Escherichia coli capsule bacteriophages. IV. Free capsule depolymerase 29.

The free host capsule depolymerase, induced by Escherichia coli capsule bacteriophage no. 29, and causing the formation of haloes around its plaques, has been purified to homogeneity. As judged from the following facts, this "enzyme" consists of free phage 29 spikes. (i) Detached phage organelles and depolymerase 29 particles exhibit the same molecular weight (about 245,000, as determined from the sedimentation equilibrium), contain polypeptide chains of the same two sizes (57,000 plus or minus 3,000 and 29,500 plus or minus 2,000, as determined by SDS-PAA gel electrophoresis), and have (within experimental error) the same sedimentation coefficient, isoelectric point, and amino acid composition. (ii) Isolated depolymerase and phage spikes in situ both catalyze the hydrolysis of glucosidic bonds in host capsular polysaccharide, leading ultimately to the formation of oligosaccharide fragments of one, two, and three hexasaccharide repeating units. (iii) Depolymerase 29 and phage 29 spikes have roughly the same electron optical dimensions. As tentatively estimated from the total and the virus-associated capsule depolymerase activity in the lysates, phage 29 infection seems to produce eight to seventeen times more free than incorporated spikes.     

Escherichia coli capsule bacteriophages. V. Lysozyme 29.

In addition to the spike-associated host capsule depolymerase, infection by Escherichia coli capsule bacteriophage no. 29 also induces the synthesis of a large bacteriolytic enzyme which has been purified to homogeneity. On incubation of isolated host murein sacculi with this enzyme, no amino groups but reducing sugar groups were liberated, and muraminitol, but no glucosaminitol, was found in the degraded sacculi after subsequent reduction with NaBH4. The bacteriolytic enzyme is thus another lysozyme (mucopeptide N-acetylmuramylhydrolase; EC 3.2.1.17). Electron optical visualization of negatively stained lysozyme specimens showed oblong particles of roughly 4.5 to 5.5 nm in diameter and 15 to 19 nm in length. Although the material tended to dissociate, a crude estimate of its molecular weight (270,000 plus or minus 30,000) could be obtained from these dimensions, from its sedimentation equilibrium, and from its behavior in gel chromatography. After disintegration of homogeneous lysozyme 29 by heating in solution with sodium dodecyl sulfate and dithiothreitol, polypeptides of one size only (about 46,000 dalton, probably six copies per molecule) were found in sodium dodecyl sulfate-polyacrylamide electrophoresis. The amino acid analysis of the enzyme accounted for more than 90% of its dry weight. One percent or less of the bacteriolytic activity in phage 29 lysates was found to be associated with the intact or disrupted virus particles, and a polypeptide of 46,000 daltons was not detected in the virions. These results strongly suggest that, in contrast to the host capsule depolymerase also induced by the same phage, and in spite of its comparatively large size, "lysozyme 29" does not constitute an integral part also of the homologous bacteriophage particles.     

Escherichia coli capsule bacteriophages. III. Fragments of bacteriophage 29.

A glycanase activity, catalyzing the depolymerization of host capsular polysaccharide, is associated with Escherichia coli capsule bacteriophage no. 29, a small virus with an isometric head, carrying a base plate with a set of spikes. The bacteriophage particles were disrupted by mild acid treatment (5 to 8 min at pH 3.5 and 37 C), and the enzymatically active fragments were isolated and subjected to sodium dodecyl sulfate-gel electrophoresis as well as to electron microscopy. Of the at least nine different polypeptide chains found in the complete virion, three (of 57,000 plus or minus 3,000, 29,500 plus or minus 2,000 and 13,500 plus or minus 1,000 daltons) were detected in detached base plates. They had the appearance of six-pointed stars of about 14 nm in outer diameter, with a central hole or prop, carrying six (or, possibly, a multiple thereof) spikes. Two sizes of polypeptide chains (57,000 and 29,500) were found in pure spikes, cylindrical particles of about 14.5 to 15 nm in length and 5 nm in diameter, and one (57,000) in -- still capsule depolymerizing -- spike subunits of roughly 5 nm in diameter. Phage 29 spike preparations, homogeneous in analytical ultracentrifugation and immunoelectrophoresis, were found to have a molecular weight of 245,000, as determined from the sedimentation equilibrium, and to contain equimolar amounts of the two polypeptides, probably three copies of each per organelle. The amino acid analysis of the isolated spikes revealed that aspartic acid, alanine, serine, and glycine are their dominant constituents; no amino sugars or other carbohydrates were detected in the preparations.     

Localization and functional role of the pseudomonas bacteriophage 2 depolymerase.

The adsorption apparatus of phage 2 consits of a symmetrical base plate of snowflake appearance, composed of six droplike spikes 7.0 to 7.5 nm in length with a maximum diameter of 4.5 to 5.0 nm. The spikes are attached by their narrow ends to a central ring 7.0 to 7.5 nm in diameter. Phage 2 deopolymerase, a phage 2-induced hydrolytic enzyme, was found to be a structural protein of phage 2 or in close association with the base plate. Pdp1, a phage 2 mutant, possesses a polypeptide that is antigenically similar to the depolymerase, but devoid of hydrolytic activity. This polypeptide was found to be located in the region of the base plate of pdp1. Treatment of intact cells of strain BI with purified phage 2 depolymerase inhibited the adsorption of phage 2. When phage receptor-containing fractions of slime glycolipoprotein and lipopolysaccharide were hydrolyzed by the depolymerase, amino sugars were released, and the phage-inactivating activities of these fractions were lost. The depolymerase was also observed to induce the lysis of strain BI cells in hypotenic medium. The phage 2 depolymerase appears to play a role in adsorption and release of phage.     

Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 μg/mouse) of depolymerase intraperitoneal injection protected 90%–100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.     

Endo-N-acetylneuraminidase associated with bacteriophage particles.

A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-). phi 1.2 is specific for the host capsular polysaccharide (colominic acid). The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues. Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate. Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated. No NeuNAc monomers, however, are formed. The capsules of E. coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme. Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D. E. Bradley, Bacteriol. Rev. 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes. By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes.     

Partial purification and characterization of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis.

An exopolysaccharide depolymerase was isolated from a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin. Purified preparations of the depolymerase showed maximum activity against the unacetylated polymannuronic acid exopolysaccharide from the same strain and little activity against commercially prepared alginic acid. The evidence suggests that the enzyme is either periplasmic in location or associated with the outer cell membrane and is released extracellularly, in the absence of cell lysis, after a reduction of the culture magnesium (Mg2+) concentration below 3.0 mM. The depolymerase is also released after the addition of sublethal concentrations of EDTA to cultures containing 3.0 mM Mg2+. A survey of additional mucoid P. aeruginosa isolates recovered from patients with cystic fibrosis showed that nearly 60% demonstrated similar depolymerase activity while none of the nonmucoid revertants of the parent strains produced detectable depolymerase activity.     

Purification and characterization of an extracellular poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Acidovorax sp. HB01

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading strain Acidovorax sp. HB01 was isolated from an activated sludge sample. A novel PHBV depolymerase with a molecular weight of 43.4 kDa was purified to homogeneity from the culture supernatant of the HB01 strain. The optimum pH and temperature of the PHBV depolymerase were 7.0 and 50 °C, respectively. The PHBV depolymerase can also degrade polyhydroxybutyrate, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), and poly(caprolactone); however, the PHBV degradation activity of the depolymerase is higher than its activity against the other polymers. Effect of metal ions and various inhibitors on the PHBV depolymerase activity was examined. The addition of Na(+), K(+), and Ca(2+) markedly increased the hydrolysis rate, whereas the enzyme activity was inhibited by Zn(2+), Mg(2+), Mn(2+), and particularly by Cu(2+) and Fe(2+). Ethylenediaminetetraacetic acid was found to have a significant inhibitory effect. The main degradation product of depolymerase was identified as the 3-hydroxybutyric acid monomer and 3-hydroxyvaleric acid monomers via mass spectrometry.     

Unmasking of surface components by removal of cell-associated emulsan from Acinetobacter Sp. RAG-1

Summary Acinetobacter calcoaceticusRAG-1 cells lacking the emulsan capsule on the cell surface were obtained by two methods; a) by selecting for mutants that lack emulsan with a specific phage and b) by removal of the emulsan capsule from wild type cells with a specific emulsan depolymerase. Emulsan deficient cells obtained by either method become deficient in the adsorption of phage ap3 and sensitive to a newly isolated bacteriophage, nø. When RAG-1 cells were first treated with emulsan depolymerase and subsequently incubated without the enzyme, regeneration of the cell-associated emulsan was correlated with an increase in phage ap3 adsorption and an inhibition in phage nø adsorption. By partial regeneration of cell surface emulsan, a physiological state was obtained in which RAG-1 cells were sensitive to and efficiently adsorbed found phages. Enzyme-treated RAG-1 cells were found to be more adherent to hexadecane than the untreated RAG-1 cells. The data indicate that in addition to its function as the ap3 receptor, cell-associated emulsan masks the expression of other cell-surface determinant(s) which function(s) as: (i) receptor for bacteriophage nø, and (ii) cell-surface sites which enhance adherence to hydrophobic surfaces.Present address: Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA     

Escherichia coli Capsule Bacteriophages II. Morphology

The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli, all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes.     

Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types

Klebsiella pneumoniae is an important human pathogen causing opportunistic nosocomial and community-acquired infections. A major public health concern regarding K. pneumoniae is the increasing incidence of multidrug-resistant strains. Here, we isolated three novel Klebsiella bacteriophages, KN1-1, KN3-1 and KN4-1, which infect KN1, KN3 and K56, and KN4 types respectively. We determined their genome sequences and conducted a comparative analysis that revealed a variable region containing capsule depolymerase-encoding genes. Recombinant depolymerase proteins were produced, and their enzymatic activity and specificity were evaluated. We identified four capsule depolymerases in these phages that could only digest the capsule types of their respective hosts. Our results demonstrate that the activities of these capsule depolymerases were correlated with the host range of each phage; thus, the capsule depolymerases are host specificity determinants. By generating a capsule mutant, we demonstrate that capsule was essential for phage adsorption and infection. Further, capsule depolymerases can enhance bacterial susceptibility to serum killing. The discovery of these phages and depolymerases lays the foundation for the typing of KN1, KN3, KN4 and K56 Klebsiella and could be useful alternative therapeutics for the treatment of K. pneumoniae infections.     

Partial purification and characterization of a polymannuronic acid depolymerase produced by a mucoid strain of Pseudomonas aeruginosa isolated from a patient with cystic fibrosis

An exopolysaccharide depolymerase was isolated from a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin. Purified preparations of the depolymerase showed maximum activity against the unacetylated polymannuronic acid exopolysaccharide from the same strain and little activity against commercially prepared alginic acid. The evidence suggests that the enzyme is either periplasmic in location or associated with the outer cell membrane and is released extracellularly, in the absence of cell lysis, after a reduction of the culture magnesium (Mg2+) concentration below 3.0 mM. The depolymerase is also released after the addition of sublethal concentrations of EDTA to cultures containing 3.0 mM Mg2+. A survey of additional mucoid P. aeruginosa isolates recovered from patients with cystic fibrosis showed that nearly 60% demonstrated similar depolymerase activity while none of the nonmucoid revertants of the parent strains produced detectable depolymerase activity.     

Different effects of sphingid and noctuid moths on the fecundity ofPlatanthera metabifolia (Orchidaceae) in Hokkaido

The fecundity ofPlatanthera metabifolia was investigated in relation to different types of behavior and morphology in both noctuid and sphingid pollinator moths. Low capsule set in the native habitats resulted from low activity of pollinating moths. From different patterns of capsule formation on spikes, pollinated spikes could be classified into sphingid-pollinated and noctuid-pollinated types. The contributions of sphingids to capsule set were nearly constant at the three study sites. High percentage capsule set at one site was linked with high noctuid activity. Different preferences of sphingids and noctuids as to spike features were detected: sphingids selected spikes with large numbers of flowers in early stages of flowering. Flowers with different spur length were equally pollinated by sphingids. On the other hand, noctuids selected spikes with short and medium-sized spurs in the late stage of flowering. The size and density of spikes scarcely affected the pollination activity of noctuids. Selection pressure upon spike size and spur length in relation to sphingid pollination was suggested. The possibility of local differentiation in spur length due to activity of noctuids was discussed.     

Enzymatic depolymerization of emulsan

Emulsan, the polyanionic emulsifying agent synthesized by Acinetobacter calcoaceticus RAG-1, was depolymerized by an enzyme obtained from a soil bacterium YUV-1. The extracellular emulsan depolymerase was produced when strains RAG-1 and YUV-1 were grown together on agar medium. The enzyme was extracted from the agar and concentrated by ultrafiltration and ammonium sulfate precipitation. The molecular weight of the enzyme was estimated to be 89,000. Emulsan depolymerase activity was due to an eliminase reaction which split glycosidic linkages within the heteropolysaccharide backbone of emulsan to generate reducing groups and alpha, beta-unsaturated uronides with an absorbance maximum of 233 nm. Deesterified emulsan was degraded by emulsan depolymerase at only 27% of the rate of the native polymer. The treatment of emulsan solutions with emulsan depolymerase for brief periods caused a rapid and parallel drop in viscosity and emulsifying activity. More than 75% of the viscosity and emulsifying activity was lost at a time when less than 0.5% of the glycosidic linkages were broken. These data indicate that (i) emulsan depolymerase is an endoglycosidase and (ii) the higher the molecular weight of emulsan, the greater its emulsifying activity. Exhaustive digestion of emulsan with emulsan depolymerase produced oligosaccharides with a number average molecular weight of about 3,000. The fractionation of the digest on Bio-Gel P-6 yielded four broad peaks. The pooled fractions from each of the peaks contained the same relative amounts of reducing sugar and had an absorbance at 233 nm. The molar ratio of esterified sugar to reducing groups was close to 2 in each fraction.     

The Tail Associated Protein of Acinetobacter baumannii Phage ΦAB6 Is the Host Specificity Determinant Possessing Exopolysaccharide Depolymerase Activity

Acinetobacter baumannii is a non-fermenting, gram-negative bacterium. In recent years, the frequency of A. baumannii infections has continued to increase, and multidrug-resistant strains are emerging in hospitalized patients. Therefore, as therapeutic options become limited, the potential of phages as natural antimicrobial agents to control infections is worth reconsidering. In our previous study, we isolated ten virulent double-stranded DNA A. baumannii phages, ϕAB1–9 and ϕAB11, and found that each has a narrow host range. Many reports indicate that receptor-binding protein of phage mediates host recognition; however, understanding of the specific interactions between A. baumannii and phages remains very limited. In this study, host determinants of A. baumannii phages were investigated. Sequence comparison of ϕAB6 and ϕAB1 revealed high degrees of conservation among their genes except the tail fiber protein (ORF41 in ϕAB1 and ORF40 in ϕAB6). Furthermore, we found that ORF40^ϕAB6 has polysaccharide depolymerase activity capable of hydrolyzing the A. baumannii exopolysaccharide and is a component of the phage tail apparatus determining host specificity. Thus, the lytic phages and their associated depolymerase not only have potential as alternative therapeutic agents for treating A. baumannii infections but also provide useful and highly specific tools for studying host strain exopolysaccharides and producing glycoconjugate vaccines.     

富养罗尔斯通氏菌菌株PHB"泄漏"机理的初探

建立了一种分离纯化聚羟基丁酸(Polyhydroxybutyrate,PHB)颗粒的改良方法。采用这种方法从Ralstonia eutropha菌株H16(野生型)、SK1489(Tn5诱变的PHB泄漏菌株)、JMP222(野生的PHB泄漏菌株)分离了PHB颗粒。进一步比较研究了不同菌株的PHB解聚酶和3-羟基丁酸脱氢酶的活性。研究结果表明,菌株SK1489的PHB解聚酶活性(48h培养后达1．82U／mg)明显高于野生型菌株H16(48h培养后达0．37U／mg),菌株JMP222的3-羟基丁酸脱氢酶活性(培养96h后达165．9U／mg)比菌株H16培养(96h后达64．0U／mg)高许多。这些结果显示,不同菌株PHB的泄漏有不同的原因,突变株SK1489导致PHB泄漏的原因是解聚酶活性高,而野生型JMP222PHB泄漏的原因主要是3-羟基丁酸脱氢酶活性高。     

Polycaprolactone depolymerase produced by the bacterium Alcaligenes faecalis

Several microorganisms were isolated as bacteria degrading polycaprolactone (PCL), and one of them, a strain B273 identified as Alcaligenes faecalis, was selected. Because this strain produced only slight PCL depolymerase activity, the hyper-producing mutant, TS22, was isolated after UV irradiation. Synthesis of PCL depolymerase was derepressed, probably based on the altered regulation of metabolic pathways in strain TS22. The partially purified enzyme hydrolyzed p-nitrophenyl fatty acids and triglycerides other than PCL, but not poly(3-hydroxybutyrate), indicating that PCL depolymerase may be a kind of lipase.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.