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GUEST COMMENTARY

Rambling and Scrambling in Bacterial Transformation— a Historical and Personal Memoir

Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000

In these retrospective comments I hope to accomplish several objectives. I shall briefly review the early history of bacterial transformation, with an emphasis on breakthroughs and personalities. Then, I shall describe my own role in the development of the field with its curious dependence on the interplay of rational experimentation and serendipity. In this context, I shall discuss the importance of mentoring, cooperation, chance, and revelation. My title refers to my good fortune in having support that enabled me to ramble over various aspects of transformation, although from time to time I was obliged to scramble to obtain grant funding, to produce publications, and to generally compete in the field. To achieve within these few pages the multiple objectives mentioned above, I shall be rambling and scrambling once again.

EARLY HISTORY

Fred Griffith in 1928 reported that heat-killed encapsulated pneumococci could transfer the ability to make a capsule and, hence, to infect mice, when injected together with live, unencapsulated and nonpathogenic pneumococci (18). He termed the phenomenon "transformation." At the time, it was not realized that bacteria contained genes, let alone that DNA was the genetic material. Griffith conjectured that a seed of capsular polysaccharide was perhaps transferred from the heat-killed bacteria, but he went one step further in wondering whether an enzymatically active protein might be the agent transferred. Few people know that Griffith, a medical doctor, was killed, together with a colleague at the Ministry of Public Health, during the German bombing of London in 1941 (5).

Soon after Griffith's discovery, Oswald Avery at the Rockefeller Institute in New York took up the problem, and most of the subsequent developments were connected to his laboratory. In the 1930s Dawson and Sia achieved transformation in vitro as opposed to in mice (8), and Alloway was able to extract the active principle from heat-killed cells (2). However, proof that the transforming principle was DNA awaited the landmark paper of 1944 (3). That work had enormous impact in demonstrating that the genetic material consisted of DNA.

QUANTITATIVE TRANSFORMATION

Although capsular transformation was effective in revealing the phenomenon of transformation, it depended on screening for smooth (encapsulated cell) and rough (unencapsulated cell) colonies. For precise quantitation, selectable markers are preferable. Rollin Hotchkiss, a member of Avery's group who carried on the investigation of pneumococcal transformation at Rockefeller, obtained drug resistance markers and devised methods for the quantitative measurement of transformation frequencies. The value of quantitative measurements for understanding molecular mechanisms of transformation, regulation of recipient cell competence, processes of DNA degradation, and genetic analysis by transformation is apparent from the results of Hotchkiss and those influenced by his laboratory. In 1957, using a streptomycin resistance marker, Hotchkiss showed that transformants increased linearly with added marker DNA, until a saturation level was obtained, thereby demonstrating a discrete number of DNA uptake sites on the recipient cells (22). Competence for DNA uptake, measured as transformation frequency, was found to vary systematically during the culture growth cycle (21). Later it was shown that competence depended on accumulation of an extracellular polypeptide (20, 55).

Particularly noteworthy was the demonstration by Marmur and Lane of strand separation on denaturation of the native, duplex DNA and the renaturation of its transforming activity after thermal denaturation by annealing the separated strands at submelting temperatures (44). Although this work was accomplished at Harvard University, Marmur had been a postdoctoral fellow and Lane had been a technician in the Hotchkiss laboratory. Another outstanding contribution from that laboratory was Maury Fox's discovery of the eclipse of donor marker transforming activity immediately after DNA uptake. When DNA is extracted from recipient cells just after uptake, it is unable to transform tester cells, but the ability is fully restored within 10 min at 37°C (14). Several investigators, including Fox (13), Goodgal and Herriott (17), and Lerman and Tolmach (39), pioneered the use of ³²P-labeled DNA to demonstrate the physical uptake of DNA in transformation. Sol Goodgal was instrumental in organizing the first Transformation Meeting in 1956. It is to Leonard Lerman that we owe the name of the Wind River Conference; he organized the Transformation Meeting at the Wind River Ranch, Estes Park, Colo., in 1959.

My own work in transformation began in 1955, as the first graduate student of Rollin Hotchkiss. Rollin fostered self-determination in his students, and he agreed to my proposal to study recombination between nutritional mutants—to see if we could analyze genetic fine structure by transformation. For my thesis I isolated and analyzed eight mutants unable to use the sugar maltose (32). Even from this small number of mutants, several significant facts emerged. Three maltose-negative mutations were multisite and ultimately shown to be deletions; one covered the entire locus, and the other two overlapped from opposite ends. Their transformation efficiencies depended inversely on deletion length. (Transformation efficiencies are expressed as the ratio of a maltose marker transformation frequency to that of a standard streptomycin resistance marker to account for variations in culture competence.) Five mutations recombined with each other and appeared to be mutated at single sites; they presumably corresponded to base changes. What was surprising was that these single-site mutations showed transformation efficiencies with wild-type DNA that varied over a 10-fold range. Later, such variation in transformation efficiency was shown to result from differential action of a base mismatch repair system (11, 25). We were able to measure production of the enzyme amylomaltase, product of the malM gene, immediately after DNA uptake by the various mutants. Surprisingly, the amount of enzyme produced was not dependent on the amount of DNA taken up but was proportional, rather, to the transformation efficiency of the marker (33). This, too, was a manifestation of the eclipse; not only was the incoming DNA unable to transform another cell, but it was also unable to direct production of a protein prior to its integration.

MOLECULAR MECHANISM OF DNA UPTAKE

In searching for a molecular explanation of the eclipse phenomenon, my initial hypothesis was that DNA was fragmented during entry and that the double-stranded fragments were later connected, presumably while aligned on a recipient chromosomal template, to restore both transforming and transcribing activity. My idea was to label the donor DNA with ³²P, break open recipient cells after uptake, and look for ³²P-DNA fragments with a methylated-albumin coated kieselguhr (MAK) column. Mandell and Hershey had recently shown that fragments of T-even coliphage DNA were separated on MAK columns in a size-dependent manner (43). However, when I carried out the experiment, 40% of the ³²P counts were immediately eluted and only 5% were eluted by the salt gradient, which eluted even the high-molecular-weight recipient DNA; most of the radioactivity was missing, and I was puzzled over the low recovery. That evening I had a moment of "eureka": the remaining counts must have been retained by the column! On returning to the laboratory, I detected, in fact, radioactivity still on the MAK column. When eluted with 0.1 N NaOH, the counts amounted to 50% of the total. Upon more carefully reexamining the paper by Mandell and Hershey, I saw that they had mentioned in passing that denatured DNA sticks to the column. Apparently, the DNA was converted to single strands on uptake. This conclusion was soon confirmed by separation of newly entered and resident DNA in a CsCl density gradient that separated single- from double-stranded DNA.

In reporting my findings in 1962, I proposed that one strand of the duplex DNA was degraded as the other strand entered the cell (23). Such conversion to a single-stranded form explained the eclipse, inasmuch as denatured DNA was known to be poorly taken up by cells and could not serve as a template for transcription. Furthermore, conversion to a single strand would facilitate interaction with the recipient chromosome. In fact, radioactive label passed rapidly from single strands into double stranded, presumably chromosomal DNA (23). Later, it was shown by density labeling that single-stranded segments of donor DNA were indeed incorporated into the transformed chromosome (15).

I considered the determination of the fate of DNA on entry to be an important new finding for the understanding of transformation. Therefore, I was quite surprised to read, afterward, in a thesis of Pierre Schaeffer published in 1961 the following concluding statement, "Une hypothèse est proposée d'après laquelle la pénétration de la molécule d'ADN dans la bactérie requiert une structure à double chaîne, alors que la synapse, dont dépend l'intégration, requiert au contraire une chaîne polynucléotidique unique" (50a). Translated, this reads "A hypothesis is proposed according to which penetration of the DNA molecule into the bacterium requires a double-stranded structure, while synapsis, on which integration depends, conversely requires a single polynucleotide strand." Schaeffer was a medical doctor, a mature investigator, who had for some years investigated the competition between different DNAs in the transformation of various bacterial species. As was the custom in France, such researchers often took an additional degree of Docteur Sciences later in life. Schaeffer based the uptake part of his hypothesis on his own experimental work, but the conclusion that a single strand was needed for integration was the result of careful thought. He reasoned that only single-stranded DNA, with its bases exposed, could readily interact with recipient DNA. It was sobering for me that these thoughts were presented before publication of my own work. In fact, it is possible that they were expressed during Schaeffer's thesis defense, which I attended in Paris, France, in 1958, when I was working in the laboratory of François Gros, another very fine mentor, at the Pasteur Institute. Despite its proposal on theoretical grounds, I comforted myself in the novelty of my work showing that the strand conversion process occurred during DNA entry. However, I never forgot that scientific understanding is a large edifice to which many contribute.

Because the amount of low-molecular-weight ³²P found in the cells approximately equalled the amount in single strands, it appeared that one strand was degraded as the other strand entered (23). However, Morrison and Guild showed that the amount of DNA breakdown inside the cell depends on the size and quality of the donor DNA; more intact DNA gave fewer breakdown products so that a larger proportion of DNA taken up was in the form of single strands (45). In the early 1970s, they and Bill Greenberg and I independently discovered that the breakdown products of the strand complementary to the one that enters appear external to the cells (28, 46). Early on, I hypothesized that a cellular DNase acted on donor DNA to facilitate entry of one strand by degrading the opposite strand (23). Investigation of the pneumococcal nucleases revealed two enzymes active on DNA (27). In vitro, EndA, which was found to be membrane bound (31), degraded DNA to give oligonucleotide products, and ExoA (like ExoIII of Escherichia coli) gave mononucleotides and single strands. To see which, if either, enzyme facilitated entry, I sought mutants lacking them. Useful in this approach was a method for detecting colonies that lacked nuclease activities by growing them in agar containing DNA and methyl green, which stains only intact DNA (26). Colonies around which the DNA was digested showed colorless zones that were absent around mutant colonies (Fig. 1).

View larger version (121K): [in this window] [in a new window]   FIG. 1. Nuclease detection in nutrient agar plates containing DNA and methyl green. Cells of S. pneumoniae wild-type strain R6 (endA+ exoA+) and mutant strain 593 (endA1 exoA+) were plated in a ratio of 1:10 and incubated at 37°C for 30 h.

The in situ detection method (Fig. 2) helped show conclusively that null mutants of EndA reduced uptake and transformation by a factor of a thousand (35). ExoA mutants had no effect on transformation. Both its membrane location and finding that the external products of in vivo degradation were oligonucleotides (34) supported a role for EndA in bringing one strand into the cell as it degraded the other.

View larger version (61K): [in this window] [in a new window]   FIG. 2. DNases in wild-type and mutant strains of S. pneumoniae detected after electrophoresis in polyacrylamide gels containing SDS and DNA. Wells contained 25 µg of protein from SDS lysates of S. pneumoniae as follows: wild-type R6 (a), exoA5 (b), endA1 (c), and endA14 (d). (e) This well contained pancreatic DNase I and micrococcal nuclease. Electrophoresis was anodal in the downward direction. After washing to remove SDS, the gel was incubated for 64 h, stained with ethidium bromide, and photographed with UV illumination. Size markers (in thousands) were from a parallel lane of proteins stained with Coomassie blue (not shown). Dark bands correspond to DNases.

Elimination of the major DNases from the pneumococcal cell still left a weak DNase activity, which gave no acid-soluble products but could be detected by its reduction of DNA viscosity. This enzyme turned out to be DpnI, an unusual restriction endonuclease that cleaves only DNA with adenine methylated at GATC sites (29, 56). Normally, restriction enzymes cleave unmethylated DNA, and DNA is protected from cleavage by methylation. The contrary is true for DpnI. Its discovery was fortuitous, in that E. coli DNA, which happens to be methylated at GATC sites, was used as the substrate for testing DNase activity, and not DNA from the pneumococcal strain, which was unmethylated. Interesting as this enzyme was, it was only the first part of the story.

The concept of serendipity, proposed by Horace Walpole in 1754, derives from an ancient tale entitled The Three Princes of Serendip. In the story, three young men from Serendip, which is an old name for Sri Lanka, set off on a trip abroad and encountered many adventures. By a combination of chance and sagacity, they fared well in their travels. In a personal instance of serendipity, a culture of a strain that should have made DpnI produced instead a different enzyme, DpnII, which is complementary to DpnI and cleaves only at unmethylated GATC. We wondered if by some genetic switch, perhaps related to DNA methylation, the DpnI-producing strain had converted itself into a DpnII-producing strain (30). This turned out not to be the case. This DpnII strain was a contaminant streptococcus that outgrew the original inoculum. However, different isolates of pneumococcus from nature could produce either DpnI or DpnII (47), and the responsible genes were found to constitute mutually replaceable genetic cassettes (38). Similar findings for capsular genes were foreshadowed by this result. An additional unexpected feature of the DpnII system was the presence of two methyltransferases (9), one of which preferentially methylated single-stranded DNA and was able to prevent the restriction of unmethylated plasmid entering a cell by the transformation pathway (6).

CLONING IN STREPTOCOCCUS PNEUMONIAE

Among the tools and methods developed in my laboratory, one of the most important was the pneumococcal cloning system using derivatives of the plasmid pMV158 (53). We were forced to turn to this system by our inability to clone many important pneumococcal genes, such as malM, endA, and hexA, in the available E. coli systems, apparently due to the strong promoters of S. pneumoniae and the toxicity of the gene products in E. coli (52). The pneumococcal cloning system was elaborated in a relatively short time by the intense effort of Diane Stassi, Paloma Lopez, and Manolo Espinosa. This followed an initial suggestion by Francis Barany in a telephone conversation with me. Walter Guild provided pMV158; he obtained it from its discoverer, Vickers Burdett. There again, we see the interwoven undergarment of scientific progress.

Cloning in pneumococcus not only allowed the analysis of otherwise unclonable genes, but it also provided a facile method for exchanging chromosomal segments present in a recombinant plasmid. Such exchange resulted from chromosomal facilitation of plasmid establishment, in which an entering linear strand from a recombinant plasmid must interact with the chromosome in order to circularize (42). That the DpnI and DpnII system genes resided at the same position in the chromosome was first discovered by the substitution of a DpnI gene segment on transformation of a DpnI cell by a recombinant plasmid containing the DpnII gene segment (38). Cloning in S. pneumoniae of a chromosomal segment containing sul-d, a mutation which conferred sulfonamide resistance, enabled us to carry out the first genetic analysis of folate biosynthesis in any organism (41).

DNA MISMATCH REPAIR

A major thrust of my work has been in DNA mismatch repair. This phenomenon was first discovered in S. pneumoniae, a decade before it was found in E. coli, by examination of transformation frequencies of different mutational markers at the amylomaltase locus. In my doctoral work (circa 1957) I had found different mutations to be transformed at characteristic frequencies or efficiencies (32). Perhaps influenced by both the periodic table, with its categories of elements, and astrophysics, with its grouping of stars by luminosity, I undertook a classificatory approach, grouping mutations of similar transformation frequency. By 1965, I had over 70 single-site mutations in the malM gene, which clustered in four efficiency groups, and I had found that reciprocal transformation of markers gave identical frequencies (24, 25). These findings allowed the attribution of the four efficiency groups, I to IV (ratios of 0.05, 0.2, 0.5, and 1.0), to the six possible classes of mutations giving base mismatches. On the basis of mutagenic origin and changes at the same site, I proposed the following correspondence of efficiency class to mutations giving rise to mismatches in the heteroduplex product of transformation: I, ATGC, GCAT; II, ATTA; III, GCCG; IV, ATCG, GCTA (25). These proposed assignments proved to be generally correct when cloning and sequencing of the malM gene was achieved (37).

I had been impressed by the colligative properties of the efficiency groups with respect to parameters other than transformation efficiency. Marker sensitivity to UV light (24, 40) and speed of recovery from eclipse (16) were both inversely correlated with efficiency. Both effects result from dependence of donor marker excision on single-strand breaks prior to ligation of the donor segment. Repair of UV damage produces subsequent strand breaks that result in additional mismatch repair (36). The apparent earlier recovery of low-efficiency markers reflects their later loss by repair, as was evident from early peaking of their recovery (51). These correlations helped support a model for mismatch repair in which an entire donor strand segment that would normally be integrated in transformation is excluded from integration (7, 25, 36).

With important contributions by various investigators, the different transformation efficiencies and other properties were ascribed to a cellular mismatch repair system. A major contributor was Harriett Ephrussi-Taylor. In 1958, she invited me to give a talk at her laboratory in Gif, near Paris. Harriett was very interested in the malM mutant transformation frequencies and recombination results. With Michel Sicard she devised another gene system, based on aminopterin resistance, for analyzing mismatch repair (11). She was the first to propose that the pneumococcal system removed mismatches by an enzymatic mechanism (10). Unfortunately, Harriett died soon afterward; however, Michel and, later, his student Jean-Pierre Claverys, continued to contribute importantly to the field. Our discovery of the hex mutants, in which all markers transformed at high efficiency (26), supported the concept of an enzymatic system for detecting and repairing DNA mismatches, in which large tracts of donor DNA containing the mismatched base were removed. Using such hex mutants, Tiraby and Fox showed that the mismatch repair mechanism also prevented spontaneous mutations (54). T. S. Balganesh in my laboratory (4) was the first to clone a mismatch repair gene, hexA, and that enabled determination of its nucleotide sequence (48). Sequence comparison with mismatch repair genes from other species indicated that the system was universally found in living cells (19, 48, 49). Strikingly, a homologous system in human cells prevents colon cancer (12) (Fig. 3).

View larger version (186K): [in this window] [in a new window]   FIG. 3. From left to right, Bill Greenberg, Sanford Lacks, and Sylvia Springhorn examine autoradiograms that reveal the DNA sequence of genes encoding the restriction enzymes of S. pneumoniae.

The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.

ACKNOWLEDGMENTS

I thank the scientists who were my mentors, my collaborators, and those who came to work in my laboratory as visiting scientists, postdoctoral associates, students, and technical associates. Although everyone made important contributions, I regret that I was able to mention only a few by name in this memoir. I am indebted to two associates who worked with me for very long periods, Bill Greenberg and Sylvia Springhorn; they are pictured in Fig. 3.

	FOOTNOTES

 
* Mailing address: Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000. Phone: (631) 344-3669. Fax: (631) 344-3407. E-mail: lacks{at}bnl.gov.

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