Toothpicks, Serendipity and the Emergence of the Escherichia coli DnaK (Hsp70) and GroEL (Hsp60) Chaperone Machines

Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove THE purpose of this essay is to retrace some of the early steps that I and a few (then) young geneticists took in the late 1960s and early 1970s to define the Escherichia coli functions used by phage to properly execute their developmental cycle. Eventually, this led to the discovery and functional understanding of the so-called DnaK (Hsp70) and GroEL (Hsp60) molecular chaperone machines, universally conserved among the biological kingdoms (Lindquist and Craig 1988; Georgopoulos and Welch 1993). Now we know that these and other molecular chaperone machines are involved in a multitude of biological processes, including protection of nascent polypeptide chains from premature aggregation, disaggregation of protein aggregates, polypeptide transport across biological membranes, and proteolysis (Bukau and Horwich 1998; Hartl and Hayer-Hartl 2002; Craig et al. 2006). Because protein denaturation and aggregation are enhanced by various environmental stresses, e.g., an increase in temperature, it is not surprising that the DnaK (Hsp70) and GroEL (Hsp60) molecular chaperone machines were also discovered independently as “heat-shock” or “stress” proteins. Because of their very short growth cycles, phage have evolved a variety of strategies to subvert and customize host functions for their own use. Phage likely have a greater need than their hosts for quick and abundant chaperone power to carry out their developmental cycle in a timely fashion. If they fail to complete their cycle before the host lyses, infectious phage progeny will not be released into the medium, risking their extinction. This differential need for chaperone power likely explains why many of the bacterial mutations found to block phage development were eventually shown to be in genes encoding the GroEL and DnaK chaperone machines. Although my major at Amherst College was physics, I became interested in microbial genetics as a profession in 1961. As an undergraduate, I had taken a part-time job as a laboratory assistant working on phage P22 transduction in the laboratory of Harold H. Plough, a former student of T. H. Morgan. This experience persuaded me to pursue a Ph.D. with Salva Luria at the Massachusetts Institute of Technology (MIT). Under the steadying influence and guidance of Helen Revel (then a senior research associate in Luria's laboratory and a mother hen to me), I finished my Ph.D. thesis on the “sweet and sour” restriction of phage T4 (Revel and Luria 1970). From T4, I moved on to phage λ in Dale Kaiser's laboratory at Stanford. The reason for this leap from the T4 world to that of λ was largely due to the presence of Ethan Signer's laboratory down the hall at MIT. It seemed to me that at that time the smartest workers in biology were those working on phage λ. The developmental cycle and exquisite regulation of this organism are very complicated, and in those early days were way over my head. Thus, I decided to become part of the almost mystical and magical world of phage λ. I simply wanted to be as smart as those guys. It was going to be a great challenge. Because of a temporary lack of bench space at Stanford, I decided to get a head start on my postdoctoral studies by finding a phage λ genetics project during my waiting period at MIT. Luria supported my decision and generously arranged for me to remain as an Instructor in Microbiology for a few extra months. I have always loved genetics because of its strong reliance on logic. The isolation of mutants and the deciphering of their phenotypes allow the elucidation of concrete biological pathways and, in many instances, the identification of specific interactions among the various participants. Although genetic systems are elegant and powerful, they are also very exacting, occasionally yielding “unexpected” results that can be explained only when one fully understands the applied selection/screen. A key person during my initial ventures into lambdology was Ira Herskowitz, at that time a second-year graduate student in Signer's laboratory. Although he was a few years my junior, he was one of my phage λ intellectual fathers simply because he knew so much about it and was always friendly and available. In the spring of 1969, most λ workers were isolating and characterizing phage mutants (mostly of the nonsense/amber, or temperature-sensitive varieties) that were unable to propagate lytically, lysogenize, or recombine (Campbell 1993; Edgar 2004). During strategy-plotting sessions, Ira and I decided to take a completely backward approach, isolating and studying E. coli mutants that block the propagation of phage λ at a step subsequent to injection of its DNA. We reasoned that since the coding capacity of phage λ is rather limited, there must be many interesting host functions that directly or indirectly participate in its developmental cycle. Our hope was to uncover such protein–protein interactions between these two well-studied organisms, shedding additional light on the physiology of important functions in E. coli. Signifying our youthful independence was the fact that Ira and I never discussed our plans or specific experiments with our respective superiors. We simply forged ahead with our collaboration. By serendipity, we chose to carry out our selection with the E. coli strain C600, carrying the supE amber (am) nonsense suppressor gene, an important detail whose significance will become obvious shortly. C600 bacteria were mutagenized and spread on LB agar plates along with an appropriate amount of phage λcI− and phage 434cI− (Figure 1). These lambdoid phage variants were chosen because, although they grow well, neither of them can lysogenize, resulting in certain host death following infection. In addition, the fact that each attaches to a different host–surface receptor reduces the frequency of “trivial” bacterial mutants unable to adsorb phage, since both phage receptor-encoding genes would have to mutate to acquire resistance. The genetic strategy used to isolate the E. coli Gro mutants. The diagram shows the effect of increasing phage concentrations on bacterial growth and colony morphology. See text and Georgopoulos (1971) for details. Normally, when a wild-type bacterium is infected, the released progeny phage will then infect neighboring bacteria in the growing colony, thus limiting the size of the colony. For our experiment, the amount of phage mixed with the bacteria on the plate was critical, since too much phage kills all members of a growing colony (Figure 1D), whereas too little phage results in both “nibbled” colonies and many false positives (Figure 1B). If the infected bacteria are defective in a host function essential for phage development, no viable phage progeny will be released. Hence, although an individual bacterium may be killed as a result of phage infection, the infecting phage are eliminated in the process, thus sparing the neighboring sibling bacteria from being killed, and allowing colony formation (Figure 1C). The procedure was first carried out at 30° in the hope that these mutations would also block bacterial growth at higher temperatures. Under the chosen conditions, wild-type E. coli bacteria formed tiny colonies (Figure 1C) due to killing of the outer cells in a colony by the phage on the plate. However, at a frequency of ∼10−4, large-colony formers appeared on such plates (Figure 1C). The functions defined by these gro bacterial mutations (named for their inability to grow phage; this nomenclature was suggested to me later by Doug Berg at Stanford) did indeed turn out to be important for host growth, as exemplified by the E. coli Gro15 and GroC3 mutants (see below), originally isolated and studied at MIT, which are unable to form colonies at 43°. While at MIT, Ira and I concentrated mostly on the E. coli mutant Gro15. Although the vast majority of λcl− phage are unable to form plaques on this strain, I noted the appearance, at an approximate frequency of 10−7, of phage mutants that were able to somehow compensate for the gro15 block. Interestingly, none of the phage mutants isolated on Gro15 formed plaques on our other mutant, GroC3. Searching for clues as to their nature, I purified many of these gro15 compensatory phage mutants and tested them for growth on standard E. coli laboratory strains, including supD, supE, supF (am-suppressing), and various sup0 (nonsuppressing) wild-type bacterial hosts. To my great surprise and delight, ∼20% of these λ compensatory mutants did not form plaques on any of the wild-type sup0 E. coli tested, but did grow on all of the various am-suppressing wild-type strains. Thus, these phage compensatory mutants carried a suppressible am mutation in some essential, yet unknown, phage gene. This finding highlighted the importance of starting with a strain carrying an am nonsense suppressor allele. Had we started with an E. coli sup0 strain, we would not have isolated such λam compensatory mutants, consequently making it much more tedious to identify the corresponding λ suppressor gene. I vividly recall our great anticipation and excitement as Ira tested Signer's laboratory collection of essential amber mutants of λ for complementation with our λam compensatory mutants. The very simple, yet elegant technique of “spot” complementation enabled us, in <6 hr, to establish that all of our λam mutants isolated on E. coli gro15 carried mutations in gene P, whose product was already known to be essential for λDNA replication. Because of my Greek origin, I baptized the λ compensatory mutants as “π” to indicate that they map in the P gene. We quickly showed that the bona fide λPam3 or λPam80 mutants of Allan Campbell also grew on the C600 supE gro15 mutant bacteria. Furthermore, recombination experiments showed that most of the λam compensatory mutations were distinct from one another, as well as from Pam3 and Pam80. Ira and I were perplexed as to how at least nine different am mutations in the λP gene allowed the phage to grow on our C600 supE gro15 mutant host. Since C600 supE gro15 mutants do not grow at high temperature (Table 1), we knew that the corresponding gro15 gene function is essential for bacterial growth, at least at 43°. Our preliminary conclusion was that, for λDNA replication, the λP protein must interact with an essential E. coli protein. Furthermore, we reasoned that our mutant host protein must be at least partially functional, since it carries out its essential bacterial function at all temperatures <43°. Perhaps by simply lowering the λP protein level (because supE is known to suppress am mutations only to ∼5–30% of the wild-type levels), the partially disabled gro15 gene product manages to carry out its function more effectively, thus resulting in some viable phage progeny and hence in plaque formation. Plating properties of groP mutant bacteria The original classification scheme devised for the groP class of bacterial mutants is depicted and is taken essentially from Table 1 of Georgopoulos and Herskowitz (1971), that the later of the are in The or phage compensatory mutants were isolated as plaque formers on the groP bacterial mutants at an approximate frequency of at (see text for The subsequent genetic of the bacterial is in Plating properties of groP mutant bacteria The original classification scheme devised for the groP class of bacterial mutants is depicted and is taken essentially from Table 1 of Georgopoulos and Herskowitz (1971), that the later of the are in The or phage compensatory mutants were isolated as plaque formers on the groP bacterial mutants at an approximate frequency of at (see text for The subsequent genetic of the bacterial is in The phage transduction experiments of Ira at from and showed that gro15 and the vast majority of the groP mutations isolated by me at in or very the of E. coli. This finding was because and had shown that λDNA was completely in certain mutants. Furthermore, we showed that known bona fide E. coli mutants our groP mutants, as they the growth of mutants to wild-type λ. Thus, Ira and I at the phage λ that, for λDNA replication, the host and phage λP must interact (Figure and this by that the purified E. coli and phage λP indeed form a and that all of known biological are in this While the preliminary results on the groP mutants that from my with Ira were only in the I by I this to be one of my very and Herskowitz The original diagram used by Ira and me during the on phage λ to the between some of the E. coli groP mutants and the corresponding phage λP compensatory It out that the only mutation of my groP collection that did not map at or the was Interestingly, E. coli the of members of both the and compensatory mutant (Table 1). The mutation was eventually during my subsequent in laboratory in at of the E. coli the and By an E. coli into a phage λ by and I was able to for the its to form plaques on the mutant host The fact that the lambdoid phage growth and colonies at high temperature that the mutation is to the wild-type and for both bacterial temperature and the block to lambdoid phage I also that most likely the protein a with the λP protein and somehow this is by the about this had independently isolated an E. coli mutant, on the of its inability to propagate both phage λ and phage In their in the as Georgopoulos et al. showed that their mutation is indeed very to and, also bacterial growth at high and for growth of an appropriate at also isolated bacterial mutations that with λDNA and these mutations of their was in the gene, another, the and Ira a in the with the and which quickly that all of the or indeed into two but distinct complementation et al. defined by and and the genes and because they host at high temperatures. members of one chaperone had The of the chaperone was on the of genetic properties of the mutation of and which at et al. The mutant was isolated as a colony former at However, subsequent showed that E. coli mutant cells do not grow at and that the gene is essential for bacterial growth at all temperatures et al. and Georgopoulos Although the mutation also host at its gene was not et al. The of the chaperone in λDNA was over the years in various including that of with our the late and in et al. The key step in both E. coli and λDNA is the of the essential E. coli at a in E. coli and in phage λ. To on the protein to the gene et al. The phage λP as then and it to the by with the protein et al. et al. However, the of by all known of in this Thus, although the at it the λP is from the This is disaggregation of the at by the chaperone resulting in the of λP and the of in et al. it was shown that the DnaK chaperone also E. coli et al. as well as whose is essential for bacterial et al. This finding the for why or mutations with bacterial at high of the in which the and the DnaK chaperone to carry out its biological function is by its very the of cycle by at the level of et al. and Horwich 1998; Hartl and Hayer-Hartl also chaperone by and specific to DnaK in Craig et al. 2006). In this it is interesting to that the original and mutations with the of their corresponding gene with DnaK et al. et al. et al. 2002; et al. whereas the original mutation with the of the protein and et al. et al. et al. The discovery of the E. coli genes encoding the GroEL chaperone was in the early the of geneticists isolating bacterial mutants that phage λ et al. or phage T4 and et al. Revel et al. studies on at were carried out in with a an important in both the early and the subsequent development of the GroEL chaperone (see key in the For of the λ compensatory mutants isolated at as plaque formers on isolated at the time as had am or temperature-sensitive mutations in the gene of λ to as Thus, the GroC3 mutant was to this The various mutants in my collection were as or on the of their to propagate various compensatory mutants (Table I that members of both the and the did not propagate other lambdoid phage did the phage and with bacterial growth at high A subsequent genetic of mutants that whereas the mutations always in the gene, the mutations in the or the gene, for of the phage This result was because Kaiser's of bacteria infected by wild-type λ showed that the protein was but in an et al. In this of bacteria by wild-type λ of wild-type E. coli sup0 bacteria by or Thus, it appeared from those early studies that the block by our mutations on phage λ was very likely at the level of In this the is a and the would have more and later showed that the GroEL indeed a in the of the an early step in λ of the the that the GroEL is the of It would have almost to in the early 1970s that the at the level of nascent polypeptide and that their subsequent into (see and Plating properties of mutant bacteria The original E. coli mutant were into A or on the of the of the various compensatory mutations to form plaques on It out later that all mutations to the mutations into both the and the in Plating properties of mutant bacteria The original E. coli mutant were into A or on the of the of the various compensatory mutations to form plaques on It out later that all mutations to the mutations into both the and the in The finding was the that one of the E. coli mutant in to not was among my in not my phage T4 as well, although I had no selection for such a in the original isolation procedure (Table However, at a frequency of T4 compensatory mutants to as be The of was that one of these phage T4 mutants, its to propagate on some of the other mutant hosts which allow growth of wild-type T4 phage (Table The inability of to grow on the mutant host led to the quick of its mutation to the gene by complementation a of known T4 am mutants with William then at et al. In this GroEL mutants a to wild-type sup0 bacteria infected by T4 am mutants et al. 1970). The effect of a mutation on both λ and T4 phage head to a between the gene product and the gene in protein about interactions were by and et al. and Revel et al. their corresponding mutations and By the first phage λ of E. coli phage were as plaque formers on various mutant hosts and and a in the isolation of a phage by the λ from whereas independently from laboratory. the product as an protein following of bacteria. The of from cells led to its by and et al. by of its and a more genetic of various led to the that there are two the GroEL and indicate the et al. of my mutant in the gene and the in of its with GroEL et al. and its of These results were by genetic suppressor studies and Georgopoulos and they why mutations in or the phenotypes on the growth of E. coli or phage λ et al. The genetic experiments of that the and genes are the only major genes essential for E. coli all tested et al. This to its to a of essential E. coli et al. It took a few more years to directly that is a bona fide GroEL et al. et al. studies showed that many of for in E. coli growth in et al. et al. However, it is not why but not is for of the protein. It is interesting to out that the of phage λ in our original selection was because it led to the isolation of mutations in both and thus the of the two If we had used only phage T4, which not need only would have these Because of the chaperone effect on phage and the fact that the GroEL is also involved in the of the protein in the that is involved in protein et al. This to when et al. that GroEL in to the nascent of a for the E. coli that the chaperone the of in a purified et al. The years in an in our of the of the (Bukau and Horwich 1998; Hartl and Hayer-Hartl protein aggregation, and interactions are (Bukau and Horwich 1998; Hartl and Hayer-Hartl 2002; et al. and Craig et al. 2006). the of the DnaK and GroEL chaperone machines in E. coli to premature nascent polypeptide aggregation, thus the gene is a abundant chaperone that to the in the of the polypeptide and thus first with the nascent the DnaK chaperone can step in and some of these nascent However, because of their additional may also interact and with with a protein The key that the and DnaK chaperone machines in protein is highlighted by the resulting of the mutant 30° et al. et al. 2004). The DnaK (Hsp70) in with the can also certain protein formed (Bukau et al. 2006). In to and the DnaK the GroEL is to mostly at a level to some The in and of the various chaperone machines is exemplified by et al. have shown that GroEL can associate with nascent chains in a in the of the GroEL or the chaperone both growth of the mutant and a of protein at temperatures et al. et al. et al. 2004). both the E. coli DnaK chaperone and its various can function both in and in with various and and Craig et al. 2006). Thus, it is likely that in some nascent polypeptide chains can be by more than one chaperone of DnaK and chaperone machines in nascent protein The diagram the the and the and chaperone machines. The diagram the various pathways that nascent may during the to at their It is essentially from Hartl and Hayer-Hartl all are to See text for details. studies have also highlighted the of the chaperone in a variety of important such as growth, and certain by protein aggregation, e.g., and In this the development of of as and 2006). The chaperone also shown to an important in the development of a form of et al. Ira a few before death in the spring of 2004). we took a along the and about the We both that we the early of the of molecular In the spring of 1969, with we on a of and discovery with no in We were that our genetic with such as and phage had to a understanding of the We were certain that of important were to be at the of by of microbial we that genetic of organisms, such as phage, and is the and way to at I all of my and have to in the chaperone for and for I am to for me on this of and for in making the text more and I am also to the of the and the of for