Enrichment of the bacteriophage PR4 membrane in phosphatidylglycerol is not essential for phage assembly and infectivity.

The membrane phospholipids of bacteriophage PR4 grown on wild-type Escherichia coli are markedly enriched in phosphatidylglycerol (PG) relative to host phospholipids. To investigate the role of PG in phage assembly and infectivity, we propagated PR4 on an E. coli mutant defective in PG synthesis. The PG content of PR4 grown on the mutant host accounted for 0.4% of the total viral phospholipids, representing a 90-fold decrease in PG relative to the PG content of phage grown on a wild-type host. Phosphatidylethanolamine and phosphatidic acid, the two major phospholipid species present in these phage preparations, accounted for 88.4 and 9.4% of the total viral phospholipids, respectively. This drastic alteration of the phage phospholipid composition had little or no adverse effect on either the stability or infectivity of the phage. We conclude that the enrichment of the PR4 virion in PG does not reflect an absolute structural requirement of the phage and is not essential for phage infectivity.     

Effects of temperature and host cell genetic characteristics on the replication of the lipid-containing bacteriophage PR4 in Escherichia coli.

The lipid-containing bacteriophage PR4 is of special intest because it can replicate in various gram-negative bacteria, including Escherichia coli, that carry one of a group of drug resistance plasmids. PR4 grown in E. coli strain PS2R contains about 10% lipid by weight, with the negatively charged phospholipid phosphatidylglycerol being the most abundant lipid in the virion. We now report the following. (i) PR4 attaches to E. coli with an attachment rate constant of Ka approximately 6.2 X 10(-10) ml/min, which is about twice that of the enveloped phage phi6 (to Pseudomonas phaseolicola), but a factor of 5 less than that of phage PM2 (to Pseudomonas BAL-31). (ii) Use of an E. coli glycerol auxotroph indicated that a normal amount of PR4 replication occurs only if glycerol starvation (inhibition of all phospholipid synthesis) begins no earlier than about halfway through the lytic cycle. (iii) Use of an E. coli fatty acid synthesis temperature-sensitive mutant and an E. coli phosphatidylethanolamine synthesis temperature-sensitive mutant indicate that PR4 replication can occur in the absence of either normal fatty acid synthesis or normal phospholipid synthesis if the infection takes place prior to the termination of overall cell growth and the onset of cell death, (iv) Whereas PR4 burst size in nutrient media at 30 degrees C to 42%C is about 40, the burst size at 20 degrees C is less than 3, Temperature-shift experiments show that the temperature late in infection determines the burst size.     

The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains.

Streptomyces cyaneus genomic DNA ligated into either lambda phage or plasmid vectors was very inefficiently cloned into standard Escherichia coli host strains. However, the same material could be efficiently cloned using Mcr-deficient E. coli strains. These results suggest that the S. cyaneus genome contains 5-methylcytosine residues, some of which occur within the recognition sequences of the E. coli Mcr restriction system.     

Phosphatidylinositol, a phospholipid of ice-nucleating bacteria.

The nature of the phospholipids of the various bacteria that have ice nucleation activity in supercooled water has been determined. The seven bacteria studied included Pseudomonas syringae, Erwinia herbicola, three Escherichia coli K-12 strains that are phenotypically Ice+ because they contain plasmids with different amounts of either P. syringae or E. herbicola cloned DNA, and two E. coli K-12 strains without cloned ice gene DNA. All five Ice+ bacterial strains contained small amounts (0.1 to 1.0% of the total phospholipids) of phosphatidylinositol (PI), a phospholipid not previously detected in E. coli, Pseudomonas, or Erwinia species. The Ice- E. coli strains also contained trace level of PI that amounted to 2 to 30% of the level found in the Ice+ E. coli strains. Extracts of Ice+ strains contained low but measurable activities of PI synthase, while the activities in Ice- strains amounted to only 8 to 12% or less of that found in extracts of Ice+ bacteria. The functioning of the ice gene apparently increased both the PI synthase activity and the PI content of Ice+ strains from low endogenous levels. The relative ice nucleation activity at -4 degrees C or above (class A nucleation activity) of all Ice+ strains was found to be proportional to their PI content. The addition of myo-inositol (5 x 10(-4) M) to synthetic culture media increased the class A nucleation activity of both Ice+ E. coli strains and P. syringae up to sevenfold but had no stimulating effect on ice nucleation at lower temperatures (class B and class C nucleation activities). If these cells after fusion with PI vesicles were incubated with an energy source, the class A nucleation activity increased 70-fold over that present before fusion. These results indicate that PI plays an important role in ice nucleation at warm temperatures and is a likely precursor or component of the class A structure.     

Inhibitory Effect of Fatty Acids on the Entry of the Lipid-Containing Bacteriophage PR4 into Escherichia coli

Various unsaturated fatty acids (notably palmitoleic acid and oleic acid) interfered with plaque production by the lipid-containing bacteriophage PR4 on lawns of Escherichia coli. Addition of fatty acid to give 50 mug/ml ( approximately 0.2 mM) at the time of infection prevented phage replication. If, however, the fatty acid was added after infection, normal amounts of phage were produced. If the fatty acid was added (to 50 mug/ml) to the host cell culture a long enough time before infection such that the fatty acid concentration in the growth medium at the time of infection was reduced to less, similar5 mug/ml (due to fatty acid incorporation by the host cells), normal phage replication occurred also. Neither palmitoleic acid nor oleic acid prevented PR4 attachment to E. coli. Several types of experiments indicated that it is the entry process of the virus that is inhibited by these fatty acids. Specifically, if the fatty acid was added at the time of infection, the host cells were not killed by the virus and no detectable amounts of viral protein were synthesized. In addition, experiments using purified radioisotope-labeled virions showed directly that entry is inhibited. Mutants of PR4 that did replicate in the presence of oleic acid arose spontaneously at a frequency of 10(-6). Three of these mutants that have been further characterized have protein and phospholipid compositions indistinguishable from those of wild-type PR4.     

Group V phospholipase A2 in bone marrow-derived myeloid cells and bronchial epithelial cells promotes bacterial clearance after Escherichia coli pneumonia

Group V-secreted phospholipase A(2) (GV sPLA(2)) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA(2) in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA(2) after pulmonary E. coli infection. GV(-/-) mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV(+/+) mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV(+/+) and GV(-/-) mice after lung E. coli infection. In contrast, GV(+/+) mice have higher levels of prostaglandin D(2) (PGD(2)), PGF(2α), and 15-keto-PGE(2) in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV(-/-) mice after lung infection with E. coli. Selective deletion of GV sPLA(2) in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA(2) in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA(2) in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.     

Structure and serologic properties of O-specific polysaccharide from Citrobacter freundii possessing cross-reactivity with Escherichia coli O157:H7

Citrobacter freundii OCU158 is a serologically cross-reactive strain with Escherichia coli O157:H7. To explore the close relationship between two strains, we have analyzed the chemical structures of O-specific polysaccharides and antigenic properties of lipopolysaccharides (LPSs) of both strains. The structure of O-specific polysaccharides from both strains was found to be identical by chemical and nuclear magnetic resonance analyses, in which D-PerNAc was 4-acetamido-4,6-dideoxy-D-mannose: [-->4)-beta-D-Glc-(1-->3)-alpha-D-PerNAc-(1-->4)-alpha-D-GalNAc-(1 --> 3)-alpha-L-Fuc-(1-->](n). The enzyme immunoassay using LPS derived either from E. coli O157 or from C. freundii could equally detect high levels of serum antibodies against LPS in patients with enterohemorrhagic E. coli (EHEC) O157 infection. Absorption of antibodies in EHEC patient serum by LPS from E. coli O157 or C. freundii, however, showed a difference in the epitopes. This difference was attributable to the epitope specificity of the core region and/or lipid A structure in LPS.     

Temporal origin of viral phospholipids of the enveloped bacteriophage phi 6.

The enveloped bacteriophage phi 6 contains a higher relative level of the negatively charged phospholipid phosphatidylglycerol than is found in the membranes of the host bacterium. During infection of Pseudomonas phaseolicola with phi 6, the level of phosphatidylglycerol synthesis increases significantly. The lipid used to form the viral envelope consists almost entirely of cellular phospholipids synthesized before infection and phosphatidylglycerol synthesized after infection. Based on these and previously published results, a speculative model for this viral envelope formation process is presented.     

Properties of R plasmid R772 and the corresponding pilus-specific phage PR772.

R plasmid R772 was isolated from a strain of Proteus mirabilis and is a self-transmissible P-1 incompatibility group plasmid having a molecular weight of about 27 x 10(6). It renders bacterial hosts resistant to kanamycin. Phage PR772 was isolated as a phage dependent on the presence of R772 in bacterial hosts. It is hexagonal-shaped with a diameter of 53 nm, has a thick inner membrane and no tail. Vaguely defined appendages are sometimes apparent at some vertices and the phage possesses double-stranded DNA. The DNA has a guanine plus cytosine molar content of 48%. The phage is sensitive to chloroform and has a buoyant density of 1.26 g cm(-3). These observations suggested that the inner membrane of the phage could contain lipid. Phage PR772 differs in morphology from the double-stranded DNA plasmid-specific phages PR4 and PRR1 which adsorb to tips and sides, respectively, of sex pili coded for by P-1 incompatibility group plasmids. Phage PR772 formed clear plaques which varied in diameter. Serologically, phages PR772 and PR4 are possibly related though very distantly, but the two phages have identical host ranges. Phage PR772 adsorbed by one of its apices to tips of sex pili coded for by plasmid R772 in Escherichia coli. It also formed plaques on Salmonella typhimurium Proteus morganii and Providence strains harbouring this plasmid as well as strains of E. coli carrying plasmids of incompatibility groups N or W. The phage produced areas of partial clearing on lawns of P. mirabilis PM5006 harbouring plasmid R772, the P-1 incompatibility group plasmid RP4, the W group plasmid RSa or the N group plasmid N3, and on lawns of Providence strain P29 carrying plasmid RP4.     

Selective activation of human intestinal mast cells by Escherichia coli hemolysin

Mast cells (MCs) are recognized to play an important role in bacterial host defense in the murine system. In this study, we studied the interaction of human MCs, isolated from the intestine and purified to homogeneity, with different Escherichia coli and Shigella flexneri strains. We show that alpha-hemolysin (Hly)-producing E. coli strains induce the release of histamine, leukotrienes, and proinflammatory cytokines in intestinal MCs. In contrast, MCs were virtually unresponsive to S. flexneri and several Hly-negative E. coli strains, including the isogenic Hly-deficient mutants of Hly(+) strains. Hly(+) E. coli but not Hly(-) E. coli caused an increase in intracellular Ca(2+) levels. Blocking of extracellular Ca(2+) and of the calmodulin/calcineurin pathway by cyclosporin A inhibited the response to Hly(+) E. coli. Furthermore, inhibition of MAPKs p38 and ERK reduces activation of MCs by Hly(+) E. coli. In addition, using an ex vivo system, we directly record the histamine release by MCs located in the lamina propria after infection with Hly(+) E. coli. Our data indicate that human intestinal mast cells interact with selected Gram-negative bacteria, establish E. coli Hly as a factor regulating MC effector functions, and argue further for a role of human MCs in innate immunity.     

O-antigen modulates infection-induced pain States

The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic E. coli (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to E. coli lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. E. coli strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among E. coli strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.     

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and phage DNA biosynthesis.     

Functional Interactions between the DNA Ligase of ESCHERICHIA COLI and Components of the DNA Metabolic Apparatus of T4 Bacteriophage

T4 phage completely defective in both gene 30 (DNA ligase) and the rII gene (function unknown) require at least normal levels of host-derived DNA ligase (E. coli lig gene) for growth. Viable E. coli mutant strains that harbor less than 5% of the wild-type level of bacterial ligase do not support growth of T4 doubly defective in genes 30 and rII (T4 30- rII- mutants). We describe here two classes of secondary phage mutations that permit the growth of T4 30- rII- phage on ligase-defective hosts. One class mapped in T4 gene su30 (Krylov 1972) and improved T4 30- rII- phage growth on all E. coli strains, but to varying degrees that depended on levels of residual host ligase. Another class mapped in T4 gene 32 (helix-destabilizing protein) and improved growth specifically on a host carrying the lig2 mutation, but not on a host carrying another lig- lesion (lig4). Two conclusions are drawn from the work: (1) the role of DNA ligase in essential DNA metabolic processes in T4-infected E. coli is catalytic rather than stoichiometric, and (2) the E. coli DNA ligase is capable of specific functional interactions with components of the T4 DNA replication and/or repair apparatus.     

A comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the German E. coli O104:H4 outbreak strain

Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors.     

Host transfer RNA cleavage and reunion in T4-infected Escherichia coli CTr5x.

T4 mutants lacking polynucleotide kinase (pnk-) or RNA ligase (rli-) do not grow on E. coli CTr5x. During the abortive infections there accumulate host tRNA fragments that match into two species severed 3' to the anticodon. The CTr5x-specific fragments appear only transiently with wt phage, implicating the affected enzymes in phosphoryl group rearrangement and religation [David et al. (1982) Virol. 123, 480]. In a search for the vulnerable host tRNAs and putative religation products, tRNA ensembles from uninfected E. coli CTr5x or cells infected with various phage strains were fractionated and compared. A tRNA species absent from rli- infected cells but present in uninfected cells or late in wt infection was thus detected. RNase T1 finger prints of this species, isolated before or after wt infection, were compared with that of an in vitro ligated pair of CTr5x-specific fragments. The results indicated that this tRNA is cleaved upon infection and later on restored to it's original or to a very similar form, by polynucleotide kinase and RNA ligase reactions. It is suggested that depletion of such vulnerable host tRNA species underlies the restriction of pnk- or rli- phage on E. coli CTr5x.     

CTXphi infection of Vibrio cholerae requires the tolQRA gene products

CTXphi is a lysogenic filamentous bacteriophage that encodes cholera toxin. Filamentous phages that infect Escherichia coli require both a pilus and the products of tolQRA in order to enter host cells. We have previously shown that toxin-coregulated pilus (TCP), a type IV pilus that is an essential Vibrio cholerae intestinal colonization factor, serves as a receptor for CTXphi. To test whether CTXphi also depends upon tol gene products to infect V. cholerae, we identified and inactivated the V. cholerae tolQRAB orthologues. The predicted amino acid sequences of V. cholerae TolQ, TolR, TolA, and TolB showed significant similarity to the corresponding E. coli sequences. V. cholerae strains with insertion mutations in tolQ, tolR, or tolA were reduced in their efficiency of CTXphi uptake by 4 orders of magnitude, whereas a strain with an insertion mutation in tolB showed no reduction in CTXphi entry. We could detect CTXphi infection of TCP(-) V. cholerae, albeit at very low frequencies. However, strains with mutations in both tcpA and either tolQ, tolR, or tolA were completely resistant to CTXphi infection. Thus, CTXphi, like the E. coli filamentous phages, uses both a pilus and TolQRA to enter its host. This suggests that the pathway for filamentous phage entry into cells is conserved between host bacterial species.     

Identification and preliminary characterization of a mutant defective in the bacteriophage T4-induced unfolding of the Escherichia coli nucleoid.

The nucleoids of Escherichia coli S/6/5 cells are rapidly unfolded at about 3 min after infection with wild-type T4 bacteriophage or with nuclear disruption deficient, host DNA degradation-deficient multiple mutants of phage T4. Unfolding does not occur after infection with T4 phage ghosts. Experiments using chloramphenicol to inhibit protein synthesis indicate that the T4-induced unfolding of the E. coli chromosomes is dependent on the presence of one or more protein synthesized between 2 and 3 min after infection. A mutant of phage T4 has been isolated which fails to induce this early unfolding of the host nucleoids. This mutant has been termed "unfoldase deficient" (unf-) despite the fact that the function of the gene product defective in this strain is not yet known. Mapping experiments indicate that the unf- mutation is located near gene 63 between genes 31 and 63. The folded genomes of E. coli S/6/5 cells remain essentially intact (2,000-3,000S) at 5 min after infection with unfoldase-, nuclear disruption-, and host DNA degradation-deficient T4 phage. Nuclear disruption occurs normally after infection with unfoldase- and host DNA degradation-deficient but nuclear disruption-proficient (ndd+), T4 phage. The host chromosomes remain partially folded (1,200-1,800S) at 5 min after infection with the unfoldase single mutant unf39 x 5 or an unfoldase- and host DNA degradation-deficient, but nuclear disruption-proficient, T4 strain. The presence of the unfoldase mutation causes a slight delay in host DNA degradation in the presence of nuclear disruption but has no effect on the rate of host DNA degradation in the absence of nuclear disruption. Its presence in nuclear disruption- and host DNA degradation-deficient multiple mutants does not alter the shutoff to host DNA or protein synthesis.     

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F(1)F(o) ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.