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D J Kushner

Publications and source records attributed to D J Kushner.

At least 19 recordsLinked to original sources
PubMed1995

Factors regulating cryIVB expression in the cyanobacterium--Synechococcus PCC 7942.

The expression of the larvicidal Bacillus thuringiensis subsp. israelensis cryIVB gene in cyanobacteria has been suggested to be an effective means of controlling mosquito populations. Using a variety of cryIVB constructs, in this study we have examined the effect of Synechococcus PCC 7942 culture age on intracellular toxin levels and have attempted to determine the mechanisms by which cryIVB gene expression is regulated. The data suggest that specific degradation of the cryIVB mRNA limits toxin production; however, the addition of cyanobacterial 3' untranslated DNA sequences to the cryIVB gene did not improve mRNA stability or toxin levels. An analysis of the cryIVB sequence and comparison of codon usage patterns with highly expressed cyanobacterial genes suggest that inefficient translation and intragenic ribosomal binding sites impede protein synthesis and result in rapid turnover of the toxin mRNA.

Bacillus thuringiensis↗
PubMed1995

Description of two new species of Halomonas: Halomonas israelensis sp.nov. and Halomonas canadensis sp.nov.

Six well-known strains of halotolerant bacteria, including two strains previously identified only as NRCC 41227 and Ba1, have been compared using 125 phenotypic characters and DNA-DNA hybridization. Although these strains represent some of the most heavily studied salt-tolerant bacteria, they have never been taxonomically compared. The data presented show that these bacteria form a relatively homogeneous group related at the genus level. The taxonomic comparison showed that these six organisms represented four distinct species all related above the 65% Jaccard coefficient level. In addition to two previously identified bacterial species, Halomonas elongata (ATCC 33173T) and Halomonas halodurans (ATCC 29686T), the strains included in this study represent two previously unnamed Halomonas species. These two new taxa have been assigned the names Halomonas israelensis (ATCC 43985T) and Halomonas canadensis (NRCC 41227T = ATCC 43984). DNA-DNA hybridization show that these two species are related to the type species H. elongata at 54.9 and 48.9%, respectively.

Culture Media↗
PubMed1993

Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp. strain PCC 7942.

The impact of promoter modification on the expression of the mosquitocidal Bacillus thuringiensis subsp. israelensis cryIVB gene when used to transform the cyanobacterium Synechococcus sp. strain PCC 7942 has been examined. Maximal transcript and protein abundances were achieved by the addition of the lacZ promoter upstream of the cryIVB sequence. Replacement of the endogenous corresponding Bacillus sequences with the Synechococcus petF1 promoter, ribosome binding site, and initiation codon also resulted in increased expression of the cryIVB gene relative to the expression obtained with the Bacillus promoter alone but decreased expression relative to the expression achieved with the tandem array of the Bacillus and lacZ promoters. Synechococcus cells carrying plasmids in which the expression of the cryIVB gene was regulated by either the lacZ or the petF1 promoter were readily consumed by first-instar Culex restuans larvae and proved to be toxic for these organisms.

Bacillus thuringiensis↗
PubMed1990

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola.

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichia coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNA(FMet) (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA(FMet) showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4+ (as glutamate). Higher salt concentrations, especially those with Cl- as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl- ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl- ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4Cl, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl- ions inhibited the attachment of initiation factors to tRNA(FMet) but had little effect on binding of initiation factors to R17 RNA.

Bacterial Proteins↗
PubMed1989

In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions.

In vitro protein synthesis in Vibrio costicola [poly(U)-directed incorporation of phenylalanine] was studied. The extent of protein synthesis was limited by the number of ribosomes present. Density gradient centrifugation experiments suggested that, after runoff of ribosomes from the artificial messenger, the 50S subunit was unable to attach to the 30S-messenger complex. As shown previously (M. Kamekura and D. J. Kushner, J. Bacteriol. 160:385-390, 1984), Cl- ions inhibited protein synthesis; indeed, the highest rate of synthesis took place in the lowest attainable Cl- concentration (37 mM). The inhibitory effects were partly reversed by glutamate and betaine, both of which are concentrated within cells of V. costicola. The strongest reversal was seen when both glutamate and betaine were present. Cl- ions can prevent binding of ribosomes to poly(U) and displace ribosomes already bound to this artificial messenger. The effects of Cl- ions on binding were also reversed by glutamate and betaine. Cl- ions did not affect accuracy of translation; they were shown previously (Kamekura and Kushner, J. Bacteriol. 160:385-390, 1984) not to affect phenylalanyl-tRNA synthetase. It was also found that washing ribosomes with inhibitory NaCl concentrations did not interfere with their ability to carry out protein synthesis later in optimal (low) salt concentrations. On the contrary, these ribosomes were more active than before they were washed. We conclude that the main site of action of Cl- in the system studied is on the binding of ribosomes to the mRNA.

Bacterial Proteins↗
PubMed1985

Proton circulation in Vibrio costicola.

The importance of proton movements was assessed in the moderate halophile Vibrio costicola. When anaerobic cells in acidic buffer (pH 6.5) were given an O2 pulse, protons were extruded regardless of the presence of Na+. At pH 8.5, however, V. costicola produced an acidic response to an O2 pulse in the absence of Na+ and an alkaline response when Na+ was present. An Na+/H+ antiport activity was confirmed at pH 8.5. All of these effects were prevented by protonophores or butanol treatment. Growth in complex medium at pH 8.5 was prevented by a high concentration (50 microM) of carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or a low concentration (5 microM) of another protonophore, 3,3',4',5-tetrachlorosalicylanilide (TCS). The relative ineffectiveness of the former protonophore was caused by the proteose peptone and tryptone ingredients of the complex medium, since 5 microM completely prevented growth in their absence. The results are explained by a primary respiratory-linked proton efflux coupled to a secondary Na+/H+ antiport operating at alkaline pH. Evidence was seen for a role of Na+ in stimulating proton influx at alkaline pH, presumably via the pH homeostasis mechanism.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1985

Growth of Vibrio costicola and other moderate halophiles in a chemically defined minimal medium.

A simple chemically defined minimal medium consisting of sodium glutamate, glucose, vitamins, and salts was devised to support growth of the moderate halophile, Vibrio costicola, over as wide a range of NaCl concentrations as the complex medium, proteose peptone + tryptone. The lag period at higher NaCl concentrations was longer in the chemically defined minimal medium than in proteose peptone + tryptone. Chemically defined minimal medium also supported the growth of an unidentified moderate halophile, HX, and of Vibrio alginolyticus and Vibrio cholerae. The Mg2+ concentration required for good growth changed with the growth temperature for both V. costicola and HX.

Culture Media↗
PubMed1984

Energetics of sodium-dependent alpha-aminoisobutyric acid transport in the moderate halophile Vibrio costicola.

The energetics of alpha-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (delta psi) and alpha-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5-9.0. Cells specifically required Na+ ions to transport alpha-aminoisobutyric acid and to maintain the highest delta psi (150-160 mV). Sodium was not required to sustain high rates of O2-uptake. Delta psi (and alpha-aminoisobutyric acid transport) recovered fully upon addition of Na+ to Na+-deficient cells, showing that Na+ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of delta psi and alpha-aminoisobutyric acid transport. Also, dissipation of delta psi with triphenylmethylphosphonium cation abolished alpha-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in alpha-aminoisobutyric acid transport, without affecting delta psi. Similarly, N,N'-dicyclohexylcarbodiimide (10 microM) stimulated respiration and alpha-aminoisobutyric acid transport and did ot affect delta psi, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (delta psi and Na+ chemical potential), we conclude that delta psi is obligatory for alpha-aminoisobutyric acid transport, and that for maximum rates of transport an Na+ gradient is also required.

Aminoisobutyric Acids↗
PubMed1984

Effect of chlorination on antibiotic resistance profiles of sewage-related bacteria.

A total of 1,900 lactose-fermenting bacteria were isolated from raw sewage influent and chlorinated sewage effluent from a sewage treatment plant, as well as from chlorinated and neutralized dilute sewage, before and after a 24-h regrowth period in the laboratory. Of these isolates, 84% were resistant to one or more antibiotics. Chlorination of influent resulted in an increase in the proportion of bacteria resistant to ampicillin and cephalothin, the increase being most marked after regrowth occurred following chlorination. Of the other nine antibiotics tested, chlorination resulted in an increased proportion of bacteria resistant to some, but a decrease in the proportion resistant to the remainder. Multiple resistance was found for up to nine antibiotics, especially in regrowth populations. Identification of about 5% of the isolates showed that the highest proportion of Escherichia coli fell in untreated sewage. Some rare and potentially pathogenic species were isolated from chlorinated and regrowth samples, including Yersinia enterocolitica, Yersinia pestis, Pasteurella multocida, and Hafnia alvei. Our results indicate that chlorination, while initially lowering the total number of bacteria in sewage, may substantially increase the proportions of antibiotic-resistant, potentially pathogenic organisms.

Anti-Bacterial Agents↗
PubMed1984

Effect of chloride and glutamate ions on in vitro protein synthesis by the moderate halophile Vibrio costicola.

Vibrio costicola grown in the presence of different NaCl concentrations contains cell-associated Na+ and K+ ions whose sum is equal to or greater than the external Na+ concentration. In the presence of 0.5 M NaCl, virtually no in vitro protein is synthesized in extracts of cells grown in 1.0 M NaCl. However, we report here that active in vitro protein synthesis occurred in 0.6 M or higher concentrations of Na2SO4, sodium formate, sodium acetate, sodium aspartate, or sodium glutamate, whereas 0.6 M NaF, NaCl, or NaBr completely inhibited protein synthesis as measured by polyuridylic acid-directed incorporation of [14C]phenylalanine. Sodium glutamate, sodium aspartate, and betaine (0.3 M) counteracted the inhibitory action of 0.6 M NaCl. The cell-associated Cl- concentration was 0.22 mol/kg in cells grown in 1.0 M NaCl. Of this, the free intracellular Cl- concentration was only 0.02 mol/kg. Cells contained 0.11 mol of glutamate per kg and small concentrations of other amino acids. All of the negative counterions for cell-associated Na+ and K+ have not yet been determined. In vitro protein synthesis by Escherichia coli was inhibited by sodium glutamate. Hybridization experiments with ribosomes and the soluble (S-100) fractions from extracts of E. coli and V. costicola showed that the glutamate-sensitive fraction was found in the soluble, not the ribosomal, part of the system. The phenylalanyl-tRNA synthetase of V. costicola was not inhibited by 0.5 M or higher concentrations of NaCl; it was slightly more sensitive to high concentrations of sodium glutamate. Therefore, this enzyme was not responsible for the salt response of the V. costicola in vitro protein-synthesizing system.

Amino Acids↗
PubMed1983

The reduction of hydroxamic acids with titanium(III) chloride: a tool for the characterization of siderophores.

Hydroxamic acid siderophores were observed to be inactivated by exposure to titanium(III) chloride. To study the reaction, a series of eight model hydroxamic acids were prepared and reacted with titanium(III) chloride. The products were shown by ir and NMR comparisons with authentic compounds to be the corresponding amides. The reduction was found to require 2 mol of titanium(III) per mol of hydroxamic acid.

Chemical Phenomena↗
PubMed1983

Development of salt-resistant active transport in a moderately halophilic bacterium.

The moderately halophilic bacterium Vibrio costicola accumulates alpha-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na(+) ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.

Aminoisobutyric Acids↗
PubMed1983

Proton motive force and Na+/H+ antiport in a moderate halophile.

The influence of pH on the proton motive force of Vibrio costicola was determined by measuring the distributions of triphenylmethylphosphonium cation (membrane potential, delta psi) and either dimethyloxazolidinedione or methylamine (osmotic component, delta pH). As the pH of the medium was adjusted from 5.7 to 9.0, the proton motive force steadily decreased from about 170 to 100 mV. This decline occurred, despite a large increase in the membrane potential to its maximum value at pH 9.0, because of the loss of the pH gradient (inside alkaline). The cytoplasm and medium were of equal pH at 7.5; membrane permeability properties were lost at the pH extremes of 5.0 and 9.5. Protonophores and monensin prevented the net efflux of protons normally found when an oxygen pulse was given to an anaerobic cell suspension. A Na+/H+ antiport activity was measured for both Na+ influx and efflux and was shown to be dissipated by protonophores and monensin. These results strongly favor the concept that respiratory energy is used for proton efflux and that the resulting proton motive force may be converted to a sodium motive force through Na+/H+ antiport (driven by delta psi). A role for antiport activity in pH regulation of the cytosol can also explain the broad pH range for optimal growth, extending to the alkaline extreme of pH 9.0.

Carrier Proteins↗
PubMed1982

Role of membrane-bound 5'-nucleotidase in nucleotide uptake by the moderate halophile Vibrio costicola.

Intact cells of Vibrio costicola hydrolyzed ATP, ADP, and AMP. The membrane-bound 5'-nucleotidase (C. Bengis-Garber and D. J. Kushner, J. Bacteriol. 146:24-32, 1981) was solely responsible for these activities, as shown by experiments with anti-5'-nucleotidase serum and with the ATP analog, adenosine 5'-(beta gamma-imido)-diphosphate. Fresh cell suspensions rapidly accumulated 8-14C-labeled adenine 5'-nucleotides and adenosine. The uptake of ATP, ADP, and AMP (but not the adenosine uptake) was inhibited by adenosine 5'-(beta gamma-imido)-diphosphate similarly to the inhibition of the 5'-nucleotidase. Furthermore, the uptake of nucleotides had Mg2+ requirements similar to those of the 5'-nucleotidase. The uptake of ATP was competitively inhibited by unlabeled adenosine and vice versa; inhibition of the adenosine uptake by ATP occurred only in the presence of Mg2+. These experiments indicated that nucleotides were dephosphorylated to adenosine before uptake. The hydrolysis of [alpha-32P]ATP as well as the uptake of free adenosine followed Michaelis-Menten kinetics. The kinetics of uptake of ATP, ADP, and AMP also each appeared to be a saturable carrier-mediated transport. The kinetic properties of the uptake of ATP were compared with those of the ATP hydrolysis and the uptake of adenosine. It was concluded that the adenosine moiety of ATP was taken up via a specific adenosine transport system after dephosphorylation by the 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1982

Polar lipids in phototrophic bacteria of the Rhodospirillaceae and Chromatiaceae families.

The polar lipids of photosynthetic purple bacteria of the genera Chromatium, Thiocapsa, Thiocystis, Ectothiorhodospira, Rhodopseudomonas, Rhodospirillum, and Rhodomicrobium were analyzed. Characteristic compositions of the polar lipids were found for most of the Rhodospirillaceae and Chromatiaceae species. Phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin were the major phospholipids in most species. Phosphatidylcholine was present as a major component in all species of the genus Ectothiorhodospira, but was not detected in the remaining Chromatiaceae. It was also present in most of the Rhodospirillaceae species. No glycolipids were found in any of the Ectothiorhodospira species. In the Rhodospirillaceae, the glycolipids mono- and digalactosyl diglycerides were generally absent. Sulfoquinovosyl diglyceride was present in significant amounts in at least three species of the Rhodospirillaceae and may have been present in most of them, but only in traces. All of the Chromatiaceae species contained several glycolipids, one of which was similar to monogalactosyl diglyceride. Ornithine lipids were found in large amounts in most Rhodospirillaceae, but were absent in Ectothiorhodospira and in the other Chromatiaceae. The species examined could be divided into three groups on the basis of their lipid composition: (i) the genus Ectothiorhodospira; (ii) the remaining Chromatiaceae; and (iii) the Rhodospirillaceae. The data presented are compared with those available in the literature, and differences from other phototrophic organisms are discussed.

Chromatiaceae↗
PubMed1981

Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.

Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).

5'-Nucleotidase↗