SummaryFunctional ribosomal RNAs are generated from longer precursor species in every organism known. Maturation of the 5Ј side of 16S rRNA in Escherichia coli is catalysed in a two-step process by the cooperative action of RNase E and RNase G. However, many bacteria lack RNase E and RNase G orthologues, raising the question as to how 16S rRNA processing occurs in these organisms. Here we show that the maturation of Bacillus subtilis 16S rRNA is also a two-step process and that the enzyme responsible for the generation of the mature 5Ј end is the widely distributed essential ribonuclease YkqC/ RNase J1. Depletion of B. subtilis of RNase J1 results in an accumulation of 16S rRNA precursors in vivo. The precursor species are found in polysomes suggesting that they can function in translation. Mutation of the predicted catalytic site of RNase J1 abolishes both 16S rRNA processing and cell viability. Finally, purified RNase J1 can correctly mature precursor 16S rRNA assembled in 70S ribosomes, showing that its role is direct.

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3- While important advances have been made toward understanding how Salmonella interacts with the intestinal epithelium to initiate disease (reviewed in references 6 and 44), investigations into the interaction of Salmonella with the human intestinal epithelium have been limited by the lack of in vitro and in vivo models which faithfully replicate the in vivo condition. In particular, it is well documented that important differences exist between the pathogenesis of Salmonella enterica serovar Typhimurium in human infections and that in widely used cell culture and animal models (34,40,47). In vitro assays using cultured mammalian epithelial cells have long been used as a model for investigating the interaction between Salmonella and the intestinal mucosa. However, there are inherent limitations associated with the use of these cultured cell lines (34), as they are not exact models of the conditions faced in vivo by Salmonella. Several characteristics of conventional tissue culture models have raised concerns regarding their overall efficacy as models for microbial infectivity in general (34) due to the dedifferentiation of these cells during conventional cell culture. Indeed, many of the physiological differences between cultured cells and their in vivo counterparts are believed to be the result of the dissociation of cells from their native three-dimensional (3-D) geometry in vivo to their propagation on a two-dimensional substrate in vitro (10). Likewise, many characteristics of animal models fail to mimic the human disease, and animal models present a complex system in which many variables cannot be controlled. A high-fidelity enteric cell culture model could provide new insights into studies of Salmonella infectivity by bridging the gap between the inherent limitations of cultured mammalian cells and intact animals.DFor humans, S. enterica serovar Typhimurium is among the most common Salmonella serotypes isolated from individuals suffering from infectious gastroenteritis and has long been recognized as a major public health problem (23). Gastroenteritis results from infection of the small intestine after ingestion with Salmonella. Indeed, the ability to colonize the intestinal epithelium is an essential feature in the pathogenicity of Salmonella infection. Moreover, the initial interactions between Salmonella and the host intestinal epithelium are believed to play a key role in mediating the intense inflammatory and secretory response which is a hallmark of serovar Typhimurium infections in humans (reviewed in reference 6). Studies with cultured intestinal epithelial cells have shown that,

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ICF (immunodeficiency, centromeric region instability and facial anomalies) is a recessive disease caused by mutations in the DNA methyltransferase 3B gene (DNMT3B). Patients have immunodeficiency, chromosome 1 (Chr1) and Chr16 pericentromeric anomalies in mitogen-stimulated lymphocytes, a small decrease in overall genomic 5-methylcytosine levels and much hypomethylation of Chr1 and Chr16 juxtacentromeric heterochromatin. Microarray expression analysis was done on B-cell lymphoblastoid cell lines (LCLs) from ICF patients with diverse DNMT3B mutations and on control LCLs using oligonucleotide arrays for approximately 5600 different genes, 510 of which showed a lymphoid lineage-restricted expression pattern among several different lineages tested. A set of 32 genes had consistent and significant ICF-specific changes in RNA levels. Half of these genes play a role in immune function. ICF-specific increases in immunoglobulin (Ig) heavy constant mu and delta RNA and cell surface IgM and IgD and decreases in Ig(gamma) and Ig(alpha) RNA and surface IgG and IgA indicate inhibition of the later steps of lymphocyte maturation. ICF-specific increases were seen in RNA for RGS1, a B-cell specific inhibitor of G-protein signaling implicated in negative regulation of B-cell migration, and in RNA for the pro-apoptotic protein kinase C eta gene. ICF-associated decreases were observed in RNAs encoding proteins involved in activation, migration or survival of lymphoid cells, namely, transcription factor negative regulator ID3, the enhancer-binding MEF2C, the iron regulatory transferrin receptor, integrin beta7, the stress protein heme oxygenase and the lymphocyte-specific tumor necrosis factor receptor family members 7 and 17. No differences in promoter methylation were seen between ICF and normal LCLs for three ICF upregulated genes and one downregulated gene by a quantitative methylation assay [combined bisulfite restriction analysis (COBRA)]. Our data suggest that DNMT3B mutations in the ICF syndrome cause lymphogenesis-associated gene dysregulation by indirect effects on gene expression that interfere with normal lymphocyte signaling, maturation and migration.

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