Genome-wide QTL mapping of nine body composition and bone mineral density traits in pigs.

BACKGROUND: Since the pig is one of the most important livestock animals worldwide, mapping loci that are associated with economically important traits and/or traits that influence animal welfare is extremely relevant for efficient future pig breeding. Therefore, the purpose of this study was a genome-wide mapping of quantitative trait loci (QTL) associated with nine body composition and bone mineral traits: absolute (Fat, Lean) and percentage (FatPC, LeanPC) fat and lean mass, live weight (Weight), soft tissue X-ray attenuation coefficient (R), absolute (BMC) and percentage (BMCPC) bone mineral content and bone mineral density (BMD). METHODS: Data on the nine traits investigated were obtained by Dual-energy X-ray absorptiometry for 551 pigs that were between 160 and 200 days old. In addition, all pigs were genotyped using Illumina's PorcineSNP60 Genotyping BeadChip. Based on these data, a genome-wide combined linkage and linkage disequilibrium analysis was conducted. Thus, we used 44 611 sliding windows that each consisted of 20 adjacent single nucleotide polymorphisms (SNPs). For the middle of each sliding window a variance component analysis was carried out using ASReml. The underlying mixed linear model included random QTL and polygenic effects, with fixed effects of sex, housing, season and age. RESULTS: Using a Bonferroni-corrected genome-wide significance threshold of P < 0.001, significant peaks were identified for all traits except BMCPC. Overall, we identified 72 QTL on 16 chromosomes, of which 24 were significantly associated with one trait only and the remaining with more than one trait. For example, a QTL on chromosome 2 included the highest peak across the genome for four traits (Fat, FatPC, LeanPC and R). The nearby gene, ZNF608, is known to be associated with body mass index in humans and involved in starvation in Drosophila, which makes it an extremely good candidate gene for this QTL. CONCLUSIONS: Our QTL mapping approach identified 72 QTL, some of which confirmed results of previous studies in pigs. However, we also detected significant associations that have not been published before and were able to identify a number of new and promising candidate genes, such as ZNF608.

Pyruvate carboxylase plays a crucial role in carbon metabolism of extra- and intracellularly replicating Listeria monocytogenes.

The human pathogen L. monocytogenes is a facultatively intracellular bacterium that survives and replicates in the cytosol of many mammalian cells. The listerial metabolism, especially under intracellular conditions, is still poorly understood. Recent studies analyzed the carbon metabolism of L. monocytogenes by the (13)C isotopologue perturbation method in a defined minimal medium containing [U-(13)C(6)]glucose. It was shown that these bacteria produce oxaloacetate mainly by carboxylation of pyruvate due to an incomplete tricarboxylic acid cycle. Here, we report that a pycA insertion mutant defective in pyruvate carboxylase (PYC) still grows, albeit at a reduced rate, in brain heart infusion (BHI) medium but is unable to multiply in a defined minimal medium with glucose or glycerol as a carbon source. Aspartate and glutamate of the pycA mutant, in contrast to the wild-type strain, remain unlabeled when [U-(13)C(6)]glucose is added to BHI, indicating that the PYC-catalyzed carboxylation of pyruvate is the predominant reaction leading to oxaloacetate in L. monocytogenes. The pycA mutant is also unable to replicate in mammalian cells and exhibits high virulence attenuation in the mouse sepsis model.

Carbon metabolism of Listeria monocytogenes growing inside macrophages.

The intracellular metabolism of Listeria monocytogenes was studied by (13)C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-(13)C(6)]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-(13)C(6)]glucose was provided during the infection period, (13)C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C(3)-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-(13)C(6)]glucose into bacterial amino acids under the same experimental settings. The (13)C pattern suggests that (i) significant fractions (50-100%) of bacterial amino acids were provided by the host cell, (ii) a C(3)-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.

Glycerol metabolism and PrfA activity in Listeria monocytogenes.

Listeria monocytogenes is able to efficiently utilize glycerol as a carbon source. In a defined minimal medium, the growth rate (during balanced growth) in the presence of glycerol is similar to that in the presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed high-level transcriptional upregulation of the genes known to be involved in glycerol uptake and metabolism (glpFK and glpD) in the presence of glycerol (compared to that in the presence of glucose and/or cellobiose). Levels of expression of the genes encoding a second putative glycerol uptake facilitator (GlpF(2)) and a second putative glycerol kinase (GlpK(2)) were less enhanced under these conditions. GlpK(1) but not GlpK(2) was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while the loss of GlpK(1) affected replication in Caco-2 cells less than did the loss of GlpK(2) and GlpD. Additional genes whose transcription levels were higher in the presence of glycerol than in the presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression control. Transcriptional downregulation in the presence of glycerol (compared to those in the presence glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N metabolism, and the biosynthesis of branched-chain amino acids. The highest level of transcriptional upregulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions, a low level of HPr-Ser-P and a high level of HPr-His-P were present in the cells, suggesting that all enzyme IIA (EIIA) (or EIIB) components of the phosphotransferase system (PTS) permeases expressed will be phosphorylated. These and other data suggest that the phosphorylation state of PTS permeases correlates with PrfA activity.

Pathogenomics of Listeria spp.

This review provides an overview of recent progress in the exploration of genomic, transcriptomic, and proteomic data in Listeria spp. to understand genome evolution and diversity, as well as physiological aspects of metabolism utilized by the bacteria when growing in diverse and varied environments.

The orphan response regulator HP1021 of Helicobacter pylori regulates transcription of a gene cluster presumably involved in acetone metabolism.

Helicobacter pylori is a gastric pathogen for which no nonhuman reservoir is known. In accordance with the tight adaptation to its unique habitat, the human stomach, H. pylori is endowed with a very restricted repertoire of regulatory proteins. Nevertheless, the three complete two-component systems of H. pylori were shown to be involved in the regulation of important virulence traits like motility and acid resistance and in the control of metal homeostasis. HP1021 is an orphan response regulator with an atypical receiver domain whose inactivation has a considerable impact on the growth of H. pylori. Here we report the identification of HP1021-regulated genes by whole-genome transcriptional profiling. We show that the transcription of the essential housekeeping genes nifS and nifU, which are required for the assembly of Fe-S clusters, is activated by HP1021. Furthermore, we demonstrate that the expression of a gene cluster comprising open reading frames hp0690 to hp0693 and hp0695 to hp0697 which is probably involved in acetone metabolism is strongly upregulated by HP1021. Evidence is provided for a direct regulation of the hp0695-to-hp0697 operon by the binding of HP1021 to its promoter region.

Regulation of expression of atypical orphan response regulators of Helicobacter pylori.

The human gastric pathogen Helicobacter pylori exhibits a remarkably small repertoire of transcriptional regulators including three complete two-component systems as well as the orphan response regulators HP1021 and HP1043. Both HP1021 and HP1043 show atypical receiver sequences and are required for the normal cell growth of H. pylori. Recently, we demonstrated that phosphorylation of HP1021 and HP1043 according to the two-component paradigm is not a prerequisite for the cell growth-associated functions of these response regulators, raising the question of how the activity of this regulatory proteins is modulated. Here, we report that strict transcriptional control of its expression is not involved in the cell-growth associated function of HP1021. We show that expression of hp1043 is controlled both on the post-transcriptional or post-translational level and by transcriptional regulation. Furthermore, we provide evidence that hp1043 can be replaced by the orthologous gene cj0355 from Campylobacter jejuni.

Phosphorylation-independent activity of atypical response regulators of Helicobacter pylori.

The genome of the gastric pathogen Helicobacter pylori harbors a remarkably low number of regulatory genes, including three and five open reading frames encoding two-component histidine kinases and response regulators, respectively, which are putatively involved in transcriptional regulation. Two of the response regulator genes, hp1043 and hp166, proved to be essential for cell growth, and inactivation of the response regulator gene hp1021 resulted in a severe growth defect, as indicated by a small-colony phenotype. The sequences of the receiver domains of response regulators HP1043 and HP1021 differ from the consensus sequence of the acidic pocket of the receiver domain which is involved in the phosphotransfer reaction from the histidine kinase to the response regulator. Using a genetic complementation system, we demonstrated that the function of response regulator HP166, which is essential for cell growth, can be provided by a mutated derivative carrying a D52N substitution at the site of phosphorylation. We found that the atypical receiver sequences of HP1043 and HP1021 are not crucial for the function of these response regulators. Phosphorylation of the receiver domains of HP1043 and HP1021 is not needed for response regulator function and may not occur at all. Thus, the phosphorylation-independent action of these regulators differs from the well-established two-component paradigm.

Genetic evidence for histidine kinase HP165 being an acid sensor of Helicobacter pylori.

Helicobacter pylori is a human gastric pathogen which is extremely well adapted to its unique habitat. Crucial for the survival under the acidic conditions prevailing in the stomach is the enzyme urease. Transcriptome analysis has shown that transcription of a large number of genes responds to the exposure of H. pylori to acid including the genes encoding the urease subunits UreA and UreB as well as several genes which have been previously identified as target genes of the two-component system HP166-HP165. Here, we provide genetic evidence that a stimulus perceived by the histidine kinase HP165 is pH since increased transcription at acidic pH from the promoters controlling the expression of the ORFs hp119 and hp1432 which belong to the HP166-HP165 regulon is strictly dependent on the presence of histidine kinase HP165. Furthermore, we show that the basal transcription from the promoter of the ureA gene is modulated by the HP166-HP165 two-component system in response to acidic pH. On the other hand, the acid-induced increase in transcription of the promoter directing the expression of the orphan response regulator HP1021 is not controlled by the HP166-HP165 two-component system, nor is it mediated by HP1021 itself.