A GWAS meta-analysis from 5 population-based cohorts implicates ion channel genes in the pathogenesis of irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) shows genetic predisposition, however, large-scale, powered gene mapping studies are lacking. We sought to exploit existing genetic (genotype) and epidemiological (questionnaire) data from a series of population-based cohorts for IBS genome-wide association studies (GWAS) and their meta-analysis. METHODS: Based on questionnaire data compatible with Rome III Criteria, we identified a total of 1335 IBS cases and 9768 asymptomatic individuals from 5 independent European genotyped cohorts. Individual GWAS were carried out with sex-adjusted logistic regression under an additive model, followed by meta-analysis using the inverse variance method. Functional annotation of significant results was obtained via a computational pipeline exploiting ontology and interaction networks, and tissue-specific and gene set enrichment analyses. KEY RESULTS: Suggestive GWAS signals (P ≤ 5.0 × 10-6 ) were detected for 7 genomic regions, harboring 64 gene candidates to affect IBS risk via functional or expression changes. Functional annotation of this gene set convincingly (best FDR-corrected P = 3.1 × 10-10 ) highlighted regulation of ion channel activity as the most plausible pathway affecting IBS risk. CONCLUSION & INFERENCES: Our results confirm the feasibility of population-based studies for gene-discovery efforts in IBS, identify risk genes and loci to be prioritized in independent follow-ups, and pinpoint ion channels as important players and potential therapeutic targets warranting further investigation.

Abdominal fat deposits determined by magnetic resonance imaging in relation to leptin and vaspin levels as well as insulin resistance in the general adult population.

BACKGROUND: Various fat depots including visceral (VAT), subcutaneous adipose tissue (SAT) or liver fat content (LFC) were supposed to have different influences on various entities including adipokine levels as well as insulin resistance/sensitivity. Therefore, the aim of the study was to investigate the associations of SAT, VAT and LFC with the levels of leptin and vaspin as well as insulin resistance in a general non-diabetic population. METHODS: In total, 1825 participants of the Study of Health in Pomerania were characterized according to body fat compartments and LFC determined by magnetic resonance imaging. Of those subjects, insulin resistance (HOMA-IR) and insulin sensitivity ([ISI(comp)) were determined in 981 participants and adipokines were assessed in 698 using enzyme-linked immunosorbent assay. Analyses of variance and linear regression models adjusted for age, sex, smoking, height, physical inactivity and alcohol consumption were used for analysis. RESULTS: Using the residual method to assess independently the effect of the various fat depots, a strong positive association of SAT (beta per standard deviation (s.d.) increase 0.54 (95% confidence interval (CI) 0.47-0.60)) but not VAT (beta 0.01 (95% CI -0.08 to 0.09)) and LFC (beta 0.01 (95% CI -0.06 to 0.08)) with log2-leptin levels was found independent of the HOMA-IR status. Moreover, a positive association of LFC (beta 0.17 (95% CI 0.07-0.26)) with log2-vaspin levels becomes apparent, which were mostly driven by subjects with a low HOMA-IR. With respect to HOMA-IR and ISI(comp) index, pronounced positive and inverse associations to all fat markers were revealed, respectively, with the strongest relation found for SAT and LFC. CONCLUSIONS: SAT and LFC were identified as predominant sites associated with leptin and vaspin levels, respectively. Residual analysis pointed towards a general adverse effect of disproportional triglyceride storage across physiological despots, in particular in ectopic sides such as the liver, with markers of insulin resistance.

Sexual dimorphism of AMBRA1-related autistic features in human and mouse.

Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1+/- mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation-inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.

Genetic effects influencing risk for major depressive disorder in China and Europe.

Major depressive disorder (MDD) is a common, complex psychiatric disorder and a leading cause of disability worldwide. Despite twin studies indicating its modest heritability (~30-40%), extensive heterogeneity and a complex genetic architecture have complicated efforts to detect associated genetic risk variants. We combined single-nucleotide polymorphism (SNP) summary statistics from the CONVERGE and PGC studies of MDD, representing 10 502 Chinese (5282 cases and 5220 controls) and 18 663 European (9447 cases and 9215 controls) subjects. We determined the fraction of SNPs displaying consistent directions of effect, assessed the significance of polygenic risk scores and estimated the genetic correlation of MDD across ancestries. Subsequent trans-ancestry meta-analyses combined SNP-level evidence of association. Sign tests and polygenic score profiling weakly support an overlap of SNP effects between East Asian and European populations. We estimated the trans-ancestry genetic correlation of lifetime MDD as 0.33; female-only and recurrent MDD yielded estimates of 0.40 and 0.41, respectively. Common variants downstream of GPHN achieved genome-wide significance by Bayesian trans-ancestry meta-analysis (rs9323497; log10 Bayes Factor=8.08) but failed to replicate in an independent European sample (P=0.911). Gene-set enrichment analyses indicate enrichment of genes involved in neuronal development and axonal trafficking. We successfully demonstrate a partially shared polygenic basis of MDD in East Asian and European populations. Taken together, these findings support a complex etiology for MDD and possible population differences in predisposing genetic factors, with important implications for future genetic studies.

Exome Variant Analysis of Chronic Periodontitis in 2 Large Cohort Studies.

Periodontitis is characterized by inflammation of the gingival tissue. The main risk factors are socioeconomic factors, sex, age, smoking, and diabetes, but periodontal disease has also a genetic background. Previous genome-wide association studies failed to reveal genome-wide significant associations of single common single-nucleotide polymorphisms with chronic periodontitis. Using the Illumina ExomeChip data of 6,576 participants of the German population-based cohort studies Study of Health in Pomerania (SHIP) and SHIP-Trend, the authors performed single variant and also gene-based association studies of rare and common exonic variations on different periodontal case definitions. Although our study comprised the largest sample size to date to assess genetic predisposition for chronic periodontitis, the authors found no significant association. This study emphasizes that for chronic periodontitis, large sample sizes will be necessary to find genetic associations, even when examining rare genetic variants.

Genome-wide association study of pathological gambling.

BACKGROUND: Pathological gambling is a behavioural addiction with negative economic, social, and psychological consequences. Identification of contributing genes and pathways may improve understanding of aetiology and facilitate therapy and prevention. Here, we report the first genome-wide association study of pathological gambling. Our aims were to identify pathways involved in pathological gambling, and examine whether there is a genetic overlap between pathological gambling and alcohol dependence. METHODS: Four hundred and forty-five individuals with a diagnosis of pathological gambling according to the Diagnostic and Statistical Manual of Mental Disorders were recruited in Germany, and 986 controls were drawn from a German general population sample. A genome-wide association study of pathological gambling comprising single marker, gene-based, and pathway analyses, was performed. Polygenic risk scores were generated using data from a German genome-wide association study of alcohol dependence. RESULTS: No genome-wide significant association with pathological gambling was found for single markers or genes. Pathways for Huntington's disease (P-value=6.63×10(-3)); 5'-adenosine monophosphate-activated protein kinase signalling (P-value=9.57×10(-3)); and apoptosis (P-value=1.75×10(-2)) were significant. Polygenic risk score analysis of the alcohol dependence dataset yielded a one-sided nominal significant P-value in subjects with pathological gambling, irrespective of comorbid alcohol dependence status. CONCLUSIONS: The present results accord with previous quantitative formal genetic studies which showed genetic overlap between non-substance- and substance-related addictions. Furthermore, pathway analysis suggests shared pathology between Huntington's disease and pathological gambling. This finding is consistent with previous imaging studies.

Relationship between APOE Genotype and Structural MRI Measures throughout Adulthood in the Study of Health in Pomerania Population-Based Cohort.

BACKGROUND AND PURPOSE: The presence of the apolipoprotein E ε4 allele is the strongest sporadic Alzheimer disease genetic risk factor. We hypothesized that apolipoprotein E ε4 carriers and noncarriers may already differ in imaging patterns in midlife. We therefore sought to identify the effect of apolipoprotein E genotype on brain atrophy across almost the entire adult age span by using advanced MR imaging-based pattern analysis. MATERIALS AND METHODS: We analyzed MR imaging scans of 1472 participants from the Study of Health in Pomerania (22-90 years of age). We studied the association among age, apolipoprotein E ε4 carrier status, and brain atrophy, which was quantified by using 2 MR imaging-based indices: Spatial Pattern of Atrophy for Recognition of Brain Aging (summarizing age-related brain atrophy) and Spatial Pattern of Abnormality for Recognition of Early Alzheimer Disease (summarizing Alzheimer disease-like brain atrophy patterns), as well as the gray matter volumes in several Alzheimer disease- and apolipoprotein E-related ROIs (lateral frontal, lateral temporal, medial frontal, and hippocampus). RESULTS: No significant association was found between apolipoprotein E ε4 carrier status and the studied ROIs or the MR imaging-based indices in linear regression models adjusted for age, sex, and education, including an interaction term between apolipoprotein E and age. CONCLUSIONS: Our study indicates that measurable apolipoprotein E-related brain atrophy does not occur in early adulthood and midlife and suggests that such atrophy may only occur more proximal to the onset of clinical symptoms of dementia.

Advanced brain aging: relationship with epidemiologic and genetic risk factors, and overlap with Alzheimer disease atrophy patterns.

We systematically compared structural imaging patterns of advanced brain aging (ABA) in the general-population, herein defined as significant deviation from typical BA to those found in Alzheimer disease (AD). The hypothesis that ABA would show different patterns of structural change compared with those found in AD was tested via advanced pattern analysis methods. In particular, magnetic resonance images of 2705 participants from the Study of Health in Pomerania (aged 20-90 years) were analyzed using an index that captures aging atrophy patterns (Spatial Pattern of Atrophy for Recognition of BA (SPARE-BA)), and an index previously shown to capture atrophy patterns found in clinical AD (Spatial Patterns of Abnormality for Recognition of Early Alzheimer's Disease (SPARE-AD)). We studied the association between these indices and risk factors, including an AD polygenic risk score. Finally, we compared the ABA-associated atrophy with typical AD-like patterns. We observed that SPARE-BA had significant association with: smoking (P<0.05), anti-hypertensive (P<0.05), anti-diabetic drug use (men P<0.05, women P=0.06) and waist circumference for the male cohort (P<0.05), after adjusting for age. Subjects with ABA had spatially extensive gray matter loss in the frontal, parietal and temporal lobes (false-discovery-rate-corrected q<0.001). ABA patterns of atrophy were partially overlapping with, but notably deviating from those typically found in AD. Subjects with ABA had higher SPARE-AD values; largely due to the partial spatial overlap of associated patterns in temporal regions. The AD polygenic risk score was significantly associated with SPARE-AD but not with SPARE-BA. Our findings suggest that ABA is likely characterized by pathophysiologic mechanisms that are distinct from, or only partially overlapping with those of AD.

Meta-analysis of genome-wide association studies of anxiety disorders.

Anxiety disorders (ADs), namely generalized AD, panic disorder and phobias, are common, etiologically complex conditions with a partially genetic basis. Despite differing on diagnostic definitions based on clinical presentation, ADs likely represent various expressions of an underlying common diathesis of abnormal regulation of basic threat-response systems. We conducted genome-wide association analyses in nine samples of European ancestry from seven large, independent studies. To identify genetic variants contributing to genetic susceptibility shared across interview-generated DSM-based ADs, we applied two phenotypic approaches: (1) comparisons between categorical AD cases and supernormal controls, and (2) quantitative phenotypic factor scores (FS) derived from a multivariate analysis combining information across the clinical phenotypes. We used logistic and linear regression, respectively, to analyze the association between these phenotypes and genome-wide single nucleotide polymorphisms. Meta-analysis for each phenotype combined results across the nine samples for over 18 000 unrelated individuals. Each meta-analysis identified a different genome-wide significant region, with the following markers showing the strongest association: for case-control contrasts, rs1709393 located in an uncharacterized non-coding RNA locus on chromosomal band 3q12.3 (P=1.65 × 10(-8)); for FS, rs1067327 within CAMKMT encoding the calmodulin-lysine N-methyltransferase on chromosomal band 2p21 (P=2.86 × 10(-9)). Independent replication and further exploration of these findings are needed to more fully understand the role of these variants in risk and expression of ADs.

Gene transcripts associated with muscle strength: a CHARGE meta-analysis of 7,781 persons.

Lower muscle strength in midlife predicts disability and mortality in later life. Blood-borne factors, including growth differentiation factor 11 (GDF11), have been linked to muscle regeneration in animal models. We aimed to identify gene transcripts associated with muscle strength in adults. Meta-analysis of whole blood gene expression (overall 17,534 unique genes measured by microarray) and hand-grip strength in four independent cohorts (n = 7,781, ages: 20-104 yr, weighted mean = 56), adjusted for age, sex, height, weight, and leukocyte subtypes. Separate analyses were performed in subsets (older/younger than 60, men/women). Expression levels of 221 genes were associated with strength after adjustment for cofactors and for multiple statistical testing, including ALAS2 (rate-limiting enzyme in heme synthesis), PRF1 (perforin, a cytotoxic protein associated with inflammation), IGF1R, and IGF2BP2 (both insulin like growth factor related). We identified statistical enrichment for hemoglobin biosynthesis, innate immune activation, and the stress response. Ten genes were associated only in younger individuals, four in men only and one in women only. For example, PIK3R2 (a negative regulator of PI3K/AKT growth pathway) was negatively associated with muscle strength in younger (<60 yr) individuals but not older (≥ 60 yr). We also show that 115 genes (52%) have not previously been linked to muscle in NCBI PubMed abstracts. This first large-scale transcriptome study of muscle strength in human adults confirmed associations with known pathways and provides new evidence for over half of the genes identified. There may be age- and sex-specific gene expression signatures in blood for muscle strength.

Atrial fibrillation and rapid acute pacing regulate adipocyte/adipositas-related gene expression in the atria.

PURPOSE: Atrial fibrillation (AF) has been associated with increased volumes of epicardial fat and atrial adipocyte accumulation. Underlying mechanisms are not well understood. This study aims to identify rapid atrial pacing (RAP)/AF-dependent changes in atrial adipocyte/adipositas-related gene expression (AARE). METHODS: Right atrial (RA) and adjacent epicardial adipose tissue (EAT) samples were obtained from 26 patients; 13 with AF, 13 in sinus rhythm (SR). Left atrial (LA) samples were obtained from 9 pigs (5 RAP, 4 sham-operated controls). AARE was analyzed using microarrays and RT-qPCR. The impact of diabetes/obesity on gene expression was additionally determined in RA samples (RAP ex vivo and controls) from 3 vs. 6 months old ZDF rats. RESULTS: RAP in vivo of pigs resulted in substantial changes of AARE, with 66 genes being up- and 53 down-regulated on the mRNA level. Differential expression during adipocyte differentiation was confirmed using 3T3-L1 cells. In patients with AF (compared to SR), a comparable change in RA mRNA levels concerned a fraction of genes only (RETN, IGF1, HK2, PYGM, LOX, and NR4A3). RA and EAT were affected by AF to a different extent. In patients, concomitant disease contributes to AARE changes. CONCLUSIONS: RAP, and to lesser extent AF, provoke significant changes in atrial AARE. In chronic AF, activation of this gene panel is very likely mediated by AF itself, AF risk factors and concomitant diseases. This may facilitate the development of an AF substrate by increasing atrial ectopic fat and fat infiltration of the atrial myocardium.

Genetic, psychosocial and clinical factors associated with hippocampal volume in the general population.

The hippocampus--crucial for memory formation, recall and mood regulation--is involved in the pathophysiology of dementia and depressive disorders. Recent genome-wide association studies (GWAS) have identified five genetic loci associated with hippocampal volume (HV). Previous studies have described psychosocial and clinical factors (for example, smoking, type 2 diabetes and hypertension) to have an impact on HV. However, the interplay between genetic, psychosocial and clinical factors on the HV remains unclear. Still, it is likely that genetic variants and clinical or psychosocial factors jointly act in modifying HV; it might be possible they even interact. Knowledge of these factors might help to quantify ones individual risk of or rather resilience against HV loss. We investigated subjects (N=2463; 55.7% women; mean age 53 years) from the Study of Health in Pomerania (SHIP-2; SHIP-TREND-0) who underwent whole-body magnetic resonance imaging (MRI) and genotyping. HVs were estimated with FreeSurfer. For optimal nonlinear model fitting, we used regression analyses with restricted cubic splines. Genetic variants and associated psychosocial or clinical factors were jointly assessed for potential two-way interactions. We observed associations between HV and gender (P<0.0001), age (P<0.0001), body height (P<0.0001), education (P=0.0053), smoking (P=0.0058), diastolic blood pressure (P=0.0211), rs7294919 (P=0.0065), rs17178006 (P=0.0002), rs6581612 (P=0.0036), rs6741949 (P=0.0112) and rs7852872 (P=0.0451). In addition, we found three significant interactions: between rs7294919 and smoking (P=0.0473), rs7294919 and diastolic blood pressure (P=0.0447) and between rs7852872 and rs6581612 (P=0.0114). We suggest that these factors might have a role in the individual susceptibility to hippocampus-associated disorders.

A genome-wide search for quantitative trait loci affecting the cortical surface area and thickness of Heschl's gyrus.

Heschl's gyrus (HG) is a core region of the auditory cortex whose morphology is highly variable across individuals. This variability has been linked to sound perception ability in both speech and music domains. Previous studies show that variations in morphological features of HG, such as cortical surface area and thickness, are heritable. To identify genetic variants that affect HG morphology, we conducted a genome-wide association scan (GWAS) meta-analysis in 3054 healthy individuals using HG surface area and thickness as quantitative traits. None of the single nucleotide polymorphisms (SNPs) showed association P values that would survive correction for multiple testing over the genome. The most significant association was found between right HG area and SNP rs72932726 close to gene DCBLD2 (3q12.1; P=2.77 × 10(-7) ). This SNP was also associated with other regions involved in speech processing. The SNP rs333332 within gene KALRN (3q21.2; P=2.27 × 10(-6) ) and rs143000161 near gene COBLL1 (2q24.3; P=2.40 × 10(-6) ) were associated with the area and thickness of left HG, respectively. Both genes are involved in the development of the nervous system. The SNP rs7062395 close to the X-linked deafness gene POU3F4 was associated with right HG thickness (Xq21.1; P=2.38 × 10(-6) ). This is the first molecular genetic analysis of variability in HG morphology.

Association of heat shock proteins with all-cause mortality.

Experimental mild heat shock is widely known as an intervention that results in extended longevity in various models along the evolutionary lineage. Heat shock proteins (HSPs) are highly upregulated immediately after a heat shock. The elevation in HSP levels was shown to inhibit stress-mediated cell death, and recent experiments indicate a highly versatile role for these proteins as inhibitors of programmed cell death. In this study, we examined common genetic variations in 31 genes encoding all members of the HSP70, small HSP, and heat shock factor (HSF) families for their association with all-cause mortality. Our discovery cohort was the Rotterdam study (RS1) containing 5,974 participants aged 55 years and older (3,174 deaths). We assessed 4,430 single nucleotide polymorphisms (SNPs) using the HumanHap550K Genotyping BeadChip from Illumina. After adjusting for multiple testing by permutation analysis, three SNPs showed evidence for association with all-cause mortality in RS1. These findings were followed in eight independent population-based cohorts, leading to a total of 25,007 participants (8,444 deaths). In the replication phase, only HSF2 (rs1416733) remained significantly associated with all-cause mortality. Rs1416733 is a known cis-eQTL for HSF2. Our findings suggest a role of HSF2 in all-cause mortality.

The androgen receptor CAG repeat polymorphism as a risk factor of low serum testosterone and its cardiometabolic effects in men.

Previous studies reported correlations of CAG repeat length with sex hormone serum concentrations and cardiometabolic risk factors, but were limited by small cross-sectional samples. We used data of 1859 men aged 20-79 years from the population-based Study of Health in Pomerania (SHIP) to investigate the direct and modulating effects of CAG repeat length on androgen action and cardiometabolic risk factors. We performed cross-sectional and longitudinal linear and Poisson regression models adjusted for age, smoking, physical activity, alcohol consumption and body mass index. The CAG repeat length was categorized into quartiles and low total testosterone (TT) defined according to the age-specific (by decades) 10th percentile, respectively. Age-adjusted cross-sectional linear regression models showed a positive association between CAG repeat length and serum testosterone concentrations [ß coefficient for TT, 0.099 (p = 0.028) and for free T, 0.002 (p = 0.001), respectively]. After a 5.0 year median follow-up period, men with CAG repeat length in the lowest quartile had an increased risk of incident low TT concentrations [relative risk (RR), 2.31; 95% confidence interval (CI), 1.18-4.55]. We found no direct association between CAG repeat length and cardiometabolic risk factors in cross-sectional and longitudinal multivariable linear regression analyses; whereas men with longer CAG repeat length and low TT concentrations showed the highest risk of incident MetS (RR, 1.51; 95% CI, 1.05-2.16). CAG repeat length is a risk factor of incident low TT concentrations and a contributing factor of testosterone-related cardiometabolic effects. The added clinical value of a combined assessment of CAG repeat length and serum TT concentrations merits further investigation.

Development of an improved protocol to analyse gene expression in temporomandibular joint condylar cartilage of rats using DNA microarrays.

PURPOSE: During recent years, gene expression analyses based on DNA chip technologies have allowed for the genome-wide identification of genes potentially associated with growth processes in a variety of organs. The present study aims to identify genes differentially expressed in the growing temporomandibular joint cartilage by means of transcriptome analyses. MATERIAL AND METHODS: In total, the condylar cartilage of 32 rats comprising 4 age groups (newborn, 10 days, 21 days, 8 weeks) were used for analysis. Transcriptome analyses were carried out using Affymetrix Expression Arrays (Rat Genome 230 2.0 Arrays). The availability of high-quality RNA preparations from homogeneous tissue samples is a fundamental precondition of successful transcriptome analyses using DNA arrays. An optimised preparation protocol allowed RNA isolation of sufficient quality which was validated using capillary electrophoresis. RNA collected from 8 test animals of the 4 age groups respectively was mixed in equimolar RNA pools which served for the transcriptome analyses using Affymetrix arrays. RESULTS: Statistical analysis of the gene expression data indicated the existence of genes differentially regulated in the growing temporomandibular cartilage. This evidence, however, requires validation by RT-PCR using individual animals' RNA. Preliminary candidate genes belong, among others, to the groups of matrix-degrading proteases, protease inhibitors and genes involved in cell growth, apoptosis and bone remodelling. CONCLUSION: These differentially expressed genes in TMJ growth identified using DNA array technology may possibly contribute to a better understanding of growth biology and provide an approach to necessary therapy.

Cold induction of the Bacillus subtilis bkd operon is mediated by increased mRNA stability.

Recently it has been demonstrated that the ptb - bcd - buk - lpdV - bkdAA - bkdAB - bkdB operon ( bkdoperon) of Bacillus subtilis, which encodes the enzymes that catalyze the degradation of branched-chain amino acids, is inducible by a temperature downshift from 37 to 18 degrees C. Deamination and oxidative decarboxylation of isoleucine generates 2-methyl-butyryl-CoA, which serves as the precursor of anteiso-branched fatty acid species. Most probably, the induction of this operon upon cold shock ensures an increase in the content of anteiso-branched fatty acids in the membrane lipids at low temperature, thus permitting maintenance of membrane fluidity at lower temperatures. In the present study, we have analyzed the mechanism of cold induction of the bkd operon and of four further cold-inducible transcriptional units in B. subtilis. We demonstrate that cold induction of these genes is mediated by an increase in the stability of the corresponding mRNAs. None of the promoters that control the five transcriptional units analyzed is actually cold-inducible. Furthermore, the results of this study indicate that the 5' leader regions are not involved in the cold-induced stabilization of the mRNAs. The structural elements that enhance mRNA stability must therefore be restricted to the 3'-ends and/or the coding regions.

Bacillus subtilis functional genomics: genome-wide analysis of the DegS-DegU regulon by transcriptomics and proteomics.

The DegS-DegU two-component regulatory system of Bacillus subtilis controls various processes that characterize the transition from the exponential to the stationary growth phase, including the induction of extracellular degradative enzymes, expression of late competence genes and down-regulation of the sigma(D) regulon. The degU32(Hy) mutation stabilizes the phosphorylated form of DegU (DegU-P), resulting in overproduction of several extracellular degradative enzymes. In this study, the pleiotropic DegS-DegU regulon was characterized by combining proteomic and transcriptomic approaches. A comparative analysis of wild-type B. subtilis and the degU32(Hy) mutant grown in complex medium was performed during the exponential and in the stationary growth phase. Besides genes already known to be under the control of DegU-P, novel putative members of this regulon were identified. Although the degU32(Hy) mutant is assumed to contain high levels of phosphorylated DegU in the exponential as well as in the stationary growth phase, many genes known to be positively regulated by DegU-P did not show enhanced expression in the mutant strain during exponential growth. This is consistent with the fact that most genes belonging to the DegS-DegU regulon are subject to multiple regulation; this is also reflected in the strong stationary-phase induction of these genes in the mutant strain. As expected, during the exponential growth phase, the sigma(D) regulon was expressed at significantly lower levels in the degU32(Hy) mutant than in the wild type.

Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon.

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Although the biochemical activity of glycolytic enzymes has been studied in detail, no information about the expression of glycolytic genes has so far been available in this organism. Therefore, transcriptional analysis of all glycolytic genes was performed. The genes cggR, gapA, pgk, tpi, pgm and eno, encoding the enzymes required for the interconversion of triose phosphates, are transcribed as a hexacistronic operon as demonstrated by Northern analysis. This gapA operon is repressed by the regulator CggR. The presence of sugars and amino acids synergistically results in the induction of the gapA operon. The transcriptional start site upstream of cggR was mapped by primer extension. Transcripts originating upstream of cggR are processed near the 3' end of cggR. This endonucleolytic cleavage leads to differential stability of the resulting processing products: the monocistronic cggR message is very rapidly degraded, whereas the mRNA species encoding glycolytic enzymes exhibit much higher stability. An additional internal constitutive promoter was identified upstream of pgk. Thus, gapA is the most strongly regulated gene of this operon. The pfk pyk operon encoding phosphofructokinase and pyruvate kinase is weakly induced by glucose. In contrast, the genes pgi and fbaA, coding for phosphoglucoisomerase and fructose-1,6-bisphosphate aldolase, are constitutively expressed.