bluuebunny/crossref_metadata_2025 huggingface dataset:
A surface polysaccharide of Escherichia coli O111 contains O-antigen and inhibits agglutination of cells by O-antiserum
Authors: Goldman R C , White D , Orskov F , Orskov I , Rick P D , Lewis M S , Bhattacharjee A K , Leive L
Published: January 2020

The repeating pentasaccharide of O-antigen from Escherichia coli O111 contains galactose, glucose, N-acetylglucosamine, and colitose, the latter representing the major antigenic determinant. Phenol extraction of this strain was previously shown to release two fractions (I and II) containing O-antigen carbohydrate, and both fractions were believed to be lipopolysaccharide. We have now characterized fractions I and II and conclude that only fraction II represents lipopolysaccharide. Fraction II contains phosphate, 2-keto-3-deoxyoctonate, beta-hydroxymyristic acid, and potent endotoxin activity, whereas fraction I was deficient in all of these properties of the lipid A and core oligosaccharide regions of lipopolysaccharide. Fractions I and II each represented 50% of the total cellular O-antigen, and both were present on the cell surface. Both fractions were metabolically stable, and no precursor-product relationship existed between them. Fraction II had a number-average molecular weight of 15,800, corresponding to an average of 12 O-antigen repeats per molecule. In contrast, fraction I had a number-average molecular weight of 354,000, corresponding to an average of 404 O-antigen repeats per molecule. Before heat treatment, cells of E. coli O111 are poorly agglutinated by O-serum; although this indicates the presence of a capsule, the corresponding K-antigen was never detected. We conclude that fraction I, when present on the cell surface, inhibits agglutination of unheated cultures of E. coli O111 by O-serum because: (i) a variant strain which lacks fraction I was agglutinated by O-serum without prior heating; (ii) erythrocytes coated with purified fraction I behaved like bacteria containing fraction I in showing inhibition of O-serum agglutination; and (iii) heat treatment released fraction I and rendered bacterial cells agglutinable in O-serum.
Novel phosphoenolpyruvate-dependent futile cycle in Streptococcus lactis: 2-deoxy-D-glucose uncouples energy production from growth
Authors: Thompson J , Chassy B M
Published: January 2020

The addition of 2-deoxy-D-glucose to cultures of Streptococcus lactis 133 that were growing exponentially on sucrose or lactose reduced the growth rate by ca. 95%. Inhibition did not occur with glucose or mannose as the growth sugar. The reduction in growth rate was concomitant with rapid accumulation of the analog in phosphorylated form (2-deoxy-D-glucose 6-phosphate) via the phosphoenolpyruvate-dependent mannose:phosphotransferase system. Within 5 min the intracellular 2-deoxy-D-glucose 6-phosphate concentration reached a steady-state level of greater than 100 mM. After maximum accumulation of the sugar phosphate, the rate of sucrose metabolism (glycolysis) decreased by only 30%, but the cells were depleted of fructose-1,6-diphosphate. The addition of glucose to 2-deoxy-D-glucose 6-phosphate preloaded cells caused expulsion of 2-deoxy-D-glucose and a resumption of normal growth. S. lactis 133 contained an intracellular Mg2+-dependent, fluoride-sensitive phosphatase which hydrolyzed 2-deoxy-D-glucose 6-phosphate (and glucose 6-phosphate) to free sugar and inorganic phosphate. Because of continued dephosphorylation and efflux of the non-metabolizable analog, the maintenance of the intracellular 2-deoxy-D-glucose 6-phosphate pool during growth stasis was dependent upon continued glycolysis. This steady-state condition represented a dynamic equilibrium of: (i) phosphoenolpyruvate-dependent accumulation of 2-deoxy-D-glucose 6-phosphate, (ii) intracellular dephosphorylation, and (iii) efflux of free 2-deoxy-D-glucose. This sequence of events constitutes a futile cycle which promotes the dissipation of phosphoenolpyruvate. We conclude that 2-deoxy-D-glucose functions as an uncoupler by dissociating energy production from growth in S. lactis 133.
Reassembly in vitro of hexagonal surface arrays in a protein-producing bacterium, Bacillus brevis 47
Authors: Tsuboi A , Tsukagoshi N , Udaka S
Published: January 2020

Bacillus brevis 47 had two protein layers (the outer and middle walls) and a peptidoglycan layer (the inner wall) and contained two major proteins with approximate molecular weights of 130,000 and 150,000 in the cell wall. Both the total and Triton-insoluble envelopes revealed a hexagonal lattice array with a lattice constant of 14.5 nm. The proteins of 130,000 and 150,000 molecular weight isolated from the Triton-insoluble envelopes were serologically different from each other and assembled in vitro on the peptidoglycan layer. A mixture of 130,000- and 150,000-molecular-weight proteins led to the formation of a five-layered cell wall structure, two layers on each side of the peptidoglycan layer, which resembled closely the Triton-insoluble envelopes. A three-layered cell wall structure, one layer on each side of the peptidoglycan layer, was reconstituted when only the 150,000-molecular-weight protein was used. Both five- and three-layered cell walls reconstituted in vitro also contained hexagonally arranged arrays with the same lattice constant as that of the total and Triton-insoluble envelopes. A mutant, strain 47-57, which was isolated as a phage-resistant colony, had a two-layered cell wall consisting of the middle and inner wall layers and contained only 150,000-molecular-weight protein as the major cell wall protein. The cell envelopes of the mutant revealed the hexagonal arrays with the same lattice constant as that of the wild-type cell envelopes. We conclude that the outer and middle wall layers consist of proteins with approximate molecular weights of 130,000 and 150,000, respectively. Furthermore, the 150,000-molecular-weight protein formed the hexagonal arrays in the middle wall layer.
Structural requirements for neuraminidase induction in Arthrobacter sialophilus
Authors: Flashner M , Kessler J , Tanenbaum S W
Published: January 2020

The effectiveness of 13 N-acetylneuraminic acid derivatives as potential inducers of Arthrobacter sialophilus neuraminidase were examined. N-Acetylneuraminic acid nitrogen and thioglycosides were not inducers, whereas 2,3-dehydro-N-acetylneuraminic acid, a transition state analog for neuraminidases, was the most effective inductive ligand. The C-4 hydroxyl function of N-acetylneuraminic acid was essential for enzyme derepression.
Rhizobium meliloti mutants altered in ammonium utilization
Authors: Osburne M S
Published: January 2020

Derivatives of Rhizobium meliloti 2011 required trace amounts of glutamate to use ammonium as the nitrogen source for growth, although they could use serine as the sole nitrogen source. Specific activities of ammonium assimilatory enzymes were similar to those in strain Rm2011. The mutants were deficient in nitrogen fixation.
Vanadate-Resistant Mutants of
Neurospora crassa
Are Deficient in a High-Affinity Phosphate Transport System
Authors: Bowman Barry J. , Allen Kenneth E. , Slayman Carolyn W.
Published: January 2020

Mutant strains of
Neurospora crassa
have been selected which grow on media containing vanadate, an inhibitor of the plasma membrane ATPase. The mutations all map to a single region (designated
van
) on the left arm of linkage group VII. The
van
mutants are unable to take up vanadate from the medium and are also deficient in the uptake of phosphate via a derepressible, high-affinity phosphate transport system. In the
van
mutants, the

K
m

for phosphate transport is elevated as much as 35-fold, indicating that the
van
locus may code for a structural component of the high-affinity phosphate transport system.
Excretion of flagellin by a short-flagella mutant of Salmonella typhimurium
Authors: Ikeda T , Kamiya R , Yamaguchi S
Published: January 2020

A nonmotile mutant of Salmonella typhimurium, SJW1254, has very short flagella (less than 0.1 micron long) due to a mutation in the structural gene of flagellin (H2). When ammonium sulfate was added to the culture medium of SJW1254 grown to the late-log phase, a large amount of protein precipitated. Gel electrophoresis and immunodiffusion showed that more than 90% (wt/wt) of the precipitated protein was flagellin. The mutant flagellin appeared to be excreted in the monomeric form, in an amount comparable to the amount in the flagellar filaments of wildtype bacteria. No such precipitate was obtained from the medium of wild-type bacteria. The mutant flagellin had the same apparent molecular weight (55,000) and isoelectric point (5.3) as the wild-type flagellin, but differed in mobility in polyacrylamide gel electrophoresis under nondenaturing conditions. Moreover, the mutant flagellin did not polymerize in vitro under various conditions in which wild-type flagellin polymerized. These results suggested that the mutant bacteria excreted flagellin because the flagellin polymerized poorly and therefore could not be trapped at the tip of the flagellar filament. This short-flagella mutant should be useful for studying the mechanism of flagellin transport.
Genetic homology between independently isolated chlorobenzoate-degradative plasmids
Authors: Chatterjee D K , Chakrabarty A M
Published: January 2020

Two chlorobenzoate-degradative plasmids were studied by the hybridization of the restriction endonuclease-generated fragments of one plasmid after transfer to a nitrocellulose filter with nick-translated radioactive DNA of the other plasmid as a probe. Two strains harboring the 3-chlorobenzoic acid-degradative plasmids were isolated in two different parts of the world at two different times. The plasmids are now found to be closely related to each other by hybridization studies. The chlorobenzoate-degradative plasmid from Pseudomonas sp. strain B13 (termed pB13) has a 6-kilobase deletion but otherwise is homologous with previously described plasmid pAC25.
Pseudomonas stutzeri and related species undergo natural transformation
Authors: Carlson C A , Pierson L S , Rosen J J , Ingraham J L
Published: January 2020

Cells of Pseudomonas stutzeri are naturally transformed by homologous chromosomal DNA; they do not require chemical treatment to become competent. This capacity to undergo natural transformation was found to be shared by the closely related species P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, but was not detectable in strains of P. aeruginosa, P. perfectomarinus, P. putida, P. fluorescens, or P. syringae. P. stutzeri could be transformed either on plates or in liquid medium. Only double-stranded chromosomal DNA was effective; single-stranded DNA and plasmid DNA were not. DNA fragments larger than 10 kilobase pairs were more effective than smaller fragments. The transformation frequency was proportional to DNA concentration from 1 ng/ml to 1 microgram/ml; higher concentrations were saturating. The maximum frequency, about 10(-4) transformants per recipient cell, was obtained with cells from a culture in the early stationary growth phase. A variety of chromosomal mutations have been transformed, including mutations to auxotrophy and to antibiotic resistance. Other systems for genetic exchange in P. stutzeri have not yet been found; transformation offers a means for the genetic analysis of this metabolically versatile organism.
Gene for Heat-Inducible Lysyl-tRNA Synthetase (
lysU
) Maps near
cadA
in
Escherichia coli
Authors: VanBogelen Ruth A. , Vaughn Vicki , Neidhardt Frederick C.
Published: January 2020

A hybrid ColE1 plasmid from the Clarke-Carbon colony bank with a 7-kilobase insertion was found to encode the inducible lysyl-tRNA synthetase along with the catabolic enzyme lysine decarboxylase. The gene for the inducible synthetase,
lysU
, must lie within 0.3 min of the lysine decarboxylase gene,
cadA
, at 92 min on the
Escherichia coli
genetic map.
Bacteriophage MS2 Lysis Protein Does Not Require Coat Protein to Mediate Cell Lysis
Authors: Coleman Jack , Inouye Masayori , Atkins John
Published: January 2020

We inserted all but the extreme 5′ end of a DNA copy of the bacteriophage MS2 lysis gene downstream of a
lac
-induced promoter on a multicopy plasmid. Upon induction, cells harboring this plasmid began to lyse, showing that phage coat protein is not required for the lytic process itself.
Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli
Authors: Morona R , Manning P A , Reeves P
Published: January 2020

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.
Adaptive response of Bacillus subtilis to N-methyl-N'-nitro-N-nitrosoguanidine
Authors: Hadden C T , Foote R S , Mitra S
Published: January 2020

Cell extracts of Bacillus subtilis contain a methyltransferase that appears to remove the O6-methyl group from O6-methylguanine in DNA in situ. This reaction proceeds in a stoichiometric fashion, as in Escherichia coli. However, the basal level of the enzyme (approximately 240 molecules per cell) is significantly higher in B. subtilis than in E. coli. In addition, the methyltransferase level increases by an order of magnitude as a result of de novo protein synthesis after adaptive treatment with a low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as in E. coli. Concomitant with adaptation, B. subtilis cells become more resistant to both killing and mutagenesis by a challenge dose of N-methyl-N'-nitro-N-nitrosoguanidine. We present evidence to support the hypothesis that the majority of N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations in B. subtilis are of the guanine-to-adenine transition type.
Binding of Streptococcal Lipoteichoic Acid to Fatty Acid-Binding Sites on Human Plasma Fibronectin
Authors: Courtney Harry S. , Simpson W. Andrew , Beachey Edwin H.
Published: January 2020

The ability ofStreptococcus pyogeneslipoteichoic acid and palmitic acid to bind to purified human plasma fibronectin was investigated. Initial studies indicated that intact fibronectin formed soluble complexes with lipoteichoic acid, resulting in a change in the mobility of fibronectin in an electrical field. Fibronectin covalently linked to agarose beads bound radiolabeled lipoteichoic acid in the acylated form but not in the deacylated form. An 18-M excess of fibronectin inhibited binding of lipoteichoic acid to the immobilized protein by 92%. Fibronectin-bound [3H]lipoteichoic acid could be specifically eluted with unlabeled lipoteichoic acid, as well as by fatty acid-free serum albumin. Serum albumin, which is known to contain fatty acid-binding sites capable of binding to the lipid moieties of lipoteichoic acid, inhibited the binding of lipoteichoic acid to fibronectin in a competitive fashion. The fibronectin-bound lipoteichoic acid could be eluted by 50% ethanol and various detergents but not by 1.0 M NaCl, various amino acids, or sugars. Similarly, radiolabeled palmitic acid adsorbed to fibronectin could be eluted with 50% ethanol but not with 1.0 M NaCl. Fibronectin adsorbed to a column of palmityl-Sepharose was eluted with 50% ethanol in 0.5% sodium dodecyl sulfate but not with 1.0 M NaCl or 1% sodium dodecyl sulfate alone. The binding of lipoteichoic acid to fibronectin followed first-order kinetics and was saturable. A Scatchard plot analysis of the binding data indicated a heterogeneity of lipoteichoic acid-binding sites similar to that previously found for serum albumin. Nevertheless, fibronectin contains at least one population of high-affinity binding sites for lipoteichoic acid. The binding affinity (nKa≃ 250 μM−1) is 2 orders of magnitude greater than the binding affinity of serum albumin. These data suggest that human plasma fibronectin contains specific binding sites for fatty acids and that lipoteichoic acid binds to these sites by way of its glycolipid moiety.
Energy coupling to nitrite respiration in the sulfate-reducing bacterium Desulfovibrio gigas
Authors: Barton L L , LeGall J , Odom J M , Peck H D
Published: January 2020

By use of a membrane fraction prepared from Desulfovibrio gigas grown in a lactate-sulfate medium, synthesis of ATP was demonstrated to be coupled to the oxidation of molecular hydrogen and reduction of either nitrite or hydroxylamine. This phosphorylation was uncoupled from electron transport by pentachlorophenol, methyl viologen, and gramicidin, but not by oligomycin. The extrusion of protons from the cells was shown to be coupled to the hydrogen-nitrite respiratory system, and, assuming the localization of nitrite reductase on the outer side of the plasma membrane, H+/2e- values of 2.0 +/- 0.3 were obtained. Energy coupling observed with this system appears to be due to electron transfer-coupled proton translocation rather than vectorial electron transfer associated with hydrogen oxidation.
N-terminal amino acid sequencing of EDP208 conjugative pili
Authors: Frost L S , Armstrong G D , Finlay B B , Edwards B F , Paranchych W
Published: January 2020

EDP208 conjugative pili contain a single polypeptide subunit of 11,500 daltons with a blocked N-terminus. This N-terminal blocking moiety was identified as an N-acetyl group by 1H nuclear magnetic resonance analysis of an N-terminal tripeptide isolated from pronase digests of EDP208 pilin. Limited acid hydrolysis of the tripeptide allowed its sequence to be determined as acetyl-NH-Thr-Asp-Leu. Trypsin digestion of EDP208 pilin resulted in the quantitative release of a fragment containing 12 residues from the N-terminus of the protein. The sequence of this dodecapeptide was determined to be acetyl-NH-Thr-Asp-Leu-Leu-Ala-Gly-Gly-Lys-Asp-Val-Asp-Lys.
Control of Synthesis of Functional mRNA Coding for Phenylalanine Ammonia-Lyase from
Rhodosporidium toruloides
Authors: Gilbert Harry J. , Stephenson John R. , Tully Michael
Published: January 2020

The regulation of functional mRNA coding for phenylalanine ammonia-lyase (PAL) from
Rhodosporidium toruloides
was investigated. Polyadenylic acid [poly(A)]-containing RNA was an efficient template for in vitro translation in rabbit reticulocyte lysate. Non-poly(A)-containing RNA did not stimulate in vitro protein synthesis. Several lines of experimental evidence indicate that mRNA from
R. toruloides
directs PAL synthesis in reticulocyte lysate: (i) the major radioactive product in immunoprecipitates when lysates, incubated with yeast poly(A)-containing RNA, were reacted with PAL-antiserum had the same molecular weight as native PAL (75,000); (ii) this major radioactive product competes with authentic PAL for binding to PAL-antiserum; and (iii) partial proteolytic peptide maps of the in vitro translation product were very similar to those of native PAL. The levels of functional mRNA coding for PAL, when
R. toruloides
was grown in different physiological conditions, were determined by quantitation of PAL synthesized in vitro when RNA was added to reticulocyte lysate. Functional PAL mRNA was six times higher in yeast grown on phenylalanine compared with glucose-phenylalanine minimal medium. No functional PAL mRNA was detected in yeast grown on glucose-ammonia minimal medium in the presence or absence of phenylalanine. These observed changes in functional PAL mRNA were similar to levels of PAL catalytic and antigenic activity. The kinetics of functional PAL mRNA synthesis and degradation were studied. Maximum levels of functional PAL mRNA were observed within 60 min of transfer to PAL-inducing growth conditions. Poly(A)-containing RNA and functional PAL mRNA were rapidly degraded when cells were transferred from phenylalanine to glucose-ammonia minimal medium, with half-lives of 25 and 10 min, respectively. Thus, it is suggested that the alterations in the amount of PAL in cells of
R. toruloides
grown in different physiological conditions primarily result from alteration in the amount of functional mRNA coding for the enzyme.
Isolation and characterization of Tn5 insertion mutations in the lexA gene of Escherichia coli
Authors: Krueger J H , Elledge S J , Walker G C
Published: January 2020

A Mu d(Ap lac)-generated fusion of lacZ to dinD, a gene induced by DNA damage, was used to isolate Tn5 insertion mutations that affect the regulation of the SOS responses. Three mutants were obtained that contained Tn5 insertions genetically linked to the lexA gene and had properties that suggested the mutants were deficient in lexA expression. The lexA protein has been shown to function as the repressor for genes involved in the SOS responses. By Southern blotting experiments, the three Tn5 insertions were physically mapped to distinct locations within the coding region of the lexA gene. The introduction of these mutations in six strains carrying lacZ fusions to different damage-inducible genes resulted in high expression of beta-galactosidase in all but one of the strains. In the dinF fusion strain, lacZ expression was reduced below that seen in a lexA+ background. Physical mapping studies of the dinF locus gave results consistent with the notion that dinF is part of the lexA transcription unit and that a lexA::Tn5 mutation has a polar effect on dinF expression. With certain din-lac fusion strains, a correlation was seen between the amount of beta-galactosidase production and the location of the particular Tn5 insertion within the lexA gene.
Molecular properties of succinate dehydrogenase isolated from Micrococcus luteus (lysodeikticus)
Authors: Crowe B A , Owen P
Published: January 2020

Succinate dehydrogenase (EC 1.3.99.1) of Micrococcus luteus was selectively precipitated from Triton X-100-solubilized membranes by using specific antiserum. The precipitated enzyme contained equimolar amounts of four polypeptides with apparent molecular weights of 72,000, 30,000, 17,000, and 15,000. The 72,000 polypeptide possessed a covalently bound flavin prosthetic group and appeared to be strongly antigenic as judged by immunoprinting experiments. Low-temperature absorption spectroscopy revealed the presence of cytochrome b556 in the antigen complex. By analogy with succinate dehydrogenase purified from other sources, the 72,000 and 30,000 polypeptides were considered to represent subunits of the succinate dehydrogenase enzyme, whereas one (or both) of the low-molecular-weight polypeptides was attributed to the apoprotein of the b-type cytochrome. A succinate dehydrogenase antigen cross-reacting with the M. luteus enzyme complex could be demonstrated in membranes of Micrococcus roseus, Micrococcus flavus, and Sarcina lutea, but not in the membranes isolated from a wide variety of other gram-positive and gram-negative bacteria.
Inverted repeats in the DNA of plasmid pCU1
Authors: Konarska-Kozlowska M , Thatte V , Iyer V N
Published: January 2020

Renaturable regions in the DNA strands of the N group plasmid pCU1 have been visualized as stem-loop structures by electron microscopy. Four such distinct structures are described, the smallest of which is within the loop of a larger one. The region of pCU1 in which these structures occur has several restriction sites. This and the availability of plasmid deletions and recombinants has permitted the mapping of these structures relative to one another and to the restriction and functional map of the plasmid. The replication and maintenance region of the plasmid is located within one of these stem-loop structures.
Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines
Authors: Knauf V C , Panagopoulos C G , Nester E W
Published: January 2020

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.
Intracellular accumulation of extracellular proteins by pleiotropic export mutants of Aeromonas hydrophila
Authors: Howard S P , Buckley J T
Published: January 2020

Pleiotropic export mutants of Aeromonas hydrophila were obtained which are unable to release protease, hemolysin, and glycerophospholipid:cholesterol acyltransferase. The synthesis of the proteins was not impaired; they were accumulated in active forms inside the mutant cells. The hemolysin could be isolated from cell contents by immunoprecipitation in a form with the same apparent molecular weight as the wild-type extracellular product, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Because both the protease and the hemolysin could be released from the mutant cells by osmotic shock, it was concluded that they were accumulated in the periplasmic space. Some mutants were missing two major outer membrane proteins, both of which reappeared in revertants with the wild-type excretory phenotype. Another mutant class had a normal outer membrane protein profile. That two different mutant classes could be obtained indicates that at least two gene products may be needed for export after protein translocation through the inner membrane. The accumulation of proteins which can be released by osmotic shock suggests that the periplasm may be part of the normal route for protein export.
Immunochemical relationship of the major outer membrane protein of Rhodopseudomonas sphaeroides 2.4.1 to proteins of other photosynthetic bacteria
Authors: Deal C D , Kaplan S
Published: January 2020

Immunoblots of sodium dodecyl sulfate-polyacrylamide gels derived from outer membrane preparations of various strains of Rhodopseudomonas sphaeroides revealed polypeptides which cross-reacted with antibody directed against the major outer membrane protein of R. sphaeroides 2.4.1. Immunochemical quantitation of the major outer membrane protein of strain 2.4.1 showed approximately 5.5 x 10(4) molecules per cell whether cells were grown chemoheterotrophically or photoheterotrophically. Rhodospirillum rubrum outer membranes contained a cross-reactive protein, whereas the outer membranes derived from Rhodopseudomonas capsulata and Paracoccus denitrificans showed no cross-reaction with the antibody prepared against the major outer membrane protein from R. sphaeroides 2.4.1.
Transformation of Kluyveromyces lactis by killer plasmid DNA
Authors: de Louvencourt L , Fukuhara H , Heslot H , Wesolowski M
Published: January 2020

Some strains of Kluyveromyces lactis contain two linear double-stranded DNA plasmids, k1 and k2. The presence of the two plasmids confer on the cell a "killer" character, due to the production of a toxin that kills the sensitive cells. We have used one of these linear DNA molecules as a gene vector to transform K. lactis cells. Hybrid plasmids containing parts of the k1 plasmid and the URA3 gene of Saccharomyces cerevisiae have been constructed. We have found that the hybrid plasmids were able to transform a uracil-requiring strain of K. lactis (uraA mutant) to a prototrophic form. The transformed phenotype cosegregated with the hybrid plasmids. The transforming plasmids contained the sequence of one or both ends of the linear k1 DNA, but they were integrated into a circular molecule.
New mutation (mmrA1) in Escherichia coli K-12 that affects minimal medium recovery and postreplication repair after UV irradiation
Authors: Sharma R C , Sargentini N J , Smith K C
Published: January 2020

After UV irradiation, Escherichia coli uvrA mutant cells show higher survival on minimal than on rich growth medium, i.e., they show minimal-medium recovery. This effect of rich growth medium on survival is not observed in a uvrA mutant carrying an mmrA1 mutation, and the uvrA mmrA strain showed the same survival rate on minimal and rich growth media as the uvrA strain did on minimal medium plates. The mmrA1 mutation was isolated as a hidden mutation from a uvrA polA mutant strain and shown to map at 84.3 min on the E. coli K-12 linkage map. In contrast to the uvrA strain, the repair of DNA daughter strand gaps was not inhibited in the uvrA mmrA strain by rich growth medium after irradiation. However, the uvrA and uvrA mmrA strains were similar in their ability to repair DNA when compared in minimal medium. These data are consistent with the idea that the mmr gene product is not involved directly in the repair of UV radiation-induced DNA damage, but rather allows rich growth medium to inhibit a portion of postreplication repair.
Streptococcus faecium ATCC 9790 penicillin-binding proteins and penicillin sensitivity are heavily influenced by growth conditions: proposal for an indirect mechanism of growth inhibition by beta-lactams
Authors: Fontana R , Canepari P , Satta G , Coyette J
Published: January 2020

The effects of variations in growth conditions on the penicillin response of Streptococcus faecium ATCC 9790 were studied. Changes in the growth temperature and medium composition were found to cause striking changes in the bacterial generation time, cellular penicillin sensitivity (minimum inhibitory concentration), sensitivity of peptidoglycan synthesis to inhibition by penicillin, rate of autolysis, and labeling pattern of penicillin-binding proteins. However, no constant relationship between these parameters and the minimum inhibitory concentration could be observed. Similar electrophoretic patterns for penicillin-binding proteins were observed in cells grown in different media at the optimal growth temperature. Inhibition of cell division by penicillin in cells grown at this temperature (but not at higher or lower temperatures) caused filamentation of the bacteria. In cells grown in a chemically defined medium at the optimal temperature (but not at temperatures above or below), complete inhibition of cell division was associated with only partial inhibition (34% after 150 min) of peptidoglycan synthesis. It is suggested that the status and physiological importance of individual penicillin-binding proteins in S. faecium are heavily influenced by growth conditions. Depending on the growth conditions, different penicillin-binding proteins may perform the cellular function, indispensible for bacterial growth.
"Nonfibrillar" chitin associated with walls and septa of Trichophyton mentagrophytes arthrospores
Authors: Pollack J H , Lange C F , Hashimoto T
Published: January 2020

Two morphologically distinct forms of chitin were found in the arthrospore walls and septa of Trichophyton mentagrophytes. Two-thirds of the total wall chitin was the microfibrillar and chitinase-sensitive form. The remaining chitin existed in a previously uncharacterized "nonfibrillar" form and was insensitive to the action of Streptomyces chitinase. Exhaustive digestion of the arthrospore walls and septa with beta (1 leads to 3)-glucanase and chitinase followed by extraction with NaOH (1 N, 100 degrees C, 3 h) resulted in a fraction which retained the original wall shape. This fraction consisted of 85% N-acetylglucosamine, 2.0% galactosamine, 2.5% glucose, and 0.4% amino acids, 74% of which were lysine. Both its infrared spectrum and its X-ray diffraction pattern were almost identical to those of authentic chitin. There was no evidence of the presence of muramic acid, hexuronic acid, phosphate, or sulfate in this fraction. Its resistance to chitinase was due neither to the presence of protective wall layers or melanin nor to its close or covalent association with beta-glucan. Aside from its nonfibrillarity, this hexosamine polymer differed from authentic chitin in that it was soluble in 6 N HCl and 7.5 N NaOH. The development of this nonfibrillar chitin layer in the cell wall during arthrosporogenesis of T. mentagrophytes may be related to the arthrospores being resistant to a variety of antifungal agents.
A complex attenuator regulates inducible resistance to macrolides, lincosamides, and streptogramin type B antibiotics in Streptococcus sanguis
Authors: Horinouchi S , Byeon W H , Weisblum B
Published: January 2020

Macrolide-lincosamide-streptogramin B resistance specified by Streptococcus sanguis plasmid pAM77 involves an adenine methylase, whose synthesis, demonstrable both phenotypically and by analysis of methionine-labeled proteins made in Bacillus subtilis minicells, is inducible by erythromycin, lincomycin, and streptogramin type B antibiotics. Localization of the methylase structural gene, including its control region in DNA fragments obtained with restriction endonucleases, has been deduced from DNA blot experiments with characterized target and probe DNAs from other streptococci, combined with DNA sequence analysis and comparison of the putative streptococcal methylase sequence with that of a cognate methylase in staphylococcal plasmid pE194. The streptococcal methylase migrates electrophoretically in polyacrylamide gels with the mobility of a 29,000-dalton protein. The sequence organization of the putative streptococcal methylase mRNA leader sequence partially resembles its staphylococcal counterpart and can support a similar mechanism of secondary structure rearrangement leading to methylase synthesis. The deduced 5' leader sequence preceding the pAM77 methylase structural gene sequence comprises approximately 155 nucleotides within which one can identify a putative control peptide 36 amino acid residues in length (in contrast to 19 in the pE194 peptide) and at least 14 possible classes of overlapping inverted complementary repeat sequences (in contrast to 3 in the pE194 control region), one of which can sequester the sequence AGGAG 7 nucleotides upstream from the putative (methionine) start codon of the streptococcal methylase. Comparison of the pAM77 and pE194 methylase amino acid sequences and their respective nucleotide sequences shows 51% conservation of amino acid residues (124 of 244) and 59% conservation of nucleotide residues (433 of 738), which suggests a common origin for the two methylase structural gene sequences. Differences in mRNA base composition associated with conserved amino acid residues occur mostly in the third nucleotide ("wobble") position of codons and may reflect adaptation of methylase genes to optimal expression in host cells with differing codon use patterns.
Expression of accumulated capacity for initiation of chromosome and minichromosome replication in dnaA mutants of Escherichia coli
Authors: LaDuca R J , Helmstetter C E
Published: January 2020

Chromosome and minichromosome replication were examined in temperature-sensitive dnaA mutants of Escherichia coli growing at temperatures between permissive and nonpermissive. Periodicities in [14C]thymidine uptake were detected as cultures incubated at intermediate temperatures approached late exponential-early stationary phase of growth. Exposure of the cultures to a nutritional shift-up caused a stimulation of chromosome replication associated with a rapid initiation of new rounds of replication, very similar to that observed after exposure to chloramphenicol. Addition of rifampin also caused a stimulation, but to a much lesser extent. The induced initiations of chromosome replication took place in two waves, as was the case when the cultures were simply shifted to permissive temperature. Minichromosomes were also stimulated to replicate by the addition of chloramphenicol at intermediate temperatures, providing further evidence that the chromosomal region which responded to the chloramphenicol treatment was in the vicinity of oriC. The findings are consistent with the conclusion that the initiations induced by chloramphenicol are consequences of the involvement of the dnaA gene product in a transcriptional step at initiation, as suggested by Orr et al. The results also suggest that the activity of the dnaA gene product is not normally involved in controlling the frequency of initiation of chromosome replication.
Calcium as a branching signal in Neurospora crassa
Authors: Reissig J L , Kinney S G
Published: January 2020

The divalent cation ionophore A23187 was found to induce apical branching in Neurospora crassa. Optimal effects were obtained by treatment with 0.1 mM ionophore for 30 min. Branching first became manifest during or shortly after treatment; successive rounds of branching could be observed at later times. Calcium starvation of the mycelium markedly reduced its subsequent response to the ionophore, whereas starvation for other divalent cations had no detectable effect. The branching response was markedly reduced in the presence of 10 to 30 mM cyclic AMP or derivatives thereof.
Lipopolysaccharide core mutants of Salmonella typhimurium containing D-glycero-D-manno-heptose
Authors: Brañes L V , Kay W W
Published: January 2020

Mutants resistant to several hydrophobic membrane antagonists were isolated from a "deep rough" (rfaC) mutant of Salmonella typhimurium. The resistance was due to an alteration in the core region lipopolysaccharide composition as evidenced by altered bacteriophage and complement sensitivity and by compositional analysis. The principal change in carbohydrate composition was the predominance of the unusual heptose isomer D-glycero-D-manno-heptose. The unusually wide pleiotropic phenotype of this organism is suggested to be due to a fundamental change in the properties of the bacterial outer membrane.
Utilization of plasmid pULB113 (RP4::mini-Mu) to construct a linkage map of Erwinia carotovora subsp. chrysanthemi
Authors: Schoonejans E , Toussaint A
Published: January 2020

We report experimental evidence that pULB113, an RP4::mini-Mu plasmid, mediates chromosome transfer in a strain of Erwinia carotovora subsp. chrysanthemi which does not accept the F episome. This allowed us to construct a genetic map of that strain by measuring the frequencies of cotransfer of different markers (thy, leu, pro, [his, trp], thyA, rpsL, ile).
Heat sensitivity of Azotobacter vinelandii genetic transformation
Authors: Doran J L , Page W J
Published: January 2020

Heating competent Azotobacter vinelandii at 37 or 42 degrees C resulted in a total loss of competence with no loss of viability. The transformation process was relatively insensitive to heating at either temperature once DNase-resistant DNA binding was nearly complete. Although competent and 42 degrees C-treated cells bound equivalent amounts of [32P]DNA in a DNase-resistant state, no donor DNA marker (nif) or radioactivity was detected in the envelope-free cell lysate of heated cells, suggesting that DNA transport across the cell envelope was a heat-sensitive event. Competence was reacquired in a 42 degrees C-treated culture after 2 h of incubation at 30 degrees C by a process which required RNA and protein syntheses. The release of a surface glycoprotein, required for competence, from cells treated at 42 degrees C occurred in an insufficient amount to account for the total loss of competence. Recovery of competence in 42 degrees C-treated cells and further transformation of competent cells were prevented by the exposure of cells to saturating amounts of transforming DNA. Further DNase-resistant DNA binding, however, still occurred, suggesting that there were two types of receptors for DNase-resistant DNA binding to competent A. vinelandii. DNase-resistant DNA binding was dependent on magnesium ions, and at least one receptor type did not discriminate against heterologous DNA.
Coordination of flagella on filamentous cells of Escherichia coli
Authors: Ishihara A , Segall J E , Block S M , Berg H C
Published: January 2020

Video techniques were used to study the coordination of different flagella on single filamentous cells of Escherichia coli. Filamentous, nonseptate cells were produced by introducing a cell division mutation into a strain that was polyhook but otherwise wild type for chemotaxis. Markers for its flagellar motors (ordinary polyhook cells that had been fixed with glutaraldehyde) were attached with antihook antibodies. The markers were driven alternately clockwise and counterclockwise, at angular velocities comparable to those observed when wild-type cells are tethered to glass. The directions of rotation of different markers on the same cell were not correlated; reversals of the flagellar motors occurred asynchronously. The bias of the motors (the fraction of time spent spinning counterclockwise) changed with time. Variations in bias were correlated, provided that the motors were within a few micrometers of one another. Thus, although the directions of rotation of flagellar motors are not controlled by a common intracellular signal, their biases are. This signal appears to have a limited range.
Two fep genes are required for ferrienterochelin uptake in Escherichia coli K-12
Authors: Pierce J R , Pickett C L , Earhart C F
Published: January 2020

Escherichia coli mutants defective in the assimilation of iron from ferrienterochelin were isolated and characterized. One mutant was able to bind ferrienterochelin to its outer membrane but could not transport it into the cell. Complementation tests with lambda hybrid phage were employed to distinguish the defective gene, which we term fepB, from fepA, the structural gene for the outer membrane ferrienterochelin receptor protein. These same physiological and genetic tests were employed to tentatively classify several previously described fep mutants as carrying either fepA or fepB. The data demonstrate the existence of fepB and provide an explanation for previous difficulties in identifying fepB mutants.
Identification of a membrane protein induced concurrently with cell filamentation by cyclic AMP in an Escherichia coli K-12 fic mutant
Authors: Utsumi R , Kawamukai M , Obata K , Morita J , Himeno M , Komano T
Published: January 2020

A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant. In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced. Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.
Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus
Authors: Kreft J , Berger H , Härtlein M , Müller B , Weidinger G , Goebel W
Published: January 2020

From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertion was recloned as a PstI fragment into pJKK3-1, a shuttle vector which replicates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no external or internal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at levels comparable to those of the B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin O-producing strain of Streptococcus pyogenes or from listeriolysin-producing strains of Listeria monocytogenes, no positive hybridization signals were obtained. These data suggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.
Iodination of Myxococcus xanthus during development
Authors: Maeba P Y
Published: January 2020

Intact cells of Myxococcus xanthus were iodinated with [125I]lactoperoxidase to permit examination of the surface components accessible to labeling during cell development. Vegetative cells, starved on a defined solid medium, aggregated, formed fruiting bodies, and produced myxospores. Cells collected at different stages were iodinated, and their proteins were analyzed by one- and two-dimensional electrophoresis and autoradiography. One-dimensional electrophoresis revealed six iodinated bands in vegetative cell extracts. During development, 10 radioactive bands were detected, 4 of which migrated to the same positions as those of vegetative cells. Only six bands were detected in purified, labeled myxospores. Of these, one band possessed mobility similar to that of labeled vegetative cell proteins, whereas the other bands possessed mobility similar to that detected in developing cells. Analysis of two-dimensional gels indicated that at least 14 proteins were iodinated in vegetative cells, one of which was intensely labeled (protein b). Another of the proteins (protein a) was labeled throughout development. During development, about 30 proteins were iodinated and the prominently labeled ones were designated c, d, e, f, and g. The latter two (proteins f and g) were not detected in purified, iodinated myxospores. The data indicated a pronounced change in surface structure during development; some of the change may be involved in cellular interaction during aggregation.
S gene product: identification and membrane localization of a lysis control protein
Authors: Altman E , Altman R K , Garrett J M , Grimaila R J , Young R
Published: January 2020

The product of the bacteriophage S gene has been previously shown to be required for an essential step in triggering host cell lysis. By using two different protein labeling systems, maxicells and UV-irradiated infected cells, we identified the S gene product as an 8,500-molecular-weight polypeptide associated with the cell envelope. The apparent molecular weight is significantly less than the 11,500 predicted from the S gene sequence. We were unable to confirm two previous identifications of S gene products, an acidic 15,000-molecular-weight polypeptide found by two-dimensional gel electrophoresis of infected cells and a 5,500-molecular-weight polypeptide in purified phage particles.