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 ].

A flagellar gene fliZ regulates the expression of invasion genes and virulence phenotype in Salmonella enterica serovar Typhimurium.

We previously reported that the fliZ gene encodes a positive regulatory factor for the class 2 flagellar operons in Salmonella enterica serovar Typhimurium. In this study, we found that the fliZ mutation reduced not only the amounts of excreted flagellar proteins, but also those of several secreted invasion proteins encoded by the genes within Salmonella pathogenicity island 1. Using the lacZ gene fused to a subset of virulence-associated genes, we show that this downregulation was caused by a decreased transcription of the hilA gene, which encodes a positive regulator for the invasion genes. We further show that the fliZ mutation reduced invasion ability of S. enterica serovar Typhimurium to HEp-2 cells. Consistent with these results, orally challenged cells of the fliZ mutant show an attenuated virulence phenotype in a mouse typhoid model. These results indicate that the fliZ gene product positively regulates the invasion genes and is necessary for expression of full virulence.

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.

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.

Substrate specificity switching of the flagellum-specific export apparatus during flagellar morphogenesis in Salmonella typhimurium.

During flagellar morphogenesis in Salmonella typhimurium, the flagellum-specific anti-sigma factor FlgM is exported out of the cells only after completion of hook assembly. In this study, we examined the export of the flagellar proteins, FlgD (hook capping protein), FlgE (hook protein), FlgK and FlgL (hook-filament junction proteins), FliD (filament capping protein), and FliC (flagellin), before and after completion of hook assembly. Like the FlgM protein, the FlgK, FlgL, FliD, and FliC proteins are exported efficiently only after completion of hook assembly. On the other hand, the FlgD and FlgE proteins are exported efficiently before, but poorly after, hook completion. These results indicate that the export properties are different between these two groups and that their export order exactly parallels the assembly order of the hook-filament structure. We propose that the substrate specificity switching occurs in the flagellum-specific export apparatus upon completion of hook assembly.

Peptidoglycan-hydrolyzing activity of the FlgJ protein, essential for flagellar rod formation in Salmonella typhimurium.

Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation in Salmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein from S. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJ mutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.

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.

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.

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.

Negative regulation by fliD, fliS, and fliT of the export of the flagellum-specific anti-sigma factor, FlgM, in Salmonella typhimurium.

The fliD operon of Salmonella typhimurium consists of three flagellar genes, fliD, fliS, and fliT, and is transcribed in this order. It has been shown that an fliD::Tn10 mutation causes an excess export of the flagellum-specific anti-sigma factor, FlgM, resulting in an overexpression of flagellar class 3 operons. In this study, using gene-disruption mutants in the individual genes in the fliD operon, we showed that mutations in any one of the genes in the operon enhanced both FlgM export and the expression of flagellar regulon. This indicates that all three genes in the operon are involved in the negative regulation of FlgM export.

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.

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.

Functional analysis of the flagellar genes in the fliD operon of Salmonella typhimurium.

The fliD genes of Salmonella typhimurium and Escherichia coli encode the filament-cap protein of the flagellar apparatus, which facilitates the polymerization of endogenous flagellin at the tips of the growing filaments. Previous sequence analysis of this operon in both organisms has revealed that the fliD gene constitutes an operon together with two additional genes, fliS and fliT. Based on the gene-disruption experiment in E. coli, both the fliS and fliT genes have been postulated to be necessary for flagellation. In the present study, we constructed S. typhimurium mutants in which either fliS or fliT on the chromosome was specifically disrupted. Both mutants were found to produce functional flagella, indicating that these genes are dispensable for motility development in S. typhimurium. However, flagellar filaments produced by the fliS mutant were much shorter than those produced by the wild-type strain. This indicates that the fliS mutation affects the elongation step of filament assembly. The excretion efficiency of flagellin was examined in the fliD-mutant background, where the exported flagellin molecules cannot assemble onto the hooks, resulting in their excretion into the culture media. We found that the amount of flagellin excreted was much reduced by the fliS mutation. Based on these results, we conclude that FliS facilitates the export of flagellin through the flagellum-specific export pathway.

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)

Isolation and characterization of FliK-independent flagellation mutants from Salmonella typhimurium.

A flagellum of Salmonella typhimurium and Escherichia coli consists of three structural parts, a basal body, a hook, and a filament. Because the fliK mutants produce elongated hooks, called polyhooks, lacking filament portions, the fliK gene product has been believed to be involved in both the determination of hook length and the initiation of the filament assembly. In the present study, we isolated two mutants from S. typhimurium which can form flagella even in the absence of the fliK gene product. Flagellar structures were fractionated from these suppressor mutants and inspected by electron microscopy. The suppressor mutants produced polyhook-filament complexes in the fliK mutant background, while they formed flagellar structures apparently indistinguishable from those of the wild-type strain in the fliK+ background. Genetic and sequence analyses of the suppressor mutations revealed that they are located near the 3'-end of the flhB gene, which has been believed to be involved in the early process of the basal body assembly. On the basis of these results, we discuss the mechanism of suppression of the fliK defects by the flhB mutations and propose a hypothesis on the export switching machinery of the flagellar proteins.

Genetic and molecular analyses of the interaction between the flagellum-specific sigma and anti-sigma factors in Salmonella typhimurium.

More than 50 genes are required for flagellar formation and function in Salmonella typhimurium. According to the cascade model of flagellar regulon, the flagellar operons are divided into three classes, 1, 2, and 3, with respect to transcriptional hierarchy. FliA is an alternative sigma factor specific for transcription of the class 3 operons, while FlgM is an anti-sigma factor which binds to FliA and prevents its association with RNA polymerase core enzyme. In the present study, we isolated a number of fliA mutants in which the altered FliA proteins become insensitive to inhibition by FlgM. Sequence analysis of their mutation sites revealed that most of them caused the amino acid substitutions in region 4 of the conserved amino acid sequences of sigma factors which lies near the C-terminal end of FliA. Using a set of fliA deletion mutants in a high-expression plasmid, we demonstrated that polypeptides containing the C-terminal portion of FliA could titrate the intracellular FlgM protein resulting in derepression of the class 3 operons. This result indicates that the C-terminal region of FliA contains the FlgM-binding domain. This was confirmed by a chemical cross-linking experiment with FlgM and truncated FliA proteins.

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.

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)