Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane.

The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein. Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorporate the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body.     

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.     

M ring, S ring and proximal rod of the flagellar basal body of Salmonella typhimurium are composed of subunits of a single protein, FliF.

The flagellar basal body, a major part of the flagellar motor, consists of a rod and four rings. When the fliF gene of Salmonella typhimurium, which was previously shown to code for the component protein of the M ring, was cloned and overexpressed in Escherichia coli, the FliF subunits formed ring structures in the cytoplasmic membrane. Electron microscopic observation of the purified ring structures revealed that each was composed of two adjacent rings and a short appendage extending from the center of the rings. Antibodies raised against the purified FliF protein decorated both the M and S rings of the intact basal body. We conclude that the FliF protein is the subunit protein of the M ring, and of the S ring and of part of the proximal rod of the flagellar basal body.     

Identification of flagellar hook and basal body gene products (FlaFV, FlaFVI, FlaFVII and FlaFVIII) in Salmonella typhimurium.

The flagellar genes flaFV, flaFVII, and flaFVIII of Salmonella typhimurium were cloned, and their presence on a given plasmid was verified by complementation of Escherichia coli mutants defective in the homologous genes. The gene products were identified by radiolabeling in a minicell system as being proteins of the following molecular masses: FlaFV, 42 kilodaltons (kDa); FlaFVI, 32 kDa; FlaFVII, 30 kDA; and FlaFVIII, 27 kDa. These data, together with isoelectric focusing data, confirm gene product assignments of flagellar components made indirectly from mutant studies. Flagellar components are transported by either a signal peptide-dependent or a flagellar-specific pathway. Consistent with its location in the outer membrane ring of the basal body, protein FlaFVIII seems to use the signal peptide-dependent pathway, since it was synthesized in a precursor form and processed, presumably by peptide cleavage, to a mature form; the maturation process was inhibited by addition of a proton ionophore. Proteins synthesized in minicells were localized as follows: FlaFVI was localized to the soluble fraction (cytoplasm); pre-FlaFVIII and FlaFVIII were localized to the particulate fraction (membrane or high-molecular-weight aggregate); FlaFV and FlaFVII were localized to both fractions. The significance of these locations in terms of known or suspected roles in the flagellar apparatus is discussed.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

Escherichia coli produces a cytoplasmic alpha-amylase, AmyA.

In the gap between two closely linked flagellar gene clusters on the Escherichia coli and Salmonella typhimurium chromosomes (at about 42 to 43 min on the E. coli map), we found an open reading frame whose sequence suggested that it encoded an alpha-amylase; the deduced amino acid sequences in the two species were 87% identical. The strongest similarities to other alpha-amylases were to the excreted liquefying alpha-amylases of bacilli, with > 40% amino acid identity; the N-terminal sequence of the mature bacillar protein (after signal peptide cleavage) aligned with the N-terminal sequence of the E. coli or S. typhimurium protein (without assuming signal peptide cleavage). Minicell experiments identified the product of the E. coli gene as a 56-kDa protein, in agreement with the size predicted from the sequence. The protein was retained by spheroplasts rather than being released with the periplasmic fraction; cells transformed with plasmids containing the gene did not digest extracellular starch unless they were lysed; and the protein, when overproduced, was found in the soluble fraction. We conclude that the protein is cytoplasmic, as predicted by its sequence. The purified protein rapidly digested amylose, starch, amylopectin, and maltodextrins of size G6 or larger; it also digested glycogen, but much more slowly. It was specific for the alpha-anomeric linkage, being unable to digest cellulose. The principal products of starch digestion included maltotriose and maltotetraose as well as maltose, verifying that the protein was an alpha-amylase rather than a beta-amylase. The newly discovered gene has been named amyA. The natural physiological role of the AmyA protein is not yet evident.     

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.     

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.     

The ompA signal peptide directed secretion of Staphylococcal nuclease A by Escherichia coli

The hybrid pre-enzyme formed by fusion of the signal peptide of the OmpA protein, a major outer membrane protein of Escherichia coli, to Staphylococcal nuclease A, a protein secreted by Staphylococcus aureus, is translocated across the cytoplasmic membrane of E. coli with concomitant cleavage of the signal peptide. A DNA fragment containing the coding sequence for the ompA signal peptide was initially ligated to a DNA fragment containing the coding sequence for nuclease A, with a linker sequence of 33 nucleotides separating the coding sequences. When this fused gene was induced, an enzymatically active nuclease was secreted into the periplasmic space; sequential Edman degradation of this protein revealed that the ompA signal peptide was removed at its normal cleavage site resulting in a modified version of the nuclease having 11 extra amino acid residues attached to the amino terminus of nuclease A. The 33 nucleotides between the coding sequences for the ompA signal peptide and the structural gene for nuclease A were subsequently deleted by synthetic oligonucleotide-directed site-specific mutagenesis. The nuclease produced by this hybrid gene was secreted into the periplasmic space and by sequential Edman degradation was identical to nuclease A. Thus, the ompA signal peptide is able to direct the secretion of fused staphylococcal nuclease A, and signal peptide processing occurs at the normal cleavage site. When the hybrid gene is expressed under the control of the lpp promoter, nuclease A is produced to the extent of 10% of the total cellular protein.     

Secretion into the culture medium of a foreign gene product from Escherichia coli: use of the ompF gene for secretion of human beta-endorphin.

The ompF gene codes for a major outer membrane protein of Escherichia coli. A plasmid was constructed in which the structural gene for human beta-endorphin is preceded by the upstream region of the ompF gene consisting of the promoter region and the coding regions for the signal peptide and the N terminus of the OmpF protein. When the plasmid was introduced into E. coli N99, and OmpF-beta-endorphin fused peptide was synthesized and secreted into the culture medium through both the cytoplasmic and outer membranes. The OmpF signal peptide was cleaved correctly during the secretion, indicating that the export of the fused protein across the cytoplasmic membrane was dependent on the signal peptide. The secretion into the culture medium was apparently selective. Neither beta-lactamase nor alkaline phosphatase (both are periplasmic proteins) appeared in the culture medium in significant amounts. The mode of passage of the fused peptide across the outer membrane is discussed.     

Assembly of the switch complex onto the MS ring complex of Salmonella typhimurium does not require any other flagellar proteins.

The cytoplasmic portion of the bacterial flagellum is thought to consist of at least two structural components: a switch complex and an export apparatus. These components seem to assemble around the MS ring complex, which is the first flagellar basal body substructure and is located in the cytoplasmic membrane. In order to elucidate the process of assembly of cytoplasmic substructures, the membrane localization of each component of the switch complex (FliG, FliM, and FliN) in various nonflagellated mutants was examined by immunoblotting. It was found that all these switch proteins require the MS ring protein FliF to associate with the cell membrane. FliG does not require FliM and FliN for this association, but FliM and FliN associate cooperatively with the membrane only through FliG. Furthermore, all three switch proteins were detected in membranes isolated from fliE, fliH, fliI, fliJ, fliO, fliP, fliQ, fliR, flhA, flhB, and flgJ mutants, indicating that the switch complex assembles on the MS ring complex without any other flagellar proteins involved in the early stage of flagellar assembly. The relationship between the switch complex and the export apparatus is discussed.     

Identification of a bacterial sensing protein and effects of its elevated expression.

The Escherichia coli flaA gene product (also called cheC) plays a crucial role in switching flagellar rotational direction during chemotactic responses. Wild-type and mutant alleles have been cloned onto plasmid vectors, and the gene product has been identified as a 37,000-dalton protein. The flaA product appeared as a soluble protein in the cytoplasm when overproduced in minicells and maxicells. The protein could not be detected in flagellar basal structures purified from a wild-type strain. To assess the effects of altered flaA expression, the gene was fused to a synthetic tac promoter that could be regulated by the addition of an inducer. Overproduction resulted in strong counterclockwise flagellar rotational bias and partial paralysis of flagellar motors. These results suggest that the flaA protein provides the interface between the flagellar machinery and the chemotaxis signaling system in a motor structure external to the basal body.     

Characterization of a targeting motif for a flagellar membrane protein in Leishmania enriettii.

The surface membranes of eukaryotic flagella and cilia are contiguous with the plasma membrane. Despite the absence of obvious physical structures that could form a barrier between the two membrane domains, the lipid and protein compositions of flagella and cilia are distinct from the rest of the cell surface membrane. We have exploited a flagellar glucose transporter from the parasitic protozoan Leishmania enriettii as a model system to characterize the first targeting motif for a flagellar membrane protein in any eukaryotic organism. In this study, we demonstrate that the flagellar membrane-targeting motif is recognized by several species of Leishmania. Previously, we demonstrated that the 130 amino acid NH(2)-terminal cytoplasmic domain of isoform 1 glucose transporter was sufficient to target a nonflagellar integral membrane protein into the flagellar membrane. We have now determined that an essential flagellar targeting signal is located between amino acids 20 and 35 of the NH(2)-terminal domain. We have further analyzed the role of specific amino acids in this region by alanine replacement mutagenesis and determined that single amino acid substitutions did not abrogate targeting to the flagellar membrane. However, individual mutations located within a cluster of five contiguous amino acids, RTGTT, conferred differences in the degree of targeting to the flagellar membrane and the flagellar pocket, implying a role for these residues in the mechanism of flagellar trafficking.     

Two flagellar genes, AGG2 and AGG3, mediate orientation to light in Chlamydomonas

Ciliary membranes have a large repertoire of receptors and ion channels that act to transduce information from the environment to the cell. Chlamydomonas offers a tractable system for dissecting the transport and function of ciliary and flagellar membrane proteins. Isolation of ergosterol and sphingolipid-enriched Chlamydomonas flagellar membrane domains identified potential signaling molecules by mass spectroscopy. These include a membrane protein and a matrix flavodoxin protein that are encoded by the AGG2 and AGG3 genes, respectively. Agg2p localizes to the proximal flagellar membrane near the basal bodies. Agg3p is distributed throughout the flagellar matrix, with an increased concentration in the proximal regions where Agg2p is located. Chlamydomonas cells sense light by using a microbial-type rhodopsin , transduce a signal from the cell body to the flagella, and alter the waveform of the flagella to turn a cell toward the light. Protein depletion by RNA interference reveals that both AGG gene products play roles in the orientation of cells to a directional light source. The depleted strains mimic the phenotype of the previously identified agg1 mutant, which swims away from light. We propose that the localization of Agg2p and Agg3p to the proximal region of the flagella may be important for interpreting light signals.     

Intergenic suppression between the flagellar MS ring protein FliF of Salmonella and FlhA, a membrane component of its export apparatus

The MS ring of the flagellar basal body of Salmonella is an integral membrane structure consisting of about 26 subunits of a 61-kDa protein, FliF. Out of many nonflagellate fliF mutants tested, three gave rise to intergenic suppressors in flagellar region II. The pseudorevertants swarmed, though poorly; this partial recovery of motile function was shown to be due to partial recovery of export function and flagellar assembly. The three parental mutants were all found to carry the same mutation, a six-base deletion corresponding to loss of Ala-174 and Ser-175 in the predicted periplasmic domain of the FliF protein. The 19 intergenic suppressors identified all lay in flhA, and they consisted of 10 independent examples at the nucleotide level or 9 at the amino acid level. Since two of the nine corresponded to different substitutions at the same amino acid position, only eight positions in the FlhA protein have given rise to suppressors. Thus, FliF-FlhA intergenic suppression is a fairly rare event. FlhA is a component of the flagellar protein export apparatus, with an integral membrane domain encompassing the N-terminal half of the sequence and a cytoplasmic C-terminal domain. All of the suppressing mutations lay within the integral membrane domain. These mutations, when placed in a wild-type fliF background, had no mutant phenotype. In the fliF mutant background, mutant FlhA was dominant, yielding a pseudorevertant phenotype. Wild-type FlhA did not exert significant negative dominance in the pseudorevertant background, indicating that it does not compete effectively with mutant FlhA for interaction with mutant FliF. Mutant FliF was partially dominant over wild-type FliF in both the wild-type and second-site FlhA backgrounds. Membrane fractionation experiments indicated that the fliF mutation, though preventing export, was mild enough to permit assembly of the MS ring itself, and also assembly of the cytoplasmic C ring onto the MS ring. The data from this study provide genetic support for a model in which at least the FlhA component of the export apparatus physically interacts with the MS ring within which it is housed.     

An essential 45 kDa yeast transmembrane protein reacts with anti-nuclear pore antibodies: purification of the protein, immunolocalization and cloning of the gene.

A yeast membrane protein was isolated by its binding to tRNA Sepharose column. The 45 kDa protein shares characteristics with rat liver nuclear pore proteins in having reactivity with a monoclonal antibody (RL1) raised against rat liver nuclear pore proteins and by the binding of wheat germ agglutinin (WGA), indicating the presence of N-acetylglucosamine (GlcNAc) moieties. Immunofluorescence microscopy and cell fractionation experiments indicate that the protein is located in the nuclear envelope and the endoplasmic reticulum of the cell. The gene for the 45 kDa protein was cloned using degenerate oligonucleotides derived from the N-terminal protein sequence and confirmed by internal peptide sequences. The gene was named WBP1. The protein coding sequence of the WBP1 gene reveals an ER entry signal peptide and a C-terminal membrane spanning domain. Topological studies indicate that the C-terminus of the protein is located in the cytoplasm. The cytoplasmic tail of the protein contains the K-K-X-X signal known to be sufficient for retention of transmembrane proteins in higher eukaryotic cells. Gene disruption experiments show that the 45 kDa protein is essential for the vegetative life cycle of the yeast cell.     

Ultrastructural analysis of flagellar development in plurilocular sporangia of Ectocarpus siliculosus (Phaeophyceae)

Flagellar development in the plurilocular zoidangia of sporophytes of the brown alga Ectocarpus siliculosus was analyzed in detail using transmission electron microscopy and electron tomography. A series of cell divisions in the plurilocular zoidangia produced the spore-mother cells. In these cells, the centrioles differentiated into flagellar basal bodies with basal plates at their distal ends and attached to the plasma membrane. The plasma membrane formed a depression (flagellar pocket) into where the flagella elongated and in which variously sized vesicles and cytoplasmic fragments accumulated. The anterior and posterior flagella started elongating simultaneously, and the vesicles and cytoplasmic fragments in the flagellar pocket fused to the flagellar membranes. The two flagella (anterior and posterior) could be clearly distinguished from each other at the initial stage of their development by differences in length, diameter and the appendage flagellar rootlets. Flagella continued to elongate in the flagellar pocket and maintained their mutually parallel arrangement as the flagellar pocket gradually changed position. In mature zoids, the basal part of the posterior flagellum (paraflagellar body) characteristically became swollen and faced the eyespot region. Electron dense materials accumulated between the axoneme and the flagellar membrane, and crystallized materials could also be observed in the swollen region. Before liberation of the zoospores from the plurilocular zoidangia, mastigoneme attachment was restricted to the distal region of the anterior flagellum. Structures just below the flagellar membrane that connected to the mastigonemes were clearly visible by electron tomography.