bluuebunny/crossref_metadata_2025 huggingface dataset:
RNA Synthesis inYersinia pestisDuring Growth Restriction in Calcium-Deficient Medium
Authors: Charnetzky Willard T. , Brubaker Robert R.
Published: January 2020

Yersinia pestisrequires 2.5 mM Ca2+for growth at 37°C but not at 26°C. After a shift from 26 to 37°C in a Ca2+-deficient medium, an ordered series of metabolic alterations occur which result in transition from a growing cell to a viable but non-proliferating cell. The earliest known alteration in normal metabolism associated with this transition is a termination of net RNA synthesis. Competitive RNA/DNA hybridizations with uniformly labeled RNA and stable RNA competitor indicated identical mRNA to stable RNA ratios in growing cells and non-proliferating Ca2+-deprived cells. Similar hybridizations with pulse-labeled RNA demonstrated that growing cells synthesized 57% mRNA, 37% rRNA, and 5% tRNA, whereas Ca2+-deprived cells synthesized 95% mRNA, 4.7% rRNA, and 0.7% tRNA. After addition of radioactive uracil and rifampin to growing and Ca2+-deprived cells, decay of approximately 40 and 90% of the newly synthesized RNA was found for growing and Ca2+-deprived cells, respectively. The half-life of the mRNA was found to be 1.5 min for growing cells and 4.5 min for Ca2+-deprived cells.Y. pestiselicited increases in the levels of guanosine tetraphosphate and guanosine pentaphosphate in response to amino acid deprivation and yielded transient increases in the levels of these phosphorylated nucleotides after a shift from 26 to 37°C. These increases were independent of Ca2+availability and preceded the alteration in RNA synthesis by more than 1 h. The levels of these phosphorylated nucleotides then stabilized at about 80 and 40 pmol for Ca2+-deprived and Ca2+-supplemented cultures, respectively, and did not increase further in the Ca2+-deprived culture at the time corresponding to the reduction in stable RNA synthesis. These findings indicate that the early lesion in RNA synthesis associated with the growth restriction of Ca2+-deprivedY. pestisreflects a block in stable RNA synthesis and that this effect is not mediated by guanosine tetraphosphate or guanosine pentaphosphate.
Isolation of a stable enzyme.14CO2.Mg2+.carboxyarabinitol bisphosphate complex with ribulosebisphosphate carboxylase from Chromatium vinosum
Authors: Brown H M , Chollet R
Published: January 2020

Higher plant-type ribulosebisphoshate carboxylase from Chromatium vinosum formed a stable, nonexchangeable complex with activator 14CO2 in the presence of Mg2+ and 2-carboxyarabinitol bisphosphate, an analog of the proposed transition-state intermediate. The response of the procaryotic enzyme to this analog was indistinguishable from that of the higher-plant carboxylase, which should permit comparative analysis of the activator site amino acid sequence in the two proteins.
Utilization of a thermosensitive episome bearing transposon TN10 to isolate Hfr donor strains of Erwinia carotovora subsp. chrysanthemi
Authors: Kotoujansky A , Lemattre M , Boistard P
Published: January 2020

A thermosensitive episome bearing the transposon Tn10, F(Ts)::Tn10 Lac+, has been successfully transferred from Escherichia coli to several wild strains of the enterobacteria Erwinia carotovora subsp. chrysanthemi, which are pathogenic on Saintpaulia ionantha. In one of these strains, all of the characters controlled by this episome (Lac+, Tetr, Tra+) were expressed, and its replication was stopped at 40 degrees C and above. At 30 degrees C, the episome was easily transferred between strains derived from E. carotovora subsp. chrysanthemi 3937j and to E coli. Hfr donor strains were obtained from a F' strain of 3937j by selecting clones which grew at 40 degrees C on plates containing tetracycline. One of these strains, Hfrq, was examined in more detail: the characters Lac+ and Tetr were stabilized and did not segregate higher than its parental F' strain. The mating was most efficient at 37 degrees C on a membrane. Hfrq transferred its chromosome to recipient strains at high frequency and in a polarized fashion, as evidenced by the gradient of transfer frequencies, the kinetics of marker entry (in interrupted mating experiments), and the analysis of linkage between different markers. The chromosome of Hfrq was most probably transferred in the following sequence: origin...met...xyl...arg...ile...leu...thr...cys...pan...ura...gal...trp...his. ..pur... Moreover, this genetic transfer system proved to be efficient in strain construction.
Another gene affecting sexual expression of Escherichia coli
Authors: Lerner T J , Zinder N D
Published: January 2020

We have examined the relationship between two chromosomal mutations of Escherichia coli K-12, fexA (0 min) and fexB (85 min), in regulating expression of the F sex factor. Together, fexA and fexB exert a pleiotropic effect on the expression of the F tra genes. F pilus synthesis, conjugal donor activity, and surface exclusion activity are all inhibited in the fexA fexB mutant. Either fex mutation alone is cryptic.
Complex glnA-glnL-glnG operon of Escherichia coli
Authors: Pahel G , Rothstein D M , Magasanik B
Published: January 2020

The glnG gene product is both a positive regulator and a negative regulator of the expression of glnA, the structural gene for glutamine synthetase, as well as a positive regulator of the expression of a number of genes whose products are involved in the uptake and degradation of nitrogen-containing compounds. The regulation of beta-galactosidase in various strains containing a Mu d1 (lac bla) insertion within glnG leads to the following conclusions regarding the expression of this gene: (i) like the synthesis of glutamine synthetase, the synthesis of the glnG product is regulated in response to the nitrogen source; (ii) high-level expression of glnG under nitrogen-limiting growth conditions depends on transcription initiated at the glnA promoter; and (iii) there is a second, glnA-distal promoter for glnG, whose activity is negatively controlled by the glnG product. Thus, the glnG product regulates the synthesis of the glnG product at two distinct promoters (positively and negatively at the glnA promoter and negatively at the glnA-distal promoter). In addition, a high level of glnG product, corresponding to the level produced by initiation of transcription at the glnA promoter under nitrogen-limiting conditions, is necessary for activation of histidase synthesis. The lower level of glnG product originating from transcription initiated at the glnA-distal promoter is not sufficient to activate histidase synthesis, but is sufficient to activate fully and to repress glnA expression.
Characterization of a gene, glnL, the product of which is involved in the regulation of nitrogen utilization in Escherichia coli
Authors: Chen Y M , Backman K , Magasanik B
Published: January 2020

DNA was prepared from a strain of Escherichia coli bearing a mutation which confers the GlnC phenotype (inability to reduce the expression of glnA and other nitrogen-regulated operons in response to ammonia in the growth medium). A fragment of this DNA carrying glnA, the structural gene for glutamine synthetase, was cloned on plasmid pBR322. By using recombination in vitro, we mapped the GlnC mutation to a region between glnA and glnG. This region defines a gene, glnL, which codes for a trans-acting product; the GlnC mutant produces an altered product. The glnL product plays a key role in the communication of information concerning the quality and abundance of the nitrogen source in the growth medium to a destination responsible for the regulation of glnA and other genes for enzymes responsible for nitrogen utilization.
Isolation of a trisaccharide containing 2-amino-2-deoxy-D-galacturonic acid from the Bordetella pertussis endotoxin
Authors: Moreau M , Chaby R , Szabo L
Published: January 2020

4-O-(2-Amino-2-deoxy-alpha-D-glucopyranosyl-6-O-(2-amino-2-deoxy-alpha-D-galactopyranuronyl)-D-glucopyranose, a branched-chain trisaccharide, was isolated after hydrolysis of Bordetella pertussis endotoxin with 4 M HCl for 1 h at 100 degrees C. The trisaccharide was present in both polysaccharide moieties of the two constituent lipopolysaccharides of this endotoxin. Its structure was established by analysis of the 400-MHz nuclear magnetic resonance spectrum and by chemical and enzymatic degradation.
Novel peptidoglycans in Caulobacter and Asticcacaulis spp
Authors: Poindexter J S , Hagenzieker J G
Published: January 2020

Peptidoglycan sacculi free of poly-beta-hydroxybutyric acid were prepared from whole cells of four species of Caulobacter and two species of Asticcacaluis and from morphological mutants of Caulobacter crescentus and Caulobacter leidyi. Acid hydrolysates of the sacculi were analyzed quantitatively, and each of the hydrolysates was found to contain significant amounts of only five ninhydrin-reactive compounds: alanine, glutamic acid, alpha , omega-diaminopimelic acid, muramic acid, and glucosamine. Four types of peptidoglycans were distinguishable on the basis of the molar ratios among these five compounds. The respective ratios were as follows: in C. leidyi, 2:1:1:1:0.8; in Asticcacaulis biprosthecum, 1.7:1.6:1.1:0.7; in the cells of the remaining species, 2:1:1:1.2:0.8; and in stalks shed by the abscission mutant 2NY66, 2:1:1:1:1.67. Thus, in addition to some species differences among these caulobacters, it was found that the peptidoglycan sacculus of the stalked C. crescentus cell is chemically differentiated; the cellular peptidoglycan is richer in muramic acid than is the peptidoglycan of typical gram-negative bacteria, and the peptidoglycan of the stalk is correspondingly rich in glucosamine. Empirical formulas for the repeating units of the peptidoglycans have been inferred on the basis of the molar ratios of their amino components.
Production and composition of an exocellular Nigeran-protein complex isolated from cultures of Aspergillus awamori
Authors: Bobbitt T F , Nordin J H
Published: January 2020

Aspergillus awamori Nakazawa (QM873) hyphae maintained on a nitrogen-deficient medium produced an exocellular complex consisting of the wall alpha-glucan, nigeran (94%), water-soluble nigeran oligosacchrides (1 to 2%), protein (2 to 4%), and a small amount of beta-glycan (less than 1%). Nigeran was not covalently linked to protein. The complex appeared in the growth medium only under those temporal or nutritional conditions where the hyphal wall content to nigeran reached at least 30% of the cell dry weight. Rates of nigeran accumulation in the hypha cell wall, scanning electron microscopy of hyphae, and pulse-chase experiments with [14C]glucose suggested that the complex arises via displacement of a portion of the hyphal wall into the medium. The nigeran portion of the complex contained lamellar crystalline domains similar to those in the intact cell wall. Enzymic digestion of nigeran in the complex indicated that it has a degree of crystallinity lower than that observed with pure nigeran crystals grown in vitro. Therefore, polymerization in vivo resulted in somewhat less chain organization in the crystallite. Since this complex was isolated without any prior chemical or exogenous enzymatic treatment, it should be useful for studies of hyphal wall biogenesis and organization in this organism.
Genetic transformation of Rhizobium leguminosarum by plasmid DNA
Authors: Bullerjahn G S , Benzinger R H
Published: January 2020

We demonstrated the genetic transformation of Rhizobium leguminosarum by R68.45 plasmid DNA by freezing and thawing cell suspensions in the presence of R68.45 plasmid DNA and 20 mM MgCl2. Clones resistant to kanamycin and tetracycline were recovered at a frequency of 10(-8) per recipient cell. No colonies that were doubly drug resistant were recovered in parallel control experiments.
Determining the phoM map location in Escherichia coli K-12 by using a nearby transposon Tn10 insertion
Authors: Wanner B L , Bernstein J
Published: January 2020

A phoR strain was constructed with transposon Tn10 inserted near the phoM+ locus. This was done without any prior knowledge of the phoM map location. Subsequently, we defined the phoM map position by screening tetracycline-sensitive (Tcs) derivatives for mutants which were both alkaline phosphatase negative (ther phoR phoM double mutant phenotype) and auxotrophic simultaneously. Some of these mutants were Thr-. Bacteriophage P1-mediated transductions were used to confirm that phoM and its nearby Tn10 insertion were closely linked to thr. Unexpectedly, 7 of 10 mutants analyzed also had mutations unlinked to the phoM-thr-Tn10 region. These may represent a new type of Tn10-promoted molecular event which is caused by transposition of a Tn10 end (IS10).
Transport and incorporation of N-acetyl-D-glucosamine in Bacillus subtilis
Authors: Mobley H L , Doyle R J , Streips U N , Langemeier S O
Published: January 2020

Bacillus subtilis 168 has been found to possess a high-affinity transport system for N-acetyl-D-glucosamine (GlcNAC). The Km for uptake was approximately 3.7 microM GlcNAc, regardless of the nutritional background of the cells. Apparent increases in Vmax were noted when the bacteria were grown in the presence of GlcNAc. The uptake of GlcNAc by B. subtilis was highly stereoselective; D-glucose, D-glucosamine, N-acetyl-D-galactosamine, D-galactose, D-mannose, and N-acetylmuramic acid did not inhibit GlcNAc uptake. In contrast, glycerol was an effective inhibitor of [3H]GlcNAc transport and incorporation. Partial inhibition of GlcNAc uptake was observed with azide, fluoride, and cyanide anions, carbonyl cyanide-m-chlorophenyl hydrazone, methyltriphenylphosphonium bromide, N,N'-dicyclohexylcarbodiimide, gramicidin, valinomycin, monensin, and nigericin. Two anions, arsenite and iodoacetate, were potent inhibitors of the uptake of GlcNAc in B. subtilis. Results from paper chromatography showed that there was no intracellular pool of free GlcNAc and that the acetylamino sugar was probably phosphorylated during transport. A modification of the Park-Hancock cell fractionation scheme indicated that cells grown on glycerol or D-glucose incorporated [3H]GlcNAc primarily into the cell wall fraction. When GlcNAc was used as the sole carbon source, label could be demonstrated in fractions susceptible to protease and nuclease, as well as lysozyme, showing that the N-acetylamino sugar was utilized in macromolecular synthesis and energy metabolism.
Relationship between cell death and altered lipid A synthesis in a temperature-sensitive lethal mutant of Salmonella typhimurium that is conditionally defective in 3-deoxy-D-manno-octulosonate-8-phosphate synthesis
Authors: Rick P D , Young D A
Published: January 2020

The relationship between the inability to synthesize a complete 3-deoxy-D-manno-octulosonate region of lipopolysaccharide and cell death was investigated in a temperature-sensitive lethal mutant of Salmonella typhimurium. The defect in lipopolysaccharide synthesis is due to a mutation in the structural gene for 3-deoxy-D-manno-octulosonate-8-phosphate synthetase (designated kdsA) and results in the synthesis of a temperature-sensitive enzyme. Expression of the kdsA lesion at elevated temperatures, at which the synthesis of 3-deoxy-D-manno-octulosonate is complete blocked, is required for expression of the temperature-sensitive lethal phenotype. However, the defect in lipopolysaccharide synthesis is not alone sufficient cause for the observed cell death. Genetic evidence if presented which indicates that the mutant possesses a second mutation, or possibly multiple mutations, whose lethal expression is dependent on the inability of the mutant to synthesize a fully acylated and 3-deoxy-D-manno-octulosonate-substituted lipid A portion of lipopolysaccharide at elevated temperatures.
Biosynthesis of Peptidoglycan in
Gaffkya homari
: Reactivation of Membranes by Freeze-Thawing in the Presence and Absence of Walls
Authors: Kalomiris Efstathia , Bardin Claudette , Neuhaus Francis C.
Published: January 2020

The reactivation of membranes from
Gaffkya homari
for the synthesis of sodium dodecyl sulfate-insoluble peptidoglycan (SDS-insoluble PG) was achieved by successive cycles of freeze-thawing (− 196 versus 25°C). The presence of
G. homari
walls during this process affected the synthesis of both SDS-soluble (nascent) and SDS-insoluble PG. At two cycles the synthesis of SDS-soluble PG decreased by 70%, whereas that of SDS-insoluble PG increased sevenfold when compared with membranes reactivated in the absence of walls but assayed in the presence of walls. Moreover, at six cycles the lag time for the synthesis of SDS-insoluble PG decreased from 15 min to 5 to 7 min. Walls from
G. homari
could not be replaced with walls from
Bacillus megaterium
or cellulose. In addition to these effects, the presence of walls from
G. homari
or
B. megaterium
or of cellulose during the incubation of membranes freeze-thawed in the absence of walls increased twofold the amount of SDS-insoluble PG. Reactivated membranes showed greater sensitivities to penicillin (an inhibitor of
dd
-carboxypeptidase) and
d
-methionine (an inhibitor of
ld
-carboxypeptidase) than did isolated membrane-walls. The percentage of cross-linking of the SDS-insoluble PG synthesized by the reactivated system was 34%, a value similar to that observed for the polymer synthesized by isolated membrane-walls. Freeze-thawing membranes and walls together gave a complex with a density different from that of either membranes or walls. Thus, the assembly system for the synthesis and processing of PG was reconstituted in a complex of membranes and walls prepared from the isolated components. Whether this complex has the exact interrelationship between membrane and wall found in the organism has not been established.
Co-curing of plasmids affecting killer double-stranded RNAs of Saccharomyces cerevisiae: [HOK], [NEX], and the abundance of L are related and further evidence that M1 requires L
Authors: Sommer S S , Wickner R B
Published: January 2020

We describe two sets of plasmid-plasmid interactions in the yeast Saccharomyces cerevisiae. [HOK], [EXL], [NEX], and [KIL-k1] are genetically defined plasmids, and M1 and L are biochemically defined double-stranded RNA plasmids. We show that (i) [HOK], [NEX], and the abundance of L are related, and (ii) under submaximal curing conditions, all colonies retaining M1 also retain L. There are three pieces of evidence that either [NEX] required [HOK] for replication or [NEX] and [HOK] are on the same plasmid. The evidence is as follows. (i) The great majority of strains containing [HOK] also contain [NEX]. However, two [HOK] [NEX-o] strains do exist. (ii) Growth at 39 degrees C or growth at 34 degrees C with 3% ethanol or 2-propanol cures [HOK] and [NEX]. In a [HOK] [NEX] strain, the two plasmids are always co-cured. (iii) [HOK] and [NEX] are both maintained in mak4, mak6, and mak27 strains (mak = maintenance of [KIL-k1]), but not in mak3, mak10, and pet18 strains. Strains containing [HOK] and [NEX] have about fourfold more L double-stranded RNA than their isochromosomal, cured derivatives. In addition, a cytoductant which has acquired [HOK] and [NEX] has fourfold more L than its parent. These results are consistent with either [HOK] being a form of L or [HOK] increasing the copy number of L. Using a K1 killer strain in which L, as well as M1, could be cured by growth at 38 degrees C, we examined the distribution of loss of M1 and L under conditions giving 98% M-o colonies and at least 50% L-o colonies. No M1L-o colonies were observed, supporting the previous suggestion by others that M1 requires L.
Isolation and characterization of Phycomyces blakesleeanus ferritin
Authors: LaBombardi V J , Pisano M A , Klavins J V
Published: January 2020

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.
Role of the catabolite activator protein in the maltose regulon of Escherichia coli
Authors: Chapon C
Published: January 2020

The maltose regulon consists of three operons controlled by a positive regulatory gene, malT. Deletions of the gene crp were introduced into strains which carried a malT-lacZ hybrid gene. From the observed reduction in beta-galactosidase activity it was concluded that the expression of malT-lacZ, and therefore of malT, is controlled by the catabolite activator protein (CAP), the product of the gene crp. Mutations were obtained which allowed a malT-lacZ hybrid gene to be expressed at a high level even in the absence of CAP. These mutations were shown to be located in or close to the promoter of the malT gene and were called malTp. The malTp mutations were transferred in the cis position to a wild-type malT gene. In the resulting strains, the expression of two of the maltose operons, malEFG and malK-lamB, still required the action of CAP, whereas that of the third operon, malPQ, was CAP independent. Therefore, in wild-type cells, CAP appears to control malPQ expression mainly, if not solely, by regulating the concentration of MalT protein in the cell. On the other hand, it controls the other two operons more stringently, both by regulating malT expression and by a more direct action, probably exerted in the promoters of these operons.
Analysis of an avtA::Mu d1(Ap lac) mutant: metabolic role of transaminase C
Authors: Whalen W A , Berg C M
Published: January 2020

Escherichia coli can synthesize alpha-ketoisovalerate, the precursor of valine, leucine, and pantothenate, by three routes: anabolically via dihydroxyacid dehydrase and catabolically via both the branched-chain amino acid transaminase (transaminase B) and the alanine-valine transaminase (transaminase C). An E. coli K-12 mutant devoid of transaminase C (avtA) was isolated by mutagenizing an isoleucine-requiring strain devoid of transaminase B (ilvE::Tn5) with Mu d1(Ap lac) and selecting for valine-requiring derivatives which were ampicillin resistant, Lac+, able to crossfeed an ilvD mutant, and unable to grow on alpha-ketoisovalerate in place of valine. Strains defective in one, two, or all three alpha-ketoisovalerate metabolic enzymes were constructed, and their properties were analyzed. The data indicated that avtA is the structural gene for transaminase C, that transaminase C is a single enzyme species, and that the sole pathway for pantothenate biosynthesis is from alpha-ketoisovalerate. The data further showed that isoelectric inhibits the transaminase B-catalyzed deamination of valine in vivo.
Evolution of the D-ribose operon on Escherichia coli B/r
Authors: Abou-Sabé M , Pilla J , Hazuda D , Ninfa A
Published: January 2020

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.
Analysis of Mycoplasma hyorhinis genome by use of restriction endonucleases and by electron microscopy
Authors: Darai G , Zöller L , Matz B , Delius H , Speck P T , Flügel R M
Published: January 2020

The chromosome of Mycoplasma hyorhinis was analyzed by using different restriction endonucleases and electron microscopy. It was found that restriction enzymes BstEII, XhoI, and SacI are the enzymes of choice for analysis and characterization of M. hyorhinis. The bands resulting from digestion of M. hyorhinis DNA with BstEII had apparent molecular weights ranging from 1.2 X 10(6) to 75 X 10(6). The apparent total molecular weight of DNA was calculated from the molecular weights of the individual bands and found to be 251 X 10(6). Electron microscopic contour length measurements of the largest DNA fragments verified the molecular weight values calculated from gel analysis. Electron microscopic contour length measurements of intact DNA of M. hyorhinis revealed a molecular weight of 5.4 +/- 5 X 10(8). The discrepancy between the values of molecular weight of M. hyorhinis DNA as determined by restriction enzyme analysis and contour length measurement is based on the fact that some of the DNA fragments which migrate as an apparent single band in the agarose gel really are double or multiple DNA fragments.
Metabolism of acetylene by Nocardia rhodochrous
Authors: Kanner D , Bartha R
Published: January 2020

A Nocardia rhodochrous strain capable of utilizing acetylene as its sole source of carbon and energy exhibited slow growth on low concentrations of acetaldehyde. Resting cells incubated with acetylene formed a product identified as acetaldehyde, but attempts to demonstrate acetylene hydrase activity in cell-free extracts were unsuccessful. Acetaldehyde dehydrogenase in N. rhodochrous was found to be NAD+ linked and nonacylating, converting acetaldehyde to acetate. Specific activities of acetaldehyde dehydrogenase, acetothiokinase, and isocitrate lyase were enhanced in cells grown on acetylene and ethanol as compared with cells grown on alternate substrates. These results suggest that acetylene is catabolized via acetaldehyde to acetate and eventually to acetyl coenzyme A. Acetylene oxidation in N. rhodochrous appears to be constitutive and is not inhibited in the presence of either ethylene, nitrous oxide, or methane.
Cation-Dependent Binding of Substrate to the Folate Transport Protein of
Lactobacillus casei
Authors: Henderson Gary B. , Potuznik Suzana
Published: January 2020

Lactobacillus casei
cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (

K
d

) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium.

K
d

measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca
2+
, Mg
2+
, and ethylenediammonium
2+
ions). Monovalent cations (e.g., Na
+
, K
+
, and Tris
+
) were also effective, but only at concentrations in the millimolar range. The

K
d

for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl
2
. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of
L. casei
may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.
Extracellular acid proteases from Neurospora crassa
Authors: Lindberg R A , Rhodes W G , Eirich L D , Drucker H
Published: January 2020

Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5.
Glucose transport system in a facultative iron-oxidizing bacterium, Thiobacillus ferrooxidans
Authors: Sugio T , Kudo S , Tano T , Imai K
Published: January 2020

Properties of a heat-labile glucose transport system in Thiobacillus ferrooxidans strain AP-44 were investigated with iron-grown cells. [14C]glucose was incorporated into cell fractions, and the cells metabolized [14C]glucose to 14CO2. Amytal, rotenone, cyanide, azide, 2,4-dinitrophenol, and dicyclohexylcarbodiimide strongly inhibited [14C]glucose uptake activity, suggesting the presence of an energy-dependent glucose transport system in T. ferrooxidans. Heavy metals, such as mercury, silver, uranium, and molybdate, markedly inhibited the transport activity at 1 mM. When grown on mixotrophic medium, the bacteria preferentially utilized ferrous iron as an energy source. When iron was exhausted, the cells used glucose if the concentration of ferrous sulfate in the medium was higher than 3% (wt/vol). However, when ferrous sulfate was lower than 1%, both of the energy sources were consumed simultaneously.
Phospholipase C regulatory mutation of Pseudomonas aeruginosa that results in constitutive synthesis of several phosphate-repressible proteins
Authors: Gray G L , Berka R M , Vasil M L
Published: January 2020

We describe here a new mutant of Pseudomonas aeruginosa PAO, strain D10C (genotype plcB), which produces phospholipase C and alkaline phosphatase constitutively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracellular proteins produced by this mutant in high- and low-Pi media revealed that the mutation resulted in a marked deficiency of one major Pi-regulated protein of 41,000 molecular weight and constitutive synthesis of all other major extracellular Pi-regulated proteins. Furthermore, the plcB mutant was deficient in phosphate transport. A plcA mutation, which also led to a loss of the 41,000-molecular-weight protein, was similarly deficient in Pi transport. The genetic loci, plcA and plcB, located at 22 to 23 min on the PAO chromosome, were indistinguishable by conjugational and transductional mapping, and may therefore be in the same gene or in a cluster of genes which regulate the synthesis of Pi-repressible proteins.
Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans
Authors: Jacobson E S , Ayers D J , Harrell A C , Nicholas C C
Published: January 2020

Stable mutants with reduced capacity to produce capsules were isolated from suspensions of Cryptococcus neoformans after treatment of the wild type with a mutagen. The mutants could be assigned one of two phenotypes, hypocapsular or acapsular. Hypocapsular mutants were immunochemically and physicochemically indistinguishable from the wild type, whereas acapsular mutants lacked a major capsular antigen and a negatively charged exterior. In genetic analysis, the mutant trait segregated as a Mendelian gene (1:1) when random basidiospores from an outcross were studied, and analysis of products of single meiotic events from outcrossed mutants was likewise consistent with meiotic segregation. Two-factor crosses yielded the expected four classes of progeny, with recombinants equal to parentals. We concluded that chromosomal genes are responsible for synthesis of the cryptococcal capsule and that random basidiospore analysis represents a useful technique for genetic analysis in this species.
Efficiency of light-driven metabolite transport in the photosynthetic bacterium Rhodospirillum rubrum
Authors: Zebrower M , Loach P A
Published: January 2020

An evaluation of the efficiency of the L-alanine and L-malate transport systems was undertaken with the photosynthetic bacterium Rhodospirillum rubrum grown on the amino acid whose uptake was measured. An all-glass apparatus was constructed for measuring transport activity under anaerobic conditions. L-Alanine transport activity decreased under conditions of Mg2+ depletion. When cells were allowed to become inactive by suspending them in the dark in Mg2+-free buffer, full activity could be restored with a few minutes by adding 20 mM Mg2+ and illuminating the cells. The transport activity was completely inhibited by carbonyl cyanide m-trifluoromethoxyphenylhydrazone and by ammonia. The quantum yield for the uptake of either L-alanine or L-malate was 0.015 molecules per photon. The results are discussed in relation to the expected efficiencies for metabolite transport and regulation by Mg2+.
Enzymes related to fructose utilization in Pseudomonas cepacia
Authors: Allenza P , Lee Y N , Lessie T G
Published: January 2020

Growth of Pseudomonas cepacia on fructose, mannitol, or sorbitol depended on formation of an inducible fructokinase (forming fructose-6-phosphate) and the presence of enzymes of the Entner-Doudoroff pathway. Mutants deficient in any of these enzymes failed to utilize the aforementioned carbohydrates. Fructokinase deficiency did not affect growth of the bacteria on glucose. Fructose was accumulated intracellularly by active transport. Mutants blocked in transport of fructose grew normally on mannitol or sorbitol despite their inability to utilize fructose. Growth on either of these hexitols or on galactitol was accompanied by induction of two hexitol dehydrogenases, one active primarily with mannitol and the other active with sorbitol and galactitol. As expected, a mutant deficient in mannitol dehydrogenase failed to utilize mannitol as a carbon and energy source but grew normally on sorbitol and galactitol. Extracts of bacteria grown on fructose, mannitol, or sorbitol and higher levels of phosphoglucose isomerase than extracts of bacteria grown on alternate carbon sources such as citrate or phthalate. The higher levels were due to appearance of a second phosphoglucose isomerase species not present in cells with the lower activity. The results indicate that the initial steps in fructose utilization by P. cepacia differ from those of most other pseudomonads, which transport fructose by phosphoenolpyruvate-dependent translocation, forming fructose-1-phosphate, and suggest that degradation of fructose, mannitol, and sorbitol occurs primarily via the Entner-Doudoroff pathway.
Rosanilins: indicator dyes for chloramphenicol-resistant enterobacteria containing chloramphenicol acetyltransferase
Authors: Proctor G N , Rownd R H
Published: January 2020

Rosanilin dyes such as crystal violet and basic fuchsin have been used as indicator dyes in solid growth medium for chloramphenicol-resistant enterobacterial colonies containing the enterobacterial resistance enzyme chloramphenicol acetyltransferase (CAT). On certain media containing rosanilins, cells containing CAT formed darker colonies than cells not containing CAT. Contrast was affected by the types and concentrations of complex nutrients, sugars salts, and rosanilin dyes present. When crystal violet was used as the indicator dye, contrast could not be obtained for strains whose growth was partially inhibited by crystal violet. Contrast could not be obtained between yeast colonies with and without the enterobacterial resistance enzyme, between Bacillus subtilis colonies with and without the staphylococcal resistance enzyme, or between enterobacterial colonies with and without the staphylococcal resistance enzyme. The darker coloration of enterobacterial colonies with the enterobacterial enzyme was due to the binding of dye to enzyme. Rosanilin dues have been used to score resistance phenotypes by colony color, to detect chloramphenicol-sensitive sectors in chloramphenicol-resistant colonies, and to screen for occasional chloramphenicol-sensitive cells in a resistant population during cloning by insertional inactivation of the chloramphenicol resistance gene.
Threonine prevents derepression of pyridoxine synthesis in Escherichia coli B
Authors: Dempsey W B
Published: January 2020

After 40 min of pyridoxal starvation, pyridoxine phosphate oxidase-less mutants of Escherichia coli B derepressed pyridoxine biosynthesis 13-fold to a rate of 1.7 X 10(-9) mol/h per mg of cells. Threonine at 100 mg/liter prevented this derepression but did not affect the continued synthesis of pyridoxine. Neither serine nor branched-chain amino acids altered the threonine effect.
Specialized transduction with lambda plac5: dependence on recB
Authors: Porter R D , Welliver R A , Witkowski T A
Published: January 2020

Genetically disabled lambda plac5 transducing phage derivatives were used to study the recB dependence of recombination during specialized transduction. The frequency of transduction was normalized to colony-forming units, and the end product of recombination was monitored by scoring for addition and substitution transductants. When a chromosomal lac gene was the recipient DNA substrate molecule, both the normalized transduction frequency and the proportion of addition and substitution transductants showed essentially no recB dependence. There was a pronounced recB dependence for both normalized transduction frequency and recombination end product formation when F42 lac was the recipient DNA substrate. recB appears to have no significant role in the recombination that occurs between the two lac regions in an addition transductant. UV irradiation of the transducing phages increased the absolute level of both addition and substitution transductants obtained with a chromosomal lac gene but resulted in a considerable change in the relative frequency of addition versus substitution transductants.
RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA
Authors: Shen V , Imamoto F , Schlessinger D
Published: January 2020

Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro.
Molecular mechanism for loss of nodulation properties of Rhizobium trifolii
Authors: Zurkowski W
Published: January 2020

Of 18 Rhizobium trifolii strains tested, 12 showed a high frequency of loss of nodulation ability after incubation in cultures at elevated temperatures. A correlation between loss of nodulation ability and loss of a large plasmid was demonstrated for R. trifolii. In some nonnodulating (Nod-) mutants, deletions occurred instead of total elimination of the plasmid molecule. The maximum curing effect was observed in bacteria incubated at 35 degrees C. After 4 or more days of incubation at this temperature, the viability of bacteria decreased markedly, and the number of nonnodulating mutants increased significantly. At the elevated temperature DNA synthesis was stopped completely after 2 h, whereas protein synthesis proceeded for a few days. Microscopic observations showed that during the first 3 days of incubation at the elevated temperature, the bacterial cells increased markedly in size. These large irregular cells then divided and produced Nod- clones. Nonnodulating clones did not result from the selection of temperature-resistant mutants. The presence of P-group plasmids in Rhizobium strains strongly inhibited the loss of nodulation ability during incubation at 35 degrees C. The observed phenomenon did not result from integrative suppression. It is possible that a product(s) of the genes of R-plasmids acts as a stabilizing agent on the replication process of the indigenous Rhizobium plasmids.
Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa
Authors: Carlson C A , Ferguson L P , Ingraham J L
Published: January 2020

Dissimilatory nitrate reductase was purified to homogeneity from anaerobic cultures of the denitrifying bacterium Pseudomonas aeruginosa. The following procedures were used in the rapid isolation of this unstable enzyme: induction by nitrate in semianaerobic cell suspension, heat-stimulated activation and solubilization from the membrane fraction, and purification by hydrophobic interaction chromatography. The molecular weight of the purified enzyme was estimated by nondenaturing polyacrylamide gel electrophoresis, sucrose density gradient sedimentation, and gel filtration chromatography. Subunit molecular weights were estimated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The active enzyme monomer, with a molecular weight of 176,000 to 260,000 (depending upon the method of determination), was composed of subunits with molecular weights of approximately 64,000 and 118,000. The monomer aggregated to form an inactive tetramer of about 800,000 molecular weight. Purified enzyme exhibited a broad pH optimum, between 6.5 and 7.5. Kinetic studies showed that the apparent Km was 0.30 mM for nitrate, and 2.2 to 2.9 microM for dithionite-reduced benzyl viologen. Azide was an effective inhibitor: the concentration required for half-maximal inhibition was 21 to 24 microM. Azide inhibition was competitive with nitrate (Ki = 2.0 microM) but uncompetitive with reduced benzyl viologen (Ki = 25 microM). Based upon spectral evidence, the purified molybdo-enzyme had no associated cytochromes but did contain nonhaem iron that responded to dithionite reduction and nitrate oxidation. The enzyme that was purified after being heat solubilized from membranes had properties essentially identical to those of the enzyme that was purified after deoxycholate solubilization.
Detection of a gonococcal endo-beta-N-acetyl-D-glucosaminidase and its peptidoglycan cleavage site
Authors: Gubish E R , Chen K C , Buchanan T M
Published: January 2020

Neisseria gonorrhoeae contains several hydrolases which may be responsible for gonococcal cell lysis. One of these enzymes, an endo-beta-N-acetyl-D-glucosaminidase, has been extracted from supernatants of sonicated gonococci and partially purified by ammonium sulfate precipitation and affinity and ion-exchange chromatography. This enzyme has a different specificity than egg white lysozyme and cleaves the beta 1 leads to 4 glycosidic linkage between N-acetylglucosamine and N-acetylmuramic acid in gonococcal peptidoglycan.
Genetic and molecular characterization of Tn21, a multiple resistance transposon from R100.1
Authors: de la Cruz F , Grinsted J
Published: January 2020

Tge transposon Tn21 has been transposed from R100.1 to plasmid pACYC184 and, from the resulting recombinants, to plasmid R388. The sites of insertion and the orientation of the element in several pACYC184::Tn21 recombinants have been examined. Restriction enzyme analysis of these recombinants has resulted in a detailed map of Tn21; this is compared with the published maps of the relevant part of R100.1. Heteroduplex analysis has shown short inverted repeat sequences at the ends of the element. With various in vitro-generated deletion mutants of Tn21, the internal gene necessary for transposition (tnpA) was localized within the terminal 4.3 kilobases of the right-hand end of the element. Genetic analysis of transposition of Tn21 suggests that the process proceeds via cointegrates. Since the end products of transposition are simple recombinants of the element and the recipient replicon, Tn21 must contain a gene that codes for a resolvase type of activity (tnpR gene).
Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens
Authors: Perry K L , Kado C I
Published: January 2020

Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA.