Bacterial ice nucleation activity after T4 bacteriophage infection.

The changes in ice nucleation activity of transformed Ina+ Escherichia coli K12 after infection with T4D bacteriophage have been examined. Within 2 min after infection class A nucleation activity (measured at -4 degrees C) fell about 100-1000-fold whilst class B (measured at -5.5 degrees C) and class C (measured at -9 degrees C) nucleation activities increased 50-100-fold and then rapidly decreased. These changes also occurred after interaction with T4D ghost particles or T4D 11-/12- particles. Since ghost particles lack DNA and 11-/12- particles lack short tail fibres, the T4D particles appear to be exerting their effect by the attachment of the phage long tail fibres to the cell. The changes were not influenced by the addition of chloramphenicol.

Components of ice nucleation structures of bacteria.

Nonprotein components attached to the known protein product of the inaZ gene of Pseudomonas syringae have been identified and shown to be necessary for the most efficient ice nucleation of supercooled H2O. Previous studies have shown that cultures of Ina+ bacteria have cells with three major classes of ice-nucleating structures with readily differentiated activities. Further, some cells in the culture have nucleating activities intermediate between those of the different classes and presumably have structures that are biosynthetic intermediates between those of the different classes. Since these structures cannot be readily isolated and analyzed, their components have been identified by the use of specific enzymes or chemical probes, by direct incorporation of labeled precursors, and by stimulation of the formation of specific classes of freezing structures by selective additions to the growth medium. From these preliminary studies it appears that the most active ice nucleation structure (class A) contains the ice nucleation protein linked to phosphatidylinositol and mannose, probably as a complex mannan, and possibly glucosamine. These nonprotein components are characteristic of those used to anchor external proteins to cell membranes of eucaryotic cells and suggest that a similar but not identical anchoring mechanism is required for efficient ice nucleation structure. The class B structure has been found to contain protein presumably linked to the mannan and glucosamine moieties but definitely not to the phosphatidylinositol. The class C structure, which has the poorest ice nucleation activity, appears to be the ice nucleation protein linked to a few mannose residues and to be partially imbedded in the outer cell membrane.

Formation of bacterial membrane ice-nucleating lipoglycoprotein complexes.

The preliminary finding that nonprotein additions to the protein product of the ice-nucleating gene of Pseudomonas syringae or Erwinia herbicola are essential for ice nucleation at the warmest temperatures has led to experiments aimed at identifying possible linkages between the ice protein and the other components. It appears that the protein is coupled to various sugars through N- and O-glycan linkages. Mannose residues are apparently bound via an N-glycan bond to the amide nitrogen of one or more of the three essential asparagine residues in the unique amino-terminal portion of the protein. In turn, these mannose residues are involved in the subsequent attachment of phosphatidylinositol to the nucleation structure. This phosphatidylinositol-mannose-protein structure is the critical element in the class A nucleating structure. In addition to sugars attached to the asparagine residues, additional sugar residues appear to be attached by O-glycan linkages to serine and threonine residues in the primary repeating octapeptide, which makes up 70% of the total ice protein. These additional sugar residues include galactose and glucosamine and most likely additional mannose residues. These conclusions were based on (i) the changes in ice-nucleating activity due to the action of N- and O-glycanases, alpha- and beta-mannosidoses, and beta-galactosidase; (ii) immunoblot analyses of ice proteins in cell extracts after enzyme treatments; and (iii) the properties of transformed Ice+ Escherichia coli cells containing plasmids with defined amino-terminal and carboxyl-terminal deletions in the ice gene. Finally, evidence is presented that these sugar residues may play a role in aggregating the ice gene lipoglycoprotein compound into larger aggregates, which are the most effective ice nucleation structures.

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.

Three separate classes of bacterial ice nucleation structures.

Studies of the properties of the ice nucleation structure exposed on the surfaces of various bacteria such as Pseudomonas syringae, Erwinia herbicola, or various strains of Ice+ recombinant Escherichia coli have shown that there are clearly three major related but chemically distinct types of structures on these cells. First, the ability of Ice+ cells to nucleate super-cooled D2O has been examined, and it has been found that this ability (relative to the ability of the same cells to nucleate super-cooled H2O) exhibited three characteristic nucleating patterns. The rarest structure, called class A, is found on only a small fraction of cells in a culture, nucleates H2O at temperatures above -4.4 degrees C, and is an effective nucleator of super-cooled D2O. A second class of structure, called class B, is found on a larger portion of the cells, nucleates H2O between -4.8 and -5.7 degrees C, and is a relatively poor nucleator of super-cooled D2O. The class C structure is found on almost all cells and nucleates at -7.6 degrees C or colder. These three classes of structures were also differentiated by their sensitivities to low concentrations of water-miscible organic solvents such as dioxane or dimethyl sulfoxide. Depending on the specific bacterial strain, the addition of these solvents to bacterial suspensions lowered the nucleation activity of the class A structure by 1,000-fold or more. The nucleation activities of class B structures in the same culture were highly resistant to these compounds and were lowered only by 20 to 40%. The class C structures were more sensitive than Class B structures were, and the nucleation activities decreased 70 to 90%. Finally, the pH sensitivity of these three classes of structures was examined. The class A structure was destroyed in buffers at pH 4.5 lower but was stable in buffers at higher pHs. The class B structure was less sensitive to acidic buffers but was destroyed at pH 5.5 or lower and was stable at higher pHs. However, the class C structure was unaffected by incubation in buffers with pHs of 3.5 to 9.0. Suggestions for the actual nucleation structures of the three classes are proposed.

Evidence for a net-like organization of lipopolysaccharide particles in the Escherichia coli outer membrane.

Cell wall LPS of Escherichia coli are organized as particles which are visible in the electron microscope, after treatment of the wall with alkali. We now describe alkali treated walls of three E. coli strains with differences in susceptibility to the T4 phage infection. Strain CR63, a usual host for the T4 phage, shows the LPS particles on the murein layer. These particles are absent in alkali treated cell walls of the strain W. Walls of this strain are broken during T4 infection and phages can be seen bearing pieces of membrane attached to their long as well as their short tail fibers. Strain AS19 which is hypersensitive to the lysis from without caused by T4 shows murein layers with no LPS particles on their surface, and networks of LPS particles with bacterial shape. This suggested that LPS are organized in a network of particles which may serve as the skeleton of the cell wall.

Iliac arterial-enteric fistulas occurring after pelvic irradiation.

Fistulas from the iliac artery to the bowel constitute a condition that is often lethal. Excluding fistulas related to vascular grafts, a review of previously reported cases shows that they are most often due to atherosclerotic iliac aneurysms. Three unusual cases of this condition that occurred after high-dose pelvic irradiation for treatment of cancer are presented; in no case was recurrent tumor evident. These cases suggest that high-dose pelvic irradiation can predispose to the formation of iliac arterial-enteric fistulas, particularly if sepsis or inflammation develops. The definitive surgical management of these fistulas entails bowel resection, arterial ligation, and extra-anatomic bypass.

Identification of T4 gene 25 product, a component of the tail baseplate, as a 15K lysozyme.

The proteins synthesized in Escherichia coli B cells after infection with various T4 bacteriophage tail baseplate mutants were analysed by the immunoblotting method for the presence of the 15 Kilodalton lysozyme found in phage T4 particles. Using three different antisera: anti-phage, anti-baseplate and anti-15K lysozyme, it has been found that the 15K lysozyme is not present in lysates of bacteria infected with T4 gene 25 amber mutants. The 15K lysozyme was also found to be expressed in E. coli B cells transformed with a plasmid containing only a small portion of the T4 genome but which included T4 gene 25. These observations indicate that the 15K lysozyme is the gene 25 product.

A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose.

An improved procedure for the electrophoretic transfer of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose is described. The use of more alkaline transfer buffers and the omission of an equilibration step before the transfer allow for the almost complete transfer of strongly basic proteins from gels to nitrocellulose without lowering the transfer efficiency for other proteins.

Timing of carotid artery endarterectomy after stroke.

Carotid endarterectomy has been advocated to prevent further neurologic deterioration in patients who have had a stroke. Previous reports have shown that endarterectomy within 2 weeks of a stroke is associated with high morbidity and mortality rates presumably from hemorrhagic complications in the brain. Some recommend a 2- to 6-week waiting period after a stroke, but the safety of operation in the interval of time beyond 2 weeks has not been documented in the literature. The present study investigated the morbidity and mortality rates of 352 consecutive carotid endarterectomies. Three hundred three endarterectomies were performed on patients with symptoms other than stroke. Forty-nine endarterectomies were performed on patients with a deficit lasting more than 24 hours. Of these, 27 carotid endarterectomies were performed in an interval less than 5 weeks after initial stroke (early interval) and 22 operations were performed in a 5- to 20-week interval after stroke (late interval). Five strokes occurred in the 27 patients operated on within 5 weeks, an incidence of 18.5%; none of the patients operated on after 5 weeks exhibited worsening of their preoperative neurologic status. With the use of Fisher's exact test to compare these two intervals, the results were found to be significant (p less than 0.05). The cause of stroke in those operated on in the early interval was investigated by postoperative CT scans; in only one instance was there a hemorrhagic infarct of the ipsilateral hemisphere. The literature suggests that a variety of intracerebral vascular changes render the brain more susceptible to reinfarction soon after stroke. This study suggests an unstable situation in the 5-week interval following stroke that contraindicates carotid endarterectomy.

Identification of bacteriophage T4D gene products 26 and 51 as baseplate hub structural components.

Products of two bacteriophage T4D genes, 26 and 51, both known to be essential for the formation of the central hub of the phage tail baseplate, have been partially characterized chemically, and their biological role has been examined. The gene 26 product was found to be a protein with a molecular size of 41,000 daltons and the gene 51 product a protein of 16,500 daltons. The earlier proposal (L. M. Kozloff and J. Zorzopulos, J. Virol. 40:635-644), from observations of a 40,000-dalton protein in labeled hubs, that the gene 26 product is a structural component of the baseplate, has been confirmed. The gene 51 product, not yet detected in phage particles, appears from indirect evidence also to be a structural component of the baseplate hub. These current conclusions about the gene 26 and 51 products are based on properties of T4 mutant particles containing altered gene 26 or 51 products and include (i) changes in heat lability, (ii) changes in adsorption rates, and (iii) changes in plating efficiencies on different hosts, and with the results of previous isotope incorporation experiments indicate that T4 particles contain three copies of the gene 26 product and possibly one or at most two copies of the gene 51 product. Properties of these mutant particles indicate that the gene 26 product, together with the other hub components such as the gene 28 product, plays a critical role in phage DNA injection into the host cell, whereas the 51 product seems essential in initiating baseplate hub assembly.

Phosphatidylinositol as a Component of the Ice Nucleating Site of Pseudomonas syringae and Erwinia herbiola.

Phosphatidylinositol has been identified as a major component of the ice nucleating site on the outer surface of two bacteria, Pseudomonas syringae and Erwinia herbicola. Plant lectins binding to inositol and a highly purified phosphatidylinositol-specific hydrolase (a C(II) lipase) inhibited or decreased the efficiency of the ice nucleating activity (INA) of both bacteria. Extracts of these two INA(+) bacteria had phosphatidylinositol synthase activity while extracts from related INA(-) Pseudomonas or Erwinia strains had no detectable synthase activity. An Escherichia coli strain acquired phosphatidylinositol synthase activity when transformed to the INA(+) phenotype with recombinant plasmids containing fragments of P. syringae DNA.

Priority of revascularization in patients with graft enteric fistulas, infected arteries, or infected arterial prostheses.

Patients with arterial infections, infected arterial prostheses, or graft enteric erosions or fistulas have high amputation and mortality rates after treatment. An unresolved therapeutic question is whether remote ("extra-anatomic") bypass should precede or follow removal of the infected artery or prosthesis. None of the ten patients reported here who had a remote bypass inserted first developed distal limb ischemia or infection of the remote bypass. Literature review of patients with aortic prosthetic infections revealed a mortality of 71% (10/14) if infected graft removal preceded remote bypass and 26% (6/23) if remote bypass was first. Patients with graft enteric erosions or fistulas had a mortality of 53% (40/75) if graft removal was first and 17% (5/29) if remote bypass was first. Subsequent infection of the remote bypass was rare. Therefore, when possible, remote bypass with a prosthetic graft should precede removal of an infected artery, an infected arterial prosthesis, a graft enteric erosion, or a graft enteric fistula.

An electrophoretic method for the identification of poly-gamma-glutamyl chain lengths in folates and related compounds.

An electrophoretic method for the identification and separation of folyl polyglutamates of different chain lengths and of the corresponding p-aminobenzoyl polyglutamate compounds has been developed. These compounds have been separated using electrophoresis in a 40% polyacrylamide gel using a higher voltage and other modifications of the standard polyacrylamide gel electrophoresis procedures used to separate larger polypeptides. Good separation has been obtained on folates containing up to 12 glutamyl residues. Further, this method has been used to investigate the nature of the products formed by the gamma-glutamyl carboxypeptidases from hog kidney and bovine liver.

Identification of bacteriophage T4D gene 29 product, a baseplate hub component, as a folylpolyglutamate synthetase.

An assay for folylpolyglutamate synthetase activity in extracts of uninfected and bacteriophage T4D-infected Escherichia coli B has been developed. T4D infection induced the formation of a new synthetase raising the total synthetase activity three-fold. Extracts obtained after infection with T4 gene 51, 27 or 28 amber mutants showed increased synthetase activities while extracts obtained from cells infected with a T4D gene 29 amber mutant did not show any increase in synthetase activity. The phage-induced synthetase was found to copurify with the gene 29 product and a 100-fold purified synthetase of molecular size of 74,000 daltons has been obtained. The purified synthetase has a folate substrate specificity different from the host synthetase since it added glutamate residues to dihydrofolate as well as to the usual tetrahydrofolate substrate.

Distribution of the T4D bacteriophage binding sites on the Escherichia coli B surface.

A study of the distribution of the T4D bacteriophage binding sites on the Escherichia coli B bacterial surface has shown that: (1) the number of binding sites per unit surface area is larger during growth period than during the division period, (2) the density of the binding sites on one-half of the bacterial cell is larger than the density of binding sites on the other half; (3) in newly-divided bacteria, the maximal binding site density is situated at one pole; (4) as bacteria grow, this maximum shifts to the middle of the cell; (5) when the septum is established, the middle of the cell becomes very poor in phage binding sites activity, and (6) phage adsorbs in clusters or in groups following curved lines around the bacterial cell.

Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola.

Chemical and biological properties of the ice nucleating sites of Pseudomonas syringae, strain C-9, and Erwinia herbicola have been characterized. The ice nucleating activity (INA) for both bacteria was unchanged in buffers ranging from pH 5.0 to 9.2, suggesting that there were no essential groups for which a change in charge in this range was critical. The INA of both bacteria was also unaffected by the addition of metal chelating compounds. Borate compounds and certain lectins markedly inhibited the INA of both types of bacterial cells. Butyl borate was not an inhibitor, but borate, phenyl borate, and m-nitrophenyl borate were, in order, increasingly potent inhibitors. These compounds have a similar order of affinity for cis hydroxyls, particularly for those found on sugars. Lentil lectin and fava bean lectin, which have binding sites for mannose or glucose, inhibited the INA of both bacteria. All other lectins examined had no effect. The inhibition of INA by these two types of reagents indicate that sugar-like groups are at or near the ice nucleating site. Sulfhydryl reagents were potent inhibitors of the INA of both bacteria. When treated with N-ethylmaleimide, p-hydroxymercuribenzoate, or iodoacetamide, the INA was irreversibly inhibited by 99%. The kinetics of inactivation with N-ethylmaleimide suggested that E. herbicola cells have at least two separate ice nucleating sites, whereas P. syringae cells have possibly four or more separate sites. The effect of infection with a virulent phage (Erh 1) on the INA of E. herbicola was examined. After multiple infection of a bacterial culture the INA was unchanged until 40 to 45 min, which was midway through the 95-min latent period. At that time, the INA activity began falling and 99% of the INA was lost by 55 min after infection, well before any cells had lysed. This decrease in INA before lysis is attributed to phage-induced changes in the cell wall.

A structural role for dihydropteroyl hexaglutamate in the tail baseplate of various bacteriophages.

A novel non-metabolic role is proposed for dihydropteroyl hexaglutamate as a critical link binding together sub-structures of the tail of Escherichia coli bacteriophage T4. Six molecules of this folate compound have been found to be components of the complex tail baseplate of the phage particle. The baseplate is assembled using a total of at least 18 viral gene products in a series of reactions in which six wedge-like elements (each 0.7 X 10(6) daltons) bind symmetrically around a central tail plug (1.55 X 10(6) daltons) to form a flat hexagonal structure. It appears likely that the pteridine portion of the folate binds to a site on a viral-induced dihydrofolate reductase molecule, a wedge component, while the glutamate residues of the folate bind to a viral-induced thymidylate synthase molecule, a central plug component. Additionally, it appears that the folyl glutamate residues play a role in forming a flexible bond between the proximal end of the phage long tail fiber and the baseplate. Two bacteriophages attacking a quite different bacterial host, Pseudomonas syringae, have been isolated and partially characterized. Both phage strains have tail structures morphologically analogous to T4. Both were irreversibly inactivated by an enzyme which cleaves the gamma-glutamyl bonds of folyl polyglutamate. It appears that these Pseudomonas phage particles also contain a folyl poly-glutamate whose integrity is essential for their infectivity.

Structural characteristics of the short-tail fibers of T4 bacteriophage.

The characteristics of pure preparations of short-tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43-nm fibers were the most abundant structure. During the process of purification of the short-tail fibers, the formation of aggregates was observed each time the material containing the short-tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short-tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short-tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well-defined bundles. The amino acid composition of the highly purified short-tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short-tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short-tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.