bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold
Authors: Tinglu G , Ghosh A , Ghosh B K
Published: January 2020

Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold-IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C.
Outer membrane protein NmpC of Escherichia coli: pore-forming properties in black lipid bilayers
Authors: Hindahl M S , Crockford G W , Hancock R E
Published: January 2020

The purified NmpC outer membrane protein from Escherichia coli, when incorporated into planar lipid bilayers, gave rise to channels with a single-channel conductance of 1.8 nS in 1 M KCl. This suggests that the NmpC protein is a porin.
Excretion of unassembled flagellin by Salmonella typhimurium mutants deficient in hook-associated proteins
Authors: Homma M , Fujita H , Yamaguchi S , Iino T
Published: January 2020

Of the flagellar filamentless mutants of Salmonella typhimurium, the flaV, flaU, and flaW mutants, which are defective in hook-associated proteins, synthesized flagellin molecules, but flagella did not polymerize at the tips of the mutant hooks and were excreted into the culture medium as intact monomers.
Correction of the nucleotide sequence of the Citrobacter freundii tryptophan operon regulatory region
Authors: Kuroda M I , Yanofsky C
Published: January 2020

We present a correction of the previously reported nucleotide sequence of the Citrobacter freundii trp operon regulatory region. The original sequence analyses were performed with a plasmid designated pCF2. We repeated the cloning of the trp regulatory region of C. freundii and concluded from the determined sequence that a DNA rearrangement had occurred within the leader region of the cloned trp DNA of pCF2. The correct sequence is homologous to the Escherichia coli sequence.
Role of inositol-containing sphingolipids in Saccharomyces cerevisiae during inositol starvation
Authors: Hanson B A
Published: January 2020

The in vitro lipid requirements of UDP-N-acetylglucosamine-dolichol phosphate N-acetylglucosamine-1-phosphotransferase for the inositol-containing sphingolipids from Saccharomyces cerevisiae were characterized in terms of concentration and specificity. The effects of combinations of lipids, especially phosphatidylinositol and the inositol-containing sphingolipids, were also tested on the transferase. Phosphatidylinositol and phosphatidylglycerol stimulated the enzyme 3.3- and 2.8-fold, respectively. The inositol-containing sphingolipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine did not stimulate the activity of the transferase. Phosphatidylcholine and phosphatidylethanolamine in combination with phosphatidylinositol had no effect on the transferase activity; however, the inositol-containing sphingolipids markedly inhibited the stimulation of the transferase by phosphatidylinositol. This inhibition by the sphingolipids was prevented if phosphatidylcholine, in addition to the other lipids, was present in the assay mixture. In addition, changes due to inositol starvation in the in vivo membrane lipid environment, i.e., phosphatidylinositol and the inositol-containing sphingolipids, were analyzed to determine whether they corresponded to the observed in vitro effects. Three hours after the beginning of inositol starvation, there were 9- and 14-fold reductions in the accumulation of phosphatidylinositol in membrane fractions IIA (vesicles) and IV (endoplasmic reticulum), respectively, although there was only a 6-fold reduction in membrane fraction I (plasma membrane). The accumulation of [14C]inositol into inositol-containing sphingolipids also reflected the differences in the cellular location of membranes.
Some properties of the nickel-containing hydrogenase of chemolithotrophically grown Rhizobium japonicum
Authors: Harker A R , Xu L S , Hanus F J , Evans H J
Published: January 2020

The uptake hydrogenase of chemolithotrophically grown Rhizobium japonicum was purified to apparent homogeneity with a final specific activity of 69 mumol of H2 oxidized per min per mg of protein. The procedure included Triton extraction of broken membranes and DEAE-cellulose and Sephacryl S-200 chromatographies. The purified protein contained two polypeptides separable only by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They comigrated on native polyacrylamide gels and sucrose density gradients. The molecular weights were ca. 60,000 and 30,000. Densitometric scans of the sodium dodecyl sulfate gels indicated a molar ratio of 1.03 +/- 0.03. Antiserum was developed against the 60-kilodalton polypeptide for use in hydrogenase detection by an enzyme-linked immunosorbent assay. The antiserum did not cross-react with the 30-kilodalton polypeptide. Native gel electrophoresis of Triton-extracted cells grown in the presence of 63Ni showed comigration of the hydrogenase and radioactive Ni.
Regulation of hutUH operon expression by the catabolite gene activator protein-cyclic AMP complex in Klebsiella aerogenes
Authors: Nieuwkoop A J , Boylan S A , Bender R A
Published: January 2020

RNA polymerase transcribed the hutUH operon of Klebsiella aerogenes if the catabolite gene activator protein (CAP) and cyclic AMP (cAMP) were present or if the DNA template was derived from a promoter mutant in which hutUH expression was independent of the need for positive effectors. In the absence of CAP or cAMP, not only was hutUH transcription absent, but transcription in the opposite direction (toward hutC) was initiated at a site (pC) ca. 70 base pairs from the site (pUH) of hutUH mRNA initiation. When the pC promoter was cloned in front of a promoterless galK gene, active expression of galK was observed. Thus, the pC promoter is active in vivo as well as in vitro. Transcription from pUH and pC may be mutually exclusive, with the major effect of CAP and cAMP being to prevent transcription from pC, thus relieving the antagonistic effect on transcription from pUH. This "double-negative" control by CAP-cAMP is supported by two observations: (i) CAP-cAMP was unable to activate transcription from pUH if RNA polymerase had been previously bound to pC and (ii) a mutation that allowed transcription from pUH in the absence of positive effectors simultaneously eliminated the activity of pC. An alternative model, in which CAP-cAMP is required for pUH expression and RNA polymerase binding at pC serves to modulate this control in some unknown way, is also considered. The physiological role of the transcript from pC other than regulation of pUH is unknown.
Cytochemistry of cytochrome oxidase in the cytoplasmic and intracytoplasmic membranes of Azotobacter vinelandii
Authors: Payne H R , Socolofsky M D
Published: January 2020

Vegetative cells of Azotobacter vinelandii contain a system of intracytoplasmic membranes in the form of numerous internal vesicles. The three-dimensional morphology of these internal vesicles was established by an examination of stereopair electron micrographs of negatively stained cells. The vesicles assumed a variety of forms ranging from nearly spherical units to short, curved tubules. These structures were found at the periphery of the cytoplasm, subjacent to the cytoplasmic membrane. Large flattened cisternae were also present in some cells. The amount of intracytoplasmic membrane varied widely even among individual cells from the same culture. The total surface area of the intracytoplasmic membranes was greater than that of the cytoplasmic membrane in many cells. To assess the possible association of cytochrome oxidase activity with the intracytoplasmic membranes, enzyme localization experiments were conducted with the cytochemical substrate 3,3'-diaminobenzidine. The results showed that a cyanide-sensitive cytochrome oxidase activity is located at the intracytoplasmic membrane. The quantity of cytochrome oxidase activity present in the internal membranes is probably less than that present in the cytoplasmic membrane.
Construction and use of a gene bank of Alcaligenes eutrophus in the analysis of ribulose bisphosphate carboxylase genes
Authors: Andersen K , Wilke-Douglas M
Published: January 2020

A gene bank of the DNA from the hydrogen bacterium Alcaligenes eutrophus ATCC 17707 was constructed in the broad host range cosmid vector pVK102 and established in Escherichia coli. A triparental replica plating procedure was developed to allow rapid screening of large numbers of isolated E. coli gene bank clones for complementation of A. eutrophus mutants. This procedure was used to identify hybrid cosmids that complemented CO2 fixation-negative (Cfx-), H2 uptake-negative (Hup-), and auxotrophic A. eutrophus mutants. The average insert DNA size in these hybrid cosmids was 22 kilobases. Nine hybrid cosmids that complemented ribulose bisphosphate carboxylase-negative (RuBPCase-) mutants were characterized. They fell into two distinct groups with respect to their restriction patterns. Complementing subclones from the two groups contained no common restriction fragments, but hybridization experiments indicated a high degree of sequence homology. Restriction fragments corresponding to one of the subclones were absent in total DNA from a cured strain that had lost plasmid pAE7, indigenous to the wild type. It is concluded that functional CO2 fixation genes in the A. eutrophus ATCC 17707 chromosome are reiterated on plasmid pAE7.
Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product
Authors: Nakata A , Amemura M , Shinagawa H
Published: January 2020

The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed.
Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli
Authors: Novotny M J , Reizer J , Esch F , Saier M H
Published: January 2020

D-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and D-glucitol-6-phosphate dehydrogenase (EC 1.1.1.140) were purified to apparent homogeneity in good yields from Escherichia coli. The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined. Both enzymes showed absolute specificities for their substrates. The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively. It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer. These two proteins differed in several fundamental respects.
Chloramphenicol-inducible gene expression in Bacillus subtilis is independent of the chloramphenicol acetyltransferase structural gene and its promoter
Authors: Mongkolsuk S , Ambulos N P , Lovett P S
Published: January 2020

cat-86 specifies chloramphenicol acetyltransferase and is the indicator gene on the Bacillus subtilis promoter cloning plasmid pPL703. Insertion of promoters from various sources into pPL703 at a site ca. 144 base pairs upstream from cat-86 activates expression of cat-86, and the expression is characteristically inducible by chloramphenicol. Thus, chloramphenicol inducibility of cat-86 is independent of the promoter that is used to activate the gene. To determine whether cat-86 or its products were involved in chloramphenicol inducibility, gene replacement studies were performed. cat-86 consists of 220 codons. The lacZ gene from Escherichia coli was inserted into a promoter-containing derivative of pPL703, plasmid pPL603E, at two locations within cat-86. pPL3lac2 contains lacZ inserted in frame after codon 2 of cat-86. pPL3lac30 contains lacZ inserted in frame after codon 30 of cat-86. In both constructions, all cat coding sequences 3' to the site of the lacZ insertion were deleted. Both plasmids exhibited chloramphenicol inducibility of beta-galactosidase in B. subtilis. These studies provide the first direct demonstration that the transcription and translation products of a chloramphenicol-inducible cat gene are uninvolved in chloramphenicol inducibility of gene expression. The results localize the region essential to inducibility to the 144-base pair segment that intervenes between the site of promoter insertion and the cat-86 gene.
Cloning of the tyrP gene and further characterization of the tyrosine-specific transport system in Escherichia coli K-12
Authors: Wookey P J , Pittard J , Forrest S M , Davidson B E
Published: January 2020

The tyrP gene which codes for a component of the tyrosine-specific transport system of Escherichia coli has been cloned on a 2.8-kilobase insert into plasmid pBR322. Transposon mutagenesis, using Tn1000, indicates that the tyrP+ gene is at least 1.1 kilobase in length. Labeling of the tyrP protein in maxicells with [35S]methionine indicates an apparent molecular weight of ca. 24,500. Sedimentation analysis reveals that the tyrP protein is associated with the cell membrane and is not free in the cytoplasm or periplasm. Strains with many copies of the tyrP+ gene show an enhanced uptake of tyrosine, but the expression of the system is still modulated by tyrosine and phenylalanine in the presence of the tyrR+ regulator protein. Accumulated radioactive tyrosine is rapidly effluxed by the addition either of energy uncouplers or of excess nonradioactive tyrosine, indicating that the transport system is energized by the proton motive force and that the internal pool is readily exchangeable. The effect of increasing expression of the tyrP gene on the steady-state level of tyrosine accumulated by cells indicates that although the transport system may be dependent on the proton motive force to drive uptake, the system never reaches thermodynamic equilibrium with it.
lon transcriptional regulation of genes necessary for capsular polysaccharide synthesis in Escherichia coli K-12
Authors: Trisler P , Gottesman S
Published: January 2020

It has previously been observed that Escherichia coli lon mutations increase the levels of enzymes involved in the synthesis of colanic acid capsular polysaccharide (A. Markovitz, p. 415-462, in I. Sutherland, ed., Surface Carbohydrates of the Prokaryotic Cell, 1977). To determine how lon regulates these enzymes, we have isolated, mapped, and characterized lac operon and lac protein fusions to genes necessary for capsule synthesis by the Mu d(lac Amp) in vivo fusion technique of Casadaban and Cohen (M. J. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979). At least five genes have been identified which share a common pattern of regulation: they are transcribed at low levels in lon+ strains and at significantly higher levels in lon strains. These genes are located in a cluster close to udk at 45 min on the E. coli map; we have named these genes cpsA, B, C, D, and E. An additional locus, cpsF, located at 90 min, is regulated in a similar manner to cpsA to E but is not essential for colanic acid synthesis. Similar studies on the transcriptional regulation of fusions in the gal and manA operons, also necessary for colanic acid synthesis, do not show significant regulation by the lon locus. Therefore, the regulatory system described here does not extend to all genes in the colanic acid synthesis pathway.
Proline uptake through the major transport system of Salmonella typhimurium is coupled to sodium ions
Authors: Cairney J , Higgins C F , Booth I R
Published: January 2020

Strains of Salmonella typhimurium deficient in one or more of the proline transport systems have been constructed and used to study the mechanism of energy coupling to transport. Proline uptake through the major proline permease (PP-I, putP) is shown to be absolutely coupled to Na+ ions and not to H+ ions as has previously been assumed. Transport through the minor proline permease (PP-II, proP), however, is unaffected by the presence or absence of Na+. The effect of Na+ on the kinetics of proline uptake shows that external Na+ increases the Vmax for transport. It seems probable that proline transport through PP-I is also coupled to Na+ ions in Escherichia coli.
Cell wall receptor for bacteriophage Mu G(+)
Authors: Sandulache R , Prehm P , Kamp D
Published: January 2020

The invertible G segment in phage Mu DNA controls the host range of the phage. Depending on the orientation of the G segment, two types of phage particles, G(+) and G(-), are produced which recognize different cell surface receptors. The receptor for Mu G(+) was located in the lipopolysaccharide (LPS) of gram-negative bacteria. The analysis of different LPS core types and of mutants that were made resistant to Mu G(+) shows that the primary receptor site on Escherichia coli K-12 lies in the GlcNAc beta 1 . . . 6Glc alpha 1-2Glc alpha 1-part at the outer end of the LPS. Mu shares this receptor site in E. coli K-12 with the unrelated single-stranded DNA phage St-1. Phage D108, which is related to Mu, and phages P1 and P7, which are unrelated to Mu but contain a homologous invertible DNA segment, have different receptor requirements. Since they also bind to terminal glucose in a different configuration, they adsorb to and infect E. coli K-12 strains with an incomplete LPS core.
Increased permeability and subsequent resealing of the host cell membrane early after infection of Escherichia coli with bacteriophage T1
Authors: Keweloh H W , Bakker E P
Published: January 2020

The addition of T1 to cells growing at 37 degrees C in a minimal medium at 0.4 mM Mg2+ rapidly induced an irreversible loss of K+ and Mg2+ and uptake of Na+ by the cells. Both the ATP pool of the cells and the transmembrane proton motive force were reduced. These cells did not lyse from within, since viral DNA replication and the maturation of the 36,000-molecular-weight phage head protein were inhibited. By contrast, cells lysed when infected at 5.4 mM Mg2+. In these cells, T1 initially induced K+ efflux and Na+ influx and lowered the cytoplasmic ATP concentration. After a few minutes, the cation gradients and ATP pool were restored to levels close to that of control cells. At 5.4 mM Mg2+, the shutoff of host protein synthesis was delayed and coincided with the restoration of the ATP pool. In an ATP synthase-negative mutant, infection with T1 did not affect the cytoplasmic ATP concentration but inhibited host protein synthesis with the same rate as it did in wild-type cells.
High incidence of transposon Tn3 insertions into a replication control gene of the chimeric R/Ent plasmid pCG86 of Escherichia coli
Authors: Picken R N , Mazaitis A J , Maas W K
Published: January 2020

Insertion of transposon Tn3 into the chimeric R/Ent plasmid pCG86 occurred preferentially into a replication control gene, generating mutants with increased plasmid copy number. In two mutants, the nucleotide sequence of the region of the gene containing the Tn3 insertion was determined.
In vitro deletions in the partition locus of plasmid pSC101
Authors: Kalla S R , Gustafsson P
Published: January 2020

Deletion mutants in the 375-base-pair EcoRI-AvaI fragment carrying the partition locus of plasmid pSC101 were formed by the combined action of exonuclease III and nuclease S1. Six deletion mutants were isolated, and the endpoints of the deletions were sequenced. One of the deletions extended 69 base pairs from the EcoRI site without impairing plasmid stability. The other five deletions caused the plasmid to be unstable and extended 199 to 251 base pairs from the EcoRI site.
Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production
Authors: Hageman J H , Shankweiler G W , Wall P R , Franich K , McCowan G W , Cauble S M , Grajeda J , Quinones C
Published: January 2020

The composition and application of a single, chemically defined medium or growth and sporulation of Bacillus subtilis is described. At 37 degrees C cells grew with a doubling time of about 40 min; cultures attained near-maximal spore formation (70 to 80% by 12 h after the end of exponential growth and produced 1 X 10(9) to 2 X 10(9) heat-resistant free spores at 24 h. Dipicolinic acid production was completed between 7 and 11 h. Cells grown in the single, chemically defined medium excreted levels of serine and neutral proteases comparable to those excreted in nutrient broth medium.
Coordinate expression of hydrogenase and ribulose bisphosphate carboxylase in Rhizobium japonicum Hupc mutants
Authors: Merberg D , Maier R J
Published: January 2020

In contrast to the wild type, H2 uptake-constitutive mutants of Rhizobium japonicum expressed both hydrogenase and ribulose bisphosphate carboxylase activities when grown heterotrophically. However, as bacteroids from soybean root nodules, the H2 uptake-constitutive mutants, like the wild type, did not express ribulose bisphosphate carboxylase activity.
Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli
Authors: Orndorff P E , Falkow S
Published: January 2020

The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12. At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression. We present evidence which indicates that one gene encodes an inhibitor of piliation. Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E. coli as unique small, compact colonies. Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation. Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell. All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp). Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene. Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation.
Molecular basis of isozyme formation of beta-galactosidases in Bacillus stearothermophilus: isolation of two beta-galactosidase genes, bgaA and bgaB
Authors: Hirata H , Negoro S , Okada H
Published: January 2020

Bacillus stearothermophilus IAM11001 produced three beta-galactosidases, beta-galactosidase I, II, and III (beta-gal I, II, and III), which are detectable by polyacrylamide (nondenatured) gel electrophoresis. By connecting restriction fragments of the chromosomal DNA to plasmid vectors, followed by transformation of Escherichia coli, two beta-galactosidase genes (bgaA and bgaB) located close to each other on the chromosome were isolated. Identification of the gene products and Southern hybridization analyses with a 2.7-kilobase-pair EcoRI fragment containing the bgaA gene as probe revealed that a single bgaA gene exists on the genome and that beta-gal II and beta-gal III consist of a common subunit (the bgaA gene product; molecular weight, 120,000), but differ in their assembly (beta-gal II is a dimer, and beta-gal III is a tetramer). The bgaB gene product (molecular weight, 70,000) in Bacillus subtilis harboring pHG5 (a hybrid plasmid consisting of pUB110 and a 2.9-kilobase-pair EcoRI fragment) was estimated to be the beta-gal I protein from its heat stability. Southern hybridization and immunological testing indicated that the two genes have no homology.
Mutants of Aspergillus nidulans blocked at an early stage of sporulation secrete an unusual metabolite
Authors: Butnick N Z , Yager L N , Hermann T E , Kurtz M B , Champe S P
Published: January 2020

Mutants of Aspergillus nidulans defective in conidiation (asexual sporulation) can be classified according to whether they are blocked before or after induction of conidiation. Mutants blocked before induction (preinduction mutants) appear to be unable to respond to the inducing stimulus and thus are defective in one of the earliest events in the sporulation process. Three preinduction mutants have been isolated and characterized. Each was found to exhibit the same pleiotropic phenotype: they also were defective in sexual sporulation and secreted a set of phenolic metabolites at a level much higher than did wild type or mutants blocked at later stages of conidiation. One of the metabolites has been identified as the antibiotic diorcinal (3,3'-dihydroxy-5,5'-dimethyldiphenyl ether) which is known to be involved in the synthesis of certain farnesyl phenols of unknown function. These results suggest that preinduction mutants are blocked in a phenolic metabolic pathway, one or more product of which participates in the initiation of sporulation.
Mechanism of adhesion of Alysiella bovis to glass surfaces
Authors: Irvin R T , To M , Costerton J W
Published: January 2020

Alysiella bovis adheres to surfaces by means of short, ruthenium red-staining, rod-like fimbriae. The fimbriae remain associated with the cell envelope of A. bovis, even when sonicated or exposed sequentially to toluene, Triton X-100, lysozyme, ribonuclease, and deoxyribonuclease. Adhesion of outer membrane-derived cell wall ghosts of A. bovis to glass was inhibited by IO4-, sodium dodecyl sulfate, urea, pronase, and trypsin. Protease treatment digested the fimbriae from the distal end, and exposure to sodium dodecyl sulfate depolymerized the fimbriae. Exposure of ghosts to 1% sodium dodecyl sulfate preferentially solubilized a 16,500-dalton protein which was subsequently purified by gel filtration and demonstrated to be a glycoprotein (ca. 17% carbohydrate). Antibodies raised against the 16,500-dalton glycoprotein agglutinated whole cells and inhibited adhesion of ghosts to glass.
Isolation of a novel protein involved in the transport of fructose by an inducible phosphoenolpyruvate fructose phosphotransferase system in Streptococcus mutans
Authors: Gauthier L , Mayrand D , Vadeboncoeur C
Published: January 2020

Fructose transport in Streptococcus mutans LG-1 is mediated by at least two distinct phosphoenolpyruvate fructose phosphotransferase systems. One system is constitutive and consists of membrane components enzyme II as well as enzyme I and heat-stable protein. The other system is inducible and requires, in addition to enzyme I and heat-stable protein, a soluble substrate-specific protein for catalytic activity. This protein factor, designated IIIfru, was purified by DEAE-cellulose chromatography, hydroxylapatite chromatography, molecular sieving on Sephadex G-75, and preparative electrophoresis. The purified preparation showed only one protein band, with a molecular weight of 12,600, on sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis, on gel electrophoresis with the discontinuous buffer Tris-glycine, and after electrofocusing in gel (pI congruent to 3.7). The molecular weight of the native protein determined by gel filtration at 4 degrees C was 51,000. Immunodiffusion experiments performed with immunoglobulins prepared against the purified IIIfru from S. mutans LG-1 suggested that other S. mutans strains possessed a IIIfru. No precipitin bands, however, were detected with extracts from S. salivarius, S. sanguis, S. lactis, S. faecalis, Staphylococcus aureus, Bacillus subtilis, Lactobacillus casei, and Escherichia coli.
Effect of culture pH on the D-alanine ester content of lipoteichoic acid in Staphylococcus aureus
Authors: MacArthur A E , Archibald A R
Published: January 2020

The lipoteichoic acid in Staphylococcus aureus growing at high pH values contained very little alanine ester, showing that high overall levels of substitution were not essential for growth. The low alanine content could have resulted from a progressive loss due to base-catalyzed hydrolysis of the labile ester linkages.
Bacteriophage T5 gene A2 protein alters the outer membrane of Escherichia coli
Authors: Snyder C E
Published: January 2020

Evidence for changes in Escherichia coli envelope structure caused by the bacteriophage T5 gene A2 protein was obtained by the use of mutant bacteriophages, envelope fractionation procedures, electrophoretic analysis, and in vitro binding studies with purified gene A2 protein. The results suggested that the T5 gene A2 protein perturbs the host envelope as it functions to promote DNA transfer.
Mating ability during chemically induced G1 arrest of cells of the yeast Saccharomyces cerevisiae
Authors: Bedard D P , Li A W , Singer R A , Johnston G C
Published: January 2020

Diploid formation by haploid cells of Saccharomyces cerevisiae was tested during and after treatment with chemical agents which bring about arrest at the cell cycle regulatory step "start." All compounds, except sinefungin, allowed efficient mating. During sinefungin treatment, zygote formation, but not karyogamy, was affected.
Molecular cloning and expression of genetic determinants for the iron uptake system mediated by the Vibrio anguillarum plasmid pJM1
Authors: Tolmasky M E , Crosa J H
Published: January 2020

Plasmid pJM1 from an invasive strain of Vibrio anguillarum encodes an iron uptake system which mediates the biosynthesis of a siderophore and a membrane receptor for the iron-siderophore complex. This system has been associated with the ability of V. anguillarum to cause hemorrhagic septicemic disease in marine fish. Recombinant derivatives containing essential regions of the pJM1-mediated iron uptake system cloned into cosmid vector pVK102 were introduced into low-virulence iron uptake-deficient V. anguillarum strains by using a trifactor mating procedure with helper plasmid pRK2013. Three recombinant clones, pJHC-T7, pJHC-T11, and pJHC-T2612, possessed genetic determinants for receptor activity. Production of receptor activity was correlated in all three cases with the presence of OM2, an 86-kilodalton outer membrane protein which was induced under iron-limiting conditions. Two of the clones, pJHC-T7 and pJHC-T2612, also coded for the production of siderophore activity, although at a much lower level than the wild type. Strains harboring either of these two clones were still unable to grow under iron-limiting conditions. This inability was overcome only when other indigenous pJM1 derivatives were present in the cells in addition to the recombinant cosmids. This restoration of high siderophore production and ability to grow under iron-limiting conditions was achieved even when the indigenous plasmids possessed lesions in genes involved in siderophore activity or in both siderophore and receptor production. Thus, another function mediated by plasmid pJM1, possibly a transacting factor, may play a role in the regulation of siderophore production. Results of experimental infections demonstrated that restoration of the ability to grow under conditions of iron limitations by introduction of an recombinant clone into one of the low-virulence V. anguillarum strains was correlated with an increase in bacterial pathogenicity.
Cell wall and phospholipid composition and their contribution to the salt tolerance of Halomonas elongata
Authors: Vreeland R H , Anderson R , Murray R G
Published: January 2020

The salt-tolerant bacterium Halomonas elongata makes a variety of physiological adaptations in response to increases in the salt concentration of its growth medium. The cell walls become more compact and internally coherent. The overall lipid pattern shows an increased amount of negatively charged lipids. In addition, the peptidoglycan composition of H. elongata, although not changing in response to increased NaCl, contains the hydrophobic amino acid leucine which is unique among bacterial species. The results suggest that H. elongata is able to live in a wide variety of salt concentrations because it alters its cell physiology in ways which increase both structural integrity and the amount of less-mobile, "structured" cell water, making the cells less susceptible to NaCl-induced dehydration.
Analysis of initiation of sites of cell wall growth in Streptococcus faecium during a nutritional shift
Authors: Gibson C W , Daneo-Moore L , Higgins M L
Published: January 2020

Three-dimensional reconstruction methods were applied to electron micrographs of Streptococcus faecium to study the initiation of cell wall growth sites during a nutritional shift experiment. Upon lowering the mass doubling time from 76 to 33 min by the addition of excess glutamate, the formation of new cell wall growth sites accelerated above the old steady-state rate at about the same time (10 to 15 min) as did mass, RNA, protein, cell numbers, and autolytic capacity but considerably before DNA (30 min) and peptidoglycan (20 min) synthesis did. During the shift, the average range of cell volumes over which new wall growth sites were introduced did not change significantly. However, upon the shift there was an increase in the frequency of cells having new sites, which was due to the faster-growing cells initiating more new sites in peripheral locations before division. After a transition period, the number of new sites per milliliter of culture increased at a rate that paralleled that of the culture mass. These findings support a model in which new sites are introduced when cells grow to a relatively constant, growth rate-independent size, while the rate at which sites form and grow increases with the growth rate. In this model, chromosome synthesis does not regulate the formation of new sites of cell wall growth, but existing sites cannot be completed until rounds of chromosome synthesis are completed.
General approach to bacterial nutrition: growth factor requirements of Moraxella nonliquefaciens
Authors: Juni E , Heym G A , Bradley R A
Published: January 2020

A general procedure was devised for the determination of growth factor requirements of heterotrophic bacteria based upon identification of individual nutrients as they are successively depleted from a limited quantity of complex medium. By using this approach, it was possible to develop a defined medium for growth of Moraxella nonliquefaciens that contained nine amino acids and three vitamins. Three of the amino acids, proline, serine, and cysteine, were required in unusually high concentrations to obtain optimal growth. Methionine had a sparing action on the requirements for serine and cysteine. Glycine could substitute for serine. Although a required nutrient, cysteine was inhibitory for growth, but this inhibitory action was antagonized by valine or leucine. The requirement for cysteine was satisfied by cystine, glutathione, or sodium sulfide. M. nonliquefaciens could not use ammonia as a nitrogen source but could use glutamate or aspartate for this purpose. With the exception of 1 auxotrophic strain, the growth factor requirements of 23 independently isolated strains of M. nonliquefaciens were essentially the same.
Sensitivity of Coxiella burnetii peptidoglycan to lysozyme hydrolysis and correlation of sacculus rigidity with peptidoglycan-associated proteins
Authors: Amano K , Williams J C
Published: January 2020

The protease-resistant proteins associated with the peptidoglycan (PG) of the phase I small-cell variant Coxiella burnetii were either partially released from the PG by boiling the PG-protein complex (PG-PC) in sodium dodecyl sulfate containing 2-mercaptoethanol and EDTA or totally released by 1 N NaOH hydrolysis at 23 degrees C. An 18,300-dalton protein was released from the PG-PC under reducing conditions, whereas 1 N NaOH treatment extracted PG-associated proteins without apparent dissolution of the PG. Purified PG was composed of muramic acid, glucosamine, glutamic acid, alanine, and meso-diaminopimelic acid in a molar ratio of 0.9:0.9:1.0:1.4:1.0. Lysozyme hydrolysis of cell walls, PG-PC, and purified PG caused an increase in reducing groups which correlated with roughly 60 to 100% digestion of disaccharides. There was no significant decrease in turbidity during lysozyme hydrolysis of cell walls and PG-PC; however, hydrolysis of purified PG caused about 90% decrease in turbidity. Approximately 60% of the meso-diaminopimelic acid groups of PG were not susceptible to dinitrophenylation, thus, demonstrating an apparent contribution of PG-associated proteins, rather than cross-linkage between peptides, to sacculus rigidity of cell wall and PG-PC. This association of PG and protease-resistant covalently bound proteins may be important structural and functional determiners of resistance to both environmental conditions and intracellular digestion of C. burnetii by eucaryotic cells.
Spiroplasma membrane lipids
Authors: Davis P J , Katznel A , Razin S , Rottem S
Published: January 2020

Membranes of six spiroplasma strains belonging to different Spiroplasma species and subgroups were isolated by a combination of osmotic lysis and sonication in the presence of EDTA to block endogenous phospholipase activity. Analysis of membrane lipids showed that in addition to free and esterified cholesterol the spiroplasmas incorporated exogenous phospholipids from the growth medium. Sphingomyelin was preferentially incorporated from phosphatidylcholine-sphingomyelin vesicles or from the serum used to supplement the growth medium. Palmitate was incorporated better than oleate into membrane lipids synthesized by the organisms during growth. The major phospholipid synthesized by the spiroplasmas was phosphatidylglycerol. The positional distribution of the fatty acids in phosphatidylglycerol of Spiroplasma floricola resembled that found in Mycoplasma species, in which the saturated fatty acids prefer position 2 in the glycerol backbone and not position 1 as found in Acholeplasma species and elsewhere in nature. Electron paramagnetic resonance analysis of spin-labeled fatty acids incorporated into S. floricola membranes exhibited homogeneous single-component spectra without immobilized regions. The S. floricola membranes were more rigid than those of Acholeplasma laidlawii and less rigid than those of Mycoplasma gallisepticum.
Cloning of a developmentally regulated element from alkalophilic Bacillus subtilis DNA
Authors: Kudo T , Yoshitake J , Kato C , Usami R , Horikoshi K
Published: January 2020

An alkalophilic Bacillus DNA bank cloned in an expression probe plasmid, pGR71, was screened for the presence of developmentally regulated genetic elements. A 508-base pair HindIII fragment isolated from this bank in plasmid pGR71-5 expressed plasmid-encoded chloramphenicol resistance only during the sporulation phase of a Bacillus subtilis host grown on Schaeffer medium. This developmentally regulated expression was altered in spo0E and spo0H mutants which had very low levels of chloramphenicol acetyltransferase activity relative to the wild type or other spo0 mutants. We determined the nucleotide sequence of the entire 508-base pair fragment and located the site of regulated transcription initiation by high-resolution S1 nuclease mapping of the in vivo transcript. The deduced promoter sequences upstream from this start site were 5'C-G-A-A-T-C-A-T-G-A3' at -10 and 5' A-G-G-A-A-T-C3' at -35. This transcript was not detected in spo0E or spo0H mutants, indicating that the products of these genes control developmentally regulated chloramphenicol acetyltransferase expression at the level of transcription.
Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa
Authors: Darzins A , Nixon L L , Vanags R I , Chakrabarty A M
Published: January 2020

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.
DNA-directed in vitro synthesis and assembly of the form II D-ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides
Authors: Chory J , Muller E D , Kaplan S
Published: January 2020

A biochemical analysis of the in vitro assembly of the form II ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides after transcription and translation from cloned DNA is presented. The predominant enzymatically active oligomeric forms of the in vitro-synthesized and -assembled ribulose-1,5-bisphosphate carboxylase are tetramers and hexamers. Assembly of the monomeric subunits to form active enzyme appears to be dependent on the presence of a minimum number of subunits in the cell extract. Assembly of ribulose-1,5-bisphosphate carboxylase also was observed when the protein-synthesizing extracts were prepared from cells which were partially derepressed for ribulose-1,5-bisphosphate carboxylase expression.
Conjugal transfer system of the IncN plasmid pKM101
Authors: Winans S C , Walker G C
Published: January 2020

The conjugal transfer system of the broad-host range IncN plasmid pKM101 was analyzed genetically. Its organization differed significantly from that of the F plasmid. The tra genes are located in three regions, each between 3 and 4 kilobases in length. All of the genes in the first two regions are required for sensitivity to "donor-specific" phage which bind to the plasmid-mediated sex pilus, and these genes therefore are involved in the synthesis, and possibly retraction, of the sex pilus. The plasmid's origin of transfer was localized to a 1.2-kilobase region at an extreme end of the transfer region. Using two different methods, we have identified 11 complementation groups required for transfer. One of these, traC, is of special interest in that mutations at this locus can be partially suppressed if, prior to mating, cells carrying a traC mutant plasmid are incubated with cells which elaborate sex pili but are unable to transfer their plasmids. One possible explanation for this is that pilus-elaborating cells can donate traC gene product to a traC mutant in a form that can be reused.
Transformation of Salmonella typhimurium with plasmid DNA: differences between rough and smooth strains
Authors: MacLachlan P R , Sanderson K E
Published: January 2020

Lipopolysaccharide-defective mutants of Salmonella typhimurium were transformed by plasmid DNA with a Ca2+ treatment method. Only those mutants with an Rc or Rd2 chemotype, due to galE or rfaF mutations, respectively, gave efficiencies greater than 10(5) transformants per microgram of DNA, frequencies 8- to 630-fold higher than with smooth strains or other rough mutants.
Cyclic AMP receptor protein from yeast mitochondria: submitochondrial localization and preliminary characterization
Authors: Rödel G , Müller G , Bandlow W
Published: January 2020

We have identified and characterized a cyclic AMP receptor protein in mitochondria of the yeast Saccharomyces cerevisiae. The binding is specific for cyclic nucleotides, particularly for cyclic AMP which is bound with high affinity (Kd of 10(-9) M) at 1 to 5 pmol/mg of mitochondrial protein. The mitochondrial cyclic AMP receptor is synthesized on cytoplasmic ribosomes and has an apparent molecular weight of 45,000 as determined by photoaffinity labeling. It is localized in the inner mitochondrial membrane and faces the intermembrane space. Cross-contamination of mitochondrial inner membranes by plasma membranes or soluble cytoplasmic proteins is excluded.