Search PubMed⌕ Search

Biomedical subjects

R M Macnab

Publications and source records attributed to R M Macnab.

At least 19 recordsLinked to original sources
PubMed1998

Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium.

Frameshift mutations in the fliK gene of Salmonella result in abnormal elongation of the hook and the failure to assemble filament (polyhook phenotype). Second-site suppressor mutations restore filament assembly, but the cells often remain defective in hook-length control (polyhook-filament phenotype). Where the suppressor mutations are intragenic, the second mutation restores the original frame, generating a region of frameshifted sequence, but restoring the natural C terminus. Some of these frameshifted sequences contain a UGA (opal) termination codon. These cells have few flagella and swarm poorly. We suspected that readthrough of UGA by tRNATrp might be the reason for the partial function. When the UGA codon was changed to the Trp codon UGG, flagellar assembly and function were restored to wild-type levels. Conversely, underexpression of the wild-type fliK gene, achieved by changing the sole Trp codon in the sequence (Trp271) to UGA, decreased both the number of flagella and the ability to swarm. These results validate the readthrough hypothesis and indicate that low levels of FliK sustain some degree of flagellation and motility. At low levels of FliK, most flagella had polyhooks. With increasing amounts, the morphology progressively changed to polyhook-filament, and eventually to wild-type hook-filament. When FliK was overproduced, the hook length was slightly shorter (46(+/-7) nm) than that of the wild-type strain (55(+/-9) nm). FliK levels were measured by immunoblotting. Wild-type levels were about 40 to 80 molecules/cell. FliK synthesized by UGA readthrough could be detected when overproduced from plasmid fliK-W271opal, and the levels indicated a probability of readthrough of 0.002 to 0.01. This value was used to estimate the cellular level of underexpressed FliK, which could partly restore function to a fliK mutant, at about 0.07 to 0.8 molecule/cell. These results suggest that FliK does not form a large structure in the cytoplasm and may function as a regulatory protein for protein export. A model for hook-length control is presented that involves feedback from the assembly point to the export apparatus.

Bacterial Proteins↗
PubMed1998

Translation of the flagellar gene fliO of Salmonella typhimurium from putative tandem starts.

The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092-6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189-197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene cheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with cheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.

Amino Acid Sequence↗
PubMed1997

Distinct regions of bacterial flagellar switch protein FliM interact with FliG, FliN and CheY.

The FliG, FliM, and FliN proteins of the bacterial flagellar motor are believed to interact with one another to form the switch complex, which in turn is thought to interact with one of the chemotaxis proteins, CheY. In particular, FliM appears to be an intermediary between CheY and FliG: the current model suggests that CheY, when phosphorylated (CheY-P), binds to FliM and produces a conformational change in FliM that is propagated to FliG. The result of these interactions is to induce clockwise rotation of the flagellar motors and tumbling of the cell. Various genetic and biochemical studies have provided evidence that the switch proteins associate with each other and that CheY-P binds to FliM. Here, we have used affinity blotting to obtain direct evidence of interaction between Salmonella typhimurium FliM and FliN, FliM and FliG, and FliM and CheY-P. We have also examined the ability of various FliM deletion and truncation mutant proteins to bind to FliN, FliG, and CheY-P. From these data, we conclude that distinct regions of the FliM protein bind to each of these other proteins. We propose a model in which the N-terminal region of FliM binds to CheY-P, the middle region of FliM binds to FliG, and the C-terminal region binds to FliN.

Bacterial Proteins↗
PubMed1997

The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III flagellar export apparatus, are located in the flagellar basal body.

Most of the structural components of the flagellum of Salmonella typhimurium are exported through a flagellum-specific pathway, which is a member of the family of type III secretory pathways. The export apparatus for this process is poorly understood. A previous study has shown that two proteins, about 23 and 26 kDa in size and of unknown genetic origin, are incorporated into the flagellar basal body at a very early stage of flagellar assembly. In the present study, we demonstrate that these basal body proteins are FliP (in its mature form after signal peptide cleavage) and FliR respectively. Both of these proteins have homologues in other type III secretion systems. By placing a FLAG epitope tag on FliR and the MS-ring protein FliF and immunoblotting isolated hook basal body complexes with anti-FLAG monoclonal antibody, we estimate (using the FLAG-tagged FliF as an internal reference) that the stoichiometry of FliR is fewer than three copies per basal body. An independent estimate of stoichiometry was made using data from an earlier quantitative radiolabelling analysis, yielding values of around four or five subunits per basal body for FliP and around one subunit per basal body for FliR. Immunoelectron microscopy using anti-FLAG antibody and gold-protein A suggests that FliR is located near the MS ring. We propose that the flagellar export apparatus contains FliP and FliR and that this apparatus is embedded in a patch of membrane in the central pore of the MS ring.

Autoradiography↗
PubMed1997

The FliO, FliP, FliQ, and FliR proteins of Salmonella typhimurium: putative components for flagellar assembly.

The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide--a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.

Amino Acid Sequence↗
PubMed1997

An extreme clockwise switch bias mutation in fliG of Salmonella typhimurium and its suppression by slow-motile mutations in motA and motB.

Pseudorevertants (second-site suppressor mutants) were isolated from a set of parental mutants of Salmonella with defects in the flagellar switch genes fliG and fliM. Most of the suppressing mutations lay in flagellar region IIIb of the chromosome. One fliG mutant, SJW2811, gave rise to a large number of suppressor mutations in the motility genes motA and motB, which are in flagellar region II. SJW2811, which has a three-amino-acid deletion (delta Pro-Ala-Ala) at positions 169 to 171 of FliG, had an extreme clockwise motor bias that produced inverse smooth swimming (i.e., swimming by means of clockwise rotation of a hydrodynamically induced right-handed helical bundle), and formed Mot(-)-like colonies on semisolid medium. Unlike previously reported inverse-swimming mutants, it did not show a chemotactic response to serine, and it remained inverse even in a delta che background; thus, its switch is locked in the clockwise state. The location of the mutation further underscores the conclusion from a previous study of spontaneous missense mutants (V. M. Irikura, M. Kihara, S. Yamaguchi, H. Sockett, and R. M. Macnab, J. Bacteriol. 175:802-810, 1993) that a relatively localized region in the central part of the FliG sequence is critically important for switching. All of the second-site mutations in motA and motB caused some impairment of motility, both in the pseudorevertants and in a wild-type fliG background. The mechanism of suppression of the fliG mutation by the mot mutations is complex, involving destabilization of the right-handed flagellar bundle as a result of reduced motor speed. The mutations in the MotA and MotB sequences were clustered to a considerable degree as follows: in transmembrane helices 3 and 4 of MotA and the sole transmembrane helix of MotB, at helix-membrane interfaces, in the cytoplasmic domains of MotA, and in the vicinity of the peptidoglycan binding region of the periplasmic domain of MotB. The potential importance of Lys28 and Asp33 of the MotB sequence for proton delivery to the site of torque generation is discussed.

Amino Acid Sequence↗
PubMed1996

Enzymatic characterization of FliI. An ATPase involved in flagellar assembly in Salmonella typhimurium.

FliI is a protein needed for flagellar assembly in Salmonella typhimurium. It shows sequence similarity to the catalytic beta subunit of the F0F1-ATPase and is even more closely related to putative ATPases in Type III bacterial secretory pathways. A His-tagged version of FliI, which was fully functional in complementation tests, was purified to homogeneity. It had an ATPase activity of 0.16 s-1 at 25 degrees C and pH 7, and a Km for ATP of 0.3 mM; Mg2+ was required. The activity was not affected by inhibitors of the F-, V- or P-type ATPases, or inhibitors of the Type I or Type II bacterial secretory pathways. Mutations K188I and Y363S decreased the ATPase activity about 100-fold, increased the Km about 10-fold, blocked flagellar assembly, and were dominant. Other FliI mutations that disrupted flagellar protein export were found near the N terminus; they permitted essentially wild-type ATPase activity, were not dominant, and showed a dosage-dependent phenotype. We propose that FliI has a C-terminal ATPase domain and an N-terminal domain that interacts with other components in the flagellum-specific export apparatus.

Adenosine Diphosphate↗
PubMed1996

Characterization of the flagellar hook length control protein fliK of Salmonella typhimurium and Escherichia coli.

During flagellar morphogenesis in Salmonella typhimurium and Escherichia coli, the fliK gene product is responsible for hook length control. A previous study (M. Homma, T. Iino, and R. M. Macnab, J. Bacteriol. 170:2221-2228, 1988) had suggested that the fliK gene may generate two products; we have confirmed that both proteins are products of the fliK gene and have eliminated several possible explanations for the two forms. We have determined the DNA sequence of the fliK gene in both bacterial species. The deduced amino acid sequences of the wild-type FliK proteins of S. typhimurium and E. coli correspond to molecular masses of 41,748 and 39,246 Da, respectively, and are fairly hydrophilic. Alignment of the sequences gives an identity level of 50%, which is low for homologous flagellar proteins from S. typhimurium and E. coli; the C-terminal sequence is the most highly conserved part (71% identity in the last 154 amino acids). The central and C-terminal regions are rich in proline and glutamine residues, respectively. Linker insertion mutagenesis of the conserved C-terminal region completely abolished motility, whereas disruption of the less conserved N-terminal and central regions had little or no effect. We suggest that the N-terminal (or N-terminal and central) and C-terminal regions may constitute domains. For several reasons, we consider it unlikely that FliK is functioning as a molecular ruler for determining hook length and conclude that it is probably employing a novel mechanism.

Amino Acid Sequence↗
PubMed1996

Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium.

Mutations in the fliK gene of Salmonella typhimurium commonly cause failure to terminate hook assembly and initiate filament assembly (polyhook phenotype). Polyhook mutants give rise to pseudorevertants which are still defective in hook termination but have recovered the ability to assemble filament (polyhook-filament phenotype). The polyhook mutations have been found to be either frameshift or nonsense, resulting in truncation of the C terminus of FliK. Intragenic suppressors of frameshift mutations were found to be ones that restored the original frame (and therefore the C-terminal sequence), but in most cases with substantial loss of natural sequence and sometimes the introduction of artificial sequence; in no cases did intragenic suppression occur when significant disruption remained within the C-terminal region. By use of a novel PCR protocol, in-frame deletions affecting the N-terminal and central regions of FliK were constructed and the resulting phenotypes were examined. Small deletions resulted in almost normal hook length control and almost wild-type swarming. Larger deletions resulted in loss of control of hook length and poor swarming. The largest deletions severely affected filament assembly as well as hook length control. Extragenic suppressors map to an unlinked gene, flhB, which encodes an integral membrane protein (T. Hirano, S. Yamaguchi, K. Oosawa, and S.-I. Aizawa, J. Bacteriol. 176:5439-5449, 1994; K. Kutsukake, T. Minamino, and T. Yokoseki, J. Bacteriol. 176:7625-7629, 1994). They were either point mutations in the C-terminal cytoplasmic region of FlhB or frameshift or nonsense mutations close to the C terminus. The processes of hook and filament assembly and the roles of FliK and FlhB in these processes are discussed in light of these and other available data. We suggest that FliK measures hook length and, at the appropriate point, sends a signal to FlhB to switch the substrate specificity of export from hook protein to late proteins such as flagellin.

Amino Acid Sequence↗
PubMed1996

Physiological and biochemical analyses of FlgH, a lipoprotein forming the outer membrane L ring of the flagellar basal body of Salmonella typhimurium.

The FlgH protein of Salmonella typhimurium, from which the outer membrane L ring of the flagellar basal body is constructed, has a consensus motif (LTG C) for lipoylation and signal peptide cleavage. We have confirmed the previous finding (M. Homma, K. Ohnishi, T. Iino, and R. M. Macnab, J. Bacteriol. 169:3617-3624, 1987) that it is synthesized in precursor form and processed to a mature form with an apparent molecular mass of ca. 25 kDa. flgH alleles with an in-frame deletion or a 3' truncation still permitted processing. The deletion permitted partial restoration of motility in complementation tests, whereas the truncation did not. Globomycin, an antibiotic which inhibits signal peptide cleavage of prolipoproteins, caused accumulation of precursor forms of FlgH. When cells transformed with a plasmid containing the flgH gene were grown in the presence of [3H]palmitate, a 25-kDa protein doublet was found to be radiolabeled; its identity as FlgH was confirmed by shifts in mobility when the internally deleted and truncated alleles of the gene were used. Hook-basal body complexes from cells grown in the presence of [3H]palmitate demonstrated that FlgH incorporated into flagellar structure was also labeled. An in-frame fusion between the leader sequence of the periplasmic protein PeIB and the mature FlgH sequence, with the putative N-terminal cysteine replaced by glycine, resulted in production of a fusion protein that was processed to its mature form. With a low-copy-number plasmid, the ability of this pelB-flgH fusion to complement a flgH mutant was poor, but with a high-copy-number plasmid, it was comparable to that of the wild type. Although lacking the N-terminal cysteine and therefore being incapable of lipoylation via a thioether linkage, the mutant protein still incorporated [3H]palmitate at low levels, perhaps through acylation of the N-terminal alpha-amino group. We conclude that FlgH is a lipoprotein and that under normal physiological conditions the lipoyl modification is necessary for FlgH to function properly as the L-ring protein of the flagellar basal body. We suggest that the N terminus of FlgH is responsible for anchoring the basal body in the outer membrane and that the C terminus may be responsible for binding to the P ring to form the L,P-ring complex.

Alleles↗
PubMed1996

Analysis of a FliM-FliN flagellar switch fusion mutant of Salmonella typhimurium.

In the course of an analysis of the three genes encoding the flagellar motor switch, we isolated a paralyzed mutant whose defect proved to be a 4-bp deletion of the ribosome binding sequence of the fliN switch gene (V. M. Irikura, M. Kihara, S. Yamaguchi, H. Sockett, and R. M. Macnab, J. Bacteriol. 175:802-810,1993). This sequence lies just before the 3' end of the coding sequence of the upstream fliM switch gene, in the same operon. This mutant readily gave rise to pseudorevertants which, though much less motile than the wild type, did exhibit significant swarming. One such pseudorevertant was found to contain a compensating frameshift such that the fliM and fliN genes were placed in frame, coding for an essentially complete FliM-FliN protein fusion. Minicell analysis demonstrated that, as expected, the parental mutant synthesized an essentially full-length FliM protein but no detectable FliN. The pseudorevertant, in contrast, synthesized a protein with the predicted size for the FliM-FliN fusion protein and no detectable FliM or FliN. Immunoblotting of minicells with antibodies against FliM and FliN confirmed the identities of these various proteins. Immunoblotting of book-basal-body complexes from the wild-type strain gave a strong signal for the three switch proteins FliG, FliM, and FliN. Complexes from the FliM-FliN fusion mutant gave a strong signal for FliG but no signal for either FIiM or FliN; a moderately strong signal for the FliM-FliN fusion protein was seen with the anti-FliM antibody, and a weaker signal was seen with the anti-FliN antibody. The cytoplasmic C ring of the structure, which is seen consistently in electron microscopy of wild-type complexes and which is known to contain the FliM and FliN proteins, was much more labile in the FliM-FliN fusion mutant, giving a fragmented and variable appearance or being completely absent. Complementation data indicated that wild-type FliM had a mild dominant negative effect over the fusion protein, that wild-type FliN and the fusion protein work much better than the fusion protein alone, and that wild-type FliM and FliN together have no major positive or negative effect on the function of the fusion protein. We interpret these data to mean that the FliM-FliN fusion protein incorporates into structure but less stably than do the FliM and FliN proteins separately, that wild-type FliM tends to displace the fusion protein, and that wild-type FliN can supplement the FliN domain of the fusion protein without displacing the FliM domain. The data support, but do not prove, a model in which FliM and FliN in the wild-type switch complex are stationary with respect to each other.

Amino Acid Sequence↗
PubMed1996

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.

Bacterial Proteins↗
PubMed1994

FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium.

FlgD is known to be absolutely required for hook assembly, yet it has not been detected in the mature flagellum. We have overproduced and purified FlgD and raised an antibody against it. By using this antibody, we have detected FlgD in substantial amounts in isolated basal bodies from flgA, flgE, flgH, flgI, flgK, and fliK mutants, in much smaller amounts in those from the wild type and flgL, fliA, fliC, fliD, and fliE mutants, and not at all in those from flgB, flgD, flgG, and flgJ mutants. In terms of the morphological assembly pathway, these results indicate that FlgD is first added to the structure when the rod is completed and is discarded when the hook, having reached its mature length, has the first of the hook-filament junction proteins, FlgK, added to its tip. Immunoelectron microscopy established that FlgD initially is located at the distal end of the rod and eventually is located at the distal end of the hook. Thus, it appears to act as a hook-capping protein to enable assembly of hook protein subunits, much as another flagellar protein, FliD, does for the flagellin subunits of the filament. However, whereas FliD is associated with the filament tip indefinitely, FlgD is only transiently associated with the hook tip; i.e., it acts as a scaffolding protein. When FlgD was added to the culture medium of a flgD mutant, cells gained motility; thus, although the hook cap is normally added endogenously, it can be added exogenously. When culture media were analyzed for the presence of hook protein, it was found only with the flgD mutant and, in smaller amounts, the fliK (polyhook) mutant. Thus, although FlgD is needed for assembly of hook protein, it is not needed for its export.

Bacterial Proteins↗
PubMed1994

Characterization of the fliL gene in the flagellar regulon of Escherichia coli and Salmonella typhimurium.

filL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli. A spontaneous fliL mutant of S. typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch. However, in-frame deletions of the E. coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis. Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the effect on motility caused by truncation of the gene is probably an indirect one.

Bacterial Proteins↗
PubMed1993

Domain organization of the subunit of the Salmonella typhimurium flagellar hook.

The deduced amino acid sequences of the family of axial proteins of the bacterial flagellum possess N and C-terminal heptad repeats of hydrophobic amino acid residues, which suggests that these proteins all fold to form bundles of alpha-helices (e.g. coiled coils). There is evidence that flagellin, which is one of the axial proteins, has an axially oriented bundle of alpha-helices that gives rise to the inner, rod-shaped domains seen in electron density maps. We present evidence that a second member of the family, the hook subunit, also has such an axially oriented, rod-shaped domain. In three-dimensional reconstructions from electron micrographs of the helical hook of Salmonella typhimurium, the rod-shaped domain has a diameter of 18 A, which is that expected for a coiled coil. The corresponding domain in the flagellin subunit of the filament, however, is larger, having a diameter of 24 A suggesting a bundle of three or more alpha-helices. In addition to the rod-shaped domain, the hook has two other domains. At a radius of 55 A is the middle spheroidal domain about 25 A in diameter and at a radius of 75 A is the outer ellipsoidal domain about 20 A by 30 A by 40 A. The flagellin subunit also has a middle and an outer domain although they appear different from those of the hook. This is no doubt a result of the lack of any sequence similarity of the hook and flagellin subunits, apart from the N and C-terminal heptad repeats. Along the hook axis, there is a 25 A wide channel, which presumably serves in the export of hook and flagellin subunits in the assembly of the filament. There is a comparably sized channel in the filaments as deduced from electron micrographs. Thus, electron microscopy consistently finds a small channel, whereas in X-ray diffraction studies of the filament, the channel size appeared to be about 60 A. At a diameter of 60 A, the channel could pass the flagellin or hook subunit in its completely folded state, but if the channel is only 25 A in diameter, the subunit would have to be at least partially unfolded in order to pass through the channel.

Flagella↗
PubMed1993

Organization of the Escherichia coli and Salmonella typhimurium chromosomes between flagellar regions IIIa and IIIb, including a large non-coding region.

Flagellar regions IIIa and IIIb of the Escherichia coli and Salmonella typhimurium chromosomes (at 40 min and 42-43 min, respectively) has been shown to be separated by DNA unrelated to flagellar function, with region IIIa being immediately followed by a gene, amyA, that encodes a cytoplasmic alpha-amylase. The chromosome between amyA and flagellar region IIIb has now been investigated. The high level of DNA similarity between the E. coli and S. typhimurium sequences that exists in flagellar region IIIa and in amyA continues initially, with three genes of unknown function; in E. coli, there may be a fourth gene. The remainder of the region, up to the start of flagellar region IIIb, lacks any obvious open reading frames, scores poorly on an algorithm for coding probability, has a high A+T content, and is totally dissimilar in the two species. We conclude that it is non-coding. In E. coli this region extends for 2.7 kb and in S. typhimurium for 0.8 kb. These values are unusually large for prokaryotes, where the non-coding regions between operons are generally quite short. The data, which are discussed in the context of a hypothesized disruption of a contiguous ancestral flagellar region, may give new insight into the organization and evolution of the bacterial chromosome.

Amino Acid Sequence↗
PubMed1993

Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens.

FliI is a Salmonella typhimurium protein that is needed for flagellar assembly and may be involved in a specialized protein export pathway that proceeds without signal peptide cleavage. FliI shows extensive sequence similarity to the catalytic beta subunit of the F0F1 ATPase (A. P. Volger, M. Homma, V. M. Irikura, and R. M. Macnab, J. Bacteriol. 173:3564-3572, 1991). It is even more similar to the Spa47 protein of Shigella flexneri (M. M. Venkatesan, J. M. Buysse, and E. V. Oaks, J. Bacteriol. 174:1990-2001, 1992) and the HrpB6 protein of Xanthomonas campestris (S. Fenselau, I. Balbo, and U. Bonas, Mol. Plant-Microbe Interact. 5:390-396, 1992), which are believed to play a role in the export of virulence proteins. Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation, supporting the hypothesis that FliI is an ATPase. FliI was overproduced and purified almost to homogeneity. It demonstrated ATP binding but not hydrolysis. An antibody raised against FliI permitted detection of the protein in wild-type cells and an estimate of about 1,500 subunits per cell. An antibody directed against the F1 beta subunit of Escherichia coli cross-reacted with FliI, confirming that the proteins are structurally related. The relationship between three proteins involved in flagellar assembly (FliI, FlhA, and FliP) and homologs in a variety of virulence systems is discussed.

Adenosine Triphosphate↗