bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of the nuclear division cycle of Aspergillus nidulans
Authors: Bergen L G , Morris N R
Published: January 2020

We have analyzed the cell cycle kinetics of Aspergillus nidulans by using the DNA synthesis inhibitor hydroxyurea (HU) and a temperature-sensitive cell cycle mutant nimT that blocks in G2. HU rapidly inhibits DNA synthesis (S), and as a consequence progression beyond S to mitosis (M) is blocked. Upon removal of HU the inhibition is rapidly reversible. Conidia (asexual spores) of nimT were germinated at restrictive temperature to synchronize germlings in G2 and then downshifted to permissive temperature in the presence of HU. This procedure synchronizes the germlings at the beginning of S in the second cell cycle after spore germination. We have measured the total duration of S, G2, and M as the time required for these cells to recover from the HU block and undergo the next nuclear division. The duration of S was defined by the time course of sensitivity to reintroduction of HU during recovery from the initial HU block. The cell cycle time was measured as the nuclear doubling time, and the duration of mitosis was determined from the mitotic index. The duration of G1 was calculated by subtracting the combined durations of S, G2, and M from the nuclear doubling time, and the length of G2 was calculated by subtracting S and M from the aggregate length of S, G2, and M. We have also determined the duration of the phases of the cell cycle during the first cycle after spore germination. In these experiments spores were germinated directly in HU without first being blocked in G2. Because the durations of G1, S, G2, and M for the first cell cycle after spore germination were identical with those previously determined for spores presynchronized at the beginning of S in the second cell cycle, we conclude that dormant conidia of A. nidulans are arrested at, or before, the start of S.
Homologous sequences other than insertion elements can serve as recombination sites in plasmid drug resistance gene amplification
Authors: Peterson B C , Rownd R H
Published: January 2020

A plasmid (pRR983) was constructed which has a gene coding for neomycin and kanamycin resistance flanked by direct repeats of regions of homology which contain no known insertion sequences. pRR983 does not have any homologous IS1 sequences. Growth of Proteus mirabilis harboring pRR983 in medium containing high concentration of neomycin resulted in cells which were highly resistant to both neomycin and kanamycin. Plasmid DNA was analyzed by using restriction endonucleases. In most cases the neomycin resistance gene had been tandemly duplicated by using the homologous DNA sequences flanking the resistance gene as recombination sites. This is analogous to tandem duplication of drug resistance genes on NR1 using the two direct repeats of IS1 as recombination sites. The amplified plasmid DNA returned to its original structure by the deletion of amplified neomycin resistance determinants when the host cells were cultured without selection for high resistance to neomycin.
Evidence for an active role of donor cells in natural transformation of Pseudomonas stutzeri
Authors: Stewart G J , Carlson C A , Ingraham J L
Published: January 2020

The transfer of chromosomal genes in a cell mat of Pseudomonas stutzeri was ca. 10(3) times more efficient per microgram of DNA if DNA was added as a constituent of intact donor cells rather than as a solution. Such intact cell-mediated transfer appears to depend on cell contact. It is independent of the presence of plasmids in donor strains and is DNase I sensitive, thus fitting the usual definition of transformation. It is bidirectional: cells of either strain in a transformation mixture served as the donor and recipients. The donor function in cell contact transformation was inhibited by nalidixic acid but was unaffected by rifampin and streptomycin at growth-inhibiting concentrations. Concentrations of nalidixic acid sufficient to inhibit donor function completely had no effect on the ability of nalidixic acid-resistant recipients to take up DNA from solution. These experiments suggest that certain cells donate DNA to others in the cell mat: they argue against the hypothesis that the function of donor cells is merely cell lysis.
Evidence for multisite growth of Escherichia coli murein involving concomitant endopeptidase and transpeptidase activities
Authors: Burman L G , Reichler J , Park J T
Published: January 2020

During diaminopimelic acid starvation of Escherichia coli W7, a large fraction of the preexisting murein cross-links are opened by murein endopeptidase and the resulting uncross-linked material is degraded. This is reflected morphologically in a general loss of rigidity of the murein sacculus long before lysis occurs. In growing cells, a dynamic situation is demonstrable. When cells whose murein sacculi are uniformly labeled with [14C]diaminopimelic acid were chased with unlabeled DAP, a significant, rapid shift of [14C]diaminopimelic acid from the donor to the acceptor half of dimers was observed. The shift can be explained by the presence of about 100 separate sites where new murein strands were being inserted between old radioactive strands of murein. Thus, the gradual loss of rigidity of the murein sacculus as endopeptidase continues to function during starvation of E. coli W7 suggests an even distribution of the active endopeptidases. This is consistent with the kinetic data which suggest that endopeptidase, along with murein synthetase and transpeptidase, acts at about 100 distinct sites to elongate the murein sacculus.
Escherichia coli CAN lacks a tRNA-processing nuclease
Authors: Asha P K , Deutscher M P
Published: January 2020

Escherichia coli strain CAN is unable to support the growth of bacteriophage T4 strains requiring the suppressor function of T4 tRNASer. Biochemical analysis of the mutant strain revealed that it is deficient in a RNase which acts on the artificial tRNA precursor tRNA-C-U.
Density gradient analysis of DNA replicated during Bacillus subtilis sporulation
Authors: Binnie C , Coote J G
Published: January 2020

Density gradient centrifugation was used to monitor DNA replication during sporulation of a 5-bromo-2'-deoxyuridine-tolerant, thymidine-requiring strain of Bacillus subtilis. DNA of heavy, intermediate, and light density was found in cells induced to sporulate in the presence of bromodeoxyuridine, but only intermediate DNA was detected in mature spores. Cells grown with bromodeoxyuridine until DNA was in the heavy form formed spores containing intermediate and light DNA when sporulated with thymidine alone.
2-Oxoacid dehydrogenase complexes of Escherichia coli: cellular amounts and patterns of synthesis
Authors: Smith M W , Neidhardt F C
Published: January 2020

The oxidative decarboxylations of pyruvate and 2-oxoglutarate in Escherichia coli are carried out by two large, multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The enzyme complexes each contain three subunits: two are unique to the individual complexes, the third is shared between them. Resolution of the polypeptide subunits on two-dimensional gels allowed quantitative analysis of their cellular levels and patterns of synthesis in growing cells. Cells growing in glucose-salts medium were found to contain roughly 85 to 136 pyruvate dehydrogenase complexes and 73 2-oxoglutarate complexes. Lipoamide dehydrogenase, the subunit shared by the two complexes, was found to be in significant excess of its stoichiometric demand in the two enzyme complexes under most growth conditions. The subunits unique to each of the complexes were coordinately regulated over a wide variety of growth conditions and a broad range of expression. The two complexes responded to different, but partially overlapping, regulatory signals. Most importantly, the shared subunit was actively regulated to accommodate its demand in both enzymes. These results are discussed with regard to possible mechanisms of regulation of the enzyme complexes in general and of the shared subunit specifically.
Eucaryote thermophily: role of lipids in the growth of Talaromyces thermophilus
Authors: Wright C , Kafkewitz D , Somberg E W
Published: January 2020

The effects of growth temperature on the fatty acid composition of the phospholipids of the fungus Talaromyces thermophilus were investigated. This thermophilic organism was unable to increase the degree of unsaturation of its fatty acids when shifted from high to low growth temperatures. Inhibition of fatty acid synthesis by the antibiotic cerulenin was reversed by the addition of a mixture of palmitic, stearic, and oleic acids and ergosterol. The data obtained were consistent with the hypothesis that the thermophilic character of T. thermophilus is due to metabolic limitations that restrict its ability to regulate membrane fluidity.
Conservation of genetic linkage in nonisogenic isolates of Candida albicans
Authors: Poulter R , Hanrahan V
Published: January 2020

A number of laboratories are now engaged in the genetic analysis of Candida albicans. This diploid yeast, the major fungal pathogen of humans, is imperfect. Parasexual techniques have been devised for complementation and recombination analysis in this organism. This paper attempts to address the question of the extent to which nonisogenic strains of C. albicans have conserved a common genetic map. This analysis is a prerequisite for the integration of work done in different laboratories and may also provide useful information on the taxonomy of the genus Candida. The paper also reports the analysis of an interspecific hybrid between C. albicans and Candida stellatoidea. The method employed in these studies was the analysis of the mitotic recombination relationships of a group of linked genes and their centromere. Strains carrying linked auxotrophic mutations were fused with isogenic and nonisogenic complementary strains to form tetraploids. The mitotic recombination analyses of these tetraploids suggest that in the isolates studied the genetic map is conserved. A comparison of tetraploid and diploid mitotic recombination analyses is also presented.
Reversal of 2-bromoethanesulfonate inhibition of methanogenesis in Methanosarcina sp
Authors: Smith M R
Published: January 2020

2-Bromoethanesulfonate (BES) inhibition of methanogenesis from methanol by resting-cell suspensions or cell extracts of Methanosarcina was reversed by coenzyme M. BES inhibition of methylcoenzyme M methylreductase activity in cell-free extracts was reversed by methylcoenzyme M but not by coenzyme M. Methanol/coenzyme M methyltransferase activity was not inhibited by 10 microM BES. Inhibition of methylreductase by BES and 3-bromopropionate was competitive with methylcoenzyme M, but inhibition by 2-bromoethanol exhibited mixed kinetics. The Ki values for the inhibitors in cell-free extracts were similar to the concentrations which inhibited intact cells. BES-resistant mutants of strain 227 were apparently permeability mutants because in vitro assays showed that mutant and parent strain methylreductases were equally sensitive to BES.
Roles of the uvsC, uvsD, uvsE, and mtcA genes in the two pyrimidine dimer excision repair pathways of Deinococcus radiodurans
Authors: Evans D M , Moseley B E
Published: January 2020

In Deinococcus radiodurans, the genes uvsC, uvsD, uvsE, and mtcA are all involved in the single-strand incision of UV-irradiated DNA, and mutations in at least two of them were required to produce an incisionless strain. One mutation must be in mtcA and one in uvsC, uvsD, or uvsE. Strains carrying single mutations in any one of the genes can incise DNA to the same extent as the wild-type strain. Neither the presence of EDTA nor the absence of protein synthesis affected the incision step. Strains deficient in DNA incision have greatly reduced DNA degradation after UV irradiation, and upon addition of chloramphenicol to the postirradiation medium, they do not undergo excessive DNA degradation as is seen in the wild-type strain and strains singly mutant in uvsC, uvsD, or uvsE. The strain singly mutant in mtcA also lacked chloramphenicol-enhanced DNA degradation and loss of viability but behaved similarly to the wild-type strain with respect to resumption of DNA synthesis and DNA degradation in the absence of chloramphenicol. It is proposed that two constitutive, cation-independent UV endonucleases are present in D. radiodurans: UV endonuclease alpha (the product of the mtcA gene), which incises in response to pyrimidine dimers, mitomycin C cross-links, bromomethylbenzanthracene adducts, and other alkylation damage, and UV endonuclease beta (the product of the uvsC, uvsD, and uvsE genes), which incises only in response to pyrimidine dimers. Both endonucleases have associated exonuclease activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Genetic analysis of transposon-induced mutations of the Rac prophage in Escherichia coli K-12 which affect expression and function of recE
Authors: Fouts K E , Wasie-Gilbert T , Willis D K , Clark A J , Barbour S D
Published: January 2020

Fourteen mitomycin-resistant revertants of a recB21 recC22 strain were isolated after Tn5 mutagenesis. Eight of the mutations (type I) were essentially inseparable from aphA+ (Kanr) of Tn5; six (type II) were not. We hypothesize that the former are Tn5 and that the latter are IS50 insertions. Because of their phenotypic similarity to sbcA and sbcB mutations, which also suppress recB21 recC22, we have called them sbc mutations. sbc-lll::Tn5 was cotransducible with nirR and has thereby been located at position 29.8 on the Escherichia coli map in the vicinity of the Rac prophage and sbcA mutations. A recB21 recC22 sbc-lll::Tn5 strain was subjected to Tn10 mutagenesis, and a mitomycin- and UV-sensitive mutant was isolated. tet+ of Tn10 was 85% cotransducible with aphA+ of Tn5, locating these two transposons 0.1 map unit apart. A three-point cross located the Tn10 mutation at position 29.7. We hypothesize that the Tn10 insertion is located in recE and that the Tn5 and IS50 insertions activate expression of this gene. sbc-lll::Tn5 was found to be cis acting and dominant to its wild-type allele as were two sbcA mutations (sbcA1 and sbcA6). Five other type I and type II insertion mutations were dominant to their wild-type alleles. We hypothesize that the sbc insertion and sbcA mutations affect transcription regulation of recE and discuss the possibility that they do so differently.
Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain
Authors: Beveridge T J , Davies J A
Published: January 2020

Exponentially growing cells of Bacillus subtilis and Escherichia coli were Gram stained with potassium trichloro(eta 2-ethylene)platinum(II) (TPt) in place of the usual KI-I2 mordant. This electron-dense probe allowed the staining mechanism to be followed and compared with cellular perturbations throughout the staining process. A crystal violet (CV)-TPt chemical complex was formed within the cell substance and at the cell surface of B. subtilis when the dye and Pt mordant were added. The ethanol decolorization step dissolved the precipitate from the cell surface, but the internal complex was retained by the cell wall and remained within the cell. This was not the case for E. coli; the ethanol decolorization step removed both surface-bound and cellular CV-TPt. During its removal, the outer membrane was sloughed off the cells until only the murein sacculus and plasma membrane remained. We suspect that the plasma membrane was also perturbed, but that it was retained within the cell by the murein sacculus. Occasionally, small holes within the murein and plasma membrane could be distinguished through which leaked CV-TPt and some cellular debris. Biochemical identification of distinct envelope markers confirmed the accuracy of these images.
Subcloning of the cloacin DF13/aerobactin receptor protein and identification of a pColV-K30-determined polypeptide involved in ferric-aerobactin uptake
Authors: Krone W J , Luirink J , Koningstein G , Oudega B , de Graaf F K
Published: January 2020

A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin.
Characterization and molecular cloning in Escherichia coli of a plasmid from the mollicute Spiroplasma citri
Authors: Mouches C , Barroso G , Bové J M
Published: January 2020

Two plasmids, pMH1 with 7 kilobase pairs and pM41 with 8 kilobase pairs, were purified from the plant pathogen Spiroplasma citri and characterized by restriction mapping. Upon in vitro DNA recombination with plasmid pBR328 as a vector, we have cloned pMH1 in Escherichia coli. A radioactive probe obtained upon nick translation of the recombinant plasmid was used to further characterize and compare pMH1 and pM41.
Mu dX, a derivative of Mu d1 (lac Apr) which makes stable lacZ fusions at high temperature
Authors: Baker T A , Howe M M , Gross C A
Published: January 2020

We describe defective Mu phage Mu dX (Mu d1 Bx::Tn9 [lac Apr Cmr]) which is useful for insertion mutagenesis and for construction of lac operon fusions in vivo. Mu dX retains the insertion properties of Mu d1 but produces temperature-resistant lysogens and transposes at a reduced frequency. A method is described to convert existing Mu d1 insertions to Mu dX.
Regulatory pattern of the Escherichia coli lysA gene: expression of chromosomal lysA-lacZ fusions
Authors: Stragier P , Borne F , Richaud F , Richaud C , Patte J C
Published: January 2020

The regulation of lysA which encodes the last enzyme for lysine biosynthesis in Escherichia coli, diaminopimelic acid-decarboxylase, was studied by using lysA-lacZ fusions. Our results indicate an absolute requirement for the LysR product for its activation, LysR protein present in a limiting amount which can be titrated by a multicopy plasmid carrying its target site and a negative regulatory role for the LysA protein itself which decreases lysA-lacZ expression 30-fold.
Requirement of the cheB function for sensory adaptation in Escherichia coli
Authors: Yonekawa H , Hayashi H , Parkinson J S
Published: January 2020

The chemotactic behavior of Escherichia coli mutants defective in cheB function, which is required to remove methyl esters from methyl-accepting chemotaxis proteins, was investigated by subjecting swimming or antibody-tethered cells to various attractant chemicals. Two cheB point mutants, one missense and one nonsense, exhibited stimulus response times much longer than did the wild type, but they eventually returned to the prestimulus swimming pattern, indicating that they were not completely defective in sensory adaptation. In contrast, strains deleted for the cheB function showed no evidence of adaptation ability after stimulation. The crucial difference between these strains appeared to be the residual level of cheB-dependent methylesterase activity they contained. Both point mutants showed detectable levels of methanol evolution due to turnover of methyl groups on methyl-accepting chemotaxis protein molecules, whereas the cheB deletion mutant did not. In addition, it was possible to incorporate the methyl label into the methyl-accepting chemotaxis proteins of the point mutants but not into those of the cheB deletion strain. These findings indicate that cheB function is essential for sensory adaptation in Escherichia coli.
Circularized copies of amplifiable resistance genes from Haemophilus influenzae plasmids
Authors: Spies T , Laufs R
Published: January 2020

Tandem repeat amplification of resistance determinants in Haemophilus influenzae plasmids is associated with the occurrence of separate circular DNA molecules. They were demonstrated to represent mono- and multimeric forms of the amplifiable segments of the plasmids which comprise the respective resistance transposons and an additional region designated as an amplification sequence. The latter region mediates the recombinational events involved in amplification. The DNA circles apparently lack the ability to replicate autonomously but most probably provide an effective means for the translocation of resistance genes from one plasmid to another.
Effect of dnaA and rpoB mutations on attenuation in the trp operon of Escherichia coli
Authors: Atlung T , Hansen F G
Published: January 2020

The rate of synthesis of tryptophan synthetase was found to be increased by heat inactivation of the dnaA protein in three dnaA mutants temperature sensitive for initiation of DNA replication. The effect of the dnaA mutations was dependent upon the presence of an intact attenuator in the tryptophan operon. The activity of the mutated dnaA protein at the tryptophan attenuator and its activity as initiator for chromosome replication decreased gradually with increasing temperature. Two rpoB mutations that suppress the temperature defect of the dnaA46 mutation in initiation of replication were tested for effects on attenuation in the tryptophan operon. One of the rpoB mutations caused increased transcription termination at the attenuator independent of the dnaA allele, whereas the other mutation had no effect. Expression of the histidine and threonine operons, which are also regulated by attenuation, was unaffected by the dnaA mutations.
Morphological and genetic characterization of a bacteriophage-resistant Bacillus subtilis macrofiber-producing strain
Authors: Saxe C L , Mendelson N H
Published: January 2020

Bacillus subtilis C6 phi R4 is an SPO1-resistant derivative of strain C6D, a left-hand macrofiber-producing strain described previously (N. H. Mendelson, Proc. Natl. Acad. Sci. U.S.A. 75:2478-2482, 1978). In addition to the phage resistance property, strain C6 phi R4 differs from its parent in macrofiber organization and formation of aggregates in liquid shake cultures. The phage resistance mutation was located in the gtaC gene. The macrofiber organization and aggregation phenotypes also appear to be controlled by the gtaC locus. Strains constructed by introduction of the gtaC mutation into C6D appear to be identical to the original C6 phi R4 strain in all phenotypic properties. In contrast, other constructs carrying either gtaA or gtaB that are resistant to SPO1 do not display the characteristic C6 phi R4 morphological phenotypes.
Potentiometric analysis of the cytochromes of an Escherichia coli mutant strain lacking the cytochrome d terminal oxidase complex
Authors: Lorence R M , Green G N , Gennis R B
Published: January 2020

A combination of potentiometric analysis and electrochemically poised low-temperature difference spectroscopy was used to examine a mutant strain of Escherichia coli that was previously shown by immunological criteria to be lacking the cytochrome d terminal oxidase. It was shown that this strain is missing cytochromes d, a1, and b558 and that the cytochrome composition of the mutant is similar to that of the wild-type strain grown under conditions of high aeration. The data indicate that the high-aeration branch of the respiratory chain contains two cytochrome components, b556 (midpoint potential [Em] = +35 mV) and cytochrome o (Em = +165 mV). The latter component binds to CO and apparently has a reduced-minus-oxidized split-alpha band with peaks at 555 and 562 nm. When the wild-type strain was grown under conditions of low aeration, the components of the cytochrome d terminal oxidase complex were observed: cytochrome d (Em = +260 mV), cytochrome a1 (Em = +150 mV) and cytochrome b558 (Em = +180 mV). All cytochromes appeared to undergo simple one-electron oxidation-reduction reactions. In the absence of CO, cytochromes b558 and o have nearly the same Em values. In the presence of CO, the Em of cytochrome o is raised, thus allowing cytochromes b558 and o to be individually quantitated by potentiometric analysis when they are both present.
Mechanism of tetanolysin-induced membrane damage: studies with black lipid membranes
Authors: Blumenthal R , Habig W H
Published: January 2020

Tetanolysin produced similar rates of leakage of K+ and hemoglobin from erythrocytes. When studied by using cholesterol-containing black lipid membranes, this hemolysin induced conductance steps with a broad frequency distribution. These findings are inconsistent with the formation of structural channels and suggest that tetanolysin acts by causing lipid perturbations.
A meiotic mutant of the fission yeast Schizosaccharomyces pombe that produces mature asci containing two diploid spores
Authors: Nakaseko Y , Niwa O , Yanagida M
Published: January 2020

A mutant of the fission yeast Schizosaccharomyces pombe grew normally in the mitotic cycle but produced two-spored asci in the meiosis cycle. These spores were diploid, and the segregation of centromere-linked markers in the dyads was mostly reductional. Only the first meiotic division appears to occur in this tws1 mutant, resulting in enclosure of diploid nuclei into spores.
Permeability of the boundary layers of Bdellovibrio bacteriovorus 109J and its bdelloplasts to small hydrophilic molecules
Authors: Cover W H , Martinez R J , Rittenberg S C
Published: January 2020

Measurements of the sucrose-permeable and -impermeable volumes during Bdellovibrio bacteriovorus attack on Escherichia coli or Pseudomonas putida showed that the volume of the bdelloplast increased over that of the substrate cell. Although the pattern of the increase differed with the two organisms, the volumes reached maximum at about 60 min into the bdellovibrio growth cycle. By this time, the cytoplasmic membranes of the attacked cells were completely permeable to sucrose. The kinetics of increase in sucrosepermeable volumes were similar to the kinetics of attachment and penetration (Varon and Shilo, J. Bacteriol. 95:744-753, 1968). These data show that the original cytoplasmic and periplasmic compartmentalization of the substrate cell ceases to exist with respect to small hydrophilic molecules during bdellovibrio attack. In contrast, the effective pore size of the outer membrane of the substrate cell to small oligosaccharides remains unaltered during bdelloplast formation as was shown by direct measurements of its exclusion limits. The major porin protein of E. coli, OmpF, was recoverable from the bdelloplast outer membrane fraction until the onset of lysis. The Braun lipoprotein was removed from the bdelloplast wall early, and OmpA was lost in the terminal part of the bdellovibrio growth cycle.
Production and release of peptidoglycan and wall teichoic acid polymers in pneumococci treated with beta-lactam antibiotics
Authors: Fischer H , Tomasz A
Published: January 2020

Autolysin-defective pneumococci treated with inhibitory concentrations of penicillin and other beta-lactam antibiotics continued to produce non-cross-linked peptidoglycan and cell wall teichoic acid polymers, the majority of which were released into the surrounding medium. The released cell wall polymers were those synthesized by the pneumococci after the addition of the antibiotics. The peptidoglycan and wall teichoic acid chains released were not linked to one another; they could be separated by affinity chromatography on an agarose-linked phosphorylcholine-specific myeloma protein column. Omission of choline, a nutritional requirement and component of the pneumococcal teichoic acid, from the medium inhibited both teichoic acid and peptidoglycan synthesis and release. These observations are discussed in terms of plausible mechanisms for the coordination between the biosynthesis of peptidoglycan and cell wall teichoic acids.
Characterization of the cloned fip gene and its product
Authors: Russel M , Model P
Published: January 2020

A DNA fragment encoding the fip (filamentous phage production) gene from Escherichia coli, when cloned in a filamentous phage vector, restored to the phage ability to assemble progeny in fip mutant hosts. The fip gene was located just upstream of and transcribed in the same direction as the rho gene. Minicells containing fip+ phage or plasmids synthesized a 12,500-dalton protein that was missing or truncated when the Fip+ phenotype was inactivated by insertion of Tn5. The fip protein was cytoplasmic and was partially purified.
Antagonistic transcriptional regulation of the putrescine biosynthetic enzyme agmatine ureohydrolase by cyclic AMP and agmatine in Escherichia coli
Authors: Satishchandran C , Boyle S M
Published: January 2020

The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (agmatinase; EC 3.5.3.11) catalyzes the conversion of agmatine to putrescine in Escherichia coli. The specific activity of AUH was determined in crude extracts prepared from wild-type strains and from strains with mutations in the adenylate cyclase gene (cya) or the cAMP receptor protein gene (crp) or both. In glucose minimal medium, a delta cya strain exhibited 70 to 90% higher AUH activity than a cya+ strain. Addition of 1 to 10 mM cAMP to cya+ and delta cya strains cultured in glucose repressed AUH activity in a dose-dependent manner. Addition of 1 to 10 mM cAMP to a delta crp strain failed to repress AUH activity. Addition of agmatine resulted in a three- to fourfold induction of AUH in delta cya and delta crp strains. This induction could be blocked by the addition of chloramphenicol. Simultaneous additions of various proportions of cAMP and agmatine resulted in reduced levels of induction and repression of AUH activity. This antagonistic regulation was shown to be exerted by independent mechanisms since AUH activity could be induced by agmatine in a delta crp strain supplemented with cAMP. These results suggest that both agmatine and cAMP antagonistically regulate AUH activity at the level of transcription. In minimal medium supplemented with 1 mM putrescine, the strains did not exhibit repression of AUH activity. In contrast, in minimal medium supplemented with 1 mM ornithine or arginine, cya+ or delta cya strains exhibited induced AUH activity as a result of conversion of these substrates to agmatine. Further experiments in vitro demonstrated that the effects observed with cAMP, agmatine, and arginine were not post-translationally mediated.
Regulation of glucose metabolism in oral streptococci through independent pathways of glucose 6-phosphate and glucose 1-phosphate formation
Authors: Keevil C W , Marsh P D , Ellwood D C
Published: January 2020

In vivo rates of glucose uptake and acid production by oral streptococci grown in glucose- or nitrogen-limited continuous culture and batch culture were compared with the glucose phosphorylation activities of harvested, decryptified cells. The strains examined contained significant phosphoenolpyruvate-phosphotransferase system (PTS) activity, measured by a glucose 6-phosphate (G6P) dehydrogenase-linked assay procedure, but this activity was insufficient to account for the in vivo glucose uptake rates. However, ATP was a superior phosphoryl donor to phosphoenolpyruvate, and unlike the PTS, phosphoryl transfer with ATP was insensitive to bacteriostatic concentrations of chlorhexidine, suggesting glucokinase-mediated G6P formation. Again, G6P formation from the PTS and glucokinase reactions was not commensurate with some of the glucose uptake rates observed, implying that other phosphorylation reactions must be occurring. Two novel reactions involving carbamyl phosphate and acetyl phosphate were identified in some of the strains. No G6P formation was detected with these potential phosphoryl donors, but in the presence of phosphoglucomutase, glucose 1-phosphate (G1P) formation was evident, which was insensitive to chlorhexidine. G1P is a precursor of glycogen, and good correlation was obtained between G1P formation activity and endogenous metabolism of washed cells measured either as a rate of acid production at a constant pH 7 or as a decrease in pH with time in the absence of titrant. A "league table" of abilities to synthesize G1P and produce acid from endogenous metabolism was compiled for oral streptococci grown in batch culture. This indicated that Streptococcus mutans Ingbritt and Streptococcus sanguis Challis were unable to form G1P or produce much acid endogenously, whereas increasing activities were obtained with Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis. In particular, S. mitis had the highest G1P formation activities and was able to decrease the pH to less than 5 in 15 min by endogenous metabolism alone. The data are consistent with the intracellular accumulation of free glucose driven by proton motive force when PTS activities are low and the subsequent phosphorylation to either G6P for metabolism via glycolysis or G1P for glycogen biosynthesis. The accumulation of acetyl phosphate during glucose-limited growth and the availability of arginine for catabolism to carbamyl phosphate provide an explanation as to why some glucose-limited oral streptococci continue to synthesize glycogen under these conditions, which might prevail in plaque.
Use of phi(glp-lac) in studies of respiratory regulation of the Escherichia coli anaerobic sn-glycerol-3-phosphate dehydrogenase genes (glpAB)
Authors: Kuritzkes D R , Zhang X Y , Lin E C
Published: January 2020

Expression of the glpA operon encoding the extrinsic membrane anaerobic sn-glycerol-3-phosphate dehydrogenase complex of Escherichia coli K-12 was studied in five strains carrying independent glpA-lac operon fusions. The location of the fusions was confirmed by transduction. Two of the strains produced an enzymatically active anaerobic sn-glycerol-3-phosphate dehydrogenase that accumulated in the cytoplasmic fraction of the cells. This suggests the loss of a specific membrane anchor subunit encoded by a distal gene, glpB, which was disrupted by the insertion. beta-Galactosidase in all five strains carrying phi(glpA-lac) was highly inducible by glycerol only anaerobically. A mutation in fnr, a pleiotropic activator gene, prevented full induction of the phi(glpA-lac), demonstrating that the Fnr protein is a positive regulator of the primary dehydrogenase as well as of the terminal reductases of anaerobic respiratory chains. Low concentrations of the respiratory poison KCN had a permissive effect on aerobic expression of phi(glpA-lac). Aerobic expression of the hybrid operon was also enhanced in isogenic derivatives of the fusion strains deficient in protoporphyrin biosynthesis (hemA). Thus, heme proteins may play a role in mediating aerobic repression of the anaerobic respiratory chain.
Bacteriophage chi sensitivity and motility of Escherichia coli K-12 and Salmonella typhimurium Fla- mutants possessing the hook structure
Authors: Kagawa H , Ono N , Enomoto M , Komeda Y
Published: January 2020

The production of hook protein and flagellin in 29 Fla- mutants of Escherichia coli K-12 was determined by the complement fixation assay. Six mutants produced hook protein, and four of them also produced flagellin. A flaE mutation was introduced into these fla mutants carrying the hook structure. All of these mutants made polyhooks and were used as hosts for a newly isolated host-range mutant of chi phage that has a high affinity for the hook structure. All except one mutant produced significant amounts of progeny phages. A flaD flaE double mutant was that exception which did not yield significant amounts of progeny by the phage propagation method. All of the flaE double mutants produced comparable amounts of polyhooks, and no qualitative difference was detected between chi-sensitive and chi-insensitive mutants by the complement fixation assay. Accordingly, it was thought that the polyhook of the flaD flaE mutant had a mechanical defect for chi phage infection. This assumption was confirmed by tethered-cell experiments; the flaD flaE mutant did not rotate. These results are well explained by a proposed regulation pathway of flagellar genes. flaE mutants can express other genes which govern the final step of the flagellar morphogenesis, whereas flaD mutants cannot rotate, possibly because the mocha operon is not expressed. The results obtained in E. coli were also found to be applicable to Salmonella typhimurium.
Exonucleases I, III, and V are required for stability of ColE1-related plasmids in Escherichia coli
Authors: Bassett C L , Kushner S R
Published: January 2020

The stability of two ColE1-related plasmids (pRSF2124 and pMB9) was examined in strains of Escherichia coli multiply deficient in exonucleases I (sbcB), III (xthA), or V (recB recC). Any combination of exonuclease I, III, and V deficiency resulted in dramatically decreased stability of both pRSF2124 and pMB9. Inactivation of the RecF pathway by introducing either recF or recJ mutations to the recB recC subcB background resulted in nearly wild-type levels of stability for both plasmids. In contrast, the introduction of uvrD3 uvr-257, uvrE100, or recL152 into the recB21 recC22 sbcB15 strain did not affect plasmid stability. Furthermore, the amount of plasmid DNA recovered from pRSF2124 or pMB9 transformants of a xthA1 sbcB15 strain was strikingly reduced relative to that of a wild-type control. Taken together, these results suggest that some aspect of DNA repair is required for stable maintenance of ColE1-related plasmids in E. coli.
Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis
Authors: Ruhfel R E , Robillard N J , Thorne C B
Published: January 2020

Bacteriophage CP-51, a generalized transducing phage for Bacillus anthracis, B. cereus, and B. thuringiensis, mediates transduction of plasmid DNA. B. cereus GP7 harbors the 2.8-megadalton multicopy tetracycline resistance plasmid, pBC16. B. thuringiensis 4D11A carries pC194, the 1.8-megadalton multicopy chloramphenicol resistance plasmid. When phage CP-51 was propagated on these strains, it transferred the plasmid-encoded antibiotic resistances to the nonvirulent Weybridge (Sterne) strain of B. anthracis, to B. cereus 569, and to strains of several B. thuringiensis subspecies. The frequency of transfer was as high as 10(-5) transductants per PFU. Tetracycline-resistant and chloramphenicol-resistant transductants contained newly acquired plasmid DNA having the same molecular weight as that contained in the donor strain. Antibiotic-resistant transductants derived from any of the three species were effective donors of plasmids to recipients from all three species.
Structural gene for the phosphate-repressible phosphate-binding protein of Escherichia coli has its own promoter: complete nucleotide sequence of the phoS gene
Authors: Surin B P , Jans D A , Fimmel A L , Shaw D C , Cox G B , Rosenberg H
Published: January 2020

The complete nucleotide sequence of the phoS gene, the structural gene for the phosphate-repressible, periplasmic phosphate-binding protein Escherichia coli K-12, was determined. The phosphate-binding protein is synthesized in a precursor form which includes an additional N-terminal segment containing 25 amino acid residues, with the general characteristics of a signal sequence. The amino acid sequence derived from the nucleotide sequence shows the mature protein to be composed of 321 amino acids with a calculated molecular weight of 34,427. The phoS gene is not part of an operon and is transcribed counterclockwise with respect to the E. coli genetic map. A promoter region has been identified on the basis of homology with the consensus sequence of other E. coli promoter regions. However, an alternative promoter region has been identified on the basis of homology with the promoter regions of the phoA and phoE genes, the structural genes for alkaline phosphatase and outer-membrane pore protein e, respectively.
Molecular cloning of a gene abundantly expressed during fruiting body initiation in Schizophyllum commune
Authors: Dons J J , Springer J , de Vries S C , Wessels J G
Published: January 2020

Complementary DNA was synthesized on polyadenylated RNA from a dikaryotic mycelium of the basidiomycete Schizophyllum commune bearing fruiting body initials. The complementary DNA was cloned into the PstI site of pBR327 by the deoxyguanidylate-deoxycytidylate tailing approach. After transformation into Escherichia coli cells, a differential screening was performed by colony hybridization with complementary [32P]DNA made on the RNAs of the monokaryon and dikaryon strains. Two clones were selected for further analysis by Northern blotting and hybrid release translation. Clone 1D10 hybridized with an mRNA of 775 nucleotides, coding for a polypeptide with an Mr of 15,000. Although this RNA was present in both monokaryotic and dikaryotic mycelia, its concentration appeared to change considerably over time and with different cultivation conditions. This mRNA is probably the most abundantly expressed sequence in S. commune. Clone 1G2 and its homologs hybridized with an mRNA of 650 nucleotides, coding for a polypeptide with an Mr of 13,000. This gene was exclusively expressed in the dikaryon strain. In liquid-grown cultures, the concentration of this mRNA was low but increased ca. 20-fold during the establishment of fruiting body primordia. A chromosomal fragment of 9 kilobase pairs which contained the 1G2 gene was cloned into pBR327 and used as a probe in Northern blot hybridization. It was found that surrounding sequences were not expressed at the same time or to the same extent as the 1G2 gene.
DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA
Authors: Toukdarian A E , Lidstrom M E
Published: January 2020

DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight.
Expression of Bacillus megaterium and Bacillus subtilis small acid-soluble spore protein genes during stationary-phase growth of asporogenous B. subtilis mutants
Authors: Mason J M , Setlow P
Published: January 2020

The small acid-soluble spore proteins alpha and beta were not detected during stationary-phase growth of asporogenous Bacillus subtilis mutants blocked in stages 0, II, or III, but mutants blocked in stages IV or V accumulated nearly wild-type levels of these small acid-soluble spore proteins. Similar results were obtained when production of Bacillus megaterium C protein (also a small acid-soluble spore protein), as well as alpha and beta, were monitored in these mutants containing a recombinant plasmid carrying the B. megaterium C protein gene. The only exception was a spo0H mutant which synthesized a small amount of C protein, but no alpha or beta.
Isolation of Bacillus subtilis mutants pleiotropically insensitive to glucose catabolite repression
Authors: Fisher S H , Magasanik B
Published: January 2020

A pleiotropic mutant of Bacillus subtilis was isolated which overproduced in the presence of glucose several enzymes whose synthesis is subject to glucose catabolite repression. Examination of intracellular metabolites suggested that the mutation may have resulted in a defect in glycolysis, increasing phosphoenolpyruvate and decreasing pyruvate, 2-ketoglutarate, and oxaloacetate.
Evidence for a nickel-containing carbon monoxide dehydrogenase in Methanobrevibacter arboriphilicus
Authors: Hammel K E , Cornwell K L , Diekert G B , Thauer R K
Published: January 2020

In growing cultures of Methanobrevibacter arboriphilicus (Methanobrevibacter arboriphilus), the synthesis of active carbon monoxide dehydrogenase required nickel. The 21-fold-purified enzyme from 63Ni-labeled cells of M. arboriphilicus comigrated with 63Ni during gel filtration. These results provide evidence that the carbon monoxide dehydrogenase of methanogens is a nickel protein.
Clustering of genes for L-fucose dissimilation by Escherichia coli
Authors: Chakrabarti T , Chen Y M , Lin E C
Published: January 2020

Aerobic and anaerobic L-fucose utilization by Escherichia coli involves an inducible trunk pathway mediated by a permease, an isomerase, a kinase, and an aldolase. Tn5 insertion mutants of a parental strain expressing this pathway constitutively were used to map the positions of the structural genes by transduction. Results from this and previous studies show that all of the structural genes of the L-fucose trunk pathway map between eno and argA at minute 60.2 of the chromosome.
Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide
Authors: Chart H , Shaw D H , Ishiguro E E , Trust T J
Published: January 2020

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the lipopolysaccharides of typical and atypical strains of the fish pathogen Aeromonas salmonicida. 32P intrinsically radiolabeled lipopolysaccharide in sarcosinate-extracted outer membrane preparations, lipopolysaccharide stained by silver in proteinase K-digested outer membrane preparations and whole cell lysates, as well as purified lipopolysaccharide, displayed O-polysaccharide chains which were unusually homogeneous with respect to chain length. Chemical analysis further revealed that the sugar composition of the smooth lipopolysaccharide purified from three typical strains was very similar. Immunoblotting and immunofluorescent staining with both polyclonal and monoclonal antibody showed that the O-polysaccharide chains were strongly immunogenic and were antigenically cross-reactive on typical and atypical strains from diverse origins. Immunofluorescence analysis and phage binding studies demonstrated that a number of these O-polysaccharide chains traversed the surface protein array of virulent strains of A. salmonicida and were exposed on the cell surface.
Anaerobiosis induces expression of ant, a new Escherichia coli locus with a role in anaerobic electron transport
Authors: Yerkes J H , Casson L P , Honkanen A K , Walker G C
Published: January 2020

Escherichia coli has a formate hydrogenlyase system which allows it to maintain an electron balance during anaerobic growth by passing electrons from formate to H+ ions, thus generating H2. The Mu d1(Ap lac) bacteriophage was used to generate mutants that were defective in passing electrons from formate to benzyl viologen, an artificial electron acceptor. A subset of these mutants was studied in which beta-galactosidase was expressed at much higher levels under anaerobic conditions than under aerobic conditions. If nitrate was present during anaerobic growth, the same levels of beta-galactosidase were seen in these fusion strains as were seen under aerobic conditions. The Mu d1(Ap lac) insertions in these mutants were genetically mapped between mutS and srl and thus define a new locus we have termed ant (anaerobic electron transport). Recombinant lambda derivatives were isolated which complemented the deficiency of the ant mutants in anaerobic electron transport and also carried a trans-acting region of DNA which reduced expression of the ant-lac fusions under anaerobic conditions; a probe to the ant region was generated from one of these recombinant lambda derivatives. Southern hybridization analysis revealed that the four independent ant::Mu d1(Ap lac) fusions we isolated spanned an approximately 5-kilobase region and that all were transcribed in the same direction, counterclockwise on the E. coli genetic map.