bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of cyclic adenosine 3',5'-monophosphate across Escherichia coli vesicle membranes
Authors: Goldenbaum P E , Hall G A
Published: January 2020

The uptake and efflux of cyclic adenosine 3',5'-monophosphate (3',5'-cAMP) by Escherichia coli membrane vesicles were studied. Metabolic energy was not required for the uptake process and was found to actually decrease the amount of 3',5'-cAMP found in the vesicles. 3',5'-cAMP uptake exhibits saturation kinetics (Km = 10 mM, Vmax = 2.8 nmol/mg of protein per min) and was competitively inhibited by a number of 3',5'-cAMP analogs. The uptake of 3',5'-cAMP was found to be sharply affected by a membrane phase transition. The excretion of 3',5'-cAMP was studied by using everted membrane vesicles. Efflux in this system was dependent upon metabolic energy and was reduced or abolished by uncouplers. Different energy sources powered efflux at different rates, showing a relationship between the degree of membrane energization and rate of excretion of 3',5'-cAMP. The efflux process also displayed saturation kinetics (Km = 10.0 mM, Vmax = 0.98 nmol/mg of protein per min) and was competitively inhibited by the same 3',5'-cAMP analogs and to the same degree as was the uptake process. 3',5'-cAMP was found to be chemically unaltered by both the uptake and excretion processes. These data are interpreted as showing that the uptake and excretion of 3',5'-cAMP in E. coli membrane vesicles are carrier-mediated phenomena, possibly employing the same carrier system. Uptake is by facilitated diffusion whereas efflux is via an energy-dependent, active transport process. Evidence is presented showing that cells can regulate the number of 3',5'-cAMP transport carriers. The rate of 3',5'-cAMP excretion is possibly regulated by both the degree of membrane energization and the number of carriers present per cells.
Poising of the arginine pool and control of bioluminescence in Beneckea harveyi
Authors: Makemson J C , Hastings J W
Published: January 2020

Arginine dramatically stimulates bioluminescence in the marine bacterium Beneckea harveyi growing in minimal media, an effect that is due to increases in both the synthesis and expression of luciferase. To elucidate the mechanism of this phenomenon, studies were made of the transport and metabolism of arginine in B. harveyi. The transport of arginine and lysine involves two kinetically distinct transport systems for the uptake of arginine and lysine. In contrast, ornithine is transported only by a system common to all three amino acids. The internal amino acid pools were measured in mutants that do not show stimulation of bioluminescence by arginine and in wild-type cells that do. In minimal media, the internal arginine pools are undetectably low. Furthermore, exogenously added labeled arginine is rapidly transported and converted to citrulline and argininosuccinate. The results can be accommodated by a model in which the internal arginine is poised at a very low concentration; the stimulatory effect of exogenous arginine on luciferase biosynthesis occurs at the transcriptional level, and the actual mediator can be either arginine or argininyl transfer ribonucleic acid.
Transfer ribonucleic acid synthesis during sporulation and spore outgrowth in Bacillus subtilis studied by two-dimensional polyacrylamide gel electrophoresis
Authors: Henner D J , Steinberg W
Published: January 2020

The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.
Deoxyribonucleic Acid Adenine and Cytosine Methylation in
Salmonella typhimurium
and
Salmonella typhi
Authors: Gómez-Eichelmann M. C.
Published: January 2020

The methylations of adenine in the sequence —GATC— and of the second cytosine in the sequence — [Formula: see text] — were studied in
Salmonella typhimurium
and in
Salmonella typhi
. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences —GATC— (
Dpn
I and
Mbo
I) and — [Formula: see text] — (
Eco
RII). The restriction patterns obtained for this plasmid isolated from transformed
S. typhimurium
and
S. typhi
were compared with those of pBR322 isolated from
Escherichia coli
K-12. In
E. coli
K-12, adenines at the sequence —GATC— and the second cytosines at — [Formula: see text] — are met hylated by enzymes coded for by the genes
dam
and
dem
, respectively. From comparison of the restriction patterns obtained, it is concluded that
S. typhimurium
and
S. typhi
contain genes responsible for deoxyribonucleic acid methylation equivalent to
E. coli
K-12 genes
dam
and
dcm
.
Aromatic aminotransferases in coryneform bacteria
Authors: Fazel A M , Jensen R A
Published: January 2020

Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) are capable of transaminating all three of the aromatic pathway intermediates; prephenate, phenylpyruvate, and 4-hydroxy-phenylpyruvate. Two molecular species of aromatic aminotransferase (denoted aminotransferase I and aminotransferase II) were partially purified from C. glutamicum and B. flavum, whereas a single aromatic aminotransferase was isolated from B. ammoniagenes. In both C. glutamicum and B. flavum, aromatic aminotransferase I and aromatic aminotransferase II have molecular weights of about 155,000 and 260,000 respectively. The two aromatic aminotransferases from C. glutamicum and B. flavum, although exhibiting a similar spectrum of overlapping specificities, differ substantially in substrate preference. Pyridoxal-5'-phosphate is tightly associated with these aminotransferases, since little loss of activity was detected when partially purified enzyme preparations were assayed in the absence of exogenous pyridoxal-5'-phosphate. The aminotransferases are quite sensitive to inhibition by phenylhydrazine. This has practical application when assay of prephenate dehydratase is desired in the presence of aromatic aminotransferase activity since potentially trivial interference can be negated by selective phenylhydrazine inhibition of aromatic aminotransferase activity. At 0.1 mM concentrations of phenylhydrazine, 90% inhibitions of aminotransferase activities were achieved in partially purified preparations of B. flavum and C. glutamicum.
Lysis of Escherichia coli mutants by lactose
Authors: Alexander J K
Published: January 2020

Growth of Escherichia coli strain MM6-13 (ptsI suc lacI sup), which as a suppressor of the succinate-negative phenotype, was inhibited by lactose. Cells growing in yeast extract-tryptone-sodium chloride medium (LB broth) were lysed upon the addition of lactose. In Casamino Acids-salts medium, lactose inhibited growth, but due to the high K+ content no lysis occurred. Lysis required high levels of beta-galctosidase and lactose transport activity. MM6, the parental strain of MM6-13, has lower levels of both of these activities and was resistant to lysis under these conditions. When MM6 was grown in LB broth with exogenous cyclic adenosine monophosphate, however, beta-galactosidase and lactose transport activities were greatly increased, and lysis occurred upon the addition of lactose. Resting cells of both MM6 and MM6-13 were lysed by lactose in buffers containing suitable ions. In the presence of MG2+, lysis was enhanced by 5 mM KCl and 100 mM NaCl. Higher slat concentrations (50 mM KCl or 200 mM NaCl) provided partial protection from lysis. In the absence of Mg2+, lysis occurred without KCl. Lactose-dependent lysis occurred in buffers containing anions such as sulafte, chloride, phosphate, or citrate; however, thiocyanate or acetate protected the cells from lysis. These data indicate that both cations and anions, as well as the levels of lactose transport and beta-galactosidase activity, are important in lysis.
Ploidy determination of Canadida albicans
Authors: Olaiya A F , Sogin S J
Published: January 2020

The dimorphic yeast Candida albicans, as a member of the fungi imperfecti, has been assumed to be in the haploid, or imperfect, state. The deoxyribonucleic acid content of this species has been measured by flow microfluorometry, a technique capable of analyzing single cells. These results were compared with flow microfluorometric deoxyribonucleic acid determinations on a series of strains of Saccharomyces cerevisiae of known ploidy (haploid, diploid, triploid, and tetraploid). These ploidy levels were readily distinguished by the flow microfluorometry procedure. By this criterion, C. albicans was found to contain a diploid amount of deoxyribonucleic acid. Ultraviolet radiation survival and chemical mutagenesis experiments support the conclusion that both clinically isolated and laboratory strains of C. albicans are diploid.
Lactose metabolism by Streptococcus mutans: evidence for induction of the tagatose 6-phosphate pathway
Authors: Hamilton I R , Lebtag H
Published: January 2020

Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway.
Synthesis and inactivation of carbamyl phosphate synthetase isozymes of Bacillus subtilis during growth and sporulation
Authors: Paulus T J , Switzer R L
Published: January 2020

Pyrimidine-repressible carbamyl phosphate synthetase P was synthesized in parallel with aspartate transcarbamylase during growth of Bacillus subtilis on glucose-nutrient broth. Both enzymes were inactivated at the end of exponential growth, but at different rates and by different mechanisms. Unlike the inactivation of aspartate transcarbamylase, the inactivation of carbamyl phosphate synthetase P was not interrupted by deprivation for oxygen or in a tricarboxylic acid cycle mutant. The arginine-repressible isozyme carbamyl phosphate synthetase A was synthesized in parallel with ornithine transcarbamylase during the stationary phase under these growth conditions. Again, both enzymes were subsequently inactivated, but at different rates and by apparently different mechanisms. The inactivation of carbamyl phosphate synthetase A was not affected in a protease-deficient mutatn the inactivation of ornithine transcarbamylase was greatly slowed.
Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome
Authors: Miki T , Kimura M , Hiraga S , Nagata T , Yura T
Published: January 2020

The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phase lambda i21 dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of lambda i21 dnaA-2 and pMCR501 revealed the following. The lambda i21 dnaA-2 DNA retained the delta sr I lambda 1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. The size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in lambda i21 dnaA-2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the lambda i21 dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the lambda i21 dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322.
Immunochemical Analysis of Triton X-100-Insoluble Residues from
Micrococcus lysodeikticus
Membranes
Authors: Owen Peter , Doherty Helen
Published: January 2020

Triton X-100-insoluble residues from
Micrococcus lysodeikticus
membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.
Isolation and properties of the protease from the wild-type and mutant strains of Pseudomonas fragi
Authors: Noreau J , Drapeau G R
Published: January 2020

A simplified procedure for the purification of the extracellular protease of Pseudomonas fragi was developed. The enzyme was isolated from a derepressed mutant producing 40 times the enzyme level of the parental organism. It was collected from culture filtrates by ammonium sulfate precipitation, and it was obtained in pure form by single chromatography on a column of diethylaminoethyl cellulose. The protease had a molecular weight of 52,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and had properties of a classical neutral endopeptidase with the exception of its substrate specificity. Mutants of P. fragi producing proteases of altered substrate specificities were isolated from plates containing elastin as the sole carbon source. The SP-Sephadex elution patterns of enzymes extracted from each mutant examined were complex, suggesting that either the enzyme was autodigested or several active forms could be generated from a common precursor. The substrate specificities of the mutant enzymes were different from that produced by the parental strain.
Synthesis of nitrogenase and heterocysts by Anabaena sp. CA in the presence of high levels of ammonia
Authors: Bottomley P J , Grillo J F , Van Baalen C , Tabita F R
Published: January 2020

Anabaena sp. CA fails to synthesize heterocysts and nitrogenase when grown with KNO3 as the nitrogen source. By contrast, both heterocysts and proheterocysts are synthesized in NH4Cl-containing media to a level nearly commensurate with cells grown in the absence of combined nitrogen. The growth rate of the organism in NH4Cl-containing media was similar to that obtained with KNO3 as the nitrogen source and was independent of the presence of N2 in the atmosphere. Thus, our results indicate that the organism assimilated nitrate and ammonium nitrogen equally well to meet the nitrogen requirements for growth. Moreover, in contrast to previous studies with other cyanobacteria, the repressor singal for heterocyst differentiation in Anabaena sp. CA is not derived from the metabolism of ammonia but appears to be involved with nitrate metabolism. Nitrogenase activity was partially expressed in NH4Cl-grown cultures. Increasing the level of nitrogenase activity to a value representative of a N2-grown culture required both the inhibition of ammonia assimilation and de novo protein synthesis. An increase in the number of mature heterocysts was not required. The fact that high levels of exogenous ammonia only partially repress the synthesis of proteins required for the maximum expression of nitrogenase activity in Anabaena sp. CA has important implications.
Pentitol metabolism in Lactobacillus casei
Authors: London J , Chace N M
Published: January 2020

Strains of Lactobacillus casei capable of growing on either ribitol or xylitol carry out a heterolactic fermentation producing ethanol, acetate, and a mixture of D- and L-lactate. Following conversion of the pentitols to ribulose 5-phosphate or xylulose 5-phosphate via enzymatic steps unique to these organisms, the intermediate products are further metabolized by enzymes of the pentose pathway. The initial enzymes of the pathway, i.e., pentitol:phosphoenolypyruvate phosphotransferase and penititol phosphate dehydrogenase, do not appear to be stringently regulated by glucose or intermediate products of glycolysis.
Thujaplicins from Thuja plicata as iron transport agents for Salmonella typhimurium
Authors: Akers H A , Abrego V A , Garland E
Published: January 2020

Strains of Salmonella typhimurium which are unable to synthesize their normal iron transport agent, enterobactin, and which must be supported with an exogenous chelator (siderophore) on certain media, were used to examine various types of wood for the presence of chelators. Western red cedar wood, Thuja plicata, was observed to contain large amounts of three substances that in low concentration would serve as chelators for S. typhimurium. The chelators from T. plicata were characterized and found to be alpha-, beta-, and gamma-thujaplicin. Other planar cyclic alpha-hydroxyketones were examined, and several were found to function as chelators for S. typhimurium.
Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance
Authors: Thomas C M , Meyer R , Helinski D R
Published: January 2020

The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determined for the BglII, PstI, and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, we deleted nonessential regions of RK2 by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained five HaeII fragments of RK2 which total 5.4 kilobases. These fragments were derived from three regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kilobases) has the properties expected of the origin of replication. The outer four fragments, located in two separate regions of RK2, were found to provide, in trans, functions that permit the replication of the HaeII fragment carrying the origin of the replication. These results indicate that at least two plasmid-encoded genes, capable of acting in trans, and a replication origin are required for RK2 replication and maintenance.
Control of arginine metabolism in Neurospora: flux through the biosynthetic pathway
Authors: Goodman I , Weiss R L
Published: January 2020

The flux into the arginine biosynthetic pathway of Neurospora crassa was investigated using a mutant strain lacking the ornithine-degrading enzyme ornithine aminotransferase (EC 2.6.1.13). Flux was measured by the increase in the sum of the radioactivity (derived from [14C]glutamic acid) in the ornithine pool, the arginine pool, and arginine incorporated into proteins. Complete cessation of flux occurred immediately upon the addition of arginine to the growth medium. This response occurred prior to expansion of the arginine pool. After short-term exposure to arginine (80 min), flux resumed quickly upon exhaustion of arginine from the medium. This took place despite the presence of an expanded arginine pool. Initiation of flux required approximately 80 min when the mycelia were grown in arginine-supplemented medium for several generations before exhaustion of the exogenous arginine. The arginine pool of such mycelia was similar to that found in mycelia exposed to exogenous arginine for only 80 min. The results are consistent with rapid onset and release of feedback inhibiton of arginine biosynthesis in response to brief exposure to exogenous arginine. The insensitivity of flux to the size of the arginine pool is consistent with a role for compartmentation in this regulatory process. The lag in initiation of flux after long-term growth in the presence of exogenous arginine suggests the existence of an additional regulatory mechanism(s). Several possibilities are discussed.
Purine-containing compounds, including cyclic adenosine 3',5'-monophosphate, induce fruiting of Myxococcus xanthus by nutritional imbalance
Authors: Manoil C , Kaiser D
Published: January 2020

Induction of Myxococcus xanthus fruiting by a number of different purine-containing compounds, including cyclic adenosine 3',5'-monophosphate, is defective in a mutant resistant to 2,6-diaminopurine. Furthermore, the purine-induced fruiting of wild-type cultures is uniquely blocked by a low concentration of added glycine. These results imply that different purine-containing compounds induce fruiting through a single mechanism involving nutritional imbalance.
Role of transcriptional regulation and enzyme inactivation in the synthesis of Escherichia coli carbamoylphosphate synthase
Authors: Piérard A , Lissens W , Halleux P , Cunin R , Glansdorff N
Published: January 2020

The question of posttranscriptional control during cumulative repression of Escherichia coli carbamoylphosphate synthase has been examined by following the kinetics of repression and by comparing messenger ribonucleic acid and enzyme levels after growth under various conditions. The data provide no evidence for control of synthesis at a level other than transcription. They suggest, however, that enzyme inactivation (or turnover) plays a significant role in the establishment of repressive conditions.
In Vitro Synthesis of
Escherichia coli
Carbamoylphosphate Synthase: Evidence for Participation of the Arginine Repressor in Cumulative Repression
Authors: Lissens Willy , Cunin Raymond , Kelker Norman , Glansdorff Nicolas , Piérard André
Published: January 2020

A deoxyribonucleic acid-directed in vitro system for the synthesis of
Escherichia coli
carbamoylphosphate synthase has been developed, and its properties have been studied. The system uses the deoxyribonucleic acid of a lambda phage carrying the
car
genes (λd
carAB
) as template and mediates the synthesis of both subunits of the enzyme. This newly synthesized enzyme exhibits the properties of native carbamoylphosphate synthase. A study of the in vitro synthetic capacities of S-30 extracts from strains containing either a mutated or the wild-type allele of gene
argR
supports earlier suggestions, based on in vivo evidence, that the
argR
product is involved in cumulative repression of carbamoylphosphate synthase by arginine and the pyrimidines. Repression in vitro is as efficient as in vivo. In keeping with such observation it is shown that in vitro synthesis of carbamoylphosphate synthase is repressed by partially purified arginine repressor. Evidence was obtained which indicates that arginine repression of carbamoylphosphate synthase mainly operates at the level of transcription. This was based on the design of an in vitro transcription system for gene
carA
, the structural gene for the light subunit of carbamoylphosphate synthase. This system also allowed us to demonstrate that free arginine is the corepressor involved in carbamoylphosphate synthase repression. The present in vitro approaches, in addition to the information they have already provided, open new possibilities for further investigations on the mechanism of cumulative repression and, in particular, on the participation of pyrimidine end products in this regulatory mechanism.
Efflux of beta-galactosidase products from Escherichia coli
Authors: Huber R E , Lytton J , Fung E B
Published: January 2020

Several different strains of Escherichia coli were grown on a variety of carbon sources under various growth conditions. Lactose was added (usually at mid-log phase), and the concentrations of the products of beta-galactosidase action on this sugar (galactose, glucose, and allolactose) were determined at various times thereafter in the total culture and in the medium. It was found that with each strain, with all carbon sources, and under all of the conditions studied, a very large proportion of the products were found in the medium. Control studies were carried out which showed that these results were not artifacts of the method of separating the cells from the medium. The results also did not arise from the secretion of beta-galactosidase into the medium, from the diffusion of substrates and products into and out of the cells due to leaks in the membrane, or from faults in the method of sugar analysis. In addition, the results showed that there were very high levels of products inside the cells under the conditions used and that the efflux of the products was rapid. The efflux might be energetically advantageous to the cell as well as being a means of storing excess products until needed.
Presence of two subunit types in ribulose 1,5-bisphosphate carboxylase from Thiobacillus intermedius
Authors: Bowman L H , Chollet R
Published: January 2020

Ribulose bisphosphate carboxylase (EC 4.1.1.39) has been purified to homogeneity from glutamate-CO2-thiosulfate-grown Thiobacillus intermedius by pelleting the protein from the 93,000 X g supernatant fluid followed by ammonium sulfate fractionation and sedimentation into a discontinuous sucrose density gradient. The molecular weight of the native protein approximated that of the higher plant enzyme (550,000) based on its relative electrophoretic mobility in polyacrylamide disc gels compared with that of standards of known molecular weight, including crystalline tobacco ribulose bisphosphate carboxylase. Sodium dodecyl sulfate electrophoresis in 12% polyacrylamide disc gels and Sephadex G-100 chromatography in the presence of sodium dodecyl sulfate indicated that the purified Thiobacillus protein, like the tobacco enzyme, consisted of two types of nonidentical subunits. The molecular weights of the large and small subunits were estimated to be about 55,000 and 13,000, respectively, by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The carboxylase activity of the protein purified from spinach leaves and T. intermedius responded similarly to the effectors reduced nicotinamide adenine dinucleotide phosphate and 6-phosphogluconate. Contrary to a previous report (K. Purohit, B. A. McFadden, and A. L. Cohen, J. Bacteriol. 127:505-515, 1976), these results indicate that ribulose bisphosphate carboxylase purified from Thiobacillus intermedius closely resembles the higher plant enzyme with respect to quaternary structure, molecular weight, and regulatory properties.
Primary structure of the Escherichia coli serotype K30 capsular polysaccharide
Authors: Chakraborty A K , Friebolin H , Stirm S
Published: January 2020

Methylation, 1H nuclear magnetic resonance, and bacteriophage degradation results indicate that the Escherichia coli serotype K30 capsular polysaccharide consists of leads to 2)-alpha-D-Manp-(1 leads to 3)-beta-D-Galp-(1 leads to chains carrying beta-D-GlcUAp-(1 leads to 3)-alpha-D-Galp-(1 leads to branches at position 3 of the mannoses.
Replication of ColE1 plasmid deoxyribonucleic acid in a thermosensitive dnaA mutant of Escherichia coli
Authors: Abe M
Published: January 2020

The replication of ColE1 deoxyribonucleic acid (DNA) took place at the restrictive temperature in a dnaA mutant, dnaA167(Ts). It proceeded at a constant rate at 42 degrees C for at least 3 h. The replication was insensitive to rifampin, which blocked replication at the permissive temperature or in the presence of chloramphenicol, even at the restrictive temperature. A linear DNA strand of ColE1 longer than unit genome size was synthesized. The structure of the replicating molecules observed by electron microscopy was mostly sigma shaped, composed of a circle of a unit genome length with a double-stranded tail. These observations suggest that the replication of ColE1 DNA proceeds via a rolling-circle type of structure in the absence of dnaA function.
Purification and properties of a binding protein for branched-chain amino acids in Pseudomonas aeruginosa
Authors: Hoshino T , Kageyama M
Published: January 2020

A binding protein for branched-chain amino acids was purified to a homogeneous state from shock fluid of Pseudomonas aeruginosa PML14. It was a monomeric protein with an apparent molecular weight of 4.3 x 10(4) or 4.0 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration, respectively. The isoelectric point was determined to be pH 4.1 by electrofocusing. Amino acid analysis of the protein showed that aspartic acid, glutamic acid, glycine, and alanine were major components and that the protein contained only one residue each of tryptophan and cysteine per molecule. The binding protein contained no sugar. The binding activity of the protein was specific for the branched-chain amino acids. The protein also bound alanine and threonine with lower affinity. The dissociation constants of this protein for leucine, isoleucine, and valine were found to be 0.4, 0.3, and 0.5 microM, respectively. Mutants defective in the production of the binding protein were identified among the mutants deficient in a transport system for branched-chain amino acids (LIV-I). The revertants from these mutants to LIV-I-positive phenotype simultaneously recovered normal levels of the binding protein. These findings suggest strongly the association of the binding protein with the LIV-I transport system.
In vivo inactivation of glycerol dehydrogenase in Klebsiella aerogenes: properties of active and inactivated proteins
Authors: Ruch F E , Lin E C , Kowit J D , Tang C T , Goldberg A L
Published: January 2020

Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation of the oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had similar isoelectric points (pH 4.7). Inactivation did, however, alter the gel filtration properties of the enzyme protein and, more importantly, reduced its affinity for the dye Cibacron F3GA and the coenzyme NAD+.
Purification and characterization of a kanamycin nucleotidyltransferase from plasmid pUB110-carrying cells of Bacillus subtilis
Authors: Sadaie Y , Burtis K C , Doi R H
Published: January 2020

The nucleotidyltransferase encoded by plasmid pUB110 was purified to greater than 95% purity with a 33% yield. The enzyme is a monomeric protein with a molecular weight of 34,000. The optimum pH for activity is 5, and the optimum MgCl2 concentration for activity is 18 mM. The enzyme, which is synthesized constitutively, is stable for several weeks at 4 degrees C. This enzyme would appear to be a good model gene product for the development of a pUB110 deoxyribonucleic acid-dependent in vitro protein-synthesizing system from Bacillus subtilis.
Characterization of Serologically Dominant Outer Membrane Proteins ofNeisseria gonorrhoeae
Authors: McDade Ralph L. , Johnston Kenneth H.
Published: January 2020

The proteins of the outer membrane ofNeisseria gonorrhoeaeplay an important role in the serotyping system defined by K. H. Johnston et al. (J. Exp. Med.143:741–758, 1976). This study attempted to delineate the molecular arrangement of the major proteins of the outer membrane of the gonococcus by using three approaches. First, natural protein-protein relationships were demonstrated by symmetrical, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, proteins exposed on the surface of outer membrane vesicles were cross-linked by using the bifunctional reagents dimethyl-3,3′-dithiobispropionimidate and dithiobis[succinimidyl propionate]. Third, specific antigen-antibody interactions on the surface of membrane vesicles were analyzed by radioautographic techniques. The major proteins of the outer membrane of the gonococcus were defined, and a nomenclature was devised to take into account the effects of heat and reducing agents on the resolution of these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results of cross-linking experiments strongly suggest that two of the major proteins of the gonococcal outer membrane (proteins 1 and 3) form a hydrophobically associated trimeric unit in situ which can be stabilized by selective cross-linking reagents. Results substantiated that these proteins are responsible for imparting serotypic specificity.
Glucose Fermentation Pathway of
Thermoanaerobium brockii
Authors: Lamed R. , Zeikus J. G.
Published: January 2020

Thermoanaerobium brockii
was shown to catabolize glucose via the Embden-Meyerhof-Parnas pathway into ethanol, acetic acid, H
2
-CO
2
, and lactic acid. Radioactive tracer studies, employing specifically labeled [
14
C]glucose, demonstrated significant fermentation of
14
CO
2
from C-3 and C-4 of the substrate exclusively. All extracts contained sufficient levels of activity (expressed in micromoles per minute per milligram of protein at 40°C) to assign a catabolic role for the following enzymes: glucokinase, 0.40; fructose-1,6-diphosphate aldolase, 0.23; glyceraldehyde-3-phosphate dehydrogenase, 1.73; pyruvate kinase, 0.36; lactate dehydrogenase (fructose-1,6-diphosphate activated), 0.55; pyruvate dehydrogenase (coenzyme A acetylating), 0.53; hydrogenase, 3.3; phosphotransacetylase, 0.55; acetaldehyde dehydrogenase (coenzyme A acetylating), 0.15; ethanol dehydrogenase, 1.57; and acetate kinase, 1.50. All pyridine nucleotide-linked oxidoreductases examined were specific for nicotinamide adenine dinucleotide, except ethanol dehydrogenase which displayed both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked activities. Fermentation product balances and cell growth yields supported the glucose catabolic pathway described. Representative balanced end product yields (in moles per mole of glucose fermented) were: ethanol, 0.94;
l
-lactate, 0.84; acetate, 0.20; CO
2
, 1.31; and H
2
, 0.50. Growth yields of 16.4 g of cells per mole of glucose were demonstrated. Both growth and end product yields varied significantly in accordance with the specific medium composition and incubation time.
Isolation and characterization of Neurospora crassa mutants impaired in feedback control of ornithine synthesis
Authors: Weiss R L , Lee C A
Published: January 2020

Thirty-two independent mutants were isolated which overcame the proline requirement of pro-3 mutations in Neurospora crassa. The mutations were not revertants, appeared to be allelic, were closely linked or allelic to arg-6, and in strains unable to degrade ornithine no longer suppressed the proline requirement. The suppressor mutations did not alter the levels of biosynthetic or catabolic enzymes, yet allowed accumulation of ornithine. Suppressed strains unable to degrade arginine still produced ornithine (as detected by growth) in arginine-supplemented medium. The results suggest that the suppressor mutants were impaired in the feedback inhibition of ornithine synthesis by arginine. The activity of the appropriate biosynthetic enzyme was less sensitive to inhibition by arginine. The potential usefulness of such mutations is discussed.
Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron
Authors: Glass T L , Hylemon P B
Published: January 2020

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.
Identification of flagellin associated with the Bacillus subtilis folded chromosome
Authors: Guérout-Fleury A M , Le Hégarat F , Hirschbein L
Published: January 2020

We have analyzed the nature and contents of a major protein, P36, in the nucleoid of the Bacillus subtilis wild type and an isogenic mutant devoid of flagella. It appears that deoxyribonucleic acid-P36 complex is flagellin present as membrane-associated flagella.
Preferential suppression of normal exoenzyme formation by membrane-modifying agents
Authors: Fishman Y , Rottem S , Citri N
Published: January 2020

Cerulenin, phenethyl alcohol, benzyl alcohol, procaine, and a series of aliphatic alcohols selectively suppressed production of active exoenzymes by various bacterial strains.
Inhibition of deoxyribonucleic acid gyrase: effects on nucleic acid synthesis and cell division in Escherichia coli K-12
Authors: Fairweather N F , Orr E , Holland I B
Published: January 2020

Mutants of Escherichia coli resistant to the antibiotic clorobiocin are also coumermycin resistant, and the mutation to resistance in at least one mutant was mapped near gyrB. We conclude, therefore, that clorobiocin inhibits deoxyribonucleic acid gyrase, and the drug was used to probe the role of this enzyme in vivo. Deozyribonucleic acid synthesis was preferentially inhibited but not completely blocked by the antibiotic. Transcription and cell division were also markedly affected. However, unlike other inhibitors of deoxyribonucleic acid synthesis, clorobiocin failed to induce the synthesis of protein X, the recA gene product. In mutants resistant to clorobiocin the replication velocity was unaffected, but initiation of deoxyribonucleic acid synthesis appeared to be delayed. We conclude that deoxyribonucleic acid gyrase, and hence the supercoiled structure of the chromosome, is important for transcription, normal initiation of deoxyribonucleic acid replication, and cell division. The possible role of deoxyribonucleic acid gyrase in the elongation of replication forks is also discussed.
Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli
Authors: Felton J , Michaelis S , Wright A
Published: January 2020

Escherichia coli K-12 has two genes, asnA+ and asnB+, either one of which is able to satisfy the need of cells for asparagine. In order for a strain to have an auxotrophic requirement for asparagine, both genes must be mutationally inactivated. We obtained mutants with Tn5 inserted in asnB. asnB was mapped by conjugation and by three-factor P1 transductions at 15 min on the E coli K-12 linkage map, between ubiF and nagB. Specialized transducing phage lamba 781 supE was shown to carry asnB, as well as supE, ubiF, nagA, and nagB. asnA is the previously mapped ilv-linked asn locus, whiich is between uncB and rbs. E. coli C also has two asn genes, corresponding to asnA and asnB.
Sporulation in
Hansenula wingei
Is Induced by Nitrogen Starvation in Maltose-Containing Media
Authors: Crandall Marjorie , Lawrence L. J.
Published: January 2020

The sexually agglutinative yeast
Hansenula wingei
lives in association with bark beetles that inhabit coniferous trees. This yeast was induced to sporulate by malt extract, which contains a high percentage of maltose (50%) and a low percentage of nitrogen (0.5%). A solution of 1.5% maltose without any growth factors also induced ascosporogenesis in
H. wingei
. Thus, only a carbon source is required for sporulation as in
Saccharomyces
. However, potassium acetate did not induce sporulation in
H. wingei
as it does in
S. cerevisiae
. Instead, disaccharides (such as maltose, sucrose, or cellobiose) promote sporulation better than either monosaccharides (such as dextrose, fructose, or mannose) or respiratory substrates (such as ethanol or glycerol). The specificity of disaccharides in promoting sporulation in
H. wingei
may be considered an adaptation since these disaccharides are present in the natural environment of this yeast. In addition, the specificity of disaccharides may be related to the induction of the disaccharidase because cells precultured on dextrose sporulate well on maltose, but cells precultured on maltose sporulate poorly on maltose. When (NH
4
)
2
SO
4
was added at a low concentration (3 mM) to synthetic sporulation medium (1.5% maltose solution), sporulation was abolished, whereas other salts and nitrogen sources inhibited to a lesser extent and vitamins and trace elements had no effect. Oxygen was required for sporulation, as expected for an obligate aerobe. Maximal sporulation was achieved in 2% malt extract broth at high cell density (10
9
cells per ml), pH 5, and 25°C. By using these optimal physiological conditions and hybrid strains selected from an extensive genetic breeding program, about 30% asci (10% tetrads) were obtained routinely. Thus, the genetics of cell recognition in this yeast can now be studied.
Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors
Authors: Bruni C B , Musti A M , Frunzio R , Blasi F
Published: January 2020

A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed.
Control of cell septation by lateral wall extension in a pH-conditional morphology mutant of Klebsiella pneumoniae
Authors: Satta G , Canepari P , Botta G , Fontana R
Published: January 2020

The pH-conditional morphology mutant of Klebsiella pneumoniae strain MirM7 grows as cocci at pH 7 and as rods at pH 5.8. The mutant has a high-level mecillinam resistance (50% lethal dose greater than 200 micrograms/ml) in both forms. When broth cultures of the rod-shaped mutant were grown with 0.7 microgram of mecillinam per ml, cells assumed a round shape and continued to divided at a higher rate than the untreated control. A MirM7 rod-shaped revertant (MirA12), when treated with the same antibiotic concentration, changed to coccal shape and stopped dividing. The penicillin-binding proteins (PBPs) of strains MirA12 and MirM7 were analyzed. K. pneumoniae had six major PBPs quite similar to those of Escherichia coli. No differences were seen in the PBPs of MirM7 cocci and rods and MirA12 cells. In particular, PBP2 was found to be present and similar in MirM7 rods and cocci and MirA12 cells. We suggest that that in gram-negative rods, a control mechanism exists which prevents further septation in the absence of lateral cell wall elongation. The unique behavior of MirM7 is due to the fact that the control mechanism is not active in this strain. This model allows us to explain the preservation of shape in bacterial rods under various conditions of growth and the mechanism of bacterial killing by mecillinam.