Hook-associated proteins essential for flagellar filament formation in Salmonella typhimurium.

The hooks of the flagella of Salmonella typhimurium were purified by a newly developed method, using a flaL mutant without a filament, and the hook components were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, we detected three protein species in addition to hook protein. We call these three proteins hook-associated proteins (HAPs). Their molecular weights were 59,000 for HAP1, 53,000 for HAP2, and 31,000 for HAP3. The HAP1/hook protein/HAP3/HAP2 molar ratio, calculated from their relative amounts and their molecular weights, was 1:10:1.1:0.53. The compositions of HAPs were analyzed in the hooks from the other filamentless mutants which were defective in H1 H2, flaV, flaU, or flaW. Hooks from the H1 H2 mutant had the same HAP composition as hooks from the flaL mutant. Hooks from the flaV mutants contained HAP1 and HAP3. Hooks from the flaU mutants contained HAP1. Hooks from the flaW mutants contained a very small amount of HAP3. From these results, the process of hook morphogenesis and the genes responsible for each step were postulated. Electron micrographs of hooks from the filamentless mutants showed that hooks which contained all three HAPs had a sharp clawlike tip, whereas hooks lacking any HAP had a flat tip. Electron micrographs of hooks treated with antibody against the hook protein showed that each claw-shaped end was not covered with antibody. These results strongly suggest that all three HAPs or at least some of them are located at the claw-shaped end and play an essential role in filament formation.     

Genetic analysis of three additional fla genes in Salmonella typhimurium

In Salmonella typhimurium, 27 fla genes responsible for formation of flagella have been identified and assigned to three regions on the genetic map, termed fla regions I to III. By genetic analysis of 1984 non-flagellate mutants obtained from a phase-1 stable strain of S. typhimurium, SJW1103, three additional fla genes were identified; one, termed flaW, was assigned to fla region I and the other two, termed flaV and flaX, to fla region III. By intergeneric complementation tests, the flaW, flaV and flaX genes were shown to be functionally homologous with flaS, flbC and flaP of Escherichia coli, respectively. Electron microscopy showed that flaW and flaV mutants carried hook-basal body structures.     

Synthesis of flagellin and hook subunit protein in flagellar mutants of Escherichia coli K12

Summary We have examined Escherichia coli K12 flagellar mutants affected in each of 29 different loci for the synthesis of flagellin and hook subunit protein. Immune precipitation experiments were employed by treating cell extracts with antiserum against each protein. Flagellin was synthesized in mutants defective in genes flaS, flaT, flaU and flbC. The flaE and flaZ mutants produced small amounts of flagellin, while all the other mutants failed to produce any detectable amount of flagellin.Hook subunit protein was found in most mutants including those defective in genes flaA, flaB, flaC, flaD, flaE, flaG, flaH, flaL, flaM, flaN, flaO, flaP, flaQ, flaR, flaS, flaT, flaU, flaV, flaW, flaX, flaY, flaZ, flbA, flbC, flbD, and hag but not in mutants of flaK, flaI, and flbB. The results conform to the predictions made by our previous indirect gene fusion study (Komeda 1982).     

Excretion of unassembled flagellin by Salmonella typhimurium mutants deficient in hook-associated proteins.

Of the flagellar filamentless mutants of Salmonella typhimurium, the flaV, flaU, and flaW mutants, which are defective in hook-associated proteins, synthesized flagellin molecules, but flagella did not polymerize at the tips of the mutant hooks and were excreted into the culture medium as intact monomers.     

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.     

Genetic and molecular characterization of the polar flagellum of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.     

Identification and characterization of the products of six region III flagellar genes (flaAII.3 through flaQII) of Salmonella typhimurium.

A portion of flagellar region III of the Salmonella typhimurium genome has been cloned and shown to contain six genes: flaAII.3, flaAIII, flaS, flaR, flaQI, and flaQII. Of these, all but flaQI were known to exist from mutant studies; the former flaQ has been renamed flaQII. The genes were shown by minicell analysis to encode proteins with apparent molecular masses of 28, 48, 15, 46, 17, and 37 kilodaltons, respectively. The presence of a flagellar-gene-specific promoter in the vicinity of flaQI was established by testing expression of the plasmid-encoded tetracycline resistance gene in artificial constructions. In minicell preparations, the flaAII.3 and flaR products were found principally in the cytoplasmic fraction; the rest were found principally in the membrane fraction. A comparison between the homologous genes of S. typhimurium and Escherichia coli confirmed that their genomic organizations were similar and that their products had similar molecular masses and isoelectric points.     

Formation of flagella lacking outer rings by flaM, flaU, and flaY mutants of Escherichia coli.

Among flagellar mutants of Escherichia coli, flaM or flaU mutants form basal bodies lacking the outer P and L rings, whereas flaY mutants predominantly form basal bodies lacking the L ring. In these mutants, hooks and filaments are occasionally assembled onto these incomplete basal bodies. When the hook protein gene, flaFV, of Salmonella typhimurium was cloned on the multicopy plasmid pBR322 and introduced into these mutants, the efficiency with which cells assembled hooks and filaments onto the incomplete basal bodies increased significantly. Such cells formed characteristic dotted swarms on semisolid plates, indicating that cells carrying flagella without the outer rings are weakly motile because of poor function of their flagella, a low flagellar number per cell, or both of these defects. FlaV mutants also produced incomplete basal bodies lacking the outer rings, but assembly of hooks and filaments did not occur in these mutants even after introduction of the plasmid carrying flaFV of S. typhimurium. The failure in the case of flaV mutants was attributed to their inability to modify the rod tip to the structure competent for assembly of hook protein.     

Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.     

Cloning and analysis of the sfrB (sex factor repression) gene of Escherichia coli K-12.

The sfrB gene of Escherichia coli K-12 and the rfaH gene of Salmonella typhimurium LT2 are homologous, controlling expression of the tra operon of F and the rfa genes for lipopolysaccharide synthesis. We have determined a restriction map of the 19-kilobase ColE1 plasmid pLC14-28 which carries the sfrB gene of E. coli. After partial Sau3A digestion of pLC14-28, we cloned a 2.5-kilobase DNA fragment into the BamHI site of pBR322 to form pKZ17. pKZ17 complemented mutants of the sfrB gene of E. coli and the rfaH gene of S. typhimurium for defects of both the F tra operon and the rfa genes. pKZ17 in minicells determines an 18-kilodalton protein not determined by pBR322. A Tn5 insertion into the sfrB gene causes loss of complementing activity and loss of the 18-kilodalton protein in minicells, indicating that this protein is the sfrB gene product. These data indicate that the sfrB gene product is a regulatory element, since the single gene product elicits the expression of genes for many products for F expression and lipopolysaccharide synthesis.     

Sequence of the phosphomannose isomerase-encoding gene of Salmonella typhimurium

The pmi gene, encoding phosphomannose isomerase, of Salmonella typhimurium, was cloned in Escherichia coli K-12, and the protein product visualised in minicells. The cloned gene was sequenced; there was 77.4% nucleotide homology between the cloned pmi gene and the analogous manA gene of E. coli K-12, and 86.2% amino acid sequence homology between their presumptive gene products.     

Excretion of unassembled hook-associated proteins by Salmonella typhimurium.

Hook-associated proteins (HAPs) were excreted into the culture medium of the Fla+ strain as well as into the growth medium of the filamentless mutants of Salmonella typhimurium. This indicates that the bacteria synthesize HAPs excessively, beyond the amount required for construction of flagella. The extra HAPs are shed into the culture medium after a definite amount of each HAP has been assembled into the flagellar structure.     

Functional Interchangeability of DNA Replication Genes in SALMONELLA TYPHIMURIUM and ESCHERICHIA COLI Demonstrated by a General Complementation Procedure

Twenty-four genes from Salmonella typhimurium that affect DNA replication were isolated from a lambda-Salmonella genomic library by lysogenic complementation of temperature-sensitive mutants of Salmonella or E. coli, using a new plaque complementation assay. The complementing lambda clones, which make red plaques in this assay, and noncomplementing mutant derivatives, which make uncolored plaques, were used to further characterize the temperature-sensitive Salmonella mutants and to establish the functional similarity of E. coli and Salmonella DNA replication genes. For 17 of 18 E. coli mutants representing distinct loci, a Salmonella gene that complemented the mutant was found. This result indicates that single Salmonella replication proteins are able to function in otherwise all E. coli replication complexes and suggests that the detailed properties of Salmonella and E. coli replication proteins are very similar. The other seven Salmonella genes that were cloned were unrelated functionally to any E. coli genes examined. --As an aid to the derivation of chromosomal mutations affecting some of the cloned genes, a general method was developed for placing a transposon in the Salmonella chromosome in a segment corresponding to cloned DNA. Chromosomal mutations were derived in Salmonella affecting a gene (dnaA) that was cloned by complementation of an E. coli mutant by using the transposon-encoded drug resistance as a selectable marker in local mutagenesis.     

Expression of the cloned Escherichia coli O9 rfb gene in various mutant strains of Salmonella typhimurium.

To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.     

Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase.

The phoN gene of Salmonella typhimurium encodes nonspecific acid phosphatase (EC 3.1.3.2), which is regulated by a two-component regulatory system consisting of the phoP and phoQ genes. We cloned the phoN region into a plasmid vector by complementation of a phoN mutant strain and determined the nucleotide sequence of the phoN gene and its flanking regions. The phoN gene could encode a 26-kDa protein, which was identified by the maxicell method as the product of phoN. Results of the enzyme assay and Southern hybridization with chromosomal DNA of Escherichia coli K-12 suggests that there is no phoN gene in E. coli. The regulatory pattern of phoN in E. coli and Southern hybridization analysis of the E. coli chromosome with the S. typhimurium phoP gene suggest that E. coli K-12 also harbors the phoP and phoQ genes.