ARTICLES Microbial Pathogenesis: and Beyond Evelyn J. Strauss* and Stanley Falkow

The growing number of complete microbial genome sequences provides a powerful quence has been used to identify candidate tool for studying the biology of . In combination with assays for virulence genes and components of the bio- function, genomic-based approaches can facilitate efficient and directed research synthetic machinery for lipopolysaccharide strategies to elucidate mechanisms of bacterial pathogenicity. As genomic information (LPS) (3, 4), a molecule known for decades accrues, the challenge remains to construct a picture of the biology that accurately to function in pathogenesis. LPS is critical reflects how individual genes collaborate to create the complex world of microbial in maintaining the integrity and function of specialization. the outer cell membrane of Gram negative and is an essential component of a successful infection. However, LPS is such a potent activator of the immune system that Entering the Genomic Era— This can assist in the immediate design of the response against it can have devastating New Challenges experiments to deduce gene function; the consequences for the host as well as the sequence may suggest possible biochemical . For many biologists, the late 1990s mark activities, implied by the presence of motifs Many pathogens, including M. geni- the beginning of the era of genomics. For or homology to genes with known activi- talium and H. influenzae, are able to con- the first time in history, we have access to ties. Conversely, if there is interest in a found the immune system by varying the the entire genetic content of a growing particular function that is suggested by a expression of bacterial envelope molecules. number and variety of living creatures. This conserved amino acid or nucleotide se- This process, called phase variation, can be information has the potential to open in- quence, candidate genes can be identified mediated by the loss or gain of one or more novative and efficient research avenues. simply by searching the complete sequence nucleotide repeating units, which alters However, homology is not function, and of the organism of interest. These motif- transcription signals or changes the reading the burden for the biologist is to translate based approaches can provide an intellec- frame of genes encoding cell surface mole- inanimate DNA sequences into cellular ac- tual framework when information about bi- cules. The Haemophilus genome was tivities. The availability of extensive ological function is sought. searched and nine novel loci with multiple genomic information frees us to develop General information about a microbe tandem tetranucleotide repeats were identi- new genetic and molecular approaches by can also be derived from examining ge- fied (3). Several of these genes encoded which to examine microbial populations in nome sequences. It is now possible to es- homologs of bacterial envelope factors, and host organisms and the environment. timate how much of an organism’s genom- another (lgtC) was shown to be involved in In this article, we discuss some of the ic content is invested in different biosyn- the phenotypic switching of an LPS promises and limitations of using genom- thetic or metabolic processes. This can epitope. Moreover, an H. influenzae strain ics—the combination of complete genome yield insight into the organism’s activities, carrying a disruption in lgtC is less virulent sequences and the informatic tools with if one assumes that the space allotted to a than the parental strain in an animal model which to analyze them—to study bacterial given function correlates with its impor- of disease. pathogenicity. We review some of the strat- tance to the organism. For example, the Despite long familiarity with the impor- egies that can be used to exploit genomics, streamlined genome of Mycoplasma geni- tance of LPS, many aspects of its biosyn- and we point out caveats of various ap- talium commits almost 5% of its content to thetic pathway in H. influenzae have re- proaches. We also survey several techniques encoding an adhesin gene and related se- mained elusive. The Haemophilus genome that are complementary to genomics, al- quences (1); it is thought that the organ- was searched for DNA and amino acid sim- lowing direct examination of the genetic ism uses these sequences to produce anti- ilarity to genes known to be involved in basis of virulence. Finally, we discuss chal- genic variants of the protein in order to LPS production in other organisms (4). lenges of the future and how genomics evade the immune response. What is ab- Targeted gene disruptions, immunochemi- may help us surmount them. Although our sent from a genome can be informative as cal techniques, tricine-T–SDS–polyacryl- focus is pathogenesis, many of the con- well. For example, Haemophilus influenzae amide gel electrophoresis, and mass spec- cepts we discuss can be applied to other appears to be missing three of the enzymes trometry were used to examine potential areas of . needed for the tricarboxylic acid cycle (2), roles in LPS biosynthesis for the 25 genes a finding that is surprising but might yield identified, and strains carrying mutations in Providing More Clues and insight into key features of the metabolic many of these genes were shown to have Saving Time strategy that this fastidious organism uses altered LPS structures. The analysis has al- to survive exclusively in humans. ready provided information of direct rele- With the aid of a database, a small amount vance to pathogenesis: The behavior of of DNA sequence can provide a view of an Using Genomics to Identify strains containing mutations in these genes entire gene and its neighbors in minutes. Genes That Are Important allowed predictions of the minimal LPS for Virulence structure required for efficient intravascu- The authors are in the Department of Microbiology and Immunology, Stanford University School of Medicine, lar dissemination of H. influenzae in the Stanford, CA 94305–5402, USA. The complete H. influenzae sequence was infant rat. *To whom correspondence should be addressed. E-mail: finished in 1995 (2), and the potential of There is a more general lesson here as [email protected] genomics is already being realized. The se- well. In the case of LPS biosynthesis, it is www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 276 ⅐ 2 MAY 1997 707 not uncommon for proteins of related ecule causes the EHEC-specific symptoms employing subtractive hybridization with function to be encoded by genes of homol- (7). Indeed, when Shiga-like toxin I was computer informatics. ogous amino acid, but divergent nucleo- expressed in the rabbit pathogen, rabbit Sequence not only provides information tide, sequence. Of the genes that were diarrheal E. coli-1 (RDEC-1), the bacteri- useful for distinguishing closely related sets described, 60% could not have been reli- um caused an illness in rabbits that closely of organisms, but it also raises some broad ably identified by DNA sequence alone resembles an EHEC-like colitis rather questions about the nature of pathogenicity. and, therefore, would not have been found than the diarrheal disease normally seen Although pathogenesis is rare in general by nucleic acid hybridization experiments, with RDEC-1(8). In this case, the pres- (when one considers the low fraction of with the use of either conventional blot- ence or absence of the toxin defined these extant organisms that are virulent), ample ting techniques or a computer. strains before elucidation of the relevant numbers of pathogenic eukaryotes and pro- genetic determinant. However, if the en- karyotes exist. In contrast, there are no Using Clues About the Essentials tire sequences of both organisms had been known members of the archaea kingdom of Life to Study Microbial complete before the initiation of these that are virulent, despite the fact that some Pathogenesis studies, the significance of the Shiga-like archaeans share our environment and are toxin I gene might have been deduced by part of our normal bowel flora. We do not Genomics has the potential for informing us about the minimal gene set required for Table 1. Genome sequencing projects cover a wide range of organisms [courtesy of C. Venter, The cellular life. The first two publicly avail- Institute for Genome Research (TIGR)]. Updated information can be found at the TIGR worldwide web able whole bacterial genomes (H. influen- site, www.tigr.org HGE indicates human granulocytic ehrlichiosis. zae and M. genitalium) were compared (5), and a catalog of genes conserved in both Organism Organization Size (Mb) organisms was compiled, adding genes to fill in the missing steps of critical meta- Bacteria Actinobacillus University of Oklahoma 2.20 bolic pathways that are encoded by dissim- actinomycetemcomitans ilar genes in the two bacteria and subtract- Aquifex aeolicus Recombinant BioCatalysis Inc. 1.50 ing apparently redundant and organism- Bacillus subtilis European and Japanese consortium 4.20 specific genes. Because such a list may Borrelia burgdorferi* TIGR 1.30 represent a minimal metabolic core of re- Caulobacter crescentus TIGR 3.80 actions for all cells (or at least for cells Deinococcus radiodurans TIGR 3.00 that live in similar environments), sub- Ehrlichia species (HGE agent) TIGR 1.40 tracting it from a test organism’s total Enterococcus faecalis† TIGR 3.00 genetic inventory should reveal factors Escherichia coli* University of Wisconsin 4.60 TIGR 1.83 that specify the phenotypic characters Haemophilus influenzae‡ Helicobacter pylori* TIGR 1.66 that make the organism unique. If this Legionella pneumophila TIGR 4.10 exercise is carried out on a pathogen, the Mycobacterium avium TIGR 4.70 resulting gene set should include traits Mycobacterium tuberculosis TIGR (clinical isolate) 4.40 required for virulence. Mycobacterium tuberculosis Sanger Center (lab strain) 4.40 Many bacterial virulence genes are Mycoplasma genitalium‡ TIGR 0.58 found as discrete segments, present in Mycoplasma pneumoniae‡ University of Heidelberg 0.81 pathogenic organisms but absent from University of Oklahoma 2.20 nonpathogenic members of the same Neisseria meningitidis TIGR and Oxford University 2.30 TIGR 2.20 genus or species (6). These pathogenicity Porphyromonas gingivalis Rickettsia prowazekii University of Uppsala 1.10 islands often display distinct codon usage Salmonella typhimurium† TIGR 4.50 and a different overall base composition Shewanella putrefaciens† TIGR 4.50 from the core chromosomal elements, Streptococcus pneumoniae TIGR 2.20 suggesting that they were acquired from a Streptococcus pyogenes University of Oklahoma 1.98 foreign source. These genetic features Synechocystis sp. ‡ Kazusa DNA Research Institute 3.57 provide possible criteria for identifying Thermotoga maritima TIGR 1.80 potential virulence genes when scanning a Treponema denticola TIGR and University of Texas 3.00 complete chromosomal sequence. Treponema pallidum* TIGR and University of Texas 1.05 Although pathogenicity is often multi- Ureaplasma urealyticum University of Alabama, Birmingham 0.76 Vibrio cholerae TIGR 2.50 factorial, the inheritance or alteration of a Archaea single virulence factor can profoundly Archaeoglobus fulgidus* TIGR 2.20 change the infectious process that a Halobacterium salinarium Max Planck Institute for Biochemistry 4.00 pathogen causes in its host. For example, Methanococcus jannaschii‡ TIGR 1.66 infection by enteropathogenic Escherichia Methanobacterium Genome Therapeutics and Ohio State University 1.70 coli (EPEC) is characterized by a watery, thermoautotrophicum often chronic, diarrheal disease. A distinct Pyrobaculum aerophilum University of California Los Angeles and 1.80 E. coli strain, enterohemorrhagic E. coli Recombinant Biocatalysis Inc. Pyrococcus furiosus Center of Marine Biotechnology and University of 2.10 (EHEC) causes bloody colitis and some- Utah times hemolytic uremia syndrome. Yet, Pyrococcus shinkaj MITI and University of Tokyo 2.00 EHEC shares many of the major virulence Sulfolobus solfataricus Canadian and European consortium 3.05 genes of EPEC. In addition, EHEC carries Thermoplasma acidophilum Max Planck Institute for Biochemistry 1.70 a Shiga-like toxin that is not present in *Expected to be completely sequenced by Spring 1997. †Application submitted, funding pending. ‡Com- EPEC; it has been proposed that this mol- pletely sequenced and published. 708 SCIENCE ⅐ VOL. 276 ⅐ 2 MAY 1997 ⅐ www.sciencemag.org ARTICLES know why the archaea are absent from the interpreted. The statistical information Uninformative and Even group of pathogenic microbes, but as we that is currently available from database Misleading Sequence continue to collect and examine microbial searches is often underutilized. Fortunate- Comparisons genome sequences, informative patterns may ly, there are numerous resources, including emerge that reveal fundamental distinctions the Internet, that can facilitate wise use of Approximately 42% of the Haemophilus between commensals and pathogens. A pop- available sequence information (12). It is genes are either orphans, not belonging to ular paradigm in the field of pathogenesis is critical to determine whether the gene of any established gene family, or are similar to one in which normally harmless bacteria interest is a true functional homolog of a genes of unknown function (2). These se- acquire virulent properties; the archaeans gene with known activities, or if they quences, therefore, do not immediately lend may be an indication that, in some cases, share only some features. Finally, even if insight into biological activity. there are more fundamental differences be- a gene contains a motif that always signi- Despite low or undetected sequence tween organisms that cause us harm and fies a particular enzymatic behavior, the similarities (even using the relatively lax those that live peacefully within us. gene’s function may remain unclear. For requirement of amino acid sequence simi- example, in vitro activity often does not larity), proteins still may share functions. Number and Types of Sequences reveal the relevant biological substrate or For example, Listeria monocytogenes and substrates. Shigella flexneri display strikingly similar Table 1 lists the organisms that have a Finally, even if a gene contains a behavior inside host cells. They both har- completed genome sequence and those recognizable motif and the motif can ness actin, using the force generated by whose genomes are currently being se- function as predicted, it is still pos- actin polymerization to infect adjacent quenced. It contains a wide range of mi- sible that the molecule’s biological rele- cells (Fig. 1) (14). In each case, one bac- crobes, including many pathogens. The vance lies in a different arena or is more terial molecule is used to initiate actin pace of research in this area is increasing subtle than expected. An example of this assembly (ActA in L. monocytogenes and rapidly. Although several of these sequenc- is the case of calmodulin, a calcium- IcsA in S. flexneri). However, ActA and es are currently proprietary, the amount of binding protein required for viability in IcsA have virtually no sequence similarity. publicly available genomic information is Saccharomyces cerevisiae. Although cal- This theme of evolutionarily unrelated growing quickly. The political issues and modulin possesses calcium-dependent func- molecules performing similar functions is practical implications surrounding private tions in yeast, a calmodulin deriva- reiterated and strengthened by the obser- ownership of genome sequences are subjects tive that no longer binds calcium is vation that vaccinia virus, which shares of great importance, but ones that are more still able to perform its essential activities. the ability to recruit host cell actin, con- appropriate for a news article (9). This was a surprising finding and one anti- tains no genes with resemblance to either The choice of genomes to examine has thetical to functional predictions based on ActA or IcsA (15). The genes used to significant impact on the potential use of sequence (13). harness actin would not be identified in a sequence information. It is sobering to realize that the full sequence of E. coli K-12, when available, is likely to have no AB more than a handful of the known viru- lence determinants of pathogenic Esche- richia. Even carefully choosing one patho- genic member of a species is insufficient. Diversity within species produces pro- foundly different outcomes. The complete sequence of one H. influenzae strain is available. Yet, of the 104 H. influenzae clones that exist, six cause about 80% of all human disease (10). Why are these clones different from any other clone? It is not immediately obvious from the genom- ic sequence of one organism why only a few emerged in recent history as major human pathogens. It is impractical and impossible, of course, to sequence every organism in existence. Technological in- novations (11) promise to allow large- scale genome comparisons, thus expand- ing the usefulness of information from the genomes that are sequenced. Once strain- or pathogen-specific genes are identified, however, the challenge remains to devise ways to study their biological functions. Fig. 1. Listeria monocytogenes and S. flexneri use actin to propel themselves within host cells. (A) Using Care When Analyzing Listeria monocytogenes in Ptk-2 cells were labeled with rabbit ␣-ActA polyclonal serum, followed by Sequences Texas red–labeled ␣-rabbit antibody (red), and actin was labeled with fluorescein-phalloidin (green) (courtesy of J. Theriot). (B) Shigella flexneri in HeLa cells were labeled with rabbit ␣-LPS polyclonal Once candidate virulence genes are iden- serum, followed by rhodamine-labeled ␣-rabbit antibody (orange), and actin was labeled with tified, the sequence information must be NBD-phalloidin (green) (reprinted with permission from Molecular Microbiology). www.sciencemag.org ⅐ SCIENCE ⅐ VOL. 276 ⅐ 2 MAY 1997 709 genomics-based approach. quired for invasion is keyed to a precise sages are produced in the experimental sys- Sequence similarity can be misleading in combination of pH, osmolarity, and oxygen tem of choice. An approach was devised to some cases. For example, Salmonella and S. concentration (19). Presumably, these con- study globally regulated genes in E. coli, flexneri share similar virulence factor secre- ditions reflect signals that the bacteria ex- using overlapping lambda clones spanning tion systems (16), and some of the secreted perience when they encounter the specific the genome to map and clone genes whose molecules functionally complement each type of epithelial cell in the Peyer’s patch transcription levels change in response to other. The S. flexneri ipaB gene product that they invade. Understanding when and various conditions (20). New methods that plays an essential role in host cell invasion where gene products act is an essential com- employ high-density nucleic acid probe ar- and subsequent escape from the endosomal ponent of elucidating mechanisms of patho- rays on chips have been used to examine vacuole. The Salmonella homolog, SipB, is genicity, and one that illustrates some of mRNA levels in several systems (21). The required for Salmonella invasion, and it can the limitations of using sequence informa- power of these techniques lies in their abil- complement the invasive defect of a S. tion alone to discern function. ity to assay expression of thousands of genes flexneri ipaB-strain (17). This functional in parallel and their semiautomated and complementation is not surprising, because Getting at Function quantitative properties. With such ap- both organisms invade epithelial cells. proaches, a series of message snapshots can What is notable is that sipB also overcomes Genomics can provide clues, but scientists be compiled that documents gene expres- the inability of the mutant Shigella strain to in the age of complete genomic sequences sion temporally, such as during an infec- escape from the vacuole into the cytoplasm: are still left with the challenge of determin- tion, or spatially, at different sites in the In contrast to Shigella, Salmonella remain ing function. Conventional genetic and host’s body. inside the vacuole throughout their intra- biochemical approaches remain vital to A genetic approach was devised for cellular phase. The Salmonella gene is thus these types of endeavors. In addition, new identifying genes that are specifically in- performing a function in Shigella that it does tools are being used to probe microbial in- duced during infection (22). The general not normally display in its parental organ- teractions within the host or environmental method of in vivo expression technology ism. Presumably SipB’s inherent ability to reservoir. The coordinate expression of a (IVET) is to make a library in which dissolve the membrane is blocked in Salmo- subset of genes in the host is often necessary random genomic fragments are ligated to a nella. In this case, sequence scanning and for an organism’s ability to colonize, sur- gene for a selectable marker that is re- functional complementation might lead to vive, and replicate successfully. Identifica- quired for survival in the host animal. the conclusion that SipB and IpaB perform tion of bacterial genes expressed preferen- Only those bacteria harboring a fusion identical functions in their native strains. tially within infected cells and animals is that contains an active promoter will sur- Just because a factor can do something does critical in understanding how bacterial vive passage through the host. Fusions not mean that it does. pathogens circumvent the immune system bearing promoters with constitutive activ- A gene’s sequence does not necessarily and cause disease. ity can be identified and discarded by reveal when its corresponding product is examining reporter activity on laboratory expressed during the life of the microorgan- Selected Modern Approaches medium. By harvesting bacteria from dif- ism. A critical feature of any successful in- ferent sites in the body, a list of genes fection, and indeed any microbial life cycle, With the complete sequence of an organism required for different stages of infection is responsiveness to extracellular cues (18). in hand, it is possible in principle, to pre- can be compiled. The general method of Such conditions can be complex, and the cisely define which genes are expressed un- IVET can be applied to a variety of situa- microbial response must be precise to en- der any particular set of conditions. This tions where a pair of experimental condi- sure successful infection. Thus, it is not can be accomplished in a number of ways, tions can be compared. The IVET system sufficient to know biochemical activity; one and the efficiency of these methods can be can also be altered to vary the selective must also uncover the circumstances under significantly enhanced by use of informa- stringency. One strategy demands only which a particular molecule acts. In Salmo- tion from genomics. that the promoter be activated briefly in nella typhimurium, expression of genes re- One technique is to survey which mes- the host (23). This will yield those genes that are required at a specific time during, rather than throughout, infection. Identifying host-induced bacterial genes with fluorescence-based methods is especially powerful because it allows high throughput and semiautomation. Green fluorescent protein (Gfp) can be expressed in a variety of microorganisms without adversely affecting their pathogenicity. A procedure called differential fluorescence induction (DFI) has been developed, in which bacteria bearing random transcrip- tional fusions to gfp are sorted by a fluo- rescence-activated cell sorter on the basis of stimulus-dependent synthesis of Gfp (24). The methodology has been em- ployed to identify genes of Salmonella that respond to an acidic environment as well as those genes that are exclusively ex- Fig. 2. RAW 264.7 cells were infected with S. typhimurium expressing Gfp fused to a macrophage- pressed within macrophages (Fig. 2). With inducible promoter (courtesy of R. Valdivia). the use of a fluorescence-activated cell 710 SCIENCE ⅐ VOL. 276 ⅐ 2 MAY 1997 ⅐ www.sciencemag.org ARTICLES sorter to select for Gfp expression, the with all the pieces of a complex jigsaw helpful in both assigning a pathogen to a selection parameters can be adjusted such puzzle; as we decipher function, new pieces particular group and in inferring properties that bacterial clones can be separated on will suddenly fit, and the picture will take based on that classification. Such informa- the basis of small differences in expression on a recognizable form. tion may suggest methods by which to levels. Because selections are independent culture the pathogen or interfere with its of nutritional requirements or drug suscep- Future Challenges growth. tibility, this method uncouples metabolic Genomics provides us with a snapshot requirements from selection parameters. The techniques discussed here are most of a microorganism in its current state of A powerful screening method has been powerful when used in conjunction with evolution. But bacteria evolve significant- used to identify genes that are required for experimental systems that include a host or ly within a single human lifetime. One survival in a host (25). This signature- host cells that accurately reflect conditions need only look at the impact of worldwide tagged transposon method (STM), like of a natural infection. Virulence genes and antibiotic use on natural selection to see IVET, requires passage through a host, but their functions will be completely under- the sudden appearance of new bacterial it does not measure promoter activity; stood only when we have elucidated the clones that differ significantly from those rather, it determines whether disrupting a signals provided by these environments. In seen in historical memory. In 50 years, the particular gene adversely affects survival. the meantime, however, IVET, STM, and sequence of a representative pathogen In STM, each member of a complex li- DFI allow us to probe bacterial-host inter- could be significantly different than it is brary of mutants is marked with a unique actions in the absence of full knowledge now. One benefit of collecting microbial oligonucleotide sequence. If a mutant is about the relevant chemical and physical sequences will be the opportunity to doc- absent after passage of the library through cues. These methods facilitate identifica- ument evolutionary changes at a level of an infected animal or another selective tion of expressed sequences or genes impor- precision previously unattainable. environment, the mutation it harbored tant under a given set of circumstances Counterintuitive as it may seem, many may be in a gene essential for survival. without requiring that the conditions be of the most notorious diseases in recent The first application of STM was highly replicated accurately in a test tube. The decades are illnesses of human progress. successful and led to the discovery of a approaches do not necessarily elucidate For example, the shift in the West from previously unknown pathogenicity island roles of the gene products. In some cases, baths to showers has had a profound effect necessary for Salmonella survival in the however, knowing when or where a factor is on microorganisms such as Legionella pneu- spleen (26). required, in combination with information mophila, which can be transmitted by aero- IVET, STM, and DFI represent the first about its sequence, can lead to reasonable solization in potable water supplies. We experimental forays to detect and follow and testable hypotheses of gene function. have created new environmental niches specific virulence factors at discrete stages Although we have developed some tools for many organisms, thus introducing of interaction between the mammalian with which to probe pathogen-host interac- them into human proximity (29). As the host and the invading microorganism. tions, we have barely begun to study what new scientific discipline of genomic anal- Complete genomic sequence information happens to pathogens in the environment. ysis of bacteria matures and our database will be helpful in using these methods Events during the environmental stage of a grows, it will permit us to better under- because, once a promoter or disrupted pathogen’s life cycle can have profound ef- stand the nature of the delicate balance gene is identified, the entire gene to fects on their potential for causing infec- that exists between us and the microor- which it corresponds can be easily isolat- tion. For example, Vibrio cholerae enters a ganisms that inhabit our bodies. ed. Furthermore, identifying candidate viable but not culturable state (VBNC) un- Just as bacteria have served as model virulence genes by comparative approach- der particular conditions (27). Organisms in systems in which to develop genetic tech- es (such as those described) may increase this state remain a serious threat to human niques, their small and manipulable ge- the efficiency of these genetic methods. populations because certain cues can trigger nomes promise to serve the global scien- The availability of such gene subsets exit from VBNC to the infectious form of tific community well as we embark on this should allow investigators to use directed, V. cholerae. It is essential to understand new genomic path. The 21st century will rather than open-ended, strategies, at least such transitions in order to gain a coherent no doubt usher in ways to tap the vast for the many pathogens in which targeted view of this pathogen’s life cycle and to resource of genomic information as we use gene disruption technology is available. hope to control cholera epidemics. our ingenuity to understand microbes and Eventually, it may be possible to build Studies of entire microbial populations their intimate relationship with us and all upon the genetic methods, adapting them in the wild aimed at identifying the inhab- life on the planet. for use in conjunction with the high-den- itants of particular environments (28) may REFERENCES AND NOTES sity probe array systems and exploiting the segue nicely into the pathogenicentric ______power of semiautomation inherent to world presented in this review. For exam- 1. C. M. Fraser et al., Science 270, 397 (1995). these techniques. 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demonstrated that it is important not only Protein Localization and Cell to understand what a protein does but Fate in Bacteria where and when it acts. Asymmetric Cell Division Lucy Shapiro and Richard Losick Differentiation in C. crescentus and B. sub- tilis results from a modified process of cell A major breakthrough in understanding the bacterial cell is the discovery that the cell is division (6, 7). Unlike the familiar process highly organized at the level of protein localization. Proteins are positioned at particular of binary fission in which a dividing cell sites in bacteria, including the cell pole, the incipient division plane, and the septum. generates identical progeny, C. crescentus Differential protein localization can control DNA replication, chromosome segregation, and sporulating B. subtilis undergo cell di- and cytokinesis and is responsible for generating daughter cells with different fates upon vision asymmetrically, each generating dis- cell division. Recent discoveries have revealed that progression through the cell cycle similar progeny with distinct fates (Fig. 1). and communication between cellular compartments are mediated by two-component Asymmetric division in C. crescentus is an signal transduction systems and signaling pathways involving transcription factor ac- obligatory feature of its cell cycle. Thus, a tivation by proteolytic processing. Asymmetric cell division in Caulobacter crescentus dividing cell generates a motile, swarmer and sporulation in Bacillus subtilis are used as paradigms for the control of the cell cycle cell, which is propelled by a rotary flagel- and cellular morphogenesis in bacterial cells. lum, and a nonmotile, stalked cell. The stalked cell replicates its chromosome and undergoes differentiation into a predivi- sional cell with a nascent swarmer cell on Temporal and spatial constraints restrict copy and fluorescence microscopy to study one side of the division plane and the how a bacterium gleans information from the subcellular organization of bacterial stalked cell on the other. Meanwhile, after its genome. These constraints dictate the cells has revealed a surprising extent of an interval in which DNA replication is ordered expression of cell cycle events, the protein compartmentalization and localiza- repressed, the swarmer sheds its flagellum three-dimensional organization of the cell, tion. In some instances, localized proteins and undergoes metamorphosis into a and the biogenesis of subcellular organelles are not essential for viability, as is the case stalked cell, which repeats the cycle. and altered cellular forms. Each of these for the flagellum and the chemosensory ap- Asymmetric division in sporulating B. general classes of cellular functions is pro- paratus (1), actin-recruiting virulence pro- subtilis, in contrast, is an environmentally grammed by a genetic network that in- teins in certain pathogenic bacteria (2), and induced adaptation to conditions of nutri- cludes a pathway of regulatory events. The certain morphogenetic and regulatory pro- tional stress. Bacillus subtilis normally prop- combination of bacterial genetics and new teins involved in the process of sporulation agates by binary fission. However, when ways of using cytological methods to exam- (3–5). In other cases, such as the proteins nutrients become limiting, it undergoes a ine bacterial cell organization has not only involved in DNA replication, chromosome switch from symmetric to asymmetric divi- led to the identification of the components segregation, or cell division, the site of pro- sion by the formation of a polar septum. of these regulatory pathways, but has placed tein deposition is an integral part of the The polar septum partitions the developing the pathway outcomes at specific sites in regulatory machinery that mediates these cell into a small, forespore cell and a large, the cell. Analysis of two bacteria separated essential functions. A critical question now mother cell. Initially, the forespore and the by large evolutionary distance, the Gram- facing the prokaryotic community is how mother cell lie side by side in the sporangi- negative, aquatic bacterium Caulobacter proteins are deployed in a targeted three- um. Later, the forespore becomes wholly crescentus and the Gram-positive, soil bac- dimensional array in the absence of known engulfed by the mother cell in a phagocytic- terium Bacillus subtilis, has elucidated com- cytoskeletal components. Notwithstanding like process to create a cell within a cell. mon mechanisms that serve as paradigms free diffusion in the microenvironment of The forespore ultimately becomes a spore for prokaryotic cell cycle control and cellu- the bacterial cell, how are proteins directed and gives rise to future progeny, whereas the lar morphogenesis. to specific sites in the cell, and how are they mother cell is discarded by lysis when mat- The use of immunogold electron micros- kept there? What controls the rearrange- uration of the spore is complete. ment of proteins during morphogenesis? As L. Shapiro is in the Department of Developmental Biolo- we will show, C. crescentus and B. subtilis Polarity and Protein Localization gy, Beckman Center, Stanford University School of Med- offer numerous and striking examples of icine, Stanford, CA 94305–5427, USA. R. Losick is in the subcellular localization of proteins. These, In C. crescentus, the cell cycle perpetuates Department of Molecular and Cellular Biology, The Bio- logical Laboratories, Harvard University, Cambridge, MA and examples from other bacteria, have asymmetry that is present before cell divi- 02138, USA. changed our view of the bacterial cell and sion. The predivisional cell is itself asym- 712 SCIENCE ⅐ VOL. 276 ⅐ 2 MAY 1997 ⅐ www.sciencemag.org