Peter Reeves

Regulation of the pho regulon of Escherichia coli K-12: Cloning of the regulatory genes phoB and phoR and identification of their gene products☆

Abstract The regulatory genes phoB and phoR of the Escherichia coli K-12 pho regulon have been cloned using two types of selection. The genes were localized on one of the hybrid plasmids, pJP50, by analysis of mutant plasmids generated by Tn5 insertions. Restriction mapping of the other plasmid, pPR20, indicated that the gene order in this region of the chromosome is phoB phoR tsx . Analysis of plasmid-coded proteins in a minicell system showed that: (1) the products of phoB and phoR are proteins with apparent molecular weights of 30,000 and 47,000, respectively; and (2) the synthesis of the phoB gene product is derepressed by phoR mutations. The implications of this observation for the model of pho regulation are discussed.

Defective growth functions in mutants of Escherichia coli K12 lacking a major outer membrane protein.

Abstract Various properties of mutants of Escherichia coli K12 lacking specific outer membrane proteins have been studied. ompA mutants are shown to grow less well than their parent strains under a variety of growth conditions, and after completion of growth to enter a decline phase in which viability is lost and the cells become heavily piliated and clump. They are defective in the uptake of amino acids, whereas the uptakes of the larger transport substrates ferrienterochelin and cyanocobalamin (vitamin B12) are normal. These ompA mutants also grow poorly at 42 °C. The implications of these results are discussed in terms of the function of the ompA. gene product. No growth or uptake defects were observed for ompB or tsx mutants.

Outer membrane of Escherichia coli K-12: Tsx mutants (resistant to bacteriophage T6 and colicin K) lack an outer membrane protein

Abstract Tsx mutants of Escherichia coli are fully resistant to a set of T6-like bacteriophage and are resistant to colicin K. We demonstrate that these mutants are missing an outer membrane protein (the tsx-protein) of molecular weight 32,000 as measured by SDS-polyacrylamide gel electrophoresis. Tsx mutants are receptor mutants which are unable to absorb either the bacteriophages or the colicin and the loss of receptor function can be demonstrated using outer membrane preparations. We suggest that the tsx-protein is the receptor for both the bacteriophage and colicin.

The role of colicin receptors in the uptake of ferrienterochelin by Escherichia coli K-12.

Abstract The colicin B-D receptor in the outer membrane of Escherichia coli K-12 is shown to function also as the receptor for the iron-siderophore complex ferrienterochelin. The outer membrane receptors for colicins I and M are not involved in ferrienterochelin uptake.

Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation

Abstract Growth of E. coli K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with tonA mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.

Outer membrane proteins of Escherichia coli K-12: Isolation of a common receptor protein for bacteriophage T6 and colicin K

Summarytsx mutants, resistant to T6-like bacteriophages and colicin K, of Escherichia coli K-12 lack an outer membrane protein of 26,000 molecular weight. This protein is shown to have receptor activity for both bacteriophage T6 and colicin K. The protein has been purified and its amino acid composition determined. Some tsx mutants appear to have an altered receptor protein, as indicated by their ability to plate extended host-range mutants of bacteriophage T6. These mutants are also cotransducible with proC and can be arranged in an order of increasing resistance to the host range phages, which appear to have differing degrees of ability to propagate on the tsx mutants.The tsx protein was shown to be catabolite repressible both by use of varying growth conditions and cya and crp mutants.

Comparison of Colicins B-K260 and D-CA23: Purification and Characterization of the Colicins and Examination of Colicin Immunity in the Producing Strains

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. Images

Molecular cloning of the tolC locus of Escherichia coli K-12 with the use of transposon Tn10

SummaryWe have cloned the tolC gene of E. coli K-12 into pSF2124 by using transposon Tn10 as the marker to first isolate the relevant DNA fragment. The gene is on a 10.5 kb EcoRI fragment, and Tn5 insertion mutagenesis locates the gene near one end of this EcoRI fragment. An EcoRI-PstI fragment has been subcloned into pBR322 to facilitate further analysis of the gene.

The TolC protein of Escherichia coli K12 is synthesised in a precursor form.

We examined the biosynthesis of the TolC protein of Escherichia coli K12 in a pulse—chase experiment, followed by immunoprecipitation with anti‐TolC antibody and SDS—PAGE of the immunoprecipitate. This showed that TolC protein was originally synthesised in a precusor form (M r 54 500) which could be chased into the mature form (M r 52 000). DNA sequencing of a portion of the cloned tolC gene showed that the N‐terminus of the mature rotein was preceded by a typical signal sequence of 22 residues (M r 2542). The initiator Met was preceded by a Shine—Dalgarno sequence, with the correct spacing.

Effect of tra Mutations on F Factor-Specified Immunity to Lethal Zygosis

SummaryHfr, 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 (Ilz). 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 Ilz is genetically complex, and present a hypothesis to account for these results.