This article intends to inform a broader audience on a fascinating class of protein toxins (bacteriocins) which usually kill only cells of the same species. Those who gained a deeper interest in bacteriocins can find a comprehensive description of the field in a recent book based on a conference (James et al. 1992), and in more specialized review articles dealing with certain aspects (Pugsley 1984a, b), or certain colicins (De Graaf and Oudega 1986; Harkness and Olschläger 1991; Lazdunski et al. 1988). The older literature has been reviewed by Brandis and Smarda (1971), Reeves (1972), Hardy (1975) and Konisky (1982).

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the TonB, ExbB and ExbD proteins. These colicins contain a sequence, called the TonB box, which has been implicated in transport via TonB. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with TonB may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193-197 and 223-252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the TonB box at the N-terminal end of colicin M must be involved in colicin uptake via TonB across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.

Colicin M (Cma) displays a unique activity that interferes with murein and O-antigen biosynthesis through inhibition of lipid-carrier regeneration. Immunity is conferred by a specific immunity protein (Cmi) that inhibits the action of colicin M in the periplasm. The subcellular location of Cmi was determined by constructing hybrid proteins between Cmi and the TEM-beta-lactamase (BlaM), which confers resistance to ampicillin only when it is translocated across the cytoplasmic membrane with the aid of Cmi. The smallest Cmi'-BlaM hybrid that conferred resistance to 50 micrograms/ml ampicillin contained 19 amino acid residues of Cmi; cells expressing Cmi'-BlaM with only five N-terminal Cmi residues were ampicillin sensitive. These results support a model in which the hydrophobic sequence of Cmi comprising residues 3-23 serves to translocate residues 24-117 of Cmi into the periplasm and anchors Cmi to the cytoplasmic membrane. Residues 8-23 are integrated in the cytoplasmic membrane and are not involved in Cma recognition. This model was further tested by replacing residues 1-23 of Cmi by the hydrophobic amino acid sequence 1-42 of the penicillin binding protein 3 (PBP3). In vivo, PBP3'-'Cmi was as active as Cmi, demonstrating that translocation and anchoring of Cmi is not sequence-specific. Substitution of the 23 N-terminal residues of Cmi by the cleavable signal peptide of BlaM resulted in an active BlaM'-'Cmi hybrid protein. The immunity conferred by BlaM'-'Cmi was high, but not as high as that associated with Cmi and PBP3'-'Cmi, demonstrating that soluble Cmi lacking its membrane anchor is still active, but immobilization in the cytoplasmic membrane, the target site of Cma, increases its efficiency. Cmi delta 1-23 remained in the cytoplasm and conferred no immunity. We propose that the immunity protein inactivates colicin M in the periplasm before Cma can reach its target in the cytoplasmic membrane.

A gene of Puccinia graminis f. sp. tritici, coding for the translation elongation factor 1 alpha (EF-1 alpha), was isolated from a P. graminis genomic library using the EF-1 alpha gene sequence of Absidia glauca. The coding region of 1389 nucleotides encodes a polypeptide of 463 amino acids and is interrupted by eight introns. An additional intron is located in the 5' untranslated region. A single transcription start point (tsp) was mapped by primer extension. A cDNA fragment corresponding to P. graminis EF-1 alpha mRNA hybridized with a 1.9-kb-long poly(A+)RNA, sufficient to encode the EF-1 alpha protein. Southern hybridization of digested genomic DNA revealed that two copies of the EF-1 alpha gene exist in the genome of P. graminis.