Bacteriophages of freshwater Brevundimonas vesicularis isolates

Research in Microbiology 157 (2006) 213–219 www.elsevier.com/locate/resmic

Bacteriophages of freshwater Brevundimonas vesicularis isolates Frauke Beilstein, Brigitte Dreiseikelmann ∗ Department of Microbiology/Genetechnology, Faculty of Biology, University of Bielefeld, 33615 Bielefeld, Germany Received 6 May 2005; accepted 21 July 2005 Available online 7 September 2005

Abstract Nine strains of Brevundimonas vesicularis were isolated from surface water of three ponds in Bielefeld, Germany. With those strains as indicators seven bacteriophages with different host ranges were isolated. Molecular characterization showed that all phages contained linear double-stranded DNA with a similar genome size of about 37 kb. Restriction analysis and hybridization of phage DNAs revealed that three of these phages are closely related to each other. These phages had morphologies typical of the family Siphoviridae. Their genomes contained cohesive ends. Four phages were classified into the family of Podoviridae. Restriction analysis of the DNAs of these phages did not reveal any similarities. The DNA of these phages were terminally redundant. All phages were unable to transduce plasmids or marker genes.  2005 Elsevier SAS. All rights reserved. Keywords: Brevundimonas vesicularis; Bacteriophage; Freshwater isolates

1. Introduction Bacteria of the genus Brevundimonas which represent a separate line in the α-subclass of Proteobacteria, are ubiquitous in the environment. They have been isolated, for example, from freshwater of Chilean salmon farms [17] from an inner Mongolian lake [16], from soil even in the Antarctic [22], and from the Russian space laboratory Mir [14]. Although Brevundimonas strains were isolated from the gastric mucosa of dogs [5] and from seriously infected humans [12] it is not yet clear whether they are true pathogens [7]. In the last two years, several new Brevundimonas strains have been detected like B. mediterranea [10] or B. nasdae [14], but still little is known about this genus. Currently the genus Brevundimonas includes 10 species (http://www.bacterio.cict.fr/b/brevundimonas.html). In this paper, we present nine freshwater isolates of Brevundimonas vesicularis and seven bacteriophages for this species. To our knowledge, this is the first description of B. vesicularis specific phages. Phages, especially transducing phages, may be useful for further biomolecular research on Brevundimonas vesicularis. They could be used, e.g., for phage typing or for the isolation of interesting mutants. Until now * Corresponding author.

E-mail address: [email protected] (B. Dreiseikelmann). 0923-2508/$ – see front matter  2005 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2005.07.005

α-Proteobacteria are not well characterized because there are no specific tools for genetic manipulations available. Phage DNAs or parts of them like promoters, operators and repressor genes may be worthy for the development of vectors for Brevundimonas species or perhaps even for other α-Proteobacteria. 2. Materials and methods 2.1. Isolation of bacterial strains and bacteriophages Freshwater was collected in October 2003 from the surface of three ponds in Bielefeld (Oetkerparkteich, Schloßhofteich and Meierteich), Germany. Samples of suitable dilutions were plated on TBY agar plates containing (per liter) 5 g tryptone, 2.5 g yeast extract, 0.5 g NaCl and 15 g agar and incubated at 26 ◦ C for 48 h. Strains were purified by three consecutive single colony passages. The isolates were numbered and given a prefix capital letter according to the name of the pond, from which they were taken. From a bacterial community of 850 members, 9 orange red isolates were chosen for further investigations (Table 1). They were used as hosts for the enrichment of bacteriophages from the same freshwater sample or samples of nearby freshwater environments (Table 2). For phage isolation, 100 µl overnight culture of the indicator strains were mixed with 0.5 ml 4 times concentrated TBY and 2 ml

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Table 1 Brevundimonas strains used in this study Strain

Source and/or reference

S121 M164 O212 O214 O217 O272 O283 O284 O287 Brevundimonas diminuta Brevundimonas vesicularis LMG 2350T

Freshwater Schloßhofteich Freshwater Meierteich Freshwater Oetkerparkteich Freshwater Oetkerparkteich Freshwater Oetkerparkteich Freshwater Oetkerparkteich Freshwater Oetkerparkteich Freshwater Oetkerparkteich Freshwater Oetkerparkteich Kindly provided by S. Fetzner (Münster, Germany), Originally isolated from Leech (Hirudo sp.), urinary bladder, epithelium, [6], Acc. no. AJ227780, kindly provided by W.-R. Abraham (Braunschweig, Germany)

Table 2 Characteristics of newly isolated phages Phage

Isolation site

Host isolate

Plaque size (mm Ø)

Family

DNA ends

φO272 φS121 φM164 φO283 φO283.6 φO283.15 φO283.12

Pond Oetkerpark, Bielefeld Pond Oetkerpark, Bielefeld Pond Oetkerpark, Bielefeld Pond Oetkerpark, Bielefeld Brook, Bielefeld River Weser, Minden Sewage

O272 S121 M164 O283 O283 O283 O283

0.8–1 0.8–1 0.8–1 4–5 4–5 4–5 4–5

Siphoviridae Siphoviridae Siphoviridae Podoviridae Podoviridae Podoviridae Podoviridae

cos ends cos ends cos ends Terminal redundant Terminal redundant Terminal redundant Terminal redundant

filtered (membranous filter 0.2 µm, Renner) freshwater sample. After overnight incubation at 26 ◦ C, bacteria were removed by centrifugation and the supernatant was plated onto indicator plates of the same strain. Starting with a single plaque, phages were multiplied on host strains. 2.2. Host range analysis of phages The host range of all seven Brevundimonas bacteriophages was assayed on the freshwater Brevundimonas isolates as well as on B. diminuta and B. vesicularis (LMG 2350T ) type strains. Dilutions (10 µl) of the phage lysates with titers between 1010 and 1011 plaque forming units (PFU) were dropped onto agar plates previously overlayed with 3 ml top agar and 100 µl logarithmic growing cells of Brevundimonas indicator strains and incubated at 26 ◦ C for 24 h. 2.3. Purification of phages and phage DNA A 500 ml bacterial culture (3 × 108 colony forming units (CFU)/ml) was infected with a multiplicity of infection (MOI) of 0.1 and incubated overnight at 26 ◦ C. Bacteria were removed from the lysate by centrifugation for 15 min at 7000 rpm. To concentrate the phages the supernatant was centrifuged for 2 h at 23 500 g. Phages were resuspended in 0.5 ml buffer (20 mM Tris–HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2 ) with DNase I at a final concentration of 20 µg/ml. After incubation at room temperature for 2 h, the phages were purified by ultracentrifugation in a discontinuous CsCl gradient (0.7 ml CsCl solution with densities of 1.6, 1.5, 1.4, 1.3) for 2 h at 15 ◦ C and 35 000 rpm in a TST 55.5 rotor. Phages were removed and dialyzed against 20 mM Tris–HCl (pH 7.5) and 50 mM NaCl.

The DNA was extracted from purified phage suspensions with phenol for three times and dialyzed against TE buffer (10 mM Tris–HCl (pH 8), 1 mM EDTA). 2.4. Digestion with restriction endonucleases and Southern hybridization For comparison of DNA fragment patterns, phage genomic DNA was digested with different restriction endonucleases according to the instructions of the manufacturer (New England BioLabs) and analyzed in a 1% agarose gel. Table 4 shows which enzymes were used. Southern blotting and hybridization were performed with standard protocols [19]. Hybridization was done at 68 ◦ C with a digoxigenin labeled probe (DIG DNA labeling kit, Roche) of φS121 DNA or φO283 DNA, respectively. 2.5. Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis (PFGE) was performed with a Chef-DR II electrophoresis device (Bio-Rad) in 1% agarose gels at 14 ◦ C in 1x TBE buffer (89 mM Tris–borate (pH 8.3), 2 mM Na2 EDTA). A constant voltage of 5 V was applied with an increasing pulse time of 5–15 s over a period of 23 h. Concatemers of bacteriophage λ and pBR328 (5-kb ladder, BioRad) were used as size standards. DNA samples were mixed 1:10 with 25% (w/v) sucrose in TBE before loading. Gels were stained with ethidium bromide.

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2.6. Identification of terminal redundancy and circular permutation of bacteriophage DNA About 1 µg of bacteriophage DNA was mixed with a buffer containing 67 mM glycine–KOH (pH 9.4), 2.5 mM MgCl2 (final concentrations) and 1 µl lambda exonuclease (5000 U/ml) in a final volume of 20 µl. After incubation at 12 ◦ C for 10– 20 min, the enzyme was inactivated by incubation for 20 min at 80 ◦ C. Subsequently the DNA was slowly cooled to a temperature of at least 40 ◦ C to support the formation of concatemers due to single-stranded terminal redundant ends. Formation of concatemeric structures was demonstrated by PFGE. Identification of circular permutation was done by time-limited digestion with Bal31 as described [15].

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To determine whether the phages were virulent or temperate, bacteria growing in the center of a plaque were isolated by four consecutive single colony passages on TBY plates. To decide between resistance and immunity isolates which were no longer sensitive to the phage were streaked onto an agar plate, irradiated with increasing doses of UV light and overlaid with a phage sensitive indicator strain. For the detection of lysogenic Brevundimonas isolates one isolate was streaked onto agar plates, irradiated with increasing doses of UV light and overlaid with all remaining Brevundimonas isolates. 3. Results and discussion 3.1. Isolation and identification of host strains

2.7. Electron microscopy Drops of 8 µl of purified bacteriophages with a concentration of 109 PFU/ml were spotted onto formvar-carbon coated copper grids. After 30 s the fluid was removed and the phages were negatively stained by a series of three overlays with 8 µl 1% uranyl acetate. The phages were visualized using a Zeiss 109 electron microscope at a magnification of x87 500 operating at 80 kV. 2.8. Transductions of genetic marker and plasmids Phage lysates were tested for their ability to transduce a chromosomal Tn5 (Omegon-Km) marker or the plasmid pSunny. The plasmid pSunny is a derivative of the broadhost-range plasmid RSF1010 with a size of 11.45 kb [13]. The chromosome was labeled by insertion of the OmegonKm transposon of pJFF350 [9]. Phages were propagated on strains either labeled with the Omegon-Km or on strains containing pSunny. The ability to transduce a marker was assayed directly by a transduction assay and indirectly by the detection of transducing particles in a lysate by PCR amplification [20]. For transduction logarithmic growing cells were infected with a MOI of 0.5. After an adsorption time of 30 min suitable samples were plated on TBY plates with kanamycin (30 mg/ml). For amplification of kanamycin resistance genes from transducing particles phages were purified by intensive DNase I digestion and by two consecutive CsCl gradient centrifugation steps. The kanamycin resistance gene aphA3 of pSunny was amplified using PCR primers 5 -GTTGGGGTATCTTTAAATACT-3 and 5 -CAAGCTTTTTAGACATCT-3 . The aphA2 gene of the Omegon-Km transposon (pJFF350) was amplified with primers 5 -GGATTGCACGCAGGTTCTC-3 and 5 -CTCGTCAAGAAGGCGATAG-3 . 2.9. Identification of plasmids and prophages Plasmids were identified by an alkaline isolation which was developed for the detection of large plasmids [18]. We have successfully applied this method for the detection of plasmids from a lot of bacterial isolates among them some Paracoccus isolates which belong to the α-Proteobacteria [3].

A bacterial community of 850 members was isolated from the surface water of three freshwater ponds in Bielefeld (Germany). About 400 of these isolates affiliated with 15 genera. The most abundant groups were those of the genus Pseudomonas (38%) and Aeromonas (20%). A small group of nine isolates was conspicuous because of the orange red color of the colonies. These isolates were Gram-negative, motile small rods. Comparison of total protein samples separated in a SDS–acrylamide electrophoresis revealed that the isolates were closely related (data not shown). 16S rDNA sequence analysis of the strain O283 showed a high degree of sequence similarity to the genus Brevundimonas (GenBank accession number DQ111026). The closest relative (99.7%) was the species B. vesicularis (type strain LMG 2350T ), until now the only red orange pigmented Brevundimonas species described. Comparison of the physiological abilities of B. vesicularis LMG 2350T with two of the Brevundimonas isolates (S121 and O283) was done in a BIOLOG GN2 microplate and showed a high agreement. From these results it was concluded that the nine isolates belong to the species B. vesicularis. This species was first isolated by Busing and coworkers [6] from a medicinal leech under the name Corynebacterium vesiculare. Later on the classification changed to Pseudomonas vesiculare [11] and finally to Brevundimonas vesicularis [21]. 3.2. Isolation and characterization of phages from freshwater samples For the isolation of bacteriophages, we used the same freshwater samples as for the isolation of the host strains and 15 additional water samples from nearby freshwater locations in Bielefeld. Surface water was chosen because in former studies the highest abundance of phages was found in the surface microlayer and it decreased in the deeper layers (reviewed in [23]). The members of the bacterial community were used as hosts for the isolation of bacteriophages. Altogether 40 different phages were isolated as described in Section 2. The phages were named according to the host originally used for the enrichment. For example, 140 Pseudomonas isolates were used for phage enrichment and eight phages were identified for this group; for 73 Aeromonas strains ten phages were identified. Seven phages

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which infected the B. vesicularis strains were isolated (Table 2). It was astonishing to find such a high number of Brevundimonas phages in comparison to the low abundance of the host strain. Further analysis of these phages should determine whether or not these are different phage types. Two different plaque morphologies of Brevundimonas phages were observed. Phages φO272, φS121 and φM164 produced small plaques with a clear center and a turbid halo of diameters from 0.8 to 1 mm, phages φO283, φO283.6, φO283.12 and φO283.15 (from different sources) formed large clear plaques with sizes from 4 to 5 mm diameters. From the center of the plaques from all phages cells could be isolated which were no longer infected by the phages. No phages could be induced by UV irradiation, that means the isolated cells probably were phage resistant mutants. O272 mutants resistant to φO272 were also resistant to φS121 and φM164 but sensitive to O283 phages. This was also the case for S121 and M164 mutants isolated from plaques of the respective phages. Thus, the three phages use the same receptor. O283 mutants isolated from φO283 plaques were only resistant to this phage. Mutants isolated from the plaques of φO283.6, φO283.12 and φO283.15 were resistant to the four O283 phages. The two different O283 mutant types may indicate that the four phages use the same receptor but different overlapping parts of the molecule. Attempts to demonstrate transduction by the phages failed. Neither transduction of a chromosomal Omegon-Km marker or plasmid pSunny (11.45 kb) nor the identification of transducing particles by PCR amplification of a packaged transposon or plasmid was successful for any phage. It should be noted that neither plasmids nor UV-inducable prophages could be identified in the Brevundimonas isolates. Although the Brevundimonas population is very small, the absence of plasmids and prophages in the isolates and the absence of temperate and transducing phages was striking because most of the other, partly similar small groups of the community contained a lot of plasmids and prophages (manuscript in preparation). On the other hand, for none of the other populations such a high number of phages was isolated.

3.3. Morphology of bacteriophages To classify the phages into morphotype groups phage particles were examined by electron microscopy (Fig. 1). Phages φS121, φM164 and φO272 showed an icosahedral head with an average diameter of about 50 nm and a 150 nm long noncontractile tail. This is the characteristic morphology for members of the family Siphoviridae [1,2]. The four phages of O283 possessed a different shape: an icosahedral head and a short tail which is typical for the family Podoviridae. Among 5000 tailed phages examined until now, only 14% belong to this family [2]. 3.4. Host range analysis of Brevundimonas phages Dilutions of stock lysates with about 1010 to 1011 PFU on the original host strain were spotted on all nine Brevundimonas isolates as well as B. diminuta and B. vesicularis (Table 3). Phages φO272, φS121 and φM164 (Siphoviridae, small plaques) had very similar host ranges and formed plaques on most of the isolates with the exception of O283, O214 and O287. Infectivity on O212 and O284 was strongly reduced probably due to restriction of the phage.

Fig. 1. Electron micrographs of B. vesicularis bacteriophages φS121 (A) and φO283.12 (B). The phages were negatively stained with uranyl-acetate. Magnification 87 500x; bars correspond to 100 nm.

Table 3 Host range of freshwater Brevundimonas bacteriophages Strain

Relative infectivity of bacteriophages

O272 S121 M164 O283 O212 O214 O217 O284 O287 B. diminuta B. vesicularis

φO272 1 2 1 <5 × 10−9 2 × 10−5 <5 × 10−9 2 2 × 10−3 <5 × 10−9 <5 × 10−9 <5 × 10−9

φS121 1 1 1 <10−9 10−6 <10−9 1 10−6 <10−9 <10−9 <10−9

φM164 10 9 1 <10−9 <10−9 <10−9 1 <10−9 <10−9 <10−9 <10−9

φO283 6 1 6 1 <10−9 <10−9 2 × 10−2 <10−9 <10−9 <10−9 <10−9

φO283.6 1 2 × 10−2 5 × 10−2 1 <10−8 <10−8 2 × 10−2 <10−8 2 × 10−1 <10−9 <2 × 10−7

φO283.12 10−3 <5 × 10−9 <5 × 10−9 1 <5 × 10−9 <5 × 10−9 <5 × 10−9 <5 × 10−9 1 <5 × 10−9 <5 × 10−9

φO283.15 2 × 10−2 4 × 10−4 10−3 1 <5−9 <5 × 10−9 3 × 10−4 <5 × 10−9 6 × 10−4 <5 × 10−9 <5 × 10−9

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Table 4 Hydrolysis of phage DNA with restriction endonucleases DNA

 φS121  φO272  φM164 φO283  φO283.6 φO283.15 φO283.12

Hydrolysis by endonucleasea

No hydrolysis by endonuclease

AscI (10), BspHI (11), EcoRV (18), MluI (10), NaeI, NruI, StuI (18), Tth111, XhoI SmaI (7), Sau3AI AccI (8), AscI (5), BglI, HindIII (9), MluI (2), NciI, Sau3AI, StuI (10), AccI (2), BspHI, HindIII (10), Sau3AI, StuI (3)

AccI, BglII, EcoRI, HindIII, KpnI, MfeI, NcoI, PmlI, PstI, PvuII, SalI, SmaI, SphI, XbaI AccI, EcoRI, EcoRV, HindIII, PstI, SphI, XhoI EcoRI, NaeI, PstI, SmaI, AscI, MluI, NaeI, PstI, SmaI, Tth111,

a Numbers of DNA fragments in parentheses, no numbers = large number of fragments (>20).

The phages originally isolated for strain O283 (Podoviridae, large plaques) from various locations showed a different host range. They were not able to grow on strains O212, O284 and O214. The most narrow host range was observed for phage φO283.12. None of the phages showed a visible lytic activity against the isolate O214 and B. diminuta. B. vesicularis type strain LMG 2350T was successfully infected only by phage φO283.6, but with a very low rate. The results of phage typing reflect that the species B. vesicularis is still diverse. 3.5. Hydrolysis of DNA with restriction endonucleases Similarities in plaque and particle morphologies, host ranges of the phages φO272, φS121 and φM164 and cross resistance of host mutants indicate that these phages were related. The four phages isolated for O283 showed an identical plaque morphology but they differed more in their host ranges. To analyze whether the similarities were also reflected on the genomic level, the DNA was isolated and hydrolyzed with restriction endonucleases. All DNAs could be cleaved with several restriction endonucleases (Table 4) indicating that the phages belong to the group of double-stranded DNA viruses. There was no indication of a type of DNA modification that prevents hydrolysis at some restriction sites. The DNAs of the phages φO272, φS121 and φM164 were hydrolyzed by the same enzymes and the patterns of DNA restriction fragments were similar but not identical (Fig. 3A). Restriction analysis of φO283.6 and φO283.15 DNA gave identical results although the phages were isolated from different freshwater sources (Fig. 2, lanes 4 and 5). The patterns of φO283 and φO283.12 DNA fragments were completely different (Fig. 2). The sub molar fragment in the pattern of φO283.6 and φO283.15 DNA was reproducible (Fig. 2, lanes 4 and 5). Sub molar fragments may indicate cohesive ends or the pac site fragment of circular permuted genomes [4]. Sub molarity due to concatemer formation based on cohesive ends should disappear after heating. DNA of O283 phages have neither cohesive ends nor are the genomes circular permutated (see below), that means the sub molar fragment derived from an incomplete hydrolysis. Southern hybridization under stringent conditions with the whole genomic DNA of φS121 as probe confirmed the strong relationship between φO272, φS121 and φM164 (Fig. 3B). Hybridization was observed with all DNA fragments, extending the sequence similarity to the whole length of the phage

Fig. 2. Comparison of phage DNAs by restriction analysis. 1% agarose gel. Lane M: λ DNA EcoRI/HindIII. Lane 1: φO283 DNA hydrolyzed with SmaI; lanes 2–5: DNA of φO283, φO283.12, φO283.6, φO283.15 hydrolyzed with HindIII.

Fig. 3. Comparison of genomes from Siphoviridae phages. (A) 0.8% agarose gel. Lane 1: φO272 DNA MluI; lane 2: φS121 DNA MluI; lane 3: φM164 DNA MluI; lane 4: φO272 DNA AscI; lane 5: φS121 DNA AscI; lane 6: φM164 DNA AscI; M: λ DNA EcoRI/HindIII. (B) Hybridization with Dig-labeled φS121 DNA at 68 ◦ C.

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Fig. 4. PFGE of bacteriophage DNA. (A) Size determination of DNA and identification of cohesive ends. Lane 1: concatemers of pBR328 (5-kb ladder, Bio-Rad) as size standard; lane 2: concatemeric DNA of bacteriophage λ; lanes 3–6: DNA of the phages φO283, φO283.6, φO283.12 and φO283.15 (monomers); lanes 7–9: DNA of the phages φS121, φM164 and φO272 (concatemeric structures). (B) Identification of terminal redundancies after partial degradation with λ exonuclease. Lanes 1–4: DNA of φO283, φO283.6, φO283.12 and φO283.15; lane 5: concatemers of pBR328. PFGE conditions as described in Section 2.

genomes. The marker lane (Fig. 3B) was cut off because it was totally blank indicating the stringency of the hybridization. This pronounced relationship of the phages was surprising because they were isolated from different sources. No cross hybridization was observed with the phages of Brevundimonas O283. As expected from host ranges and restriction analysis no hybridization with any of the other phage DNAs could be detected with a probe of φO283 (data not shown). 3.6. Size and terminal structure of bacteriophage DNA Pulsed-field gel electrophoresis (PFGE) is an often used method for the determination of the genomic size of phages, e.g., [8]. Furthermore concatemeric structures can be detected by PFGE indicating cohesive ends of the DNA. DNA of all Brevundimonas phages was analyzed by PFGE (Fig. 4A) They all showed the same sizes which was estimated at about 37 kb in comparison with a λ concatemeric DNA standard and a 5-kb ladder. In addition, the PFGE analysis revealed that DNA of Brevundimonas phages φS121, φM164 and φO272 were able to form concatemers (Fig. 4A; lanes 7–9). These structures disappeared after heating of the DNA samples (10 min at 80 ◦ C) before loading onto the gel (data not shown). As shown in Fig. 4A (lanes 3–6) phages of O283 did not form concatemers. The following assay was used to determine if the double stranded ends of these phages are terminally redundant. The DNA was partially hydrolyzed with λ exonuclease which catalyzes the removal of 5 mononucleotides from the 5 end of duplex DNA. In the case of terminal redundancy concatemers may be formed via the remaining single strands. Concatemer formation was visualized again by pulsed-field gel electrophoresis (Fig. 4B). The DNAs of phages φO283, φO283.6 (identical with φO283.15) and φO283.12 were able to form concatemers af-

Fig. 5. Time-limited digestion of phage DNA with Bal31 followed by digestion with restriction endonucleases. (A) φO283 DNA, SmaI; (B) φO283.6 DNA, HindIII, (C) φO283.12 DNA, HindIII. Incubation time with Bal31 above the lanes. M; λ DNA EcoRI/HindIII. 1% agarose gel.

ter λ exonuclease treatment. To determine whether the DNAs were circularly permutated or not the DNAs were partially digested with exonuclease Bal31 followed by complete hydrolysis with restriction endonuclease SmaI or HindIII (Fig. 5). The truncation of two specific DNA fragments indicate that all DNA molecules of the respective phages had the same ends and were not circularly permutated. Together with the failure of transduction this supports a hypothesis of Zimmer and coworkers [24] that general transduction appears to be correlated with terminal redundancy and circular permutation. Acknowledgements We thank Peter Heimann for his expert instruction and help with electron microscopy, Ulrike Harke for technical assistance, and Ralph Sorenson for critical reading of the manuscript. References [1] H.W. Ackermann, Frequency of morphological phage descriptions in the year 2000, Arch. Virol. 146 (2001) 843–857. [2] H.W. Ackermann, Bacteriophage observations and evolution, Res. Microbiol. 154 (2005) 245–251. [3] A. Battermann, C. Disse-Krömker, B. Dreiseikelmann, A functional plasmid-borne rrn operon in soil isolates belonging to the genus Paracoccus, Microbiology 149 (2003) 3587–3593. [4] L.W. Black, DNA packaging in dsDNA bacteriophages, Ann. Rev. Microbiol. 43 (1989) 267–292. [5] S. Buczolits, R. Rosengarten, R. Hirt, H.-J. Busse, Classification of a Brevundimonas strain detectable after PCR with a Helicobacter-specific primer pair, Syst. Appl. Microbiol. 24 (2001) 368–376. [6] K.J. Busing, W. Doll, K. Freytag, Die Bakterienflora der medizinischen Blutegel, Arch. Mikrobiol. 19 (1953) 52–68. [7] C.Y. Chi, C.P. Fung, W.W. Wong, C.Y. Liu, Brevundimonas bacteremia: Two case reports and literature review, Scand. J. Infect. Dis. 36 (2004) 59–61. [8] J. Doškaˇr, P. Pallov˘a, R. Pant˚ucˇ ek, S. Rosypal, V. R˚užiˇcková, P. Pant˚ucˇ ková, J. Kailerová, K. Klepárnik, Z. Maiá, P. Boˇcek, Genomic relatedness of Staphylococcus aureus phages of the international typing set and

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