bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutants of Plasmid Prophage P1 with Elevated Copy Number: Isolation and Characterization
Authors: Scott June R. , Kropf Martha M. , Padolsky Linda , Goodspeed James K. , Davis Richard , Vapnek Daniel
Published: January 2020

A series of mutations of the P1 plasmid prophage that lead to increased copy number was isolated and analyzed. The copy number of the mutants was elevated at least five- to eightfold relative to wild-type P1, as determined by single-cell resistance to antibiotics, activity of enzymes, content of superhelical DNA, and reassociation kinetics. The copy number of two of the mutants was temperature dependent. Based on dominance tests, the mutants fell into two classes,
cis
specific and recessive. The latter class included a temperature-sensitive copy mutant. The existence of a class of recessive mutants suggests that the replication of the P1 plasmid is negatively regulated.
The cloacin receptor of ColV-bearing Escherichia coli is part of the Fe3+-aerobactin transport system
Authors: Bindereif A , Braun V , Hantke K
Published: January 2020

Escherichia coli strains which contain the Fe3+-aerobactin transport system specified by the ColV plasmid became deficient in aerobactin-dependent iron transport when they were converted to cloacin-resistant derivatives. An outer membrane protein with a molecular mass of 74,000 daltons was overproduced under iron-limiting growth conditions and was absent in cloacin-resistant mutants. Fe3+-aerobactin protected cells against cloacin. These results suggest that the cloacin receptor protein, controlled by the colV plasmid, also participates in Fe3+-aerobactin transport.
Replication terminus of the Bacillus subtilis chromosome
Authors: O'Sullivan M A , Anagnostopoulos C
Published: January 2020

Bidirectional replication of the Bacillus subtilis chromosome terminates at a point on the circular chromosome which is symmetrically opposite to the replication origin. Since replication rates are similar in both "halves" of the chromosome, termination presumably occurs at the meeting point of the two replication forks. To investigate whether the DNA sequence of this region of the chromosome contributes to the termination event, we have determined the latest replicating region of a chromosome in which this DNA sequence is no longer symmetrically opposite to the origin. The merodiploid strain GSY1127 has a very large nontandem duplication (approximately 25% of the total chromosome length) in the left-hand half of the chromosome, so that size and symmetry of this chromosome are grossly different from those of normal strains. We have examined the replication order of genetic markers in this strain by measuring subtilis terminal marker for replication remains a terminal marker in the merodiploid, i.e., replicates later than a marker situated symmetrically opposite to the replication origin. These results were supported by replication orders determined by pulse-density transfer experiments during synchronous replication. The data obtained indicate that there is a preferred site for the termination of replication in the B. subtilis chromosome.
Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes
Authors: Friedman S
Published: January 2020

5-Azacytidine was found to be bactericidal to Escherichia coli carrying plasmids specifying EcoRII restriction-modification systems, but not to the same strains lacking these plasmids. Of other base analogs tested, only 5(beta-D-ribofuranosyl)isocytidine had similar, although weaker, effects. Plasmids that had lost the EcoRII restriction-modification system did not confer sensitivity to 5-azacytidine. Mutants defective in the restriction function remained sensitive to the toxic effects of the drug; however, a mutant defective in the modification function lost most of the sensitivity to 5-azacytidine. For the bactericidal effect to be seen, the cells had to be growing; cells in the stationary phase of growth were not killed by the drug. The drug inhibited the methylase enzyme, and an inhibitor of the enzyme could be detected in vitro in extracts of cells that had been treated with 5-azacytidine. This nalidixic acid inhibited its formation. Coumermycin but not nalidixic acid antagonized the bactericidal effect of the drug; however, coumermycin was more effective in preventing the inhibition of the methylase by 5-azacytidine than was nalidixic acid.
Gliding motility of Cytophaga sp. strain U67
Authors: Lapidus I R , Berg H C
Published: January 2020

Video techniques were used to analyze the motion of the gliding bacterium Cytophaga sp. strain U67. Cells moved singly on glass along the long axis at a speed of about 2 micrometers/s, advancing, retreating, stopping, pivoting about a pole, or flipping over. They did not flex or roll. Cells of different lengths moved at about the same speed. Cells sometimes spun continuously about a pole at a frequency of about 2 HZ, the body moving in a plane parallel to that of the glass or on the surface of a cone having either a large or a small solid angle. Polystyrene latex spheres moved to and fro on the surfaces of cells, also at a speed of about 2 micrometers/s. They moved in the same fashion whether a cell was in suspension, gliding, or at rest on the glass. Two spheres on the same cell often moved in opposite directions, passing by one another in close proximity. Small and large spheres and aggregates of spheres all moved at about the same speed. An aggregate moved down the side of a cell with a fixed orientation, even when only one sphere was in contact with the cell. Spheres occasionally left one cell and were picked up by another. Cell pretreated with small spheres did not adhere to glass. When the cells were deprived of oxygen, they stopped gliding, and the spheres stopped moving on their surfaces. The spheres became completely immobilized; they no longer moved from cell to cell or exhibited Brownian movement. Cytophaga spp. are known to have a typical gram-negative cell envelope: an inner (cytoplasmic) membrane, a thin peptidoglycan layer, and an outer (lipopolysaccharide) membrane. Our data are consistent with a model for gliding in which sites to which glass and polystyrene strongly adsorb move within the fluid outer membrane along tracks fixed to the rigid peptidoglycan framework.
Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B
Authors: Eaton R W , Ribbons D W
Published: January 2020

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.
Determination of the transcription direction of the exuT gene in Escherichia coli K-12: divergent transcription of the exuT-uxaCA operons
Authors: Hugouvieux-Cotte-Pattat N , Robert-Baudouy J
Published: January 2020

The exuT gene of Escherichia coli, coding for the hexuronate transport system, was fused to lac genes by the use of Mu d(Apr lac) insertions (M. J. Casadaban and S. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979). The method of chromosome mobilization with F' lac::Mu episomes (J. B. Zeldis, A. I. Bukhari, and D. Zipser, Virology 55:289-294, 1974) made it possible to determine the transcription direction of the exuT gene from the orientation of the Mu d(Apr lac) insertion in the fusion strains. Our results for a exuT-lac fusion strain suggest that the direction of transcription of other single gene operon exuT is clockwise on the standard E. coli map and confirm that the direction of transcription of uxaC is counterclockwise. The two close operons exuT and uxaCA are thus transcribed in opposite directions.
Effects of Growth Temperature on Ribosomes and Other Physiological Properties of
Escherichia coli
Authors: Zaritsky Arieh
Published: January 2020

The amount of ribosomal protein relative to total protein remained constant when
Escherichia coli
B/r was grown at different temperatures. Some effects of growth temperature on bacterial physiology are discussed.
Marker rescue by a homologous recipient plasmid during transformation of gonococci by a hybrid Pcr plasmid
Authors: Biswas G D , Graves J F , Sox T E , Tenover F C , Sparling P F
Published: January 2020

A 42-kilobase hybrid Pcr plasmid (pFA14) was formed when the naturally occurring 7.2-kilobase Pcr plasmid pFA3 was introduced by transformation into a competent gonococcal recipient containing the 36-kilobase conjugative plasmid pFA2 (Sox et al., J. Bacteriol. 138:510-518). Analysis of the structure of pFA14 showed that it was a stable recombinant between pFA3 and pFA2. The transformation efficiency of pFA14 was increased 300- to 10,000-fold by the presence in isogenic recipients of the homologous plasmid pFA2. The presence of a homologous plasmid in the recipient also markedly increased the likelihood of recovery of intact donor-size Pcr plasmids in the transformants. The presence of pFA2 had no effect on the competence of piliated or nonpiliated gonococci for transformation by either linear chromosomal DNA or a nonhomologous Pcr plasmid. Increased transformation efficiency of the hybrid Pcr plasmid pFA14 may have been due to recombination between the nicked or linearized donor plasmid and the homologous recipient plasmid (marker rescue).
Evidence that Bacillus subtilis sporulation induced by the stringent response is caused by the decrease in GTP or GDP
Authors: Ochi K , Kandala J , Freese E
Published: January 2020

Partial amino acid deprivation of Bacillus subtilis, which evokes the stringent response, initiates sporulation not because the highly phosphorylated guanine nucleotides guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) increase but because GTP decreases. This was shown with a mutant (Myc) partially resistant to mycophenolate, an inhibitor of IMP dehydrogenase. Upon amino acid deprivation, the Myc mutant (62032) showed the usual increase in ppGpp and pppGpp but a reduced decrease in GTP, and only few cells sporulated. Extensive sporulation was restored by the addition of mycophenolate or decoyinine, and inhibitor of GMP synthetase, which caused a further decrease in GTP.
Genetic and enzymatic characterization of the inducible glycerol dissimilatory system of Neurospora crassa
Authors: Denor P F , Courtright J B
Published: January 2020

The glycerol dissimilatory system in Neurospora crassa was analyzed through the characterization of 18 Glp- mutants which were isolated after inositol-less death and filtration enrichment. All mutants obtained by this procedure could be assigned to one of three complementation groups. The subsequent genetic characterization of these glp mutations revealed lesions on the I, II, and VI chromosomes at the glp-1, glp-2, and glp-4 loci, each of which was subjected to fine-structure analysis. Evidence from the enzymatic characterization of these mutants indicated that glp-2 and glp-4 were the structural genes encoding the mitochondrial glycerol-3-phosphate dehydrogenase and cytosolic glycerokinase, respectively. Additional evidence, obtained from studies of the inducibility of glycerokinase by glycerol, cold treatment, or deoxyribose, suggests that glp-1 is involved in controlling the expression of glp-4.
Lysine biosynthesis in Rhodotorula glutinis: properties of pipecolic acid oxidase
Authors: Kinzel J J , Bhattacharjee J K
Published: January 2020

Pipecolic acid oxidase from Rhodotorula glutinis, which converts pipecolic acid to alpha-aminoadipic-delta-semialdehyde, an intermediate of the biosynthetic pathway of lysine, was purified 290-fold. The enzyme from the crude extract and purified preparation exhibited a molecular weight of approximately 43,000 and was composed of a single subunit. The purified enzyme was heat labile and exhibited a pH optimum of 8.5 and an apparent Km for L-pipecolic acid of 1.67 X 10(-3) M. L-Proline acted as a competitive inhibitor for the enzyme. The enzyme was inhibited by the sulfhydryl agents p-chloromercuribenzoate and mercuric chloride. The in vitro enzyme activity required oxygen and upon oxidation of pipecolic acid, oxygen was reduced to hydrogen peroxide.
Genetic analysis of Candida albicans: identification of different isoleucine-valine, methionine, and arginine alleles by complementation
Authors: Kakar S N , Magee P T
Published: January 2020

By using the spheroplast fusion technique as a tool for genetic analysis, we have demonstrated complementation among three of four isoleucine-valine mutants, two of three methionine mutants, and two arginine mutants of independent origin from two different Candida albicans isolates. The two adenine mutants derived from the same parent strain did not complement. Complementation resulted predominantly from heterokaryon formation and, in some cases, from heterozygote formation. In either case, most fusion products were unstable and showed nuclear as well as chromosomal segregation, in a few cases resulting in recombination of parental auxotrophic markers. However, some fusion products were fairly stable.
Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12
Authors: Stewart V
Published: January 2020

I used a chlC-lac operon fusion to study regulatory mutations which affect nitrate reductase expression in Escherichia coli. A NarL- mutant apparently lacks a nitrate-specific positive regulatory component. Furthermore, an fnr (nirR) mutation prevented enzyme induction under any conditions. These data are consistent with a two-step, positive control model for nitrate reductase regulation.
Differential polypeptide synthesis of the proton-translocating ATPase of Escherichia coli
Authors: Brusilow W S , Klionsky D J , Simoni R D
Published: January 2020

We investigated the regulation of the synthesis of the eight polypeptides of the Escherichia coli proton-translocating ATPase. A plasmid carrying the eight genes of the unc operon was used to direct in vivo and in vitro protein synthesis of the eight polypeptides. Analysis of these data indicates that the ATPase polypeptides are synthesized in unequal amounts both in vitro and in vivo. We identified several regions within the unc operon at which expression of a gene is either increased or decreased from that of the preceding gene. Since genetic information indicates a single polycistronic mRNA for all eight genes of this operon, the observed differential synthesis of the polypeptides is most likely the result of translational regulation. The effect of varying the temperature suggests that the secondary structure in the mRNA may affect the rate of translation initiation in the region between uncE and uncF.
2-micrometers DNA-like plasmids in the osmophilic haploid yeast Saccharomyces rouxii
Authors: Toh-e A , Tada S , Oshima Y
Published: January 2020

DNA plasmids were detected in two independent strains of Saccharomyces rouxii among 100 yeast strains other than Saccharomyces cerevisiae tested. The plasmids, pSR1 and pSR2, had almost the same mass (approximately 4 X 10(6) daltons) as 2-micrometers DNA of S. cerevisiae. pSR1 and pSR2 gave identical restriction maps with restriction endonucleases BamHI, EcoRI, HincII, HindIII, and XhoI, and both lacked restriction sites for PstI, SalI, and SmaI. These maps, however, differed significantly from that of S. cerevisiae 2-micrometers DNA. Restriction analysis also revealed two isomeric forms of each plasmid and suggested the presence of a pair of inverted repeat sequences in the molecules where intramolecular recombination took place. DNA-DNA hybridization between the pSR1 and pSR2 DNAs indicated significant homology between their base sequences, whereas no homology was detected between pSR1 and pJDB219, a chimeric plasmid constructed from a whole molecule of 2-micrometers DNA, plasmid pMB9, and a 1.2-kilobase DNA fragment of S. cerevisiae bearing the LEU2 gene. A chimeric plasmid constructed with pSR1 and YIp1, the larger EcoRI-SalI fragment of pBR322 ligated with a 6.1-kilobase DNA fragment of S. cerevisiae bearing the HIS3 gene, could replicate autonomously in an S. cerevisiae host and produced isomers, presumably by intramolecular recombination at the inverted repeats.
Formation of 9-nm filaments from pilin monomers obtained by octyl-glucoside dissociation of Pseudomonas aeruginosa pili
Authors: Watts T H , Scraba D G , Paranchych W
Published: January 2020

Pili isolated from Pseudomonas aeruginosa PAK were solubilized in the detergent octyl-glucoside. Subsequent removal of the detergent by dialysis resulted in the formation of short rods of about 9 nm in diameter and various lengths. The aggregation process was followed by ultracentrifugation, viscometry, and electron microscopy to show that the aggregates produced in this way are distinct from pili produced by the bacteria, both in diameter, as measured by electron micrographs, and in their inability to compete in a biological assay for pili.
Box-shaped halophilic bacteria
Authors: Javor B , Requadt C , Stoeckenius W
Published: January 2020

Three morphologically similar strains of halophilic, box-shaped procaryotes have been isolated from brines collected in the Sinai, Baja California (Mexico), and southern California (United States). Although the isolates in their morphology resemble Walsby's square bacteria, which are a dominant morphological type in the Red Sea and Baja California brines, they are probably not identical to them. The cells show the general characteristics of extreme halophiles and archaebacteria. They contain pigments similar to bacteriorhodopsin which apparently mediate light-driven ion translocation and photophosphorylation.
Oxygen inactivation and recovery of nitrogenase activity in cyanobacteria
Authors: Pienkos P T , Bodmer S , Tabita F R
Published: January 2020

Exposure of nitrogen-fixing cultures of Anabaena spp. to 100% oxygen resulted in the rapid decline of nitrogenase activity. When oxygen-treated cells were transferred to 100% argon, nitrogenase activity was quickly restored in a process that required protein synthesis. Anaerobiosis was not essential for the recovery process; in fact, cells of Anabaena sp. strains CA and 1F will recover nitrogenase activity after prolonged incubation in 100% oxygen. Oxygen treatment acted directly on the intracellular nitrogenase and did not affect other metabolic processes. Examination of crude extracts of oxygen-treated Anabaena sp. strain CA indicated that both components of nitrogenase are inactivated. However, several lines of evidence suggest that oxygen treatment does not result in irreversible denaturation of nitrogenase, but rather results in a reversible inactivation which may serve as a protection mechanism. Nitrogenase present in crude extracts from cells of Anabaena sp. strain 1F which had been incubated for a prolonged period in 100% oxygen was less sensitive to oxygen in vitro than was nitrogenase of a crude extract of untreated cells.
Expression of a Streptococcus mutans glucosyltransferase gene in Escherichia coli
Authors: Robeson J P , Barletta R G , Curtiss R
Published: January 2020

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrose-hydrolyzing activity by selection for growth on raffinose in the presence of isopropyl-beta-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded beta-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes the formation of fructose and a glucan with an apparent molecular weight of 1,500. We detected the gtfA protein in S. mutans cells with antibody raised against the cloned gtfA enzyme. Immunologically identical gtfA protein appears to be present in S. mutans cells of serotypes c, e, and f, and a cross-reacting protein was made by serotype b cells. Proteins from serotype a, g, and d S. mutans cells did not react with antibody to gtfA enzyme. The gtfA activity was present in the periplasmic space of E. coli clones, since 15% of the total gtfA activity was released by cold osmotic shock and the clones were able to grow on sucrose as sole carbon source.
Role of primary structure and disulfide bond formation in beta-lactamase secretion
Authors: Pollitt S , Zalkin H
Published: January 2020

Plasmid pBR322-encoded beta-lactamase was shown to contain a single disulfide bond, which caused the protein to migrate faster in sodium dodecyl sulfate-polyacrylamide gels than the fully reduced form. A similar difference in mobility of the in vitro synthesized precursor before and after reduction indicates that it also contained a disulfide bond. Formation of the disulfide bond in vivo, however, occurred concomitant with processing. In vivo accumulation of the precursor by inhibition of secretion did not allow disulfide bond formation to occur. This result is consistent with post-translational translocation of the precursor. Synthesis of a fragment of beta-lactamase lacking the carboxy terminus was obtained by insertion of a foreign DNA segment into the PstI site of bla. Processing and secretion of the protein did not appear to be greatly affected, indicating that the carboxy terminus is not required for secretion.
Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2
Authors: Shanabruch W G , Rein R P , Behlau I , Walker G C
Published: January 2020

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.
Constitutive uptake and degradation of fatty acids by Yersinia pestis
Authors: Moncla B J , Hillier S L , Charnetzky W T
Published: January 2020

Yersinia pestis was found to utilize palmitic acid as a primary carbon and energy source. No inhibition of growth by palmitic acid was observed. Comparison of palmitic acid uptake by cells pregrown either with or without palmitic acid demonstrated that fatty acid uptake was constitutive. High basal levels of two enzymes of beta-oxidation, beta-hydroxyacyl-coenzyme A dehydrogenase and thiolase, and the two enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, were found in cells grown in defined medium with glucose. Elevated levels of all four enzymes were found when cells were grown with acetate as a primary carbon and energy source, and even higher levels were observed when palmitic acid was provided as a primary carbon and energy source. High-pressure liquid chromatography was used to demonstrate that, in the presence of glucose, uniformly labeled [14C]palmitic acid was converted to intermediates of the tricarboxylic acid cycle and glyoxylate shunt. Pregrowth with palmitic acid was not required for this conversion. Strains lacking the 6- or the 47-megadalton plasmid did not take up [3H]palmitic acid but did possess levels of enzyme activity comparable to those observed in the wild-type strain.
CFA/I-ST plasmids: comparison of enterotoxigenic Escherichia coli (ETEC) of serogroups O25, O63, O78, and O128 and mobilization from an R factor-containing epidemic ETEC isolate
Authors: Murray B E , Evans D J , Penãranda M E , Evans D G
Published: January 2020

Colonization factor antigen I (CFA/I) plays an important role in the pathogenesis of diarrhea due to enterotoxigenic Escherichia coli. In this study, we examined 11 CFA/I+ enterotoxigenic E. coli from serogroups O25, O63, O78, and O128 and found that with all strains, spontaneous loss of CFA/I was associated with the loss of heat-stable toxin (ST) and with the loss of a single plasmid ranging in size from 54 to 60 megadaltons; when heat-labile toxin was lost, this was associated with the loss of another plasmid. The R factor of one strain, TX432 (O78:H12:CFA/I+; ST+), was found to mobilize the CFA/I-ST plasmid into E. coli K-12 at a frequency of 20%. These studies provide further evidence that CFA/I production is plasmid mediated in enterotoxigenic E. coli belonging to serogroups O25, O63, O78, and O128.
Location on the
Salmonella typhimurium
Chromosome of the Gene Encoding Nucleoside Diphosphokinase (
ndk
)
Authors: Rodriguez Susan B. , Ingraham John L.
Published: January 2020

The gene encoding nucleosidediphosphate kinase (
ndk
) was located at 55 units on the
Salmonella typhimurium
chromosome. The
ndk
locus was 83% cotransducible with
hisS
and 2% cotransducible with
glyA
in phage P22-mediated crosses. A nucleosidediphosphate kinase mutant that produced only 10% of the wild-type enzyme activity (
ndk-1
) grew normally and produced a heat-labile enzyme.
Molecular cloning and expression of a gene that controls the high-temperature regulon of Escherichia coli
Authors: Neidhardt F C , VanBogelen R A , Lau E T
Published: January 2020

The high-temperature production (HTP) regulon of Escherichia coli consists of a set of operons that are induced coordinately by a shift to a high temperature under the control of a single chromosomal gene called htpR or hin. To identify more components of this regulon, the rates of synthesis of many polypeptides resolved on two-dimensional polyacrylamide gels were measured in various strains by pulse-labeling after a temperature shift-up. A total of 13 polypeptides were found to be heat inducible only in cells bearing a normal htpR gene on the chromosome or on a plasmid; on this basis these polypeptides were designated products of the HTP regulon. Several hybrid plasmids that contain segments of the E. coli chromosome in the 75-min region were found to carry the htpR gene. A restriction map of this region was constructed, and selected fragments were subcloned and tested for the ability to complement an htpR mutant. The polypeptides encoded by these fragments were detected by permitting expression in maxicells, minicells, and chloramphenicol-treated cells. Complementation was accompanied by production of a polypeptide having a molecular weight of approximately 33,000. This polypeptide, designated F33.4, was markedly reduced in amount in an htpR mutant expected to contain very little htpR gene product. Polypeptide F33.4 is postulated to be the product of htpR and to be an effector that controls heat induction of the HTP regulon.
Escherichia coli
Kasugamycin Dependence Arising from Mutation at the
rpsI
Locus
Authors: Dabbs Eric R.
Published: January 2020

Escherichia coli
mutants with alterations in the electrophoretic mobility of ribosomal protein S9 were used to locate
rpsI
, the gene for this protein, on the linkage map.
rpsI
was located at about 70 min, roughly halfway between
argG
and
fabE
. It was very close to the gene for ribosomal protein L13,
rplM
. Another mutation at the
rpsI
locus gave rise to a phenotype of kasugamycin dependence and resistance. In this mutant, dependence on antibiotic came from kasugamycin being necessary to slow the rate of protein synthesis.
Construction of hybrid plasmids containing the Escherichia coli uxaB gene: analysis of its regulation and direction of transcription
Authors: Blanco C , Mata-Gilsinger M , Ritzenthaler P
Published: January 2020

The uxaB gene of Escherichia coli, encoding for altronate oxidoreductase involved in the hexuronate degradative pathway, was isolated on a ColE1-uxaB hybrid plasmid from the Clarke and Carbon bank. The restriction map of this plasmid was established. The uxaB gene was mapped on a 1.5-megadalton HindIII-KpnI DNA fragment. Use of an in vitro gene fusion between uxaB and lacZ genes led to the determination that uxaB is transcribed from the KpnI towards the HindIII restriction sites. Gene amplification in cells containing various uxaB hybrid plasmids allowed us to show a gradation in the level of repression of exu operator sites by the exuR regulatory gene product.
Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes
Authors: Coleman D C , Chopra I , Shales S W , Howe T G , Foster T J
Published: January 2020

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.
Localization of the major antigenic determinant of EDP208 pili at the N-terminus of the pilus protein
Authors: Worobec E A , Taneja A K , Hodges R S , Paranchych W
Published: January 2020

Trypsin digestion of pilin monomers from EDP208 conjugative pili causes cleavage of Lys12 to yield an N-terminal dodecapeptide, ET1 (Mr approximately equal to 1,500), and the remaining C-terminal fragment, ER (Mr approximately equal to 10,000). Using the amino acid sequence for ET1 provided by Frost et al. (J. Bacteriol. 153:950-954), we synthesized the N-terminal dodecapeptide chemically, conjugated it to bovine serum albumin, and subjected it to immunological studies. Antisera prepared against intact EDP208 pili as well as against the synthetic ET1-BSA conjugate were used in experiments involving an enzyme-linked immunosorbant assay and electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Both experimental approaches showed strong reactivity between the synthetic dodecapeptide and antiserum raised against whole pili. It was also found that antiserum raised against the synthetic peptide was reactive against intact pilus protein, indicating that the N-terminal dodecapeptide is an important antigenic determinant of the EDP208 pilus protein. Additional studies showed that the C-terminal fragment, ER, may contain one or two additional antigenic sites.
Cell elongation and division probability during the Escherichia coli growth cycle
Authors: Kubitschek H E , Woldringh C L
Published: January 2020

A new method is presented for determining the growth rate and the probability of cell division (separation) during the cell cycle, using size distributions of cell populations grown under steady-state conditions. The method utilizes the cell life-length distribution, i.e., the probability that a cell will have any specific size during its life history. This method was used to analyze cell length distributions of six cultures of Escherichia coli, for which doubling times varied from 19 to 125 min. The results for each culture are in good agreement with a single model of growth and division kinetics: exponential elongation of cells during growth phase of the cycle, and normal distributions of length at birth and at division. The average value of the coefficient of variation was 13.5% for all strains and growth rates. These results, based upon 5,955 observations, support and extend earlier proposals that growth and division patterns of E. coli are similar at all growth rates and, in addition, identify the general growth pattern of these cells to be exponential.
Two alanine racemase genes in Salmonella typhimurium that differ in structure and function
Authors: Wasserman S A , Walsh C T , Botstein D
Published: January 2020

Mutations were isolated in a previously undescribed Salmonella typhimurium gene encoding an alanine racemase essential for utilization of L-alanine as a source of carbon, energy, and nitrogen. This new locus, designated dadB, lies within one kilobase of the D-alanine dehydrogenase locus (dadA), which is also required for alanine catabolism. The dadA and dadB genes are coregulated. Mutants (including insertions) lacking the dadB alanine racemase do not require D-alanine for growth unless a mutation is introduced at a second locus, designated dal. Two genes specifying alanine racemase activity were cloned from S. typhimurium. The two cloned DNA sequences do not cross-hybridize with each other; one was shown to contain the dadB gene.
cea-kil operon of the ColE1 plasmid
Authors: Sabik J F , Suit J L , Luria S E
Published: January 2020

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.
Differentiation after premature release of intraperiplasmically growing Bdellovibrio bacteriovorous
Authors: Ruby E G , Rittenberg S C
Published: January 2020

Bdellovibrio bacteriovorous attacks and penetrates other gram-negative bacteria, creating a growth chamber termed a bdelloplast. We have found that exposing the bdelloplasts to EDTA, followed by treatment with a lytic enzyme concentrate derived from bdellovirio cultures, prematurely released the intraperiplasmically growing bdellovibrios at any time during their growth cycle. Upon release, the growth-form bdellovibrios terminated any initiated rounds of DNA synthesis and differentiated into motile attack-form cells. The ability of growth-form cells to synthesize DNA appears to depend upon an initiation signal that is not received until about 60 min after attack. Each subsequent round of DNA synthesis by the growing bdellovibrio filaments seems to require an additional initiation signal that is provided by their intraperiplasmic environment. Differentiation included fragmentation into multiple progeny cells to a degree proportional to the extent of intraperiplasmic growth. This differentiation could be performed totally at the expense of cellular reserves. The significance of these data to an understanding of the regulation of differentiation in bdellovibrios is discussed.
Maintenance of the 2 microns circle plasmid in populations of Saccharomyces cerevisiae
Authors: Futcher A B , Cox B S
Published: January 2020

The 2 microns circle plasmid is maintained at high frequencies in populations of yeast cells. To find out how the plasmid is maintained, three forces were measured: the selective advantage or disadvantage conferred by 2 microns circles, the rate of generation of [Cir0] cells, and the rate of illegitimate transfer of 2 microns circles from cell to cell. It was found that under the conditions used, 2 microns circles confer a selective disadvantage of about 1%, that [Cir0] cells are generated at the rate of 7.6 x 10(-5) per [Cir+] cell per generation, and that illegitimate transfer of 2 microns circles occurs at a rate less than 10(-7) per recipient cell per generation. The most likely explanation of 2 microns circle maintenance is that the plasmid is sexually transmitted at such a rate that it spreads through populations despite selection against it.
Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry
Authors: Skarstad K , Steen H B , Boye E
Published: January 2020

The cell cycle kinetics of Escherichia coli B/r A and B/r K cells were studied by flow cytometry. Three-dimensional histograms of cell cultures show the number of cells as a function of cellular DNA and protein contents and give detailed pictures of the cell cycle distribution with regard to these parameters. Histograms of slowly growing chemostat cultures showed that cell cycle periods B and C + D increase with a decreasing growth rate and that the B period occupies an increasing fraction of the cycle. The DNA replication patterns of B/r A and K were found to be quite similar. At extremely low growth rates (doubling time [T] = 17 h), B/r A cells had a B period of 0.8 T, a C period of 0.1 T, and a D period of 0.1 T, and B/r K cells (T = 16 h) had a B period of 0.6 T, a C period of 0.15 T, and a D period of 0.25 T. Mass increase, i.e., essentially protein synthesis, was seen in all three periods of the cell cycle. For B/r A cells, the average rate of mass increase was 11 times greater in the D period than in the B period, whereas for B/r K cells the rate of mass increase was twice as great in the D period as in the B period. The DNA and cell size distributions of batch cultures in exponential growth were found to vary with time, indicating that such cultures are not suitable for studies of cell cycle kinetics.
Inhibition of DNA replication by transformation in a Haemophilus influenzae mutant carrying an altered Rec-1 protein
Authors: Kooistra J , van Boxel T , Venema G
Published: January 2020

In the HM5 mutant of Haemophilus influenzae, which carries a mutation in the rec-1 gene region and in which the replication of donor-recipient DNA complexes formed in transformation is inhibited, the transformation frequency could be greatly enhanced by inhibition of protein synthesis during transformation, indicating that transformation in the HM5 mutant induces the synthesis of a protein that inhibits the replication of the donor-recipient DNA complexes. This induction occurred in an early step of the recombination. Synthesis of the wild-type Rec-1 protein after transformation of the HM5 mutant with wild-type DNA could diminish the inhibiting effect on DNA replication. The HM5 mutant synthesized an altered Rec-1 protein (molecular weight, 38,000) whose pI differed from that of the wild type. As a result of the mutation in the rec-1 gene, two other proteins (molecular weights, 37,500 and 43,000) are lacking in the HM5 mutant.
Effects of aerobic and anaerobic shock on catabolite repression in cyclic AMP suppressor mutants of Escherichia coli
Authors: Lee J H , Dobrogosz W J
Published: January 2020

Cultures of Escherichia coli K-12 grown on glucose or gluconate under aerobic conditions exhibited catabolite repression of beta-galactosidase synthesis. Depression occurred when these cultures were subjected to anaerobic shock. These states of repression and depression were found to be associated with low and high differential rates of cyclic AMP synthesis, respectively. This observation is consistent with the view that cyclic AMP plays a central role in the catabolite repression phenomenon. We report here, however, that identical stages of repression and derepression occur in mutant strains possessing cya crp(Csm) genotypes and therefore unable to synthesize cyclic AMP. These results suggest that cyclic AMP is not the sole regulator involved in catabolite repression.
Plasmid marker rescue transformation in Bacillus subtilis
Authors: Weinrauch Y , Dubnau D
Published: January 2020

We constructed an 18-megadalton plasmid (pBD221) carrying resistance determinants for kanamycin, chloramphenicol, and erythromycin, as well as the hisH determinant from the Bacillus licheniformis chromosome. This plasmid has a copy number of about one and can be stably maintained in Bacillus subtilis. Linear fragments of pBD221 DNA were used to transform competent cultures carrying mutant variants of the same plasmid. Rescue transformation did not proceed by recircularization and replication of the donor DNA. Rescue transformation exhibited first-order dependence on DNA concentration, and the concentration dependence curve was virtually identical to the curve obtained with chromosomal DNA. The donor DNA molecular weight dependence of plasmid marker rescue transformation obtained by using restriction fragments was not distinguishable from previously published data obtained by using fractionated sheared chromosomal DNA. Plasmid rescue transformation, like chromosomal transformation, was dependent on the recE, recA, recB, and recD gene products. Plasmid rescue transformation, like chromosomal transformation, proceeded with few exchanges. Linkage data obtained with the plasmid rescue system fit a quantitative model based on studies with chromosomal transformation. We conclude that plasmid marker rescue transformation probably proceeds by a mechanism similar to the mechanism used during the formation of chromosomal transformants and hence may be considered an appropriate general model for the study of transformational recombination.
6-Aminonicotinamide-resistant mutants of Salmonella typhimurium
Authors: Hughes K T , Cookson B T , Ladika D , Olivera B M , Roth J R
Published: January 2020

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.
Model for bacterial culture growth rate throughout the entire biokinetic temperature range
Authors: Ratkowsky D A , Lowry R K , McMeekin T A , Stokes A N , Chandler R E
Published: January 2020

The "square-root" relationship proposed by Ratkowsky et al. (J. Bacteriol. 149:1-5, 1982) for modeling the growth rate of bacteria below the optimum growth temperature was extended to cover the full biokinetic temperature range. Two of the four parameters of this new nonlinear regression model represent minimum and maximum temperature bounds, respectively, for the predicted growth of the culture. The new model is easy to fit and has other desirable statistical properties. For example, the least-squares estimators of the parameters of the model were almost unbiased and normally distributed. The model applied without exception to all bacterial cultures for which we were able to obtain data. Results for 30 strains are reported.
Distribution of polyamines in methanogenic bacteria
Authors: Scherer P , Kneifel H
Published: January 2020

Members of all four families of methanogenic bacteria were analyzed for polyamine concentrations. High-performance liquid chromatography analysis of dansylated cell extracts revealed typical polyamine patterns for each family. Members of Methanobacteriaceae (family I) were characterized by very low polyamine concentrations; members of Methanococcaceae (family II) were characterized by putrescine and high spermidine concentrations; members of Methanomicrobiaceae (family III) were characterized by the presence of putrescine, spermidine, and sym-homospermidine; and members of Methanosarcinaceae (family IV) contained only high concentrations of sym-homospermidine in addition to putrescine. The highest polyamine concentration was found in Methanosarcina barkeri Jülich, with 0.35% putrescine in the dry cell material. The polyamine distribution found coincides with the dendrogram based on comparative cataloguing of 16S rRNA and offers a new, rapid chemotaxonomic method for characterizing methanogenic bacteria. Variation of the growth substrates (H2-CO2, methanol, acetate, and trimethylamine) for M. barkeri resulted in quantitative but not qualitative differences in polyamine composition.
Possible role of lectins in mycoparasitism
Authors: Elad Y , Barak R , Chet I
Published: January 2020

Lectin activity in a host-mycoparasite relationship was demonstrated with Rhizoctonia solani and Trichoderma harzianum. Attachment of O but not A and B erythrocytes to hyphae occurred on R. solani but not on its mycoparasite. This phenomenon, which was Ca2+ and Mn2+ dependent, was prevented by galactose, present in T. harzianum cell walls, and by fucose.