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Protein E, a 91-residue membrane protein of phiX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis. In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene slyD (sensitivity to lysis) that block the lytic effects of E. The role of slyD, which encodes a FK506 binding protein-type peptidyl-prolyl cis-trans isomerase, is not fully understood. However, E mutants referred to as Epos (plates on slyD) lack a slyD requirement, indicating that slyD is not crucial for lysis. To identify the gene encoding the cellular target, we selected for survivors of Epos. In this study, we describe the isolation of dominant mutations in the essential host gene mraY that result in a general lysis-defective phenotype. mraY encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis. The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis.

Recessive mutations in the slyD (sensitivity to lysis) gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage phi X174 [1]. The slyD- mutation, transduced into the normal phi X174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function. slyD encodes a protein with 196 residues. A segment corresponding to the first 142 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans isomerases, or rotamases. The C-terminal 46 codons of slyD encode a remarkable histidine-rich peptide which is a metal-binding domain [2]. This sequence is dispensable for slyD function in E-mediated lysis. Although there is no obvious phenotype associated with the slyD- genotype other than the resistance to E-mediated lysis, overexpression of slyD causes cells to filament and to increase significantly in diameter. Mutations in phi X174 can restore the plaque-forming ability of the phage on a slyD- host. These pos (plates on slyD) mutants plate on E. coli C wild-type and slyD-. A model for SlyD involvement in E function and the role of SlyD in the cell is discussed.

Recessive mutations in the slyD gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage phi X174 (Maratea, D., Young, K., and Young, R. (1985) Gene (Amst.) 40, 39-46). The slyD1 mutation, transduced into the normal phi X174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function rather than for expression from the plasmid vector. The cloning, sequencing, and deletion analysis of a 1-kilobase pair genomic fragment containing the slyD locus, mapping at 73.5', is reported. Three reading frames, orf72, orf159, and orf196, are contained within this fragment, with the latter two reading frames occupying the same DNA on opposite strands. Deletion analysis shows that the complementing activity is restricted to the orf159/orf196 DNA. Complementation of the SlyD phenotype was observed irrespective of the orientation of the orf159/orf196 DNA with respect to a vector promoter, indicating that a cryptic promoter serves slyD on this fragment. Using site-directed mutagenesis, nonsense mutations were created in each reading frame which were silent in the opposing frame. Both orf196 nonsense alleles failed to complement slyD1, whereas both orf159 nonsense alleles retained complementation, demonstrating rigorously that orf196 is slyD. A segment corresponding to the first 150 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans-isomerases or rotamases. The COOH-terminal 46 codons of slyD encode a remarkable histidine-rich peptide sequence which is at least partly dispensable for slyD function in E-mediated lysis. Overexpression of slyD in E. coli is toxic. These findings are discussed in terms of a model for SlyD involvement in E function and in terms of a model for SlyD involvement of the ubiquitous FKBP rotamases.

Plasmids have an important role in the pathogenicity of certain bacterial species, and Escherichia coli provides the most complete example of the relationship involved. Enterotoxigenic strains of E. coli, in addition to producing heat-stable and/or heat-labile enterotoxins, may also produce a haemolysin and fimbriate cell surface antigens which facilitate the adherence of the bacterial cell to the mucosa of the small bowel. Numerous studies have shown that these properties are plasmid-mediated and that the plasmids act in concert to confer on the host bacterium the ability to produce enteric disease in man and in animals. Moreover, studies with invasive strains of E. coli have shown that the Col V plasmid, which codes for the synthesis of colicin V, significantly enhances the pathogenicity of its host bacterium. Although the relationship between Col V plasmids and virulence is unclear, reports indicate that Col V-containing strains of E. coli are better able to survive in the alimentary tract and that colicine V itself inhibits macrophage function. It is probable that bacterial virulence is a complex phenomenon involving both chromosomal and plasmid genes. We describe here a virulence plasmid which mediates tissue invasiveness in human pathogenic strains of Yersinia enterocolitica.

1. The two polypeptide chains that comprise aspartate carbamoyltransferase in Escherichia coli are encoded by adjacent cistrons expressed in the order, promoter-leader-catalytic cistron-regulatory cistron (p-leader-pyrBI). These two cistrons and their single control region have been cloned as a 2,800 base pair (bp) fragment (The minimal coding requirement for the catalytic and regulatory polypeptides is about 1,350 bp plus control regions). The genes contained by this fragment are subject to normal repression controls and thus possess the intact control regions. 2. By deleting an internal fragment with specific restriction endonucleases, it was possible to construct shortened fragments which no longer produced the regulatory polypeptide. In these cases the expression of the catalytic cistron was normal and subject to repression upon growth in the presence of uracil. Since the pyrB cistron retained transcriptional control, the regulatory polypeptide was not required for expression or control of the catalytic cistron. As expected, the catalytic trimer (Mr = 100,000 daltons) from these deletion mutants had no effector response nor did it exhibit homotropic kinetics for aspartate. The enzyme was identical to the c3 trimer purified from the native holoenzyme by neohydrin dissociation. 3. Insertion of Mu d1(lac Apr) into the structural region of pyrB had a negative effect on the expression of pyrI. This supports the idea that the catalytic and regulatory polypeptide chains of aspartate carbamoyl-transferase are encoded by a single bicistronic operon. Detailed restriction analysis of the cloned pyrBI region has produced a genetic map of restriction sites which is colinear with the published amino acid sequences of the two polypeptides. These maps indicate that the 3'-terminus of the catalytic cistron is adjacent to the 5'-terminus of the regulatory cistron and separated by 10-20 bp. 4. DNA sequence analysis of the 5'-proximal regions of pyrBI revealed that an extensive leader sequence separated the promoter and first structural gene pyrB. This leader of approximately 150 bp contains an attenuator sequence and the translational signals required for the production of a leader polypeptide of 43 amino acids. In this paper we describe the structural organization of pyrBI, and provide a detailed analysis of its regulatory region including its DNA sequence.