Nanoscopic analysis of oxygen segregation at tilt boundaries in silicon ingots using atom probe tomography combined with TEM and ab initio calculations.

We have developed an analytical method to determine the segregation levels on the same tilt boundaries (TBs) at the same nanoscopic location by a joint use of atom probe tomography and scanning transmission electron microscopy, and discussed the mechanism of oxygen segregation at TBs in silicon ingots in terms of bond distortions around the TBs. The three-dimensional distribution of oxygen atoms was determined at the typical small- and large-angle TBs by atom probe tomography with a low impurity detection limit (0.01 at.% on a TB plane) simultaneously with high spatial resolution (about 0.4 nm). The three-dimensional distribution was correlated with the atomic stress around the TBs; the stress at large-angle TBs was estimated by ab initio calculations based on atomic resolution scanning transmission electron microscopy data and that at small-angle TBs were calculated with the elastic theory based on dark-field transmission electron microscopy data. Oxygen atoms would segregate at bond-centred sites under tensile stress above about 2 GPa, so as to attain a more stable bonding network by reducing the local stress. The number of oxygen atoms segregating in a unit TB area NGB (in atoms nm-2 ) was determined to be proportional to both the number of the atomic sites under tensile stress in a unit TB area nbc and the average concentration of oxygen atoms around the TB [Oi ] (in at.%) with NGB ∼ 50 nbc [Oi ].

Nucleotide sequence of the flgD gene of Salmonella typhimurium which is essential for flagellar hook formation.

We determined the complete nucleotide sequence of the flgD gene of Salmonella typhimurium which is essential for flagellar hook formation. The sequence predicts a protein of 232 amino acids and a calculated molecular mass of 23,987 Da. However, the N-terminal 86 amino acids of FlgD were found sufficient to complement all the flgD mutations examined. The predicted secondary structure suggested that FlgD has a high content of beta structure.

Control by phosphatidylglycerol of expression of the flhD gene in Escherichia coli.

We reported elsewhere that mutation in the pgsA gene, responsible for the synthesis of phosphatidylglycerol, repressed the synthesis of flagellin and caused the loss of motility of Escherichia coli (Tomura et al., FEBS Letters 329, 287-290, 1993). We now describe evidence for a decrease in promoter activity of the flhD gene, a master gene for flagellum synthesis, in the pgsA3 mutant. We constructed a plasmid with a promoter region of the flhD gene connected with the structure region of the lacZ gene. The activity of beta-galactosidase in the extract prepared from the pgsA3 mutant harboring the fusion plasmid was 30% of that in the wild type cells. This result means that phosphatidylglycerol is likely to be required for the initiation of transcription of the flhD gene. We also found that the motility-less phenotype of the mutant was partially suppressed by elevating incubation temperature. This suppression is caused by restoration of transcription of the flhD gene by high temperature. As the content of phosphatidylglycerol did not increase by elevating incubation temperature, we proposed that this suppression is caused by alternation of a physical structure of phospholipid bilayers in cytoplasmic membranes.

FlgB, FlgC, FlgF and FlgG. A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium.

The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.

Definition of additional flagellar genes in Escherichia coli K12.

Twenty-nine flagellar genes in Escherichia coli K12 have previously been assigned to three regions of the genome. Flagellar region I is located between pyrC and ptsG, region II between aroD and uvrC, and region III between uvrC and his. In this study, flagellar mutants in Escherichia coli K12 were obtained by selection for resistance to the flagellotropic phage, chi. They were analyzed in complementation tests using P1 phage-mediated transduction. In addition to the fla genes already described, eight more flagellar genes were identified. This analysis defined six more fla genes in region I (flaU, etc.), one more in region II (flbB) and one more in region III (flbC). Region I was shown to include at least 12 fla cistrons. Complementation analysis with polar Mu phage-induced Fla- mutants and with lambda fla phage defined four transcriptional units in region I. These were: flaU, flbA-flaW-flaV-flaK-flaX-flaL-flaY-flaM, flaZ and flaS- flaT, with transcription proceeding from left to right. The flB gene was found to be part of an operon: flB-flaI in region II. In region III, a previously unidentified gene flbC was located between hag and flaN.

Identification of a novel Escherichia coli gene whose expression is dependent on the flagellum-specific sigma factor, FliA, but dispensable for motility development.

FliA is an alternative sigma factor specific for class 3 flagellar operons. Using a promoter-probe vector, we randomly cloned Escherichia coli DNA fragments, which showed FliA-dependent promoter activities. Among the DNA fragments cloned, one was found to be derived from a non-flagellar region. Hybridization analysis with the Kohara E. coli library indicated that this DNA fragment is located at around 35.4 min on the E. coli chromosome where no flagellar gene has been reported yet. DNA sequence analysis revealed that it contains an FliA-dependent promoter-like sequence followed by an open reading frame (ORF) that can encode a 110-amino-acid protein. A rho-independent terminator-like sequence follows this ORF. This putative gene was named flxA. A gene disruptant was constructed by inserting the kan gene cassette into the flxA gene on the chromosome. This mutant was found to be actively motile, suggesting that this gene is unlikely to be involved in the motility phenotype of E. coli.

A gene for DNA invertase and an invertible DNA in Escherichia coli K-12.

An assay system for the pin gene function, which suppresses the vh2 mutation of Salmonella, was developed and used to show that most strains of Escherichia coli K-12 are Pin+, whereas all the strains of E. coli C examined are Pin-. An E. coli host strain was constructed and used for detection of DNA fragments carrying the E. coli K-12 pin gene cloned in the plasmid vector pBR322. Restriction analysis of the cloned fragments showed that the invertible DNA (designated P region) is adjacent to the pin gene and that its inversion is mediated by the pin gene product. The pin gene was found to be functionally homologous to the gin gene of Mu phage and the cin gene of P1 phage. The P region most probably resides within the cryptic prophage e14, and the Pin- phenotype is likely to be associated with the loss of e14.

Sequence analysis of the flgA gene and its adjacent region in Salmonella typhimurium, and identification of another flagellar gene, flgN.

The flagellar genes flgA and flgM are located at the terminus of the region-I flagellar gene cluster on the chromosome of Salmonella typhimurium. The flgA gene is involved in P-ring formation of the flagellar basal body, whereas flgM encodes the anti-sigma factor which acts as a negative regulator of the flagellar regulon. The nucleotide sequence of the DNA fragment containing these flagellar genes and the adjacent region was determined. The flgA gene was found to encode a 219-amino-acid (aa) protein of 23,556 Da. The N-terminal region of FlgA has the characteristics of a typical signal sequence, suggesting that FlgA may function in the periplasmic space where P-ring assembly takes place. The flgM gene was found to constitute an operon together with an ORF which encodes a 140-aa protein of 15,899 Da. A gene disruption mutant was constructed by inserting a cat gene cartridge into the ORF on the chromosome. This mutant showed only weak motility, indicating that the product of the ORF is involved in flagellar formation. Therefore, this ORF was designated as flgN. Electron microscopic observation revealed that most of the flagellar structures produced by the flgN mutant are hook-basal body complexes lacking the filament portions. Based on these results, we concluded that the flgN product is required for the efficient initiation of filament assembly.

Participation of the hup gene product in site-specific DNA inversion in Escherichia coli.

The closely related Escherichia coli genes hupA and hupB each encode a bacterial histone-like protein HU. We report here that DNA inversion mediated by hin, gin, pin and rci but not by cin is blocked in a hupA hupB double mutant, although inversions in these systems occur in the hupA or hupB single mutant as efficiently as in the wild-type strain. These findings show that HU protein participates in site-specific DNA inversion in E. coli and that only one subunit, either HU-1 or HU-2, is sufficient for this inversion.

Sequence analysis of operator mutants of the phase-1 flagellin-encoding gene, fliC, in Salmonella typhimurium.

In phase-2 cells of diphasic Salmonella strains, expression of the phase-1 flagellin-encoding gene, fliC, is repressed by the repressor encoded by the fljA gene. Nine operator-constitutive (Oc) mutants of fliC were isolated from S. typhimurium by selecting those which could express fliC in the presence of the repressor. Among them, eight mutants could express fliC both in the presence and the absence of the repressor, whereas the ninth one could express only in the presence of the repressor. Nucleotide sequence analysis revealed that the Oc mutations of the former type were all located between bp 7 and 20 upstream from the coding region of fliC, which suggests that this region may correspond to the operator for fliC. The latter mutant was found to have a tandem duplication of 28 bp which contains a part of the operator sequence, and seems to require the repressor to activate fliC expression.

Reevaluation of the promoter structure of the class 3 flagellar operons of Escherichia coli and Salmonella.

Flagellar class 3 operons of Escherichia coli and Salmonella are transcribed by RNA polymerase containing sigma 28. The consensus sequence of the sigma 28-dependent promoters was believed to be TAAA N15 GCCGATAA. In this study, we found that the E. coli genome contains a large number of sequences homologous to this consensus. However, we showed that they do not always exert a sigma 28-dependent promoter activity. We compare more carefully the sequences of the class 3 flagellar promoters and propose a revised structure of the sigma 28-dependent promoters as TAAAGTTT N11 GCCGATAA.

Two novel regulatory genes, fliT and fliZ, in the flagellar regulon of Salmonella.

The flagellar operons of Salmonella are divided into three classes with respect to their transcriptional hierarchy. Expression of the class 2 operons requires the class 1 gene products, FlhD and FlhC, and is increased by mutation in the flgM gene, which encodes a class 3-specific anti-sigma factor. Here we report the identification of two novel regulatory genes for class 2 transcription. Presence of the fliZ and fliT genes on multicopy plasmids enhanced and inhibited, respectively, transcription from a chromosomal class 2 promoter. Disruption of the fliZ and fliT genes on the chromosome decreased and increased, respectively, class 2 expression. These results suggest that the fliZ and fliT genes may encode positive and negative regulatory factors, respectively, for class 2 expression. Enhancement of class 2 expression by the flgM mutation was cancelled by the coexisting fliZ mutation, indicating that FliZ is essential for this enhancement.

Promoter analysis of the class 2 flagellar operons of Salmonella.

The Salmonella flagellar operons are divided into three classes with reference to their relative positions in the transcriptional hierarchy. Expression of the class 2 operons requires the class 1 gene products, FlhD and FlhC, and is enhanced by an unknown mechanism in the presence of the class 3-specific sigma factor, FliA, and in the absence of its cognate anti-sigma factor, FlgM. In this study, the transcriptional start site mapping was performed by primer extension analysis for five class 2 operons, flgA, flgB, flhB, fliE and fliL. In all cases, one or a few major transcriptional start sites were identified. These start signals disappeared in the flhDC-mutant background, and their intensity decreased and increased in the fliA-mutant and flgM-mutant backgrounds, respectively. Therefore, we conclude that the FlhD/FlhC-dependent transcription is responsible for the FliA-dependent enhancement. Sequence comparison revealed that an imperfect inverted repetitious sequence is conserved upstream of the class 2 operons. Truncation of this sequence from the flgB promoter reduced its transcriptional activity to the background level, indicating that this is an essential cis-acting element for transcription of the class 2 operons.

Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium.

The flhD and flhC genes constitute the flagellar master operon whose products are required for expression of all the remaining flagellar operons in Salmonella typhimurium. Here we report the molecular structure and in vivo and in vitro expression of the flhD operon. Nucleotide sequence analysis revealed that the upstream region of this operon contains the consensus sequence for the cAMP-CRP binding site. Primer extension analysis demonstrated six possible transcription start sites for this operon. They include CRP-dependent and CRP-repressible transcription start sites. The CRP-dependent transcription start site is located 203 bp upstream of the initiation codon of the flhD gene and preceded by the consensus sequences of the -10 and -35 regions of the sigma 70-dependent promoter. The putative cAMP-CRP binding site is located centered 70 bp upstream of this start site. The CRP-repressible transcription start site is located within this putative cAMP-CRP binding site. These two start sites were confirmed by in vitro transcription experiments using sigma 70-RNA polymerase with or without cAMP-CRP.

Excretion of the anti-sigma factor through a flagellar substructure couples flagellar gene expression with flagellar assembly in Salmonella typhimurium.

More than 50 genes are required for flagellar formation and function in Salmonella typhimurium. According to the cascade model of the flagellar regulon, the flagellar operons are divided into three classes, 1, 2, and 3, with reference to their relative positions in the transcriptional hierarchy. This sequential transcription is coupled to the assembly process of the flagellar structure, that is, genes involved in formation of the hook-basal body complex belong to the class-2 operons, whereas those involved in formation of filament belong to the class-3 operons. The fliA gene encodes an alternative sigma factor specific for transcription of the class-3 operons. A negative regulatory gene, flgM, which is responsible for the coupling of expression of class-3 operons to flagellar assembly, encodes an anti-sigma factor that binds to FliA and prevents its association with RNA polymerase core enzyme. In the present study, we showed that the flgM gene is transcribed from two different promoters: one is its own class-3 promoter and the other is the class-2 promoter for the upstream gene, flgA. Furthermore, we showed that FlgM is excreted into culture medium from cells of the wild-type strain and of class-3 mutants that can produce complete hook-basal body structures. On the other hand, FlgM is not excreted from mutants defective in the hook-basal body genes. These results indicate that FlgM is excreted from the cells through the flagellar substructures that are formed by the function of the hook-basal body genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Autogenous and global control of the flagellar master operon, flhD, in Salmonella typhimurium.

Expression of the flagellar master operon, flhD, is known to be affected by growth conditions and by mutations in a variety of genes. In the present work, the transcriptional control of the Salmonella typhimurium flhD operon was investigated in various genetic backgrounds. First, we examined the effect of mutations in the global regulators cAMP-CRP, H-NS, OmpR and RpoS. Mutations in the cya, crp or hns gene reduced but did not eliminate flhD expression. However, expression was completely inhibited in the cya hns and crp hns double mutants. These results indicate that cAMP-CRP and H-NS independently activate the flhD operon and that maximal expression is attained in the presence of both regulators. On the other hand, the ompR and rpoS mutations did not affect either the motility phenotype or flhD expression. We next examined the expression of a chromosomal flhD-lac fusion gene in the presence of a plasmid carrying the wild-type flhD operon. It was found that under this condition the chromosomal flhD operon was repressed or activated, depending on the intracellular activity of FliA, an alternative sigma factor specific for late flagellar operons. In the absence of FliA or in the presence of both FliA and its cognate anti-sigma factor FlgM, the flhD operon was autogenously repressed, whereas in the flgM mutant background it was autogenously activated in the presence of FliA. This autoregulation was still observed in the crp or hns mutant background, indicating that the autogenous control is achieved by a mechanism that is independent of the cAMP-CRP and H-NS regulatory pathways.

Hook-length control of the export-switching machinery involves a double-locked gate in Salmonella typhimurium flagellar morphogenesis.

During flagellar morphogenesis in Salmonella typhimurium, the genes involved in filament assembly are expressed fully only after completion of hook-basal body assembly. This coupling of gene expression to morphogenesis is achieved by exporting the flagellum-specific anti-sigma factor, FlgM, out of the cell through the mature hook-basal body structure. Therefore, the flagellum-specific export apparatus must be able to sense the assembly state of the flagellar structure and to turn on FlgM export at a specific stage of hook assembly. It has been suggested that FlhB may act as the molecular switch which mediates this ordered export. Here, I report genetic evidence that in addition to FlhB, the product of a newly identified gene, rflH, is involved in the negative regulation of FlgM export. FlgM is released through the basal body structure lacking the hook and the filament only when the flhB and rflH genes are both defective. Therefore, the export gate for FlgM should be double locked by FlhB and RflH. The rflH gene is located at around 52 min, where no flagellum-related gene has been found. I propose a revised model of the export-switching machinery which consists of two systems, the hook-length signal transduction pathway and the double-locked gate for FlgM export.

Molecular dissection of the flagellum-specific anti-sigma factor, FlgM, of Salmonella typhimurium.

In the flagellar regulon of Salmonella typhimurium, the flagellar operons are divided into three classes, 1, 2 and 3, with respect to transcriptional hierarchy. Class 3 operons are controlled positively by FliA, a flagellum-specific sigma factor, and negatively by FlgM, an anti-sigma factor which binds to FliA and inhibits its activity. The sequential expression of flagellar operons is coupled to the assembly process of flagellar structures. This coupling is achieved by the fact that FlgM is exported out of the cell through the flagellar structures that are formed by the functions of the class 1 and 2 genes. Therefore, FlgM has a dual function: it can bind to FliA and is capable of being exported through the flagellar structure. In this study, using a set of deletion mutants of flgM in high-expression plasmids, we demonstrated that polypeptides containing the C-terminal portion of FlgM could inhibit the FliA-dependent transcription of the class 3 genes. Loss of amino acids near the N-terminus eliminated the export of the protein, while loss of C-terminal amino acids did not affect this function. These results indicate that the domain essential for export lies in the N-terminal region and that for FliA-binding in the C-terminal region.

Role of the FliA-FlgM regulatory system on the transcriptional control of the flagellar regulon and flagellar formation in Salmonella typhimurium.

In the flagellar regulon of Salmonella typhimurium, the flagellar operons are divided into three classes, 1, 2, and 3, with respect to transcriptional hierarchy. The class 2 operons are controlled positively by the class 1 genes, flhD and flhC. The class 3 operons are controlled positively by fliA and negatively by flgM. It has been shown that FliA is a sigma factor specific for class 3, whereas FlgM is an anti-sigma factor which binds FliA to prevent its association with RNA polymerase core enzyme. Therefore, the FliA-FlgM regulatory system has been believed to control specifically the class 3 operons. In the present study, we showed that the flgM mutation enhanced the expression of class 2 by more than fivefold. When a fliA mutation was present simultaneously, this enhancement was not observed. These results indicate that the FliA-FlgM regulatory system is involved not only in the expression of class 3 but also in that of class 2. However, though neither flhD nor flhC mutants could express the class 2 operons, the fliA mutants permitted the basal-level expression of those operons. Therefore, FlhD and FlhC are indispensable for the expression of class 2, whereas FliA is required only for its enhancement in the FlgM-depletion condition. Furthermore, we showed that the flgM mutation resulted in a two- to threefold increase in flagellar number. On the basis of these results, we propose that the relative concentration of FliA and FlgM may play an important role in the determination of flagellar numbers produced by a single cell.

Transcriptional analysis of the flgK and fliD operons of Salmonella typhimurium which encode flagellar hook-associated proteins.

In Salmonella typhimurium, three hook-associated proteins, HAP1, HAP2 and HAP3, are known to be essential for formation of flagellar filament. HAP1 and HAP2 are encoded by the flgK and flgL genes, respectively, which together constitute an operon, called the flgK operon. HAP3 is encoded by the fliD gene which forms part of the fliD operon together with the fliS and fliT genes. In the flagellar regulon, the operons are divided into three classes, 1, 2 and 3, based on their positions within a transcriptional hierarchy. Transcriptional analysis suggested that the flgK and fliD operons should belong to class 3, whose expression is dependent on the flagellum-specific sigma factor FliA. However, biochemical data indicated that these HAP proteins are detectable even in the hook-basal body structures produced by the fliA mutant. This work was carried out to resolve this discrepancy. More careful examination of transcription revealed that the fliA mutation reduces but does not eliminate the expression of these operons, whereas a mutation in the flhD operon, which encodes activator proteins for the class 2 operons, eliminates their expression. This suggests that the flgK and fliD operons may be transcribed from both class 2 and class 3 promoters. Primer extension analysis indicated that the promoter region of fliD contains both class 2 and class 3 promoters, while that of flgK contains only a class 3 promoter. Transposon insertion into the flgB operon, which belongs to class 2 and lies upstream of the flgK operon, was found to decrease the expression of the flgK operon to the basal level.(ABSTRACT TRUNCATED AT 250 WORDS)