Major proteins of the Escherichia coli outer cell envelope membrane as bacteriophage receptors.

Three Escherichia coli phages, TuIa, TuIb, and TuII, were isolated from local sewage. We present evidence that they use the major outer membrane proteins Ia, Ib, and II, respectively, as receptors. In all cases the proteins, under the experimental conditions used, required lipopolysaccharide to exhibit their receptor activity. For proteins Ia and II, an approximately two- to eightfold molar excess of lipopolysaccharide (based on one diglucosamine unit) was necessary to reach maximal receptor activity. Lipopolysaccharide did not appear to possess phage-binding sites. It seemed that the lipopolysaccharide requirement reflected a protein-lipopolysaccharide interaction in vivo, and lipopolysaccharide may thus cause the specific localization of these proteins. Inactivation of phage TuII by a protein II-lipopolysaccharide complex was reversible as long as the complex was in solution. Precipitation of the complex with Mg2+ led to irreversible phage inactivation with an inactivation constant (37 degrees C)K = 7 X 10-2 ml/min per microgram. With phages TuIa and TuIb and their respective protein-lipopolysaccharide complexes, only irreversible inactivation was found at 37 degrees C. The activity of the three proteins as phage receptors shows that part of them must be located at the cells surface. In addition, the association of proteins Ia and Ib with the murein layer of the cell envelope makes this pair trans-membrane proteins.     

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. II. Genetic structure and mechanism of polylysogen formation for lambda, phi 80 and the lambda att80 hybrid

Summary The frequency of occurrence and the genetic structure of polylysogens were studied for phages , 80 and att80. In the case of , frequency of polylysogenization is high (0.20 to 0.41) with a tandem integration of prophages at the primary att site (att). With 80 and att80, this frequency is about 10 times lower, and usually one prophage becomes integrated at the primary att site (att80-I) while another (sometimes two others) integrates at one of the secondary sites. At least four secondary att80 sites have been found in wild-type Escherichia coli , two of which (near the his and tolC loci) are preferred. The frequency of secondary integration of 80 and att80 does not differ significantly in the wild-type host and in that deleted for the primary att site (0.041 and 0.045, respectively, among surviving cells at an MOI of 10).Homoimmune superinfection has revealed a constitutive cI-independent expression of the 80 int gene in the prophage state. The only 80 tandem detected proved to be unstable. With the 80int ^- mutant, we observed stabilization of 80 tandems; as a consequence, their frequency of occurrence during coinfection with 80int ⁺ was up to the level and no nontandem insertions were found. A model is proposed for the 80 and att80 nontandem integration.Abbreviations TP transducing phage(s) - PFU plaque-forming units - PC pink clear-resistant colonies on EMBO plates - MOI multiplicity of infection - O origin of Hfr transfer

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Alterations in the cell envelope of Escherichia coli late in bacteriophage T4 infection

The cell envelope of Escherichia coli was examined for changes during late stages of bacteriophage T4 infection. Late events in T4 infection are shown to result in (i) a reduction in the effectiveness of membrane separation procedures employing either isopycnic sucrose gradient centrifugation or selective solubilization of inner membrane by detergent (Sarkosyl or Triton X-100), (ii) the appearance of a 54 000 dalton host protein in membrane preparations, (iii) the adventitious presence of detergent-resistant phage morphogenetic structures in membrane preparations, and (iv) a decrease in the activity of NADH oxidase and an apparent alteration in its association with inner membrane. These modifications occur regardless of the state of the $\text{e}$ and $\text{t}$ genes of T4.     

Location of binding sites on common type 1 fimbriae from Escherichia coli.

Common type 1 fimbriae were isolated from Escherichia coli and their length distribution profile was determined before and after treatment with ultrasound. As fimbriae were shortened, so their haemagglutinating capacity decreased, but their ability to bind to erythrocytes did not decrease to the same extent. Isolated fimbriae did not agglutinate inside-out vesicles prepared from horse erythrocytes or liposomes, suggesting that the binding mechanism was not based on non-specific hydrophobic interactions. The results support a lateral rather than a terminal location for the fimbrial binding site responsible for haemagglutination.     

The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor

Although the lambdoid bacteriophage phi 80 and P22 possess site-specific recombination systems analogous to bacteriophage lambda, they have different attachment (att) site specificities. We have identified and determined the nucleotide sequences of the att sites of phi 80 and P22 and have examined the interaction of these sites with purified Escherichia coli integration host factor (IHF). The sizes of the homologous core regions of the att sites vary greatly: P22 has a 46-base pair core, while phi 80 and lambda have 17- and 15-base pair cores, respectively. The core sequences of the three phage show no significant homology, although dispersed regions of homology in arm sequences indicate that the three phage att sites are related. All three att sites have a high A + T composition, and restriction fragments carrying these sites migrate anomalously upon polyacrylamide gel electrophoresis. IHF binds to a site to the left of the common core in the phi 80 and P22 phage att sites (attP) and to a site to the right of the core in P22 attP and attB (the bacterial att site). In the lambda system, IHF interacts with three regions on attP (designated H1, H2, and H') and none on attB (Craig N., and Nash, H.A. (1984) Cell 39, 707-716). Alignment of the IHF sites of all three phage results in a consensus sequence for IHF binding, Pyr-AANNNNTTGATAT. Among the three phage, the number of IHF sites differs; however, the location and orientation of the binding sites in relation to the respective core regions are well conserved. An IHF site analogous to lambda H2 is present in both phi 80 and P22 attP, while a site analogous to lambda H' is present in P22 attP. This conservation suggests that IHF plays a very similar role in the site-specific recombination pathways of all three phage, and that the flanking arm sequences are necessary for phi 80 and P22 attP function, as is the case for lambda attP function. These structural similarities presumably reflect a conservation of the mechanism of site-specific recombination for the three phage.     

Integration of bacteriophages lambda and phi 80 in wild-type Escherichia coli at secondary attachment sites. I. Formation of secondary lysogens

Summary The family of lambdoid phages displays a varying specificity of integration into the host chromosome. The phage DNA failed to get inserted at the secondary site(s) of the gal operon (frequency <2.6x10^-8) in the presence of the primary (normal) att site. By contrast, 80 and the att80 hybrid (x80) became integrated into wild-type Escherichia coli at at least two secondary att sites of the btuB locus, and the latter near purE and purC as well (frequency 2x10^-3-10^-4). The integration of 80 and att80 into btuB occurred with about the same frequency as in cells in which the normal insertion site had been deleted (0.7-4.0x10^-6). An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found at the primary att site. We also failed to observe the integration into other loci of 80 and att80 with the formation of secondary monolysogens (frequency <0.0035 at MOI-10^-3 or 10). It is presumed that these prophages become integrated at secondary att sites only if the primary site is occupied.Abbreviations MOI multiplicity of infection (PFU/cell) - PFU plaque-forming unit - TP transducing phage - P1/HfrH P1vir multiplied on HfrH - Rif-R rifampin-resistance - Int int protein     

Growth of the Escherichia coli cell envelope

The growth pattern of the Escherichia coli envelope was studied by immunoelectron microscopy, using the outer membrane protein LamB specifically labelled by a double antibody gold particle technique. An operon fusion placing the lamB gene under lac promoter control permitted rapid turn-off of LamB synthesis. In the generation following turn-off no lamB-free regions appeared, strongly suggesting that bulk outer membrane material is not inserted in restricted growth zones.     

Regulation of Escherichia coli carbamyl phosphate synthetase. Evidence for overlap of the allosteric nucleotide binding sites

Regulation of Escherichia coli carbamyl phosphate synthetase by UMP and IMP was examined in studies with various analogs of these nucleotides. Whereas UMP inhibits enzyme activity, the arabinose analog of UMP was found to be an activator. dUMP neither activates nor inhibits, but binds to the enzyme in a manner similar to UMP as evaluated by direct binding studies, sedimentation behavior, and ultraviolet difference spectral measurements. dUMP decreases inhibition by UMP and activation by IMP, but has no effect on activation by L-ornithine. The findings are in accord with the view that IMP and UMP bind to the same region of the enzyme; a possible general model for such overlapping binding sites is considered. Additional evidence is presented that inorganic phosphate can modulate regulation of the activity by nucleotides. Phosphate (and arsenate) markedly increase inhibition by UMP, decrease activation by IMP, but do not affect activation by L-ornithine. The extent of activation by IMP and by L-ornithine and that of inhibition by UMP are decreased when Mg2+ concentrations are increased relative to a fixed concentration of ATP. The findings suggest that the allosteric effectors may affect affinity of the enzyme for divalent metal ions as well as, as previously shown, the affinity of the enzyme for Mg-ATP.     

Penicillin-binding site on the Escherichia coli cell envelope.

The binding of 35S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the "cell envelope" obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. At low pH, PBPs 1b, 1c, 2, and 3 demonstrated the greatest amount of binding. At high pH, these PBPs bound the least amount of penicillin. PBPs 1a and 5/6 exhibited the greatest amount of binding at pH 10 and the least amount at pH 4. With the exception of PBP 5/6, the effect of pH on the binding of penicillin was direct. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin. These observations suggest that a molecule of penicillin forms simultaneous bonds between its S at position 1 and sulfhydryl groups of PBP 5 and between its C-7 and free epsilon amino groups of PBP 5.     

Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174

Escherichia coli VC30 is a temperature-sensitive mutant which is defective in autolysis. Strain VC30 lyses at 30 degrees C when treated with beta-lactam antibiotics or D-cycloserine or when deprived of diaminiopimelic acid. The same treatments inhibit growth of the mutant at 42 degrees C but do not cause lysis. Strain VC30 was used here to investigate the mechanism of host cell lysis induced by bacteriophage phi X 174. Strain VC30 was transformed with plasmid pUH12, which carries the cloned lysis gene (gene E) of phage phi X174 under the control of the lac operator-promoter, and with plasmid pMC7, which encodes the lac repressor to keep the E gene silent. Infection of strain VC30(pUH12)(pMC7) with phage phi X174 culminated in lysis at 30 degrees C. At 42 degrees C, intracellular phage development was normal, but lysis did not occur unless a temperature downshift to 30 degrees C was imposed. Similarly, induction of the cloned phi X174 gene E with isopropyl-beta-D-thiogalactoside resulted in lysis at 30 degrees C but not at 42 degrees C. Temperature downshift of the induced culture to 30 degrees C resulted in lysis even in the presence of chloramphenicol. These results indicate that host cell lysis by phage phi X174 is dependent on a functional cellular autolytic enzyme system.     

DNA sequence analysis. Terminal sequences of bacteriophage phi80.

Sequences of the cohesive ends and the 3'-terminal regions of phi80 DNA have been determined. Sequences of the cohesive ends were obtained through the use of two standard methods. The first method involved the incorporation of all four labeled deoxyribonucleotides into the phi80 cohesive ends using DNA polymerase I. The DNA was then partially digested with micrococcal nuclease or pancreatic DNase. The products were separated by two-dimensional electrophoresis and characterized by composition, 3'-terminal, and nearest neighbor analyses. The second method involved partial incorporation using one, two, or three labeled deoxyribonucleotides followed by similar analyses. Sequences of the double-stranded regions adjacent to the cohesive ends were determined by three new methods. These methods were: (a) the DNA was specifically labeled at the 3' terminus and then partially degraded. Labeled oligonucleotide products were sequenced by their mobilities on various separation systems. (b) The cohesive ends were enlarged by limited degradation with exonuclease III. After this treatment, the DNA was partially repaired with labeled nucleotides, digested, and the products were analyzed. (c) A synthetic ologonucleotide primer was bound to phi80 DNA which had been repaired with DNA polymerase I, and then partially digested with lambda-exonuclease. The primer was extended into the region of interest by partial repair with labeled nucleotides. The extended primer was isolated and analyzed.     

Protein complexes of the Escherichia coli cell envelope

Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.     

Functions in outer and inner membranes of Escherichia coli for ferrichrome transport.

Mutants of the fhuA gene of Escherichia coli K-12, which encodes a receptor protein in the outer membrane, took up ferrichrome after exposure to pronase, whereas fhuB mutants remained transport negative. The latter finding supports our previous proposal that fhuB mutants are defective in a function that residues in the cytoplasmic membrane. Cells remained completely viable after treatment with pronase, although they became sensitive to the antibiotic actinomycin.     

New genes in the left arm of the bacteriophage phi 80 chromosome.

We describe the isolation and partial genetic characterization of 247 amber (suppressor-sensitive) mutants of temperate bacteriophage phi 80 of Escherichia coli. Of these 247 mutants, the mutations of 201 mapped to the left arm of the phi 809 chromosome and the mutations of 39 mapped to the right arm of the genome. Complementation tests among these and previously described left arm mutants defined five additional genes in the left arm of the chromosome. The positions of these genes are consistent with the hypothesis that four of them represent functions essential for phi 80 tail assembly and one represents a capsid assembly function, probably the major coat protein. The identification of these genes brings the phi 80 genome into closer correspondence with the organization of the phage lambda genome. Two- and three-factor crosses performed between mutants with defects in each of the previously identified genes and mutants with defects in the five new genes allowed us to construct a consistent, roughly additive recombination map of the left arm of the bacteriophage phi 80 genome.     

Integration of the related prophages lambda, phi 80 and their hybrid lambda att80 into the secondary chromosomal att sites of wild-type Escherichia coli

The family of lambdoid phages displays a varying specificity of integration into the host chromosome. The lambda phage DNA failed to get inserted at the secondary attachment site(s) of the gal operon (frequency less than 2.6 X 10(-8)) in the presence of the primary (normal) one. By contrast, phi 80 and the lambda att80 hybrid integrated into wild-type Escherichia coli at least, at two secondary att sites of the btuB locus, the latter phage being also capable of integration in the vicinity of purE and purC (frequency 2 X 10(-3) to 10(-4)). Integration of phi 80 and lambda att80 into btuB occurred with about the same frequency as in cells deleted for normal insertion site (0.7 divided by 4.0 X 10(-6)). An analysis of the secondary lysogens with the prophage in btuB showed them to be polylysogens; the additional prophage(s) was found in the primary att site. We also failed to observe integration of phi 80 and lambda att80 with formation of secondary monolysogens into other foci (frequency less than 0.0035, if multiplicity of infection was 10(-3) or 10). It is presumed that phi 80 and lambda att80 prophages get only integrated at secondary att sites in case the primary site is occupied.     

Anatomy of Escherichia coli ribosome binding sites

During translational initiation in prokaryotes, the 3' end of the 16S rRNA binds to a region just upstream of the initiation codon. The relationship between this Shine-Dalgarno (SD) region and the binding of ribosomes to translation start-points has been well studied, but a unified mathematical connection between the SD, the initiation codon and the spacing between them has been lacking. Using information theory, we constructed a model that treats these three components uniformly by assigning to the SD and the initiation region (IR) conservations in bits of information, and by assigning to the spacing an uncertainty, also in bits. To build the model, we first aligned the SD region by maximizing the information content there. The ease of this process confirmed the existence of the SD pattern within a set of 4122 reviewed and revised Escherichia coli gene starts. This large data set allowed us to show graphically, by sequence logos, that the spacing between the SD and the initiation region affects both the SD site conservation and its pattern. We used the aligned SD, the spacing, and the initiation region to model ribosome binding and to identify gene starts that do not conform to the ribosome binding site model. A total of 569 experimentally proven starts are more conserved (have higher information content) than the full set of revised starts, which probably reflects an experimental bias against the detection of gene products that have inefficient ribosome binding sites. Models were refined cyclically by removing non-conforming weak sites. After this procedure, models derived from either the original or the revised gene start annotation were similar. Therefore, this information theory-based technique provides a method for easily constructing biologically sensible ribosome binding site models. Such models should be useful for refining gene-start predictions of any sequenced bacterial genome.     

Organization of the early region of bacteriophage phi 80. Genes and proteins

An EcoRI segment containing the early region of bacteriophage phi 80 DNA that controls immunity and lytic growth was identified as a segment whose presence on a plasmid prevented growth of infecting phi 80cI phage. The nucleotide sequence of the segment (EcoRI-F) and adjacent regions was determined. Based on the positions of amber mutations and the sizes of some gene products, the reading frames for five genes were identified. From the relative locations of these genes in the genome, the properties of some isolated gene products, and the analysis of the structures of predicted proteins, the following phi 80 to lambda analogies are deduced: genes cI and cII to their lambda namesakes; gene 30 to cro; gene 15 to O; and gene 14 to P. An amber mutation by which gene 16 was defined is a nonsense mutation in the frame for gene 15 protein, excluding the presence of gene 16. An amber mutation in gene 14 or 15 inhibits phage DNA synthesis, as is the case with their lambda analogues, gene O or P. Some characteristics of proteins from the early region predicted from their primary structures and their possible functions are discussed.     

Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein.

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.