Identification of Salmonella somatic and flagellar antigens by modified serological methods.

This report describes two modified methods for the identification of Salmonella somatic (O) and flagellar (H) antigens. Over a period of 2 years, both modified methods were found to be approximately three times less labor intensive than the standard methods while requiring no more technical skill. The modified methods were as accurate as the standard methods in identifying the O and H antigens of 350 Salmonella isolates. Furthermore, 43 O antisera reacted exclusively with organisms possessing homologous O antigens when the modified and two standard methods were used. At the antiserum dilutions used for H antigen identification, H antisera did not react with O antigens or heterologous H antigens by either the modified or the standard method. Compared with the standard method for H antigen identification, the modified method was approximately 20 times more economical with respect to antisera and usually generated a 1.5- to 4-fold higher titer. Since the antisera stored for use in the modified method for H antigen identification were usually 100-fold more dilute than the antisera stored for the standard method, an antibody-stabilizing buffer was incorporated in the diluted antisera, allowing these reagents to be used for at least 9 to 16 months.     

Identification of Salmonella somatic and flagellar antigens by modified serological methods.

This report describes two modified methods for the identification of Salmonella somatic (O) and flagellar (H) antigens. Over a period of 2 years, both modified methods were found to be approximately three times less labor intensive than the standard methods while requiring no more technical skill. The modified methods were as accurate as the standard methods in identifying the O and H antigens of 350 Salmonella isolates. Furthermore, 43 O antisera reacted exclusively with organisms possessing homologous O antigens when the modified and two standard methods were used. At the antiserum dilutions used for H antigen identification, H antisera did not react with O antigens or heterologous H antigens by either the modified or the standard method. Compared with the standard method for H antigen identification, the modified method was approximately 20 times more economical with respect to antisera and usually generated a 1.5- to 4-fold higher titer. Since the antisera stored for use in the modified method for H antigen identification were usually 100-fold more dilute than the antisera stored for the standard method, an antibody-stabilizing buffer was incorporated in the diluted antisera, allowing these reagents to be used for at least 9 to 16 months.     

Substructure of the flagellar basal body of Salmonella typhimurium.

The Salmonella typhimurium basal body, a part of the flagellar rotary motor, consists of four rings (denoted M, S, P and L) and a coaxial rod. Using low-dose electron microscopy and image averaging methods on negatively stained and frozen-hydrated preparations, we examined whole basal body complexes and subcomplexes obtained by dissociation in acid. Dissociation occurs in steps, allowing us to obtain images of substructures lacking the M ring, lacking the M and S rings, and lacking the M and S rings and the proximal portion of the rod. We obtained images of the L and P ring subcomplex. The existence of a subcomplex missing only the M ring suggests either that the S and M rings derive from two different proteins, or that the M ring is a labile domain of a single protein, which makes up both rings. At the 25 to 30 A resolution of our averaged images, the L, P and S rings appear cylindrically symmetric. Images of the M ring show variability that may be due to differences in angular orientation of the grid, but equally could be due to structural variations. Three-dimensional reconstructions of these structures from the averaged images reveal the internal structure and spatial organization of these components.     

Specific inactivator of flagellar reversal in Salmonella typhimurium.

Specific inhibition of flagellar rotation reversal was observed after exposure of chemotactic Salmonella typhimurium to citrate autoclaved at neutral pH. The presence of a rotation reversal inactivator was established in autoclaved citrate-containing media and nutrient broth. Since modulation of flagellar rotation by attractants and repellents is the basis of chemotactic behavior, a specific inhibitor of rotation reversal, which is essential for tumble generation, provides a useful probe into the molecular mechanism of bacterial chemotaxis. The inactivator inhibits clockwise rotation without affecting counterclockwise rotation, speed of rotation, or the capacity of the cells to grow and divide. Inactivation of clockwise rotation is gradual and irreversible, differing from the transient inhibition of clockwise rotation by attractants, which is characterized by an immediate suppression followed by a return to normal rotation patterns. The rotation reversal inactivator is stable to acidification, rotary evaporation, lyophilization, and rehydration.     

Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium.

Incomplete flagellar structures were detected in osmotically shocked cells or membrane-associated fraction of many nonflagellate mutants of Salmonella typhimurium by electron microscopy. The predominant types of these structures in the mutants were cistron specific. The incomplete basal bodies were detected in flaFI, flaFIV, flaFVIII, and flaFIX mutants, the structure homologous to a basal body in flaFV mutants, the polyhook-basal body complex in flaR mutants, and the hook-basal body complex in flaL and flaU mutants. No structures homologous to flagellar bases or their parts were detected in the early-fla group nonflagellate mutants of flaAI, flaAII, flaAIII, flaB, flaC, flaD, flaE, flaFII, flaFIII, flaFVI, flaFVII, flaFX, flaK, and flaM. From these observations, a process of flagellar morphogenesis was postulated. The functions of the early-fla group are essential to the formation of S ring-M ring-rod complexes bound to the membrane. The completion of basal bodies requires succeeding functions of flaFI, flaFIV, flaFVIII, and flaFIX. Next, the formation of hooks attached to basal bodies proceeds by the function of flaFV and by flaR, which controls the hook length. Flagellar filaments appear at the tips of hooks because of the functions of flaL, flaU, and flagellin genes.     

How Salmonella typhimurium measures the length of flagellar filaments

We present a mathematical model for the growth and length regulation of the filament of the flagellar motor of Salmonella Typhimurium. Under the assumption that the molecular constituents are translocated into the nascent filament by an ATPase and then move by molecular diffusion to the growing end, we find a monotonically decreasing relationship between the speed and the velocity of growth that is inversely proportional to length for a large length. This gives qualitative but not quantitative agreement with data of the velocity of growth. We also propose that the length of filaments is “measured” by the rate of secretion of the σ²⁸-antifactor FlgM, using negative feedback, and present a mathematical model of this regulatory network. The combination of this regulatory network with the length-dependent rate of growth enable the bacterium to detect length shortening and regrow severed flagellar filaments.     

Deletion analysis of the FliM flagellar switch protein of Salmonella typhimurium.

The flagellar switch of Salmonella typhimurium and Escherichia coli is composed of three proteins, FliG, FliM, and FliN. The switch complex modulates the direction of flagellar motor rotation in response to information about the environment received through the chemotaxis signal transduction pathway. In particular, chemotaxis protein CheY is believed to bind to switch protein FliM, inducing clockwise filament rotation and tumbling. To investigate the function of FliM and its interactions with FliG and FliN, we engineered a series of 34 FliM deletion mutant proteins, each lacking a different 10-amino-acid segment. We have determined the phenotype associated with each mutant protein, the ability of each mutant protein to interfere with the motility of wild-type cells, and the effect of additional FliG and FliN on the function of selected FliM mutant proteins. Overall, deletions at the N terminus produced a counterclockwise switch bias, deletions in the central region of the protein produced poorly motile or nonflagellate cells, and deletions near the C terminus produced only nonflagellate cells. On the basis of this evidence and the results of a previous study of spontaneous FliM mutants (H. Sockett, S. Yamaguchi, M. Kihara, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 174:793-806, 1992), we propose a division of the FliM protein into four functional regions: an N-terminal region primarily involved in switching, an extended N-terminal region involved in switching and assembly, a middle region involved in switching and motor rotation, and a C-terminal region primarily involved in flagellar assembly.     

Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium.

The hook-basal body complex of Salmonella typhimurium, a major component of its flagellar apparatus, was subjected to detailed analysis by electron microscopy and gel electrophoresis. The study was facilitated by the development of an improved protocol for isolation of the complexes in high yield and purity. Nine proteins were identified with the structure. These proteins had apparent molecular weights of 65,000 (65K), 60K, 42K, 38K, 32K, 30K, 27K, 16K, and 14K. Small but reproducible shifts in the apparent molecular weights of specific proteins from conditionally nonflagellate mutants indicated the following gene-polypeptide correspondences: flaFV, 42K; flaFVI, 32K; flaFVII, 30K; flaFIX, 38K; flaAII.1, 65K. Several new morphological features of hook-basal body complexes were recognized, including a clawlike structure on the cytoplasm-proximal M ring and additional material at the cytoplasmic face of the M ring. Based on this study and the work of others, we suggest that the morphological features of the hook-basal body complex correspond to the following proteins: hook-filament junction, 60K; hook, 42K; rod, 30K and 32K; L ring and outer cylinder wall, 27K; P ring, 38K; S ring, unknown; M ring 65K.     

Molecular analysis of the flagellar switch protein FliM of Salmonella typhimurium.

Defects in the chemotaxis proteins CheY and CheZ of Salmonella typhimurium can be suppressed by mutations in the flagellar switch, such that swarming of a pseudorevertant on semisolid plates is significantly better than that of its parent. cheY suppressors contribute to a clockwise switch bias, and cheZ suppressors contribute to a counterclockwise bias. Among the three known switch genes, fliM contributes most examples of such suppressor mutations. We have investigated the changes in FliM that are responsible for suppression, as well as the changes in CheY or CheZ that are being compensated for. Ten independently isolated parental cheY mutations represented nine distinct mutations, one an amino acid duplication and the rest missense mutations. Several of the altered amino acids lie on one face of the three-dimensional structure of CheY (A. M. Stock, J. M. Mottonen, J. B. Stock, and C. E. Schutt, Nature (London) 337:745-749, 1989; K. Volz and P. Matsumura, J. Biol. Chem. 266:15511-15519, 1991); this face may constitute the binding site for the switch. All 10 cheZ mutations were distinct, with several of them resulting in premature termination. cheY and cheZ suppressors in FliM occurred in clusters, which in general did not overlap. A few cheZ suppressors and one cheY suppressor involved changes near the N terminus of FliM, but neither cheY nor cheZ suppressors involved changes near the C terminus. Among the strongest cheY suppressors were changes from Arg to a neutral amino acid or from Val to Glu, suggesting that electrostatic interactions may play an important role in switching. A given cheY or cheZ mutation could be suppressed by many different fliM mutations; conversely, a given fliM mutation was often encountered as a suppressor of more than one cheY or cheZ mutation. The data suggest that an important factor in suppression is a balancing of the shift in switch bias introduced by alteration of CheY or CheZ with an appropriate opposing shift introduced by alteration of FliM. For strains with a severe parental mutation, such as the cheZ null mutations, adjustment of switch bias is essentially the only factor in suppression, since the attractant L-aspartate caused at most a slight further enhancement of the swarming rate over that occurring in the absence of a chemotactic stimulus. We discuss a model for switching in which there are distinct interactions for the counterclockwise and clockwise states, with suppression occurring by impairment of one of the states and hence by relative enhancement of the other state. FliM can also undergo amino acid changes that result in a paralyzed (Mot-) phenotype; these changes were confined to a very few residues in the protein.     

Structural genes for flagellar hook-associated proteins in Salmonella typhimurium.

The flaW, flaU, and flaV genes of Salmonella typhimurium LT2 were cloned into pBR322. These genes were mapped on the cloned DNA fragments by restriction endonuclease analysis and construction of the deletion derivatives. Their gene products were identified, by the minicell method, as proteins whose molecular weights were estimated to be 59,000 for the flaW product, 31,000 for the flaU product, and 48,000 for the flaV product. These values are identical to those of three species of hook-associated proteins (HAPs), namely, HAP1, HAP3, and HAP2. Furthermore, antibodies against HAP1, HAP3, and HAP2 specifically reacted with the gene products of flaW, flaU, and flaV, respectively. Therefore, we concluded that they are structural genes for HAPs. The antibodies against HAP1 and HAP3 also specifically reacted with the gene products of flaS and flaT of Escherichia coli, respectively. This indicates that these gene products are HAPs in E. coli. This result is consistent with the demonstration that flaS and flaT of E. coli are functionally homologous with flaW and flaU of S. typhimurium.     

Genetic and behavioral analysis of flagellar switch mutants of Salmonella typhimurium.

At the interface between the sensory transduction system and the flagellar motor system of Salmonella typhimurium, the switch complex plays an important role in both sensory transduction and energy transduction. To examine the function of the switch complex, we isolated from 10 cheY mutants 500 pseudorevertants with a suppressor mutation in one of the three genes (fliG, fliM, and fliN) encoding the switch complex. Detailed mapping revealed that these suppressor mutations were localized to several segments of each switch gene, suggesting localization of functional sites on the switch complex. These switch mutations were introduced into the wild-type background and into a chemotaxis deletion background. Behavior of the pseudorevertants and their derivatives (1,500 strains in all) was observed by light microscopy. In the chemotaxis deletion background, about 70% of the switch mutants showed smooth swimming and the rest showed more or less tumbly swimming. There was some correlation between the mutational sites and the swimming patterns in the chemotaxis deletion background, suggesting that there is segregation of functional sites on the switch complex. The interaction of the switch complex with the chemotaxis protein, CheY, and the stochastic nature of switching in the absence of CheY are discussed.     

Locations of hook-associated proteins in flagellar structures of Salmonella typhimurium.

Hooks of the flagella of Salmonella typhimurium were purified from an flaL mutant. Hook-associated proteins, namely HAP1, HAP2, and HAP3, were separated from them, and the antibody against each HAP was prepared. By immunoelectron microscopic observation, these three kinds of antiHAP antibodies were found to bind on the distal ends of hooks of filamentless mutants consistently with their composition of HAPs. The antiHAP2 antibody bound to the very tops of the claw-shaped ends of the hooks which contain all three HAPS. The antibodies against HAP1 and HAP3 bound to the basal areas and the middle areas, respectively, of the claw-shaped ends. The order of disassembly of the component proteins by heat treatment of the hook structure from the filamentless mutants was (HAP2, HAP3) greater than HAP1 greater than hook protein. These observations were consistent with our layered structure model: HAP1, HAP3, and HAP2 are assembled at the distal end of the hook in this sequence. All three HAPs were detected in the hook-filament complexes prepared from a flagellate strain. When the hook-filament structure was treated with antibody against HAP1 and with the anti-rabbit immunoglobulin G antibody, the antibody aggregate was observed in the region corresponding to the boundary between filament and hook. This observation strongly suggests that HAP1 is the protein connecting filament with hook. The locations of HAP2 and HAP3 in the hook-filament structure were not clarified with the same procedure.