Trans-dominant mutation affecting restriction and modification in Escherichia coli K-12

Summary We have analysed the mechanism of action of a ts mutation in E. coli, which has an effect on the expression of the restriction and modification phenotype. The frequencies of recombinants obtained in transduction experiments support the idea that the temperature sensitive mutation is located outside the hsd operon in the gene denoted hsd. X. Complementation experiments demonstrated the trans-dominant nature of the temperature sensitive mutation. The possible role of the hsd.X product in the formation of EcoR.K and EcoM.K complexes and their interaction with the recognition site on the DNA is discussed.     

The nucleotide sequence recognized by the Escherichia coli K12 restriction and modification enzymes.

The sites recognized by the Escherichia coli K12 restriction endonuclease were localized to defined regions on the genomes of phage φXsK1, φXsK2, and G4 by the marker rescue technique. Methyl groups placed on the genome of plasmid pBR322 by the E. coli K12 modification methylase were mapped in HinfI fragments 1 and 3, and HaeIII fragments 1 and 3. A homology of seven nucleotides in the configuration: 5′-A-A-C .. 6N .. G-T-G-C-3′, where 6N represents six unspecified nucleotides, was found among the DNA sequences containing the five EcoK sites of φXsK1, φXsK2, G4, and pBR322. Three lines of evidence indicate that this sequence constitutes the recognition site of the E. coli K12 restriction enzyme. The C in 5′-A-A-C and the T in 5′-G-T-G-C are locations of mutations leading to loss or gain of the site and thus are positions recognized by the enzyme. This sequence does not occur on φXam3cs70, simian virus 40 (SV40), and fd DNAs which do not possess EcoK sites, and occurs only once on φXsK1, φXsK2, and G4 DNAs, and twice on pBR322 DNA. In order to prove that all seven conserved nucleotides are essential for the recognition by the E. coli K12 restriction enzyme, the nucleotide sequences of φX174, G4, SV40, fd, and pBR322 were searched for sequences differing from the sequence 5′-A-A-C .. 6N .. G-TG-C-3′ at only one of the specified positions. It was found that sequences differing at each of the specified positions occur on DNA sequences that do not contain the EcoK sites. Thus, the recognition site of the E. coli K12 restriction enzyme has the same basic structure as that of the EcoB site (Lautenberger et al., 1978). In each case there are two domains, one containing three and the other four specific nucleotides, separated by a sequence of unspecified bases. However, the unspecified sequence in the EcoK site must be precisely six bases instead of the eight found in the EcoB site. Alignment of the EcoK and EcoB sites suggests that four of the seven specified nucleotides are conserved between the sequences recognized by these two allelic restriction and modification systems.     

Characterization of a restriction enzyme from Escherichia coli K carrying a mutation in the modification subunit.

The restriction enzyme from a restriction and modification-deficient strain of Escherichia coli K mutated in the modification gene (hsdM) has been purified using an in vitro complementation assay with a mutant restriction enzyme from a strain lacking only restriction. The restriction enzyme from the hsdM mutant lacks all of the activities that are associated with the wild type enzyme: binding of unmodified DNA to filters, cleavage, or methylation of unmodified DNA and ATP hydrolysis. It is shown that the enzyme from this hsdM mutant cannot bind S-adenosylmethionine, an allosteric effector in the restriction reaction. In the absence of enzyme activation by S-adenosylmethionine, no binding to unmodified DNA takes place. A comparison with other mutant restriction enzymes allows us to outline the biochemical role of the subunits of the E. coli K restriction endonuclease.     

Identification of a trans-dominant mutation affecting proline dehydrogenase in Escherichia coli

L-Proline dehydrogenase catalyzes the oxidation of L-proline to delta 1-pyrroline-5-carboxylate, a reaction that is an important step in the utilization of proline as a carbon or nitrogen source by bacteria. A mutant of Escherichia coli K-12 lacking L-leucyl-tRNA:protein transferase had been found previously to contain about five times as much proline dehydrogenase activity as its parent strain. This difference has now been shown to be due to the presence in the parent strain of a previously unrecognized mutation. This mutation, which has been designated put-4977, specifically affects proline dehydrogenase rather than proline uptake. Although proline dehydrogenase remains inducible by L-proline in strains carrying the mutation, there is a premature cessation of differential synthesis during induction that results in a lower specific activity. The mutation shows about 50% P1-mediated cotransduction with pyrC and is therefore located at about 22 min on the E. coli chromosome. Merodiploids containing a normal F' factor still exhibit decreased enzyme activity, indicating that the put-4977 mutation is trans-dominant. The mutation cannot be detected in present stocks of the transferase-deficient mutant, suggesting that this mutant is a revertant for put-4977.     

Isolation and characterization of a new temperature-sensitive polynucleotide phosphorylase mutation in Escherichia coli K-12

Polynucleotide phosphorylase (PNPase) has been studied in detail since its discovery in 1955 [1]. In an attempt to determine what role, if any, it has in mRNA decay in Escherichia coli, we have isolated and characterized a temperature-sensitive mutation, pnp-200, in the pnp gene. In vitro phosphorolysis, polymerization and exchange activities of the partially purified Pnp-200 enzyme are all reduced to 30-40% of wild-type activity at 50 degrees C compared to 32 degrees C. The pnp-200 mutation alone does not affect cell growth or mRNA stability. A triple mutant strain containing pnp-200 in combination with other temperature-sensitive mutations in genes known to affect mRNA metabolism (rnb-500 and ams-1) is conditionally lethal and shows increased mRNA stability after shift to the non-permissive temperature.     

Isolation and characterization of a temperature-sensitive amber suppressor mutant of Escherichia coli K12

Summary By mutagenizing an E. coli strain carrying an amber suppressor supD ^- (or su _I ⁺), we isolated a mutant whose amber suppressor activity was now temperature-sensitive. The mutant suppressor gene was named sup-126, which was found to be cotransduced with the his gene by phage P1vir at the frequency of ca. 20%. At 30° C it suppresses many amber mutations of E. coli, phage T4, and phage . At 42° C, however, it can suppress none of over 30 amber mutations tested so far. The sup-126 mutation is unambiguous and stable enough to be useful for making production of an amber protein temperature-sensitive.     

A mutation that converts serine340 of the HsdSK polypeptide to phenylalanine and its effects on restriction and modification in Escherichia coli K-12

A hybrid hsdS gene, encoding the HsdSts + d polypeptide, was constructed by joining the proximal region of the wild-type (wt) hsdS sequence with the distal region of the hsdSts + d sequence, at the hsdS BglII site. The hybrid hsdS-Sts + d gene exerts a trans-dominant effect on restriction and modification, which points to the location of the temperature-sensitive (ts) trans-dominant (+ d) mutation in the gene hsdSts + d distal region. Sequencing of the region downstream from the HindIII target in the Escherichia coli K-12 hsdSts + d mutant was carried out. It is identical to the wt hsdS sequence (GenBank/EMBL accession number ECHSDK LV00288), except for a single base-pair transition C1245----T. The results obtained support the idea that the trans-dominant effect of the ts mutation described earlier is related to the single base-pair transition in the nonhomologous region of the hsdSts + d sequence.     

The nature of ompA mutants of Escherichia coli K12 exhibiting temperature-sensitive bacteriophage resistance

Summary A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed. The mutants produce detectable levels of the protein at 42°C but not at 30°C (Manning and Reeves 1976). They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene. Several previously characterized mutants possessing insertions or a deletion in the non-translated 5 area of the gene also exhibited a similar temperature-sensitive phage resistance. This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al. 1984).     

Hybrid plasmids complement a putP mutation in Escherichia coli K12.

Clarke and Carbon have prepared a colony bank of 2000 Escherichia coli strains each containing a random segment of the Escherichia coli chromosome inserted into the EcoR1 restriction site of the plasmid ColE1. We have screened the colony bank by conjugation and have identified three strains bearing hybrid plasmids that complement a defective putP gene. Each of these strains shows increased L-proline uptake activity in comparison with the unmodified host or with the host bearing noncomplementing hybrid plasmids. However, CS520, the DNA source strain employed in constructing the hybrid plasmids, is a putP mutant. Since Escherichia coli possesses two L-proline porters, a variety of possible complementation mechanisms are discussed.     

Map location of the ssd mutation in Escherichia coli K-12.

A pleiotropic mutation at the ssd locus was mapped at 86 min near rha. A mutation at the ssd locus resulted in elevated L-serine deaminase activity, inability to grow with succinate as the carbon source, and inability to grow anaerobic conditions.