~Crude extracts of Rhizobium trifolii strain 7000 contained enzymes of the Entner-Doudoroff and pentose phosphate pathways. No phosphofructokinase (EC 2.7.1 . 1 1) activity and only a low activity of fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) were found, suggesting that the Embden-Meyerhof-Parnas pathway was not physiologically important in this strain.Independent carbohydrate-negative mutants of R. trifolii were isolated and characterized as deficient in glucokinase (glk; EC 2.7.1 .2), fructose uptake (fup), the Entner-Doudoroff pathway (edp) and pyruvate carboxylase (pyc; EC 6.4.1 .1). Glucokinase was essential for glucose phosphorylation in R. trifolii and was also required for growth on sucrose. The edp mutant was impaired in growth on all hexoses tested except galactose, suggesting that the ED pathway was the major pathway used by R. trgolii for the catabolism of these sugars.Galactose may be catabolized via a different pathway, possibly involving an NADP+-linked galactose dehydrogenase. Pyruvate carboxylase was an important anaplerotic enzyme in R. trifolii required for growth on all carbon sources tested, except succinate.All the mutants, including a glk fup double mutant, formed an effective symbiosis on red clover, suggesting that neither glucose, fructose nor sucrose are used by the bacteroids to provide ATP and reductant for nitrogen fixation. The bacteroids probably receive a supply of tricarboxylic acid cycle intermediates from the plant cytosol, and these may be their major source of energy.

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A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 x lo6.This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5 % chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColEl DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.The advent of recombinant DNA technology has facilitated the routine isolation of specific nucleic acid sequences of both prokaryotes and eukaryotes. This technology relies heavily on the use of vectors and for most cloning experiments bacterial plasmids are the vectors of choice. Plasmid DNA rarely constitutes as much as 50% of the total DNA present in a bacterial cell, even under growth conditions most conducive to plasmid replication [l]. Purification of plasmid DNA therefore requires the selective removal of chromosomal DNA. A very effective initial pmification is achieved by the preparation of a cleared lysate [2]. Methods for the further purification of plasmid DNA take advantage of the closed-circular nature of the DNA [3,4]. The most widely used method relies on the greater density of the closed-circular DNA relative to sheared chromosomal DNA in ethidium bromide/caesium chloride density gradients [5]. While this technique provides a high degree of purification it suffers from a number of disadvantages: it is expensive in both centrifuge time and materials, has limited capacity, and complete removal of the ethidium bromide is both difficult and tedious.In this paper we describe a purification procedure which does not depend on CsCl centrifugation and which is rapid, inexpensive and yields DNA of high purity. Our method takes advantage of the selective and reversible retention by hydroxyapatite of small double-stranded DNA molecules under conditions known to prevent the binding of all RNA and protein [6].

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S U M M A R YEscherichia coli, C-M7, a His+Nif+ hybrid obtained by intergeneric mating with a Klebsiella pneumoniae donor strain, also inherited the unselected markers gnd and rfb. The R factor, R144drd3, which had been used to confer fertility on the donor, was present, detectable as covalently closed circular DNA of molecular weight 69 x lo6 daltons. No other species of supercoiled DNA were isolated and the elimination of R144drd3 did not result in the loss of Klebsiella genes. Segregation analysis of donor markers indicated that the Klebsiella DNA was integrated at the his region of the E. coli chromosome in the probable order his-gnd-nif-rfb. Strain C-M7 produced a nitrogenase physiologically identical to that of K. pneumoniae, but synthesized a heteromeric species of gluconate-6-phosphate dehydrogenase.