Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.     

Cloning of the uvrD gene of E. coli and identification of the product

The uvrD gene has been cloned from Escherichia coli chromosomal DNA into phage lambda, cosmid, and low-copy-number plasmid vectors. Comparison of the proteins encoded by the cloned fragments with those encoded by fragments in which the uvrD gene is inactivated by transposon insertion or by deletion shows that the uvrD gene product is a protein of Mr = 73000.     

Molecular cloning and amplification of the gene for thymidylate synthetase of E. coli

The thyA gene of Escherichia coli, which directs the synthesis of the enzyme thymidylate synthetase, has been subcloned from a recombinant λ phage (Hickson et al., 1982) into the multicopy plasmid pBR325 to give the plasmid pPE245. To identify the thyA gene product, the transposon Tn1000 was inserted into pPE245 and derivative plasmids isolated that were no longer able to complement thyA mutations. When proteins synthesised by these plasmids and by pPE245 were labelled and analysed on SDS-polyacrylamide gels a protein of 33000 M_r, presumably the thyA⁺ gene product was absent whenever the thyA gene was inactivated. On assaying cell extracts prepared from cells harbouring pPE245 for thymidylate synthetase, the level of this enzyme was found to be elevated by a factor of at least 25.     

Characterization of the phospholipase C gene of Pseudomonas aeruginosa cloned in Escherichia coli

We have cloned a 4.9-kb fragment of Pseudomonas aeruginosa DNA containing the structural gene of phospholipase C (PLC), by inserting it into the BamHI site of plasmid pBR322. Strains of Escherichia coli carrying this recombinant plasmid produce PLC, but expression of the gene differs from that in P. aeruginosa in two respects: (i) synthesis of the enzyme appears to be constitutive, i.e., not repressible by the presence of inorganic phosphate in the growth medium, and (ii) most of the enzyme remains associated with the outer membrane instead of being secreted. Insertion mutagenesis at a unique restriction site within the PLC gene destroyed the ability of the plasmid to code, in maxicells, for phospholipase C activity and for an Mr 80000 polypeptide.     

Analysis of the cya locus of Escherichia coli

A 9500-bp DNA segment containing the adenylate cyclase gene (cya) of Escherichia coli has been isolated and analyzed. Four large proteins are encoded within this fragment - the adenylate cyclase protein (92 kDal), two proteins of unknown function (37 and 32 kDal), and a part of the uvrD-coded protein. Various truncated adenylate cyclase proteins, made from cya genes having as much as 60% of their carboxy-terminal end deleted, are sufficient to complement cya- hosts. When these truncated cya genes are present on a multicopy plasmid in a cya- host, the synthesis of beta-galactosidase is still regulated by glucose. The "maxicell" technique was used to visualize the four proteins encoded by this region and some of the truncated adenylate cyclase proteins.     

Effect of lexA and ssb genes, present on a uvrA recombinant plasmid, on the UV survival of Escherichia coli K-12

The recombinant plasmid pJA01 contains, besides the uvrA gene, the genes lexA, ubiA and ssb. This plasmid does not fully complement a uvrA mutation in a Rec+ background. Plasmids which contain the uvrA and ssg genes, but not the lexA gene, show a higher but still only partial complementation. Full complementation achieved when the ssb gene us inactivated by insertion of Tn5. Furthermore, it appears that the presence of the ssb gene on a multicopy plasmid sensitizes wild-type cells to UV light. The effect of Ssb (single-strand DNA binding protein) overproduction on UV survival is discussed.     

Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae

Summary A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.     

Characterization of a plasmid carrying the Escherichia coli K-12 entD, fepA, fes, and entF genes

Abstract The plasmid pMS101 carries Escherichia coli K-12 genes ( entD, fes, entF ) essential for enterochelin-mediated iron transport [Laird, A.J. and Young, I.G., Gene 11 (1980) 359–366]. We have further characterized pMS101 and shown that it also contains the gene ( fepA ) for the 81 000 Da outer membrane ferrienterochelin receptor. Subcloning experiments in conjunction with complementation and maxicell studies demonstrated the gene order to be entD fepA fes entF . The entF - and fes -encoded polypeptides were found to be 115 000 and 42 000-Da soluble proteins, respectively. Plasmid-borne enterochelin cluster genes were overexpressed in iron-deficient conditions and their products were undetectable under iron-replete conditions.