Studies on Temperate Phages of Lactobacillus salivarius

Twenty-three temperate phages of Lactobacillus salivarius isolated from human feces were studied as to their morphological, biological, and serological properties. (1) Among 30 strains of L. salivarius tested, 23 strains were lysed by induction with mitomycin C (MC). In all these lysates, phage particles were detected by electron microscopic examination. (2) These phages were morphologically divided into three groups: particles with a regular hexagonal head and a long flexible tail; particles having a regular hexagonal head with or without a short tail-like structure; particles with an elongated head and a long noncontractile tail. (3) Only two, phage 223 having an elongated head and phage 227 with a regular hexagonal head and a long noncontractile tail, produced tiny and very turbid plaques on several host bacteria. Six phages could produce only inhibition zones, ranging from complete inhibition through partial inhibition to normal growth by a serial dilution spot test. (4) All these killer particles could also inhibit the growth of their producer cells. (5) A serological relationship was observed between temperate phages and killer particles, and this was somewhat consistent with the morphological groupings.     

2株可感染大肠埃希菌工程菌的噬菌体的鉴定与分析

大肠埃希菌来源的基因工程菌是应用最为频繁的工程菌,但在基因工程菌规模化制备生物活性制剂的过程中常常会被噬菌体感染。通过对鸡粪中噬菌体大量筛选及鉴定,对工程菌防御相应噬菌体感染机制开展基础研究。实验以大肠埃希菌工程菌为宿主菌（CICC编号：10424）,采用双层琼脂平板法从鸡场粪样中分离噬菌体,结果获得2株噬菌体,对其进行形态学鉴定。经透射电镜观察发现一株(CX)为短尾噬菌体,其头部外廓呈长六角形,非收缩性尾部,其噬菌斑清晰透亮,周围无晕环,裂解性较强;另一株(B1X)为长尾科噬菌体,其噬菌斑呈双层环状,中心澄清透明,直径约0.8~1.3 mm,外环呈半透明,云雾状区域,宽约0.8~1.3 mm。可进一步研究这2株噬菌体的侵染机制。     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The morphological properties of the twelve previously described HM-phages were examined by electron microscopy. Specimens were prepared by air-drying and shadow-casting method using purified phage suspensions. As a result, the HM-phages were classified into three morphologically distinct groups, 1, 11 and 111. Group 1 phages were HM 1, HM 2, HM 8, HM 9, HM 10, HM 11 and HM 12. These phages had a spherical head about 100 mμ in diameter and a rudimentary tail. Group 11 phages were HM 3, HM 4, HM 5 and HM 6. These phages had a spherical head about 100 mμ in diameter and a tail with contractile sheath, and the normal tail of these phages was about 100 mμ in length, and the contracted sheath was about 50 mμ in length, Group 111 phage was HM 7 alone. This phage had a spherical head about 120 mμ in diameter and a relatively long tail about 350 mμ in length.     

2-酮基-D-葡萄糖酸产生菌荧光假单胞菌K1005抗噬菌体菌株的选育

从２－酮基－Ｄ－葡萄糖酸产生菌荧光假单胞菌Ｋ１００５的异学发酵液中分离到两种噬菌体,分别将其命名为ＫＳ５０２和ＫＳ５０３。电镜观察表明ＫＳ５０２呈微球形,直径为６１ｎｍ;ＫＳ５０３呈蝌蚪形,具有直径为６８ｎｍ的六角形头部及８５ｎｍ长的尾部。利用紫外线诱变的方法,经多次分离筛选,获得了２株抗性稳定且产酸水平超过对照敏感菌的抗噬菌体菌株,可以应用于生产。     

Two Novel Bacteriophages of Thermophilic Bacteria Isolated from Deep-Sea Hydrothermal Fields

Bacteriophages of thermophiles are of great interest due to their important roles in many biogeochemical and ecological processes. However, no virion has been isolated from deep-sea thermophilic bacteria to date. In this investigation, two lytic bacteriophages (termed Bacillus virus W1 and Geobacillus virus E1) of thermophilic bacteria were purified from deep-sea hydrothermal fields in the Pacific for the first time. Bacillus virus W1 (BVW1) obtained from Bacillus sp. w13, had a long tail (300nm in length and 15 nm in width) and a hexagonal head (70 nm in diameter). Another virus, Geobacillus virus E1 (GVE1) from Geobacillus sp. E26323, was a typical Siphoviridae phage with a hexagonal head (130 nm in diameter) and a tail (180 nm in length and 30 nm in width). The two phages contained double-stranded genomic DNAs. The genomic DNA sizes of BVW1 and GVE1 were estimated to be about 18 and 41 kb, respectively. Based on SDS-PAGE of purified virions, six major proteins were revealed for each of the two phages. The findings in our study will be very helpful to realize the effect of virus on thermophiles as well as the communities in deep-sea hydrothermal fields.     

Morphology and General Characteristics of Bacteriophages Infectious to Robinia pseudoacacia Mesorhizobia

Four phages infectious to Mesorhizobium strains were identified in soil samples taken from local Robinia pseudoacacia stands. Based on their polyhedral heads and short noncontractile tails, three of the phages, Mlo30, Mam12, and Mam20, were assigned to group C of Bradley’s classification, the Podoviridae family, while phage Mlo1, with its elongated hexagonal head and a long flexible tail represented subgroup B2 bacteriophages, the Siphoviridae family. The phages were homogeneous in respect of their virulence, as they only lysed Mesorhizobium strains, but did not affect strains of Rhizobium or Bradyrhizobium. On the basis of one-step growth experiments, the average virus yield was calculated as approximately 10–25 phage particles for phages Mlo30, Mam12 and Mam20, and as many as 100–120 for phage Mlo1. The rate of phage adsorption to heat-treated cells showed differences in the nature of their receptors, which seemed to be thermal sensitive, thermal resistant, or a combination of the two. Only the receptor for phage Mlo30 was likely to be an LPS molecule, which was supported by a neutralization test. The smooth LPS with O-antigenic chains of the phage-sensitive M. loti strain completely reduced the bactericidal activity of virions at a concentration of 1 μg/ml. The molecular weights of phage DNAs estimated from restriction endonuclease cleavage patterns were in the range from ~39 kb for group C phages to ~80 kb for B2.     

Some Properties of Five New Salmonella Bacteriophages

Five bacteriophages were isolated from lysogenic strains of Salmonella potdam. On the basis of plaque morphology, thermostability, serology, host range, one-step growth parameters, and phage morphology, they were divided into three groups: group A, phages P4 and P9c; group B, phages P3 and P9a; and group C, phage P10. Group A phages had a hexagonal head 55 nm in diameter with a short tail 15 nm long. These phages were particularly characterized by high thermostability, lack of serological relationship with any of the other phages, and restriction of lysis to other Salmonella strains of Kauffmann-White group C(1). Group B phages had a head identical in size and shape to that of the A phages, but they possessed a tail 118 nm long with a contractile sheath. A unique feature was the occurrence of tail fibers at the end of the core rather than at the base of the sheath. These phages were considerably less thermostable, had extended host ranges, and were serologically distinct from each other but unrelated to the A phages. The group C phage, P10, had a head identical to that of the A and B phages. It had a tail 95 nm in length, with tail fibers attached to a base plate at the end of a contractile sheath. P10 was highly sensitive to heat, lysed only smooth strains of Salmonella, and showed a degree of serological relationship to both B phages. The relationship of these phage groups to previous Salmonella phage grouping schemes is discussed.     

A bacteriophage of a moderately halophilic bacterium

A bacteriophage of an aerobic, gram-negative, rod-shaped halophilic bacterium, provisionally named Pseudomonas sp. G3, is described. The phage has a head and a tail and is similar in appearance to Salmonella phage Beccles. It infects its bacterial host at all salt concentrations in which the bacteirum is able to grow. In contrast to phages of halophilic archaebacteria, the newly-described phage is relatively stable in the absence of salt. It also infects Vibrio costicola and two unidentified halophilic eubacteria.Abbreviations PPT proteose peptone-tryptone medium - pfu plaque-forming unit - G+C guanine + cytidine content, mol %     

Comparison of Vero-cytotoxin-encoding phages from Escherichia coli of human and bovine origin

Phages encoding production of Vero cytotoxins VT1 or VT2 were isolated from strains of Escherichia coli of human and bovine origin. Two human strains of serotype O157: H7 produced both VT1 and VT2 and each carried two separate phages encoding either VT1 or VT2. The phages were morphologically similar to each other and to a VT2 phage previously isolated from a strain of serotype O157: H-; all had regular hexagonal heads and short tails. The phages had similar genome sizes and DNA hybridization and restriction enzyme digestion showed that the DNAs were very closely related. This contrasts with another report that one of the strains tested (933) released two clearly distinguishable phages separately encoding VT1 and VT2. The O157 phages differed from a VT1 phage isolated from a bovine E. coli strain belonging to serotype O26: H11 and from the reference VT1 phage isolated previously from a human strain, H19, of serotype O26: H11. The two O26 phages were morphologically similar with elongated heads and long tails. They had similar genome sizes and DNA hybridization indicated a high level of homology between them. Hybridization of an O157 phage DNA probe to DNA of the O26 phages, and vice versa, showed there was some cross-hybridization between the two types of phage. A phage from a bovine strain of serotype O29: H34 had a regular hexagonal head and short tail resembling those of the O157 phages. The DNA was distinguishable from that of all the other phages tested in restriction digest patterns but hybridized significantly to that of an O157 phage. Hybridization of the phage genomes with VT1 and VT2 gene probes showed that sequences encoding these toxins were highly conserved in the different phages from strains belonging to the three serogroups.     

Relationship between phage resistance and emulsan production,interaction of phages with the cell-surface of Acinetobacter calcoaceticus RAG-1

The hydrocarbon-degrading strain Acinetobacter calcoaceticus RAG-1 produces an extracellular emulsifying agent capable of forming stable oil-in-water emulsions. The bioemulsifier, termed emulsan, is a polyanionic heteropolysaccharide (M.W. 10⁶) composed mainly of N-acyl D-galactosamine and an N-acyl hexosamine uronic acid. In order to probe the interaction of emulsan with the cell surface prior to its release into the growth medium, two new virulent bacteriophages for A. calcoaceticus RAG-1 were isolated from sewage and the properties of phage resistant mutants were studied. The two phages, ap-2 and ap-3, were differentiated on the basis of plaque morphology, electron microscopy and buoyant density. Isolated mutants of A. calcoaceticus RAG-1 which were resistant to one of the two phages retained sensitivity to the other phage. Resistance to phage ap-3 was accompanied by a severe drop in emulsan production. Independently isolated derivatives of A. calcoaceticus RAG-1 with a defect in emulsan production also turned out to be resistant towards phage ap-3. Antibodies prepared against purified emulsan specifically inhibited phage ap-3 adsorption to the cell surface of the parental strain.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The fine structure of phage HM 2 (group I) active on Clostridium saccharoperbutylacetonicum was studied by an electron microscopy with a negative-staining technique, and compared with those of more conventional types, phages HM 3 (group II) and HM 7 (group III), whose tails were clearly observed by a shadow-casting technique. This study revealed that phage HM 2 had an intricate tail which was not observed by a shadow-casting technique.

Phage HM 2 has an icosahedral head about 450 Å in diameter and a non-contractile tail about 300 Å long. The distal 130 Å of the tail axis has a width of 80 Å which is wider than the upper portion of the tail (50 to 60 Å). The distal enlargement is not seen in the hollow tail. Twelve fibrous-shaped appendages are attached symmetrically at the upper portion of tail axis and extend toward the distal base of the tail. Their length is a little shorter than 300 Å. They combine with divalent cations in the phage dilution medium, and also adsorb the host cell debris.

Phage HM 3 has an icosahedral head about 770 Å in diameter and a tail about 1000 Å long and 150 Å wide with contractile sheath. Phage HM 7 has an icosahedral head about 750 Å in diameter and a long non-contractile tail about 2000 Å long and about 120 Å wide with forked tip.

The structure of the tail of phage HM 2 is quite different from those of phages HM 3 and HM 7 hitherto described and those of the various phages of other bacteria.     

Phages of Pseudomonas aeruginosa: response to environmental factors and in vitro ability to inhibit bacterial growth and biofilm formation

Aims: To examine effects of various environmental factors on adsorption and inactivation of Pseudomonas aeruginosa‐specific phages: δ (family Podoviridae), J‐1, σ‐1 and 001A (family Siphoviridae) and their ability to inhibit bacterial growth and biofilm formation. Methods and Results: The phages examined in the study were clonally different, as revealed by RFLP. The temperature in the range 7–44°C had no influence on the adsorption of Podoviridae, but did affect Siphoviridae adsorption, particularly 001A. All phages were significantly stable at pH 5–9, and phages δ and 001A even at pH 3. Most of the examined carbohydrates and exopolysaccharides of the original host efficiently inactivated phage δ, while phages σ‐1 and J‐1 were inactivated considerably only by the amino acid alanine. Silver nitrate efficiently inactivated all the phages, while Siphoviridae were more resistant to povidone‐iodine. Serum of nonimmunized rats had no influence on phage inactivation and adsorption. Only phage δ showed ability to effectively inhibit in vitro bacterial growth and biofilm formation. Conclusions: The examined environmental parameters can significantly influence the adsorption and viability of Ps. aeruginosa‐specific phages. The phage δ is a good candidate for biocontrol of Ps. aeruginosa. Significance and Impact of the Study: The study provides important data on Ps. aeruginosa‐specific phage adsorption, inactivation and in vitro lytic efficacy.     

Ten new temperate bacteriophages of<Emphasis Type="Italic">Citrobacter youngae</Emphasis>

In a cross-test, we examined 55 strains of Citrobacter youngae against each other as potential producers of temperate bacteriophages and as potential sensitive indicators for them. Ten strains (18.2 %) showed the production of phages. Seven different strain-specific spectra of activity (from 1 to 11 strains each) were found. Phage production by 6 strains was inducible with mitomycin C, in 4 strains it was not inducible. The plaques of the phages were more or less turbid, without a lytic halo, tiny to small, 0.2-1.3 mm in diameter. Using a polyclonal, specific anti-lambda serum, all 10 phages were found to be clearly distinct from E. coli lambda phage, the phage 31/47 showing the highest neutralization titre of all. Interspecific tests with 15 strains of 8 species of Enterobacteriaceae revealed not a single case of activity of Citrobacter phages towards any of them. Five phage-immune clones lysogenized with 5 of the phages kept their remaining phage sensitivity spectra, though extended by sensitivity to 1-3 phages; 2 of these strains acquired also sensitivity to phage lambda. The phages belong to the morphotypes of Myoviridae (6 phages) and Siphoviridae (4 phages), with head diameters of 51-58 nm and tail length of 97-173 nm. Three strains produced corpuscular bacteriocins.     

Structure and function of a novel coliphage-associated sialidase

A coliphage named 63D, isolated previously, associated sialidase as a component of phage particles. In order to localize the enzyme in phage particles, phages were partially destroyed by sonication, and the disrupted particles were size fractionated using a sucrose density gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay and electron micrography of the fractions revealed the enzyme to be composed of four identical subunits with a molecular mass of 90 kDa, and the subunits were cross-linked by disulfide bonds. Electron micrographic observation indicated that six enzyme molecules were localized in a phage tail plate as a hexagonal array.     

Morphology and General Characteristics of Lytic Phages Infective on Strains of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Biological characteristics of three isolated phages (SR1, SR2, and SR3) lytic against three Bradyrhizobium japonicum strains were studied. These phages had no cross-infectivity among the host strains. Phage morphology indicates that they belonged to Siphoviridae (long noncontractile tail; SR1 and SR2) and Podoviridae (short tail; SR3) classes of bacteriophages. Lytic cycle of phages studied under identical conditions showed a distinct adsorption rate (67.3–99.1%), latent period (150–300 min), rise period (60–150 min), and burst size (110–200 pfu/cell). Stability in liquids and inactivation by osmotic shock, thermal, and ultraviolet irradiation were also distinct in this heterogeneous phage group. Influence of soil factors such as temperature, soil moisture, soil pH, and degree of phage adsorption to the soil on phage survival was determined. Major percent of free infective phages were obtained after desorption of phages from soil. Overall, temperature appeared to be the most important parameter affecting rhizobiophage survival in the soil.     

Morphological study of five bacteriophages of yellow-pigmented enterobacteria

Two bacteriophages isolated onEnterobacter cloacae (C2, C2F) and three isolated onErwinia herbicola (E3, E16P, E16B) were purified by D₂O gradient centrifugation. Phage-containing fractions were negatively stained and examined by electron microscopy. Phages C2, C2F, E3, and E16P showed an elongated head 153×51 nm and a short noncontractile tail 12 nm long terminated by at least two short fibers. These phages correspond to the rate taxonomic group C3. Big capsomeres composing the phage head were evidenced when phage suspensions in D₂O were stained. Phage E16B showed an elongated head 97×40.5 nm, and a contractile tail 89 nm long. This phage corresponds to the extremely rate group A3.     

Electron microscopy of virulent phages for Streptococcus lactis.

Electron microscopic studies were made on eight virulent Streptococcus lactis bacteriophages. These phages were taken as representative of eight host range groups established in a study of 75 phage isolates and 253 hosts (213 S. lactis, 22 S. cremoris, 18 S. diacetilactis). The phages studied were shown to have an isometric hexagonal head and noncontractile tails, usually several times longer than the head diameter. The virus heads were octahedral. The phages investigated represented three morphological types on the basis of head diameter , tail thickness, and tail length. These dimensions were approximately: for type I phages, 63, 172, and 11 nm, respectively; type II, 73, 200, and 20 nm, respectively; and type III, represented here by a single phage, 98, 551, and 12 nm, respectively. The tail surface revealed a different arrangment of the structural subunits which lent a helical appearance to the tails of type I and II phages and a guaffered tube appearance to the tail of type III phage. The number of turns along the tail axis, turn length, axial pitch, and helix angle were: type I, 32, 12 to 13 nm, 7.14 nm, and 11 degrees 43', respectively; type II, 24, 24, to 28 nm, 40.00 nm, and 32 degrees 30', respectively; and type III, 120, 12 nm, and no visible slope towards the axis. The morphology types showed complete correlation with serological groups, but not with groups based on host range pattern.     

Isolation and characterization of a virus (RL 1) infective on Rhizobium leguminosarum

A virus (RL 1) that infects Rhizobium leguminosarum was isolated and studied. The virus has phagelike morphology; it has a hexagonal head and a long, flexible, noncontractile tail with a baseplate. The edgeto-edge diameter of head is 760 Å. The tail is 1515 Å long and 115 Å wide. RL 1 is stable at 4° C in distilled water, with only 20% loss in the titer after one month storage. It does not require any ion for stability, and is stable between pH 6.0 and 8.0. The virus is composed of two components; one is thermal sensitive and the other is relatively thermal resistant. Adsorption and one step growth experiments under normal growth conditions showed a slow adsorption rate (0.82×10^-9 cm³ min^-1) followed by a 90 min latent period. The burst size was approximately 100 virus particles per cell.     

Characterization of a virulent Lactobacillus sake phage PWH2

A virulent bacteriophage PWH2 was isolated from fermented sausage. Out of 14 strains of Lactobacillus sake and 5 strains of L. curvatus tested as potential hosts, only L. sake Ls2 was sensitive. The plaques had a diameter of 0.5-1 mm. One-step growth kinetics of bacteriophage PWH2 revealed the following characteristics: a latent period of 1.5 h, a rise period of 1 h and a burst size of 110 (⁺ _– 10) phages per cell. From electron micrographs it was deduced that bacteriophage PWH2 has an icosahedral head, a long non-contractile tail and a complex base plate. The genome is linear and 35 kbp in length. The structural proteins consist of three major and two minor proteins. After an infection of L. sake Ls2, isolates were obtained that were resistant to PWH2. By treatment with mitomycin C, isolate R4a was found to be lysogenic. DNA/DNA hybridisation proved homology of phage PWH2 with the chromosomal DNA of strain R4a. Correspondence to: W. P. Hammes     

Structure of native and chloroform-methanol-treated mycobacteriophage R1.

The morphologies of native and chloroform-methanol-treated mycobacteriophage R1 were compared by electron microscopy, utilizing three negative stains. R1 was determined to be a complex phage. The head appears as an elongated cylinder with a pointed end (93 +/- 3 by 42 +/- 3 nm) constructed from an orderly arrangement of capsomeres. The phage tail measures 209 +/- 11 by 11 +/- 1 nm and possesses a striated surface with two base plates at its distal end. Treatment of R1 with chloroform-methanol resulted in disruption of both the head and tail structures and was accompanied by loss of infectivity. However, because no likely lipid-containing structure was observed in native phages, there is the possibility that the mechanism of chloroform-methanol inactivation is something other than lipid extraction.