No abstract

Colicin V-treated Escherichia coli was inhibited in its capacity to carry out active transport of proline and was unable to generate a membrane potential. Colicin V also prevented membrane potential formation by isolated cytoplasmic membrane vesicles. We conclude that a primary effect of this colicin involves the cytoplasmic membrane as a target.Although studies on bacteriocins have been initiated after Gratia's discovery of colicin V (7, 8), this colicin displays several properties which distinguish it from colicins which have been subsequently described. In particular, colicin V is of a small size, having a molecular weight of ca. 4,000 (5); furthermore its synthesis is not inducible (9). These characteristics are in contrast to those of other colicins, which range in size from 27,000 to 80,000 daltons and syntheses of which are induced by conditions that activate SOS repair (11,21). These differences suggest that colicin V may be more closely related to a class of antibiotic polypeptides, termed microcins, which are produced by some Escherichia coli strains (1, 2).Characterization of colicin V has been hampered by several factors, including instability of colicin V, the low amounts produced by colicinogenic cells, difficulty in purification, and concomitant production of colicin I in cells that produce colicin V (17, 23). We have previously reported the cloning of colicin V structural and immunity genes originating from plasmid pColV-B188 (5). On the basis of the availability of this clone and our ability to stabilize colicin V activity, we decided to initiate studies on the mode of action of colicin V despite its having so far proven refractory to purification.Colicin V was prepared from the supernatant of a tryptone broth culture of E. coli K-12 strain 294 carrying plasmid pBQ41. This plasmid carries the colicin V structural gene derived from plasmid pColV-B188 (5). A late-log culture (100 Klett units as measured in a Klett-Summerson colorimeter, blue filter) grown at 37°C with shaking was harvested, and the supematant fraction (50 ml) was placed in a steam oven for 30 min. The steam treatment was found to substantially stabilize the preparation. Whereas unsteamed samples often lost 90% of their activity in 1 week, steamed samples retained full killing activity over 2 to 3 months when stored at 5 to 10°C. After being steamed, the preparation was allowed to come to room temperature and wvas then mixed determined immunity of Escherichia coli K-12 to colicin Ia is mediated by a plasmid-encoded membrane protein.

The chromosome of Streptomyces lividans shares 154 kb homology with one end of the linear plasmid SLP2, consisting of a 101 kb terminal sequence followed by the 53 kb transposable element Tn4811. The 101 kb terminal sequence was determined. The mean GMC content of this sequence is 679 mol % with a striking G vs C bias in the last kb. The terminal 232 nt contained 10 palindromic sequences with potential to form complex secondary structures. One typical Streptomyces coding sequence (designated ORF1) of 2643 bp was predicted in the determined sequence. The amino acid sequence of the ORF1 product contained a DEAH helicase motif, and exhibited similarity to type I restriction enzyme HsdR subunits in the database, suggesting a possible role in replication of the telomeres. However, all the ORF1 sequences on the chromosome and SLP2 could be simultaneously knocked out by targeted recombination without affecting the viability of the cells and the linearity of the chromosome and SLP2. This ruled out ORF1 as an essential component in the maintenance of the linear chromosome and plasmids.

Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5′-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3′-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.

Single-stranded gaps at the 3 ends of Streptomyces linear replicons are patched by DNA synthesis primed by terminal proteins (TP) during replication. We devised an in vitro system that specifically incorporated dCMP, the first nucleotide at the 5 ends, onto a threonine residue of the TP of Streptomyces coelicolor.Chromosomes of soil bacteria Streptomyces spp. are notable for their linear structure (13), with covalently attached terminal protein (TP) and terminal inverted repeats of various lengths. Linear plasmids with the same structural features are also abundant in Streptomyces spp. Unlike the cases for the well-characterized TP-capped linear genomes of 29 phage (reviewed in reference 15) and adenoviruses (reviewed in reference 14), which initiate replication at both ends by using the TP as the primer, replication of the linear chromosomes and plasmids of Streptomyces is initiated from an internal origin and proceeds to the termini (6, 16). This leaves single-stranded gaps of 200 to 300 nucleotides at the 3Ј ends on various Streptomyces linear plasmids and chromosomes (6; C.-H. Huang, unpublished results) and presumably on the Streptomyces chromosomes. These telomere sequences contain extensive palindromes with potential to form complex and thermodynamically stable secondary structures, which presumably are important for structural integrity and for the patching of the single-strand gaps (12). Several mechanisms have been proposed for the end patching (7). Experimental evidence suggests a patching DNA synthesis using the TP as a primer (18).The TPs of several linear Streptomyces chromosomes and plasmids have been isolated and sequenced (see, for example, references 3 and 20). They are conserved in amino acid sequences and sizes (184 or 185 amino acids) and contain a putative helix domain that resembles part of the DNA-binding "thumb" domain of human immunodeficiency virus reverse transcriptase and a putative amphiphilic beta-sheet that may be involved in the observed self-aggregation of the TP and/or in membrane binding. In addition, these proteins are rich in positively charged residues, which give rise to very high predicted pI values (11 to 12). There is no clear similarity between the TPs of Streptomyces chromosomes and those of 29 phage, adenoviruses, or other TP-capped linear replicons.In the Streptomyces chromosomes and some (but not all) linear plasmids, the gene encoding TP (tpg) is located downstream from tap, which encodes an 80-kDa telomere-associated protein, Tap (1). Both Tpg and Tap proteins are essential for replication of linear chromosomes (1, 3). Tap interacts with Tpg and specific motifs in the 3Ј overhangs and is proposed to function in recruiting TP to the replication intermediates (1).To investigate the mechanism of end patching, we devised an in vitro assay in which TP from Streptomyces coelicolor was specifically labeled with [ 32 P]dCMP, the first nucleotide at the 5Ј ends of the Streptomyces linear replicon. For a substrate for such deoxynucleotidylation, a TP expression vector was ...