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N Groman

Publications and source records attributed to N Groman.

At least 19 recordsLinked to original sources

DNA sequence homology between attB-related sites of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and the attP site of gamma-corynephage.

Chromosomal restriction fragments of Corynebacterium ulcerans and C. diphtheriae, containing an integration site for corynephages of the beta family, show homology on Southern blots. Homologous DNA in also found in the soil isolate C. glutamicum, although this strain is not susceptible to beta-corynephages. Three of these DNA fragments, one for each bacterial strain, and a fragment of gamma-corynephage DNA previously shown to contain the phage integration site, were cloned and sequenced. Alignment of the 3 bacterial sequences shows a very high degree of homology in a stretch of ca 120 nucleotides, whereas the rest of the sequences is generally non-homologous. Within this common bacterial portion, a segment of ca. 96 nucleotides (core sequence) is also highly homologous to the phage sequence. The first half (ca. 50 bp) of the core sequence is identical in all aligned sequences whereas the second half, which is largely occupied by a stem-and-loop structure, contains point mutations peculiar to each clone. The described sequences are likely to be involved in phage integration/excision processes.

Attachment Sites, Microbiological↗

Transformation of Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium glutamicum, and Escherichia coli with the C. diphtheriae plasmid pNG2.

The transfection and transformation of members of two species of pathogenic corynebacteria, Corynebacterium diphtheriae and Corynebacterium ulcerans, is described. Protoplasts were produced by treatment with lysozyme following growth in glycine, and a medium was defined on which a significant fraction of the osmotically sensitive cells were regenerated. Transfections were carried out with DNA from corynephage 782, a member of the beta family of converting phages, and transformations were performed with DNA of plasmid pNG2, a 9500-kDa plasmid that was isolated from an erythromycin-resistant strain of C. diphtheriae and carries the resistance gene. Strains of Corynebacterium glutamicum and Escherichia coli were also successfully transformed with pNG2 DNA. Transfection frequencies were in the range of 3-8 X 10(3) plaque-forming units/micrograms of phage DNA, and transformation frequencies were in the range of 0.2-150 colony-forming units/micrograms of plasmid DNA. Plasmid pNG2 replicated and was stably maintained in all transformants both in the presence or absence of erythromycin. Thus, it displayed the ability to replicate in strains of both Gram-positive and Gram-negative bacteria without the intervention of genetic engineering. pNG2 DNA isolated from any of the transformed strains was able to transform all parental strains. The host range of pNG2 suggests its possible utility in or as a shuttle vector for the study and manipulation of genes from corynebacterial strains of animal origin.

Bacteriophages↗

Detection of homology to the beta bacteriophage integration site in a wide variety of Corynebacterium spp.

In toxigenic conversion of Corynebacterium diphtheriae C7, beta bacteriophage DNA integrates into either of two chromosomal attachment sites, attB1 or attB2. These attB sites share a 96-base-pair sequence with the attP sites of beta-related phages. The distribution of attB-related sites in other species of Corynebacterium was assessed by hybridization with a DNA probe containing both attB sites of the C7 strain and a second DNA probe containing the attP site of a beta-related phage. All but one of the 15 C. diphtheriae strains tested, regardless of origin or colonial type, contained at least two BamHI fragments that hybridized strongly to both of these probes under conditions of high stringency. Strains of C. ulcerans and C. pseudotuberculosis, species in which conversion to toxinogeny has also been demonstrated, also had one or two hybridizing BamHI fragments. The functionality of these sites as integration sites was demonstrated by isolating lysogens of all three species following single infection with one or more beta-related phages. As predicted, following lysogenization one of the DNA fragments that had exhibited homology with the attB1-attB2 probe was replaced by two hybridizing fragments. Other species of Corynebacterium, including pathogens and nonpathogens from animals, plant pathogens, and soil isolates also carried at least one BamHI fragment that hybridized with the attB1-attB2 and attP probes. The data indicate that sequences homologous to the beta phage integration sites in C. diphtheriae have been conserved in members of the genus Corynebacterium.

Attachment Sites, Microbiological↗

A beta-related corynebacteriophage which lacks a tox allele but can acquire it by recombination with converting phage.

Corynebacteriophage 782, a phage highly related to the beta family of corynebacteriophages but lacking a tox allele, was isolated from a nontoxinogenic clinical isolate of Corynebacterium diphtheriae. Phage 782 exhibits beta immunity but has a wider host range than beta, forming plaques on strains of C. ulcerans and C. pseudotuberculosis as well as on C. diphtheriae. Phage 782 and beta differed in their DNA mass and in their restriction endonuclease digest patterns, but were similar in possessing cos (cohesive) and attP (phage attachment) sites. Moreover, all the BamHI fragments of 782 and beta except one hybridized with a DNA probe of the other. The exception in both cases was the attP-containing fragment, which in beta also carries the tox gene. Recombinants between phage 782 and pi phage, a tox+ beta-related phage, were isolated which contained ca. 70% of phage 782 DNA but carried the attP-tox-bearing fragment of pi and were thus now converting phages. The recombinants had lost the wide-host-range phenotype of 782 and had the narrower host range of pi. The significance of the tox-less, beta-related phages to the natural history of diphtheria is discussed.

Alleles↗

DNA relationships among some tox-bearing corynebacteriophages.

The DNA genomes of a number of tox-bearing, temperate corynebacteriophages isolated from strains of Corynebacterium diphtheriae and Corynebacterium ulcerans were compared. With one exception, these phages displayed similarities in their restriction enzyme digest profiles and extensive homology with prototypic beta converting phage. The exception, phage delta, had a unique restriction profile and exhibited homology with beta over a limited portion of its genome. DNAs of phages from each host contained cohesive ends and integrated as prophage by a mechanism analogous to that employed by coliphage lambda. It is proposed that these tox-bearing phages belong to a common family, the beta family. The role of the beta family in the movement of the tox gene between strains of C. diphtheriae and C. ulcerans is discussed.

Bacteriophages↗

Corynebacterium ulcerans and Corynebacterium pseudotuberculosis responses to DNA probes derived from corynephage beta and Corynebacterium diphtheriae.

Strains of Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (Corynebacterium ovis) were examined for the production of diphtheria toxin. A majority of C. ulcerans strains (25 of 37) and 1 C. pseudotuberculosis strain (1 of 14) gave a positive Elek test for diphtheria toxin, and for all strains but 1, production of diphtheria toxin was inhibited at the same level of Fe2+ as was the Corynebacterium diphtheriae control. All Elek-positive cultures as well as two Elek-negative isolates of C. ulcerans gave a positive signal when hybridized with a DNA probe unambiguous for the diphtheria toxin gene (tox) under conditions of high stringency. The majority of probe-positive C. ulcerans strains contained three or more DNA restriction fragments that hybridized with converting corynephage beta, suggesting that in C. ulcerans as in C. diphtheriae there may be a relationship between toxinogeny and carriage of beta-related phage. Selected strains of C. diphtheriae, C. ulcerans, and C. pseudotuberculosis were examined for DNA homology by a semiquantitative technique. There was very little homology between C. diphtheriae and members of the other two species. Strains of C. ulcerans and C. pseudotuberculosis, although more closely related, appeared to belong to distinct species as well.

Bacteriophages↗

Relationship between pNG2, an Emr plasmid in Corynebacterium diphtheriae, and plasmids in aerobic skin coryneforms.

Erythromycin-resistant (Emr) coryneforms from cutaneous lesions and erythromycin-susceptible (Ems) coryneforms from normal skin sites were screened for plasmids. Approximately one-third of the 40 isolates carried one or more plasmids ranging in mass from 2.5 to 36 megadaltons, all exhibiting different restriction enzyme digest patterns. In contrast, only Corynebacterium diphtheriae strains comprising a single cohort of apparently identical Emr, pNG2-carrying isolates have been identified as plasmid carriers. Homology was demonstrated between pNG2 and a number of fragments in restriction enzyme digests of plasmids from both Emr and Ems skin coryneforms under high-stringency conditions. However, none was detected between pNG2 and the genomic or plasmid DNAs of Emr staphylococci or streptococci isolated concurrently with the Emr coryneforms. One coryneform plasmid, pNG34, exhibited extensive homology with pNG2, and many comigrating fragments were observed. Very little relationship was observed between C. diphtheriae and the skin coryneforms when their genomic DNAs were hybridized. The origin and presence of pNG2 in Emr C. diphtheriae is discussed in relation to these findings.

Bacteria, Aerobic↗

Detection and expression of DNA homologous to the tox gene in nontoxinogenic isolates of Corynebacterium diphtheriae.

Three probes have been described which can be used to detect the presence of DNA sequences homologous to the tox gene of Corynebacterium diphtheriae. Probes "A" and "B" detected sequences coding for A and B fragments of diphtheria toxin, respectively. The third "A-B" probe contained both the A and B coding sequences. The B probe was completely unambiguous in detecting only toxin-related sequences, and the A probe was only slightly less so. The efficacy of the probes was tested on a series of toxinogenic and nontoxinogenic isolates of C. diphtheriae. All isolates which were toxinogenic as characterized by the gel immunodiffusion technique gave positive reactions with the probes. Of particular interest was the finding that 14 of 43 nontoxinogenic isolates also carried DNA homologous to both the A and B probes. All 14 isolates were nontoxinogenic by the rabbit intracutaneous test as well as by the gel immunodiffusion test; however, 12 of them produced ADP-ribosylating activity, whereas two were negative. The isolates producing ADP-ribosylating activity belonged to a cohort of cultures, of which 11 were isolated in South Dakota and 1 was isolated in Montana. Genomic DNAs of all 12 appeared to be identical when restriction enzyme digest patterns were compared, and the same fragment carried the tox gene in all of them. The tox-bearing nontoxinogenic isolates from Alaska and Florida each had unique restriction patterns and did not produce ADP-ribosylating activity. A number of genomic fragments of all the tox-bearing nontoxinogenic isolates hybridized with beta converting phage DNA. The significance of these observations to the natural history of diphtheria was discussed.

Bacteriophages↗

Gene responsible for superinfection exclusion of heteroimmune corynebacteriophage.

Wild-type beta and gamma corynebacteriophages are heteroimmune and infect lysogens of each other productively. Unlike their wild-type counterparts, the bin mutants of each phage are excluded in lysogens carrying the heteroimmune phage. The wild-type phages overcome exclusion by means of the bin gene product which appears to act as an antirepressor. When repression is lifted, exclusion of bin mutants is abolished (N. Groman and M. Rabin, J. Virol. 28:28-33, 1978; J. Virol. 36:526-532, 1980). It has not been clear whether the excluding compound is the immune repressor itself or one whose synthesis is positively regulated by repressor. We have isolated beta exclusion mutants (xcl) that as prophage exhibited normal immune repression but no longer excluded gamma-bin mutants. Furthermore, we have shown that an xcl phage with an active immune repressor acted in trans to continue the positive regulation of exclusion by a second xcl+ prophage whose immune repressor was inactivated. From these results it was concluded that there is a gene distinct from the imm gene which is directly or indirectly responsible for exclusion. The xcl gene, mapped in prophage crosses, was located between imm and bin, that is, in the regulatory region of the phage genome. The simplest hypothesis compatible with the established observations is that beta immune repressor regulates the expression of the xcl and bin genes, the former positively and the latter negatively. It is likely that an analogous regulatory model applies to gamma phage since it has already been shown that both beta and gamma have bin alleles.

Actinomycetales↗

Plasmids in Corynebacterium diphtheriae and diphtheroids mediating erythromycin resistance.

Plasmids were isolated from erythromycin-resistant Corynebacterium diphtheriae and skin coryneforms. Six erythromycin-resistant C. diphtheriae strains, isolated from cutaneous lesions, all contained a 9.5-megadalton (Mdal) plasmid. Loss of resistance was associated with the deletion of a 1-Mdal segment from the plasmid or, less frequently, with loss of the plasmid. Two erythromycin-resistant diphtheroids were isolated from similar skin lesions. One contained a 38-Mdal plasmid that was lost in the conversion to erythromycin susceptibiliy. The other diphtheroid contained a 30- and a 14-Mdal plasmid. Erythromycin-susceptible derivatives of this strain were not recovered. Restriction enzyme analysis indicated that the 9.5-Mdal plasmids in the C. diphtheriae strains are very similar, if not identical, and that each of the deleted plasmids has lost the same 1-Mdal segment. However, the restriction patterns of the plasmids in the two diphtheroids are not closely related to each other nor to the plasmids in the C. diphtheriae strains.

Corynebacterium diphtheriae↗

Effect of metal ions on diphtheria toxin production.

The effect of several metal ions on the production of diphtheria toxin was tested. By using the gel immunodiffusion system for detecting toxin, a wide range of metal ion concentrations was conveniently surveyed. Five divalent cations, Fe2+, Cu2+, Co2+, Ni2+, and Mn2+ inhibited toxin production within a range of concentrations that did not inhibit growth of the producing strain. Growth and toxin production were inhibited at identical concentrations by both Cd2+ and Zn2+, whereas Al3+ and Sr2+ affected neither growth nor toxin production over the range of concentrations tested. The data showed that Fe2+ was the most effective inhibitor on an equivalence basis, followed by Cu2+, Co2+, and Ni2+ in descending order. All eight strains of Corynebacterium diphtheriae chosen from diverse ecological origins responded similarly to all metals at similar concentrations. A mutant strain which produces toxin at Fe2+ concentrations 500 times greater than are inhibitory for the parent strain had simultaneously acquired resistance to inhibitory concentrations of Cu2+, Co2+, Ni2+, and Mn2+. This suggests that there is at least one common point in the activity of all these metal ions, and that toxin may respond broadly to changes in metal ion concentrations in the environment.

Aluminum↗

Control of corynebacteriophage reproduction by heteroimmune repression.

Corynebacteriophages beta and gamma are closely related but heteroimmune; hence, gamma reproduces in C7(beta). A series of gamma mutants, designated gamma-bin (beta-inhibited), has been isolated. They reproduce in only 2 to 14% of infected C7(beta) cells, and, as a result, plaque with an efficiency of 10(-4) to 10(-5) on this strain. The proportion of C7(beta) cells in which gamma-bin phage can replicate is increased to 30 to 80% when immunity is lifted by UV induction of C7(beta) or by heat induction of C7(beta-tsr3). The gamma-bin mutants carry out a normal vegetative or lysogenic cycle in strain C7 and thus do not appear to be defective in any essential phage function. Infection of C7(beta) by gamma-bin results in cell killing whether the infection is productive or nonproductive. The data support the hypothesis that inhibition of gamma-bin is due to the direct or indirect action of a beta prophage gene. The simplest hypothesis is that gamma-bin phages have sustained mutations in an operator site and that beta repressor now combines with the mutated operator to inhibit normal replication in a significant proportion of infected cells.

Bacteriophages↗

Heat-inducible mutants of corynebacteriophage.

Heat-inducible mutants of temperate cornebacteriophage beta and gamma, called temperature-sensitive repression (tsr) mutants, were isolated and characterized. Lysogens carrying these mutants were induced at 38 degrees C, produced a normal or slightly increased yield of phage, and underwent extensive lysis at this temperature. In some cases mutation to heat inducibility had altered the UV inducibility of the phage, the changes ranging from loss to enhancement of this trait. Complementation tests showed that all five beta-tsr strains had mutated in the same cistron and suggested that these mutations were in the gene responsible for repressor production.

Bacteriophages↗

Bacteriophage production by doubly lysogenic Corynebacterium diphtheriae.

Parental and recombinant phage production by tandem, double lysogens of Corynebacterium diphtheriae was studied in strains in which the coupling of prophage markers and the order of prophage was established. The results from studies of mass lysates and single bursts showed that the recombinant class of phage, designated R1, was predominant in UV-induced lysates followed by the parental, P1 class and to a lesser extent the P2 and R2 classes. Single bursts of UV-treated cells contained phage from one to all four of the phage classes, and this appeared to reflect the action of two excision processes. The data indicate that recombinant phages R1 and R2 are formed by a process of general recombinational excision and that this is the primary event leading to phage production in both UV-irradiated and spontaneously induced double lysogens. This process, which depends on exchange between homologous genes and is reciprocal, accounts for the excision of R1 phage from the host chromosome. A second excision process, probably site-specific excision, also occurs in many of the same cells and accounts for the excision of P1, P2, and R2 phages. The significance of these results for the spread of toxinogenicity in strains of C. diphtheriae is discussed.

Bacteriophages↗

Prophage map of converting corynebacteriophage beta.

A prophage map for corynebacteriophage beta consisting of seven markers has been constructed and compared with the vegetative map. The mapping system utilizes heteroimmune double lysogens and capitalizes on the fact that these double lysogens are very unstable and throw off monolysogenic segregants. The prophage map, produced by characterizing the recombinant phage in these monolysogenic segregants, appears to be a cyclic permutation of the vegetative map with the gene for toxin at one end of the prophage map and the gene for phage immunity at the other. This permutation is in accord with the Campbell model for insertion of lambda phage if a site between the toxin and immunity genes in the vegetative map is designated as the phage attachment site. The position of the gene for toxin in the prophage map suggests that converting phages may have originated as specialized transducing phages for this gene.

Bacteriophages↗