The Assembly of Flagella in Enteropathogenic Escherichia coli Requires the Presence of a Functional Type III Secretion System.

In enteropathogenic Escherichia coli (EPEC), the production of flagella and the type III secretion system (T3SS) is activated in the presence of host cultured epithelial cells. The goal of this study was to investigate the relationship between expression of flagella and the T3SS. Mutants deficient in assembling T3SS basal and translocon components (ΔespA, ΔespB, ΔespD, ΔescC, ΔescN, and ΔescV), and in secreting effector molecules (ΔsepD and ΔsepL) were tested for flagella production under several growth conditions. The ΔespA mutant did not produce flagella in any condition tested, although fliC was transcribed. The remaining mutants produced different levels of flagella upon growth in LB or in the presence of cells but were significantly diminished in flagella production after growth in Dulbecco's minimal essential medium. We also investigated the role of virulence and global regulator genes in expression of flagella. The ΔqseB and ΔqseC mutants produced abundant flagella only when growing in LB and in the presence of HeLa cells, indicating that QseB and QseC act as negative regulators of fliC transcription. The ΔgrlR, ΔperA, Δler, Δhns, and Δfis mutants produced low levels of flagella, suggesting these regulators are activators of fliC expression. These data suggest that the presence of an intact T3SS is required for assembly of flagella highlighting the existence in EPEC of a cross-talk between these two virulence-associated T3SSs.

Nuclear Cytometry: Analysis of the Patterns of DNA Synthesis and Transcription Using Flow Cytometry, Confocal Microscopy, and RNA Sequencing.

Eukaryotes are defined by cells that contain a nucleus and other membrane-bound organelles. Cytometric analysis in situ, utilizing imaging, provides a useful understanding of the structure and function of the various subcellular components, particularly when combined with methods that preserve the living state. In terms of information provided by the observation of eukaryotic nuclei, imaging has provided a wealth of information about cellular multiplication. When organisms are present in multicellular form (tissues and organs), this property does not generally confound imaging cytometry. Multicellular eukaryotic species present immediate problems when being considered for analysis using flow cytometry which requires suspensions of single particles. Although some eukaryotic cell types exist as natural single cell suspensions (cf. the erythropoietic system), for other tissues and organs, strategies are required to produce single particle suspensions. This chapter illustrates the application of flow cytometry combined with confocal microscopy to analyze complex organs, focusing on properties of the plant nucleus, and then goes on to describe how suspensions of nuclei can be prepared from tissues and organs, and used for flow cytometric analysis of cellular and transcriptional states. The application of these techniques to animal species is also discussed with the implication that this strategy is universally applicable for the characterization of nuclei within tissues that cannot readily be converted into suspensions of cells.

Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states.

The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis. © 2016 International Society for Advancement of Cytometry.

Nonrandom attrition of the naive CD8+ T-cell pool with aging governed by T-cell receptor:pMHC interactions.

Immunity against new infections declines in the last quartile of life, as do numbers of naive T cells. Peripheral maintenance of naive T cells over the lifespan is necessary because their production drastically declines by puberty, a result of thymic involution. We report that this maintenance is not random in advanced aging. As numbers and diversity of naive CD8(+) T cells declined with aging, surviving cells underwent faster rates of homeostatic proliferation, were selected for high T-cell receptor:pMHC avidity, and preferentially acquired "memory-like" phenotype. These high-avidity precursors preferentially responded to infection and exhibited strong antimicrobial function. Thus, T-cell receptor avidity for self-pMHC provides a proofreading mechanism to maintain some of the fittest T cells in the otherwise crumbling naive repertoire, providing a degree of compensation for numerical and diversity defects in old T cells.

The Escherichia coli ycbQRST operon encodes fimbriae with laminin-binding and epithelial cell adherence properties in Shiga-toxigenic E. coli O157:H7.

The human pathogen Shiga-toxigenic Escherichia coli (STEC) O157:H7 contains a ycbQRST fimbrial-like operon, which shares significant homology to the family of F17 fimbrial biogenesis genes f17ADCG found in enterotoxigenic E. coli. We report that growth of STEC O157:H7 strain EDL933 in minimal Minca medium at 37°C and during adherence to epithelial cells led to the production of fine peritrichous fimbriae, which were found to be composed of a major subunit of 18 kDa whose N-terminal amino acid sequence matched the predicted protein product of the ycbQ gene; and showed significant homology to the F17a-A fimbrin. Similar to the F17 fimbriae, the purified STEC fimbriae and the recombinant YcbQ protein fused to a His peptide tag bound laminin, but not fibronectin or collagen. Thus, we propose the name E. coli YcbQ laminin-binding fimbriae (ELF) to designate the fimbriae encoded by the ycbQRST operon. The role of ELF as an adherence factor of STEC to cultured epithelial cells was investigated. We provide compelling evidence demonstrating that ELF contributes to adherence of STEC to human intestinal epithelial cells and to cow and pig gut tissue in vitro. Deletion in the fimbrin subunit gene elfA (or ycbQ) in STEC strain EDL933 led to an isogenic strain, which showed significant reduction (60%) in adherence to HEp-2 cells in comparison with the parental strain. In addition, antibodies against the purified ELF also partially blocked adherence of two STEC O157:H7 strains. These observations suggest that ELF functions as an accessory adherence factor that, along with other known redundant adhesins, contributes to the overall adhesive properties of STEC O157:H7 providing these organisms with ecological advantages to survive in different hosts and in the environment.

OsRecQ1, a QDE-3 homologue in rice, is required for RNA silencing induced by particle bombardment for inverted repeat DNA, but not for double-stranded RNA.

Based on the nucleotide sequence of QDE-3 in Neurospora crassa, which is involved in RNA silencing, rice (Oryza sativa) mutant lines disrupted by the insertion of the rice retrotransposon Tos17 were selected. Homozygous individuals from the M(1) and M(2) generations were screened and used for further analyses. The expression of the gene was not detected in leaves or calli of the mutant lines, in contrast to the wild type (WT). Induction of RNA silencing by particle bombardment was performed to investigate any effects of the OsRecQ1 gene on RNA silencing with silencing inducers of the GFP (green fluorescence protein)/GUS (beta-glucuronidase) gene in the mutant lines. The results showed that OsRecQ1 is required for RNA silencing induced by particle bombardment for inverted-repeat DNA, but not for double-stranded RNA (dsRNA). The levels of transcripts from inverted-repeat DNA were much lower in the mutant lines than those in the WT. Furthermore, no effects were observed in the accumulation of endogenous microRNAs (miR171 and miR156) and the production of the short interspersed nuclear element retroelement by small interfering RNA. On the basis of these results, we propose that OsRecQ1 may participate in the process that allows inverted repeat DNA to be transcribed into dsRNA, which can trigger RNA silencing.

Gene expression enhancement mediated by the 5' UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants.

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.

Transcriptional and post-transcriptional enhancement of gene expression by the 5' UTR intron of rice rubi3 gene in transgenic rice cells.

Introns play a very important role in regulating gene expression in eukaryotes. In plants, many introns enhance gene expression, and the effect of intron-mediated enhancement (IME) of gene expression is reportedly often more profound in monocots than in dicots. To further gain insight of IME in monocot plants, we quantitatively dissected the effect of the 5' UTR intron of the rice rubi3 gene at various gene expression levels in stably transformed suspension cell lines. The intron enhanced the GUS reporter gene activity in these lines by about 29-fold. Nuclear run-on experiments demonstrated a nearly twofold enhancement by the 5' UTR intron at the transcriptional level. RNA analysis by RealTime quantitative RT-PCR assays indicated the intron enhanced the steady state RNA level of the GUS reporter gene by nearly 20-fold, implying a strong role of the intron in RNA processing and/or export. The results also implicated a moderate role of the intron in enhancement at the translational level ( approximately 45%). Moreover, results from a transient assay experiment using a shortened exon 1 sequence revealed an important role of exon 1 of rubi3 in gene expression. It may also hint a divergence in IME mechanisms between plant and animal cells. These results demonstrated transcriptional enhancement by a plant intron, but suggested that post-transcriptional event(s) be the major source of IME.

Effects of indole and caffeine on cAMP in the ind1 and cfn1 mutant strains of Schizophyllum commune during sexual development.

The ind1 and cfn1 mutations of Schizophyllum commune express resistance to high concentrations of indole and caffeine respectively, and also affect sexual development. To clarify molecular events caused by the mutations, it was investigated how cAMP levels in S. commune strains respond to externally supplied indole and caffeine. Both compounds increased the cAMP levels in wild-type strains under several culture conditions. During sexual development of the ind1 mutant, the cAMP level in an early stage (hyphal aggregation) was highly increased by addition of indole, and the phenomenon disappeared in a later stage (fruit body formation). For the cfn1 mutants, the incremental increase in cAMP levels by addition of caffeine was smaller than that of wild-type strains.