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L M Kozloff

Publications and source records attributed to L M Kozloff.

At least 37 records · Page 2Linked to original sources
PubMed1981

Defective packing of an unusual DNA in a virulent Erwinia phage, Erh 1.

A newly isolated bacteriophage, Erh 1, for Erwinia herbicola, has been characterized. This virulent phage has been found to have an elongated rod-like head and a short complex tail structure. One major protein and 5 minor proteins have been identified as phage components. The head structure was found to be transparent, flexible and could be twisted or flattened by various treatments. The DNA, isolated from highly purified phage particles, was linear, double-stranded, had a G-C content of 46-47% and displayed two unique features. (1) The isolated phage DNA molecules were highly heterogeneous in contour lengths; the most prevalent molecules had a length of 6-12 mu, but molecules have been measured with lengths ranging from 2.3 mu to 37 mu. (2) One or two long single-stranded regions, "gaps," ranging in length from 0.3 to 3.1 mu with an average length of 1.4 +/- 0.7 mu, were found in about 25% of the phage DNA molecules. Upon density gradient centrifugation of p32 labeled phage, it was found that most of the DNA was contained in apparently noninfectious, defective particles, with densities ranging from 1.34 to 1.41 while most of the infectious particles were found in fractions of density of 1.44 +/- 0.02. When used to multiply infect cells, the lower density particles were able to complement each other and form infectious centers. Further, it was found that infectious particles themselves were heterogeneous and had sedimentation constants varying from 600 S to 1400 S. From the distribution of DNA sizes in these particles, the variations in sedimentation behavior, and the flexibility of the head structure of most particles, it appears that the head structure is formed first and then the DNA is packed inefficiently into this head structure. Apparently, most phage particles are only partly filled and do not contain a complete genome while a few others may contain a large amount of redundant viral DNA.

Bacteriophages↗
PubMed1981

Polymerization of Escherichia coli bacteriophage T4D short tail fibers into filaments and filament bundles.

Crude preparations of short tail fibers of E. coli T4D bacteriophage were found to be readily soluble in 0.01 M EDTA, but after dialysis against H2O, and a 10-fold concentration, the fibers aggregated into long filaments and filament bundles. However, since the filaments from these crude preparations were associated with nonfilamentous material, the fibers were purified in the presence of 0.01 M EDTA. After dialysis of the purified short tail fiber preparation against water, and 10-fold concentration, a precipitate was again formed that could be readily separated by low speed centrifugation. In micrographs, this precipitated material showed large aggregates of short tail fibers but did not show well-defined filaments. However, when these aggregates were incubated with 0.02 M MgSO4 and 0.06 M KC1, well-defined filaments were again formed.

Electrophoresis, Polyacrylamide Gel↗
PubMed1980

Bacteriophage T4D receptor and the Escherichia coli cell wall structure: binding of endotoxin-like particles to the cell wall.

A variety of degradative treatments have been used to investigate the nature of the structure and components of the cell walls of Escherichia coli B. The binding and localization of the endotoxin-like particles found on the cell walls were of special interest because some of them are associated with the site where the inner tail tube of bacteriophage T4D penetrates the cell wall. Modified cell walls were obtained by heating a suspension of bacterial cells originally in 0.1 M phosphate, pH 7.0, after the addition of 12.5 M NaOH to a final concentration of 0.25 M. With regard to the endotoxin-like particles, it was found that: (i) at least part of them still remained bound to the modified cell wall after the alkali treatment; (ii) the subsequent incubation of alkali-treated cell walls with lysozyme destroyed the bacterial form and released a complex of endotoxin-like particles together with a fibrous material; (iii) on the other hand, treatment with 45% phenol at 70 degrees C removed the endotoxin-like particles from the surface of the alkali-treated cell walls, but most of the fibrous material was left on the cell wall; and (iv) incubation of alkali-treated cell walls with 5 mM ethylenediaminetetraacetic acid at 20 degrees C also removed the endotoxin-like particles, but did not disrupt the rodlike bacterial form. However, if the ethylenediaminetetraacetic acid treatment was performed at 55 degrees C, the bacterium-like form was destroyed. These differential sensitivities to ethylenediaminetetraacetic acid suggested that loosely bound divalent metal ions normally hold these endotoxin-like particles on the cell wall surface, but that probably more tightly bound metal ions are involved in the determination of cell shape. Analysis of the protein components of the alkalitreated cell walls showed that only one protein was present in significant amounts, and this protein had an electrophoretic mobility similar to that of the Braun lipoprotein. This protein was released from the alkali-treated cell walls upon heating with 2% sodium dodecyl sulfate at 100 degrees C. Phospholipids were also absent from this structure. The distribution of the remaining cell wall components on the alkali-treated cell walls is discussed.

Bacterial Proteins↗
PubMed1979

Bacteriophage T4D receptors and the Escherichia coli cell wall structure: role of spherical particles and protein b of the cell wall in bacteriophage infection.

The nature of the interaction of bacteriophage T4D and the outer cell wall of its host, Escherichia coli B, has been investigated. Bacteria with altered or modified cell walls have been obtained by two different growth procedures: (i) growth in high osmolarity medium or (ii) growth in broth in the presence of divalent heavy metal ions. When these altered host cells were washed and subsequently added to regular growth medium, they interacted with added phage particles, but successful infection did not occur. Most of the phage particles released from these treated cells were observed to have full heads and an altered tail structure. The altered phage tails had contracted sheaths and unusual pieces of the bacterial cell wall attached to the distal portion of the exposed phage tail tube. Phage released from bacteria grown in the high osmolarity medium had attached cell wall pieces of two major types, these pieces being either 40 or 21 nm in diameter. The smaller-type cell wall pieces (21 nm) were formed by three spheres each measuring 7 nm in diameter. Phage particles released from cells previously exposed to the divalent metal ions had only one 7-nm cell wall sphere attached to the distal end of the tail tube. It was found that these 7-nm spheres (i) are normal components of the cell wall and are morphologically similar to endotoxin, (ii) are held in place on the cell wall by a component of the cell wall called protein b, and (iii) are most likely the site of penetration of the phage tail tube through which the phage DNA enters the host cell.

Bacterial Proteins↗
PubMed1979

Structural role of the polyglutamate portion of the folate found in T4D bacteriophage baseplate.

Three types of reagents were used to determine the structural role and location of the polyglutamate portion of the Escherichia coli T4D bacteriophage baseplate dihydropteroyl hexaglutamate. These reagents were examined for their effect in vitro on some of the final steps in phage baseplate morphogenesis. The reagents were (i) a series of oligopeptides composed solely of glutamic acid residues but with various chemical linkages and chain lengths; (ii) a homogeneous preparation of carboxypeptidase G1, an exopeptidase that hydrolyzes carboxyl-terminal glutamates (or aspartates) from simple oligopeptides, including the gamma-glutamyl bonds on folyl polyglutamates as well as the bond between the carboxyl group of the p-aminobenzoyl moiety and the amino group of the first glutamic acid residue of folic acid; and (iii) antisera prepared against a polyglutamate hapten. All three types of reagent markedly inhibited the attachment of the phage long tail fibers to the baseplate. Other steps in baseplate assembly such as the addition of T4D gene 11 or gene 12 products were not affected by any of these reagents. These results indicate that the polyglutamate portion of the folate is located near the attachment site on the bacteriophage baseplate for the long tail fibers.

Carboxypeptidases↗
PubMed1978

Zinc uptake and incorporation into proteins in T4D bacteriophage-infected Escherichia coli.

The metabolism of Zn2+ in Escherichia coli infected with T4D bacteriophage and various T4D mutants has been examined. E. coli B infected with T4D, and all T4D mutants except T4D 12-, took up zinc ions at a rate identical to that of uninfected cells. E. coli B infected with T4D 12- had a markedly decreased rate of zinc uptake. The incorporation of zinc into proteins of infected cells has also been studied. T4D phage infection was found to shut off the synthesis of all bacterial host zinc metalloproteins while allowing the formation of viral-induced zinc proteins. The amount of zinc incorporated into viral proteins was affected by the absence of various T4D gene products. Cells infected with T4D 12-, and to a much less extent those infected with T4D 29-, incorporated the least amount of zinc into proteins, while cells infected with T4D 11- and T4D 51- incorporated increased amounts of zinc into the zinc metalloproteins. In cells infected with T4D 11- and 51- most of the zinc protein was found to be the product of gene 12. The marked effect of infection of E. coli with T4D 12- on both zinc uptake and zinc incorporation into protein supports the conclusion that T4D gene 12 protein is a zinc metalloprotein. Additionally, these observations have indicated that this metalloprotein interacts with host cell membrane.

Biological Transport↗
PubMed1978

Folate polyglutamates in T4D bacteriophage and T4D-infected Escherichia coli.

The folate compound which is a structural component of the Escherichia coli T-even bacteriophage baseplates, has been identified as the hexaglutamyl form of folic acid using a new chromatographic procedure (Baugh, C.M., Braverman, E. and Nair, M.G. (1974) Biochemistry 13, 4952-4957). It has also been found that the host cell contains a variety of polyglutamyl forms of folic acid. The major form is the triglutamate (about 50%) but small amounts of higher molecular weight folates including the octaglutamate (1.8%) have been identified. Upon infection with wild-type T4D bacteriophage there is a shift in the distribution of the folate compounds so that the folyl polyglutamyl compounds having the higher molecular weights are increased. Infection of E. coli with baseplate mutants of T4D containing an amber mutation in gene 28 resulted in the formation of significant amounts (over 7%) of folate compound(s) of molecular weight much higher than those observed either in uninfected cells or cells infected with wild-type T4D. It is suggested that the T4D gene 28 product functions to cleave glutamate residues from high molecular weight folyl polyglutamates to increase the availability of the folyl hexaglutamate for virus assembly.

Coliphages↗
PubMed1977

Bacteriophage T4 virion baseplate thymidylate synthetase and dihydrofolate reductase.

Additional evidence is presented that both the phage T4D-induced thymidylate synthetase (gp td) and the T4D-induced dihydrofolate reductase (gp frd) are baseplate structural components. With regard to phage td it has been found that: (i) low levels of thymidylate synthetase activity were present in highly purified preparations of T4D ghost particles produced after infection with td(+), whereas particles produced after infection with td(-) had no measurable enzymatic activity; (ii) a mutation of the T4D td gene from td(ts) to td(+) simultaneously produced a heat-stable thymidylate synthetase enzyme and heat-stable phage particles (it should be noted that the phage baseplate structure determines heat lability); (iii) a recombinant of two T4D mutants constructed containing both td(ts) and frd(ts) genes produced particles whose physical properties indicate that these two molecules physically interact in the baseplate. With regard to phage frd it has been found that two spontaneous revertants each of two different T4D frd(ts) mutants to frd(+) not only produced altered dihydrofolate reductases but also formed phage particles with heat sensitivities different from their parents. Properties of T4D particles produced after infection with parental T4D mutants presumed to have a deletion of the td gene and/or the frd gene indicate that these particles still retain some characteristics associated with the presence of both the td and the frd molecules. Furthermore, the particles produced by the deletion mutants have been found to be physically different from the parent particles.

Coliphages↗
PubMed1975

Bacteriophage T4 baseplate components. I. Binding and location of the folic acid.

Two different proteins with high affinities for the pteridine ring of folic acid have been used to determine the location of this portion of the folate molecule in the tail plate of T4D and other T-even bacteriophage particles. The two proteins used were (i) antibody specific for folic acid and (ii) the folate-binding protein from bovine milk. Both proteins were examined for their effect on various intact and incomplete phage particles. Intact T2H was weakly inactivated by the antiserum but not by the milk protein. No other intact T-even phage, including T4D, was affected by these two proteins. When incomplete T4D particles were exposed in an in vitro morphogenesis system, it was found that neither of the two proteins affected either the addition of the long tail fibers to fiberless particles or the addition of tail cores to tail plates. On the other hand, these two proteins specifically blocked the addition of T4D gene 11 product to the bottom of T4D baseplates. After the addition of the gene 11 protein, these two reagents did not inhibit the further addition of the gene 12 protein to the baseplate. It can be concluded that the phage folic acid is a tightly bound baseplate constituent and that the pteridine portion of the folic acid is largely covered by the gene 11 protein.

Animals↗
PubMed1975

Bacteriophage T4 baseplate components. II. Binding and location of bacteriophage-induced dihydrofolate reductase.

The location of T4D phage-induced dihydrofolate reductase (dfr) has been determined in intact and incomplete phage particles. It has been found that phage mutants inducing a temperature-sensitive dfr (dfrts) procude heat-labile phage particles. The structural dfr produced by these ts mutants was shown to assume different configurations depending on the temperature at which the phage is assembled. Morphogenesis of incomplete phage particles lacking the gene 11 protein on their baseplates was found to be inhibited by reagents binding to dfr, such as antibodies to dfr. Further, cofactor molecules for dfr, such as reduced nicotinamide adenine dinucleotide phosphate and reduced nicotinamide adenine dinucleotide, also inhibited the step in morphogenesis involving the addition of gene 11 product. On the other hand, inhibitors of dfr, such as adenosine dephosphoribose, stimulated the addition of the gene 11 protein. It has been concluded that the phage-induced dfr is a baseplate component which is partially covered by the gene 11 protein. The properties of phage particles produced after infection of the nonpermissive host with the one known T4D mutant containing a nonsense mutation in its dfr gene suggested that these progeny particles contained a partial polypeptide, which was large enough to serve as a structural element.

Adenine Nucleotides↗
PubMed1975

Bacteriophage T4 baseplate components. III. Location and properties of the bacteriophage structural thymidylate synthetase.

Two T4D thymidylate synthetase (td) temperature-sensitive mutants have been isolated and characterized. Both mutants produce heat-labile phage particles. This observation supports the view that this viral-induced protein is a phage structural component. Further, antiserum to td has been shown to block a specific step in tail plate morphogenesis. The results indicated that the td protein is largely covered by the T4D tail plate gene 11 protein. Since the phageinduced dihydrofolate reductase (dfr) also is partially covered by the gene 11 protein, it appears that td was adjacent to the tail plate dfr. This location has been confirmed by constructing a T4D mutant which is dfrtstdts and showing that these two tail plate constituents interact and give altered physical properties to the phage particles produced. A structural relationship for the tail plate folate, dfr, and td has been reported.

Coliphages↗
PubMed1973

Bacteriophage tail components. IV. Pteroyl polyglutamate synthesis in T4D-infected Escherichia coli B.

The nature of pteroyl polyglutamates in uninfected and T4D bacteriophage-infected Escherichia coli B has been examined. (3)H-p-aminobenzoic acid has been used to label the folate compounds and gel permeation chromatography on glass beads to separate the folate compound by molecular size. It has been found that, although the major folate compound in uninfected bacteria is pteroyl triglutamate, E. coli B cells also contain folate compounds having as many as six glutamate residues. Infection with T4D stimulated the addition of glutamate residues to the lower-molecular-weight host pteroyl compounds, resulting in the conversion of the host compounds into the hexaglutamate form. This viral-induced conversion is chloramphenicol sensitive and appears to be due to a late phage gene product. The phage gene responsible for this conversion has not been identified. In cells infected with a T4D mutant defective in gene 28, there was an apparent production of the large pteroyl polyglutamates equivalent in size to pte(glu)(9-12). These high-molecular-weight forms were converted into pte(glu)(6) by incubation with bacterial extracts made after infection with T4D 28(+). Apparently, the product of T4D gene 28(+) is capable of specifically cleaving the high-molecular-weight polyglutamates to the form necessary for phage tail assembly.

Aminobenzoates↗