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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Main text |
---|
![]() ![]() ![]() ![]() |
---|
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.
|
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 913 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.
|
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
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Bertani, G. & Baresi, L. (1987). Genetic transformation in the methanogen Methanococcus voltae PS. Journal of Bacteriology 169, 2730-2738.[Medline]
Bertani, L. E. & Six, E. W. (1988). The P2-like phages and their parasite, P4. In The Bacteriophages, vol. 2, 73143. Edited by R. Calendar. New York & London: Plenum Press.
Fox, G. E., Magrum, L. J., Balch, W. E., Wolfe, R. S. & Woese, C. R. (1977). Classification of methanogenic bacteria by 16S ribosomal RNA characterization. Proceedings of the National Academy of Sciences, USA 74, 4537-4541.
Hayashi, M., Aoyama, A., Richardson, D. L.Jr & Hayashi, M. N. (1988). Biology of the bacteriophage X174. In The Bacteriophages, pp. 1-71. Edited by R. Calendar. New York & London: Plenum Press.
Jarrell, K. F. & Koval, S. F. (1989). Ultrastructure and biochemistry of Methanococcus voltae. Critical Reviews in Microbiology 17, 53-87.[Medline]
Kalmokoff, M. L., Jarrell, K. F. & Koval, S. F. (1988). Isolation of flagella from the archaebacterium Methanococcus voltae by phase separation with Triton X-114. Journal of Bacteriology 170, 1752-1758.[Medline]
Karam, J. (editor) (1995). Molecular Biology of Bacteriophage T4. Washington, DC: American Society for Microbiology.
Meile, L., Abenschein, P. & Leisinger, T. (1990). Transduction in the archaebacterium Methanobacterium thermoautotrophicum Marburg. Journal of Bacteriology 172, 3507-3508.[Medline]
Michel, M. R., Hirt, B. & Weil, R. (1967). Mouse cellular DNA enclosed in polyoma viral capsids (pseudovirions). Proceedings of the National Academy of Sciences, USA 58, 1381-1388.[Medline]
Olsen, G. J. & Woese, C. R. (1997). Archaeal genomics: an overview. Cell 89, 991-994.[Medline]
Valentine, R. C., Shapiro, B. M. & Stadtman, E. R. (1968). Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry 7, 2143-2152.[Medline]
Wood, A. G., Whitman, W. B. & Konisky, J. (1989). Isolation and characterization of an archaebacteral viruslike particle from Methanococcus voltae A3. Journal of Bacteriology 171, 93-98.[Medline]
Yen, H. C., Hu, N. T. & Marrs, B. L. (1979). Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsulata. Journal of Molecular Biology 131, 157-168.[Medline]
Zillig, W., Reiter, W.-D., Palm, P., Gropp, F., Neumann, H. & Rettenberger, M. (1988). Viruses of archaebacteria. In The Bacteriophages, pp. 517-558. Edited by R. Calendar. New York & London: Plenum Press.
Received 9 February 1999;
accepted 31 August 1999.