A method, which we call localized mutagenesis, is described for the isolation of temperaturesensitive and other types of mutations in any specific small region (about 1%) of the bacterial chromosome. The principle of this method is to mutate the transducing DNA rather than the bacterial DNA. One can select for the introduction of this mutated DNA into any particular region of the bacterial chromosome by transducing an auxotrophic marker in that region to prototrophy, thereby introducing new mutations in the neighborhood. We have used this method to isolate many different temperaturesensitive mutations in genes of unknown function in particular regions of the chromosome. Since the method is very simple, it can be used to saturate any region of the map with mutations in essential genes, or for various types of genetic manipulations. Although we have used hydroxylamine-mutagenized phage P22 and Salmonella typhimurium, the method should be applicable to other mutagens and bacteria and transducing phage.Temperature-sensitive (ts) mutations in bacteria are extremely useful because they can be obtained in genes that code for proteins that are essential for the growth of the cell. Since the pioneering work of Horowitz and Leupold (1), temperature-sensitive mutants in bacteria have been used to study activating enzymes (2), ribosomes (3-5), DNA synthesis (6), and other components and aspects of macromolecular syntheses.A number of workers have isolated ts mutants by mutagenizing Escherichia coli or Salmonella typhimurium. Although the isolation of these mutants is easy, the mapping of them involves a considerable amount of work since they may map anywhere on the chromosome. We have developed a simple method, which we call localized mutagenesis, for the easy isolation of ts and other mutants in any specific small region of the chromosome. MATERIALS AND METHODSBacterial Strains. All strains were derived from S. typhimurium strain LT-2.Phage. Phage used for mutagenesis and transduction was P22-int-4, a nonintegrating mutant of P22 (7). The phage sensitivity test was done with P22-H5, a clear (c2) mutant of P22. Phage were stored in T2 buffer (8).Media. Nutrient broth (Difco) was used as maximally supplemented medium. E medium (9) (made 0.4% in glucose) Abbreviation: ts, (i.e., heat-sensitive).was used as minimal-salts medium. Solid medium contained 1.5% agar.Transducing Phage Mutagenesis. Phage (P22-int-4) was grown on wild-type strain LT-2 at 370C with shaking in a medium composed of 100 parts of nutrient broth, 3.5 parts of 40% glucose, and 1.3 parts of concentrated (50X) medium E. The phage, a mixture of phage and bacterial DNA in phage heads, usually had a titer of 1-3 X 1011/ml. Concentrated phage (1012/ml) in 0.9% saline was mutagenized at 370C with hydroxylamine (10). To 1 part of phage in saline were added 5 parts of 0.1 M sodium phosphate buffer (pH 6.0) containing 1 mM EDTA, and 4 parts of 1 M hydroxylamine * HCl (adjusted to pH 6.0 with NaOH) containing 1 mM EDTA. At the desired time the phage were collec...
The glutamine permease operon encoding the high-affinity transport system of glutamine in Escherichia coli could be cloned in one of the mini F plasmids, but not in pBR322 or pACYC184, by selection for restoration of the Gln+ phenotype, the ability to utilize glutamine as a sole carbon source. We determined the nucleotide sequence of the glutamine permease operon, which contains the structural gene of the periplasmic glutamine-binding protein (glnH), and indispensable component of the permease activity. The N-terminal amino acid sequence and the overall amino acid composition of the purified glutamine-binding protein were in good agreement with those predicted from the nucleotide sequence, if the N-terminal 22 amino acid residues were discounted. The latter comprised two Lys residues (nos. 2 and 6) followed by 16 hydrophobic amino acid residues and was assumed to be a signal peptide for transport into the periplasmic space. There were two additional reading frames (glnP and glnQ) downstream of glnH sharing a common promoter. It was concluded that the glnP and glnQ proteins as well as the glnH protein are essential for glutamine permease activity.
In Escherichia coli, acetyl phosphate can be formed from acetyl-CoA via the phosphotransacetylase (phosphate acetyltransferase; acetyl-CoA:orthophosphate acetyltransferase, EC 2.3.1.8) reaction and from acetate (plus ATP) via the acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) reaction. By restricting acetyl phosphate formation to the phosphotransacetylase reaction alone, through the use of metabolic inhibitors, we were able to show that, with pyruvate as a source of energy, mutants defective in phosphotransacetylase are unable to transport glutamine, histidine, and methionine. However, with the same energy source, mutants defective in acetate kinase are normal in the transport of these amino acids. The inability of the phosphotransacetylase mutants to transport is due to their presumed inability to form acetyl phosphate, because pyruvate is found to be metabolized to acetyl-CoA in these mutants. Thus acetyl phosphate has been implicated in active transport. Evidence is also presented that neither the protonmotive force nor the ecf gene product is required for the shock-sensitive transport systems.Many active transport systems in bacteria are coupled to the circulation of protons across the cytoplasmic membrane (refs. 1-3; for review, see ref. 4). However, it has been demonstrated by Berger (5) and Berger and Heppel (6) that, in Escherichia coli, ATP or some form of phosphate bond energy, not the proton gradient, may be used to drive osmotic shock-sensitive active transport systems, permeases that require the periplasmic binding proteins for activity.Berger and Heppel reached this conclusion on the basis of the following observations: (i) arsenate almost completely abolishes shock-sensitive but not shock-resistant transport activity; (ii) mutants with a nonfunctional Ca,Mg-ATPase, which are thus defective in oxidative phosphorylation, cannot drive shock-sensitive transport with the respiratory substrate D-lactate or the artificial electron donor ascorbate/phenazine methosulfate as a source of energy, whereas shock-resistant transport is active with these electron donors; and (iii) glucose, owing to substrate-level phosphorylation, stimulates shock-sensitive transport in the same mutants, and this effect is not strongly inhibited by anaerobiosis or the uncoupler 2,4-dinitrophenol.At variance with this conclusion are the findings that ATP or phosphate bond energy alone cannot be the direct energy donor for these transport systems as observed with E. coli cells defective in the energy coupling factor (ECF) protein (7) and that the electrochemical proton gradient is required in E. coli cells treated with colicin K (8) or in Salmonella cells deficient in cytochromes (9).In this paper, we show that acetyl phosphate is required for shock-sensitive transport, and that neither the electrochemical proton gradient nor the ecf gene product is required. (13). Temperature-sensitive (ts) mutants were isolated as fluoroacetate-resistant colonies at 420C. Those colonies that exhibited ts growth on acetate a...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.