Defining a role for Hfq in Gram-positive bacteria: evidence for Hfq-dependent antisense regulation in Listeria monocytogenes

Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species studied thus far. Here, we provide evidence for Hfq-dependent translational repression in the Gram-positive human pathogen Listeria monocytogenes, which is known to encode at least 50 sRNAs. We show that the Hfq-binding sRNA LhrA controls the translation and degradation of its target mRNA by an antisense mechanism, and that Hfq facilitates the binding of LhrA to its target. The work presented here provides the first experimental evidence for Hfq-dependent riboregulation in a Gram-positive bacterium. Our findings indicate that modulation of translation by trans-encoded sRNAs may occur by both Hfq-dependent and -independent mechanisms, thus adding another layer of complexity to sRNA-mediated riboregulation in Gram-positive species.     

Mannose 6-Phosphate/Insulin-like Growth Factor–II Receptor Targets the Urokinase Receptor to Lysosomes via a Novel Binding Interaction

The urokinase-type plasminogen activator receptor (uPAR) plays an important role on the cell surface in mediating extracellular degradative processes and formation of active TGF-β, and in nonproteolytic events such as cell adhesion, migration, and transmembrane signaling. We have searched for mechanisms that determine the cellular location of uPAR and may participate in its disposal. When using purified receptor preparations, we find that uPAR binds to the cation-independent, mannose 6-phosphate/insulin-like growth factor–II (IGF-II) receptor (CIMPR) with an affinity in the low micromolar range, but not to the 46-kD, cation-dependent, mannose 6-phosphate receptor (CDMPR). The binding is not perturbed by uPA and appears to involve domains DII + DIII of the uPAR protein moiety, but not the glycosylphosphatidylinositol anchor. The binding occurs at site(s) on the CIMPR different from those engaged in binding of mannose 6-phosphate epitopes or IGF-II. To evaluate the significance of the binding, immunofluorescence and immunoelectron microscopy studies were performed in transfected cells, and the results show that wild-type CIMPR, but not CIMPR lacking an intact sorting signal, modulates the subcellular distribution of uPAR and is capable of directing it to lysosomes. We conclude that a site within CIMPR, distinct from its previously known ligand binding sites, binds uPAR and modulates its subcellular distribution.     

Loss and Gain of Natural Killer Cell Receptor Function in an African Hunter-Gatherer Population

Modulating natural killer cell functions in human immunity and reproduction are diverse interactions between the killer cell immunoglobulin-like receptors (KIR) of Natural Killer (NK) cells and HLA class I ligands on the surface of tissue cells. Dominant interactions are between KIR2DL1 and the C2 epitope of HLA-C and between KIR2DL2/3 and the C1 epitope of HLA-C. KhoeSan hunter-gatherers of Southern Africa represent the earliest population divergence known and are the most genetically diverse indigenous people, qualities reflected in their KIR and HLA genes. Of the ten KhoeSan KIR2DL1 alleles, KIR2DL1*022 and KIR2DL1*026 likely originated in the KhoeSan, and later were transmitted at low frequency to the neighboring Zulus through gene flow. These alleles arose by point mutation from other KhoeSan KIR2DL1 alleles that are more widespread globally. Mutation of KIR2DL1*001 gave rise to KIR2DL1*022, causing loss of C2 recognition and gain of C1 recognition. This makes KIR2DL1*022 a more avid and specific C1 receptor than any KIR2DL2/3 allotype. Mutation of KIR2DL1*012 gave rise to KIR2DL1*026, causing premature termination of translation at the end of the transmembrane domain. This makes KIR2DL1*026 a membrane-associated receptor that lacks both a cytoplasmic tail and signaling function. At higher frequencies than their parental allotypes, the combined effect of the KhoeSan-specific KIR2DL1*022 and KIR2DL1*026 is to reduce the frequency of strong inhibitory C2 receptors and increase the frequency of strong inhibitory C1 receptors. Because interaction of KIR2DL1 with C2 is associated with risk of pregnancy disorder, these functional changes are potentially advantageous. Whereas all other KhoeSan KIR2DL1 alleles are present on a wide diversity of centromeric KIR haplotypes, KIR2DL1*026 is present on a single KIR haplotype and KIR2DL1*022 is present on two very similar haplotypes. The high linkage disequilibrium across their haplotypes is consistent with a recent emergence for these KIR2DL1 alleles that have distinctive functions.     

Human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent NK and T cell activation

The intestinal microbiota is essential for homeostasis of the local and systemic immune system, and particularly strains of lactic acid bacteria and Escherichia coli have been shown to have balancing effects on inflammatory conditions such as allergy and inflammatory bowel disease. However, in vitro assessment of the immunomodulatory effects of distinct strains may depend strongly on the cell type used as a model. To select the most appropriate model for screening of beneficial bacteria in human cells, the response to strains of intestinal bacteria of three types of antigen-presenting cells (APC) was compared; blood myeloid dendritic cells (DC), monocyte-derived DC and monocytes, and the effector response of natural killer cells and na?ve T cells was characterized. Maturation induced by gut-derived bacteria differed between APC, with blood DC and monocytes responding with the production of IL-6 and tumour necrosis factor-alpha to bacteria, which elicited mainly IL-10 in monocyte-derived DC. In contrast, comparable IFN-gamma production patterns were found in both natural killer cells and T cells induced by all bacteria-matured APC. An inhibitory effect of certain strains on this IFN-gamma production was also mediated by all types of APC. The most potent responses were induced by monocyte-derived DC, which thus constitute a sensitive screening model.     

Genome-wide homozygosity mapping localizes a gene for autosomal recessive non-progressive infantile ataxia to 20q11-q13

Autosomal recessive ataxias represent genetic and clinical heterogeneity. Unsteady gait is often accompanied by poor coordination of limbs, speech, and eye movements. To date, seven genes have been identified. In addition, five chromosomal loci have been localized in non-related families. Here, we report homozygosity mapping of a novel locus to a 19.5-cM region on chromosome 20q11–q13 in a large inbred Norwegian family with infantile non-progressive ataxia.L. Tranebjaerg, T.M. Teslovich, and M. Jones contributed equally to this work     

Correlation of SEC/MALLS with ultracentrifuge and viscometric data for chitosans

Attempts have been made to correlate estimates of molecular weight for a group of cationic polysaccharides known as chitosans between the highly popular technique of size-exclusion chromatography coupled to multi-angle laser light scattering, "SEC-MALLS", and the less convenient but more established technique of sedimentation equilibrium in the analytical ultracentrifuge. Four pharmaceutical grade chitosans of various molecular weights and degrees of acetylation (4-30%) were chosen. Better correlation than previous was achieved, although some batch variability was observed. Despite the broad spectrum in degree of acetylation, a log s degrees(20,w) versus log Mw scaling plot appeared to fit a straight line with power-law exponent b=0.25 +/- 0.04, i.e. between the limits of rod (0.15) and coil (0.4-0.5), although this may be the average of a lower b value at low Mw and higher b at high Mw. With regard to viscosity, a logeta versus logMw scaling plot appeared to also fit a straight line with power-law exponent a=0.96 +/- 0.10, again between the coil (0.5-0.7) and rod (1.8) limits.     

Concerted Action of Endogenous and Heterologous Phytase on Phytic Acid Degradation in Seed of Transgenic Wheat (Triticum aestivum L.)

Expression of heterologous phytases in crops offers a great potential for improving phosphate and mineral bioavailability in food and feed. In this context it is of relevance to describe the concerted action of endogenous and hetrologous phytases on the transgenic seed inositol phosphate profile. Here we report metal-dye detection HPLC analysis of inositol phosphate degradation in flour from transgenic wheat materials possessing wheat endogenous 6-phytase [EC 3.1.3.26] and Aspergillus 3-phytase [EC 3.1.3.8] activities under the control of the maize ubiquitin-1 promoter and the wheat high molecular weight glutenin subunit 1DX5 promoter respectively. During 50 min incubation there is an accumulation of InsP5 to InsP2 breakdown products in non-transgenic material. Aspergillus niger phytase specific breakdown products are transiently detected in transgenic material but after 50 min incubation virtually all InsP5, InsP4 and InsP3 isomers are hydrolysed.     

Engineering crop plants: getting a handle on phosphate

In plant seeds, most of the phosphate is in the form of phytic acid. Phytic acid is largely indigestible by monogastric animals and is the single most important factor hindering the uptake of a range of minerals. Engineering crop plants to produce a heterologous phytase improves phosphate bioavailability and reduces phytic acid excretion. This reduces the phosphate load on agricultural ecosystems and thereby alleviates eutrophication of the aquatic environment. Improved phosphate availability also reduces the need to add inorganic phosphate, a non-renewable resource. Iron and zinc uptake might be improved, which is significant for human nutrition in developing countries.     

An expanded role for microbial physiology in metabolic engineering and functional genomics: moving towards systems biology

Microbial physiology has traditionally played a very important role in both fundamental research and in industrial applications of microorganisms. The classical approach in microbial physiology has been to analyze the role of individual components (genes or proteins) in the overall cell function. With the progress in molecular biology it has become possible to optimize industrial fermentations through introduction of directed genetic modification - an approach referred to as metabolic engineering. Furthermore, as a consequence of large sequencing programs the complete genomic sequence has become available for an increasing number of microorganisms. This has resulted in substantial research efforts in assigning function to all identified open reading frames - referred to as functional genomics. In both metabolic engineering and functional genomics there is a trend towards application of a macroscopic view on cell function, and this leads to an expanded role of the classical approach applied in microbial physiology. With the increased understanding of the molecular mechanisms it is envisaged that in the future it will be possible to describe the interaction between all the components in the system (the cell), also at the quantitative level, and this is the goal of systems biology. Clearly this will have a significant impact on microbial physiology as well as on metabolic engineering.