bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosome Replication in Sporulating Cells of
Bacillus subtilis
Authors: Sargent Michael G.
Published: January 2020

A method of specifically labeling the chromosomal terminus of
Bacillus subtilis
is described. When sporulating cultures were pulse-labeled with [
3
H]thymidine and then treated with 6-(
p
-hydroxyphenylazo)uracil, a drug which inhibits deoxyribonucleic acid (DNA) synthesis rapidly and completely, the only labeled spores formed were those that had completed replication during the pulse period. DNA-mediated transformation was used to show that the DNA of spores formed in the presence of 6-(
p
-hydroxyphenylazo)uracil had the same ratio of origin to terminus markers as DNA from untreated spores. Furthermore, spores formed in the presence of 6-(
p
-hydroxyphenylazo)uracil had the same DNA content as untreated spores. These two observations indicated that spores formed in the presence of 6-(hydroxyphenylazo)uracil contained completed chromosomes. The rate of termination of chromosomes destined to be packaged into spores was determined by this method, using the Sterlini-Mandelstam replacement system and a single medium exhaustion system for inducing sporulation. With both systems the rate of termination reached a broad peak 2 h after the start of sporogenesis. This was measured from the time of resuspension by using the replacement system and from the point where exponential growth ceased in the exhaustion system. The amount of spore DNA synthesized in the Sterlini-Mandelstam sporulation-inducing medium was very close to one-half the amount of the DNA present in mature spores. This suggests that chromosomes destined to be packaged into spores were replicated from close to the origin and possibly initiated in the sporulation-inducing medium. A method was devised for estimating the time taken to complete replication of the chromosomes destined to be packaged into spores. This was probably no more than 50 min. Whereas starvation must have occurred almost simultaneously in most cells in the population, the chromosome replication that was essential for sporogenesis was distributed over a wide time span. Thus, in some cells, replication started within 10 min of the nutritional step-down, but the peak rate was not reached for 1 h; thereafter replication continued at a substantial rate.
Physical location of the ilvO determinant in Escherichia coli K-12 deoxyribonucleic acid
Authors: Subrahmanyam C S , McCorkle G M , Umbarger H E
Published: January 2020

A plasmid carrying the 4,6-kilobase (kb) HindIII-derived fragment from an ilvO mutant derivative of lambda h80dilv imparted a valine-resistant phenotype on strains it carried. This fragment carries a small amount of the promoter-proximal end of ilvE, the ilvO determinant, and apparently the entire ilvG gene, which specifies the valine-insensitive acetohydroxy acid synthase. Comparable deoxyribonucleic acid (DNA) from the original lambda h80dilv did not carry the valine resistance marker. The valine-resistant phenotype was always correlated with the formation of the resistant enzymes. The ilvO determinant was shown to be carried within an approximately 600-based-pair region lying between the SalI and KpnI sites on the HindIII fragment and perhaps within the ilvG gene itself. Ribonucleic acid that hybridizes with the DNA corresponding to the ilvG gene is formed in wild-type K-12 cells. This fact, coupled with the fact that ilvG is transcribed from the same DNA strand as the ilvE, D, and A genes, led to the idea that transcription is normally initiated upstream from ilvG in both wild-type and ilvO strains. In wild-type strains either the formation or the translation of the transcript would be terminated with the ilvG gene, thus preventing expression of that gene.
Iron uptake with ferripyochelin and ferric citrate by Pseudomonas aeruginosa
Authors: Cox C D
Published: January 2020

Pyochelin is an iron-binding compound produced by Pseudomonas aeruginosa and demonstrates siderophore activity by its involvement in iron transport. During the transport process, an energy-independent association of [55Fe]ferripyochelin with bacteria occurred within the initial 30 s of reaction, followed by an energy-dependent accumulation of iron. The energy-independent association with iron appeared to be at the surface of the bacteria because the iron could be washed from the cells with thioglycolate, whereas accumulated iron was not washed from the bacteria. Energy-independent association of iron with bacteria and energy-dependent accumulation of iron in the presence of ferripyochelin varied concomitantly in cells grown under various conditions, but pyochelin synthesis appeared to be controlled separately. 55Fe complexed with citrate was also taken up by P. aeruginosa with a lower level of initial cell association. Bacterial mechanisms for iron uptake from ferric citrate were present in cells grown in a variety of media and were in lowest levels in cells grown in citrate. The synthesis of bacterial components for iron uptake from ferric citrate and from ferripyochelin was inhibited by high concentrations of iron supplied in growth media.
Deoxyribonucleic acid sequence of araBAD promoter mutants of Escherichia coli
Authors: Horwitz A H , Morandi C , Wilcox G
Published: January 2020

The controlling site region for the araBAD operon is defined, in part, by two classes of cis-acting constitutive mutations. The aralc mutations allow low-level constitutive expression of ara-BAD in the absence of the positive regulatory protein coded for by the araC gene, whereas the araXc mutations allow expression of araBAD in the absence of the cyclic adenosine monophosphate receptor protein. Six independently isolated aralc mutations and three independently isolated araXc mutations were cloned onto the plasmid pBR322 using in vitro recombinant deoxyribonucleic acid techniques and in vivo recombination between plasmid and chromosomal deoxyribonucleic acid. The location of these mutations was determined by deoxyribonucleic acid sequence analysis. All of the aralc mutations occurred at position -35 within the araBAD promoter (+1 = messenger ribonucleic acid start for araBAD) and resulted from an AT leads to GC transition. All of the araXc mutations occurred at position -10 within the araBAD promoter and resulted from a GC leads to AT transition. Models are presented to explain the mode of action of the aralc and araXc mutations.
Strain variation and morphogenesis of yeast- and mycelial-phase Candida albicans in low-sulfate, synthetic medium
Authors: Manning M , Mitchell T G
Published: January 2020

A low-sulfate synthetic medium was developed in which pure cultures of yeast- and mycelial-phase Candida albicans could be cultivated for investigations of the molecular biology of dimorphism. The medium contained ammonium ions, phosphate buffer, salts, glucose, and biotin. Morphogenesis was found to be dependent upon the strain of C. albicans. Of six strains tested in the low-sulfate medium at 37 degrees C, three formed mixed cultures of yeasts, true mycelium and pseudomycelium, two formed pure cultures of true mycelium, and one maintained yeast growth. All six strains produced pure cultures of yeasts at 24 degrees C. The buffering capacity of the medium maintained the pH at 6.9 even at high-density cell growth. The low concentration of sulfate and the absence of amino acids in the medium provided conditions in which to radiolabel cellular constituents with [35S]sulfate. For molecular investigations, the use of two strains is suggested, one forming yeasts and one forming true mycelium in low-sulfate medium at 37 degrees C, thus providing controls for both strain variation and for molecular changes induced by environmental change but unrelated to morphogenesis.
Processing of alkaline phosphatase precursor to the mature enzyme by an Escherichia coli inner membrane preparation
Authors: Chang C N , Inouye H , Model P , Beckwith J
Published: January 2020

An inner membrane preparation co-translationally cleaved both the alkaline phosphatase and bacteriophage f1 coat protein precursors to the mature proteins. Post-translational outer membrane proteolysis of pre-alkaline phosphatase generated a protein smaller than the authentic monomer.
Comparison of outer membrane porin proteins produced by Escherichia coli and Salmonella typhimurium
Authors: Lee D R , Schnaitman C A
Published: January 2020

The OmpC, OmpF, and Lc (NmpC) porin proteins of Escherichia coli K-12 have been shown to be similar to the OmpC (36K), OmpF (35K) and OmpD (34K) porin proteins of Salmnella typhimurium LT2 in terms of function, regulation of expression, and, in the case of OmpC and OmpF proteins, equivalence of the genetic loci determining their production. However, the corresponding pairs of proteins from these two species showed only limited similarity in peptide maps and no similarity in terms of migration on polyacrylamide gels.
Effect of hexoses on the levels of pyruvate decarboxylase in Mucor rouxii
Authors: Barrera C R , Corral J
Published: January 2020

Pyruvate decarboxylase activity in the dimorphic fungus Mucor rouxii increased 25- to 35-fold in yeastlike and mycelial cells grown in the presence of glucose as compared to the activity observed in mycelial cultures grown in the absence of glucose.
Fructose transport in Neurospora crassa
Authors: Rand J B , Tatum E L
Published: January 2020

A specific fructose uptake system (Km = 0.4 mM) appeared in Neurospora crassa when glucose-grown mycelia were starved. Fructose uptake had kinetics different from those of intramycelial fructose phosphorylation, and uptake appeared to be carrier mediated. The only sugar which competitively inhibited fructose uptake was L-sorbose (Ki = 9 mM). Glucose, 2-deoxyglucose, mannose, and 3-O-methyl glucose were noncompetitive inhibitors of fructose uptake. Incubation of glucose-grown mycelia with glucose, 2-deoxyglucose, or mannose prevented derepression of the fructose transport system, whereas incubation with 3-O-methyl glucose caused the appearance of five times as much fructose uptake activity as did starvation conditions.
Dissociation of
tsl-tif
-Induced Filamentation and
recA
Protein Synthesis in
Escherichia coli
K-12
Authors: Huisman Olivier , D'Ari Richard , George Jacqueline
Published: January 2020

In
Escherichia coli
, expression of the
tif-1
mutation (in the
recA
gene) induces the “SOS response” at 40°C, including massive synthesis of the
recA
(
tif
) protein, cell filamentation, appearance of new repair and mutagenic activities, and prophage induction. Expression of the
tsl-1
mutation (in the
lexA
gene) induces massive synthesis of the
recA
protein and cell filamentation at 42°C, although other SOS functions are not induced. In this paper we show that the septation inhibition induced in
tif
and
tsl
strains at 42°C is not due to the presence of a high concentration of
recA
protein since (i) no
recA
mutants (≤10
−8
) were isolated among thermoresistant nonfilamenting revertants of a
tif-1 tsl-1
strain, (ii) in a
tsl-1 zab-53
strain, only the low basal level of
recA
protein was synthesized at 42°C, yet cell division was inhibited, and (iii) in a
tsl-1 recA99
(amber) strain, no
recA
protein could be detected at 42°C, yet cell division was inhibited. Among suppressors of
tsl-tif
-induced lethality are mutations at a locus which we call
infB
, located in the 66- to 83-min region. The
infB1
mutation confers a highly pleiotropic phenotype, which is suggestive of a regulatory defect; it suppressed
tsl-tif
-induced filamentation but not
recA
protein synthesis, it did not suppress ultraviolet-induced filamentation (in a
lon
derivative), and it reduced but did not abolish
tif
-mediated induction of λ prophage and bacterial mutagenesis. The dissociation of
tsl-tif
-induced septation inhibition and
recA
protein synthesis in the
tif-1 tsl-1 infB1
strain suggests that the control of SOS filamentation may not be strictly identical to the control of
recA
protein synthesis.
Initiation of deoxyribonucleic acid replication in Escherichia coli B: uncoupling from mass/deoxyribonucleic acid ratio
Authors: Grossman N , Ron E Z
Published: January 2020

In Escherichia coli growing at different rates, the ratio of cell mass to the number of chromosome origins tended to be constant at the time of the initiation of deoxyribonucleic acid (DNA) replication. This observation led to the assumption that the initiation event is controlled in some way by cell mass, e.g., by a growth-dependent synthesis of an initiator or dilution of a repressor. We have now found that the initiation of DNA synthesis can be uncoupled from cell mass. We used a synchronous culture of newly divided cells of E. coli B which was obtained by the membrane elution technique (C.E. Helmstetter, J. Mol. Biol. 24: 417-427, 1967) and was starved for an amino acid. Upon restoration of the amino acid, the cells not only divided at a size that was smaller than normal, but also initiated DNA replication long before they could increase their masses to reach the expected ratio of mass/DNA presumably required for initiation.
Glutamine and related analogs regulate guanosine monophosphate reductase in Salmonella typhimurium
Authors: Garber B B , Jochimsen B U , Gots J S
Published: January 2020

The addition of a glutamine analog, 6-diazo-5-oxo-L-norleucine, or an inhibitor of glutamine synthetase, L-methionine-dl-sulfoximine, to the growth media of most Salmonella typhimurium strains resulted in a marked elevation of guanosine monophosphate reductase levels. The elevation caused by either compound required protein synthesis and could be antagonized by exogenous glutamine. In addition, when glutamine auxotrophs were grown in suboptimal concentrations of glutamine, the guanosine monophosphate reductase levels were increased. It is postulated that glutamine or a product of its metabolism may function under normal conditions as a negative regulatory element in the control of guanosine monophosphate reductase and that decreased effective intracellular levels of glutamine result in an increase in the level of the enzyme.
Chromosomal rearrangements induced by temperate bacteriophage D108
Authors: Faelen M , Toussaint A
Published: January 2020

As previously shown for mutator phage Mu-1, to which it is closely related, temperate bacteriophage D108 induces chromosomal rearrangements (replicon fusion and transposition of chromosomal segments) in its host genome.
Reversal by trypsin of the inhibition of active transport by colicin E1
Authors: Dankert J , Hammond S M , Cramer W A
Published: January 2020

The time course for inhibition of proline transport and irreversible loss of cell viability after treatment with colicin E1 was measured as a function of temperature between 13 and 33 degrees C, using a thermostatted flow dialysis system. Complete inhibition of proline transport at 33 and 13 degrees C occurred in 0.5 min and 3 to 5 min, respectively, after addition of colicin E1 at an effective multiplicity of about 4. At these times, the fractional cell survival, assayed by dilution directly from the flow dialysis vessel into trypsin, ranged from 35 to 80%, with viability always greater than 50% at the lower incubation temperatures. Further studies were carried out at 15 degrees C. Complete inhibition of proline transport, which required 2 to 3 min, occurred much more rapidly at 15 degrees C than did the decay of trypsin rescue, which required 10 to 15 min to reach a survival level of 10 to 20%. The direct addition of trypsin to the flow dialysis vessel, after an addition of colicin E1 that caused complete inhibition of proline or glutamine transport, resulted in restoration of net transport. The restored level was typically about 40% of the control rate, and was very similar to the fractional cell viability measured after incubation in trypsin in the same vessel. It is concluded that trypsin can restore active transport to a significant fraction of a cell population in which transport has been initially inhibited by colicin E1.
Cloning of deoxyribonucleic acid regions encoding a heat-labile and heat-stable enterotoxin originating from an enterotoxigenic Escherichia coli strain of human origin
Authors: Yamamoto T , Yokota T
Published: January 2020

A heat-labile and heat-stable enterotoxin (LT+ ST+) plasmic (62.7 kilobases in size) was isolated from an enterotoxigenic Escherichia coli human strain, H10407, and used for analysis of the LT+ and ST+ deoxyribonucleic acid (DNA) regions. A DNA segment containing the LT+ and ST+ DNA regions, which consisted of two restriction endonuclease EcoRI fragments (E1 and E2), was inserted into the cloning vehicle ColE1::Tn5 by EcoRI digestion and subsequent ligation. Further cloning experiments localized the LT+ DNA region on a 5.1-kilobase restriction endonuclease PstI fragment present over the junction between the E1 and E2 fragments, as seen in the original LT+ ST+ plasmid, and the ST+ DNA region on a 1.5-kilobase PstI fragment present in either the E1 or E2 fragment. A change in the relative orientation of the E1 and E2 fragments resulted in altered levels of LT production. The relative orientation of the ColE1::Tn5 fragment to the E1 and E2 fragments also markedly influenced both LT and ST production levels. The LT+ ST+ E1-E2 region contained two unique DNA sequences consisting of a DNA segment flanked by inverted repeats which were readily distinguished from each other by size. The cloned ST+ PstI fragment was structurally very similar to one of these unique DNA sequences present in the LT+ ST+ E1-E2 region.
Molecular cloning, correlation of genetic and restriction maps, and determination of the direction of transcription of gnd of Escherichia coli
Authors: Nasoff M S , Wolf R E
Published: January 2020

Expression of the gene gnd of Escherichia coli, which encodes 6-phosphogluconate dehydrogenase, is regulated by growth rate. Using deoxyribonucleic acid from the specialized transducing phage lambda h80 dgnd his as the source of gnd, we cloned restriction fragments carrying the complete gene and portions of it on the plasmid vector pBR322. A hybrid plasmid carrying a 3.7-megadalton HindIII restriction fragment from the phage was prepared and found to be gnd+. Through restriction mapping of this fragment and subcloning segments of it, we prepared a gnd+ hybrid plasmid which carried only 1.85 megadaltons of E. coli deoxyribonucleic acid. A cleavage site for the restriction endonuclease PstI was located on the genetic map of gnd by cloning adjacent EcoRI-PstI restriction fragments and crossing the resulting hybrid plasmids with previously mapped gnd deletion and bacteriophage Mu insertion mutants. A maxicell experiment was used to determine the direction of transcription of gnd, to identify which EcoRI-PstI fragment contains the gnd promote, and to localize th beginning of the structural gene to a region about 850 +/- 150 base pairs from the PstI cleavage site. A fine-structure restriction map surrounding the PstI cleavage site was prepared for endonucleases KpnI, HincII, HaeIII, HpaII, and TaqI.
Genetic locus (nmp-1) affecting the principal outer membrane protein of Neisseria gonorrhoeae
Authors: Cannon J G , Klapper D G , Blackman E Y , Sparling P F
Published: January 2020

An increase in the apparent molecular weight of the principal outer membrane protein (POMP) of Neisseria gonorrhoeae is associated with introduction of the penB2 genetic marker, which results in low-level, relatively nonspecific antibiotic resistance. Limited proteolysis of the two forms of POMP showed that they had few if any peptides in common. The nonspecific antibiotic resistance of penB2 was separated from the change in POMP by genetic transformation and by isolation of spontaneous penB mutants that showed no change in POMP. The genetic locus involved in the change from one POMP to another, which we have designated nmp-1, is closely linked to, but not identical with, penB2.
Utilization of 2,6-diaminopurine by Salmonella typhimurium
Authors: Garber B B , Gots J S
Published: January 2020

The pathway for the utilization of 2,6-diaminopurine (DAP) as an exogenous purine source in Salmonella typhimurium was examined. In strains able to use DAP as a purine source, mutant derivatives lacking either purine nucleoside phosphorylase or adenosine deaminase activity lost the ability to do so. The implied pathway of DAP utilization was via its conversion to DAP ribonucleoside by purine nucleoside phosphorylase, followed by deamination to guanosine by adenosine deaminase. Guanosine can then enter the established purine salvage pathways. In the course of defining this pathway, purine auxotrophs able to utilize DAP as sole purine source were isolated and partially characterized. These mutants fell into several classes, including (i) strains that only required an exogenous source of guanine nucleotides (e.g., guaA and guaB strains); (ii) strains that had a purF genetic lesion (i.e., were defective in alpha-5-phosphoribosyl 1-pyrophosphate amidotransferase activity); and (iii) strains that had constitutive levels of purine nucleoside phosphorylase. Selection among purine auxotrophs blocked in the de novo synthesis of inosine 5'-monophosphate, for efficient growth on DAP as sole source of purine nucleotides, readily yielded mutants which were defective in the regulation of their deoxyribonucleoside-catabolizing enzymes (e.g., deoR mutants).
Pleiotropic effects of alkaline phosphatase regulatory mutations phoB and phoT on anaerobic growth of and polyphosphate synthesis in Escherichia coli
Authors: Zuckier G , Ingenito E , Torriani A
Published: January 2020

The appearance during anaerobiosis of spontaneous phoT phoB double mutants in a phoT background is described. During both exponential growth and stationary phase, selection against the phoT mutants relative to the wild type was evident. This reduction in viability of phoT mutants was suppressed in phoT phoB double mutants. Sensitivity to anaerobiosis was shown to be correlated with polyphosphate overproduction. A possible pleiotropic function of phoT and phoB is suggested.
Generalized transduction in the enterobacterial phytopathogen Erwinia chrysanthemi
Authors: Chatterjee A K , Brown M A
Published: January 2020

Bacteriophages induced by mitomycin treatment of Erwinia chrysanthemi KS612 produced plaques on lawns of E. chrysanthemi EC183 and KS605. Bacteriophage Erch-12, purified from one such plaque, transferred an array of chromosomal genes (arg, leu, his, ser, thr, trp, ura) to appropriate recipient strains derived from E. chrysanthemi EC 183. Recombinants were formed in the absence of cellular contact between donor and recipient bacteria and in the presence of deoxyribonuclease. Ultraviolet irradiation of the bacteriophage stimulated transductional frequency. Linkage was detected in two-factor crosses between the loci thr and ser and between rif and ade; several closely linked mutations in ser were mapped with respect to thr.
In situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase
Authors: Honda S , Toraya T , Fukui S
Published: January 2020

The catalytic properties of coenzyme B12-dependent glycerol dehydratase and diol dehydratase were studied in situ with Klebsiella pneumoniae cells permeabilized by toluene treatment, since the in situ enzymes approximate the in vivo conditions of the enzymes more closely than enzymes in cell-free extracts or cell homogenates. Both dehydratases in situ underwent rapid "suicidal" inactivation by glycerol during catalysis, as they do in vitro. The inactivated dehydratases in situ, however, were rapidly and continually reactivated by adenosine 5'-triphosphate (ATP) and Mn2+ in the presence of free adenosylcobalamin, although in cell-free extracts or in cell homogenates they could not be reactivated at all under the same reaction conditions. ATP was partially replaced by cytidine 5'-triphosphate or guanosine 5'-triphosphate but not by the beta, gamma-methylene analog of ATP in the in situ reactivation. Mn2+ was fully replaced by Mg2+ but only partially by Co2+. Hydroxocoblamin could not replace adenosylcobalamin in reactivation mixtures. The ability to reactivate the glycerol-inactivated dehydratases in situ was only seen in cells grown anaerobically in glycerol-containing media. This suggests that some factor(s) required for in situ reactivation is subject to induction by glycerol. Of the two possible mechanisms of in situ reactivation, i.e., the regeneration of adenosylcobalamin by Co-adenosylation of the bound inactivated coenzyme moiety (B12-adenosylation mechanism) and the displacement of the bound inactivated coenzyme moiety by free adenosyl-cobalamin (B12-exchange mechanism), the former seems very unlikely from the experimental results.
Possible involvement of a plasmid in arginine auxotrophic mutation of Streptomyces kasugaensis
Authors: Nakano M M , Ozawa K , Ogawara H
Published: January 2020

Streptomyces kasugaensis gave arginine auxotrophic mutants at high frequency, The coupled loss and reappearance of plasmid deoxyribonucleic acid with arginine auxotrophy suggested that the insertion of the plasmid into chromosomal deoxyribonucleic acid caused the arginine auxotrophy.
Cloning of the URA1 gene of Saccharomyces cerevisiae
Authors: Guerry-Kopecko P , Wickner R B
Published: January 2020

A 5.7-kilobase segment of Saccharomyces cerevisiae deoxyribonucleic acid which complements both the yeast ura1 and Escherichia coli pyrD mutations in dihydroorotate dehydrogenase has been cloned in plasmid YRp7.
Gene-product relationships of the nif regulon of Klebsiella pneumoniae
Authors: Roberts G P , Brill W J
Published: January 2020

Products of nifV, M, A, and F of Klebsiella pneumoniae have been identified, and suggestive evidence exists for the assignment of products for nifS, U, and L. These assignments confirm previous descriptions of transcriptional polarity and suggest that existence of several more nif genes whose products are apparently not essential for nitrogen fixation under standard laboratory conditions of growth. Although the nifA product is essential for expression of other nif transcripts, it is not involved in the regulation of its own transcript, nifRLA. The stability of nif-encoded proteins under anaerobic conditions was examined in a variety of mutants, confirming previously reported protein-protein interactions and suggesting several new ones. The stability of nif-encoded proteins in ammonia was found to be comparable to that in ammonia-free conditions, whereas all nif-gene products appeared to be labile in the presence of oxygen.
Isolation and mapping of a mutation in Escherichia coli with altered levels of ribonuclease H
Authors: Carl P L , Bloom L , Crouch R J
Published: January 2020

A mutant of Escherichia coli with altered levels of ribonuclease (RNase) H was isolated after mutagenesis with ethyl methane sulfonate. A procedure for assaying RNase H in partially purified extracts was used to screen approximately 1,500 colonies for variations in RNase H activity. Confirmation of a lower level of RNase H in the mutant was accomplished by analysis of RNase H in sodium dodecyl sulfate-polyacrylamide gels. By Hfr, F', and P1 transduction mapping, the genetic locus responsible for the lower levels of RNase H was located at 5.1 min on the E. coli chromosome. This mutation (rnh) represents a new locus on the E. coli chromosome. The only phenotypic characteristic of this mutation which has been observed to date is the lower level of RNase H (30% of parental values).
Timing of swarmer cell cycle morphogenesis and macromolecular synthesis by Hyphomicrobium neptunium in synchronous culture
Authors: Wali T M , Hudson G R , Danald D A , Weiner R M
Published: January 2020

The swarmer cycle of Hyphomicrobium neptunium consists of a temporal sequence of discrete developmental events. To time morphogenesis and to investigate modulations in macromolecular synthesis, we attempted methods for synchronous culture. During synchrony, swarmer maturation occurred over 32%, hyphal growth occurred over 36%, and bud maturation occurred over 32% of the time required to complete the swarmer cycle. Daughter cells were released after 265 min. Deoxyribonucleic acid replication was discontinuous, having a G1 period of approximately 180 min. In addition, ribonucleic acid and protein syntheses were depressed during the earlier phases of development.
Synthesis and secretion of hemolysin by Escherichia coli
Authors: Springer W , Goebel W
Published: January 2020

Hemolytic Escherichia coli cells were found to synthesize and secrete significant amounts of hemolysin into a mineral salt-glucose medium containing hemoglobin. The release of de novo-synthesized hemolysin was stopped in the presence of energy metabolism inhibitors such as 2,4-dinitrophenol, sodium azide, or potassium cyanide, resulting in an accumulation of intracellular hemolysin. A similar effect was observed in the presence of procaine, a neuroactive drug which inhibits the processing of exoproteins. Small amounts of hemolysin were secreted into the medium within approximately 10 min of inhibition of protein synthesis by chloramphenicol. This represented the final release of preformed periplasmic hemolysin en route to secretion through the outer membrane and was not caused by adsorption of external hemolysin to the cell surface. This secretion was not energy dependent but was inhibited above pH 8 and at low temperatures (10 to 20 degrees C). We concluded that two transport processes are involved in hemolysin secretion. De novo-synthesized hemolysin is extruded by an energy-dependent process through the cytoplasmic membrane and probably requires processing. In the periplasmic space a small internal pool of preformed hemolysin is accumulated temporarily before being transported through the outer membrane. Release of hemolysin through the outer membrane does not require energy or de novo protein synthesis.
Differences in the architecture of cytoplasmic and intracytoplasmic membranes of three chemotrophically and phototrophically grown species of the Rhodospirillaceae
Authors: Golecki J R , Oelze J
Published: January 2020

Freeze-fracture faces of membranes of either chemotrophically or phototrophically grown Rhodospirillum rubrum, Rhodopseudomonas sphaeroides, and Rhodospirillum tenue were analyzed. All three species differed from each other with respect to size as well as numerical density (number per square micrometer) of intramembrane particles. In R. rubrum the number of particles on exoplasmic fracture faces of the cytoplasmic membrane stayed nearly constant (about 900 particles per microns2), but on the plasmic fracture face there were 4,700 and 6,264 particles per microns2, respectively, under chemotrophic and phototrophic conditions. The increase in number was largely a result of an enhanced occurrence of particles 10 nm in diameter. This diameter corresponds to the mean diameter of the predominant class of particles visible on the plasmic fracture faces of intracytoplasmic membrane formed under phototrophic conditions. In R. sphaeroides the number of particles on both of the fracture faces of cytoplasmic membranes stayed nearly constant. The mean diameter of articles appeared to be slightly increased under phototrophic conditions. Particles of cytoplasmic and intracytoplasmic membranes of phototrophically grown cells were of similar diameter. The number of particles, however, on plasmic fracture faces of intracytoplasmic membranes (6,674/microns2) was significantly higher than that on cytoplasmic membranes (5,708/microns2). R. tenue, on the other hand, which does not produce intracytoplasmic membranes, showed on exoplasmic fracture faces 543 and 3,765 particles per micron2 under chemotrophic and phototrophic conditions, respectively, whereas the corresponding numerical densities of plasmic fracture faces were 4,043 and 3,711 particles per microns2. The increased number of articles on exoplasmic fracture faces was mainly the result of an increased occurrence of particles with diameters greater than or equal to 10 nm. The results are interpreted to allow for the different modes of intractyoplasmic membrane development in Rhodospirillum rubrum and Rhodopseudomonas sphaeroides, respectively.
Organization of transfer ribonucleic acid genes in the Escherichia coli chromosome
Authors: Campen R K , Duester G L , Holmes W M , Young J M
Published: January 2020

The arrangement of transfer ribonucleic acid (RNA) genes in the chromosome of Escherichia coli K-12 (C600) was examined with the techniques of restriction endonuclease digestion and Southern blotting. The number and size of restriction fragments containing transfer or ribosomal RNA sequences or both were estimated by a variety of restriction endonucleases, including EcoRI, BglI, SmaI, SalI, BamHI, and PstI. EcoRI liberated a minimum of 27 fragments which hybridized to transfer RNA and 16 which hybridized to ribosomal RNA. Enzymes which did not cut within the ribosomal RNA operons (PstI and BamHI) liberated 16 and 13 fragments, respectively, which hybridized to transfer RNA. Five PstI and six BamHi fragments also hybridized to ribosomal RNA, suggesting that there may be at least 11 chromosomal locations distinct from ribosomal RNA operons which encode transfer RNA genes. In addition, our data indicated that several transfer RNA genes may be very close to the 5' proximal ends of certain ribosomal RNA operons and close to the 3' distal ends of all seven ribosomal RNA operons. Similar studies have been carried out with 22 purified species of transfer RNA, and we report here the number and size of EcoRI restriction fragments which hybridize to these transfer RNA species.
Isolation and characterization of temperature-sensitive mak mutants of Saccharomyces cerevisiae
Authors: Guerry-Kopecko P , Wickner R B
Published: January 2020

The K1 killer plasmid of Saccharomyces cerevisiae is a 1.5-megadalton linear double-stranded ribonucleic acid molecule. Using simplified screening and complementation procedures, we have isolated mutants in three chromosomal genes that are temperature sensitive for killer plasmid maintenance or replication. One of these genes, mak28-1, was located on chromosome X. Two of the temperature-sensitive mutants rapidly lost the wild-type killer plasmid of A364A during spore germination and outgrowth at nonpermissive temperatures, but during vegetative growth, they only lowered the plasmid copy number. These two mutants did not lose two other wild-type K1 killer plasmids, indicating a heterogeneity of the killer plasmids in laboratory yeast strains.
Mechanism of additive genetic transformation in Haemophilus influenzae
Authors: Stuy J H
Published: January 2020

Transforming deoxyribonucleic acid (DNA) preparations from Haemophilus influenzae Rd strains carrying a chromosomally integrated, conjugative, antibiotic resistance transfer (R) plasmid were exposed to ultraviolet radiation and then assayed for antibiotic resistance transfer on sensitive wild-type Rd competent suspensions and on similar suspensions of a uvr-1 mutant unable to excise pyrimidine dimers. No host cell reactivation of resistance transfer (DNA repair) was observed. Parallel experiments with ethanol-precipitated, heated, free R plasmid DNA preparations gave much higher survival when assayed on the wild-type strain compared to the survival on the uvr-1 strain. These observations indicate that additive genetic transformation (in this case, the addition of the integrated R plasmid to the recipient genome) involves single-strand insertion.
Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation
Authors: Botta G A , Park J T
Published: January 2020

Furazlocillin (1 microgram/ml) and piperacillin (5 microgram/ml) bound specifically to penicillin-binding protein 3 (PBP-3) and not to the other major PBPs in intact Escherichia coli cells. The effect of this specific binding to PBP-3 on murein synthesis of elongating and synchronously septating cells was investigated in two thermosensitive division mutants, E. coli BUG6 and E. coli JE10730, the latter possessing a thermolabile PBP-3. Synchronous cell division was induced by shifting the cultures from the nonpermissive temperature (42 degrees C) to 30 degrees C. Both [14C]diaminopimelic acid incorporation into murein of intact cells and [14C]N-acetylglucosamine incorporation into murein of cells permeabilized with ether was inhibited by an average of 42% in septating cells. In filaments growing at the nonpermissive temperature, we detected no inhibition and, frequently, a 10 to 15% stimulation of murein synthesis. The two drugs, at concentrations used in the above experiments, bound exclusively to PBP-3 both in elongating and septating intact cells and in ether-treated cells. These results support the hypothesis that PBP-3 activity is exclusively required for septal murein synthesis.
Biosynthesis of membrane proteins of Pseudomonas aeruginosa: effects of various antibiotics
Authors: Yasumura M , Nakamura K , Mernoff D , Inouye M
Published: January 2020

The biosynthesis of membrane proteins of Pseudomonas aeruginosa was examined using various antibiotics (puromycin, streptomycin, chloramphenicol, tetracycline, and rifampin). Among six major membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the biosynthesis of two membrane proteins (proteins I and II) was found to be unusually resistant to these antibiotics. The biosynthesis of protein I (apparent molecular weight of 6,500) was completely resistant to puromycin, streptomycin, chloramphenicol, and tetracycline at conditions which severely inhibited the biosynthesis of all the other membrane proteins except for protein II. Under the same conditions, the biosynthesis of protein II (apparent molecular weight of 9,000) was also resistant to puromycin, streptomycin, and tetracycline, but was sensitive to chloramphenicol. The effect of rifampin on the biosynthesis of proteins I and II indicated that their messenger RNAs are extremely stable; their functional half-lives are 16 and 8 min for proteins I and II, respectively, in contrast with 2.0 and 3.5 min for the average half-lives of the cytoplasmic and membrane proteins, respectively. Protein II was identified as the lipoprotein of the outer membrane from its amino acid composition and mobility in gel electrophoresis. Protein I is a cytoplasmic membrane protein lacking histidine. From the content of arginine residues, the number of protein I molecules per cell was estimated to be as many as, and most likely more than, that of the lipoprotein (protein II). Therefore, protein I is the most abundant protein in P. aeruginosa.
Distribution of Tn551 insertion sites responsible for auxotrophy on the Staphylococcus aureus chromosome
Authors: Pattee P A
Published: January 2020

A method was devised to efficiently select isolates of Staphylococcus aureus 8325 in which Tn551, a transposon originating on the pI258 plasmid responsible for erythromycin resistance (Emr), had translocated to the host chromosome. This method consisted of selecting for Emr at 43 degrees C with a strain in which the pI258 plasmid was unable to replicate at 43 degrees C because of a temperature-sensitive plasmid mutation. By selecting isolates that were Emr at 43 degrees C and auxotrophic for nutrients not required by the parent strain. Tn551-induced auxotrophic mutants were readily isolated. The incidence of auxotrophic classes was not random; 80% of the isolates in one experiment were Trp-, whereas only a single example of each of some of the other classes was isolated. Among the Trp- mutants, the distribution of trp genes affected and the frequency of precise excision of Tn551 from individual sites varied. When analyzed by transformation, the Tn551-induced ala, his, ilv, lys, rib, thrA, thrB, and trp mutations were shown to occupy sites previously defined by nitrosoguanidine-induced mutations. Tn551-induced mutagenesis provided three previously unrecognized classes of auxotrophs (tyr, met, and thrC), and the Tn551 integration sites resulting in these mutations have been identified. In addition, a chromosomal region (uraB) was identified by Tn551 mutagenesis that is distinct from uraA (previously defined by chemical mutagenesis). Some Tn551-induced mutations (most notably pur) could not be linked to the known linkage groups of the chromosome by transformation. With the exception of two pur mutations, all of the Tn551-induced auxotrophic mutational sites cotransformed at unity with Tn551 and, in cases in which they were selected, prototrophic transformants were always Ems. Thus, the Tn551 and auxotrophic sites are identical.
Isolation and properties of pili from spores of Bacillus cereus
Authors: DesRosier J P , Lara J C
Published: January 2020

Structures whose morphology is identical to that of bacterial pili have been isolated from spores of Bacillus cereus. The structures are absent from log-phase and sporulating cells. The pili are 6.8 nm in diameter, are of variable length, and appear to emanate randomly from the exosporium. Examination of spores from 12 Bacillus species showed that only those from B. cereus and B. thuringiensis have pili. Although isolated spore pili were shown to be composed of protein, their subunit nature was not discernible due to the extreme insolubility of the structure. An antiserum to spore pili was labeled with ferritin and used to examine the distribution of pilus antigen on the outer spore surface.
Use of cir-lac operon fusions to study transcriptional regulation of the colicin Ia receptor in Escherichia coli K-12
Authors: Worsham P L , Konisky J
Published: January 2020

We describe cir-lac operon fusions constructed by using phage Mu d(Apr lac). Expression of beta-galactosidase in these fusion strains is analogous to known regulatory properties of cir gene expression. It is concluded that the observed regulation by iron of the cir gene is under transcriptional control.
Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme
Authors: Harris C L
Published: January 2020

Cysteine has been shown to inhibit growth in Escherichia coli strains C6 and HfrH 72, but not M108A. Growth inhibition was overcome by inclusion of isoleucine, leucine, and valine in the medium. Isoleucine biosynthesis was apparently affected, since addition of this amino acid alone could alter the inhibitory effects of cysteine. Homocysteine, mercaptoethylamine, and mercaptoethanol inhibited growth to varying degrees in some strains, these effects also being prevented by addition of branched-chain amino acids. Cysteine, mercaptoethylamine, and homocysteine were inhibitors of threonine deaminase but not transaminase B, two enzymes of the ilvEDA operon. Cysteine inhibition of threonine deaminase was reversed by threonine, although the pattern of inhibition was mixed. These results suggest a relationship between the growth-inhibitory effects of cysteine and other sulfur compounds and the inhibition of isoleucine synthesis at the level of threonine deaminase.
Comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase as an indicator of taxonomic relatedness in pseudomonad genera
Authors: Whitaker R J , Byng G S , Gherna R L , Jensen R A
Published: January 2020

Recently, an analysis of the enzymological patterning of L-tyrosine biosynthesis was shown to distinguish five taxonomic groupings among species currently named Pseudomonas, Xanthomonas, or Alcaligenes (Byng et al., J. Bacteriol. 144:247--257, 1980). These groupings paralleled with striking consistency those previously defined by ribosomal ribonucleic acid-deoxyribonucleic acid homology relationships. The comparative allostery of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase has previously been shown to be a useful indicator of taxonomic relationship at about the level of genus. The comparative allostery of DAHP synthetase was evaluated in relationship to data available from the same pseudomonad species previously studied. Species of Xanthomonas and some named species of Pseudomonas, e.g., P. maltophilia, were unmistakably recognized as belonging to group V, having a DAHP synthetase sensitive to sequential feedback inhibition by chorismate. This control pattern is thus far unique to group V pseudomonads among microorganisms. Group V organisms were also unique in their possession of DAHP synthetase enzymes that were unstimulated by divalent cations. Group IV pseudomonads (P. diminuta) were readily distinguished by the retro-tryptophan pattern of control for DAHP synthetase. Activity for DAHP synthetase was not always recovered in group IV species, e.g., P. vesicularis. The remaining three groups exhibited overlapping patterns of DAHP synthetase sensitivity to both L-phenylalanine and L-tyrosine. Individual species cannot be reliably keyed to group I. II, or III without other data. However, each group overall exhibited a different trend of relative sensitivity to L-tyrosine and L-phenylalanine. Thus, although enzymological patterning of L-tyrosine biosynthesis alone can be used to separate the five pseudomonad groups, the independent assay of DAHP synthetase control pattern can be used to confirm assignments. The latter approach is, in fact, the easiest and most definitive method for recognition of group V (and often of group IV) species.
Characterization of a site-specific restriction endonuclease from Streptomyces aureofaciens
Authors: Timko J , Horwitz A H , Zelinka J , Wilcox G
Published: January 2020

A new type II sequence-specific restriction endonuclease, SauI, was isolated from Streptomyces aureofaciens IKA18/4. The purified enzyme was free of contaminating exonuclease and phosphatase activities. SauI cleaved lambda DNA at two sites, but did not cleave pBR322, simian virus 40, or phi X174 DNA. SauI recognized the septanucleotide sequence 5'-CCTNAGG-3' and cleaved at the position indicated by the arrow, producing a trinucleotide 5'-terminal extension.
Natural heterozygosity in Candida albicans
Authors: Whelan W L , Magee P T
Published: January 2020

We subjected 16 Candida albicans clinical isolates to ultraviolet radiation and tested the survivors for auxotrophy. Six isolates displayed strongly biased auxotroph spectra: three yielded methionine auxotrophs, two yielded both isoleucine-valine and adenine auxotrophs, and one yielded lysine auxotrophs. We present evidence that auxotrophs arise by segregation from naturally occurring heterozygous states. The remaining isolates yielded few or no auxotrophs in an arbitrary sample (greater than 2,500) of survivors of irradiation. Our experiments indicate that C. albicans is diploid, although aneuploidy (2n + i) cannot be rigorously excluded. We discuss the possible utility of heterozygosity as a marker in epidemiological studies, and we discuss a rationale for the frequent occurrence of heterozygosity.
Comparative bioenergetics of sulfate reduction in Desulfovibrio and Desulfotomaculum spp
Authors: Liu C L , Peck H D
Published: January 2020

Extracts of Desulfotomaculum nigrificans, Desulfotomaculum orientis, and Desulfotomaculum ruminis exhibit low levels of inorganic pyrophosphatase but were found to have high levels of pyrophosphate:acetate phosphotransferase. Conversely, extracts of Desulfovibrio gigas, Desulfovibrio vulgaris, and Desulfovibrio desulfuricans Norway 4 were shown to have high levels of inorganic pyrophosphatase but negligible amounts of pyrophosphate:acetate phosphotransferase. Both enzymes are reductant activated and appear to have an analogous function in removing pyrophosphate formed during the activation of sulfate. Conservation of the bond energy of pyrophosphate in Desulfotomaculum eliminates the necessity for invoking electron-transfer-coupled phosphorylation to account for the growth of these bacteria on lactate plus sulfate. Relative growth yields of Desulfovibrio vulgaris and Desulfotomaculum orientis on lactate plus sulfate indicate that the latter does not carry out significant electron-transfer-coupled phosphorylation in this mode of growth.