Characterization of the sequence-specific interaction of mouse c-myb protein with DNA.

We have examined parameters that affect sequence-specific interactions of the mouse c-myb protein with DNA oligomers containing the Myb-binding motif (CA/CGTTPu). Complexes formed between these oligomers and in vitro translated c-myb proteins were analysed by electrophoresis on non-denaturing polyacrylamide gels using the mobility-shift assay. By progressive truncation of c-myb coding sequences it was demonstrated that amino acids downstream of a region of three imperfect 51-52 residue repeats (designated R1, R2 and R3), which are located close to the amino terminus of the protein, had no qualitative or quantitative effect on the ability to interact specifically with this DNA motif. However, removal of only five amino acids of the R3 repeat completely abolished this activity. The contribution of individual DNA-binding domain repeats to this interaction was investigated by precisely deleting each individually: it was demonstrated that a combination of R2 and R3 was absolutely required for complex formation while the R1 repeat was completely dispensible. c-myb proteins showed quantitatively greater interaction with oligomers containing duplicated rather than single Myb-binding motif, in particular where these were arranged in tandem. Moreover, it was observed that c-myb protein interacted with these tandem motifs as a monomer. These findings imply that a single protein subunit straddles adjacent binding sites and the implications for c-myb activity are discussed.

Suppression by the ColV,I-K94 plasmid of inhibitor sensitivities in ompA mutants of Escherichia coli.

A series of ompA mutants derived from Escherichia coli K12 strains showed increased sensitivity (compared with the ompA+ parents) to aminoglycoside antibiotics and to other cationic agents including polymyxin B. One tested mutant also showed increased sensitivity to nafcillin and fusidic acid, but not to the hydrophilic ampicillin. All these inhibitor sensitivities in the ompA mutants were suppressed by ColV, I-K94 and by certain other ColV plasmids, but not by any of the other tested large plasmids. Suppression correlated with the production of the VmpA protein, but transfer and colicin components were not needed for suppression. Further comparison of the ompA and vmpA genes and their products was made and it indicated that there is little if any homology between the genes, that the synthesis of their products is regulated by quite different mechanisms, and that regions of these gene products exposed at the cell surface show different susceptibility to protease attack after denaturation.

Increased sensitivity to erythromycin in Escherichia coli associated with the presence of the ColV,I-K94 virulence plasmid.

Introduction of the virulence plasmid, ColV,I-K94, into Escherichia coli strains led to increased sensitivity to erythromycin. This was the result of increased passage of antibiotic into ColV,I-K94+ organisms because the plasmid effect was abolished in bacteria which had been made permeable by chemical treatment. Full sensitivity in ColV+ strains appears to depend on the simultaneous presence of transfer and colicin components. Increased erythromycin sensitivity associated with the plasmid was demonstrated in organisms grown at 37 degrees C; the sensitivity of ColV,I-K94+ organisms grown at 25 degrees C was similar to that of the parent strain. Added Mg++ or Ca++ ions reversed erythromycin inhibition in strains with the basal level of sensitivity (i.e., the Col- parent grown at 25 degrees C or 37 degrees C or the ColV,I-K94+ derivative grown at 25 degrees C) and in those with the plasmid-associated increase in sensitivity. Addition of phosphate or EDTA to broth increased erythromycin sensitivity in Col- and ColV,I-K94+ strains although the latter was affected most. Erythromycin was more inhibitory at pH 8.5 than at pH 7.4. This enhanced activity was more marked against the ColV,I-K94+ strain than against the Col- strain. The effects of growth in phosphate-containing medium and at alkaline pH were partially additive. We suggest that ColV,I-K94+ strains may be sensitive to erythromycin because ColV-specified proteins are extruded by a process of "self-promoted transfer" and that the effects of these proteins on the lipopolysaccharide component of the outer membrane facilitates antibiotic influx.

Abortive infection by bacteriophage Me1 of Escherichia coli K12 strains bearing the plasmid ColV, I-K94.

Bacteriophage Me1 is unable to grow on Escherichia coli strains harbouring the ColV,I-K94 plasmid. The nature of this inhibition was investigated, and it was found not to be due to restriction, superinfection exclusion or receptor-mediated resistance, but to be a new example of plasmid-mediated abortive infection. Investigation of events occurring during abortive Me1 infection revealed some differences from previously described cases, especially with regard to late protein synthesis, which did occur, albeit showing abnormal amounts of some proteins. No major differences were observed in membrane permeability of productively and abortively infected cells. Phage-directed DNA synthesis was reduced in abortively infected cells. Comparative studies of Me1 and T4 revealed a striking similarity despite some minor differences.

Envelope protein changes, autoagglutination, sensitivity to hydrophobic agents and a conditional division lesion in Escherichia coli strains carrying ColV virulence plasmids.

The presence of the virulence plasmids ColV,I-K94 or ColV-K30 in Escherichia coli produces a number of cell membrane and envelope changes. The most striking of these are (1) the presence of the 33K VmpA outer membrane protein and (2) the ColV-associated occurrence of autoagglutination. The VmpA protein is a plasmid-encoded outer membrane protein which is synthesized from a larger precursor. It is distinct from the chromosomally-encoded OmpA protein but resembles it in a few respects. The VmpA protein does not appear to be involved in colicin synthesis or immunity, or in plasmid transfer. This protein was found in 6 out of 8 new ColV+ isolates, but not in 2 ColIa+ strains. ColV-induced autoagglutination occurred for strains grown in static culture at 37 but not at 25 degrees C. Detergents prevented agglutination, as did the presence in a ColV+ strain of a fi+ plasmid, ColB. Autoagglutination may be a virulence phenotype. Associated with the ability of ColV+ bacteria to agglutinate was inhibition of motility. ColV+ bacteria also showed changes in envelope permeability indicated by inhibitor sensitivity and by a ColV-associated suppression of the lac Y lesion. Some ColV,I-K94+ strains showed a mucoid colonial phenotype and this ability to form mucoid colonies was efficiently transferred with ColV but apparently not without it. The mucoid ColV+ strains resembled lon mutants in UV-sensitivity, division behaviour and sensitivity to lambda phage.