Phage Facts Martha Chase Began Their Experiments, Little Was Known About the Early Steps of Phage Infection

The 1966 Phage and the origins of molecular microbiologists. Nonetheless, historical cautions us to keep a broad view of molecu- biology6, edited by John Cairns, Gunther interpretations that emphasized these var- lar biology’s past — and its future. Stent, and James Watson, offered a geneal- ious ‘origins’ tended to demarcate molec- ogy of molecular biology with Delbrück as ular biology firmly from pre-existing Angela N. H. Creager is in the Department founding father. The book’s implicit mes- fields, especially biochemistry (Delbrück of History and Program in History of sage that molecular biology derived from famously disparaged biochemistry). Science, Princeton University, Princeton, the phage group drew fire from some. John By all these accounts, Hershey makes an New Jersey 08544, USA. email: Kendrew argued that structural biologists odd revolutionary. He is described by [email protected]

(such as those in his unit at Cambridge) friends as a “biochemist’s biochemist” as 1. As quoted in Judson, H.F. The eighth day of had also played a crucial role in the found- well as a pioneering molecular geneticist. creation: The makers of the revolution in biology, 7 275 (Simon and Schuster, New York; 1979). ing of molecular biology . His research defied any dichotomy between 2. Hershey, A.D. & Rotman, R. Genetics 34, 44–71 Gunther Stent subsequently offered a informational and structural approaches, (1949). 3. Hershey, A.D. & Chase, M. J. Gen. Physiol. 36, 39–56 conciliatory view of the origins of molecu- and he retained a skeptical attitude towards (1952). lar biology that combined the ‘informa- the supremacy of nucleic acids. Attention to 4. Avery, O.T., MacLeod, C.M. & McCarty, M. J. Exp. Med. 79, 137–158 (1944). tional’ school of phage genetics with the Hershey as one of the original molecular 5. Hershey, A.D., ed. The bacteriophage lambda. (Cold ‘structural’ school of X-ray crystallogra- biologists complicates the story, showing Spring Harbor Laboratory Press, Cold Spring Harbor, New York; 1971). phers, a meeting of the minds that was the limitations of views that emphasize a 6. Cairns, J., Stent, G.S. & Watson, J.D. Phage and the conveniently personified by Watson and single approach, dogma, or charismatic origins of molecular biology. (Cold Spring Harbor 8 Press, Cold Spring Harbor, New York; 1966). Crick . This enlarged family tree still did leader. By portraying a trailblazing biologist 7. Kendrew, J. Sci. Am. 216, 141–43 (1967). not satisfy everyone; Origins of molecular who combined structural, biochemical, and 8. Stent, G.S. Science 160, 390–395 (1968). 9 9. Lwoff, A. & Ullmann, A. Origins of molecular biology: A tribute to Jacques Monod , genetic methods in his quest to understand biology: A tribute to Jacques Monod. (Academic stressed the early importance of French life at the molecular level, We can sleep later Press, New York; 1979).

history

Later, when Alfred D. Hershey and Phage facts Martha Chase began their experiments, little was known about the early steps of phage infection. What was known was Often the simplest experiments lead to — induced immunity and selection of that after the phage adsorbed to the bac- © http://structbio.nature.com Group 2001 Nature Publishing the most remarkable insights. So it was spontaneous mutations — were rigorous- teria there was a latent period of ∼10 with the famous fluctuation experiments ly tested both theoretically and experi- minutes after which time infectious virus of Luria and Delbrück and the Waring mentally by Max Delbrück and Salvadore particles were made, ultimately leading blender experiments of Hershey and E. Luria, respectively, in 1943. to host cell lysis and phage release. Chase for which they were awarded the The two hypotheses lead to different pre- Hershey and Chase reasoned that if they Nobel Prize in Physiology or Medicine in dictions regarding the distribution of resis- knew the fate of the viral protein and the 1969. While the results of these experi- tant bacteria in a series of parallel cultures. nucleic acid at the beginning of phage ments are permanently etched into every The hypothesis of acquired or induced infection they would understand more first year biology student’s brain, it is immunity predicts that the number of about the nature of those early steps. worth recalling what was known at the mutants in each culture would be clustered They labeled the phage with either 35S or time these experiments were conducted around the mean with little variation 32P, allowed them to adsorb to the bacteria and the conclusions the authors drew. between cultures. According to the selection and then the cells were separated from the In the early 20th century, the role of hypothesis, resistant bacteria could arise at unadsorbed material by centrifugation. chance mutations in the genetic variation any point in the life of the culture. This The cells were resuspended and the sus- of higher organisms was generally accept- would result in very different numbers of pension was spun in a Waring blender to ed. However, bacterial cultures seemed to mutants per culture and thus the variability separate the phage from the infected bacte- be plastic. Exposed to adverse conditions, from culture to culture would be high. ria. They found that ∼80% of the 35S label cultures of bacteria could give rise to resis- Luria found that in every experiment the was removed from the bacteria whereas tant variants that remained resistant “to “fluctuation of the numbers of resistant only ∼20% of the 32P label was removed. the action of the virus even if subcultured bacteria is tremendously higher than could They concluded that “most of the phage through many generations in the absence be accounted for by the sampling errors, in DNA enters the cell, and a residue contain- of the virus”1. At the time, many scientists striking contrast to the results of plating ing at least 80% of the sulfur-containing believed that the “virus by direct action from the same culture and in conflict with protein of the phage remains at the cell induced the resistant variants”1. Others the expectations from the hypothesis of surface”2. Together with the 1944 experi- believed that the “resistant bacterial vari- acquired immunity”1. Luria and Delbrück ments of Oswald Avery3 in which nucleic ants are produced by mutation in the cul- concluded that the “resistance to virus is acid, not protein, was shown to have ture prior to the addition of virus. The due to a heritable change of the bacterial cell “transforming” properties, these findings virus merely brings the variants into which occurs independently of the action of persuaded most scientists that DNA car- prominence by eliminating all sensitive the virus”1. In other words, the mutation ried the genetic information. Curiously, bacteria”1. These two alternative theories was there before the virus was added. however, Hershey himself remained skep-

tical. Hershey and Chase concluded their mots from and about Hershey can be 1. Luria, S.E. & Delbrück, M. Genetics 28, 491–511 (1943). paper by saying “the sulfur-containing found in “We can sleep later: Alfred D. 2. Hershey, A.D. & Chase, M. J. Gen. Physiol. 36, 39–56 protein has no function in phage multipli- Hershey and the origins of molecular (1952). 2 3. Avery, O.T., MacLeod, C.M. & McCarty, M. J. Exp. cation, and the DNA has some function” . biology” which is reviewed on page 18 of Med. 79, 137–156 (1944). Talk about an understatement. More bons this issue. Boyana Konforti

picture story

A DNA wormhole

The bacteriophage φ29 is a virus that view; top panel). The narrowest part of infects the bacterium Bacillus subtilis. The the channel is ∼36 Å in diameter; a double mature φ29 contains a head region that stranded DNA could easily pass through encloses its genome and a tail region that the channel without any steric problems. contacts the cell surface to initiate the To characterize how the connector fits infectious cycle. In the late stage of the mat- into the phage head, Simpson et al. gener- uration process from which functional ated a three-dimensional reconstruction phage particles emerge, the phage genome of the prohead from cryo-electron — a linear double-stranded DNA of microscopy data and then fit the crystal ∼19,000 base pairs — is packaged into a structure into the electron density. The preassembled empty head (called the pro- result shows that the narrow part of the head). This process requires condensing connector protrudes outside of the viral the viral genome, which would span shell (bottom panel, a thin section across ∼6,400 Å if it were completely extended, the center of the prohead along the axis of into the phage head, which is only ∼500 Å the connector central channel is shown; in length. How the phage performs this the red mesh wire represents the electron remarkable task is not yet fully understood. density from the prohead and the DNA is © http://structbio.nature.com Group 2001 Nature Publishing At one end of the phage head is a small modeled in the superposition for illustra- opening through which the viral genome tion purposes). This region of the con- enters and exits. A protein complex called nector interacts with a phage-encoded the ‘connector’ — so named because the RNA (marked by green mesh wire) and tail of the phage is connected to the head possibly a viral ATPase (not present in through this complex — sits at the open- this reconstruction), both of which had ing and participates in the DNA packaging been shown to be important for DNA process. To understand the structural packaging. basis of how the connector regulates DNA With the translocating DNA as the packaging, Simpson et al. (Nature, 408, movable central shaft, the connector, the 745–750; 2000) have determined its high viral RNA and ATPase appear to consti- resolution structure. tute a ‘motor’, with the viral ATPase The connector complex consists of 12 powering conformational changes in the identical subunits (top and middle panels, connector complex. The energy of ATP one monomer is colored red to illustrate hydrolysis is converted into a transla- how the subunits are assembled in the tional motion of DNA. This study thus complex). The overall shape of the com- provides a glimpse of the initial event in plex is like a bottle stopper (side view; the DNA packaging process of bacterio- middle panel) with a central channel (top phage φ29. Hwa-ping Feng Adapted from Simpson et al.

20 nature structural biology • volume 8 number 1 • january 2001