Origin of Polysaccharide Depolymerase Associated with Bacteriophage Infection

Analyses, by construction of phage growth curves, indicated that the polysaccharide depolymerase was synthesized by Pseudomonas aeruginosa strains B and BI after infection with phage 2. The kinetics of biosynthesis of the depolymerase were found to parallel closely the rate of formation of phage-directed virions, and alterations in the experimental conditions of infection were reflected by alterations in the production of enzyme. Infection with other Pseudomonas phages, 84 and 1197, did not result in the synthesis of depolymerase. The enzyme was not detectable in uninfected cultures, and no evidence was obtained for the existence of inhibitors or activators of enzyme activity in extracts of uninfected or infected cells. The results of experiments employing chloramphenicol or an auxotorphic mutant (BI arg(-)) suggested that protein synthesis de novo was essential for production of the enzyme. Various mutants of phage 2 (pdp(1), pdp(2)), which alter the synthesis of the polysaccharide depolymerase, have been isolated. These experimental results strongly support the role of the phage genome in the synthesis of this enzyme.     

Factors Influencing the Adsorption of Bacteriophage 2 to Cells of Pseudomonas aeruginosa

Phage 2 adsorbed to Pseudomonas aeruginosa strain BI in 5 mM Tris buffer, providing that cations like Na(+), Mg(2+), or Ca(2+) were present. Adsorption was observed over a broad pH range, reaching a maximum level around pH 7.5, which coincided with the pH required for maximal activity of the phage 2-associated slime polysaccharide depolymerase. Mutants of strain BI and other strains of P. aeruginosa possessing slime layers that were devoid of phage 2 depolymerase substrate were incapable of adsorbing phage 2. On the other hand, those strains containing substrate for the phage 2 depolymerase in the slime layer were capable of adsorbing phage 2. The same relationship of phage depolymerase-substrate interaction to phage adsorption was observed with Pseudomonas phage 8, which possesses a depolymerase that differs in its specificity from the phage 2 depolymerase. The receptor-like activity of purified slime containing the specific substrate for the phage-associated depolymerase was demonstrable by its ability to inactivate phage. However, receptor-like activity or phage inactivation was not observed with those slimes that were devoid of the depolymerase substrate.     

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.     

Characterisation of coliphage K30, a bacteriophage specific for Escherichia coli capsular serotype K30

Abstract Coliphage K30, a bacteriophage specific for strains bearing the Escherichia coli serotype K30 capsular polysaccharide, produced plaques surrounded by extensive haloes, a characteristic of phage which produce capsule depolymerase (glycanase) enzymes. Klebsiella K20, a strain producing a capsular polysaccharide chemically identical to that of E. coli K30, was not lysed by coliphage K30, although the bacteriophage encoded glycanase enzyme did degrade the K20 polysaccharide. Morphologically, coliphage K30 belonged to Bradley group C. The coliphage K30 particle comprised 20 structural polypeptides which varied from 9.5–136 kDa and genomic DNA of 38.7 ± 1.0 kb.     

An alginate lysate from Azotobacter vinelandii phage.

The alginate depolymerase associated with bacteriophage infection of Azotobacter vinelandii has been used in the analysis of sodium alginate. The enzyme degraded the polysaccharide to a series of oligouronides each containing a terminal 4-deoxy-alpha-L-erythro-hex-4-enopyranuronosyl residue. Analysis of these oligouronides, together with kinetic information, indicated that the enzyme was specific for mannuronic acid-containing regions of the polyuronide. The specificity of the enzyme made it possible to determine the primary structure of the macro-molecule. The phage-induced enzyme was shown to be distinct from the alginate lyase elaborated by the host organisms by its pH optimum, molecular weight, Michaelis constant and stability.     

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.     

Bacteriophage and associated polysaccharide depolymerases – novel tools for study of bacterial biofilms

Bacteriophage for three representative strains of Gram-negative biofilm bacteria have proved to be of widespread occurrence. Lytic bacteriophage have been isolated from local sewage for the bacterium 1·15, an exopolysaccharide (EPS)-producing pseudomonad found originally as a component of biofilms in a local river, and for two Enterobacter agglomerans strains from industrial biofilms. Representative examples of all three bacteriophage possess a relatively low burst size and on solid media, exhibit very large plaques surrounded by a wide halo (5–20 mm) indicative of polysaccharide depolymerase action. The bacteriophage are thus similar to other viruses for EPS-producing bacteria in inducing the synthesis of enzymes degrading the polymers which occlude the bacterial cell surface. In each preparation, the polysaccharase activity was associated both with sedimented phage particles and with the supernate of bacterial lysates. The enzymes have been partially purified and used to prepare polysaccharide digests in which the major products from each polysaccharide are the presumed repeat units of the polymers or oligomers of these. The soluble phage enzymes each degrade their substrate by acting as endo -glycanohydrolases. The phage and their associated enzymes thus provide very useful highly specific tools for studies of biofilms incorporating the bacterial host strains. Their potential applications in studies on bacterial biofilms are discussed.     

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. 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.     

The adsorption of Pseudomonas aeruginosa bacteriophage φKMV is dependent on expression regulation of type IV pili genes

Pseudomonas aeruginosa bacteriophage φKMV requires type IV pili for infection, as observed from the phenotypic characterization and phage adsorption assays on a phage infection-resistant host strain mutant. A cosmid clone library of the host ( P. aeruginosa PAO1) genomic DNA was generated and used to select for a clone that was able to restore φKMV infection in the resistant mutant. This complementing cosmid also re-established type IV pili-dependent twitching motility. The correlation between bacteriophage φKMV infectivity and type IV pili, along with its associated twitching motility, was confirmed by the resistance of a P. aeruginosa PAO1Δ pilA mutant to the phage. Subcloning of the complementing cosmid and further phage infection analysis and motility assays suggests that a common regulatory mechanism and/or interaction between the ponA and pilMNOPQ gene products are essential for bacteriophage φKMV infectivity.     

Distinct Slime Polysaccharide Depolymerases of Bacteriophage-Infected Pseudomonas aeruginosa: Evidence of Close Association with the Structured Bacteriophage Particle

Five new polysaccharide depolymerases were isolated from cultures of Pseudomonas aeruginosa infected with phages 6, 7, 8, 9, and 10. The production of enzyme paralleled the release of phage. Depolymerase associated with phage 8 was active on slime polysaccharide A, whereas depolymerases associated with phages 6, 7, 9, and 10, like pseudomonas phage 2, hydrolyzed slime polysaccharide B. None of the depolymerases was active on slime polysaccharide C. Despite exhaustive purification, depolymerase activity was found to band with the phage particles at a density of 1.49 to 1.51 g/ml in a density gradient composed to cesium chloride. These results suggest that the depolymerases are firmly bound to the phage particles.     

Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm of Klebsiella pneumoniae

Bacteria have evolved multiple mechanisms, such as biofilm formation, to thwart antibiotic action. Yet antibiotics remain the drug of choice against clinical infections. It has been documented that young biofilm of Klebsiella pneumoniae could be eradicated significantly by ciprofloxacin treatment alone. Since age of biofilm is a decisive factor in determining the outcome of antibiotic treatment, in the present study biofilm of K. pneumoniae, grown for extended periods was treated with ciprofloxacin and/or depolymerase producing lytic bacteriophage (KPO1K2). The reduction in bacterial numbers of older biofilm was greater after application of the two agents in combination as ciprofloxacin alone could not reduce bacterial biomass significantly in older biofilms (P > 0.05). Confocal microscopy suggested the induction of structural changes in the biofilm matrix and a decrease in micro-colony size after KPO1K2 treatment. The role of phage associated depolymerase was emphasized by the insignificant eradication of biofilm by a non-depolymerase producing bacteriophage that, however, eradicated the biofilm when applied concomitantly with purified depolymerase. These findings demonstrate that a lytic bacteriophage alone can eradicate older biofilms significantly and its action is primarily depolymerase mediated. However, application of phage and antibiotic in combination resulted in slightly increased biofilm eradication confirming the speculation that antibiotic efficacy can be augmented by bacteriophage.     

Isolation of a Bacteriophage Specific for a New Capsular Type of Klebsiella pneumoniae and Characterization of Its Polysaccharide Depolymerase

Background

Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed.

Methodology/Principal Findings

To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS⁻ mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis.

Conclusions/Significance

Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.     

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.     

Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora

Erwinia amylovora infected with bacteriophage ERA103 produced an enzyme which degraded the extracellular polysaccharide of noninfected cells. The depolymerase enzyme was purified 15-fold by a procedure which included ammonium sulfate precipitation, ultracentrifugation, CM-Sephadex batchwise separation, Sephadex G-50 column chromatography, and Sephacryl S-200 column chromatography. The enzyme had a molecular weight of approximately 21,000 and a pH optimum of 6.0. Activity was enhanced by supplements of 2-mercaptoethanol or dithiothreitol.     

Bacteriophage ecology in Escherichia coli and Pseudomonas aeruginosa mixed-biofilm communities

Phage therapy is being reexamined as a strategy for bacterial control in medical and other environments. As microorganisms often live in mixed populations, we examined the effect of Escherichia coli bacteriophage λW60 and Pseudomonas aeruginosa bacteriophage PB-1 infection on the viability of monoculture and mixed-species biofilm and planktonic cultures. In mixed-species biofilm communities, E. coli and P. aeruginosa maintained stable cell populations in the presence of one or both phages. In contrast, E. coli planktonic populations were severely depleted in coculture in the presence of λW60. Both E. coli and P. aeruginosa developed phage resistance in planktonic culture; however, reduced resistance was observed in biofilm communities. Increased phage titers and reduced resistance in biofilms suggest that phage can replicate on susceptible cells in biofilms. Infectious phage could be released from mixed-culture biofilms upon treatment with Tween 20 but not upon treatment with chloroform. Tween 20 and chloroform treatments had no effect on phage associated with planktonic cells, suggesting that planktonic phage were not cell or matrix associated. Transmission electron microscopy showed bacteriophage particles to be enmeshed in the extracellular polymeric substance component of biofilms and that this substance could be removed by Tween 20 treatment. Overall, this study demonstrates how mixed-culture biofilms can maintain a reservoir of viable phage and bacterial populations in the environment.     

快速从医院废水中大规模分离铜绿假单胞杆菌噬菌体

目的:从医院废水中快速分离多株不同的铜绿假单胞杆菌噬菌体,研究其生物学特性,为建立铜绿假单胞杆菌噬菌体库做准备。方法:利用噬菌斑法从未经处理的医院污水中分离和鉴定铜绿假单胞杆菌噬菌体,根据感染谱的不同确定它们为不同的铜绿假单胞杆菌噬菌体;重点研究其中一株宿主谱较广的噬菌体的生物学特性,采用负染法电镜观察噬菌体的形态和大小,提取该噬菌体的基因组并进行酶切电泳分析,测定噬菌体感染复数并观察其一步生长曲线。结果:通过噬菌斑法分离出90株铜绿假单胞杆菌噬菌体。电镜观察显示,噬菌体Pa27P1头部呈立体对称,有一长尾;酶切结果显示,噬菌体Pa27P1的基因组为双链DNA;生长曲线表明噬菌体Pa27P1感染宿主菌的潜伏期为25 min,爆发时间为25 min,裂解量为514。结论:90株铜绿假单胞杆菌噬菌体中有5株具有较广的噬菌谱,其组合能裂解所有18株铜绿假单胞杆菌,为深入研究铜绿假单胞杆菌噬菌体的生物学特性及其功能提供了依据。     

Purification and properties of the myo-inositol-binding protein from a Pseudomonas sp.

Emulsan, the extracellular polyanionic emulsifying agent produced by Acinetobacter calcoaceticus RAG-1, has been implicated as a receptor for a specific virulent RAG-1 bacteriophage, ap3. Aqueous solutions of emulsan did not interfere with phage ap3 adsorption to RAG-1 cells. However, binding of phage ap3 occurred at the interfaces of hexadecane-in-water emulsions specifically stabilized by emulsan polymers. Binding of ap3 to emulsions was inhibited either in the presence of anti-emulsan antibodies or in the presence of a specific emulsan depolymerase. Moreover, when the phage was first bound to emulsan-stabilized emulsions and the emulsions subsequently treated with emulsan depolymerase, viable phage was released, indicating that phage ap3 DNA ejection was not triggered by binding. The results indicate that emulsan functions as the ap3 receptor and suggest that to function as a receptor, emulsan assumes a specific conformation conferred on it by its specific interaction with hydrophobic surfaces.     

Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.     

Evaluation of a rapid microbial detection method via phage lytic amplification assay coupled with Live/Dead fluorochromic stains

AIMS: To develop a method for rapid detection of bacteria via bacteriophage amplification coupled with exogenous fluorochromic stains. METHODS AND RESULTS: A method for the rapid detection of bacteria was developed which consisted of exposing the sample suspected to contain target cells to host-specific phage. After at least one infection cycle, bacteria known to be infected by the phage (helper cells) were added and the number of nascent phage particles was estimated using the Live/Dead BacLight Bacterial Viability kit. Using Pseudomonas aeruginosa, it was shown that the dead helper cell population following phage infection was proportional to the initial number of target cells present in the original sample. Approximately 1 x 10(1) CFU per ml of P. aeruginosa could be detected within 4 h without the need for enrichment. CONCLUSIONS: The phage lytic amplification assay coupled with exogenous fluorochromic stains was able to detect approx. 1 x 10(1) CFU per ml of the target bacterium within 4 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A method to detect low number of bacterial cells in a sample within 4 h without the need for enrichment was developed.