Biomedical subjects
Publications and source records attributed to P Reeves.
After a short description of the discovery of bacteriocins, especially the colicins in this review the following points are discussed: the classification of colicins especially with the aid of resistant mutants of sensitive indicator strains, the bacteriocin-receptors, the bacteriocin-specificity, and the possible ways of transport of colicins across the outer membrane.
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Colicins B-K260 and D-CA23 were purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography and were compared with respect to a number of physical and chemical properties. Both colicins were shown to be proteins and were found to have similar molecular weights, isoelectric points and amino acid compositions. The two colicins also have substantial antigenic similarities but are distinguished by the presence of non-cross-reacting antigens and by differences in stability and in sensitivity to heat and reducing conditions. In addition, strains of Escherichia coli K-12 producing colicins B-K260 and D-CA23 are not cross-immune. The similarities noted between the two colicins are compatible with their use of a common cell surface receptor while having different modes of action.
The uptake of the siderophore-iron complex ferrienterochelin was found to be strongly dependent upon an energized membrane state, as demonstrated by its sensitivity to dinitrophenol, azide, and cyanide. Ferrienterochelin uptake may also be dependent upon phosphate bond energy, as indicated by sensitivity to arsenate and iodoacetic acid. Although the adenosine triphosphatase does not appear to be involved in this energy coupling mechanism, ferrienterochelin uptake was shown to be less dependent upon phosphate bond energy than was glutamine uptake. Sensitivity of ferrienterochelin uptake to osmotic shock was shown to be due to the release of a ferrienterochelin binding compound located in the outer membrane of the cells and probably identical to the colicin B receptor protein.
We extended the study of Escherichia coli mutants defective in conjugation and showed that the mutants with altered lipopolysaccharide, which are defective as recipients with F-like donors, are also defective with the I-like plasmid R64-11. However, the extent of reduction in recipient ability for I-like donors does not correlate either with the effect on recipient ability for F-like donors or with the degree of alteration to the lipopolysaccharide.
Protein 1 was shown to be the receptor for phage PA-2 by the observations that the purified protein inactivates the phage, mutants lacking the protein are resistant to the phage, and mutants selected for PA-2 resistance have altered protein. Protein 1 appears as two bands (1a and 1b) on high-resolution polyacrylamide gels. The most abundant classes of mutants (ParI and ParII) selected for PA-2 resistance were found to lack band 1b. The mutations responsible for the ParI and ParII phenotypes were mapped at a locus termed par, which is near nalA on the Escherichia coli chromosome. The cyanogen bromide peptides of proteins 1a and 1b are similar, suggesting that these bands represent modified forms of the same polypeptide. Strains carrying the tolF mutation produce only band 1b. When a par tolF double mutant was constructed, this strain produced only band 1a. These results suggest that genes at the par and tolF loci are involved in modification of protein 1, or regulation of such modification, and are not structural genes for protein 1.
Three multiply colicin-tolerant mutants in Escherichia coli K-12 from the TolIV, TolXIV, and TolXV phenotypic groups, all lacking or having only trace amounts of protein 1, a major outer-membrane protein, were mapped by Hfr crosses, and the position on the chromosome was confirmed by cotransduction with nearby markers. The mutations were located near malQP in the 74-min region of the E. coli chromosome. This locus is designated ompB, and analysis of data from two three-point crosses determined the linear sequence of genes to be aroB-ompB-malQP-glpD.
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Hfr, F+, and F-prime cells are, unlike F- cells, insensitive to an excess of Hfr donor cells, indicating that there is an F factor mediated immunity to lethal zygosis (I1z). Results with Flac episomes carrying traJ, traS or various polar mutations in the tra region indicate that this immunity is independent of surface exclusion, of traJ control, and of all known genes within the tra operon. However, analysis of a series of strains with deletions in the F factor, extending from the right into the tra region, suggests that a gene for immunity to lethal zygosis is located within the tra region. We therefore conclude that I1z is genetically complex, and present a hypothesis to account for these results.
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Four classes of colicin B-resistant mutants of Escherichia coli K-12 were examined for defects in iron uptake. All four mutant classes (cbt, exbC, exbB, and tonB) were defective in the uptake of ferri-ennterochelin. The tonB mutant was also defective in citrate-, ferrichrome-, and rhodoturulic acid-mediated iron uptake. The defects in iron transport were reflected in increased sensitivity to iron chelators and to chromium and aluminium salts, and in hypersecretion of enterochelin. One of the mutants (cbt) was apparently defective in outer membrane ferri-enterochelin receptor activity. aroE derivatives (unable to synthesize enterochelin) of the four mutant classes and the parent strain produced increased amounts of two outer membranes polypeptides when grown under iron stress. These polypeptides are implicated in ferri-enterochelin receptor activity.
Cross-resistance between bacteriophages and colicins was studied using collections of bacteriophage- and colicin-resistant mutants of Escherichia coli K-12. No new examples were found of highly specific one-to-one cross-resistance of the type suggestive of common receptors. However, several groups of mutants showed tolerance to colicins and resistance to bacteriophages. Mutants known to be very defective in lipopolysaccharides composition were found to commonly show tolerance to certain colicins in addition to their bacteriophage resistance. Another group of mutants showed varying patterns of resistance to colicins E2, E3, K, L, A, S4, N, and X and bacteriophages E4, K2, K20, K21, K29, and H+. However, many bacteriophage-resistant mutants were fully colicin sensitive, and most colicin-resistant mutants were fully sensitive to bacteriophages.
Nine classes of group B colicin-resistant mutants were examined to study the role of enterochelin in colicin resistance. Four of the mutants studied (cbt, exbC, exbB, and tonB) hypersecreted enterochelin. Enterochelin hypersecretion was apparently responsible for resistance of the exbC mutant to colicins G and H and for resistance of the exbB mutant to colicins G, H, Ia, Ib, S1, and V. All four mutants scored as colicin B tolerant, even in the absence of enterochelin synthesis. The mutants produced substantially increased amounts of two high-molecular-weight outer membrane polypeptides when grown under limiting iron conditions. The presence of these polypeptides was correlated with increased colicin B-neutralizing activity in the outer membrane preparations.
Bacteriophage-resistant mutants isolated and classified in a previous study were examined for alterations in their lipopolysaccharide (LPS) composition, and properties likely to be affected by alterations in LPS composition were studied. It was found that many of the mutants of the Ktw (K2-resistance), Ttk (T2, T4, or K19 resistance), Bar (bacteriophage), Wrm (wide-range mutants), and miscellaneous resistance groups were altered in their response to a series of antibiotics and to two LPS-specific bacteriophages, C21 and U3. Furthermore, many of the bacteriophages to which these mutants were resistant adsorbed to LPS preparations. By direct sugar analysis of the mutant LPS preparations, it was shown that the mutants fitted into six distinct classes, which are readily derived from LPS core with a structure resembling that of Salmonella or Escherichia coli O100. A number of the mutants were shown to map between pyrE and mtl, which has been previously shown to be the site of a cluster of rfa genes in both Salmonella and E. coli. Outer membrane protein composition was studied in the above mutants using polyacrylamide gel electrophoresis. Some strains were shown to have alterations in the amount of major proteins. The nature of the bacteriophage receptors involved and the alterations leading to resistance are discussed.
Two classes of mutants, con and tolG, that appeared to be very similar in a number of respects have been shown to be identical and cotransducible with pyrD. By diethylaminoethyl-cellulose chromatography of the outer membranes, we have shown that the mutants are missing only protein 3A and retain protein 3B. Using con mutants, we were thus able to identify protein 3B on the pH 7.2 gel system of Maizel where it runs separately from protein 3A if unheated samples are used. tolG mutants were shown to be identical to con mutants in being conjugation defective with most F-like plasmid donors but not with I-like plasmid donors, and in their resistance pattern to bacteriophages and colicins. During the course of this study, it was observed that the bacteriocin produced by Serratia marcescenc JF246 was identical in its activity spectrum to colicin L-398 and is now considered to be a colicin of type L.