Authors: De Vos G F , Finan T M , Signer E R , Walker G C
Published: January 2020

Transposon Tn5 encodes streptomycin resistance in addition to kanamycin-neomycin resistance. This resistance was not detectable in Escherichia coli but was efficiently expressed in Rhizobium meliloti and certain other strains. By analysis of cloned Tn5 restriction endonuclease fragments, the streptomycin resistance (str) gene was located in the right-hand side of the central region as the transposon is conventionally drawn. Transcription of str appeared to originate at pL, the promoter for the neo gene (neomycin phosphotransferase type II). Expression of streptomycin resistance in E. coli was obtained after cloning of the neo-str region downstream of a strong E. coli promoter. A construct in which PL was deleted also showed differential expression of streptomycin resistance.
Two tuf genes in the cyanobacterium Spirulina platensis
Authors: Tiboni O , Di Pasquale G , Ciferri O
Published: January 2020

Probes derived from the tufA gene of Escherichia coli have been utilized to detect homologous sequences on Spirulina platensis DNA. A 6-kilobase-pair fragment of S. platensis DNA appears to contain two sequences homologous to the E. coli gene. Thus, as reported for gram-negative bacteria, the cyanobacterium presumably contains two tuf genes.
Two unlinked lysine genes (LYS9 and LYS14) are required for the synthesis of saccharopine reductase in Saccharomyces cerevisiae
Authors: Borell C W , Urrestarazu L A , Bhattacharjee J K
Published: January 2020

Three lysine auxotrophs, strains AU363, 7305d, and 8201-7A, were investigated genetically and biochemically to determine their gene loci, biochemical lesions, and roles in the lysine biosynthesis of Saccharomyces cerevisiae. These mutants were leaky and blocked after the alpha-aminoadipate step. Complementation studies placed these three mutations into a single, new complementation group, lys14. Tetrad analysis from appropriate crosses provided evidence that the lys14 locus represented a single nuclear gene and that lys14 mutants were genetically distinct from the other mutants (lys1, lys2, lys5, and lys9) blocked after the alpha-aminoadipate step. The lys14 strains, like lys9 mutants, accumulated alpha-aminoadipate-semialdehyde and lacked significant amounts of saccharopine reductase activity. On the bases of these results, it was concluded, therefore, that LYS9 and LYS14, two distinct genes, were required for the biosynthesis of saccharopine reductase in wild-type S. cerevisiae.
Fructose catabolism in Azospirillum brasilense and Azospirillum lipoferum
Authors: Goebel E M , Krieg N R
Published: January 2020

The pathways for catabolism of fructose were investigated in the type strains of Azospirillum lipoferum and Azospirillum brasilense grown aerobically with (NH4)2SO4 as the nitrogen source. When grown on fructose, the former species possessed a complete Entner-Doudoroff pathway, whereas the latter species lacked activity for glucose-6-phosphate dehydrogenase. Both species possessed a complete catabolic Embden-Meyerhof-Parnas pathway. Neither species possessed the key enzyme of the hexose monophosphate pathway, 6-phosphogluconate dehydrogenase. Both species could phosphorylate fructose to fructose-1-phosphate by means of a phosphoenolpyruvate-phosphotransferase system, and high activities of 1-phosphofructokinase occurred. Both species possessed glucokinase activity, but only A. lipoferum had hexokinase activity; moreover, the cells of A. brasilense were nearly impermeable to glucose, accounting for the inability of this species to grow on glucose. Both species possessed pyruvate dehydrogenase, a complete tricarboxylic acid cycle, a glyoxylate shunt, and malic enzyme. Analysis of the acidic end products for both species indicated the formation of only small amounts of various organic acids, and most of the titratable acidity was due to utilization of the ammonium ions of the medium. Gluconic acid was not formed during growth of either species on fructose but was detected during growth of A. lipoferum on glucose; this species also possessed an NADP-linked glucose dehydrogenase and gluconokinase.
K88ab gene of Escherichia coli encodes a fimbria-like protein distinct from the K88ab fimbrial adhesin
Authors: Mooi F R , van Buuren M , Koopman G , Roosendaal B , de Graaf F K
Published: January 2020

The K88ab adhesin operon of Escherichia coli encodes for a fimbrial protein (the K88ab adhesin) which is involved in colonization of the porcine intestine. We characterized a structural gene (gene A) which is part of the K88ab adhesin operon and codes for an as yet unidentified polypeptide (pA). A mutation in gene A resulted in accumulation of K88ab adhesin subunits inside the cell. The nucleotide sequence of gene A was determined, and the deduced amino acid sequence suggested that pA is synthesized as a precursor containing a typical N-terminal signal peptide. The molecular weight of pA was calculated to be ca. 17,600. Gene A is preceded by a sequence showing homology with the consensus promoter. Fimbrial subunits from a number of E. coli strains have significant homology at their N- and C-termini. pA also contained some of these conserved sequences and showed a number of other similarities with fimbrial subunits. Therefore, it seems likely that the K88ab adhesin operon codes for a fimbrial subunit (pA) distinct from the K88ab adhesin subunit.
Major extracellular protease of Neurospora crassa
Authors: Abbott R J , Marzluf G A
Published: January 2020

The inducible extracellular alkaline protease of Neurospora crassa was demonstrated to be a glycoprotein containing D-galactose residues by use of the enzyme-lectin conjugate horseradish peroxidase-Ricinus communis-agglutinin-120. The carbohydrate moiety of the protease appears to be a poor antigen since an antiserum made to the native enzyme recognizes epitopes determined only by the polypeptide portion of the enzyme. Immunochemical techniques were used to quantitatively precipitate protease labeled in vivo for electrophoretic analysis. Protease synthesis could not be detected in control, uninduced cultures, whereas ca. 0.4% of total cellular protein synthesis is devoted to protease formation under inducing conditions.
Biosynthesis of phospholipids in Clostridium butyricum: kinetics of synthesis of plasmalogens and the glycerol acetal of ethanolamine plasmalogen
Authors: Koga Y , Goldfine H
Published: January 2020

The biosynthesis of the plasmalogen forms of phosphatidylethanolamine (plasmenylethanolamine) and phosphatidylglycerol (plasmenylglycerol) and of the glycerol acetal of plasmenylethanolamine has been studied in cultures of Clostridium butyricum IFO 3852. When growing cells were pulsed with [32P]orthophosphate, there was a lag of 5 to 7 min between the rapid incorporation of label into the acylphosphatides and the rapid incorporation of label into the corresponding plasmalogens. The labeling of the glycerol acetal of plasmenylethanolamine was even slower. In pulse-chase experiments with 32Pi, the kinetics of labeling indicated precursor-product relationships between phosphatidylethanolamine and plasmenylethanolamine and between the latter and its glycerol acetal. A precursor-product relationship was also seen between phosphatidylglycerol and cardiolipin, but the kinetics of labeling of the alkenyl-containing forms of these lipids were not consistent with direct precursor-product relationships with the acyl lipids. In the presence of hydroxylamine and 32Pi, both phosphatidylserine and plasmenylserine accumulated 32P in a ratio of ca. 15:1. Upon release of the inhibition of phosphatidylserine decarboxylase, label appeared in the following sequence: phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine. Acyl phosphatidylglycerol was identified as a major phospholipid (17% of lipid phosphorus) in C. butyricum grown in low-phosphate (1.13 mM) medium with 50 mM Tris buffer. Of the acyl phosphatidylglycerol, 13% was acid labile. There appear to be two plasmalogen forms of acyl phosphatidylglycerol. One of these has a single alkenyl ether group, and the other has alkenyl ether groups on both glycerols.
Mutation causing reverse osmoregulation of synthesis of OmpF, a major outer membrane protein of Escherichia coli
Authors: Nara F , Inokuchi K , Matsuyama S , Mizushima S
Published: January 2020

Supplementation of growth media with high concentrations of substances like sucrose results in the induction of OmpC synthesis and the suppression of OmpF synthesis. We isolated a novel mutant in which OmpF synthesis is in the opposite direction from normal osmoregulation. By transductional mapping, the mutation was localized at 75 min between malA and aroB on the Escherichia coli chromosome map where the ompR-envZ region is. The mutation was suppressed by a plasmid carrying the ompR gene but not by a plasmid carrying the envZ gene alone. The mutation also resulted in the almost complete suppression of OmpC synthesis. However, the remaining OmpC synthesis was osmoregulated normally. Based on these observations, the mechanism of osmoregulation of OmpF-OmpC synthesis is discussed.
Effect of bacteriophage P1 lysogeny on lipopolysaccharide composition and the lambda receptor of Escherichia coli
Authors: Tomás J M , Kay W W
Published: January 2020

The outer membrane of Escherichia coli was altered as a consequence of lysogeny by bacteriophages P1 and P1 cmts. The predominant change was a reduction in the size of lipopolysaccharide to a heptose-deficient form. P1 cmts lysogens were still sensitive to several bacteriophages but were resistant to lambda vir. Neither whole cells nor solubilized outer membranes from P1 cmts lysogens were able to inactivate lambda vir, and 32P-labeled lambda vir was unable to adsorb to P1 cmts lysogens. P1 cmts lysogens were also affected in maltose transport. The level of periplasmic maltose-binding protein was reduced somewhat, but there was no significant reduction in the level of the outer membrane lambda receptor (LamB). These membrane abnormalities were all corrected in strains cured of P1 cmts. It is suggested that P1 cmts affects lipopolysaccharide biosynthesis by a phage conversion mechanism and consequently the function of the lambda receptor.
Deletion of plasmid sequences during Saccharomyces cerevisiae transformation
Authors: Clancy S , Mann C , Davis R W , Calos M P
Published: January 2020

Saccharomyces cerevisiae was transformed with DNA by the lithium acetate method. Mutation of nonselected markers on the transforming vector was observed at a frequency several orders of magnitude higher than spontaneous mutation frequencies. These mutations were shown to be deletions. Linearization of the vector before transformation stimulated deletion formation.
IncFII plasmid incompatibility product and its target are both RNA transcripts
Authors: Womble D D , Dong X , Wu R P , Luckow V A , Martinez A F , Rownd R H
Published: January 2020

The region of DNA coding for incompatibility (inc) and copy number control (cop) of the IncFII plasmid NR1 is transcribed in both the rightward and leftward directions. The rightward transcripts serve as mRNA for the repA1 protein, which is required for replication. A small, 91-base leftward transcript is synthesized from the opposite DNA strand and is complementary to a portion of the rightward mRNA near its 5' end. A 262-base-pair Sau3A restriction fragment that encodes the small leftward transcript, but does not include the rightward transcription promoters, was cloned into the vector pBR322 or pUC8. The same fragment was cloned from an Inc- mutant of NR1 that does not make the small leftward transcript. Transcription through the cloned fragments in these derivatives was under control of the tetracycline resistance gene in pBR322 or the lac promoter-operator in pUC8. In one orientation of the inserted DNA, a hybrid transcript containing rightward NR1 RNA sequences was synthesized. In the other orientation, a hybrid transcript containing leftward NR1 RNA sequences was synthesized. These plasmids were used to vary the intracellular levels of the rightward or leftward NR1 RNA transcripts and to test their effects in trans on various coresident derivatives of NR1. An excess of rightward NR1 RNA in trans stimulated expression of the essential repA1 gene and caused an increase in the copy number of a coresident NR1 plasmid. An excess of leftward NR1 RNA in trans inhibited the expression of the repA1 gene and lowered the coresident NR1 copy number, thereby causing incompatibility. A pBR322 derivative with no transcription through the cloned NR1 DNA had no effect in trans. These results suggest that the small leftward transcript is the incompatibility inhibitor of NR1 and that its target is the complementary portion of the rightward mRNA.
Transport of mevalonate by Pseudomonas sp. strain M
Authors: Gill J F , Beach M J , Rodwell V W
Published: January 2020

Pseudomonas sp. M, isolated from soil by elective culture on R,S-mevalonate as the sole source of carbon, possessed an inducible transport system for mevalonate. This high-affinity system had a pH optimum of 7.0, a temperature optimum of 30 degrees C, a Km for R,S-mevalonate of 88 microM, and a V max of 26 nmol of mevalonate transported per min/mg of cells (dry weight). Transport was energy dependent since azide, cyanide, or m-chlorophenylhydrazone caused complete cessation of transport activity. Transport of mevalonate was highly substrate specific. Of the 16 structural analogs of mevalonate tested, only acetoacetate, mevinolin, and mevaldehyde significantly inhibited transport. Growth of cells on mevalonate induced transport activity by 40- to 65-fold over that observed in cells grown on alternate carbon sources. A biphasic pattern for cell growth, as well as for induction of mevalonate transport activity, was observed when mevalonate was added to a culture actively growing on glucose. The induction of transport activity under these conditions began within 30 min after the addition of mevalonate and reached 60% of maximal activity during phase I. A further increase in mevalonate transport activity occurred during phase II of growth. Glucose was the preferred carbon source for growth during phase I, whereas mevalonate was preferred during phase II. Only one isomer of the R,S-mevalonate mixture appeared to be utilized, since growth ceased after 45 to 50% of the total mevalonate was depleted from the medium. However, nearly 30% of the preferred mevalonate isomer was depleted from the medium during phase I without significant metabolism to CO2. These results suggest that mevalonate or a mevalonate catabolite may accumulate in cells of Pseudomonas sp. M during phase I and that glucose metabolism may inhibit or repress the expression of enzymes further along the mevalonate catabolic pathway.
Comparison of T-DNA oncogene complements of Agrobacterium tumefaciens tumor-inducing plasmids with limited and wide host ranges
Authors: Buchholz W G , Thomashow M F
Published: January 2020

The T-DNA oncogene complements of the limited-host-range tumor-inducing plasmid pTiAg63 and the wide-host-range plasmid pTiA6 were compared. The resulting data indicate that pTiAg63 has DNA sequences related to most of the genes encoded by the oncogene region, the TL-DNA, of pTiA6 and that these sequences are divided between two T-DNA regions, the TA-DNA, which encoded sequences related to pTiA6 genes 4 (the cytokinin independence gene) and 6a, as well as to a pTiA6 TL-DNA fragment that encoded gene 6b and a portion of gene 3, and the TB-DNA, which encoded sequences related to genes 1 and 2 (the auxin independence genes). Tumor tissues of Nicotiana rustica incited by Agrobacterium tumefaciens harboring either pTiA6 or pTiAg63 grew axenically in vitro on phytohormone-free medium. The morphologies of the tissues, however, differed; whereas those incited with pTiA6 grew as loose, friable, unorganized callus, the tumors incited by pTiAg63 grew as clumps of rootlike structures. Thus, the T-DNA oncogene complements of these plasmids were not equivalent. The results are discussed in relation to the A. tumefaciens host range.
Host range encoded by the Agrobacterium tumefaciens tumor-inducing plasmid pTiAg63 can be expanded by modification of its T-DNA oncogene complement
Authors: Buchholz W G , Thomashow M F
Published: January 2020

Agrobacterium tumefaciens harboring pTiA6 incite unorganized tumors on Nicotiana rustica, sunflowers, carrots, and tomatoes, whereas isogenic strains of agrobacteria harboring pTiAg63 form "rooty" tumors on N. rustica and are essentially avirulent on sunflowers, carrots, and tomatoes. In this report we show that the different host range characteristics of these two plasmids were due, in part, to differences in the T-DNA oncogene complements of the plasmids. Specifically, we constructed derivatives of pTiAg63 that contained pTiA6 oncogenes 4, 6a, and 6b inserted into the TB-DNA region and found that agrobacteria harboring these plasmids could incite unorganized tumors on N. rustica, tomatoes, carrots, and the inbred sunflower line HA202R. Undefined host factors, however, also appeared to be involved in determining A. tumefaciens host range since three inbred sunflower lines, HA303B, HA89B, and HA290B, were susceptible to tumor formation by agrobacteria harboring pTiA6 but not by strains harboring pTiAg63 or the modified pTiAg63 plasmids.
Novel inducers of the xylan-degrading enzyme system of Cryptococcus albidus
Authors: Biely P , Petráková E
Published: January 2020

A series of compounds structurally related to xylan and 1,4-beta-xylobiose were tested as inducers of the xylan-degrading enzyme system of Cryptococcus albidus. Washed, glucose-grown cells were incubated with alpha- and beta-linked xylobioses, 4-O-beta-D-xylopyranosyl-L-arabinopyranose, 3-O-beta-D-xylopyranosyl-xylobiose, 6-O-beta-D-xylopyranosyl-cellobiose, cellobiose, and methyl beta-D-xylopyranoside. All alpha-xylobioses and cellobiose were inactive as inducers of the xylan-degrading enzyme system. Other compounds served as inducers of varying efficiency, depending on their concentration in the induction medium and the time of incubation of cells. The most rapid response of the cells, i.e., the shortest induction period of beta-xyloside permease, beta-xylosidase (EC 3.2.1.37), and beta-xylanase (EC 3.2.1.8), was observed with 1,4-beta-xylobiose, which was the most efficient inducer at low concentrations (0.1 to 0.2 mM). At higher concentrations (2 to 10 mM) and after long incubations, the highest enzyme yields were obtained with 1,2-beta-xylobiose. The results represent a new example of efficient induction of polysaccharide-degrading enzyme systems by positional isomers of dimers derived from the polysaccharide.
Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5
Authors: Yakobson E A , Guiney D G
Published: January 2020

We report here a novel system for the conjugal transfer of bacterial chromosomes which utilizes the transfer origin (oriT) of plasmid RK2 cloned into transposon Tn5. Tn5 with oriT was inserted by transposition into the chromosomes of Escherichia coli and Rhizobium meliloti. The oriT sequence then served as the origin of high-frequency chromosome transfer when a helper RK2 plasmid was present in the same cell. The broad host range features of RK2 make this system of oriented chromosome mobilization applicable to most gram-negative bacteria.
Respiratory system of vegetative and sporulating Bacillus cereus
Authors: Escamilla J E , Benito M C
Published: January 2020

The composition and organization of the Bacillus cereus respiratory system were studied. The abolition of NADH-dependent respiration in vegetative and sporulating cell membranes by near-UV light (360 nm) indicated that electrons reduce oxygen only through a quinone-cytochrome pathway. Difference spectroscopy demonstrated the presence of cytochromes b555, c548, aa3, b562, and a2. This composition and studies with respiratory inhibitors suggested that cytochromes are organized in at least two branches, one being highly sensitive to cyanide.
Effect of castanospermine on the structure and secretion of glycoprotein enzymes in Aspergillus fumigatus
Authors: Elbein A D , Mitchell M , Molyneux R J
Published: January 2020

Aspergillus fumigatus secretes a number of glycosidases into the culture medium when the cells are grown in a mineral salts medium containing guar flour (a galactomannan) as the carbon source. At least some of these glycosidases have been reported to be glycoproteins having N-linked oligosaccharides. In this study, we examined the effect of the glycoprotein processing inhibitor, castanospermine, on the structures of the N-linked oligosaccharides and on the secretion of various glycosidases. Cells were grown in the presence of various amounts of castanospermine; at different times of growth, samples of the media were removed for the measurement of enzymatic activity. Of the three glycosidases assayed, beta-hexosaminidase was most sensitive to castanospermine; and its activity was depressed 30 to 40% at 100 micrograms of alkaloid per ml and even more at higher alkaloid concentrations. On the other hand, beta-galactosidase activity was hardly diminished at castanospermine levels of up to 1 mg/ml, but significant inhibition was observed at 2 mg/ml. beta-Galactosidase was intermediate in sensitivity. Cells were grown in the presence or absence of castanospermine and labeled with [2-3H]mannose, [6-3H]glucosamine, or [1-3H]galactose to label the sugar portion of the glycoproteins. The secreted glycoproteins were digested with pronase to obtain glycopeptides, and these were identified on Bio-Gel P-4 (Bio-Rad Laboratories). The glycopeptides were then digested with endoglucosaminidase H to release the peptide portion of susceptible structures, and the released oligosaccharides were reisolated and identified on Bio-Gel P-4. The oligosaccharides from control and castanospermine-grown cells were identified by a combination of enzymatic and chemical studies. In control cells, the oligosaccharide appeared to be mostly Man8GlcNAc and Man9GlcNAc, whereas in the presence of alkaloid, the major structures were Glc3Man7GlcNAc and Glc3Man8GlcNAc. These data fit previous observations that castanospermine inhibits glucosidase I.
Infrequent cleavage of cloned Anabaena variabilis DNA by restriction endonucleases from A. variabilis
Authors: Herrero A , Elhai J , Hohn B , Wolk C P
Published: January 2020

A cosmid vector has been constructed, using a lambda replicon. A library of cosmids from Anabaena variabilis ATCC 29413 based on use of this vector is shown to be highly deficient in sites for the two type II restriction endonucleases found in that organism.
Overproduction of tryptophanyl-tRNA synthetase relieves transcription termination at the Escherichia coli tryptophan operon attenuator
Authors: Das A , Yanofsky C
Published: January 2020

Overproduction of tryptophanyl-tRNA synthetase increased trp operon expression by reducing transcription termination at the trp attenuator. The total cellular level of charged tRNATrp was not affected by increased levels of the synthetase. We propose that excess synthetase binds charged tRNATrp and reduces the concentration available for translation.
Shuttle cloning vectors for the marine bacterium Vibrio parahaemolyticus
Authors: Datta A R , Kaper J B , MacQuillan A M
Published: January 2020

Two cosmid cloning vectors containing lambda cos sequences and a 42-base-pair multipurpose cloning sequence were constructed. pAD22 also contains a 1.4-kilobase TRP-ARS fragment from Saccharomyces cerevisiae. These cosmids transformed Escherichia coli and S. cerevisiae cells and could be mobilized into Vibrio parahaemolyticus strains with a conjugative plasmid, pRK2013. The cosmid pAD22 was genetically and structurally stable during passage through V. parahaemolyticus and E. coli strains.