Paul A. Manning

Cell-cell interactions in conjugating Escherichia coli: Con− mutants and stabilization of mating aggregates

SummaryConjugation-deficient (Con-) mutants of Escherichia coli K-12 have been previously described which were defective in recipient ability. Such Con- mutants were obtained from several laboratories and retested by a standardized set of procedures. Many of the mutants did not satisfy minimal criteria for conjugation-deficiency and were discarded. The remaining mutants included 11 ConF- mutants mutated in or near the ompA cistron, 3 ConF- mutants synthesizing a heptose-deficient lipopolysaccharide and 1 ConI- mutants synthesizing a defective lipopolysaccharide. This set of mutants was tested for resistance to a variety of bacteriophages and colicins; the only phenotype fully correlated with the ConF- phenotype was that of resistance to colicin L. No simple correlation existed between the protein profile (on SDS polyacrylamide gel electrophoresis) of cell envelope outer membrane preparations and conjugation deficiency. However, many ConF- mutants did not synthesize detectable levels of outer membrane protein II* and protein II* may have been nonfunctional in the remainder. All the ConF- mutants were conjugation-deficient when matings were conducted in liquid but (with one exception) were conjugation-proficient on the surfaces of membrane filters. None of the ConF- mutants formed stable mating aggregates in liquid with (Flac)+ donor cells although all bound purified F pili. The ConF- phenotype associated with a II*-deficient recipient could be mimicked by the addition of purified protein II* (solubilized with lipopolysaccharide). In both cases, the formation of stable mating aggregates (analyzed with an improved Coulter counter technique) was inhibited whereas unstable mating aggregates were detected by electron microscopy. F pilus and wall to wall contacts were both observed under these conditions by electron microscopy. These results were used to define a stage in F-promoted conjugation, the stabilization stage, which requires the functional interaction of protein II* and lipopolysaccharide in the outer membrane of the recipient cell.

Genetic analysis of F sex factor cistrons needed for surface exclusion in Escherichia coli

Abstract The structural genes of the F plasmid which encode surface exclusion have previously been cloned in the vector plasmid pSC101 to yield the chimeric plasmid pRS31. Point mutations which partially inactivate surface exclusion have been generated in pRS31 by in vitro mutagenesis. These mutations have been used to demonstrate that two independent cistrons, traS and traT , each encodes only one of the two aspects of the surface exclusion phenomenon. When these mutations were moved genetically to F lac , they affected surface exclusion but did not affect either the synthesis of F pili or the ability to act as a donor in conjugation. 60 mutants of F lac were isolated which were either totally or only partially defective in producing surface exclusion. Those mutants which were only partially defective carried either traS or traT mutations. Most of the other mutations were in the traJ cistron, which is involved in transcriptional regulation of the tra operon. These latter mutants were defective in F piliation and in DNA transfer as well. Still other mutants carried mutations which were polar on the surface exclusion cistrons or carried multiple mutations. These results demonstrate that the F plasmid contains two structural cistrons for surface exclusion and one cistron which is involved in transcriptional regulation.

Serum Resistance in E.coli

Pathogenic bacteria that cause generalized infections or meningitis invade the blood stream and are thereby distributed throughout the body. Blood, or serum, contains a number of nonspecific (complement, lysozyme, phagocytes, iron-binding proteins, etc.) and specific (antibodies, lymphocytes) agents that alone or in combination lyse, kill or prevent the growth of the majority of bacteria with which they make contact. Abilities to resist, evade or inactivate these host defences constitute major components of the virulence of invasive bacteria. At present little is known about these bacterial properties or their molecular interactions with host defences.

RNA-polymerase binding sites within the tra region of the F factor of Escherichia coli K-12 ☆

Chimeric plasmids containing the tra operon of the Escherichia coli K-12 F factor were used to map by electron microscopy the RNA polymerase binding sites within the contiguous F EcoRI restriction fragments f6, f16, f1, f17, f19 and f2. [These fragments have been previously cloned in the EcoRI site of pSC101 to give the chimeric plasmids pRS27 (f6, f15), pRS29 (f15, f1) and pRS31 (f17, f19 and f2)]. The results may reflect the presence of a number of previously unrecognized promoters within the traY----Z operon.

Mutants that overproduce TraTp, a plasmid-specified major outer membrane protein of Escherichia coli

SummaryThe isolation of a series of plasmid mutant derivatives that overproduce the traT outer membrane protein, TraTp, is described. Some of the mutants directed the synthesis of 10-fold more TraTp (200,000 copies/cell) than did the parental plasmid (20,000 copies/cell). The proteins specified by all mutant plasmids except one were correctly inserted into the outer membrane and exposed on the cell surface. The TraTp that was not correctly inserted did not mediate the expected levels of surface exclusion and serum resistance, suggesting that surface localization is a requirement of TraTp function. The overproduction of TraTp was deleterious to bacterial growth, particularly that of minicell mutants of E. coli K-12.