We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.

An Escherichia coli heme-requiring, heme-permeable mutant had no detectable 5-aminolevulinate dehydratase or porphobilinogen deaminase activities. The gene which complemented this mutation was cloned to a high-copy-number plasmid, and porphobilinogen deaminase activity was restored to normal levels, but the synthesis of 5-aminolevulinate dehydratase increased 20- to 30-fold. A maxicell procedure confirmed that the gene cloned was hemB.

A hemA mutant of Escherichia coil containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-'4CJglutamate was substantially incorporated into ALA by this strain, whereas [2-'4CJglycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNAGIU, and pyridoxal phosphate. tRNAGIU stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. Sa Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.

An auxotrophic strain of E. coli K12 treated with CaCl2 was transformed for several markers at a frequency of up to 10-6 per recipient cell by a DNA preparation isolated from a prototrophic strain. The transforming activity of the DNA preparation was eliminated by treatment with DNase, heat, or sonication, whereas RNse or Pronase treatment had little effect. Two closely linked genetic markers (leu and ara) showed a high degree of cotransformation linkage when high molecular weight DNA was used, but the linkage was almost completely eliminated when sheared, smaller molecular weight DNA was used. There is genetic evidence that the transformation is a result of the replacement of the preexisting genetic marker on the chromosome by that of the donor DNA.Since its discovery in Pneumococcus (1, 2), genetic transformation has been reported among several other bacterial species such as Haemophilus influenzae (3) and Bacillus subtilis (4). In Escherichia coli, especially in its K12 strain, however, attempts to demonstrate genetic transformation have been unsuccessful. Several unique systems are known in E. coli in which the biological activity of isolated DNA can be assayed directly, such as transformations by bacteriophage X DNA with helper phages (5). More recently, an efficient transfection system was developed by Mandel and Higa using CaClrtreated cells (6). In most examples of transfection and transformation, the DNA is either phage or plasmid (7) DNA, which is circular (or converts to circular forms in the cells).During studies on the mechanism of ATP-dependent DNase (8-11) from E. coli in this laboratory, we have observed that the enzyme does not attack double-stranded circular DNA with or without nicks, whereas it causes extensive degradation of linear DNA molecules. Since fragments of bacterial chromosome are the source of genetic material in other general transformation systems, one of the reasons for the failure of genetic transformation in E. coli cells might be extensive degradation by the enzyme of linear DNA molecules lacking any protection mechanism. These characteristics of the enzyme plus reports of a mutant that lacks ATP-dependent DNase yet retains the capacity for genetic recombination (12) prompted us to examine the possibility of genetic transformation in E, coli K12. In this paper, we report the genetic transformation of E. coli K12 and some of its basic characteristics. MATERIALS AND METHODSBacterial Strains used in this study are listed in Table 1. Genetic symbols are those used by Taylor (13).Reagents. RNase (pancreas, crystalline RNase A), DNase (pancreas, crystalline), and calf-thymus DNA were purchased from Worthington Biochemical Co. Pronase (grade B) was purchased from Calbiochem.Isolation of Transforming DNA. Cells were grown in Pen assay broth (Difco), chilled, and harvested at the late exponential or early stationary phase by centrifugation. They were then suspended in one-fortieth of the original volume of cold 0.1 M NaCl-0.1 M EDTA (pH 8.5). 1 M Tris HCl (pH 9.0)-10% SDS (sodi...

The mutants eventually recover at lower concentrations. There is no evidence of D-serine toxicity in rich media. Toxicity is partially reversed by L-serine. D-Serine does not interfere with L-serine activation, one-carbon metabolism, or (Cronan, personal communication) formation of phosphatidylserine. Pizer (personal communication) finds, however, that it is a powerful feedback inhibitor of the first enzyme of L-serine biosynthesis. In the presence of L-serine, the residual toxicity is largely and noncompetitively overcome by pantothenate, indicating that D-serine inhibits growth by affecting two targets: pantothenate biosynthesis and L-serine biosynthesis. L-Serine causes transient growth inhibition in E. coli K-12. Contaminating L-serine in D-serine preparations contributes to the D-serine inhibitory response.

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