Bacteriophage-like particles associated with the gene transfer agent of Methanococcus voltae PS

F. Eiserling1, A. Pushkin1, M. Gingery1 and G. Bertani2

Department of Microbiology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA1
Jet Propulsion Laboratory 125-224, California Institute of Technology, Pasadena, CA 91109, USA2

Author for correspondence: Giuseppe Bertani. Fax +1 818 393 4057. e-mail gbertani{at}lalc.k12.ca.us


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The methanogenic archaeobacterium Methanococcus voltae (strain PS) is known to produce a filterable, DNase-resistant agent (called VTA, for voltae transfer agent), which carries very small fragments (4400 bp) of bacterial DNA and is able to transduce bacterial genes between derivatives of the strain. Examination by electron microscopy of two preparations of VTA that were concentrated and partially purified by different methods showed virus-like particles with isometric heads, about 40 nm in diameter, and with 61 nm long tails. These particles co-sedimented with the minute bacteriophage {phi}X174 in a sucrose density gradient.


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Like all known methanogenic bacteria, Methanococcus belongs to the archaeobacteria (Fox et al., 1977 ), a grouping that has revealed exciting new possibilities in the study of early biological evolution (Olsen & Woese, 1997 ). Methanococcus lacks a typical bacterial cell wall. Only a thin protein S-layer covers the plasma membrane. Although cultural work with methanogens (which are strictly anaerobic) is laborious and slow, some genetic information has been accumulating. One of us (Bertani, 1999 ) has described in M. voltae strain PS a system of gene transfer which resembles generalized transduction, except that the bacteriophage component (in terms of virus replication) is defective or absent. The filterable agent responsible for the transfer, called VTA (for voltae transfer agent), is resistant to DNase and contains 4·4 kb fragments of DNA, derived exclusively or almost exclusively from the bacterial chromosome. VTA is unfortunately very unstable and is very sensitive to certain manipulations, e.g. high-speed centrifugal pelleting or suspension in CsCl solutions. We report here the results of two independent attempts to demonstrate the VTA particles by electron microscopy, using VTA samples concentrated and partially purified by different methods.

Technical details concerning mutant strains, media, culturing, measurement of VTA activity (as frequency of transfer of histidine independence, the his+ marker) and filtration have been published (Bertani, 1999 ; Bertani & Baresi, 1987 ). For preparation I, 70 ml of a sterile filtrate from cultures of M. voltae strain PS-6 was spun (4 h, 6 °C) at 25000 r.p.m. in a Beckman SW28 rotor over a cushion of highly concentrated sucrose solution. A volume just above the cushion was collected, applied to a Bio-Rad P-10 Biogel column and eluted (with 0·15 M NaCl, 0·015 M sodium citrate, pH 7) to remove most of the sucrose. The fractions expected to contain the VTA activity were pooled and concentrated in Centricon 30 (Amicon) centrifugal concentrators. The procedure reduced the original filtrate volume about 1300-fold. The his+ VTA activity in the concentrate was only 9x105/ml, corresponding to a recovery of about 3%. The concentrate was stored frozen at -70 °C with 10% glycerol until used for electron microscopy 4 months later. A phenol extract from this preparation was examined by gel electrophoresis and confirmed the enrichment of DNA of VTA size. For preparation II, small volumes of filtrates of strain PS-2, highly concentrated by the PEG-bag method (Bertani, 1999 ), were layered onto sucrose step gradients (0·9 ml each of 45, 40, 35, 30%, and 0·85 ml of 25%, w/w, sucrose) in buffer (0·3 M NaCl, 20 mM Tris, 1 mM EGTA, pH 7·6) and centrifuged (Beckman SW50.1 rotor, 40000 r.p.m., 2 h, 6 °C). Fractions were collected dropwise from the bottom of the tubes. An earlier identical run (see Fig. 2) served as the guide in choosing the fractions corresponding to the VTA peak. These fractions were pooled and further concentrated with Centricon 100 (Amicon) concentrators. The total his+ VTA activity finally recovered (about 2·8x106) was about 20% of the original input, and about 250-fold more concentrated. The VTA activity was well localized on the gradient and well separated from the bulk of UV-absorbing material present in the preparation. This preparation was not frozen.



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Fig. 2. Sedimentation of his+ VTA and two Escherichia coli phages in a sucrose step gradient (described in the text). Combined data from two tubes spun simultaneously, one tube being loaded with 0·2 ml VTA from strain PS-2, the other with a mixture of E. coli phages {phi}X174 and P2 lg del1 del2. Fractions, collected dropwise from the bottom of the tubes, were slightly different in average volume for the two tubes (0·186 ml and 0·221 ml). –––, density calculated from refraction measurements for the tube containing the marker phages. ----, absorbance at 260 nm for the VTA tube. The VTA titre in the gradient fractions is given as his+ colonies in the standard VTA assay (Bertani, 1999 ). The VTA activity recovered was 38% of input. Peak titres (p.f.u.) for P2 lg del1 del2 and {phi}X174 were 2x106/ml and 6x104/ml, respectively.

 
With preparation I, samples for electron microscopy were applied directly to the surface of carbon-coated 400 mesh copper grids and allowed to adhere for 2 min. The droplet was then removed, the grid surface rinsed with several drops of distilled water, and a negative stain of 0·5% (w/v) uranyl acetate applied. Excess stain was removed with a capillary and the remainder wicked dry with filter paper. A preparation of bacteriophage T4 was added to the concentrated filtrate to serve as a size reference. With preparation II, the sample (about 0·08 ml) was diluted in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7·5) to fill tubes of the Beckman SW41 rotor, and centrifuged for 2 h at 30000 r.p.m.. The invisible pellet was resuspended in 0·25 ml 20 mM ammonium acetate, pH 7·4. The samples were prepared for electron microscopy by the single carbon method of Valentine et al. (1968) , in which a layer of carbon is floated onto the sample from a strip of mica, then contrasted with uranyl acetate and mounted on 400 mesh grids. The samples were examined in a Hitachi H-7000 electron microscope at 75 kV.

Our first electron microscopy observations were made on other less concentrated and less pure VTA preparations and showed mostly ‘membrane vesicles’ and bacterial flagella. The ‘membrane vesicles’ were between 50 and 100 nm in diameter and showed in places a regular structural array on the surface (similar to the S-layer; see Jarrell & Koval, 1989 ). On the other hand, the highly concentrated preparation I showed numerous regular, near-spherical and polyhedral particles about 40 nm in diameter (Fig. 1). These sometimes showed an attached tail, but more often the tail structure was detached. Most of the particles were penetrated by the stain. Similarly, the better purified preparation II showed numerous typical bacteriophage-like particles consisting of a head of diameter about 40 nm and a (usually attached) tail (Fig. 1). No contracted tails were observed. A series of 32 particles from both preparations was measured using T4 phage tails (whose 4·1 nm spacing is known from both electron microscopy and X-ray diffraction; see Karam, 1995 ) as reference. The averages were: 40 nm for the head diameter (measured perpendicularly to the head-tail axis), 61 nm for the tail length and 12 nm for the tail width (measured at half length). In all preparations the largest structures present were the M. voltae flagella, which have been thoroughly studied (Kalmokoff et al., 1988 ; Jarrell & Koval, 1989 ) and would offer an internal size-standard, their diameter having been estimated at 13 nm. Unfortunately, their apparent diameter is strongly affected by local staining conditions, flattening, etc. The size of the head of the bacteriophage-like particles would reasonably fit a condensate of double-stranded DNA of 4·4 kb, as expected for VTA (Bertani, 1999 ). Several other structures were observed in preparations I and II: (a) larger, irregular structures (‘membrane vesicles’) of highly variable size; (b) ‘small particles’, of 9–13 nm diameter; (c) circular structures (‘buttons’) of 17 nm diameter; and (d) fimbriae, about 4 nm in diameter. With the exception of the vesicles, it seems rather unlikely that any of these other structures may fulfil the requirements for encapsulating 4·4 kb of double-stranded DNA.



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Fig. 1. Electron micrographs of partially purified, concentrated filtrates from Methanococcus voltae PS (see text) showing vesicles, flagella and virus-like particles. The much larger, tailed particles are bacteriophage T4, added to preparation I as a size reference. Capsid-like particles found in preparation I are shown in the top panel (indicated by arrows), and in the central row (a tailed particle is indicated by the arrow). Tailed particles found in preparation II are shown in the bottom row.

 
Additional inferences on the size of the VTA particles could be drawn from a comparison (Fig. 2) of sedimentation rates for VTA, detected as his+ marker transfer, and two well studied DNA-containing bacteriophages, P2 and {phi}X174 [see reviews by Bertani & Six (1988) and by Hayashi et al. (1988) ]. The larger P2 bacteriophage has a 33 kb (more precisely, 28·5 kb for the deletion mutant used in these experiments) double-stranded DNA molecule in a 58 nm diameter head, with a 1330x65 nm tail, while the tail-less {phi}X174 is 25–30 nm in diameter, with 5·4 kb of single-stranded DNA, closer to the size of the virus-like particles described above. As is evident from Fig. 2, the VTA his+ activity sedimented at a rate very close to that of the {phi}X174 marker.

As discussed by Bertani (1999) , 4·4 kb of DNA seem too small to carry all the viral genes necessary for the formation of a structurally complex particle, the control of replication and maintenance in the bacterium, and DNA size-measuring or other cutting specificity in transduction. Other defective systems with virus-like particles carrying host cell DNA are known both for eubacteria and for eukaryotes. To our knowledge, in only two other cases – the gene transfer agent in Rhodopseudomonas capsulata (now Rhodobacter) (Yen et al., 1979 ), among eubacteria, and polyoma-related virus (Michel et al., 1967 ) among eukaryotes – is the size of the DNA fragments incorporated as small as in VTA. The existence in M. voltae of larger particles with a larger nucleic acid molecule, representing the viral component, is not excluded, but, if present, they would have to occur at a much lower frequency than is the case for classical transduction systems, like P1 or P22 in the eubacteria, or {psi}M1 in another methanogen, Methanobacterium thermoautotrophicum (Meile et al., 1990 ). The only other virus-like particles reported to-date for a Methanococcus strain are those of Wood et al. (1989) : tail-less, ovoidal, much larger (52x70 nm) than ours, and without any known biological activity. While the particles involved in gene transfer in Rhodobacter resemble the ones described here, except for their shorter tails, it does not seem that particles of this shape and size have been found to-date in other archaeobacteria (see Zillig et al., 1988 ).


   Acknowledgments
 
The work at UCLA was supported by institutional research funds to F. Eiserling. The work at JPL was supported in part by the Biocatalysis Program of the Energy Conversion and Utilization Technology Division of the U.S. Department of Energy, and by NASA Code E Contract 961524.


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Received 9 February 1999; accepted 31 August 1999.