Many Faces, Many Microbes: Personal Reflections in Microbiology

The author Cynthia A. Needham and her coworkers are all motivated by the same vision—a general public with a high level of scientific literacy, and share the same commitment—one of translating scientific discovery and principles into terms and stories that are intellectually accessible to everyone. The author had the privilege of leading a coalition of organizations known as the Microbial Literacy Collaborative on behalf of the American Society for Microbiology. The goal of the Collaborative was to increase the level of scientific literacy among the members of the general public, with a particular focus on the microbial world. The centerpiece of the initiative's effort is a four-hour documentary broadcast by PBS entitled Intimate Strangers: Unseen Life on Earth. The success of the Microbial Literacy Collaborative's initiative is due in large part to the combined energy of talented producers, educators, and scientists. Their insights and advice provided the key link between the leading edges of research and its application and the translation of this highly technical area into accessible language and imagery. Popular media such as television, radio, print, and the Internet provide the opportunity to build a more comprehensive appreciation for both the scientific process and the contemporary scientific basis for understanding complex issues. The success of these media in expanding science literacy will depend, however, on input from the scientific community.

Life is an interconnected web of organisms, and the main struts—the main fibers—in this web are evolutionary relationships. Today, microbiologists are developing a biologically meaningful understanding of microorganisms, so that we can realize how really important, and diverse the microbial world is. Many earlier molecular evolutionists knew that the technical power existed in molecular sequencing to determine distant phylogenetic relationships. Some understood that with the right molecules—for example, transfer RNAs—the full breadth of phylogeny could be spanned. Yet from their actions, it appeared that none of them appreciated the importance of determining a universal phylogenetic tree, what powerful evolutionary knowledge would emerge there from. The old prokaryote-eukaryote dichotomy was short on understanding and long on dividing biology into two camps, which more or less went their separate ways. The emphasis was on the ways in which prokaryotes and eukaryotes differ, not on why they differ and what sort of common ancestor they share. The archaea are central to a real revolution that is still occurring in microbiology. They epitomize the importance of phylogeny and the power of molecular biology to reveal evolutionary history.

The author was exhilarated by the opportunity at MIT to work with an extremophile (Clostridium thermocellum). The DNA-based biotechnology revolution is growing, and Cambridge is at the heart. The author's thesis described the regulation of active oxygen production by the fungus Phanerochaete chrysosporium. Her job was to test the biodegradability by mixed bacterial cultures of consumer product ingredients such as those used in fabric softeners, laundry detergents, and diapers. Hydrogen was produced by the Big Bang and is the most abundant element in the universe. The proximal electron donor for life three to four billion years ago on prebiotic earth was iron, which forms the core of earth and other terrestrial planets. The author suspects that it is the ability of nitrogen to form flat, stable aromatic compounds with carbon that is critical to nitrogen's importance; configuration is important to proper alignment of molecules. The theory that eukaryotes arose by endosymbiosis between bacteria and archaeans is tremendously heartening. The rise of different eukaryotic types by the mixing and matching of cell organelles, as described by Margulis, renews the author's faith in probability. The rapid evolution of microorganisms-because of their short generation times-will produce a corollary to the Law of Microbial Infallibility.

The author had a firsthand opportunity to see the world of microbiology come alive with astonishing relevance as physicians came to the clinical microbiology laboratory to learn about the infections in their patients and, more important, about which antimicrobial agents to select for therapy. After reading several dozen Kirby-Bauer disk diffusion plates, the author's fascination with antimicrobial resistance began. A better understanding of antimicrobial resistance mechanisms and the exchange genetic information among bacteria became a key research imperative. There were several different plasmid fingerprints observed among the many isolates that were collected, but more important, there appeared to be a common pattern among the "outbreak-related’’ strains. The strain from the author's tofu did not match the outbreak pattern, but the fact that plasmid fingerprinting worked was joyous news. Although the plasmid fingerprinting technique could be applied almost universally to most bacterial species, it was common at conferences to hear sometimes congenial, but at other times heated, discussions about how to interpret the results.

Researchers determined which cytokines induced the two activated antimicrobial macrophage effector reactions-resistance to infection and intracellular killing-and which cytokines shut down the killing. The author made inroads in the understanding of Francisella infections, devised a polymerase chain reaction-based detection system and an attenuated and subunit vaccine, and explored the use of immunomodulation with bacille-Calmette-Guerin (BCG) with great success. In this chapter the author seeks additional philanthropic funds to assist companies and academic investigators in the clinical development of new anti-TB drugs. The path is set for the foundation, and there is great excitement both within the foundation and the research community. Sequella, Inc., is working with investigators in universities around the world and has licensed some innovative, useful products for diagnosis and treatment of tuberculosis (TB). It is currently raising money to fund the development of its products and hopes to have at least one of its new TB products in the marketplace by the year 2001.

The author worked in the Plant Breeding Department at Cornell University, and his project involved a forage crop called bird's foot trefoil. There was work in the field scoring plants for desirable agronomic characters, and there was work in the laboratory examining chromosomes for possible cytogenetic aberrations. Beadle and Tatum had done their groundbreaking research using the red bread mold Neurospora crassa. The yeast Saccharomyces cerevisiae is not only the best-known model system for fungal life, it is also one of the best-understood model organisms for eukaryotic life. The Southern Regional Research Laboratory conducted targeted research on economically important agricultural problems. Aflatoxins are carcinogenic metabolites produced by several filamentous fungi in the genus Aspergillus. Aflatoxin contamination of food crops is an international health hazard. The fungi that make aflatoxin lack sexual phases (mycologists call them "imperfect"), and in the days of pre-recombinant DNA, it was almost impossible to conduct genetic studies on imperfect fungi. Scientists at MIT had just initiated research on the biosynthesis of aflatoxin and showed that the chemical skeleton came from acetate units. More recently, with the help of Brendlyn Faison, a group at Tulane branched out to apply fungal degradative metabolism to environmental problems, characterizing new species for bioremediation. The author's ardor for microbiology, feminism, and fungi is not appropriate for everyone, but it illustrates how in science, an ordinary but focused person can lead an extraordinary life.

One expects bacterial growth to be slow based on chemical principles, but at a certain low temperature--the minimum temperature for growth—growth stops completely. The author's studies showed that many things go wrong simultaneously at the minimum temperature of growth, and metabolism therefore stops completely. By isolating and studying mutant strains with an increased minimum temperature of growth (called as cold-sensitive mutants), it was possible to determine what single change increased the minimum temperature of growth of one particular mutant strain. The author isolated cold-sensitive mutants of Escherichia coli that were unable to grow below 20°C (the minimum temperature for growth of wild type E. coli is 8°C). In these mutants, biosynthesis of histidine was cold sensitive. The mutations causing this type of cold sensitivity lay in hisG—the gene encoding the enzyme that catalyzes the first step of the pathway, the one sensitive to feedback inhibition by free histidine. The phenomenon of change in regulatory responses of proteins with temperature proved to be a general one; however, one cannot predict whether regulation becomes more or less severe as temperature is lowered. From the study of cold-sensitive mutants, the author concluded that bacteria stops growing at low temperature because weakening of hydrophobic bonds causes conformational changes in proteins that preclude growth largely by distorting regulation or stopping assembly processes.

During the early 1960’s the author’s arrival in Boston as a scientific immigrant to work in Shattuck Laboratories led to development of his interest in microbiology. He assumed responsibility for research projects with students and also taught a laboratory course, fielding difficult questions from medical students. Later he worked on a research project on the biochemical mode of action of streptomycin was moving along, when he learnt about Wally Gilbert's work on the separation of Escherichia coli ribosomal subunits and the demonstration that transfer RNA bound to the large subunit. The author did a lot of experiments on poly U-stimulated translation in vitro, using a variety of antibiotics and radioactively labeled amino acids; and the author says that it was awesome to see the counts increase when an aminoglycoside was added. This led to the discovery of antibiotic-induced errors and the role of the 30S ribosomal subunit in the translation of messenger RNA into protein. Many of his works and achievements were due to his contacts with renowned researchers at parties.

Harvard's paleontologist Stephen Jay Gould said in a lecture on evolution at Woods Hole that the 3.5-billion-year-old microorganisms will also be the ultimate survivors on this planet. For the author, the detour via deep-sea studies was necessary to discover novel types of symbioses between chemosynthetic bacteria and marine invertebrates. The author and his coworkers demonstrated that the produced biomass could be used for feeding mussels in aquaculture. Also, this well-defined carbohydratious material may be a useful base material for fermentations to alcohols as synthetic fuels or for other industrial applications. Marine microbiology is a healthy and always exciting mix of interdisciplinary activities—both classical and modern microbiological approaches.

As interesting as the author’s thesis work on plasmid replication was, the author was especially intrigued by the mechanisms by which pathogens, like Salmonella, Shigella, and Mycobacterium tuberculosis, caused disease. The author’s almost-buried primal dream of a medical career began to resurface, and the author began to think that a career in infectious diseases might be a perfect marriage of microbiology and medicine. As part of a training program, the author elected to do research project in the Falkow laboratory, which had moved from Georgetown to the University of Washington in the early 1970s. At that point, recombinant DNA techniques, gene sequencing, cloning, agarose gel electrophoresis, endonuclease restriction enzyme analysis, and Southern hybridization were all being used in the lab. The author’s project was to study the mechanisms of multiple antibiotic resistance of Serratia strains that had been isolated from patients at the veterans administration hospital (VAH). The most exciting discovery of the author’s career resulted from an encounter with a patient for whom the author was asked to consult as an infectious disease physician. It is the dream of every microbiologist and infectious diseases clinician to discover a new disease or a new pathogen, but very few of us have this opportunity.

Michael A. Pfaller continued to pursue applied research in terms of evaluation of in vitro test systems in the clinical laboratory. In addition, the drug-bug work continued with evaluation of new antifungal agents and antibacterial agents. The topic of antimicrobial resistance became very popular in the 1990s, and the author's microbiology group at Iowa has been in the thick of this field of investigation. The close collaboration between clinical microbiology and hospital epidemiology is one that should be sought out by all concerned. From the perspective of the clinical microbiology laboratory, one can become involved in all of the most interesting infectious disease cases. Problems in diagnosis and control of infectious diseases provide a never-ending array of material for investigation and publication. Applied research in this area invariably leads to improvements in how the clinical laboratory can serve its clients and, in many cases, can serve as a springboard to more basic investigation.

On discharge from the military, the author enrolled in the graduate school of Syracuse University because he was interested in fermentation and industrial microbiology, which were major interests of the microbiology department there. The author’s interest stemmed from his observations of the use of penicillin in the miraculously successful treatment of serious bacterial infections in the military and civilian population in France, Germany, and elsewhere. His research interests were broad rather than focused on one disease or one organism or group of organisms. The author's research work showed that group B phemolytic streptococci were capable of producing serious systemic disease in children and postpartum women. He was also interested in systemic fungal diseases that were peculiar to the geographical area. Two such diseases were histoplasmosis and blastomycosis- both endemic to the Missouri Valley. The presence of Histoplasma capsulatum was confirmed in hen house droppings and soil. The author and his research team also showed that blastomycosis occurs frequently in dogs that may have transmitted the infection to humans by contact, but they were unable to prove conclusively that soil is the natural habitat of Blastomyces dermatitidis. During the early 1960s, the National Committee for Clinical Laboratory Standards (NCCLS) became prominent. It was a direct outgrowth of the collective recognition for standardization in clinical laboratories.

In this chapter the author talks about her doctoral research that involved studying interactions of Salmonella with phagocytes in vitro. The aim of this work was to study the cellular immune response to salmonellae, first by using mouse cells and then moving on to a human system. At that time, knowledge of the intricacies and complexities of the immune system was rudimentary, and the author's work did not clarify the inconsistencies already present in the literature regarding the intracellular fate of microorganisms. Early on, several clinical microbiologists began to assess critically microbiological procedures that either were already in place or coming into use. They also championed the publication of journals and manuals specifically devoted to this discipline. The areas in which the author was especially interested were blood culture instruments and automated and nonautomated rapid identification systems, especially for blood culture isolates and anaerobes. These types of studies were important for determining which systems were accurate, cost-effective, and easily integrated into the work flow of the laboratory. Clearly, the field of clinical microbiology is undergoing changes that could not have been foreseen when the author first entered it. The author expects that like the forerunners who were active in the early days of her career, the new generation of clinical microbiologists will find ways to meet the challenges and bring further regard and advancement to this discipline.

Within microbiology, Ellen Jo Baron discovered the visual aesthetics of microscopy and the ability to describe and identify bacteria. A diagnostic microbiology laboratory at a county public health department had an opening for a person with a B.S. in microbiology. Ellen Jo Baron smelled the autoclave and the unique odors of a busy microbiology laboratory. She rotated through many areas of the laboratory, learning the basics from the ground up-Mycobacterium tuberculosis laboratory, urines, susceptibilities, wounds, genital cultures, and others. The author talks about the time when she found herself at the bottom rung of the clinical profession. An incident involving an anesthesiologist led her to seek a higher academic degree. This anesthesiologist controlled the postappendectomy care for a young patient and demanded antibiotic susceptibility tests on all organisms recovered from the patient's respiratory secretion. Ellen Jo Baron knew that the organisms recovered were simply the patient's normal mucosal and saliva flora and that antibiotic therapy to try to remove them was inappropriate. Nevertheless, the anesthesiologist would not listen to a lowly laboratory technologist, and the pathologists did not have the microbiological background to refuse the doctor's requests. Multiple successive antibiotics were prescribed to treat that patient's "pathogens," and as a result the patient developed pneumonia with a panresistant Pseudomonas aeruginosa and died. At this point the author thought that if she had a master of science degree she could gain credibility and respect from clinicians. This incident urged the author to enter a master's program in medical microbiology.

Humankind has suffered many afflictions-some from the plagues of invisible microbes, some from natural disasters, and some from man's inhumanity to man. The author and his research colleagues used Legionella pneumophila as a model system and studied immune responses of mice to this infectious organism. They developed a susceptible versus resistant mouse model to study Legionella and investigated the role of B cells, T cells, and macrophages, as well as cytokine responses to infection by this bacteria. In addition to studying the immune response to Legionella, they soon began to examine the effect of marijuana on the immune response system. These studies originated with the recognition that drug abusers were especially prone to HIV infection and the development of AIDS. It is obvious that further investigation of the host-parasite relationship as it relates to immune responses against microorganisms will result in what was thought to be an unattainable understanding of how the human species can survive in the "sea of microbes" that can use humans as their hosts.

The excitement of being on his own in research in Italy groomed the author for other research experiences. With Raymond, who became both mentor and friend, the author worked independently in the area of radiation genetics. During his hematology fellowship, he followed up some research that he had begun with Charlotte Friend, placing leukemic spleens subcutaneously back into leukemic mice and noting prolongation of their lives. Stemming from the author’s experience with Watanabe, he also investigated the ecology of drug resistance and resistance plasmids. Bacteria have responded to the widespread applications of antibiotics by finding ways to become resistant, insensitive to the killing effects of these powerful drugs. The overuse of antibacterial agents kills off susceptible bacteria, enabling competitor flora in the environment to proliferate and cause infection, as well as causing some bacteria to actually develop mutations conferring resistance. The author is a voice for the proper use of antibiotics, and carries the message around the globe that we must make peace with the bacteria.

The concept that viruses could remain latent in human cells and cause cancer, propelled the author to examine the potential role of viruses in human diseases, particularly immune deficiency and cancer. The author was at the time fascinated by the field of lysogeny, in which bacterial viruses (phages) remain hidden within the organism and can make products that are toxic to the host the bacteria invade (e.g., diphtheria toxin). At the Wistar, the author helped derive human lymphoid B cell lines in the early days when only a few papers reported the establishment of such cell lines. It was then fortuitous for the author to be with the Henles when they showed the induction of prolonged growth of B cells by Epstein-Barr virus (EBV). The irony came when the author found EBV in the cell lines derived at the Wistar. Further virus research training was obtained at the National Institutes of Health (NIH) from 1967 to 1970. This experience certainly laid the foundation for the major focus of the author's research career-retroviruses. With Robertson Parkinson, the author found that mouse embryo cells, once transformed by the murine sarcoma viruses (MSV), could not grow into a visible focus of transformed cells without the spread of new progeny virus to other cells. MSV-infected mouse cells usually died.

For the last 30 years the author has been chasing outbreaks of disease—viral diseases that are as rare and as deadly as any that have ever plagued humankind. In this section the author highlights that virus hunters like him would no longer be needed. One of today's challenges is to solve the interfaces and integrations of the multiple disciplines that are needed to attack the most challenging problems. The authors narrate his experience with the navy who needed vaccine to protect the lab crew from a deadly disease. The head of the lab gave the author free rein, and the crew was vaccinated. The cause for this outbreak was discovered and the author and his team could even predict them to some degree through satellite remote sensing. They had a vaccine proven to protect against the virus, and they began setting things up so that when it hits again, they will have an experimental drug ready to be tested in the most severely ill patients. In 1985 the author made several forays into central Africa as part of a team looking for Marburg, Ebola, and other hemorrhagic fever viruses. The team needed to find out the sources of these viruses and find out why the outbreaks suddenly appeared and just as suddenly disappeared.

At the National Institutes of Health (NIH), the author focused on animal viruses and was the first to isolate a polymerase encoded by an animal virus. His research for the past twenty-eight years has focused on the molecular mechanisms underlying the replication of a cryptic human virus, adeno-associated virus (AAV). Although all of us are infected by AAV, the virus has never been associated with any human disease and thus was little known until recently. It was not discovered until 1965, when it was found as a contaminant of supposedly pure preparations of adenovirus, a common human pathogen. It was quickly shown that AAV required coinfection with an adenovirus for productive infection in cell culture. On purification the complementary strands would anneal to form duplex DNA. Virus particles containing normal-density DNA were mixed with particles containing heavy-density DNA. When the DNA was purified, the double-stranded DNA we isolated had a hybrid density. As it does not cause disease, AAV was not initially of great medical interest; this situation changed with the recognition that the virus could integrate its genome. Coupled with the virus's persistence, this characteristic made it a strong candidate to serve as a vector for human gene therapy. Now clinicians and entrepreneurs in large numbers are interested.

Under the leadership of the author, the Rega Institute, which celebrated its fiftieth anniversary in 2004, has grown to be a world-renowned center for research on nucleoside analogues, antivirals, anti-HIV drugs, cytokines, and chemokines. Today it is one of the world's leading academic centers for antiviral research. This section talks about the 1983 description of the synthesis and antiviral properties of aminoacyl esters of acyclovir, which led to the development of the valyl ester (valacyclovir) for the oral treatment of herpes simplex virus (HSV) and VZV infections, and also talks about the 1985 description of the principle of combined gene therapy/chemotherapy of cancer by using antiviral agents such as ganciclovir and BVDU that are highly and specifically cytostatic to tumor cells transfected by the HSV-1 (HSV-2 or VZV) thymidine kinase gene. The collaborative centers are located in five continents and help to establish and maintain a leading position in the field. The author's most tangible and most rewarding contributions are his discoveries that have led to a panoply of new antiviral drugs that have met medical needs and helped alleviate the symptoms, if not saved the lives, of patients suffering from the most severe and often life-threatening viral infections.

Molecular biology is so young a set of techniques and ideas that a number of current practitioners have lived through its entire development. It was easy to make discoveries and get jobs and grants in those days because everything was wide open and everyone was a raw recruit. It is a source of wistful amusement to think that the author made the first pure preparations of 30S, 50S, and 70S ribosomes from Escherichia coli and measured their molecular weights and that his Ph.D. research also included one of the first functional in vitro systems for bacterial protein synthesis. Results that the author obtained with subcellular systems provided some of the indications that RNA was involved in directing protein formation. With his colleagues David Apirion and Giorgio Mangiarotti, the author analyzed the dynamics of ribosome metabolism, facilitating the study with fragile mutants of E. coli that could be lysed gently enough to preserve the polysomal structures. As for the author's own work, involvement in discussions for the planning of microbial sequencing initiatives has been accompanied by a more interventive participation in the expansion of human genome approaches.

The author found the logic of Koch's approach for proving that a particular organism caused a particular disease particularly appealing, as well as the description of figuring out how to make single colonies on a potato slice. The author's postdoctoral project was on the site-specific excision of bacteriophage lambda. Mark Shulman, who had preceded her in the lab, had developed a lambda derivative that allowed an intramolecular excision reaction. The aim was to reconstitute the reaction in vitro; Howard Nash was using similar approaches to look at the integration reaction. Some progress was made, and the researchers found out something about the in vivo reaction as well. The researchers wanted to understand what sort of cellular substrates Lon degraded and how proteolysis is used by the cell to regulate gene expression. The ultimate test was the demonstration that suspected targets were rapidly degraded but were stabilized in lon mutants.

The expressed sequence tags (ESTs) method identified more than thirty new G protein-coupled receptors; however, these sequences were also of great interest to Human Genome Sciences, Inc. (HGS) and its partner, Smith-Kline Beecham (SB). Haemophilus influenzae is of historical significance in science as well, being the source from which Ham Smith first isolated restriction endonucleases, which led to his Nobel Prize in Medicine in 1978. No complete genome sequence for a free-living organism had ever been deciphered, so we all realized that this would be a landmark achievement if it could be accomplished. Since TIGR reported the first complete microbial genome sequence in 1995, the sequences of more than twenty bacterial and archaeal species have been published, and at least sixty other genome projects are in progress in laboratories around the world. Most species cannot be cultured in the laboratory, yet these species likely play some of the most important roles in the ecology of our planet. Breakthroughs in genomics technology and bioinformatics will continue to allow us to push back the frontiers of whole genome analysis. Breakthroughs in genomics technology and bioinformatics will continue to allow us to push back the frontiers of whole genome analysis. But at the same time, new technologies for functional genomics present exciting opportunities to begin to study the dynamic nature of the microbial cell.

The study of Bacillus subtilis transformation might be more fruitful than a continuation of the investigation on Escherichia coli. This was an important decision for a postdoc who would be looking for a job in a few years. Joshua and Esther Lederberg determined that genes of tryptophan synthesis were closely linked to a gene of histidine biosynthesis. More extensive analysis of this region demonstrated that additional genes of aromatic acid biosynthesis were closely linked to each other and to the his locus. They also identified a new form of allosteric inhibition of aromatic acid synthesis and published a number of papers on the genetics, biochemistry, and regulation of the pathway. The author also focused on studying the mechanism by which donor DNA is taken up by competent cells of B. subtilis. The author also focused on Agrobacterium and the disease that it caused in plants, crown gall tumors, for a number of reasons, all of which seem to have crystallized simultaneously. From the very beginning, Joshua and Esther Lederberg believed that Schilperoort was correct in concluding that DNA was transferred from Agrobacterium into plant cells. Using techniques of DNA-DNA hybridization, they demonstrated that many strains of Agrobacterium that were not lysogenic for this phage nevertheless caused crown gall tumors.

The College of Agriculture at Cornell University was virtually cost-free to residents of New York State and so the author decided to go there. Although there were laboratories in zoology, botany, and geology (and the author took them all), the microbiology laboratories were "user friendly." Perhaps what was so exciting was the hands-on approach. Results were in real time and experiments could be performed, even when they were not part of the formal laboratory exercise. This was certainly not true in other areas. During Srb's course Euphrusi's early work with Drosophila and McClintock's work with corn were discussed. However, it was the Neurospora genetics of Beadle and Tatum-to which Euphrusi had contributed-and Euphrusi's work with yeast and the beginnings of Escherichia coli genetics or bacteriophage T4 that seemed more meaningful. Lectures by the late Wolfe Vishniac dealt with "funny bugs," an anachronism describing a collection of bacteria that were then far outside the mainstream-for example, methane, sulfur, and photosynthetic bacteria. The photosynthetic bacteria were particularly interesting to the author because he was amazed that nonsulfur purple bacteria could grow heterotrophically and photosynthetically or not, depending on the absence or presence of oxygen, switching metabolic modes based on levels of oxygen. To investigate the photosynthetic membranes of the purple nonsulfur bacteria, it was essential to develop a genetic system for Rhodobacter sphaeroides. Such a system could be applied to study the control of gene expression by oxygen and light.

In this section the author expresses his primordial awe for one of the simplest experiments in bacterial physiology that is done over and over in laboratories. It is the measurement of the growth of a bacterial culture in a liquid medium. One can get an instantaneous reading simply by determining the turbidity of the culture at different times using a common light-measuring device such as a colorimeter. In addition to enjoying the delights of Copenhagen, the author became involved in research on bacterial growth physiology using the enteric bacterium Salmonella typhimurium. The increase in the number of cells, on the other hand, did not proceed at the new rate until quite some time later. Cultures were set up in a collection of different media that supported various growth rates, from the slowest to the fastest attainable in that laboratory. It was found that the concept that the polymerizing machinery of bacteria performs at unit rates is also true for the biosynthesis of DNA, RNA, and cell wall constituents. This finding demonstrates the economy that bacteria exhibit in adapting to different growth environments.

By isolating the first glutamine and glutamate auxotrophs of Klebsiella aerogenes, the author's research group discovered that the glutamate dehydrogenase activity could be eliminated without major consequence to cell growth. They showed that an additional mutation was needed to inactivate a second enzyme, glutamate synthase, to create a glutamate requirement. Recombinant DNA methods changed more than our ability to unravel the mysteries of microbial genetics. Microorganisms were used to make a panoply of products from interleukins for treating cancer to phenylalanine for manufacturing the sweetener aspartame to enzymes for cleaning drains. The study of cold-active enzymes could lend insight into the basic features that set an enzyme's thermostat for activity. Some isolates belong to previously undiscovered bacterial genera and species based on their physiological properties and 16S ribosomal RNA gene sequences. The group screened the isolates for cold-active enzymes such as glycosidases, phosphatases, and proteases. In one case, three different genes were cloned, each encoding an unusual β-galactosidase, from one Arthrobacter isolate. The study of psychrophilic organisms is just one example of an understudied but important area just waiting for future microbiologists to probe. Discoveries through basic research of unique lifestyles continually amaze us, and new biotechnology products await adventurers with the skill to develop them.

Based on a suggestion of setting up a small consulting company to assist young biotechnology companies in getting ready for FDA inspections, setting up culture collections, and identifying actinomycetes and fungi, the author formed The Biotic Network which soon led to the founding of Blue Sky Research Service, which specialized in designing fermentation conditions for unusual or novel microorganisms and developing new extraction methods. The author also worked part-time at Biosource Technologies with Barry Holtz on finding ways to decrease the compost prep time for mushroom farms and increasing the flavor of mushrooms early in their growth stages. Thermophilic actinomycetes proved to be the key ingredient for a compost inoculum. The Center for Environmental Biotechnology (CEB) has three research focus areas: environmental diagnostics for water quality, biowarfare diagnostics, and environmental monitoring; biogeochemical transformations for bioremediation and synchrotron assessment and validation; and environmental risk assessment based on bioavailability through ingestion, bioabsorption, and inhalation. Natural products will always play an important role in medicine, food, and the environment. The author feels that the twenty-first century will bring more integration of physiology and molecular biology and use the physicists’ tools more to elucidate and validate the structure and function of microorganisms in our world.

At the University of Illinois the author quickly moved to his work in Dr. Gunsalus' laboratory on the isolation and characterization of transducing phages for P. putida. Dr. Gunsalus-known as Gunny-was extremely interested in knowing how and why P. putida exhibited a broad degradative capability. During his postdoctoral years, the author and Dr. Gunsalus came up with some very exciting observations in rapid succession, which showed that in P. putida, the genes for the degradation of rather exotic organic compounds-such as camphor, octane, and naphthalene-occur on plasmids. The researchers had made a significant advance in their basic understanding about the genes involved in the biodegradation of exotic compounds, one that also would have significant practical implications. There are natural substrates in soil, such as lignocellulosic materials, that are often preferred by the organisms rather than the toxic chemicals, whose effective concentrations, such as that of 2,3,7,8-tetrachloro-dibenzo-p-dioxin, might be extremely low to be of meaningful nutritional value to the organisms. Such mechanisms, observed first in Pseudomonas, appear to be universal and may contribute to one's understanding of how other pathogens take over the host cell machinery.

One of the Ph.D. topics on offer for the author at the Cambridge University was the actinomycete Streptomyces. In the pneumococcus, Escherichia coli and Salmonella typhimurium, incomplete genomes were transferred from donor to recipient strains by one of three bizarre processes (transformation, conjugation, and transduction) to yield incomplete zygotes, whereas fungi and higher organisms had life cycles, including a complete diploid stage and meiosis. Lewis Frost gave the author some Streptomyces cultures. The author streaked them out and chose one that produced a striking blue pigment. He set about isolating auxotrophic mutants from this Streptomyces coelicolor strain in order to look for genetic recombination in the way it was done for E. coli. By the early 1950s, few microbiologists regarded the actinomycetes as fungi, but many still thought of them as intermediate between fungi and bacteria. For Streptomyces there were biochemical pointers to a bacterial affinity, but their cellular architecture was unclear. In the 1970s, the author's research group added artificial protoplast fusion to natural, plasmid-mediated conjugation as a means of doing Streptomyces genetics and then, much more important, the ability to transform protoplasts with plasmid or bacteriophage DNA, thus ushering in the in vitro years of gene cloning, which really put Streptomyces genetics on the map. Soon, numerous antibiotic resistance genes had been isolated, and basic aspects of gene expression worked out. They could manipulate the production of antibiotics by streptomycetes, which had enormous potential importance for the emergence of biotechnology in the pharmaceutical industry.

The author took a course in microbiology with C. B. van Niel at the Hopkins Marine Station in Pacific Grove, California. He decided to study the behavior of bacteria and then ultimately broaden out to the behavior of all other organisms. He showed that bacteria indeed do have sensory receptors. Some chemicals are not attractants for motile bacteria, even though the bacteria can use them perfectly well, simply because sensory receptors are not there for those chemicals. The author's research group isolated mutants that cannot respond to this or that attractant because of missing this or that receptor (like people who can't smell or taste certain things). Next they isolated other mutants that are not attracted or repelled by anything at all, though fully motile; they are missing the pathway from sensory receptors to the organs of locomotion-the flagella (like a person defective in the part of the nervous system that carries information from the nose or tongue to the legs). It was then the sensory receptors were identified as structures specialized for sensing an attractant or a repellent (methyl-accepting chemotaxis proteins). Some of the components that carry the information from the sensory receptors to the flagella (chemotaxis, or Che, proteins) were identified.

Cathy Squires worked on the regulation of ribosomal RNA (rRNA) synthesis and felt very fortunate to have had excellent students, postdoctoral fellows, and associates to share this project. They applied genetics, biochemistry, molecular biology, and physiology-the patchwork of his training and interests-to their studies of ribosome synthesis. Transcription regulation was studied using gene fusions to characterize a special feature of rRNA synthesis-transcription antitermination. Cell physiology and genetics were also studied as they relate to copies of ribosomal genes. A strain of Escherichia coli with no intact chromosomal rRNA operons was successfully constructed. The cell's only source of ribosomal genes is a plasmid. By exchanging coli sequences on the plasmid with those of other microbes, the author and his team were able to manipulate a cell's ribosome content in many unexpected ways and thereby pose novel questions about the cell's translation machinery.

As a graduate student, the author was attracted to microbial physiology and metabolism. Techniques such as sonic oscillation had been developed for the rupture of bacterial cell walls; enzyme activity in cell extracts could also be studied. The author worked on the enzyme hippuricase (from Streptococcus), a hydrolytic enzyme that cleaved a peptide-like bond and was considered of interest because the mechanism of peptide bond synthesis was unknown at the time. In 1961, the author began to study methanogenic bacteria, and these studies, over a twenty-year period, led to work on six new coenzymes of methanogenesis: coenzyme M, coenzyme F420, tetrahydromethanopterin, coenzyme F430, methanofuran, and coenzyme B. A major portion of the author's research career was devoted to enzyme-coenzyme relationships in the reduction of carbon dioxide to methane. The first new coenzyme of methanogenesis, coenzyme M (CoM), was required as a vitamin by Methane-bacterium ruminantium. In the summer of 1954, the author became an observer in van Niel's famous course at Pacific Grove. This fantastic experience enabled him to start his own organisms course. Through assignment of special problems, both the students and the author learned something new about an unusual organism each time the course was given. The author had a continuing interest in unusual organisms: Gallionella, Beggiatoa, magnetic bacteria, photosynthetic bacteria, acetogens, and methanogens.

The author's work focused on bacteria associated with marine animals-specifically invertebrates, including shellfish, both mollusks and crustaceans. One of the studies compared microorganisms associated with marine animals from the Rongelap and Eniwetok atolls after the atomic bomb tests. It was a fascinating study because it demonstrated concentration of radioactive elements by microorganisms, work that was confirmed by other investigators in later years and that has relevance for bioremediation in today's society—that is, microorganisms can be used to concentrate and remove radioactive elements from radioactive wastes. The author's interest in marine microbiology expanded to a curiosity about the genetics of marine microorganisms. The initial work demonstrated the presence of plasmids in marine bacteria, especially those bacteria found in harbors and coastal areas into which effluent from sewage treatment plants and industry was discharged. A major limitation to research in microbial ecology has been the inability to isolate, grow, and culture the vast majority of bacteria that are present in the environment. Researchers reported that selected human pathogens, such as Vibrio cholerae, lost the ability to grow on laboratory media after incubation in oligotrophic ocean water or in seawater microcosms in the laboratory for short periods of time (less than one day to three weeks), although cell numbers, by direct microscopic counts, changed little.

Microbiology in Munich was headed by Otto Kandler, a botanist and microbiologist whose scientific emphasis in microbiology was on the physiology and taxonomy of gram-positive bacteria. Previous analysis of peptidoglycan already pointed toward the discrepancy between classification based on classical phenotypic properties and grouping according to chemotaxonomy. A logical next step was to investigate the taxonomic potential of other characteristics, such as the qualitative and quantitative formation of ethanol, acetate, and lactic acid and the reconstruction of the glycolytic and the pentose phosphate pathways. The results of this study had no major impact on one's understanding of the relationships among the coryneform bacteria. The author spent years to understand that all the approaches used in taxonomic studies were different jigsaw puzzles using pieces that did not yet fit together. The rate of evolution between the genotype (as measured by the 16S rDNA) and the phenotype (representing many different genes) does not run isochronically, and clusters defined by high 16S rDNA similarity values may show significant differences in phenotype, which does not necessarily correlate with results of DNA reassociation studies. The introduction of molecular sequencing into systematics and the consequent stability of prokaryotic taxa raised the interest of scientists from different bacteriological disciplines. Derived and catalyzed by the development of polymerase chain reaction (PCR) technology, diagnostic methods were developed that began to change bacterial identification in natural samples. The last twenty years have witnessed radical changes in one's understanding about the role and importance of prokaryotic organisms.

The author along with John Taylor, studied the host's cellular and immune response to inhaled conidia and the development of conidia into spherules (named adiaspores, coined to indicate that these structures showed no evidence of cell division) of Emmonsia parva in host lung tissue. They published together two papers and four abstracts describing the immune response of mice to inhaled conidia. They also noticed that the adiaspores showed definite signs of reproduction as they developed buds. In fact, the adiaspores looked much like the tissue phase of Paracoccidioides brasiliensis and its multiply budded spores, which resemble a ship's wheel. With this observation as the motivating force, they toiled over the proper experimental design to obviate environmental contamination of the amber samples. To further reduce the possibility of contamination, gut tissue was extracted and cultured under stringent containment conditions. From this experiment, a culture of Bacillus sphaericus was isolated that, to this date and after numerous verifying experiments, is thought to have originated from the amber inclusion.

The research program in Amy Cheng Vollmer's own lab focused on bacterial stress response, and she and her team of researchers have been studying the role of the universal stress protein (UspA), which they believe, based on recent work, acts as a set of "brakes" on most of the stress responses in the cell. Absence of this protein results in an "over-reaction" to stress and drains Escherichia coli of its precious energy reserve. Presently, they are investigating the roles of the two genes that show homology to UspA to determine if they also serve in similar capacities. They are also investigating bacterial responses to ultrasound in E. coli as part of a study to determine how ultrasound can be best used to sterilize water and contaminated objects. The stress response field is exploding with new ideas and discoveries in the areas of microbial ecology, pathogenesis, genetics, physiology, and general microbiology. In the author's field of research (bacterial stress response), she meet interesting and dynamic people who work on ecological, medical, and evolutionary problems. The author's professional society, the American Society for Microbiology (the ASM), represents a diverse community of people and works to further the professional development of its members, as well as to encourage potential new members.

The author sets some goals for himself at the start of every new academic year, which include: to make himself more accessible to his students and to make his office a place that students enjoy visiting and to give increased emphasis to active and collaborative learning. The process of learning science should model the methods practiced by scientists. The author attempts to serve all his students well while keeping an eye open for the exceptional; to encourage thinking, not recitation. The author plans to assign problems that challenge and exercise the minds of his students, not their capacity to memorize. He is convinced of the central position of teaching in the field of microbiology. Teaching is scholarship, not peripheral to it. As a microbiologist one owes his/her profession to one or more mentors who took an interest in them as developing scientists. The author considers it as great pleasure when his former students visit him and to find them excited about their work and to know that he had a part in their success.

Among the many outstanding teachers, the author acknowledges the extraordinary efforts of Sterling Wallace, an uppergrade school teacher, and Mr. Sanders and Ms. Hope, my high school chemistry and physics and algebra-geometry teachers, respectively. With little to no financial resources, the author initially attended Tuskegee Institute (now Tuskegee University) as a five-year "work-study" student and subsequently transferred to Miles College, a small historically black college in Birmingham. This multiyear experience, along with excellent professors in organic chemistry, biochemistry, genetics, classical structural studies, and histology, afforded the author a productive and enjoyable undergraduate tenure. The author had the distinct pleasure to join the laboratory of Fred Neidhardt as a graduate student, and his research focused on the regulation of amino acid and aminoacyl-tRNA synthetase synthesis in Escherichia coli. This was a rare opportunity to learn microbial genetics, biochemistry, and physiology under the supervision of an intellectually demanding, professionally sophisticated, and seasoned mentor. In the spring of 1969, the author joined the faculty of Atlanta University. In a very short period of time, he was soon mentoring a very large pool of graduate students. The most important of the activities in which we engaged was the creation of an intellectual ambiance that was nurturing to the students who came with different preparations, different interests, and different perceptions of their self-worth—they were placed in an environment that simply disallowed those differentials.

The author started to build a research program on biodegradation without the preparation of a postdoctoral experience. This made for a fairly slow start. As the department’s only microbiologist, the author found himself involved in a variety of projects, including drinking water safety, virus detection techniques, and pesticide analysis. His research group began developing techniques that used radiolabeled compounds to measure pollutant metabolism in a variety of environmental matrices. According to the author, for microbial transformations to occur in any particular environment, there needed to be a combination of the right organism, the presence of the substrate chemical in a form the microbes could access, and a set of favorable environmental conditions that will dictate whether and how fast degradation may occur. The goal of our research in diverse environments has been to understand how the environmental component regulates what happens. An early manifestation of this goal was the work on community adaptation. Our microbiology, combined with Russ' knowledge of soil organic materials, offers the potential to understand how these two major components of the soil microenvironment relate to each other.

Looking back, the author can see the path with all its twists and turns that has led him to his current position in microbiology. It has been a career path full of serendipity and surprises. His fascination with science and microorganisms began early. A career as a physician, not as a scientist, seemed the likely career path as the author went off to college. During his graduate years, the author learned a great deal about what was involved in being a microbiologist. There were the courses, but, more important, there was the laboratory. Initially, the author tried to work on two projects—one a physiologic project on the requirement for nickel by hydrogen-utilizing bacteria and the other an ecological project on the microbial utilization of petroleum hydrocarbons. The author's interest in teaching has also led him to write textbooks. When he began teaching microbial ecology, only a few textbooks had been written in that field, and the author tried teaching using only handouts and assigned readings. The students objected, and the author soon found himself writing a microbial ecology textbook with Richard Bartha, which was completed after years of hard work.