bluuebunny/crossref_metadata_2025 huggingface dataset:
Fermentation and Anaerobic Respiration by
Rhodospirillum rubrum
and
Rhodopseudomonas capsulata
Authors: Schultz J. E. , Weaver P. F.
Published: January 2020

Rhodospirillum rubrum
and
Rhodopseudomonas capsulata
were able to grow anaerobically in the dark either by a strict mixed-acid fermentation of sugars or, in the presence of an appropriate electron acceptor, by an energy-linked anaerobic respiration. Both species fermented fructose without the addition of accessory oxidants, but required the initial presence of bicarbonate before fermentative growth could begin. Major products of
R. rubrum
fermentation were succinate, acetate, propionate, formate, hydrogen, and carbon dioxide;
R. capsulata
produced major amounts of lactate, acetate, succinate, hydrogen, and carbon dioxide.
R. rubrum
and
R. capsulata
were also capable of growing strictly through anaerobic, respiratory mechanisms. Nonfermentable substrates, such as succinate, malate, or acetate, supported growth only in the presence of an electron acceptor such as dimethyl sulfoxide or trimethylamine oxide. Carbon dioxide and dimethyl sulfide were produced during growth of
R. rubrum
and
R. capsulata
on succinate plus dimethyl sulfoxide. Molar growth yields from cultures grown anaerobically in the dark on fructose plus dimethyl sulfoxide were 3.8 to 4.6 times higher than values obtained from growth on fructose alone and were 56 to 60% of the values obtained from aerobic, respiratory growth with fructose. Likewise, molar growth yields from anaerobic, respiratory growth conditions with succinate plus dimethyl sulfoxide were 51 to 54% of the values obtained from aerobic, respiratory growth with succinate. The data indicate that dimethyl sulfoxide or trimethylamine oxide as a terminal oxidant is approximately 33 to 41% as efficient as O
2
in conserving energy through electron transport-linked respiration.
Internal pH and ATP-ADP pools in the cyanobacterium Synechococcus sp. during exposure to growth-inhibiting low pH
Authors: Kallas T , Castenholz R W
Published: January 2020

Y-7c-s Synechococcus thermophilic strain grew at its maximum rate at pH 8 and above. The growth rate of this strain was inhibited at pH 7.0 and below, and at pH 6.0 there was no sustained growth. At a suboptimal pH, high light intensity further depressed the growth rate. The inhibition of growth resulted neither from pheophytinization nor from a low chlorophyll content. At pH 5.0 a loss of viability preceded the appearance of pheophytin. Cells exposed to low, growth-inhibiting external pH levels continued to maintain a high internal pH (pH 7.1 to 7.3, as determined at moderate light intensities by 31P nuclear magnetic resonance spectroscopy). Even during exposure to pH 4.8, cells retained a relatively high internal pH. Thus, it appeared that the inhibition of growth at low pH was not caused by acidification of the cytoplasm. Darkened cells maintained a slightly lower internal pH than irradiated cells. The ATP/(ATP + ADP) ratio decreased from 0.80 to 0.82 at pH 8.0 to about 0.6 when growth was limited by exposure to pH 6.0 or by low light intensity. It is possible, but not likely, that a limitation of the energy supply may slow or stop growth when the external pH is lowered.
Rapid transient growth at low pH in the cyanobacterium Synechococcus sp
Authors: Kallas T , Castenholz R W
Published: January 2020

The thermophilic cyanobacterium Synechococcus sp. strain Y-7c-s grows at its maximum rate at a high pH (pH 8 and above) the does not show sustained growth below pH 6.5. However, rapidly growing, exponential-phase cells from high-pH cultures continued to grow rapidly for several hours after transfer to pH 6.0 or 5.0. This transient growth represented increases in mass and protein, but cells failed to complete division. Viability loss commenced well before the cessation of growth, and cells at pH 5.0 showed no net DNA synthesis. When irradiated by visible light, cells at pH 6.0 and 5.0 maintained and internal pH of 6.9 to 7.1 (determined by 31P nuclear magnetic resonance spectroscopy) and an extremely high ATP/(ATP + ADP) ratio even after growth had ceased. Cells exposed to a low pH did not show an increase in the spontaneous mutation rate, as measured by mutation to streptomycin resistance. However, cells already resistant to streptomycin were more resistant to viability loss at a low pH than the parental type. Cultures that could grow transiently at a low pH had higher rates of viability loss than nongrowing cultures in light or darkness. The retention of a high internal pH by cells exposed to a low pH suggested that a low pH acted initially on the cell membrane, possibly on solute transport.
Induction of cellulose in Schizophyllum commune: thiocellobiose as a new inducer
Authors: Rho D , Desrochers M , Jurasek L , Driguez H , Defaye J
Published: January 2020

Several mono-, di, tetra-, and polysaccharides were screened for their ability to induced cellulase production by the tetrapolar hymenomycete Schizophyllum commune. Out of 21 carbohydrates screened, 4 (thiocellobiose, carboxymethylcellulose, cellobiose, and xylan) induced all three enzymes tested (carboxymethylcellulase, beta-glucosidase, and xylanase). The inducing effect increased with rising concentrations of the inducers up to a certain value, beyond which there was either a leveling off or a decrease of the enzymatic activities. The most powerful inducer, thiocellobiose, showed the highest activity at 0.5 mM. Cellobiose, carboxymethylcellulose, and xylan showed their highest activities at 1 mM and 1%, respectively. Surprisingly, sophorose did not enhance enzyme production. The enzymatic activities were monitored over a period of 24 h. Thiocelloboise elicited a response immediately after incubation, but with all other inducers there was a latency period before their effect could be measured. High-performance liquid chromatography showed no hydrolysis of thiocellobiose when incubated in the presence of S. commune extracellular enzymes.
Formation of Molecular Complexes Between a Structurally Defined M Protein and Acylated or Deacylated Lipoteichoic Acid ofStreptococcus pyogenes
Authors: Ofek Itzhak , Simpson W. Andrew , Beachey Edwin H.
Published: January 2020

The orientation of lipoteichoic acid (LTA) molecules on the surface of bacterial cells undoubtedly is determined by the ability of the LTA, during its transit through the cell wall, to bind via its polyglycerophosphate backbone or its glycolipid moieties to other constituents of the cytoplasmic membrane and the cell wall. We have investigated the possibility that LTA may become anchored to the cell surface by binding through its polyanionic backbone to positively charged regions of cell wall proteins. LTA was found to prevent the precipitation of partially purified HCl extracts of several strains of streptococci as well as a structurally defined streptococcal M protein molecule (pep M24) in 83% solutions of ethanol. The formation of complexes between LTA and M protein was demonstrated further by immunoelectrophoresis of pep M24 protein with increasing concentrations of radiolabeled LTA and by using antiserum against pep M24 to develop precipitin arcs. Pep M24 electrophoresed alone produced a single precipitin arc close to the origin. In contrast, when electrophoresed as a mixture with LTA or deacylated LTA, the M protein produced a second precipitin arc toward the anode coinciding with the area of migration of the radioactive LTA. Increasing concentrations of LTA or deacylated LTA shifted increasing amounts of the pep M24 antigen to the region of the second arc. Maleylation of M protein to block the positively charged free amino groups before mixing it with LTA prevented the formation of complexes. The complexes formed by the M protein with LTA, but not with deacylated LTA, showed the capacity to bind bovine serum albumin; LTA had been shown previously to bind to the fatty acid binding sites on bovine serum albumin. These results indicate that the LTA molecule is able to bind via its polyanionic backbone to positively charged residues of surface proteins of cells ofS. pyogenes. The implications of such interaction as to the orientation of LTA molecules on the surface of cells ofS. pyogenesare discussed.
Purification of a soluble and a wall-bound form of beta-glucosidase from Mucor racemosus
Authors: Borgia P I , Mehnert D W
Published: January 2020

beta-Glucosidase activity in crude extracts of Mucor racemosus exists in a soluble form and in a wall-bound form which sediments at 3,500 x g. The soluble form and a wall-bound form were purified to homogeneity by ammonium sulfate fractionation. DEAE-Sephadex chromatography, and SP-Sephadex chromatography. Both forms were identical in all parameters measured. Each enzyme is a glycoprotein of 91,000 daltons, with an identical amino acid composition and N-terminal amino acid of lysine; both contain about 10% carbohydrate. Both forms catalyze the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside with identical kinetic constants.
Spread of a "Pseudomonas-specific" beta-lactamase to plasmids of enterobacteria
Authors: Medeiros A A , Hedges R W , Jacoby G A
Published: January 2020

Eleven isolates including Escherichia coli, Salmonella enteritidis, and Shigella sonnei, obtained in Brazil, Hong Kong, Indonesia, Thailand, and the United States, were found to produce beta-lactamase of the PSE-1 type, which was previously considered to be Pseudomonas specific. The enterobacterial strains produced a beta-lactamase with the same isoelectric point, immunological reactions, and substrate profile as those of the prototype PSE-1 enzyme determined by Pseudomonas plasmid RPL11. The producer strains were resistant to multiple antibiotics, and all contained plasmids, ranging in size from 37 x 10(6) to 130 x 10(6), that belonged to at last six incompatibility groups. Plasmids of IncH2 and IncFIme were shown to contain 8 x 10(6)-molecular-weight transposons Tn1401 and Tn1402 that encoded PSE-1 beta-lactamase production, resistance to streptomycin and spectinomycin via AAD(3"), and resistance to sulfonamide. PSE-1 beta-lactamase was not Pseudomonas specific and appeared to have spread among plasmids found in enterobacteria by transposition.
Physical and genetic localization of ilv regulatory sites in lambda ilv bacteriophages
Authors: Gray J E , Bennett D C , Umbarger H E , Calhoun D H
Published: January 2020

A set of nine lambda dilv phages were used to transduce bacterial recipients containing point mutations or deletions in the ilv genes located at 84 min on the Escherichia coli K-12 chromosome. This genetic analysis indicated that two phages carry the entire ilvGEDAC cluster; others carry the complete ilvC gene and, in addition, bacterial DNA that extends to a termination point between ilvA and ilvC, within ilvD, within ilvE, or within ilvG. DNA extracted from the lambda dilv phages was digested with EcoRI, HindIII, KpnI, PstI, SalI, and SmaI. The restriction maps revealed that these phages were generated after insertion at four distinct insertion sites downstream (clockwise) of ilvC. The physical relationships between the various phages were further examined by electron microscopic heteroduplex analysis. The physical maps of the phages thus generated were straightforward and in complete accord with the genetic data. No evidence for genetic rearrangements of ilv DNA in the phage was obtained, thus validating conclusions based on the use of these phages in previous and ongoing research projects. Bacterial cells with deletions of the ilv genes were made lysogenic with lambda dilv phage to examine the regulation of ilv genes present in the phage. The results confirm previous studies showing that one site for control by repression and derepression is upstream (counterclockwise) of ilvG. It was shown, in addition, that the activities of dihydroxy acid dehydrase and threonine deaminase were increased when the prototrophic lysogens were grown with 20 mM leucine. Since this increase was exhibited even when the ilvG-linked control region was not carried by the lambda dilv phage, additional control sites must be located within the ilvEDA region of the ilvGEDA transcription unit.
Penicillin is an active-site inhibitor for four genera of bacteria
Authors: Yocum R R , Amanuma H , O'Brien T A , Waxman D J , Strominger J L
Published: January 2020

The hypothesis that penicillin acts as an active-site inhibitor cell wall biosynthesis was tested by a method of partial proteolytic mapping of penicillin-binding sites versus substrate-binding sites in cell wall D-alanine carboxypeptidases. This enzyme was obtained from four genera of bacteria, purified, and tested.
Fixation of dinitrogen derived from denitrification of nitrate in a photosynthetic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans
Authors: Dunstan R H , Kelley B C , Nicholas D J
Published: January 2020

Studies with 15N demonstrated that the phototrophic bacterium Rhodopseudomonas sphaeroides forma sp. denitrificans strain IL106 cannot assimilate NO-3 but rather denitrifies it to N2. This strain also fixed N2 into cell protein, although nitrogenase activity was partially inhibited in the presence of NO-3. Strain IL106 did not assimilate NO-3, but growing cultures and washed cell suspensions incorporated the tracer from 15NO-3 via denitrification to 15N2 and then via nitrogenase into cell nitrogen. This incorporation was inhibited in cells supplied with NH4+ or in the absence of light, thus confirming the participation of nitrogenase in the assimilation of nitrogen from nitrate. This represents a novel type of N2 recycling in a photodiazotrophic denitrifying bacterium.
Fusions of flagellar operons to lactose genes on a mu lac bacteriophage
Authors: Komeda Y
Published: January 2020

Previous studies have defined 29 genes necessary for synthesis of the Escherichia coli flagellar apparatus. This study analyzed the transcriptional control of flagellar genes, using Mu d (Apr lac) phage to generate flagellar mutants by insertion. These mutants contained operon fusions of flagellar genes to the lac genes of the Mu d phage and allowed the measurement of flagellar operon expression by detection of beta-galactosidase activity. These fusion mutants expressed the enzyme activity constitutively, and an autogenous regulation mechanism was not revealed. Lambda transducing phages carrying these chromosomal fla-lac fusions were also isolated and used to examine the effect of different fla mutations on expression of each flagellar operon. The results showed that flagellar operons are divided into six classes; (class 1) the flbB operon, which controls all of the other flagellar operons; (class 2) the flaU and flbC operons, which are controlled by the flbB operon gene products and are not required for the expression of other Fla operons; (class 3) the flbA, flaG, flaD, flaN, flaB, and flaA operons, which are under flbB operon control and are required for the expression of other fla operons; (class4) the flaZ operon, which is controlled by the gene products of the group 1 and 3 operons and is required for hag transcription; (class 5) the mocha and flaS operons, which are controlled by the gene products of the group 1 and 3 operons; and (class 6) the hag operon. These results are discussed with respect to the possible assembly sequence of the fla gene products.
TOL plasmid pWW0 in constructed halobenzoate-degrading Pseudomonas strains: prevention of meta pathway
Authors: Reineke W , Jeenes D J , Williams P A , Knackmuss H J
Published: January 2020

The hybrid pathway for chlorobenzoate metabolism was studied in WR211 and WR216, which were derived from Pseudomonas sp. B13 by acquisition of TOL plasmid pWW0 from Pseudomonas putida mt-2. Chlorobenzoates are utilized readily by these strains when meta cleavage of chlorocatechols is suppressed. When WR211 utilizes 3-chlorobenzoate (3CB), the expression of catechol 2,3-dioxygenase (C23O) and the catabolic activities for chloroaromatics via the ortho pathway coexist as a consequence of inactivation of the meta cleavage activity by 3-chlorocatechol. Utilization of 4-chlorobenzoate (4CB) by WR216 presupposes the suppression of C23O by a spontaneous mutation in the structural gene, so that 4-chlorocatechol is not misrouted into the meta pathway. Such C23O- mutants were also selected when WR211 was grown continuously on 3CB. Our data explain why the phenotypic characters 3CB+ and Mtol+ (m-toluate) are compatible, whereas 4CB+ and Mtol+ are incompatible.
Role of glnA-linked genes in regulation of glutamine synthetase and histidase formation in Klebsiella aerogenes
Authors: Rothman N , Rothstein D , Foor F , Magasanik B
Published: January 2020

We isolated mutants of Klebsiella aerogenes with insertions of transposon Tn5 linked to the structural gene for glutamine synthetase, glnA. We found that K. aerogenes, like Escherichia coli and Salmonella typhimurium, contains a regulatory gene, glnG, which is closely linked to but distinct from glnA. The product of glnG is essential for normal regulation of the synthesis of glutamine synthetase and histidase. We analyzed two mutations which affected the regulation of the formation of these enzymes and appeared to be outside glnG. The results of our studies suggest the presence in K. aerogenes of at least two regulatory genes located in the glnA region, namely, glnG and another gene, the transcription of which can be initiated at the promoter of glnA.
Isolation and partial characterization of Bacillus subtilis mutants impaired in DNA entry
Authors: Mulder J A , Venema G
Published: January 2020

Transformation-deficient mutants of Bacillus subtilis have been identified either by screening for a nuclease-deficient phenotype on methyl green-DNA agar or for nontransformability on transforming DNA-containing agar. After purification of the mutations causing a reduction in the entry of DNA, a set of isogenic entry-deficient strains was obtained. In addition to being entry deficient to various extents, the strains usually were less capable of association with DNA than the entry-proficient parent. Likewise, the specific transforming activity in the purified mutant strains continued to be less than that in the wild type. With the possible exception of one strain, no evidence was obtained that the mutant strains were impaired in recombination. Since the breakdown of transforming DNA to acid-soluble products correlated fairly well with the residual capacity of the strains to take up DNA, nucleolytic activity is likely to be involved in the entry of DNA in B. subtilis.
Bacillus megaterium spore protease: purification, radioimmunoassay, and analysis of antigen level and localization during growth, sporulation, and spore germination
Authors: Loshon C A , Setlow P
Published: January 2020

The protease which initiates the massive protein degradation early in bacterial spore germination has been purified from Bacillus megaterium spores. The enzyme has a molecular weight of 160,000 and contains four apparently identical subunits, but only the tetramer is enzymatically active. A radioimmunoassay has been developed for this enzyme and has been used to show that the protease is absent from growing cells, but appears early in sporulation within the developing forespore. In contrast, the protease antigen disappears rapidly during spore germination, in parallel with the loss in enzyme activity.
Promoter-distal region of the tra operon of F-like sex factor R100 in Escherichia coli K-12
Authors: Hansen B S , Manning P A , Achtman M
Published: January 2020

The distal region of the tra (transfer) operon of F-like plasmid R100 was investigated, using small plasmids derived from R100, primarily the plasmid pSM6. The transposon Tn5 (which confers kanamycin resistance) was inserted at different positions into pSM6, and the transposition derivatives were tested for ability to complement defined tra mutants of the F sex factor. Thus, the tra genes traH, G, T, and D were localized on the plasmid R100. A restriction map of pSM6 was constructed, and the locations of the insertions were mapped, using restriction endonuclease digestion of the plasmid DNA and exploiting the fact that several restriction sites are localized in the inverted repeat regions of the transposon. The gene products of the genes traG, S, T, and D were identified by radioactive labeling of proteins synthesized in minicells carrying the various insertion plasmids followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of another transfer gene, traI, was inferred from these data. Another protein, the r2-A protein, was also identified, and its gene was mapped. On the basis of the data, a best-fit physical map of this region of the tra operon of R100 was constructed. The results confirmed that the general order and size of the distal transfer genes is as in the F sex factor, but showed that differences exist with respect to all of the gene products. The significance of these differences are discussed in the light of the genetic and physical homology (Manning et al., J. Bacteriol. 150:76-88) of the transfer regions.
Facilitation of Plasmid Transfer in
Streptococcus pneumoniae
by Chromosomal Homology
Authors: Lopez Paloma , Espinosa Manuel , Stassi Diane L. , Lacks Sanford A.
Published: January 2020

The frequency of plasmid establishment in the transformation of
Streptococcus pneumoniae
by plasmid DNA was increased more than 10-fold when the plasmid carried DNA homologous to the host chromosome. Perfect homology was not necessary for such facilitation; small additions or deletions were tolerated, but extensive deletions in the homologous segment of either plasmid or chromosome reduced or eliminated facilitation. The facilitated plasmid transfer showed a linear dependence on monomeric plasmid concentration rather than the quadratic dependence found in the absence of homology, which indicated that entering plasmid fragments interacted with the chromosome rather than with each other to establish a plasmid replicon. Restriction enzyme cleavage of the plasmid in the nonhomologous segment destroyed its activity, but cleavage in the homologous segment or even enzymatic removal of part of that segment did not prevent plasmid transfer, and plasmids of the original size were established. In facilitated transfer, chromosomal markers (additions and deletions as well as single-site mutations) entered the plasmid with a frequency ranging from 10 to 90% depending on the marker location. Several possible mechanisms for the establishment of plasmids in the presence of chromosomal homology and for the transfer of chromosomal information are considered. They depend on synapsis of the newly entered single-strand plasmid fragment with the host chromosome and subsequent copying of, donation from, or integration into the homologous chromosomal segment. After plasmid establishment, equilibration of donor and chromosomal markers between the chromosome and the plasmid pool, presumably by homologous recombination events, was observed.
Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa
Authors: Drainas C , Weiss R L
Published: January 2020

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors or respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors of glycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its association with the mobilization process are discussed.
Extracellular siderophores of rapidly growing Aspergillus nidulans and Penicillium chrysogenum
Authors: Charlang G , Horowitz R M , Lowy P H , Ng B , Poling S M , Horowitz N H
Published: January 2020

The highly active extracellular siderophores previously detected in young cultures of Aspergillus nidulans and Penicillium chrysogenum have been identified as the cyclic ester fusigen (fusarinine C), and its open-chain form, fusigen B (fusarinine B).
Repression and induction of flocculation interactions in Saccharomyces cerevisiae
Authors: Miki B L , Poon N H , Seligy V L
Published: January 2020

The biological control of flocculation interactions by factors related to growth under different conditions of aeration was documented with a new assay for flocculence. The degree of flocculence expressed in a genetically defined Saccharomyces cerevisiae strain (FLO1/FLO1 ade1/ade1) remained constant during aerobic growth but varied with aeration. Flocculence was repressed in anaerobically growing cells but was induced in stationary cells or cells returned to aerobic growth. Repression was correlated with the selective inactivation of cell surface lectin-like components. The changes in flocculence were accompanied by changes in 16 extractable proteins separated by electrophoresis; however, a clear correlation between specific protein bands and flocculence could not be established. The study clearly demonstrated that the phenotypic expression of FLO1 could be reproducibly manipulated for experimental purposes by aeration alone.
Evidence that alpha-isopropylmalate synthase of Saccharomyces cerevisiae is under the "general" control of amino acid biosynthesis
Authors: Hsu Y P , Kohlhaw G B , Niederberger P
Published: January 2020

The specific activity and the immunoreactive amount of alpha-isopropylmalate synthase were more than three times above wild-type values in a Saccharomyces cerevisiae mutant (cdr1) with constitutively derepressed levels of enzymes known to be under the "general" control of amino acid biosynthesis. The specific activity was also higher in lysine- and arginine-leaky strains when these were grown under limiting conditions, and in wild-type cells grown in the presence of 5-methyltryptophan. A low specific activity was found in a mutant (ndr1) unable to derepress enzymes of the general control system. Neither isopropylmalate isomerase nor beta-isopropylmalate dehydrogenase responded to general control signals.
Nitrate reductase from Rhodopseudomonas sphaeroides
Authors: Kerber N L , Cardenas J
Published: January 2020

The facultative phototroph Rhodopseudomonas sphaeroides DSM158 was incapable of either assimilating or dissimilating nitrate, although the organism could reduce it enzymatically to nitrite either anaerobically in the light or aerobically in the dark. Reduction of nitrate was mediated by a nitrate reductase bound to chromatophores that could be easily solubilized and functioned with chemically reduced viologens or photochemically reduced flavins as electron donors. The enzyme was solubilized, and some of its kinetic and molecular parameters were determined. It seemed to be nonadaptive, ammonia did not repress its synthesis, and its activity underwent a rapid decline when the cells entered the stationary growth phase. Studies with inhibitors and with metal antagonists indicated that molybdenum and possibly iron participate in the enzymatic reduction of nitrate. The conjectural significance of this nitrate reductase in phototrophic bacteria is discussed.
Characterization of proteins in flagellates and growing amebae of Naegleria fowleri
Authors: Woodworth T W , Keefe W E , Bradley S G
Published: January 2020

Polypeptides of whole-cell extracts of Naegleria fowleri flagellates and growing amebae were resolved by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms of the [35S]methionine-labeled polypeptides of amebae and flagellates were analyzed by two dimensional densitometry to determine whether there were correlations between intracellular concentration of a protein and subunit size or charge. The majority of the polypeptides of amebae and flagellates had molecular sizes in the range of 20 to 60 kilodaltons. The radioactivity per polypeptide species in the size range of 20 to 60 kilodaltons was greater in amebae than in flagellates. The greatest number of polypeptides detected in amebae and flagellates was in the isoelectric focusing range of pH 6 to 7. The radioactivity per polypeptide species in the isoelectric focusing gradient below 6.3 was greater in amebae than in flagellates. Polypeptides in the size range of 20 to 60 kilodaltons had a median isoelectric point below pI 6.3, whereas those larger than 60 kilodaltons had a median pI value above 6.3. These data indicated that molecular size and charge were not entirely independent variables and that the size and charge of a polypeptide might have an important influence in determining its intracellular concentration in both amebae and flagellates. Autoradiograms were also compared so that changes in intracellular protein complement and concentrations occurring during differentiation could be recognized. The relative amounts of a limited number of polypeptides increased markedly, and others decreased markedly, during enflagellation.
Ribosomal protein S4 acts in trans as a translational repressor to regulate expression of the alpha operon in Escherichia coli
Authors: Jinks-Robertson S , Nomura M
Published: January 2020

Ribosomal protein (r-protein) S4 is the translational repressor which regulates the synthesis rates of r-proteins whose genes are in the alpha operon: r-proteins S13, S11, S4, and L17. In a strain having a mutation in the gene for r-protein S4 (rpsD), the mutant S4 fails to regulate expression of the alpha operon, resulting in specific and significant overproduction of r-proteins S13, S11, and S4. This confirms and extends similar observations made with rpsD mutants (M. O. Olsson and L. A. Isaksson, Mol. Gen. Genet. 169:271-278, 1979) before post-transcriptional regulation of r-protein synthesis was proposed and is consistent with the established regulatory role of r-protein S4. The rpsD mutant has been used to study the question of whether regulatory r-proteins function in trans or strictly in cis as translational repressors. The mutant strain was lysogenized with one or two specialized transducing phages carrying a wild-type S4 gene to obtain strains which were diploid or triploid with respect to the alpha operon. The wild-type and mutant forms of S4 were separated by two-dimensional polyacrylamide gel electrophoresis, which allowed accurate measurement of the relative contributions of r-proteins from different alpha operons within a single cell. We found that expression of r-proteins from the chromosomal alpha operon containing the rpsD allele was reduced when the wild-type S4 was present, with the effect being greater in the triploid strain than in the diploid strain. We conclude that the wild-type S4 acts in trans as a translational repressor to regulate expression from the chromosomal alpha operon.
Two complementation groups mediate tetracycline resistance determined by Tn10
Authors: Curiale M S , Levy S B
Published: January 2020

The structural and regulatory tetracycline resistance genes of transposon Tn10 are located on a 2,700-base pair HpaI fragment. We have used eight tetracycline-sensitive mutations in the 2,700-base pair fragment, cloned into two compatible plasmids, to demonstrate that two complementation groups are required for tetracycline resistance. By genetic recombination with plasmids containing the regulatory or structural regions for resistance, we have determined that both complementation groups reside within the structural region. The complementation groups, designated tetA and tetB, are proximal and distal, respectively, to the promoter for the tetracycline resistance structural region. The tetB mutations are in the portion of the structural region that is known to encode the 36,000-molecular-weight, inner-membrane TET protein. The levels of tetracycline resistance expressed during complementation suggest a complex interaction between the products of the tetA and tetB loci.
Active transport of ferric schizokinen in Anabaena sp
Authors: Lammers P J , Sanders-Loehr J
Published: January 2020

The cyanobacterium Anabaena sp. strain ATCC 27898 was found to utilize the siderophore schizokinen to accumulate iron from the environment. This organism had previously been shown to produce schizokinen under low-iron conditions, and we observed that the iron-transport capability is also increased in response to iron limitation. Uptake activity was specific for ferric schizokinen displayed kinetics typical of a protein-mediated process with an apparent Km of 0.04 microM and saturation at high concentrations of substrate. Light-driven transport was blocked by uncouplers and by ATPase inhibitors. Transport in dark-adapted cells was additionally blocked by inhibitors of respiration. We conclude that ATP serves as an energy source for the cellular uptake of ferric schizokinen.
Identification of the Salmonella typhimurium cysB gene product by two-dimensional protein electrophoresis
Authors: Baptist E W , Hallquist S G , Kredich N M
Published: January 2020

Examination of two-dimensional electropherograms of proteins from wild-type Salmonella typhimurium and 16 different cysB strains permitted the identification of a single 34,500-dalton polypeptide chain with a pI of 7.6 that was the product of cysB. Exclusion chromatography indicated that the native cysB protein is a multimer of at least two and probably four or more such subunits.
Manganese binding and oxidation by spores of a marine bacillus
Authors: Rosson R A , Nealson K H
Published: January 2020

Mature, dormant spores of a marine bacillus, SG-1, bound and oxidized (precipitated) manganese on their surfaces. The binding and oxidation occurred under dormant conditions, with mature spores suspended in natural seawater. These heat-stable spores were formed in the absence of added manganese in the growth medium. The rate and amount of manganese bound by SG-1 spores was a function of spore concentration. Temperatures greater than 45 degrees C, pH values below 6.5, or the addition of EDTA or the metabolic inhibitors sodium azide, potassium cyanide, and mercuric chloride inhibited manganese binding and oxidation. However, SG-1 spores bound and oxidized manganese after treatment with glutaraldehyde, formaldehyde, ethylene oxide gas, or UV light, all of which killed the spores. Manganese oxidation never occurred in the absence of manganese binding to spores. The data suggest that Mn2+ was complexed by a spore component, perhaps an exosporium or a spore coat protein: once bound, the manganese was rapidly oxidized.
Isolation and properties of Bacillus brevis mutants unable to produce tyrocidine
Authors: Symons D C , Hodgson B
Published: January 2020

Mutants of Bacillus brevis ATCC 10068 were isolated which produced less than 1/100 of the amount of tyrocidine produced by the parent strain. These mutants produced spores at the same frequency and which were as resistant to heating at 80 degrees C for up to 3 h as were those produced by the parent strain. A partially purified tyrocidine synthetase from strain ATCC 10068 catalyzed [32P]PPi-ATP exchange reactions dependent on added tyrocidine-constituent amino acids. These activities were separated into three groups (I, II, and III) by fractionation on an Ultrogel AcA34 column. Each group was similar to one of the three components (heavy, intermediate, and light, respectively) found previously for strain ATCC 8185 except that glutamate-dependent activity was not detected in the group I activities and some amino acyl-tRNA synthetase activities were associated with the group III activities. Some of the mutants were shown to have defective tyrocidine synthetase enzymes. Mutant BH30 was defective in two of the group II amino acid-dependent [32P]PPi-ATP exchange reactions, mutant BH16 was defective in one of the group I and one of the group II reactions, and mutant BH34 had alterations to activities in all of the groups. It is unlikely that any of these mutants could synthesise tyrocidine. We conclude that tyrocidine is not involved in either the sporulation process or the resistance of spores of B. brevis ATCC 10068 to heating at 80 degrees C for up to 3 h.
D-tagatose 1,6-diphosphate aldolase from lactic streptococci: purification, properties, and use in measuring intracellular tagatose 1,6-diphosphate
Authors: Crow V L , Thomas T D
Published: January 2020

Two D-ketohexose 1,6-diphosphate aldolases are present in Streptococcus cremoris E8 and S. lactis C10. One aldolase, which was induced by growth on either lactose or galactose, was active with both tagatose 1,6-diphosphate (TDP) and fructose 1,6-diphosphate (FDP), having a lower Km and a higher Vmax with TDP as the substrate. This enzyme, named TDP aldolase, had properties typical of a class I aldolase, being insensitive to EDTA and showing substrate-dependent inactivation by sodium borohydride. Sodium dodecyl sulfate-gel electrophoresis indicated a subunit molecular weight of 34,500. The amino acid composition of TDP aldolase is reported. When the enzyme was incubated with either triose phosphates or FDP, the equilibrium mixture contained an FDP/TDP ratio of 6.9:1. The other aldolase, which had properties typical of a class II aldolase, showed activity with FDP but not with TDP. The intracellular TDP concentration, measured with the purified TDP aldolase, was 0.4 to 4.0 mM in cells growing on lactose or galactose and was lower (0 to 1.0 mM) in cells growing on glucose. The intracellular concentration of FDP was always higher than that of TDP. The role of ketohexose diphosphates in the regulation of end product fermentation by lactic streptococci is discussed.
Cloning and expression of the distal portion of the histidine operon of Escherichia coli K-12
Authors: Grisolia V , Carlomagno M S , Bruni C B
Published: January 2020

The operator-distal genes hisBHAFI(E) of the Escherichia coli K-12 histidine operon were mapped on a DNA fragment 4,500 base pairs long. This fragment, originally present in a lambda transducing phage, was cloned in the vector plasmid pBR313. A restriction map was determined, allowing identification of the orientation of the genes in the fragment. The cloned genes were expressed in appropriate hosts, independent of the orientation of the DNA fragment, as shown by transformation tests and by enzyme assays of one of the gene products, hisB, histidinol phosphatase. An internal transcription initiation site was identified by isolation of the cellular RNA, hybridization to specific DNA probes, and mapping by S1 nuclease.
thiK and thiL loci of Escherichia coli
Authors: Imamura N , Nakayama H
Published: January 2020

Mutants of Escherichia coli K-12 auxotrophic for thiamine phosphates were produced in stepwise fashion from the polyauxotrophic F- strain JC1552, via intermediate production of thiamine auxotrophs that had lost the enzymatic activity of either phosphomethylpyrimidine kinase or thiamine phosphate pyrophosphorylase. They include two types: one responds to thiamine monophosphate or thiamine pyrophosphate, and the other responds to thiamine pyrophosphate only; the former lacks thiamine kinase activity, and the latter lacks thiamine monophosphate kinase activity, in addition to the enzymatic defects caused by the first mutations. We found two genes, for which we propose the designations thiK and thiL, which govern the activities of thiamine kinase and thiamine monophosphate kinase, respectively. By conjugation and P1 transduction, the thiK locus was mapped at about 25 min, between pyrC and purB and close to fabD. The relative order of thiK with respect to nearby genes was tentatively established as pyrC-ptsG-fabD-thiK-purB. In the case of thiL, the locus was situated at about 9 min, between tsx and acrA and probably 0.2 min clockwise from the former.
Involvement of cyclic AMP and its receptor protein in filamentation of an Escherichia coli fic mutant
Authors: Utsumi R , Nakamoto Y , Kawamukai M , Himeno M , Komano T
Published: January 2020

Cyclic AMP (cAMP) inhibited septum formation in Escherichia coli PA3092 and induced cell filamentation at elevated temperatures. This phenomenon was first observed in E. coli PA3092 and is due to a temperature-sensitive mutation. We tentatively named this mutation fic (filamentation induced by cAMP). The fic gene was located near rpsL (formerly strA) on the E. coli K-12 map. the inhibitory effect of cAMP on cell division and filamentation in a fic mutant was not observed in a crp mutant. When cAMP was removed from the culture medium, filaments were divided into rods as the intracellular cAMP level decreased. These results suggest that the cAMP-cAMP receptor protein complex causes filamentation in the fic mutant, E. coli PA3092.
Reproducible and Rapid Methods for the Isolation and Assay of
a
-Factor, a Yeast Mating Hormone
Authors: Strazdis Joan R. , MacKay Vivian L.
Published: January 2020

The
Saccharomyces cerevisiae
mating hormone
a
-factor has been difficult to isolate reproducibly in sufficient yields by methods using ion-exchange chromatography, probably because of its pronounced hydrophobicity. In this work, a hydrophobic adsorbent (Amberlite XAD-2), in an insoluble bead from, was used to isolate larger (up to sixfold greater than previous reports) and quite reproducible (12% standard deviation) quantities of
a
-factor by adsorption from cell-free filtrates of
a
cultures. Moreover, when the beads were added to the cultures at the time of inoculation, sixfold greater yields were obtained than when
a
-factor was adsorbed to the beads from cell-free filtrates.
a
-Factor was readily eluted from the beads with 1-propanol. The same adsorbent could also be used in the partial purification of the less hydrophobic α-factor. Adsorption of both hormones by Amberlite XAD-2 gave a degree of purification comparable to that obtained by the first steps of previously published methods while providing larger yields of hormones. The present procedure is shorter, simpler, and, for
a
-factor, more reproducible. The activities of both hormones were quantitated by using an assay in which the size distribution of cells in the population was monitored after the addition of hormone of the opposite mating type. The extent of increase in cell size which accompanies hormone treatment is a function of the hormone concentration. To ensure solubilization of
a
-factor in the aqueous bioassay system, samples were diluted into bovine serum albumin solutions and sonicated before assaying. The resulting assay is most sensitive at hormone concentrations between 0.05 and 2 U/ml, can reliably detect as little as 0.16 ng of hormone, gave results reproducible within 16%, and is convenient for a large number (>100) of samples.
Pleiotropic control of five eucaryotic genes by multiple regulatory elements
Authors: Turoscy V , Cooper T G
Published: January 2020

We have previously shown that allophanate acts as an inducer for five structural genes whose products participate in the degradation of allantoin by Saccharomyces cerevisiae. This observation led us to hypothesize that these genes might be controlled in common and to test the hypothesis by searching for mutants unable to induce production of the allantoin-degrading enzymes. Such mutants have been found. These strains grew poorly when provided with any of the allantoin pathway intermediates, but used other nitrogen sources normally. The mutations carried in these strains were recessive to wild-type alleles and complemented mutations in all known loci associated with the allantoin pathway. The locus containing the most thoroughly studied mutation (dal81-1) was not fund to be tightly linked to any of the allantoin pathway structural genes. The low basal levels of allantoin pathway enzymes observed in Dal81- strains remained the same whether or not the inducer was present in the growth medium. However, the levels of enzyme increased moderately when mutants were grown on poor nitrogen sources. From these observations, we conclude that dal81 mutant strains possess a defect in the induction of enzyme synthesis; enzyme production due to relief of nitrogen catabolite repression, however, appears normal. The observed epistatic relationships of mutations in the DAL80 and DAL81 loci suggest that their products may possess a reasonable degree of functional independence.
Tiamulin resistance mutations in Escherichia coli
Authors: Böck A , Turnowsky F , Högenauer G
Published: January 2020

Forty "two-step" and 13 "three-step" tiamulin-resistant mutants of Escherichia coli PR11 were isolated and tested for alteration of ribosomal proteins. Mutants with altered ribosomal proteins S10, S19, L3, and L4 were detected. The S19, L3, and L4 mutants were studied in detail. The L3 and L4 mutations did not segregate from the resistance character in transductional crosses and therefore seem to be responsible for the resistance. Extracts of these mutants also exhibited an increased in vitro resistance to tiamulin in the polyuridylic acid and phage R17 RNA-dependent polypeptide synthesis systems, and it was demonstrated that this was a property of the 50S subunit. In the case of the S19 mutant, genetic analysis showed segregation between resistance and the S19 alteration and therefore indicated that mutation of a protein other than S19 was responsible for the resistance phenotype. The isolated ribosomes of the S19, L3, and L4 mutants bound radioactive tiamulin with a considerably reduced strength when compared with those of wild-type cells. The association constants were lower by factors ranging from approximately 20 to 200. When heated in the presence of ammonium chloride, these ribosomes partially regained their avidity for tiamulin.
Purification and characterization of the conidial laccase of Aspergillus nidulans
Authors: Kurtz M B , Champe S P
Published: January 2020

Conidial laccase of Aspergillus nidulans was purified by standard protein purification methods. Although the purified material showed a cluster of several protein bands on a nondenaturing gel, each of these protein bands had laccase activity. All bands of activity, however, were absent in a strain carrying a mutation in the structural gene for laccase. Concentrated solutions (greater than 1 mg/ml) were bright blue, suggesting that, like other laccases, this enzyme contains copper. The enzyme contained asparagine-linked carbohydrate (12% by weight) which could be removed by digestion with endo-beta-N-acetylglucosaminidase H. The molecular weight of native enzyme as determined by gel filtration was 110,000, but the largest component in a sodium dodecyl sulfate gel was 80,000. Several smaller components (55,000 and 36,000 molecular weight) were also visible. We present evidence which suggests that the smaller components are in vivo cleavage products tightly associated with enzymatically active molecules. Comparison of the laccase from a white-spore (wA) and a green-spore (wA+) strain showed, surprisingly, that the enzymes differed in electrophoretic pattern, in vitro heat stability, and in vivo metabolic stability. The difference was manifested for enzymes isolated from cultures after conidial pigmentation of the wA+ strain had occurred. If examined earlier, before pigmentation, the enzymes were indistinguishable. Since wA strains lack the precursor of the wild-type green pigment, i.e., the laccase substrate, we suggest that the transformation of the enzyme of the wA strain is due to its failure to interact with its normal substrate.