bluuebunny/crossref_metadata_2025 huggingface dataset:
Excretion of glutamic acid in Citrobacter intermedius C3 associated with plasmid deoxyribonucleic acid
Authors: Jofre J , Prieto M J , Tomás J , Parés R
Published: January 2020

Several mutants of Citrobacter intermedius C3 lacking both the ability to synthesize proline and the ability to excrete glutamic acid were isolated by treatment with nitrosoguanidine. No revertants for either characteristic were obtained from these mutants. The ability to excrete glutamic acid was transferred to those mutants with very high frequencies in mating experience by using auxotropic excreting strains as donors. Moreover, the ability to synthesize proline was transferred together with the ability to excrete glutamic acid when an excreting strain was used as donor. The transconjugants showed a rapid spontaneous curing of both genetic markers. It was shown by two different methods that a band of covalently closed circular deoxyribonucleic acid is present in the cesium chloride gradients corresponding to the wild type and excretor mutants. Nonexcretor mutants described herein lacked such a band. Pro + transformants that were also excretors were obtained with plasmid deoxyribonucleic acid isolated either from wild type or from an excretor mutant. These data strongly indicate that glutamic acid excretion in C. intermedius C3 is related to the presence of extrachromosomal deoxyribonucleic acid.
Obligatory biosynthesis of L-tyrosine via the pretyrosine branchlet in coryneform bacteria
Authors: Fazel A M , Jensen R A
Published: January 2020

Species of coryneform bacteria (Corynebacterium glutamicum, Brevibacterium flavum, and B. ammoniagenes) utilize pretyrosine [beta-(1-carboxy-4-hydroxy-2,5-cyclohexadien-1-yl) alanine] as an intermediate in L-tyrosine biosynthesis. Pretyrosine is formed from prephenate via the activity of at least one species of aromatic aminotransferase which is significantly greater with prephenate as substrate than with either phenylpyruvate or 4-hydroxyphenylpyruvate. Pretyrosine dehydrogenase, capable of converting pretyrosine to L-tyrosine, has been partially purified from all three species. Each of the three pretyrosine dehydrogenases is catalytically active with either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as cofactors. The Km values for nicotinamide adenine dinucleotide phosphate in C. glutamicum and B. flavum are 55 microM and 14.2 microM, respectively, and corresponding Km values for nicotinamide adenine dinucleotide are 350 microM and 625 microM, respectively. The molecular weights of pretyrosine dehydrogenase in C. glutamicum and in B. flavum are both about 158,000, compared with 68,000 moleculr weitht in B. ammoniagenes. In all three species the enzyme is not feedback inhibited by L-tyrosine. Results obtained with various auxotropic mutants, which were used to manipulate internal concentrations of L-tyrosine, suggest that pretyrosine dehydrogenase is expressed constitutively. Pretyrosine dehydrogenase is quite sensitive to p-hydroxymercuribenzoic acid, complete inhibition being achieved at 10 to 25 microM concentrations. This inhibition is readily reversed by thiol reagents such as 2-mercaptoethanol. Coryneform organisms, like species of blue-green bacteria, appear to lack the 4-hydroxyphenylpyruvate pa thway of L-tyrosine synthesis altogether. The loss of pretyrosine dehydrogenase in extracts prepared from a tyrosine auxotroph affirms the exclusive role of pretyrosine dehydrogenase in L-tyrosine biosynthesis. Other reports in the literature, in which the presence in these organisms of prephenate dehydrogenase is described, appear to be erroneous.
Use of 8-hydroxyquinoline to enrich for temperature-sensitive mutants of Tetrahymena
Authors: Martindale D W , Gardonio E C , Pearlman R E
Published: January 2020

The effects of the chelating agent 8-hydroxyquinoline (Hq) on Tetrahymena thermophila were examined. Cell division was completely inhibited by 5 micrograms of Hq per ml. At this concentration deoxyribonucleic acid, ribonucleic acid, and protein syntheses were also completely and nonselectively inhibited. The inhibition was reversible after 6 h of Hq treatment. At concentrations above 20 micrograms/ml a 10,000-fold decrease in survival as seen after 2 h in the drug. The sensitivity of Tetrahymena to Hq was found to be dependent upon cell concentration, wild-type strain, medium, and length of time the culture is at 38 degrees C before Hq is added. Mutants of Tetrahymena that are unable to divide at the restrictive temperature, but which continue macromolecular synthesis, were found to be resistant to Hq treatment. Conditions were obtained in which more than a 1,000-fold difference in survival was seen between this class of mutant and the wild type. The effect of Hq on three other classes of temperature-sensitive mutants was examined, and the results are discussed.
Three types of mycolic acid from Mycobacterium tuberculosis Brévanne: implications for structure-function relationships in pathogenesis
Authors: Toubiana R , Berlan J , Sato H , Strain M
Published: January 2020

Saponification of the chloroform-soluble wax from Mycobacterium tuberculosis Brévanne led to the isolation of three classes of mycolic acid containing characteristic functional groups along the methylene backbone: type alpha (two cyclopropane rings); type beta (methoxyl, methyl, and cyclopropane); and type gamma (ketone, methyl, and cyclopropane). The structures of these acids were elucidated principally by mass spectrometry. The high mass region of the keto mycolate is presented showing the meromycolal and molecular ion regions. This is first time a molecular peak for this mycolic acid has been reported. The structure of the keto mycolate was further substantiated by study of the mass spectral fragmentation of its dithioketal derivative. Within each type of acid, a series of homologs was encountered, varying according to the number of methylene units in the backbone chain. Chromatographic and infrared spectrophotometric evidence is presented for the alkali-induced isomerization of the three types of mycolates.
Solubilization and Properties of a Particulate Hydrogenase from
Methanobacterium
Strain G2R
Authors: McKellar R. C. , Sprott G. D.
Published: January 2020

Mechanical disruption of cells of
Methanobacterium
strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-β-
d
-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77°C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5′-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by
p
-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.
Transaminase B from
Escherichia coli
: Quaternary Structure, Amino-Terminal Sequence, Substrate Specificity, and Absence of a Separate Valine-α-Ketoglutarate Activity
Authors: Lee-Peng Fong-Chia , Hermodson Mark A. , Kohlhaw Gunter B.
Published: January 2020

Transaminase B (branched-chain amino acid aminotransferase, EC 2.6.1.42), the
ilvE
gene product, was purified to apparent homogeneity from an
Escherichia coli
K-12 strain which carries the
ilvE
gene both on the host chromosome and on a plasmid. The oligomeric structure of the enzyme, as determined by analytical ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was confirmed to be that of a hexamer with a molecular weight of about 182,000 and apparently identical subunits. Cross-linking with dimethylsuberimidate yielded trimers, dimers, and monomers, but essentially no species of higher molecular weight. These results are consistent with a double-trimer arrangement of the subunits in native enzyme. The amino-terminal sequence was found to be: Gly Thr Lys Lys Ala Asp Tyr Ile (Trp) Phe Asn Gly (Thr) (Met) Val. Purified transaminase B catalyzed transamination between α-ketoglutarate and
l
-isoleucine,
l
-leucine,
l
-valine, and, to a lesser extent,
l
-phenylalanine and
l
-tyrosine, the latter reacting very sluggishly. The enzyme was free of aspartate transaminase and of transaminase C. The apparent

K
m

values for the branched-chain α-ketoacids were smaller than those for the corresponding amino acids. The lowest

K
m

was recorded for
dl
-α-keto-β-methyl-
n
-valerate, and the highest was recorded for
l
-valine. The ratio of the valine- and isoleucine-α-ketoglutarate activities did not change significantly during purification, and both activities were quantitatively removed from crude extract by antibody raised against purified transaminase B. These observations argue against the existence of a separate valine-α-ketoglutarate transaminase. Anti-
E. coli
transaminase B antibody cross-reacted with crude extract from
Salmonella typhimurium
, but not with extract obtained from
Pseudomonas aeruginosa
.
Mutants defective in the 33K outer membrane protein of Salmonella typhimurium
Authors: Stocker B A , Nurminen M , Mäkelä P H
Published: January 2020

Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.
Isolation and partial characterization of protein E, a major protein found in certain Escherichia coli K-12 mutant strains: relationship to other outer membrane proteins
Authors: Chai T J , Foulds J
Published: January 2020

Escherichia coli outer membrane protein E was purified, and its amino acid composition and N-terminal amino acid were determined. The purified protein was shown to be immunologically and electrophoretically identical to proteins Ic (U. Henning, W. Schmidmayr, and I. Hindennach, Mol. Gen. Genet. 154:293-298, 1977) and e (W. van Alphen, N. van Selm, and B. Lugtenberg, Mol. Gen. Genet. 159:75-83, 1978). Proteins E, e, and Ic were also immunologically related to E. coli outer membrane protein Ia. Lugtenberg and co-workers (B. Lugtenberg, R. van Boxtel, C. Verhoef, and W. van Alphen, FEBS Lett. 96:99-105, 1978) have shown that electrophoretically identical peptides were generated by cyanogen bromide treatment of proteins E, e, and Ic.
Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium
Authors: Sato T , Yura T
Published: January 2020

The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.
Response of Rhodopseudomonas capsulata to illumination and growth rate in a light-limited continuous culture
Authors: Aiking H , Sojka G
Published: January 2020

Rhodopseudomonas capsulata was grown under anaerobic, photosynthetic conditions in a continuous culture device. Under light limitation, at a constant dilution rate, it was shown that cell composition, including photopigment (bacteriochlorophyll and carotenoids) and ribonucleic acid content, was not affected by incident light intensity; however, steady state culture density varied directly and linearly with light intensity. On the other hand, photopigment and ribonucleic acid levels were affected by growth rate regardless of light intensity. Additional experiments indicated a high apparent Ks for growth of R. capsulata with respect to light. These results were interpreted to mean that near the maximum growth rate (D = 0.45 h-1) some internal metabolic process became the limiting factor for growth, rather than the imposed energy limitation. A mathematical expression for the relation between steady-state culture density and dilution rate was derived and was found to adequately describe the data. A strong correlation was found between continuous cultures limited either by light or by a chemical energy source.
Localization of dolichyl phosphate- and pyrophosphate-dependent glycosyl transfer reactions in Saccharomyces cerevisiae
Authors: Marriott M , Tanner W
Published: January 2020

Membranes from Saccharomyces cerevisiae protoplasts were fractionated on a continuous sucrose gradient. Six bands were obtained, which contained altogether about 15% of the total cell protein. From their densitites, their behavior in the presence and absence of Mg2+ ions, and the distribution of marker enzymes, it was possible to identify fractions enriched in rough and smooth endoplasmic reticulum and in mitochondria. All glycosyl transfer reactions investigated where dolichyl phosphates served as glycosyl acceptors or where dolichyl phosphate- and pyrophosphate-activated sugars served as glycosyl donors showed the highest specific activity and up to 75% of the total activity in the endoplasmic reticulum. This was the case for the reactions involved in the formation of O-glycosidic as well as N-glycosidic linkages in yeast glycoprotein biosynthesis. Membrane fractions enriched in plasmalemma contained less than 3% of the corresponding activities.
Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase
Authors: Hass D , Evans R , Mercenier A , Simon J P , Stalon V
Published: January 2020

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.
Isolation and characterization of Escherichia coli K-12 mutants unable to induce the adaptive response to simple alkylating agents
Authors: Jeggo P
Published: January 2020

When Esherichia coli cells are exposed to a low level of simple alkylating agents, they induce the adaptive response which renders them more resistant to the killing and the mutagenic effects of the same or other alkylating agents. This paper describes the isolation of one strain that was deficient in mutagenic adaptation and five that were deficient in both mutagenic and killing adaptation, confirming previous suggestions that killing and mutagenic adaptation are, at least to some extent, separable. These six strains have been called Ada mutants. They were more sensitive to the killing and mutagenic effects of N-methy-N'-nitro-N-nitrosoguanidine (MNNG) than the unadapted Ada+ parent. Thus, the adaptation pathway is responsible for circumventing some alkylation-induced damage even in cells that are preinduced. The increase in mutation frequency seen in Ada cells treated with MNNG was the same whether the cells were lexA+ or lexA, showing that the extra mutations found in Ada- strains do not depend upon the SOS pathway. Ada strains accumulated more O6-methyl guanine lesions than the Ada+ parent on prolonged exposure to MNNG, and this supports the idea that O6-methyl guanine is the most important lesion for MNNG-induced mutagenesis. The ada mutations have been shown to map in the 47 to 53-min region of the E. coli chromosome.
Nicotinamide adenine dinucleotide metabolism in Candida albicans
Authors: Chaffin W L , Barton R A , Jacobson E L , Jacobson M K
Published: January 2020

The functional pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis and their regulation were studied in the dimorphic fungus Candida albicans. The presence of a functional endogenous pathway of NAD biosynthesis from tryptophan was demonstrated. In addition, nicotinamide served as an efficient salvage precursor for NAD biosynthesis but nicotinate was not utilized. The pathway for nicotinamide utilization involved nicotinate and nicotinate nucleotides as intermediates, suggesting that the failure to utilize nicotinate involves a transport defect. The mechanisms that regulate NAD levels during exponential growth operated to maintain constant NAD levels when NAD biosynthesis occurred exclusively from endogenous or salvage pathways or from a combination of the two. The regulation also operated such that the salvage pathway was preferentially utilized.
Transposon A-generated mutations in the mercuric resistance genes of plasmid R100-1
Authors: Foster T J , Nakahara H , Weiss A A , Silver S
Published: January 2020

A series of 23 transposon 801(Tn801)-induced mutations of plasmid R100-1 from mercuric salts resistance to sensitivity was studied. Although Tn801 transposed frequently into the mer region of the plasmid, fine structural analysis showed that the site of insertion within mer varied. About one-half of the Tn801 insertion events also caused a deletion of greater than 1 megadalton. Genetic and restriction endonuclease EcoRI and BamHI analysis of the mutant plasmid deoxyribonucleic acid elucidated the organization of the mer operon and suggested the existence of a trans-acting regulatory factor governing resistance to mercuric salts. Tn801 insertions leading to mercuric sensitivity occurred in the restriction endonuclease fragments EcoRI-H and EcoRI-I. Regulatory mutations leading to a 50-fold-reduced synthesis of mercuric reductase enzyme occurred in two complementation classes thought to represent the gene for a trans-acting inducer molecule and a cis-acting operator-promoter sequence. Mutations leading to total loss of the enzyme mercuric reductase occurred on both the EcoRI-H and EcoRI-I fragments, showing that the structural gene for this enzyme (merA) bridges the EcoRI cleavage site separating the segments. Hypersensitivity to mercuric salts resulted when Tn801 insertion occurred in the reductase gene in the operatordistal portion of the operon. Hypersensitive cells inducibly bound three to five times more Hg2+ at low concentrations than did sensitive (plasmidless) cells. This finding led to the proposal that another gene (merT) controls uptake of Hg2+ by the cells. Transcription of the operon was deduced to start in the EcoRI-H fragment and to move into the EcoRI-I fragment of the plasmid genome.
Mutants of Anabaena strain CA altered in their ability to grow under nitrogen-fixing conditions
Authors: Gotto J W , Tabita F R , Van Baalen C
Published: January 2020

Mutants of Anabaena strain CA impaired in nitrogenase activity and growth on N2 were isolated and characterized. One mutant was selected for resistance to L-methionine-DL-sulfoximine, and others were selected for resistance to DL-7-azatryptophan or for requirements for combined nitrogen. The mutants varied in sensitivity of growth and nitrogenase activity to atmospheric 02. Several of the mutants whose growth on N2 was impaired under aerobic conditions could grow and reduce acetylene at rates comparable to the wild type when grown microaerobically under N2-CO2 (99:1). The acetylene reduction activity of some of the strains grown under N2-CO2 was immediately and completely lost upon exposure to atmospheric O2, but in at least one strain this loss was reversed when the O2 concentration was lowered, even after 10 h of exposure to air. The characteristics of the O2-sensitive mutants suggest that there may be several sites sensitive to O2 and that the protective mechanism involves several different phenomena.
Defective transport and other phenotypes of a periplasmic "leaky" mutant of Escherichia coli K-12
Authors: Anderson J J , Wilson J M , Oxender D L
Published: January 2020

A mutant of Escherichia coli K-12 deficient in high-affinity leucine transport and related binding proteins was obtained by selecting for azaleucine resistance after bacteriophage Mu mutagenesis. We determined that the cause was a generalized loss of periplasmic binding proteins and a sharp decrease in the activity of transport systems requiring them. Other transport systems resistant to osmotic shock and present in membrane vesicles, were affected to a lesser degree or not at all. The mutation, designated lky::Mucts, was shown to be a pleiotropic envelope mutation, rendering the mutant sensitive to ionic and nonionic detergents, antibiotics, and ethylenediaminetetraacetic acid: the strain had also acquired tolerance to colicins E1, E2, and E3, while remaining normally sensitive to a variety of bacteriophages. An analysis of the lipopolysaccharide of parent and mutant strains revealed a twofold reduction in the neutral sugar content of the core oligosaccharide of the lky strain, but no change in sensitivities to phages which utilize lipopolysaccharide or outer membrane proteins for absorption. The lky::Mucts locus was mapped by transduction and found to be located near, or in, the tolPAB gene cluster linked to gal. Secondary mutations suppressing the detergent sensitivity of lky arose at a frequency of 10(-7), yielding a variety of new phenotypes. The lky::Mucts mutation did not give rise to obvious alterations in the gross morphology of the cell or in cell division.
Regulation of Phosphate Accumulation in the Unicellular CyanobacteriumSynechococcus
Authors: Grillo John F. , Gibson Jane
Published: January 2020

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acid-insoluble polyphosphate were lowered inSynechococcusin proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10% of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparentKmof about 0.5 μM, butVmaxwas 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 15°C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity.
Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation
Authors: Pato M L
Published: January 2020

Inhibition of ribonucleic acid synthesis in Escherichia coli 15 TAU bar with rifampin or streptolydigin leads to large increases in the sizes of cellular ribonucleoside and deoxyribonucleoside triphosphate pools. Inhibition of protein synthesis leads to increases in the sizes of all nucleoside triphosphate pools except the guanosine triphosphate and deoxyguanosine triphosphate pools; a decrease in the size of the latter pool may be responsible for the slowing of deoxyribonucleic acid replication fork movement observed in this strain in the absence of protein synthesis. Analysis of the kinetics of incorporation of labeled precursors into deoxyribonucleic acid and into cellular pools suggests that functional compartmentation of nucleotide pools exists, allowing the incorporation of exogenously supplied precursors into deoxyribonucleic acid without prior equilibration with the cellular pools.
Chemotaxis of Bdellovibrio bacteriovorus toward pure compounds
Authors: Straley S C , LaMarre A G , Lawrence L J , Conti S F
Published: January 2020

Positive chemotaxis by Bdellovibrio bacteriovorus strain UKi2 was measured for 139 compounds. Twenty-one compounds were attractants; sensitive attraction was elicited by acetate, propionate, thioacetate, malonate, cis-oxalacetate, D-glucose-6-phosphate, acetyl coenzyme A, ammonium ion, barium ion, manganous ion, and potassium ion. Several of the attractants for B. bacteriovorus strain UKi2 also were attractants to strains 6-5-S and 114; however, strains 109D and 109J were not attracted by the compounds tested. Of 33 compounds tested, 8 were repellents for B. bacteriovorus strain UKi2: n-caproate, alanine, isoleucine, leucine, phenylalanine, tyrosine, cobaltous chloride, and hydronium ion. None of the organic repellents for strain UKi2 elicited repulson from strains 114 or 109D. However, all three strains of Bdellovibrio show aerotaxis. Several compounds were tested for their effects on viability and predacious growth of B. bacteriovorus strain UKi2. No simple correlation was found between attraction or repulsion and benefit or harm to bdellovibrios. The data are consistent with the view that in nature, the greatest survival value of chemotaxis for bdellovibros may be in aerotaxis, attraction to certain inorganic ions and acetate, and repulsion by hydronium ion.
Regulation and solubilization of Candida albicans chitin synthetase
Authors: Braun P C , Calderone R A
Published: January 2020

A cytoplasmic component which inhibited the activation of chitin synthetase was studied in the dimorphic fungus Candida albicans. The inhibitor was found to be heat stable and trypsin sensitive and was only effective when incubated with a vacuolar protease, an activator of chitin synthetase, before the activation of chitin synthetase. In addition, the particulate chitin synthetase from the yeast form of C. albicans was solubilized by a sodium cholate-digitonin extraction and subsequently was purified approximately 30-fold by Sepharose column chromatography and Amicon XM 100 filtration. Activity of the soluble enzyme was increased by the addition of trypsin or phosphatidyl serine. The molecular weight of the enzyme was estimated to be 400,000.
Evolution of multiple-antibiotic-resistance plasmids mediated by transposable plasmid deoxyribonucleic acid sequences
Authors: Rubens C E , McNeill W F , Farrar W E
Published: January 2020

Two plasmid deoxyribonucleic acid sequences mediating multiple antibiotic resistance transposed in vivo between coexisting plasmids in clinical isolates of Serratia marcescens. This event resulted in the evolution of a transferable multiresistance plasmid. Both sequences, designated in Tn1699 and Tn1700, were flanked by inverted deoxyribonucleic acid repetitions and could transpose between replicons independently of the Excherichia coli recA gene function. Tn1699 and Tn1700 mediated ampicillin, carbenicillin, kanamycin, and gentamicin resistance but differed in the type of gentamicin-acetyltransferase enzymes that they encoded. The structural genes for these enzymes share a great deal of polynucleotide sequence similarity despite their phenotypic differences. The transposition of Tn1699 and Tn1700 to coresident transferable plasmids has contributed to the dissemination of antibiotic resistance among other gram-negative bacteria. These organisms have recently caused nosocomial infections in epidemic proportions.
Local anesthetics block induction of the Pseudomonas alk regulon
Authors: Benson S A
Published: January 2020

The local anesthetics procaine and piperocaine blocked induction of the plasmid-determined enzymatic activities involved in the metabolism of n-alkanes in Pseudomonas putida. Procaine reversibly inhibited existing alkane hydroxylase activity. Induction of a soluble aliphatic amidase activity was not affected. These results support the hypothesis that induction of the plasmid-determined alkane metabolic system in P. putida involves a membrane component(s).
Lactose metabolism in Streptococcus lactis: phosphorylation of galactose and glucose moieties in vivo
Authors: Thompson J
Published: January 2020

Starved cells of Streptococcus lactis ML3 grown previously on lactose, galactose, or maltose were devoid of adenosine 5'-triphosphate contained only three glycolytic intermediates: 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate (PEP). The three metabolites (total concentration, ca 40 mM) served as the intracellular PEP potential for sugar transport via PEP-dependent phosphotransferase systems. When accumulation of [14C]lactose by iodoacetate-inhibited starved cells was abolished within 1 s of commencement of transport, a phosphorylated disaccharide was identified by autoradiography. The compound was isolated by ion-exchange (borate) chromatography, and enzymatic analysis showed that the derivative was 6-phosphoryl-O-beta-D-galactopyranosyl (1 leads to 4')-alpha-D-glucopyranose (lactose 6-phosphate). After maximum lactose uptake (ca. 15 mM in 15 s) the cells were collected by membrane filtration and extracted with trichloroacetic acid. Neither free nor phosphorylated lactose was detected in cell extracts, but enzymatic analysis revealed high levels of galactose 6-phosphate and glucose 6-phosphate. The starved organisms rapidly accumulated glucose, 2-deoxy-D-glucose, methyl-beta-D-thiogalactopyranoside, and o-nitrophenyl-beta-D-galactopyranoside in phosphorylated form to intracellular concentrations of 32, 32, 42, and 38.5 mM, respectively. In contrast, maximum accumulation of lactose (ca. 15 mM) was only 40 to 50% that of the monosaccharides. From the stoichiometry of PEP-dependent lactose transport and the results of enzymatic analysis, it was concluded that (i) ca. 60% of the PEP potential was utilized via the lactose phosphotransferase system for phosphorylation of the galactosyl moiety of the disaccharide, and (ii) the residual potential (ca. 40%) was consumed during phosphorylation of the glucose moiety.
Escherichia coli mutants incapable of supporting replication of F-like plasmids at high temperature: isolation and characterization of mafA and mafB mutants
Authors: Wada C , Yura T
Published: January 2020

Mutants of Escherichia coli K-12 defective in replication of F-like plasmids at a high temperature (42 degrees C) were found among threonine-independent (Thr+) revertants of a threonine-requiring F' stain after localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Transduction experiments with phage P1 permitted us to divide these mutations into two classes with respect to man location; some mutations were located between thr and ara at about 0.8 min, very close to maf-1 reported previously (Wada et al., J. Mol. Biol. 108:25-41, 1976 and the others probably were located between leu and azi at about 1.8 min. The former class of mutants designated mafA exhibited the same plasmid specificity as maf-1; replication of plasmids F and ColVB trp, but not R386 or R222, were affected at a high temperature. By contrast, the latter mutants designated mafB were defective in replication of nay of these plasmids at a high temperature. When a culture of mafA mutants carrying an F' plasmid was transferred from 30 to 42 degrees C, the plasmid replication as determined by incorporation of [3H]thymidine into covalently closed circular F DNA was markedly inhibited. Under certain conditions, the temperature shift-up caused severe growth inhibition of the mutant cells. Examination of merodiploids (mafA/FmafA+) for plasmid maintenance suggested that the two mafA mutations tested (mafA23 and mafA36) were both dominant, at least partially, over the wild-type mafA+ allele. These properties of the mafA mutants, manifested at the restrictive temperature, are similar to those previously reported for the maf-1 mutant. Taken together with other evidence it is likely that these mutations affect either the same gene (mafA) or a set of closely linked genes, playing a specific role in autonomous plasmid replication in E. coli.
Cytochrome c3 from the sulfate-reducing anaerobe Desulfovibrio africanus Benghazi: purification and properties
Authors: Singleton R , Campbell L L , Hawkridge F M
Published: January 2020

Cytochrome c3 was purified from Desulfovibrio africanus Benghazi by extraction with alkaline deoxyribonuclease, fractionation with ammonium sulfate, batch elution from carboxymethyl Sephadex followed by chromatography on the same resin, and gel filtration on Sephadex G-75. The preparation was judge homogeneous by a variety of criteria. The molecular weight was determined in an analytical ultracentrifuge, and values between 14,400 and 15,490 were obtained, depending upon the presumed value of partial specific volume. Gel filtration on a calibrated column of Sephadex G-75 gave a value of 14,900 daltons. The amino acid composition was very similar to that observed for the cytochrome from other species of Desulfovibrio, with the exception of increased levels of ThR and PhE. S-Carboxymethylation of the protein before and after heme removal by HgCl2 demonstrated eight Cys molecules involved in heme binding or four heme sites per molecule. Titration with sodium dithionite under N2 gave an electrochemical potential (E' 0) of -276 mV relative to the normal hydrogen electrode. Electrochemical titration of the cytochrome gave a Nernst plot with two linear regions with E' 0 values of -0.376 and -0.534 V. The spectra produced at various potentials exhibited shifts in isosbestic points upon reduction, suggesting changes in conformation during the reaction.
Control of Plasmid R1 Replication: Kinetics of Replication in Shifts Between Different Copy Number Levels
Authors: Gustafsson Petter , Nordström Kurt
Published: January 2020

Plasmid R1 replication was studied in shifts between two steady states of copy number. The copy number was varied in two ways. First, we utilized the fact that it decreases with increasing growth rate. To minimize the metabolic effects of changes in the growth rate, the downshifts were obtained by adding α-methylglucoside to cultures growing in glucose-minimal medium, and the upshifts were obtained by adding glucose to cultures growing in the presence of glucose plus α-methylglucoside. Second, we used a temperature-dependent copy mutant of plasmid R1 (pKN301). Plasmid pPK301 shows a threefold higher copy number at 40 than at 30°C. In both types of shift, plasmid replication immediately adjusted to the postshift differential rate. The copy number asymptotically adjusted to the new steady state. Hence, the system that controls plasmid R1 replication sets the frequency of replication without measuring the actual copy number. It has been suggested that plasmid R1 replication is under negative control by an R1-mediated repressor protein. Among the replication control models that involve negative control, the Pritchard inhibitor dilution model, the Sompayrac-Maaløe autorepressor model, and the plasmid λ
dv
system all predict gene dose-independent copy number control.
Bacillus cereus autolytic endoglucosaminidase active on cell wall peptidoglycan with N-unsubstituted glucosamine residues
Authors: Kawagishi S , Araki Y , Ito E
Published: January 2020

An autolytic glycosidase from a lysozyme-resistant strain of Bacillus cereus capable of cleaving the glycosidic linkages of N-unsubstituted glucosamine in the cell wall peptidoglycan was studied. This glycosidase activity, together with N-acetylmuramyl-L-alanine amidase activity, was found in an autolytic enzyme preparation obtained from the 20,000 x g precipitate fraction by means of autolysis followed by ammonium sulfate fractionation. The major saccharide fragments resulting from digestion of the untreated, non-N-acetylated, cell wall peptidoglycan of B. cereus with the autolytic enzyme preparation were identified as N-acetylmuramyl-glucosamine and its dimer. The peptidoglycan N-acetylated with acetic anhydride could also be digested with the same enzyme preparation, giving N-acetylmuramyl-N-acetylglucosamine and its dimer as the major saccharide fragments.
Chemical Analysis of Major Outer Membrane Proteins ofNeisseria meningitidis: Comparison of Serotypes 2 and 11
Authors: Tsai Chao-Ming , Frasch Carl E.
Published: January 2020

Most of the 15 protein serotypes found in group BNeisseria meningitidishave distinct major outer membrane protein patterns when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the Weber-Osborn system. Both serotypes 2 and 11 contain major outer membrane proteins with apparent molecular weights of 41,000 and 28,000 (41K and 28K). The 41K and 28K proteins were purified from the prototype strains of these two serotypes (M986 type 2 and M136 type 11) by preparative slab SDS-PAGE and were chemically characterized. No hexosamine was found in the purified 41K and 28K proteins. Although the two 41K proteins had similar amino acid compositions, their mobilities in Laemmli SDS-PAGE and their fragmentation patterns on SDS-PAGE after cyanogen bromide cleavage were different. The two 28K proteins differed in their amino acid composition, mobilities in Laemmli SDS-PAGE, and cyanogen bromide cleavage products. Peptide maps following chymotrypsin digestion of radioiodinated 41K and 28K proteins revealed distinct peptide maps for all four proteins. Comparison of the peptide maps of two 41K or two 28K proteins indicated that most of the unique peptides were hydrophilic, whereas most of the common peptides were hydrophobic. These results indicated that both of the 41K proteins and the 28K proteins from serotypes 2 and 11 were chemically different, although the proteins having the same molecular weights appeared to share common peptides.
Escherichia coli mutants with altered control of alcohol dehydrogenase and nitrate reductase
Authors: Clark D , Cronan J E
Published: January 2020

Mutants of Escherichia coli which overproduce alcohol dehydrogenase were obtained by selection for the ability to use ethanol as an acetate source in a strain auxotrophic for acetate. A mutant having a 20-fold overproduction of alcohol dehydrogenase was able to use ethanol only to fulfill its acetate requirement, whereas two mutants with a 60-fold overproduction were able to use ethanol as a sole carbon source. The latter two mutants produced only 25% of the wild-type level of nitrate reductase, when grown under anaerobic conditions. Alcohol dehydrogenase production was largely unaffected by catabolite repression but was repressed by nitrate under both aerobic and anaerobic conditions. The genetic locus responsible for alcohol dehydrogenase overproduction was located at min 27 on the E. coli genetic map; the gene order, as determined by transduction, was trp tonB adh chlC hemA. The possible relationship of alcohol dehydrogenase to anaerobic redox systems such as formate-nitrate reductase is discussed.
Iron reductases from Pseudomonas aeruginosa
Authors: Cox C D
Published: January 2020

Cell-free extracts of Pseudomonas aeruginosa contain enzyme activities which reduce Fe(III) to Fe(II) when iron is provided in certain chelates, but not when the iron is uncomplexed. Iron reductase activities for two substrates, ferripyochelin and ferric citrate, appear to be separate enzymes because of differences in heat stabilities, in locations in fractions of cell-free extracts, in reductant specificity, and in apparent sizes during gel filtration chromatography. Ferric citrate iron reductase is an extremely labile activity found in the cytoplasmic fraction, and ferripyochelin iron reductase is a more stable activity found in the periplasmic as well as cytoplasmic fraction of extracts. A small amount of activity detectable in the membrane fraction seemed to be loosely associated with the membranes. Although both enzymes have highest activity reduced nicotinamide adenine dinucleotide, reduced glutathione also worked with ferripyochelin iron reductase. In addition, oxygen caused an irreversible loss of a percentage of the ferripyochelin iron reductase following sparge of reaction mixtures, whereas the reductase for ferric citrate was not appreciably affected by oxygen.
Physiological control of phosphorylation ribosomal protein S6 in Mucor racemosus
Authors: Larsen A , Sypherd P S
Published: January 2020

The level of phosphorylation of ribosomal protein S6 increased with accelerating rates of growth and protein synthesis in Mucor racemosus. Lowered levels of phosphorylation were seen under conditions of metabolic shift-down or the onset of stationary phase, and no phosphorylation was detected in sporangiospores. Changing metabolic states, changing intracellular levels of adenosine triphosphatase, and the level of phosphorylation of protein S6 were correlated in M. racemosus.
Genetic relationship between Treponema pallidum and Treponema pertenue, two noncultivable human pathogens
Authors: Miao R M , Fieldsteel A H
Published: January 2020

Genetic relationships among two strains of Treponema pallidum (Nichols and KKJ) and a strain of T. pertenue were determined by measuring the degree of deoxyribonucleic acid sequence homology. The results in indicated that these three virulent, noncultivable treponemes were genetically indistinguishable. Like T. pallidum (Nichols), T. pertenue (Gauthier) had no detectable deoxyribonucleic acid sequence homology with T. phagedenis (biotype Reiter), T. refringens (biotype Noguchi), or with salmon sperm.
Carbon monoxide:methylene blue oxidoreductase from Pseudomonas carboxydovorans
Authors: Meyer O , Schlegel H G
Published: January 2020

The enzyme carbon monoxide:methylene blue oxidoreductase from CO autotrophically grown cells of Pseudomonas carboxydovorans strain OM5, was purified to homogeneity. The enzyme was obtained in 26% yield and was purified 36-fold. The enzyme was stable for at least 6 days, had a molecular weight of 230,000, gave a single protein and activity band on polyacrylamide gel electrophoresis, and was homogeneous by the criterion of sedimentation equilibrium. Sodium dodecyl sulfate gel electrophoresis revealed a single band of molecular weight 107,000. Carbon monoxide:methylene blue oxidoreductase did not catalyze reduction of pyridine or flavin nucleotides but catalyzed the oxidation of CO to CO2 in the presence of methylene blue, thionine, toluylene blue, dichlorophenolindophenol, or pyocyanine under strictly anaerobic conditions. The visible spectrum revealed maxima at 405 and 470 nm. The millimolar extinction coefficients were 43.9 (405 nm) and 395.5 (275 nm), respectively. Absorption at 470 nm decreased in the presence of dithionite, and the spectrum was not affected by the substrate CO. Maximum reaction rates were found at pH 7.0 and 63 degrees C; temperature dependence followed the Arrhenius equation, with an activation energy (delta H degree) of 36.8 kJ/mol (8.8 kcal/mol). The apparent Km was 53 microM for CO. The purified enzyme was incapable of oxidizing methane, methanol, or formaldehyde in the presence of methylene blue as electron acceptor.
Four proteins synthesized in response to deoxyribonucleic acid damage in Micrococcus radiodurans
Authors: Hansen M T
Published: January 2020

Four proteins, alpha beta, gamma, and delta, preferentially synthesized in ultraviolet light-treated cells of Micrococcus radiodurans, were characterized in terms of their molecular weights and isoelectric points. Within the sublethal-dose range, the differential rate of synthesis for these proteins increased linearly with the inducing UV dose. The degree of induction reached 100-fold, and the most abundant protein beta, amounted to approximately 2% of the total newly synthesized protein after irradiation. Damage caused by ionizing radiation or by treatment with mitomycin C also provoked the synthesis of the four proteins. The proportions between the individual proteins, however, varied strikingly with the damaging agent. In contrast to treatments which introduced damage in the cellular deoxyribonucleic acid, the mere arrest of deoxyribonucleic acid replication, caused by nalidixic acid or by starvation for thymine, failed to elicit the synthesis of either protein. Repair of deoxyribonucleic acid damage requires that a number of versatile and efficient processes by employed. It is proposed that the induced proteins participate in deoxyribonucleic acid repair in M. radiodurans. Mechanisms are discussed which would allow a differentiated cellular response to damages of sufficiently distinctive nature.