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N B Groman

Publications and source records attributed to N B Groman.

At least 19 recordsLinked to original sources
PubMed1997

Characterization of bacteriophages from tox-containing, non-toxigenic isolates of Corynebacterium diphtheriae.

Non-toxigenic strains of Corynebacterium diphtheriae continue to cause disease within immunized populations. A subset of these corynebacteria carry the diphtheria toxin gene but in a cryptic form. To determine whether such strains might contribute to the re-emergence of functional toxin genes, the phages and tox mutations within three clone types were examined. tox-containing, beta-related phages were isolated from two of the strain types. The third isolate appeared to harbour a defective prophage. One of the tox- phages encoded truncated, yet enzymatically-active, forms of diphtheria toxin, suggesting that it had sustained a point mutation within the latter half of its toxin gene. In contrast, the other mutant phage did not elicit the production of either a cross-reacting material or an ADP-ribosylating activity. Complementation tests employing a series of double lysogens confirmed that the mutations responsible for the non-toxigenic phenotype of all of the phages were cis dominant. Given these findings, it is reasonable to hypothesize that tox+ genes can arise within human populations by either homologous recombination between two distinct tox- phages or spontaneous reversion within a single mutant allele.

Bacteriophages↗
PubMed1996

Extended host range of a beta-related corynebacteriophage.

Unlike most beta-related phages isolated from Corynebacterium diphtheriae, phage 782 readily plaqued on strains of C. ulcerans and C. pseudotuberculosis. The extended host range of phage 782 was not, however, due to a greater ability of the phage to absorb to bacterial surfaces. Using chemical mutagenesis, a number of phage mutants were isolated which had diminished capacities to infect C. ulcerans, suggesting the existence of a locus (ehr) for extended host range. Pre-lysogenization of C. ulcerans strains with phage 782, but not its mutant form or other beta-related phages, rendered them susceptible to infection by previously excluded phages. An examination of recombinant between phages 782, pie, and beta localized ehr to a 7 kilobase region of DNA including attP. The data are compatible with the notion that ehr encodes an anti-restriction function.

Bacteriophages↗
PubMed1993

Laboratory review of reference strains of Corynebacterium diphtheriae indicates mistyped intermedius strains.

All biotyped strains of Corynebacterium diphtheriae from the American Type Culture Collection (ATCC) were compared for morphology and biochemical reactions. Biotypes of all gravis strains and most mitis strains were confirmed, but intermedius strains were found to be misclassified. New lipid-dependent intermedius strains have been deposited with the ATCC.

Bacterial Typing Techniques↗
PubMed1990

Localization of an origin of replication in Corynebacterium diphtheriae broad host range plasmid pNG2 that also functions in Escherichia coli.

Subcloning and protoplast transformation studies identified a 2.6 kb fragment of Corynebacterium diphtheriae plasmid pNG2 which contains an origin of replication (oriR). Molecular combination of the 2.6 kb oriR cartridge with Escherichia coli plasmid pUC18CmR enabled the E. coli cloning vector to replicate within several species of Corynebacterium host cells. A 2.6 kb plasmid formed from the oriR cartridge alone is capable of replicating in E. coli. This suggests that a single origin could be used in vectors shuttling between Corynebacterium spp. and E. coli.

Cloning, Molecular↗
PubMed1989

The molecular epidemiology of three biotypes of Corynebacterium diphtheriae in the Seattle outbreak, 1972-1982.

Between 1972 and 1982 Seattle experienced a diphtheria outbreak involving 1,100 cases, primarily adults with cutaneous lesions. Biotyping revealed three consecutive overlapping outbreaks including 433 toxinogenic intermedius cases. Isolates from each quarter year were examined for DNA restriction fragment patterns and hybridization patterns with three DNA probes. All intermedius outbreak isolates appeared identical in all analyses and most stock cultures had the outbreak DNA fragment pattern, but DNA probes detected six patterns within 11 intermedius stock cultures. The mitis outbreak was heterogeneous, involving at least five restriction fragment patterns. In contrast, DNA restriction fragment analyses indicated that 22 of 25 gravis outbreak isolates belonged to a single strain, whereas there were seven strains within eight gravis stock cultures. DNA probe analyses of gravis outbreak isolates detected seven different patterns, five involving copy numbers of the toxB fragment and attB sites that presumably reflected lysogenic events that occurred during the outbreak.

Bacterial Typing Techniques↗
PubMed1986

Mapping and cloning of Corynebacterium diphtheriae plasmid pNG2 and characterization of its relatedness to plasmids from skin coryneforms.

The relationship of plasmid pNG2, isolated from an erythromycin-resistant strain of Corynebacterium diphtheriae, to plasmids isolated from skin coryneforms was examined. The extent of homology between plasmids from erythromycin-resistant and -susceptible skin coryneforms and pNG2 varied, but in aggregate homology was observed with all six BstEII fragments of pNG2. The data support the hypothesis that pNG2 originated in skin coryneforms. Intact plasmid pNG2 and some of its restriction fragments were cloned into Escherichia coli JM109. The erythromycin resistance phenotype was expressed in clones carrying intact pNG2 as well as in some of its fragments and appeared to depend on a C. diphtheriae promoter for expression. A 2.5-megadalton EcoRI fragment, the smallest expressing resistance, contained the 1.2-megadalton region of pNG2 which is deleted when the erythromycin-resistant strain of C. diphtheriae reverts spontaneously to the susceptible state.

Chromosome Mapping↗
PubMed1984

Conversion by corynephages and its role in the natural history of diphtheria.

The conversion of non-toxinogenic Corynebacterium diphtheriae to toxinogeny has been reviewed. The biology of converting phage and the relationship of converting phages to nonconverting phages are summarized. The significance of these findings to the natural history and evolution of diphtheria is assessed.

Bacteriophages↗
PubMed1982

Immunological cross-reactivity in the absence of DNA homology between Pseudomonas toxin A and diphtheria toxin.

The immunodominant determinant of Pseudomonas toxin A was shown to cross-react with a normally inaccessible determinant in fragment A of diphtheria toxin. Trypsin-treated diphtheria toxin and fragment A of diphtheria toxin inhibited binding of toxin A antibody to whole toxin A, whereas whole diphtheria toxin did not inhibit this reaction. However, even at the lowest stringency no hybridization was detected between diphtheria tox probe and Pseudomonas aeruginosa DNA.

ADP Ribose Transferases↗
PubMed1981

Physical mapping of beta-converting and gamma-nonconverting corynebacteriophage genomes.

Deoxyribonucleic acids (DNAs) from wild-type and mutant strains of beta-converting and gamma-nonconverting corynebacteriophages were isolated and physically characterized. The data obtained from DNA heteroduplexes, restriction enzyme banding profiles, and restriction maps reinforce the conclusion that beta and gamma phages are very closely related. The major physical differences seen in the DNA heteroduplexes are a small substitution bubble and one or two insertions which are present on the gamma phage genome. The insertions account for the differences in the genome sizes of beta and gamma phages, and with the substitution they are responsible for most of the differences in the restriction endonuclease profiles and maps of the corynebactriophage genomes, two special sites and the DNA fragments carrying them were identified. These were the cohesive (cos) sites and the specific attachment (attP) site of the vegetative phage genome. The behavior of these sites indicated that the transition of phage DNA from the vegetative to the prophage state involves the circularization of vegetative DNA through the cos sites and its integration into the bacterial chromosome via the attP site. The mechanism of corynebacteriophage integration was similar to that employed by Escherichia coli phage gamma. From the data assembled the physical and genetic maps of beta and gamma phage were oriented with respect to one another. The extensive similarity in their maps provides additional confirmation of a close evolutionary relationship.

Attachment Sites, Microbiological↗
PubMed1981

Genetic elements novel for Corynebacterium diphtheriae: specialized transducing elements and transposons.

It was shown in an accompanying paper (Buck and Groman, J. Bacteriol. 148: 131-142, 1981) that gamma-tsr-1 phage stocks produced by heat induction of lysogens are a mixture of two phages which differ in the content of their deoxyribonucleic acid (DNA). This difference is evidenced by the appearance of "heterogeneous" (HET) fragments in restriction enzyme digests of gamma-tsr-1 phage DNA. It was estimated that 20 to 80% of the phage in these lysates produced HET fragments. The appearance of HET fragments correlated with the appearance of a DNA insertion (DI-1) in the gamma phage genome as revealed in heteroduplexes of DNA from gamma-tsr-1 and beta corynebacteriophages. The HET fragments were seen in DNA from heat-induced lysates, but not in DNA from phage stocks produced by lytic infection. By DNA-DNA hybridization analysis it was shown that a fraction of gamma-tsr-1 phages from heat-induced lysates carried an insertion of bacterial DNA in the vegetative phage attachment site (attP), and that this insertion was responsible for the formation of HET fragments. Since the phage produced by this event carried a complete phage genome plus a small segment of bacterial DNA, they were called transducing elements. On the basis of these facts it was concluded that heat-induced gamma-tsr-1 prophage was excised at an abnormal site at a very high frequency. Abnormal excision was highly specific, and the change in excision specificity occurred simultaneously with the spontaneous mutation of the phage to heat inducibility. From this and other data it was postulated that a mutation in the immune repressor was reponsible for an alteration in the specificity of the normal excision process. This distinguishes the mechanism of formation of gamma-tsr-1 transducing elements from that employed by other phages. A second DNA insertion (DI-2) in the tox (diphtheria toxin) gene of gamma-tsr-1 and gamma-tsr-2 was also identified as an insertion of bacterial DNA. The DI-2 insertion had a stem-and-loop structure similar to that seen in heteroduplexes visualizing transposons or insertion elements. It seems likely that gamma wild-type phage, which is mutant for tox, was originally tox(+), but that transposition of bacterial DNA into the gene inactivated it.

Attachment Sites, Microbiological↗
PubMed1981

Identification of deoxyribonucleic acid restriction fragments of beta-converting corynebacteriophages that carry the gene for diphtheria toxin.

Deoxyribonucleic acid fragments bearing the gene for diphtheria toxin have been identified in restriction enzyme digests of deoxyribonucleic acids from beta-converting and gamma-nonconverting corynebacteriophages. A combination of physical and genetic evidence has established that the Bam HI band C fragment of beta phage deoxyribonucleic acid, which carries the specific phage attachment site (Buck and Groman, J. Bacteriol. 148:131-142, 1981), also carries most, and probably all, of the gene for diphtheria toxin. A detailed restriction map of this tox-bearing Bam HI fragment has been developed, and the locations and orientation of the tox gene and the attP site within this fragment have been established.

Attachment Sites, Microbiological↗
PubMed1980

Superinfection exclusion by heteroimmune corynebacteriophages.

Superinfection of Corynebacterium diphtheriae C7(beta) by heteroimmune phage gamma is productive, whereas superinfection by gamma-bin mutants is for the most part nonproductive. Exclusion of gamma-bin phage occurred after its DNA had penetrated and was partially expressed in the heteroimmune lysogen. All of the infected cells were killed, and lysis was observed. The beta inhibitor causing exclusion was produced during the prophage state and appeared to be distinct from immune repressor. The ability of gamma-bin phage to superinfect C7(beta) productively could be restored by recombination with beta phage, indicating that both beta and gamma phages contain either indentical or similar alleles of the bin gene. The bin gene was mapped by vegetative and prophage crosses and found to be located in the region of the phage genome concerned with regulation. Both beta and gamma wild-type phages induced the resident prophage in a significant fraction of superinfeted heteroimmune lysogens. This, coupled with the fact that induction of C7(beta) abolished exclusion, suggests that the bin gene product acts as antirepressor, i.e., it reduces the level of heteroimmune repressor either directly or indirectly. The gamma-bin mutants either failed to produce antirepressor or did so with reduced efficiency. Antirepressor activity was negatively controlled by homoimmune repressor. The isolation of beta mutants that appeared bin-like suggests that beta and gamma phages contain homologous systems of exclusion and antiexclusion. Exclusion of gamm-bin by beta phage in gram-positive C. diphtheriae exhibited striking parallels to the sieB exclusion described for phages P22 and lambda in gram-negative organisms. The extended similarities of these coryngephages to lambda bacteriophage is noted.

Bacteriophages↗
PubMed1973

Toxinogeny in Corynebacterium diphtheriae after loss of catalase, cystinase, or deoxyribonuclease activity.

The relationship of catalase, cystinase, and deoxyribonuclease activity to toxinogeny in Corynebacterium diphtheriae was examined. Mutants deficient in each activity were isolated after mutagenization of strain C4 with nitrosoguanidine. All mutants were converted to toxinogeny after lysogenization with beta-converting phage, thus establishing that there is no absolute link between toxin production and these enzymatic activities. No differences were observed in the rate of lysogenization of the mutants by beta-converting phage over that of the parental strain. However, the data suggest that catalase mutants lysogenic for beta phage are generally induced at a higher rate than the parental strain after irradiation with ultraviolet light. Cystinase mutants vary widely in their rate of induction whereas the deoxyribonuclease mutants are similar to the parental strain. The relationship of these results to the production of toxinogenic strains is discussed.

Bacteriological Techniques↗
PubMed1969

Activity of diphtheria toxin. II. Early events in the intoxication of HeLa cells.

The initial steps in the interaction of diphtheria toxin with HeLa cells were studied. It was demonstrated that lethal doses of toxin are rapidly adsorbed to the cell. The kinetics of uptake, as measured by lethality, indicated that a single toxin molecule is able to cause cell death. Studies on the effect of pH on intoxication showed that adsorption of toxin occurred over a wide pH range but was partially inhibited at high pH values. Experiments to determine the influence of the ionic environment on intoxication indicated that adsorption of toxin did not take place in the absence of salts and was partially inhibited in the presence of a polyanion. The evidence indicates that the initial binding of toxin to the cell is electrostatic in nature, involving positively charged surface groups. Attempts to demonstrate specific receptors for the attachment of toxin to cells were unsuccessful, suggesting that toxin adsorption may be a nonspecific process. The effect of energy inhibitors on intoxication was examined. Sodium fluoride, an inhibitor of glycolysis, almost completely prevented intoxication in HeLa cells, whereas inhibitors of respiration and oxidative phosphorylation had no effect. Sodium fluoride did not prevent adsorption of toxin but appeared to inhibit a later step in the intoxication process, perhaps the transport of toxin to subsurface or intracellular levels.

Binding Sites↗
PubMed1967

Studies of the activity of diphtheria toxin. I. Poliovirus replication in intoxicated HeLa cells.

It has been demonstrated that a saturating dose of diphtheria toxin produced a 90% inhibition of polio-virus replication in HeLa cells. This inhibition was reflected in infectious viral RNA synthesis and in mature virus production. Toxin had no direct effect on virus particles or I-RNA, and poliovirus adsorption and eclipse appeared to be carried out normally in intoxicated cells. When toxin was given at various time intervals after infection, the amount of inhibition depended on the time of toxin addition. Toxin given before or immediately after infection gave maximum inhibition, while toxin given several hours after infection had little effect. The data suggest that toxin inhibits viral replication through its effect on protein synthesis. It is likely that a critical step in the viral replication cycle, the production of poliovirus-induced RNA polymerase, is inhibited, and possibly the synthesis of capsid protein. Ammonium salts and the aliphatic amines, glycamine and prolamine, prevented the inhibition of viral replication by toxin. The kinetics of the protective action of ammonium chloride and diphtheria antitoxin are remarkably similar.

Diphtheria Toxin↗
PubMed1966

Inhibition of coliphage reproduction after superinfection of induced lysogens.

Brenner, Don J. (University of Washington, Seattle), and Neal B. Groman. Inhibition of coliphage reproduction after superinfection of induced lysogens. J. Bacteriol. 92:1727-1734. 1966.-Purified preparations of phages lambda and lambda112 inhibited lysis, phage reproduction, and endolysin synthesis by ultraviolet-induced strains of K-12 (lambda) and K-12 (lambda112). Structural and partial functional integrity of phage was required for inhibition, and the role of phage deoxyribonucleic acid (DNA) in inhibition was demonstrated. Both ultraviolet-irradiated and host-modified lysates of lambda were inhibitory, suggesting that replication of superinfecting phage DNA was not essential for inhibition. Using various combinations of superinfecting and resident phage, including lambda, 434, 434hy, and C-mutant derivatives, we observed that all three C region cistrons played some role in inhibition. Of the three, the C(1) cistron was the most critical. With certain phage combinations, inhibition was observed even though the resident and superinfecting phage differed in immune specificity. Both the medium and the method of superinfection determined whether lysis or lysis inhibition would occur. These and other observations with K-12(lambda)thy(-) indicated that many factors influence the outcome of superinfection. The data are compatible with the view that superinfection inhibition is due to the establishment or re-establishment of phage repressor activity, with the result that replication of both superinfecting and resident phage is blocked.

Antibodies↗