]'g-~EVIEWS 4 Edwards, G.E. and Walker, D.A. (1983) C3, C4: 5

Mechanisms and Cellular and Environmental Regulation of Photosynthesis, Blackwell Scientific Publications Furbank, R.T. and Foyer, C.H. (1988) NewPhytol. 109,

265-277 6 Brown, W.V. (1975) Am.J. Bot. 62, 395-402 7 Kirchanski, S. J. (1975) Am.J. Bot. 62, 695-705 8 Edwards, G.E. and Huber, S.C. (1979) in Encyclopedia of Plant PbysioloRy, New Serfes Vol. 6 (Gibbs, M. and Latzko, E., eds), pp. 102-112, Springer-Verlag 9 Steeves, T.A. and Sussex, 1.M. (1988) Patterns in Plant Development, Cambridge University Press 10 Sharman, B.C. (1942) Ann. Bot. 6, 245-282 11 Poethig, R.S. (1984) in Contemporary Problems in Plant Anatomy (White, R.A. and Dickison, W.C., eds), pp. 235-259, Academic Press 12 Sylvester, A.W., Cande, W.Z. and Freeling, M. (1990) Development 110, 985-1000 13 Maksymowych, R. (1973)Analysis of Leaf Development, Cambridge University Press 14 Langdale, J.A., Metzler, M.C. and Nelson, T. (1987) Dev. BioL 122, 243-255 15 Langdale, J.A., Rothermel, B.A. and Nelson, T. (1988) Genes Dev. 2, 106--115 16 Langdale, J.A., Zelitch, I., Miller, E. and Nelson, T. (1988) ~ B O J . 7, 3643-3651 17 Esau, K. (1943) Hilgardia 15, 327-368 18 Langdale, J.A., Lane, B., Freeling, M. and Nelson, T, (1989) Dev. Biol. 133, 128--139 19 Fahn, A. (1982) PlantAnatom~ (3rd edn), Pergamon Press 20 Antonielli, M. and Venanzi, G. (1979) Plant Sci. Lett. 15, 301-304 21 Dengler, R.E. and Dengler, N.G. (1990) Can.J. Bot. 68, 1222-1232

F l a g e l l a are reversible rotary devices, driven by protonmotive force, that propel bacteria through liquid environments 1. Changes in the direction of flagellar rotation produce turning movements that can be modulated by sensory systems to elicit selective migration or taxis. As well as having important roles in microbial behavior, flagella are themselves of intrinsic interest to microbiologists. They are also of general interest as models for a variety of cellular processes, including regulation of expression of a large gene system, and assembly of a structure that mostly exists beyond the cell surface. We concentrate here on these two topics and the genetic methods that have been used to elucidate them in the c o m m o n enteric bacteria Escherichia coli and Salmonella typhimurium. As a subject for study, the flagellar system has an enormous advantage over many systems of comparable sophistication and complexity because flagellation, motility and tactic responses are all dispensable functions for the cell, and thus readily amenable to genetic dissection. Genes associated with the motile behavior of bacteria were early on divided into three phenotypic categories: those necessary for flagellation (Fla), motility (Mot) and chemotactic responsiveness (the). As more became known about the mechanism of motility and taxis, mot genes were more precisely defined as being

22 Hattersley, P.W., Watson, L. and Osmond, C.B. (1976) Aust. J, Plant Physiol. 4, 523-539 23 Sheen, J-Y. and Bogorad, L. (1986) EMBOJ. 5, 3417-3422 24 Cheng, S-H. et al. (1989) Plant Physiol. 89, 1129-1135 25 Hudspeth, R.L., Glackin, C.A., Bonner, J. and Grula, J.W. (1986) Proc. Natl Acad. Sci. USA 83, 2884-2888 26 Glackin, C.A. and Grula, J.W. (1990) Proc. NatlAcad. Sci. USA 87, 3004-3008 27 Borsch, D. and Westhoff, R (1990) FEBSLett. 273, 111-115 28 Nelson, T. and Langdale, J.A. (1989) Plant Cell 1, 3--13 29 Berry, J.O., Nikolau, B.J., Carr, J.R and Klessig, D.F. (1986) MoL Cell. Biol. 6, 2347-2353 30 Langdale, J.A., Taylor, W.C. and Nelson, T. Mol. Gen. Genet. (in press) 31 Nelson, T., Harpster, M.H., Mayfield, S.R and Taylor, W.C. (1984)J. Cell Biol. 98, 558-564 32 Metzler, M.C., Rothermel, B.A. and Nelson, T. (1989) Plant Mol. Biol. 12, 713--722 33 Kuhlemeier, C., Green, P.J. and Chua, N-H. (1987) Ann. Rev. Plant. Physiol. 38, 221-257 34 Sheen, J-Y. (1990) Plant Cell 2, 1027-1038 35 Haberlandt, G. (1914) Physiological Plant Anatomy, Macmillan 36 Gordon-Kamm, W.J. et al. (1990) Plant Cell 2, 603-618 37 Rosche, E. and Westhoff, R (1990) FEBSLett. 273, 116-121 J.A. LANGDALE IS IN THE PLANT SCIENCES DEPARTMENT, UNIVERSITY OF OXFORI~ SOUTH PARKS ROA~ OXFORD O X l 3RA~ UK AND T. NELSON IS IN THE BIOLOGY DEPARTMENT, YALE UNIVERSITY, P O Box 6666, NEW HAVEN,

CT 06511, USA.

Genetic analysis of the bacterial flagellum ROBERT M. MACNAB AND JOHN S. PARKINSON Escherichia coli and Salmonella typhimurium invest considerable resources in makingflageila, motor organelles that function much like the propellers on a ship. Both classical and molecular genetic studies have begun to reveal how flagellar genes are regulated and how their products build and operate these remarkable devices. necessary for motor rotation and che genes as being necessary for proper control of switching between anticlockwise and clockwise directions of rotation. With the important exceptions of three genes whose products are essential for flagellar structure and also participate in the mechanisms for rotation and switching (see below), a given gene falls into one category only. Over 40 flagellar genes have now been identified, making it one of the most complex genetic systems in the cell. In contrast, the number of motility genes (two) and chemotaxis genes (six, plus specific chemoreceptor genes) is relatively small. Of the flagellar and motility genes, at least 13 are known on biochemical grounds

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[I~EVIEWS to encode structural components and another half dozen or so are suspected on genetic grounds of doing so. Several others are known or believed to control gene expression, and others still are believed to control processes of export and assembly. Finally, there are about ten flagellar genes whose functions have not been identified.

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Figure 1 illustrates our current understanding of the structure and genetic origin of the flagellum of S. typhimurium. How were the various structural genes for the flagellar apparatus assigned to a particular component, location and function? The gene assignment for flagellin, the protein from which the propulsive helical filament is constructed, was made in 19532 . Salmonella strains were found to express one of two antigenically distinguishable forms of flagellin, and mapping of the loci responsible resulted in identification of two flagellin structural genes. (Incidentally, the p h e n o m e n o n underlying the alternative expression, called phase variation, is n o w regarded as a classical example of site-specific genetic rearrangement3.) Identification of the gene for the next most abundant component, the hook protein, did not occur until 25 years later 4, when a mutation causing synthesis of an antigenically altered, hook protein was mapped. The genes for the other components of the apparatus have gradually been identified via a variety of methods, including abnormal eleetrophoretic mobility of temperature-sensitive mutant proteins 5, detection of partial structures in mutants< 7, and correlation of the products of cloned flagellar genes with k n o w n structural components 8. However, even now, direct knowledge of structural genes is restricted to those for the filament hook-basal b o d y apparatus. Although many others have been cloned, sequenced and their products identified, these products have still not been detected as part of the flagellar apparatus, which seems to consist of some quite labile components as well as the more rugged ones of the filament h o o k basal b o d y apparatus, Major examples of such labile components include the machinery of the flagellar switch and the apparatus for export of external flagellar components. What has genetics told us of the function of the various flagellar components? Here function has to be thought of somewhat differently from, for example, in a biosynthetic pathway. In many instances, we need to think almost in engineering terms about transmission shafts, mounting plates and bushings - unfamiliar ground for the microbial geneticist. Also, because mutations usually block the assembly process (see below), the function often has to be inferred from the location and morphology of the structure rather than from any specific functional defect. Nonetheless, plausible roles for most of the known structures have been assigned, and are indicated in Fig. 1. The functions of the gene products involved in motor rotation and switching are of particular interest, although still poorly understood. Defects in the motA and motB genes cause flagellar paralysis, and their products, both cytoplasmic membrane proteins, play roles in coupling protonmotive force to motor rotation.

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FIGH Schematic illustration of the flagellum of S. typhimurium or E. coli (the two are essentially identical). The structure recognized by electron microscopy - the filament hook-basal body complex - is shown in solid outline; structures inferred on genetic or other grounds are tinted. Locations of gene products are indicated, along with the name given to the substructure and its known or presumed function. The FIgK, FIgLand HiD proteins are also known as hook-associated proteins (HAPs) 1, 3 and 2, respectively (see Fig. 3). A flagellum-specific export apparatus is believed to discriminate between exported flagellar proteins (solid circle) and other cellular proteins and molecules (()pen circle). After export, the flagellar proteins are believed t~ travel down a central channel (coarse dotted outline) in the nascent structure. MotA appears to be a proton channel 9, whereas MotB may anchor the rotor to the cell walP 0, providing a solid support against which thrust can be exerted. Together, they comprise the force-generating units that turn the flagellar filaments H,le. Each motor has about eight such units, which may correspond to stud-like structures seen encircling the basal complex in freezefracture electron micrographs 13. Some mutations in the ./giG, fliM and fliN genes cause tlagellar paralysis, whereas others produce biased or unidirectional rotation. The FIiG, FliM and FliN proteins are thus believed to comprise the switch machinery that controis the direction of flagellar rotationl~L

The flagellar regulon Nearly all of the flagellar, motility and chemotaxis genes are located in four clusters not far from the terminus of chromosome replication (Fig. 2). A few - tsr and trg, which encode chemoreceptors, and fi/A and fljB, which encode components of the phase variation machinery in Salmonella- are located outside these clusters. The original names of homologous fia genes in E. coil and S. typhimurium were different, so the literature abounds with an alphabet soup of g e n t

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[~EVIEWS encoding the switch, basal body and export apparatus components (tinted in Fig. 2), which are needed at the initial stages of the assembly process, are expressed early in the hierarchy. Genes for filament structure, motor rotation and chemotactic signaling (shown in white in Fig. 2), whose products are only needed once the basal body-hook complex is complete, are expressed late in the hierarchy. All of the 'early' genes must be expressed to obtain transcription of the 'late' genes. Somehow, a functional defect in any one of the early genes can prevent expression of the late operons, whose products would obviously be useless in the absence of a functional flagellum. The mechanisms behind this elegantly parsimonious regulatory scheme have begun to emerge. A key player is the FliA protein, an alternative ~ factor specifically required for efficient expression from late promoters20, 21, which contain unusual sequences at their -10 and -35 regions2E (Although the expression patterns of tsr, trg and fljAB have not been directly examined, their promoters also exhibit the 'late gene' consensus motif.) However, this mechanism alone does not explain why all of the early gene products are needed for late expression. In some way, intact early flagellar structure seems to be required for activating late expression, either by enabling a positive regulator to function or by disabling a negative regulator. One possibility is that an early structural component blocks late expression (perhaps by antagonizing FliA action?) whenever a defect in the assembly process prevents its incorporation into the nascent basal body TM. The early-late controls are only part of the flageUar regulon story, however. The expression of the early operons themselves is dependent on the products ,,f the flhDC 'master operon' (shown in black in Fig. 2). Here, the regulatory mechanism is less apparent, but sequence comparisons have led to the suggestion that ~r FIhD and FlhC may comprise a composite ~ factor needed for early gene transcription 23. Interestingly, the -10 regions of early promoters contain the I E. coli/S, typh~nuriummap ~ ~i/ ~ same unorthodox consensus sequence found in late promoters, but their -35 regions are different (in fact no obvious consensus can be seen), consistent with the idea that novel factors may be required for their transcription 22. FIGEt The hierarchical nature of these transcriptional controls Genetic organization of the flagellar regulon in S. typhimurium and E. coil The flagellum-related genes in the two species are mainly clustered in four regions. [The ensures that flagellation is subdivision of region lII into regions Illa and IIIb is based on the recent finding that there positively regulated by the are several kilobases of DNA (dashed line), not involved in flagellation, between the fliD flhDC operon. Since the and fliE operons (I. Kawagishi, V. MOiler, A. Williams and R. Macnab, unpublished)]. A few flhDC promoter is dissimilar genes are present only in S. typhimurium (round brackets) or E. coli (square brackets). The to all other flagellar progenes in each region are listed in clockwise order relative to the standard chromosomal moters, it is presumably map. (The locations of oriC and terC, the origin and terminus of replication, respectively, are subiect to a unique set of provided as landmarks.) Cotranscribed genes are listed inside arrows that point in the regulatory inputs that control direction of transcription. The shading indicates the position of their promoters in the flagellar production. What regulatory hierarchy:flhDC (black) encode master positive regulators needed for expression sorts of environmental or of the 'early' operons (tinted), whose products in turn are needed for expression of the 'late' physiological conditions might operons (white).

symbols. In this article we use a recently devised uniform nomenclature 15 in which flagellar genes in region I are designated fig, those in region II are designated flh, and those in regions IIIa and IIIb are designated fli. The correspondence between the old and new nomenclature systems is summarized in Table 1 of Ref. 15. The overall arrangement of flagellar genes into clusters parallels their structural and functional roles. Genes in region I mainly encode the structural components of the basal body (flgBCDFGH1) and hook (flgEKL) (Fig. 1). Genes in region Ilia encode flagellin (fliC) and its capping protein (fliD). Genes in region IIIb encode components of the switch mechanism, and possibly a special export apparatus (see below). Region II contains the mot and che genes, several chemoreceptor genes (tar, tap) and flagellar genes with regulatory (flhC, flhD) or unknown (flhA, flhB, JibE) roles. Within each cluster, genes with similar functions are typically arranged into transcriptional units 16. This organization conceivably ensures that gene products needed at the same step in the assembly sequence, or those that must interact with one another, are made at the same time and in the correct relative amounts. The expression patterns of flagellar genes have been extensively characterized with lacZ operon fusions, whose ~-galactosidase levels provide a convenient measure of transcriptional activity from flagellar promoters under physiologically different conditions and in different genetic backgrounds 17-19. The possibility of post-transcriptional regulatory mechanisms has not yet been investigated, but the major control mechanisms probably operate at the transcriptional level. They regulate expression of the flagellar genes in a hierarchy that parallels their roles in the assembly pathway. Operons

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HcI! Morphogenetic assembly pathway for the flagellum of S. typhimuriumor E. coli, illustrating the progressive proximal-to-distal naluR' of the process: Genes needed to reach any given stage are indicated in italics; where it is known that a gene product is added at that stage, it is indicated in roman letters. The flhDCoperon is needed for expression of all other operons (see Fig. 2). Genes that have not been assigned to any specific stage earlier than the inner ring-rod complex, or 'rivet', are shown in square brackets. Reproduced, with permission, from Ref. 28. influence flhDC expression? Flagellar production has long been known to be subject to catabolite repression 24 and more recently has been shown to be tied to the cell division cycle 25. With the aid of reporter genes fused to the ,flhDC promoter, it should be possible to discern additional regulatory inputs and to explore their underlying mechanisms.

Assemblyof the flagellum Mutants have been used in several ways to elucidate the assembly pathway of the flagellum (Fig. 3). One approach involved the scoring by electron microscopy of partial structures assembled by mutants defective in different flagellar genes 6. This allowed a description of the following successive events: (1) assembly of an inner ring-rod complex, or 'rivet'; (2) addition of outer rings; (3) addition of hook; (4) addition of filament. Later studies of flagellin excretion in various mutants26, 2v led to the realization that between stages 3 and 4 lay the obligatory addition of three hook-associated proteins (HAPs), and that during stage 4 flagellin monomers are inserted between the tip of the growing filament and a cap made out of the most distal of the three hook-associated proteins. Most

recently, the pattern of incorporation of radiolabel into flagellar structures of temperature-sensitive mutants subjected to a temperature-shift protocol has clarified some of the earlier stages, inw)lving assembly of the inner rings and then of the rod 28. Several interesting general conclusions have emerged from these studies. The first is that ~'ven t]lc simplest partial structures we know of require re:my genes (such as the switch genes) in addition to thosu encoding the proteins of that partial structure (Fig. 3). This is further evidence that the structures seen in the electron microscope lack various labile features. Efforts are being made to isolate more extended structures. Another conclusion is that assembly proceeds, subunit by subunit, via substructures of increasing complexity rather than via modules that arc then joincd into a final structure. Incremental assembly is probably the only feasible pathway for a structure that is external to the cell. Imagine the problems in, for example, exporting a hook and a filament and then joining them together outside the cell. A final conclusion is that addition is distal. Indeed, even at the level of the individual ~,ubunit, di,',tal addition has been demonstrated for the filament

"riGJL'NE1991 VOL.7 NO. 6

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]]~EVIEWS protein, flagellin 29,3°. This implies the existence of special machinery for exporting structural subunits to the assembly site.

A flageUum-specirlcexport pathway Biochemical analysis reveals that, apart from a couple of special examples (the outer pair of rings of the basal body31), flagellar proteins that lie beyond the cell m e m b r a n e are not subject to signal peptide cleavage and, therefore, presumably are not exported by the primary export pathway of the celP 2,33. The best guess for the route is through a central channel in the nascent structure. Such a channel has been shown to exist for both the filament34 and the hook3S and probably exists for the rod also. But at some location, presumably in the vicinity of the cell membrane, there must be an apparatus that discriminates between flagellar proteins and all other molecules in the cell. There may also be a need for active protein transport in order to ensure reasonable rates of export (a single filament contains about 20 000 subunits of flagellin and assembles in about 10 minutes). How can one investigate such a putative export apparatus, when it is labile yet is necessary for assembly of the known structure of the filament hook-basal body? Once again, temperature-sensitive mutants have proved to be useful, and have led to the identification of a small number of genes that seem to be essential for regrowth of sheared flagellar filaments (A. Vogler and R. Macnab, unpublished). Interestingly, the sequence of one of these gene products (M. Homma, V. Irikura and R. Macnab, unpublished) resembles that of the catalytic subunit of the FoF 1 and other related proton-translocating ATPases. The significance of this recent result is unclear but it suggests some energetic role within the flagellum beyond that of transducing protonmotive force into motor rotation. Might it be a protein pump? Assuming that the putative ATPase subunit interacts with other flagellar proteins as part of a multisubunit complex, intergenic suppression analysis may give clues to the identities of the other components. This could be the beginning of an exciting new aspect of investigation into flagellar function.

Futureprospects We are gradually beginning to understand how flagellar genes are regulated, how their products assemble into a complex extracellular organelle, and how these fascinating rotary devices operate. Genetic methods will continue to play an important role in working towards the molecular answers to these questions.

Acknowledgements Work carried out in our laboratories has been supported by USPHS grants AI12202 and GM40335 (to RM.M.) and GM19559 and GM43098 (to J.S.P.).

References I Macnab, R.M. (1990) in BioloRv of the Cbemotactic Response (3),rap. Soc. Gen. Microbial. VoL 463 (Armitage, J. and Lackie, J., eds), pp. 77-106, Cambridge University Press

2 Lederberg, J. and Edwards, P.R. (1953)J. Immunol. 71, 232-240 3 Simon, M. et al. (1980) Science 209, 1370-1374 4 Komeda, Y., Silverman, M. and Simon, M. (1978) J. Bacteriol. 133, 364-371 5 Aizawa, S-I. et al. (1985) J. Bacteriol. 161,836-849 6 Suzuki, T. and Komeda, Y. (1981)J. Bacteriol. 145, 1036--1041 7 Jones, CJ., Homma, M. and Macnab, RM. (1987) J. Bacteriol. 169, 1489-1492 8 Homma, M., Kutsukake, K. and Iino, T. (1985) J. Bacteriol. 163, 464-471 9 Blair, D.E and Berg, H.C. (1990) Cell60, 439-449 10 Chun, S.Y. and Parkinson, J.S. (1988) Science 239, 276-278 11 Block, S.M. and Berg, H.C. (1984) Nature 309, 470-472 12 Blair, D.F. and Berg, H.C. (1988) Science 242, 1678--1681 13 Khan, S., Dapice, M. and Reese, T.S. (1988)J. Mol. Biol. 202, 575-584 14 Yamaguchi, S. et al. (1986) J. Bacteriol. 168, 1172-1179 15 Iino, T. et al. (1988) Microbiol. Rev. 52, 533-535 16 Kutsukake, K., Ohya, Y., Yamaguchi, S. and Iino, T. (1988) Mol. Gen. Genet. 214, 11-15 17 Komeda, Y. (1982)./. Bacteriol. 150, 16--26 18 Komeda, Y. (1986)J. Bacteriol. 168, 1315-1318 19 Kutsukake, K., Ohya, Y. and Iino, T. (1990) J. Bacteriol. 172, 741-747 20 Ohnishi, K., Kutsukake, K., Suzuki, H. and lino, T. (1990) Mol. Gen. Genet. 221, 139-147 21 Arnosti, D.N. and Chamberlin, M.J. (1989) Proc. Nail Acad. Sci. USA 86, 830--834 22 Bartlett, D.H., Frantz, B.B. and Matsumura, P. (1988) J. Bacteriol. 170, 1575-1581 23 Helmann, J.D. and Chamberlin, M.J. (1987) Proc. Nail Acad. Sci. USA 84, 6422~6424 24 Silverman, M. and Simon, M. (1974).L Bacteriol. 120, 1196-1203 25 Nishimura, A. and Hirota, Y. (1989) Mol. Gen. Genet. 216, 340-346 26 Homma, M., Fujita, H., Yamaguchi, S. and Iino, T. (1984) J. Bacteriol. 159, 1056-1059 27 Homma, M. and Iino, T. (1985)J. Bacteriol. 164, 1370-1372 28 Jones, c.J. and Macnab, R.M. (1990) J. BacWrioL 172, 1327-1339 29 lino, T. (1969) I. Gen. Microbiol. 56, 227-239 30 Emerson, S.U., Tokuyasu, K. and Simon, M.I. (1970) Science 169, 190-192 31 Homma, M., Komeda, Y., lino, T. and Macnab, R.M. (1987) J. Bacteriol. 169, 1493-1498 32 Joys, T.M. and Rankis. V. (1972).1. Biol. Chem. 247, 5180-5193 33 Jones, C.J., Macnab, R.M., Okino, H. and Aizawa, S-I. (1990) J. Mol. Biol. 212, 377-387 34 Namba, K., Yamashita, i. and Vonderviszt, F. (1989) Nature 342, 6484554 35 Wagenknecht, T., DeRosier, DO., Aizawa, S-I. and Macnab, R.M. (1982)J. Mol. Biol. 162, 69-87

K M . MACNAB IS IN THE DEPARTMENT OF MOLECUtAR BIOPHYSICS AND ~IOCttEMISTRY, YALE UNIVERSITY,, P O BOX 6666, NEW HAVEN, CT 06511, USA ANDJ.So PARKINSON IS IN I TttE BIOLOGY DEPARTMENT, UNIVERSITY OF UTAtt, SALT LAKE

Crn4, UT84112, USA.

rIG Jt'xe 1991 VOL. 7 XO. 6 21t(]