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S Iyoda

Publications and source records attributed to S Iyoda.

11 recordsLinked to original sources
PubMed2001

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.

Animals↗
PubMed2000

Inducible stx2 phages are lysogenized in the enteroaggregative and other phenotypic Escherichia coli O86:HNM isolated from patients.

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.

Bacterial Adhesion↗
PubMed1999

Evaluation of AFLP, a high-resolution DNA fingerprinting method, as a tool for molecular subtyping of enterohemorrhagic Escherichia coli O157:H7 isolates.

The amplified fragment-length polymorphism (AFLPTM) technique is based on the selective PCR amplification of restriction fragments. We investigated the utility of AFLP in the molecular subtyping of enterohemorrhagic Escherichia coli serotype O157:H7 isolates. We analyzed a total of 46 isolates of E. coli O157:H7 along with other serotypes, O26:H11, 0114:H19 and 0119:NT. Isolates of E. coli O157:H7 derived from the same outbreak showed an identical AFLP-banding pattern and were subtyped into the same group, giving results almost consistent with those of a pulsed-field gel electrophoresis (PFGE) study, while other serotypes showed clearly different patterns from those of E. coli O157:H7. These results suggest that the AFLP technique has potential as an alternative tool for the molecular epidemiology of E. coli O157:H7.

Bacterial Typing Techniques↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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.

Bacterial Proteins↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

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.

Amino Acid Sequence↗