Morton Mandel

Multiple copies of a DNA sequence from Pseudomonas syringae pathovar phaseolicola abolish thermoregulation of phaseolotoxin production

Phaseolotoxin, a phytotoxin of Pseudomonas syringae pv. phaseolicola, is produced at 18°C but not at 28°C. Here we report that a fragment (24.4 kb) cloned from the wild‐type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox− mutant at both temperatures. A subclone harbouring a 465bp fragment contains motifs that are characteristic of DNA‐binding sites. In mobility shift assays we have detected a protein(s) from the wild‐type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA‐binding motifs. We propose that the binding protein is a repressor which is ‘titrated’ by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.

Genetic transformation of Pseudomonas phaseolicola by R-factor DNA

SummaryThe bacterium Pseudomonas phaseolicola was successfully transformed, for the first time, with R-factors RSF1010 and pBR322 DNA by a calciumshock and heat-pulse technique. Frequency of transformation for RSF1010 ranged from 0.8×10-7 to 3.1×10-6 and was ca. 0.4×10-8 for pBR322.

Proton exchange of nucleic acids: The amino protons of 5′-AMP and poly A

Abstract Approximate values of the hydronium and hydroxyl second order rate constants for the exchange of the amino protons of 5′-AMP and Poly A were obtained from the observed variation with pH of the 100 MHz PMR resonance signal line width for the amino protons. These values are several orders of magnitude lower than corresponding rate constants for amino protons and purine hydrogens, which may resolve some important interpretative difficulties that are encountered both in the hydrogen exchange of helical polynucleotides and in recent data on the exchange of the non-hydrogen bonded amino protons of DNA.

Actinomycin sensitive mutants of Escherichia coli K-12.

SummaryActinomycin sensitive mutants of E. coli K12 have been isolated and shown to have pleiotropic defects in the fermentation of sugars. The locus of a gene controlling actinomycin resistance is very close to that of the lactose gene.

Physical properties of phage 299.

SummaryPhage 299, on equilibrium sedimentation in CsCl, gives 3 major bands whose relative proportions depend on growth conditions. One band is whole heads without tails, the other two are infectious phage of differing degrees of disarray and stability. Electron micrographs show that the infectious phage has a head about 60 nm across, probably icosahedral in shape, and a straight tail of approximately 140 nm in length. The tail assemblies appear defective and incomplete. Sedimentation in a sucrose gradient of the DNA extracted from phage 299 is monodisperse with a molecular weight of 20.6±1.5×106 daltons based on comparison with λ and 186 phage DNA’s. The DNA has a base composition of 51.7% guanine and cytosine as determined by bouyant density in CsCl. A comparison of its denaturation behavior by analysis of the hyperchromic shift at 260 nm with that of phage P2 suggests a considerable number of common characteristics, and an absence of a low guanine and cytosine portion on the part of 299 which amounts to approximately 10% of the total DNA.

Length and sequence polymorphisms in the ribosomal gene spacer of the Hawaiian sea urchin, T. gratilla

Blot-hybridization of sea urchin (Tripneustes gratilla) genomic DNA with a cloned rDNA probe revealed individual variation in the length of the rDNA repeat unit and also in the non-transcribed spacer sequences. The number of distinct rDNA repeat subclasses distinguishable within any one sea urchin was limited and usually 2 to 3. However, examination of a number of sea urchins indicated a large number of distinct rDNA repeat types in the population as a whole; all of the rDNA repeat types in nine individuals were different. The presence of limited heterogeneity in the rDNA repeats of single individuals, with may different repeat types in the population as a whole, suggests that rDNA variants can be rapidly and selectively propagated within a chromosomal lineage.

Characterization of genes involved in phaseolotoxin production and its thermal regulation

Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of bean, produces phaseolotoxin, a nonspecific chlorosis-inducing toxin. The wild-type strain of P.s. pv. phaseolicola G50-1 produces phaseolotoxin at 18°C but not at 28°C. When a derivative of a previously reported genomic clone isolated from G50-1 which complements only EMS and UV (but not Tn5) mutants is mobilized into these mutants, as well as in the wild type strain, the transconjugants produce toxin at both 18°C and 28°C. Further characterization of the insert in this clone showed that a 485 bp fragment containing motifs characteristic of DNA binding sites, when present in multiple copies in the transconjugant, relieves repression of phaseolotoxin production at 28°C. Results of gel retardation assays showed that a protein(s) present in extracts of cells grown at 28°C binds specifically to the 485 bp fragment and to a 260 bp subfragment that contains these DNA binding motifs. We suggest that thermoregulation of phaseolotoxin production at 28°C by the wild type and perhaps by the Tox- mutant at both temperatures, involves a regulatory protein(s) that represses transcription of one or more phaseolotoxin structural genes, and that the DNA binding site(s) in the fortuitously cloned fragment derepresses toxin synthesis by titrating this protein. We previously showed that a genomic cosmid clone, pHK120, containing a 24 kb fragment of DNA from the wild-type strain of the pathogen restores toxin production to all EMS, UV and Tn5 Tox- mutants. Tn5 mutagenesis of pHK120, marker exchange of pHK120::Tn5 in the wild-type strain and pair-complementation analysis revealed that a minimum of 8 genes (A through H) are clustered in the insert of pHK 120.

Molecular Analysis of Phaseolotoxin Production in Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight of bean (Phaseolus vulgaris L.). The disease is manifested at cool temperatures when the bacteria enter the plant through stomata in the leaves. At the site of infection a green-yellow chlorotic zone or halo, ornithine accumulation and growth inhibition (Patil, 1974) occur. Systemic infection results in venetion of leaves and stunting of the growing point (Hoitink et al., 1966; Rudolph et al., 1966). The chlorosis, growth inhibion, and venetation are caused by an extracellular, nonspecific Phytotoxin known as phaseolotoxin (Fig. 1) (Mitchell, 1984; Moore et al., 1984). It is not required for pathogenicity of P.s. pv. phaseolicola since naturally occurring toxin-deficient strains as well as Tox- mutants are still capable of producing water-soaking but not the other symptoms. (Rudolph et al., 1966; Patil, et al., 1974; Russell, 1975; Quigley et al., 1985; Peet et al., 1986). During the past 15 years, extensive studies on the physiology, biochemistry, chemical structure and mode of action of phaseolotoxin have been reported (Mitchell, 1984). However, little is known about the biochemistry or genetics of its production. Phaseolotoxin is a potent inhibitor of the enzyme, ornithine carbamoyltransferase (OCT) of bean in vitro (Patil et al., 1970; Kwok et al., 1979).