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.     

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.     

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.     

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).     

Ordering of the flagellar genes in Pseudomonas aeruginosa by insertions of mercury transposon Tn501.

The flagellar genes of Pseudomonas aeruginosa PAO cluster on the chromosome at two distinct regions, region I and region II. The order of the flagellar cistrons in this organism was established by using transducing phage G101 and plasmids FP5 and R68.45. A method to insert transposon Tn501 near the fla genes was devised. We obtained two strains in which Tn501 was inserted at sites close to the flagellar cistrons in region II. We isolated Fla mutants in which the chromosomal segment between the two Tn501 insertion sites was deleted. Using Tn501-encoded mercury resistance as an outside marker, we determined the order of 9 of the 11 flagellar cistrons in region II as follows: puuF-region I-flaG-flaC-flaI-flaH-flaD-flaB-flaA-flaF-flaE-pur-67. By using phage G101-mediated transduction, the mutation converting monoflagellated bacteria into the multiflagellated (mfl) form was closely linked to the five fla cistrons in region I. Using mfl as an outside marker, we determined the order of the five cistrons as follows: puuF-flaV-flaZ-flaW-flaX-flaY-region II. The mfl mutation was shown to be either located within the flaV cistron or linked very closely to this cistron. No linkage was observed in transductions between any of the fla cistrons in region I and any of the fla cistrons in region II.     

Operon structure of flagellar genes in Salmonella typhimurium

Summary In Salmonella typhimurium, more than 40 genes have been shown to be involved in flagellar formation and function and almost all of them have been assigned to three regions of the chromosome, termed region I, region II, and region III. In the present study, a large number of transposon-insertion mutants in these flagellar genes were isolated using Tn10 and Mud1. The flaV gene was found to be a strong hot spot for Tn10 insertion. Complementation analysis of the polarity effects exerted by the transposon-insertion mutants defined 13 different flagellar operons; 3 in region I, 4 in region II, and 6 in region III. These results are compared with the reported arrangement of the corresponding genes in Escherichia coli.     

Two classes of region III flagellar genes in Escherichia coli

We infected various nonflagellated mutants of Escherichia coli with fla-transducing phages and followed the kinetics of the appearance of motility. Our analysis revealed two distinct classes of region III fla genes. Class II fla genes (hag, flaD) functioned 15 min later than class I fla genes (flaN, flaB, flaC, flaO, flaA, flbD, flaQ, flaP) in flagellar morphogenesis. We suggest that the two classes of fla genes are involved in two different stages, initiation (class I) and completion (class II), of flagellar formation.     

Genes for the hook-basal body proteins of the flagellar apparatus in Escherichia coli.

Of the more than 30 genes required for flagellar function, 6 are located between pyrC and ptsG on the Escherichia coli genetic man. This cluster of genes is called flagellar region I. Four-point transductional crosses were used to establish the position and order of the region I flagellar genes with respect to the outside markers ptsG and pyrC. Bacteriophage lambda-E. coli hybrids that contained most of the genes necessary for flagellar formation were constructed. The properties of specific hybrids that carried the region I fla genes were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the lambda hybrids demonstrated the existence of at least six flagellar-specific cistrons. These directed the synthesis of polypeptides with the following apparent molecular weights: flaV, 11,000; flaK, 42,000; flaL, 30,000 and 27,000; flaM, 38,000; flS, 60,000; and flaT, 35,000. Plasmid ColE1-E. coli hybrids with region I flagellar genes were also used to program the synthesis of polypeptides in minicell-producing strains. The polypeptides synthesized in these experiments were identical to polypeptides of the hook-basal body structure and helped to confirm the assignment of genes to specific polypeptides. The synthesis of all of these polypeptides was regulated by the same mechanism that regulates the synthesis of other flagellar-related structural components.     

Motility development of Salmonella typhimurium cells with flaV mutations after addition of exogenous flagellin

Cells of Salmonella typhimurium with flaV mutations developed motility after exogenous addition of flagellin, similar to what is observed with flbC mutants of Escherichia coli K-12.     

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.     

Structural basis of C3b binding by glycoprotein C of herpes simplex virus.

Glycoproteins C (gC) from herpes simplex virus type 1 (HSV-1) and HSV-2, gC-1 and gC-2, bind the human complement fragment C3b, although the two glycoproteins differ in their abilities to act as C3b receptors on infected cells and in their effects on the alternative complement pathway. Previously, we identified three regions of gC-2 (I, II, and III) which are important for C3b binding. In this study, our goal was to identify C3b-binding sites on gC-1 and to continue our analysis of gC-2. We constructed a large panel of mutants by using the cloned gC-1 and gC-2 genes. Most of the mutant proteins were transported to the surface of transiently transfected L cells and reacted with one or more monoclonal antibodies to discontinuous epitopes. By using 31 linker insertion mutants spread across the coding region of gC-1, we identified four regions in the ectodomain of gC-1 which are important for C3b binding, three of which are similar in position to C3b-binding regions I, II, and III of gC-2. Region III shares some similarities with the short consensus repeat found in CR1, the human complement receptor. These were, in part, the targets for construction of 20 single amino acid changes in region III of gC-1 and gC-2. These mutants identified similarities and differences in the C3b-binding properties of gC-1 and gC-2 and suggest that the amino half of region III is more important for C3b binding. However, our results do not support the concept of a structural relationship between the short consensus repeat of CR1 and gC, since mutations of some of the conserved residues, including three of four cysteines in region III, had no effect on C3b binding. Finally, we constructed four deletion mutants of gC-1, including one which lacked residues 33 to 123, as well as residues 367 to 449. This severely truncated molecule, lacking four cysteines and five potential N-linked glycosylation sites, was transported to the cell surface and retained its ability to bind monoclonal antibodies as well as C3b. Thus, the four distinct C3b-binding regions of gC-1 and several epitopes within two different antigenic sites are localized within residues 124 to 366.     

APPLICATION OF A TRANSPOSON FOOTPRINTING TECHNIQUE FOR RAPID IDENTIFICATION OF SALMONELLA TYPHIMURIUM TN5 MUTANTS REQUIRED FOR SURVIVAL UNDER DESICCATION STRESS CONDITIONS

Salmonella spp. are one of the foodborne pathogens that can be isolated in the environments of poultry houses and desiccation is a potential stress condition that can influence the survival of Salmonella spp. in this environment. In order to investigate the desiccation survival mechanism of Salmonella spp. the genome of S. typhimurium ATCC 14028 was screened for the genes potentially required for survival during desiccation using a novel method based on Tn5 mutagenesis previously developed in our laboratory. This method, termed transposon footprinting, simultaneously amplifies the Tn5-flanking sequences in a complex pool of the Tn5 mutants. As the length of the amplified DNA fragment should be unique for each distinct Tn5 mutant, the polymerase chain reaction (PCR) products separated on an agarose gel generate transposon footprints with each band in the footprint representing the corresponding Tn5 mutant. By comparing the transposon footprints from the pools of S. typhimurium Tn5 mutants before and after exposure to desiccation, Tn5 mutants that were not recovered after the selection were rapidly identified that would be easily isolated for further genetic analysis.     

Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters

By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.     

Genetic Analysis of Flagellar Mutants in Escherichia coli

Flagellar mutants in Escherichia coli were obtained by selection for resistance to the flagellotropic phage chi. F elements covering various regions of the E. coli genome were then constructed, and, on the basis of the ability of these elements to restore flagellar function, the mutations were assigned to three regions of the E. coli chromosome. Region I is between trp and gal; region II is between uvrC and aroD; and region III is between his and uvrC. F elements carrying flagellar mutations were constructed. Stable merodiploid strains with a flagellar defect on the exogenote and another on the endogenote were then prepared. These merodiploids yielded information on the complementation behavior of mutations in a given region. Region III was shown to include at least six cistrons, A, B, C, D, E, and F. Region II was shown to include at least four cistrons, G, H, I, and J. Examination of the phenotypes of the mutants revealed that those with lesions in cistron E of region III produce "polyhooks" and lesions in cistron F of region III result in loss of ability to produce flagellin. Mutants with lesions in cistron J of region II were entirely paralyzed (mot) mutants. Genetic analysis of flagellar mutations in region III suggested that the mutations located in cistrons A, B, C, and E are closely linked and mutations in cistrons D and F are closely linked.     

Subdivision of flagellar region III of the Escherichia coli and Salmonella typhimurium chromosomes and identification of two additional flagellar genes.

The many genes involved in flagellar structure and function in Escherichia coli and Salmonella typhimurium are located in three major clusters on the chromosome: flagellar regions I, II and III. We have found that region III does not consist of a contiguous set of flagellar genes, as was thought, but that in E. coli there is almost 7 kb of DNA between the filament cap gene, fliD, and the next known flagellar gene, fliE; a similar situation occurs in S. typhimurium. Most of this DNA is unrelated to flagellar function, since a mutant in which 5.4 kb of it had been deleted remained fully motile and chemotactic as judged by swarming on semi-solid agar. We have therefore subdivided flagellar region III into two regions, IIIa and IIIb. The known genes in region IIIa are fliABCD, all of which are involved in filament structure and assembly, while region IIIb contains genes fliEFGHIJKLMNOPQR, all of which are related to formation of the hook (basal-body)-complex or to even earlier assembly events. We have found that fliD, the last known gene in region IIIa, is immediately followed by two additional genes, both necessary for flagellation, which we have designated fliS and fliT. They encode small proteins with deduced molecular masses of about 15 kDa and 14 kDa, respectively. The functions of FliS and FliT remain to be determined, but they do not appear to be members of the axial family of structural proteins to which FliD belongs.     

Cobalamin-dependent 1,2-propanediol utilization by Salmonella typhimurium

The enteric bacterium Salmonella typhimurium utilizes 1,2-propanediol as a sole carbon and energy source during aerobic growth, but only when the cells are also provided with cobalamin as a nutritional supplement. This metabolism is mediated by the cobalamin-dependent propanediol dehydratase enzyme pathway. Thirty-three insertion mutants were isolated that lacked the ability to utilize propanediol, but retained the ability to degrade propionate. This phenotype is consistent with specific blocks in one or more steps of the propanediol dehydratase pathway. Enzyme assays confirmed that propanediol dehydratase activity was absent in some of the mutants. Thus, the affected genes were designated pdu (for defects in propanediol utilization). Seventeen mutants carried pdu::lac operon fusions, and these fusions were induced by propanediol in the culture medium. All of the pdu mutations were located in a single region (41 map units) on the S. typhimurium chromosome between the his (histidine biosynthesis) and branch I cob (cobalamin biosynthesis) operons. They were shown to be P22-cotransducible with a branch I cob marker at a mean frequency of 12%. Mutants that carried deletions of the genetic material between his and cob also failed to utilize propanediol as a sole carbon source. Based upon the formation of duplications and deletions between different pairs of his::MudA and pdu::MudA insertions, the pdu genes were transcribed in a clockwise direction relative to the S. typhimurium genetic map.