Tetsuyoshi Inoue

Genome-Wide Screening of Genes Required for Swarming Motility in Escherichia coli K-12

Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.

Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae.

Fimbriae preparation from Actinobacillus actinomycetemcomitans was found to contain an abundant low‐molecular‐weight protein (termed Flp) with an apparent molecular mass of approximately 6.5 kDa, in addition to a small amount of 54‐kDa protein. Immunogold electron microscopy localized the Flp protein at the bacterial fimbriae but not at the cell surface. The DNA fragment including the flp gene was cloned from A. actinomycetemcomitans 304‐a and its nucleotide sequence was determined. An open reading frame of the flp gene was composed of 225 bp encoding a protein of 75 amino acids. Comparison of the translated amino acid sequence with the sequence of native Flp determined by Edman degradation indicated that the N‐terminal part of 26 amino acids is leader peptide. The N‐terminal sequence of mature Flp exhibited some similarity to type‐IV pilin. Furthermore, the processing site of premature Flp is also similar to that of type‐IV prepilin, and a gene encoding a protein homologous to type‐IV prepilin‐like protein leader peptidase was found downstream of the flp gene. These findings indicate that Flp is the major component protein of A. actinomycetemcomitans fimbriae.

Inhibition of swarming motility of Pseudomonas aeruginosa by branched‐chain fatty acids

Pseudomonas aeruginosa is capable of moving by swimming, swarming, and twitching motilities. In this study, we investigated the effects of fatty acids on Pseudomonas aeruginosa PAO1 motilities. A branched-chain fatty acid (BCFA)--12-methyltetradecanoic acid (anteiso-C15:0)--has slightly repressed flagella-driven swimming motility and completely inhibited a more complex type of surface motility, i.e. swarming, at a concentration of 10 microg mL(-1). In contrast, anteiso-C15:0 exhibited no effect on pili-mediated twitching motility. Other BCFAs and unsaturated fatty acids tested in this study showed similar inhibitory effects on swarming motility, although the level of inhibition differed between these fatty acids. These fatty acids caused no significant growth inhibition in liquid cultures. Straight-chain saturated fatty acids such as palmitic acid were less effective in swarming inhibition. The wetness of the PAO1 colony was significantly reduced by the addition of anteiso-C15:0; however, the production of rhamnolipids as a surface-active agent was not affected by the fatty acid. In addition to motility repression, anteiso-C15:0 caused 31% repression of biofilm formation by PAO1, suggesting that BCFA could affect the multiple cellular activities of Pseudomonas aeruginosa.

Biofilm Formation by a Fimbriae-Deficient Mutant of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans strain 310‐TR produces fimbriae and forms a tight biofilm in broth cultures, without turbid growth. The fimbriae‐deficient mutant 310‐DF, constructed in this study, was grown as a relatively fragile biofilm at the bottom of a culture vessel. Scanning electron microscopy revealed that on glass coverslips, 310‐TR formed tight and spherical microcolonies, while 310‐DF produced looser ones. These findings suggest that fimbriae are not essential for the surface‐adherent growth but are required for enhancing cell‐to‐surface and cell‐to‐cell interactions to stabilize the biofilm. Treatment of the 310‐DF biofilm with either sodium metaperiodate or DNase resulted in significant desorption of cells from glass surfaces, indicating that both carbohydrate residues and DNA molecules present on the cell surface are also involved in the biofilm formation.

Fermentable-sugar-level-dependent regulation of leukotoxin synthesis in a variably toxic strain of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans, a Gram-negative periodontopathic bacterium, produces a leukotoxin belonging to the RTX family. The production of leukotoxin varies greatly among different strains of this species and under different culture conditions. A toxin-production-variable strain, 301-b, stably produces significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures, but does not do so in the presence of excess fructose. This communication describes the cloning and sequencing of the leukotoxin promoter region from 301-b, showing that this strain has a promoter region similar to that from strain 652, a moderately toxic strain. Northern blot analysis using a leukotoxin gene probe demonstrated that change in toxin production in response to the level of external fructose was due to alteration in the transcriptional level of the leukotoxin gene. Pulsing of fructose into the fructose-limited chemostat culture remarkably reduced the intracellular cAMP level from 40 pmol (mg dry wt cells)(-1) to 3.1 pmol (mg dry wt cells)(-1), which was restored when the culture was returned to fructose-limited conditions. Further, it was found that addition of external cAMP to the culture with excess fructose resulted in an apparent recovery of leukotoxin production. Taken together, these findings indicate that a cAMP-dependent mechanism, possibly a catabolite-repression-like system, may be involved in the regulation of leukotoxin production in this bacterium.

Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer

We speculated whether or not the expression level of telomerase reverse transcriptase (hTERT) would be modulated by agents targeting epigenetics in oral cancer cell lines. Although hTERT is known to be targeted by epigenetic changes, it remains unclear how chemoagents targeting epigenetics work on hTERT transcription. In the present study, the epigenetic effects of the histone deacetylase (HDAC) inhibitor FR901228 on hTERT transcription in oral cancer cell lines were analyzed by RT-PCR. The mRNA expression of hTERT was upregulated after exposure to FR901228 in hTERT-negative Hep2 cells, and even SAS and KB cells expressed high levels of hTERT. Moreover, cotreatment of protein synthesis inhibitor cycloheximide (CHX) resulted in the induction of hTERT transcription by FR901228. This suggests that the induction of hTERT by FR901228 requires de novo protein synthesis to some extent and is more likely a direct than an indirect effect on epigenetic changes such as histone acetylation/deacetylation. We further examined the effect of FR901228 on c-myc protein, which is one of the main hTERT transcription activators. FR901228 repressed c-myc protein only in the absence of CHX, and depended on the enhancement of de novo protein synthesis. Our results indicate that c-myc protein is repressed indirectly by FR901228 but may not contribute to FR901228-induced hTERT transcription. The present study showed that the HDAC inhibitor FR901228 induced the hTERT gene by a complex mechanism that involved transcription factors other than c-myc, in addition to inhibition of histone deacetylation.

Heterogeneous Post-Translational Modification of Actinobacillus actinomycetemcomitans Fimbrillin

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle‐forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography‐electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310‐a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post‐translationally modified. Modification of the fimbrial peptides was also suggested by an N‐terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C‐terminal region. A periodate oxidation/biotin‐hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.

Cell Surface-Associated Enolase in Actinobacillus actinomycetemcomitans

Cell surface‐associated materials of Actinobacillus actinomycetemcomitans were extracted by a short incubation of the cell suspension in a Tris‐buffered saline in the presence and absence of a restriction enzyme, EcoRI. The supernatants (which we termed EcoRI extract and surface extract, respectively) contained a number of extracellularly released proteins. Of these proteins, four major proteins were identified by N‐terminal sequencing to be the 34 and 39 kDa outer membrane proteins, the GroEL‐like protein, and a 47 kDa protein homologous to Haemophilus influenzae enolase. Enolase activity was found in the extracts and its relative amount of activity in the EcoRI extract from a culture of the mid‐exponential growth phase was estimated as 5.7% of total enzyme activity. In contrast, the relative amount of activity of another cytosolic enzyme, lactate dehydrogenase, was extremely low in the extracts and also in the culture supernatant. These results suggest the external localization of enolase in this bacterium.

Attenuation of the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Porphyromonas gingivalis Gingipains RgpA, RgpB, and Kgp

Background: The significance of gingipains on the PI3K/Akt signaling pathway is poorly understood. Results: The inactivation of PI3K and Akt was induced by P. gingivalis, which was associated with gingipains. Conclusion: Gingipains are critical for suppressing the PI3K/Akt signaling pathway via extracellular proteolysis independent of P. gingivalis invasion. Significance: This study shows the first evidence that the gingipains negatively regulate the PI3K/Akt signaling pathway. Porphyromonas gingivalis is a major pathogen of periodontal diseases, including periodontitis. We have investigated the effect of P. gingivalis infection on the PI3K/Akt (protein kinase B) signaling pathway in gingival epithelial cells. Here, we found that live P. gingivalis, but not heat-killed P. gingivalis, reduced Akt phosphorylation at both Thr-308 and Ser-473, which implies a decrease in Akt activity. Actually, PI3K, which is upstream of Akt, was also inactivated by P. gingivalis. Furthermore, glycogen synthase kinase 3α/β, mammalian target of rapamycin, and Bad, which are downstream proteins in the PI3K/Akt cascade, were also dephosphorylated, a phenomenon consistent with Akt inactivation by P. gingivalis. However, these events did not require direct interaction between bacteria and host cells and were independent of P. gingivalis invasion into the cells. The use of gingipain-specific inhibitors and a gingipain-deficient P. gingivalis mutant KDP136 revealed that the gingipains and their protease activities were essential for the inactivation of PI3K and Akt. The associations between the PI3K regulatory subunit p85α and membrane proteins were disrupted by wild-type P. gingivalis. Moreover, PDK1 translocation to the plasma membrane was reduced by wild-type P. gingivalis, but not KDP136, indicating little production of phosphatidylinositol 3,4,5-triphosphate by PI3K. Therefore, it is likely that PI3K failed to transmit homeostatic extracellular stimuli to intracellular signaling pathways by gingipains. Taken together, our findings indicate that P. gingivalis attenuates the PI3K/Akt signaling pathway via the proteolytic effects of gingipains, resulting in the dysregulation of PI3K/Akt-dependent cellular functions and the destruction of epithelial barriers.

Cloning and sequencing of major capsid protein (mcp) gene of a vibriophage, KVP20, possibly related to T-even coliphages

A large, tailed, prolate-headed vibriophage designated KVP20 was isolated from seawater. KVP20 was morphologically very similar to the previously described vibriophage, KVP40 (Matsuzaki, S., Inoue, T., Tanaka, S., 1998. Virology, 242, 314-318). However, they showed entirely different host specificities and could easily be differentiated from each other by their patterns of DNA restriction fragments. The major capsid protein (mcp) gene of KVP20 encoding the precursor of major capsid protein (pro-Mcp) was cloned and sequenced. The deduced amino-acid (aa) sequence of KVP20 pro-Mcp was compared with the reported aa sequences of KVP40 pro-Mcp, as well as of the equivalent proteins (gp23s) of coliphages T4 and RB49. There was 96.7, 57.5, and 55.2% homology to the corresponding proteins of KVP40, T4, and RB49, respectively. These data strongly suggest that the two vibriophages are closely related to each other and that they are both distantly, but definitely, related to coliphages T4 and RB49.