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Shin-Ichi Aizawa

Publications and source records attributed to Shin-Ichi Aizawa.

15 recordsLinked to original sources
PubMed2006

Characterization of two sets of subpolar flagella in Bradyrhizobium japonicum.

Bradyrhizobium japonicum is one of the soil bacteria that form nodules on soybean roots. The cell has two sets of flagellar systems, one thick flagellum and a few thin flagella, uniquely growing at subpolar positions. The thick flagellum appears to be semicoiled in morphology, and the thin flagella were in a tight-curly form as observed by dark-field microscopy. Flagellin genes were identified from the amino acid sequence of each flagellin. Flagellar genes for the thick flagellum are scattered into several clusters on the genome, while those genes for the thin flagellum are compactly organized in one cluster. Both types of flagella are powered by proton-driven motors. The swimming propulsion is supplied mainly by the thick flagellum. B. japonicum flagellar systems resemble the polar-lateral flagellar systems of Vibrio species but differ in several aspects.

Amino Acid Sequence↗
PubMed2006

Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay.

As part of our attempt to map the impact of acetyl phosphate (acetyl approximately P) on the entire network of two-component signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl approximately P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl approximately P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl approximately P produced no capsule under normally permissive conditions, while those that accumulated acetyl approximately P synthesized capsule under conditions previously considered to be non-permissive. Acetyl approximately P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl approximately P-deficient cells. Similarly, acetyl approximately P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl approximately P-sensitive activation of capsular biosynthesis and acetyl approximately P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl approximately P) in the transition of motile, planktonic individuals into sessile biofilm communities.

Bacterial Capsules↗
PubMed2006

Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus.

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.

Bdellovibrio↗
PubMed2006

Hundreds of flagellar basal bodies cover the cell surface of the endosymbiotic bacterium Buchnera aphidicola sp. strain APS.

Buchnera aphidicola is the endosymbiotic bacterium of the pea aphid. Due to its small genome size, Buchnera lacks many essential genes for autogenous life but obtains nutrients from the host. Although the Buchnera cell is nonmotile, it retains clusters of flagellar genes that lack the late genes necessary for motility, including the flagellin gene. In this study, we show that the flagellar genes are actually transcribed and translated and that the Buchnera cell surface is covered with hundreds of hook-basal-body (HBB) complexes. The abundance of HBB complexes suggests a role other than motility. We discuss the possibility that the HBB complex may serve as a protein transporter not only for the flagellar proteins but also for other proteins to maintain the symbiotic system.

Bacterial Proteins↗
PubMed2005

Flagellar filaments of the deep-sea bacteria Idiomarina loihiensis belong to a family different from those of Salmonella typhimurium.

The helical filaments of the bacterial flagella so far studied seem to be universal in the bacterial kingdom. Despite the variation in flagellin molecular masses, which range from 24 kDa to 62 kDa in different species, there are only two forms: either the so-called Normal (left-handed) or the Curly (right-handed). The Normal and Curly helical forms are asymmetric; the two characteristic helical parameters, which are the pitch and diameter, of Normal filaments are twice those of Curly filaments. Both the universality of these two helical forms and their asymmetry are biological puzzles. We found that the marine bacteria Idiomarina loihiensis have flagella with left-handed Curly-like filaments. Analysis of the polymorphic forms under different pH conditions showed that the Curly-like filaments are actually Normal filaments having a smaller pitch and diameter than those of Salmonella typhimurium. A minor modification of Calladine's model for a filament lattice can explain the variant helical forms. Pseudomonas aeruginosa filaments also belong to the family of I.loihiensis filaments. Thus, there are at least two families of flagella filaments.

Amino Acid Sequence↗
PubMed2005

N-terminal signal region of FliK is dispensable for length control of the flagellar hook.

The length of the flagellar hook is regulated; it is 55 +/- 6 nm long in Salmonella. Five genes involved in hook-length regulation are fliK, flhB, fliG, fliM and fliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented a fliK null (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system by the FliK C-terminal domain, and that the N-terminal region is dispensable for hook length control.

Amino Acid Sequence↗
PubMed2005

Magnesium promotes flagellation of Vibrio fischeri.

The bacterium Vibrio fischeri requires bacterial motility to initiate colonization of the Hawaiian squid Euprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences on V. fischeri motility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg2+. At concentrations naturally present in seawater, Mg2+ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.

Agar↗
PubMed2004

Genome sequence of the deep-sea gamma-proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy.

We report the complete genome sequence of the deep-sea gamma-proteobacterium, Idiomarina loihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Loihi submarine volcano, Hawaii. The I. loihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. loihiensis to the genomes of other gamma-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. loihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem.

Amino Acids↗
PubMed2003

Integration host factor (IHF) mediates repression of flagella in enteropathogenic and enterohaemorrhagic Escherichia coli.

The flagellar apparatus consists of components that function as a type III secretion system (TTSS). Enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC, respectively) produce an additional TTSS, which is involved in virulence via the translocation of effector proteins into infected host cells. This system is encoded by the locus of enterocyte effacement (LEE). The authors observed that EPEC and EHEC grown in Dulbecco's modified Eagle's medium to the mid- and late-exponential growth phase at 37 degrees C are non-motile. At the same time these conditions trigger the expression of the LEE-encoded TTSS. Furthermore, it was found that EPEC with an inactivated ihfA, which encodes the IHFalpha subunit of the integration host factor (IHF), becomes hyperflagellated and motile. Similar hypermotility was seen upon inactivation of the ihfA of EHEC strains. IHF-mediated repression of the EPEC flagella involves down-regulation of flhDC, which encodes a positive regulator of the flagellar regulon. IHF indirectly mediates flhDC repression, via a putative EPEC-unique regulator which is not encoded by LEE.

Culture Media↗
PubMed2003

Growth phase-coupled alterations in cell structure and function of Escherichia coli.

Escherichia coli cultures can be fractionated into more than 20 cell populations, each having a different bouyant density and apparently representing a specific stage of cell differentiation from exponential growth to stationary phase (H. Makinoshima, A. Nishimura, and A. Ishihama, Mol. Microbiol. 43:269-279, 2002). The density increase was found to be impaired at an early step for a mutant E. coli with the disrupted rpoS gene, which encodes the RNA polymerase RpoS (sigma-S) for stationary-phase gene transcription. This finding suggests that RpoS is need for the entire process of cell density increase. In the absence of RpoF sigma factor, the flagella are not formed as observed by electron microscopy, but the growth phase-coupled density increase takes place as in wild-type E. coli, confirming that the alteration in cell density is not directly correlated with the presence or absence of flagella. In the stationary-phase cells, accumulation of electron-dense areas was observed by electron microscopic observation of bacterial thin sections. By chemical determination, the increase in glycogen (or polysaccharides) was suggested to be one component, which contributes to the increase in weight-to-volume ratio of stationary-phase E. coli cells.

Bacterial Proteins↗
PubMed2002

Identification and localization of flagellins FlaA and FlaB3 within flagella of Methanococcus voltae.

Methanococcus voltae possesses four flagellin genes, two of which (flaB1 and flaB2) have previously been reported to encode major components of the flagellar filament. The remaining two flagellin genes, flaA and flaB3, are transcribed at lower levels, and the corresponding proteins remained undetected prior to this work. Electron microscopy examination of flagella isolated by detergent extraction of whole cells revealed a curved, hook-like region of varying length at the end of a long filament. Enrichment of the curved region of the flagella resulted in the identification of FlaB3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and N-terminal sequencing, and the localization of this flagellin to the cell-proximal portion of the flagellum was confirmed through immunoblotting and immunoelectron microscopy with FlaB3-specific antibodies, indicating that FlaB3 likely composes the curved portion of the flagella. This could represent a unique case of a flagellin performing the role of the bacterial hook protein. FlaA-specific antibodies were used in immunoblotting to determine that FlaA is found throughout the flagellar filament. M. voltae cells were transformed with a modified flaA gene containing a hemagglutinin (HA) tag introduced into the variable region. Transformants that had replaced the wild-type copy of the flaA gene with the HA-tagged version incorporated the HA-tagged version of FlaA into flagella which appeared normal by electron microscopy.

Amino Acid Sequence↗
PubMed2002

Serial transplantation of p53-deficient hemopoietic progenitor cells to assess their infinite growth potential.

Thirty-five years ago, Siminovitch et al. (Siminovitch L, Till JE, McCulloch EA. J Cell Com Physiol 64:23-32, 1964), using serially transplanted mouse spleens at 14-day intervals, observed a markedly progressive decline in the proliferative capacity of bone marrow (BM) cells, with the loss of clonogenicity by the fourth transplant generation. Using the same protocol, we assessed the proliferative capacity of p53-deficient mouse BM cells transplanted serially at the same 14-day intervals into lethally irradiated mice, which was a useful tool for understanding the characteristics of hemopoietic stem cells lacking solely the p53 gene function. BM cells from p53-deficient homozygous (p53(-/-)), p53-heterozygous (p53(+/-)), and wild-type (p53(+/+)) C57BL/6 mice were transplanted into lethally irradiated C57BL/6 recipients. Fourteen days later, the repopulated spleens were harvested, and 10(7)cells were retransplanted into secondary recipients. Serial transplantation was continued at 14-day intervals until hemopoietic repopulation failure. The number of heterozygous and homozygous p53-deficient spleen cells increased logarithmically up to the fourth and fifth passages, respectively, whereas wild-type spleen cells ceased to proliferate by the third passage. The number of macroscopic spleen colonies increased logarithmically until the third passage in recipients of heterozygous and homozygous p53-deficient cells, but ceased to grow by the second passage in recipients of wild-type cells. The numbers of heterozygous and homozygous p53-deficient colony forming units in spleen (CFUs-S) remained stable during the first four transplant generations, whereas that of wild-type CFUs-S decreased progressively from the first transplant generation onward. The clonogenicity of p53-deficient cells was lost when the number of CFUs-S per spleen decreased to below 10. This suggests that one out of 10 CFUs-S might be long-term repopulating cells (LTRCs), and that p53-deficient LTRCs may proliferate more rapidly than wild-type LTRCs. Longer passages that were possible in the p53-deficient groups were considered to be due to the faster cell cycle of the p53-deficient hemopoietic progenitor cells, as determined by bromodeoxyuridine incorporation with purging by UV light exposure, followed by hemopoietic colony assay (BUUV assay).

Animals↗