Characterization of an lrp-like (IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4?kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.?coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition.     

Phenotypic Analysis of Random hns Mutations Differentiate DNA-Binding Activity from Properties of fimA Promoter Inversion Modulation and Bacterial Motility

H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of the proU and fimB genes, increased fimA promoter inversion rates, and repression of the flhCD master operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proU expression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimB occurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hns mutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.     

Characterization of an lrp -like ( IrpC ) gene from Bacillus subtilis

In the course of the Bacillus subtilis genome sequencing project, we identified an open reading frame encoding a putative 16.4 kDa protein. This protein shows, respectively, 34% and 25% identity with the Escherichia coli regulatory proteins Lrp and AsnC. Phylogenetic analysis suggests that it represents a new group in the AsnC-Lrp family. Sequence comparisons, as well as immunodetection experiments, lead to the conclusion that the product of this B. subtilislrp-likegene is a bona fide Lrp protein – the first one to be detected in gram-positive bacteria. When expressed in E.␣coli, the B. subtilis Lrp-like protein is able to repress, by about two-fold, the expression of the ilvIH operon which is normally regulated by E. coli Lrp, indicating functional similarity in their regulatory targets. Vegetative growth of a B. subtilis lrp-like mutant is not affected in rich medium. However, the lrp-like mutation causes a transitory inhibition of growth in minimal medium in the presence of valine and isoleucine, which is relieved by leucine. This points to a possible role in regulation of amino acid metabolism. In addition, sporogenesis occurs earlier in the lrp-like mutant than in the reference strain, implying that the B. subtilis Lrp-like protein plays a role in the growth phase transition. Received: 28 January 1997 / Accepted: 18 April 1997     

Ectodomains of the LDL Receptor-Related Proteins LRP1b and LRP4 Have Anchorage Independent Functions In Vivo

Background

The low-density lipoprotein (LDL) receptor gene family is a highly conserved group of membrane receptors with diverse functions in developmental processes, lipoprotein trafficking, and cell signaling. The low-density lipoprotein (LDL) receptor-related protein 1b (LRP1B) was reported to be deleted in several types of human malignancies, including non-small cell lung cancer. Our group has previously reported that a distal extracellular truncation of murine Lrp1b that is predicted to secrete the entire intact extracellular domain (ECD) is fully viable with no apparent phenotype.

Methods and Principal Findings

Here, we have used a gene targeting approach to create two mouse lines carrying internally rearranged exons of Lrp1b that are predicted to truncate the protein closer to the N-terminus and to prevent normal trafficking through the secretary pathway. Both mutations result in early embryonic lethality, but, as expected from the restricted expression pattern of LRP1b in vivo, loss of Lrp1b does not cause cellular lethality as homozygous Lrp1b-deficient blastocysts can be propagated normally in culture. This is similar to findings for another LDL receptor family member, Lrp4. We provide in vitro evidence that Lrp4 undergoes regulated intramembraneous processing through metalloproteases and γ-secretase cleavage. We further demonstrate negative regulation of the Wnt signaling pathway by the soluble extracellular domain.

Conclusions and Significance

Our results underline a crucial role for Lrp1b in development. The expression in mice of truncated alleles of Lrp1b and Lrp4 with deletions of the transmembrane and intracellular domains leads to release of the extracellular domain into the extracellular space, which is sufficient to confer viability. In contrast, null mutations are embryonically (Lrp1b) or perinatally (Lrp4) lethal. These findings suggest that the extracellular domains of both proteins may function as a scavenger for signaling ligands or signal modulators in the extracellular space, thereby preserving signaling thresholds that are critical for embryonic development, as well as for the clear, but poorly understood role of LRP1b in cancer.     

Limited functional conservation of a global regulator among related bacterial genera: Lrp in <Emphasis Type="Italic">Escherichia</Emphasis>, <Emphasis Type="Italic">Proteus</Emphasis> and <Emphasis Type="Italic">Vibrio</Emphasis>

Background  

Bacterial genome sequences are being determined rapidly, but few species are physiologically well characterized. Predicting regulation from genome sequences usually involves extrapolation from better-studied bacteria, using the hypothesis that a conserved regulator, conserved target gene, and predicted regulator-binding site in the target promoter imply conserved regulation between the two species. However many compared organisms are ecologically and physiologically diverse, and the limits of extrapolation have not been well tested. In E. coli K-12 the leucine-responsive regulatory protein (Lrp) affects expression of ~400 genes. Proteus mirabilis and Vibrio cholerae have highly-conserved lrp orthologs (98% and 92% identity to E. coli lrp). The functional equivalence of Lrp from these related species was assessed.     

LRP5 deficiency down‐regulates Wnt signalling and promotes aortic lipid infiltration in hypercholesterolaemic mice

Low-density lipoprotein receptor-related protein 5 (LRP5) is a member of the LDLR family that orchestrates cholesterol homoeostasis. The role of LRP5 and the canonical Wnt pathway in the vascular wall of dyslipidaemic animals remains unknown. In this study, we analysed the role of LRP5 and the Wnt signalling pathway in mice fed a hypercholesterolaemic diet (HC) to trigger dyslipidaemia. We show that Lrp5^−/− mice had larger aortic lipid infiltrations than wild-type mice, indicating a protective role for LRP5 in the vascular wall. Three members of the LDLR family, Lrp1, Vldlr and Lrp6, showed up-regulated gene expression levels in aortas of Lrp5^−/− mice fed a hypercholesterolaemic diet. HC feeding in Lrp5^−/− mice induced higher macrophage infiltration in the aortas and accumulation of inflammatory cytokines in blood. Wnt/β-CATENIN signalling proteins were down-regulated in HC Lrp5^−/− mice indicating that LRP5 regulates the activation of Wnt signalling in the vascular wall. In conclusion, our findings show that LRP5 and the canonical Wnt pathway down-regulation regulate the dyslipidaemic profile by promoting lipid and macrophage retention in the vessel wall and increasing leucocyte-driven systemic inflammation.     

The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12°C and 25°C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS^*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS^* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.