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Pulmonary function measures reflect respiratory health and are used in the diagnosis of chronic obstructive pulmonary disease. We tested genome-wide association with forced expiratory volume in 1 second and the ratio of forced expiratory volume in 1 second to forced vital capacity in 48,201 individuals of European ancestry with follow up of the top associations in up to an additional 46,411 individuals. We identified new regions showing association (combined P < 5 × 10(-8)) with pulmonary function in or near MFAP2, TGFB2, HDAC4, RARB, MECOM (also known as EVI1), SPATA9, ARMC2, NCR3, ZKSCAN3, CDC123, C10orf11, LRP1, CCDC38, MMP15, CFDP1 and KCNE2. Identification of these 16 new loci may provide insight into the molecular mechanisms regulating pulmonary function and into molecular targets for future therapy to alleviate reduced lung function.

Carotid intima media thickness (cIMT) and plaque determined by ultrasonography are established measures of subclinical atherosclerosis that each predicts future cardiovascular disease events. We conducted a meta-analysis of genome-wide association data in 31,211 participants of European ancestry from nine large studies in the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. We then sought additional evidence to support our findings among 11,273 individuals using data from seven additional studies. In the combined meta-analysis, we identified three genomic regions associated with common carotid intima media thickness and two different regions associated with the presence of carotid plaque (P < 5 × 10(-8)). The associated SNPs mapped in or near genes related to cellular signaling, lipid metabolism and blood pressure homeostasis, and two of the regions were associated with coronary artery disease (P < 0.006) in the Coronary Artery Disease Genome-Wide Replication and Meta-Analysis (CARDIoGRAM) consortium. Our findings may provide new insight into pathways leading to subclinical atherosclerosis and subsequent cardiovascular events.

Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.

Thyroid disorders such as goiters represent important diseases, especially in iodine-deficient areas. Sibling studies have demonstrated that genetic factors substantially contribute to the interindividual variation of thyroid volume. We performed a genome-wide association study of this phenotype by analyzing a discovery cohort consisting of 3620 participants of the Study of Health in Pomerania (SHIP). Four genetic loci were associated with thyroid volume on a genome-wide level of significance. Of these, two independent loci are located upstream of and within CAPZB, which encodes the β subunit of the barbed-end F-actin binding protein that modulates actin polymerization, a process crucial in the colloid engulfment during thyroglobulin mobilization in the thyroid. The third locus marks FGF7, which encodes fibroblast growth factor 7. Members of this protein family have been discussed as putative signal molecules involved in the regulation of thyroid development. The fourth locus represents a "gene desert" on chromosome 16q23, located directly downstream of the predicted coding sequence LOC440389, which, however, had already been removed from the NCBI database as a result of the standard genome annotation processing at the time that this study was initiated. Experimental proof of the formerly predicted mature mRNA, however, demonstrates that LOC440389 indeed represents a real gene. All four associations were replicated in an independent sample of 1290 participants of the KORA study. These results increase the knowledge about genetic factors and physiological mechanisms influencing thyroid volume., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described "minimal Tat translocase" consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate.

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor-binding protein-3 (IGFBP-3) are involved in cell replication, proliferation, differentiation, protein synthesis, carbohydrate homeostasis and bone metabolism. Circulating IGF-I and IGFBP-3 concentrations predict anthropometric traits and risk of cancer and cardiovascular disease. In a genome-wide association study of 10 280 middle-aged and older men and women from four community-based cohort studies, we confirmed a known association of single nucleotide polymorphisms in the IGFBP3 gene region on chromosome 7p12.3 with IGFBP-3 concentrations using a significance threshold of P < 5 × 10(-8) (P = 3.3 × 10(-101)). Furthermore, the same IGFBP3 gene locus (e.g. rs11977526) that was associated with IGFBP-3 concentrations was also associated with the opposite direction of effect, with IGF-I concentration after adjustment for IGFBP-3 concentration (P = 1.9 × 10(-26)). A novel and independent locus on chromosome 7p12.3 (rs700752) had genome-wide significant associations with higher IGFBP-3 (P = 4.4 × 10(-21)) and higher IGF-I (P = 4.9 × 10(-9)) concentrations; when the two measurements were adjusted for one another, the IGF-I association was attenuated but the IGFBP-3 association was not. Two additional loci demonstrated genome-wide significant associations with IGFBP-3 concentration (rs1065656, chromosome 16p13.3, P = 1.2 × 10(-11), IGFALS, a confirmatory finding; and rs4234798, chromosome 4p16.1, P = 4.5 × 10(-10), SORCS2, a novel finding). Together, the four genome-wide significant loci explained 6.5% of the population variation in IGFBP-3 concentration. Furthermore, we observed a borderline statistically significant association between IGF-I concentration and FOXO3 (rs2153960, chromosome 6q21, P = 5.1 × 10(-7)), a locus associated with longevity. These genetic loci deserve further investigation to elucidate the biological basis for the observed associations and clarify their possible role in IGF-mediated regulation of cell growth and metabolism.

Background: C-reactive protein (CRP) is a heritable marker of chronic inflammation that is strongly associated with cardiovascular disease. We sought to identify genetic variants that are associated with CRP levels., Methods and Results: We performed a genome-wide association analysis of CRP in 66 185 participants from 15 population-based studies. We sought replication for the genome-wide significant and suggestive loci in a replication panel comprising 16 540 individuals from 10 independent studies. We found 18 genome-wide significant loci, and we provided evidence of replication for 8 of them. Our results confirm 7 previously known loci and introduce 11 novel loci that are implicated in pathways related to the metabolic syndrome (APOC1, HNF1A, LEPR, GCKR, HNF4A, and PTPN2) or the immune system (CRP, IL6R, NLRP3, IL1F10, and IRF1) or that reside in regions previously not known to play a role in chronic inflammation (PPP1R3B, SALL1, PABPC4, ASCL1, RORA, and BCL7B). We found a significant interaction of body mass index with LEPR (P<2.9×10(-6)). A weighted genetic risk score that was developed to summarize the effect of risk alleles was strongly associated with CRP levels and explained ≈5% of the trait variance; however, there was no evidence for these genetic variants explaining the association of CRP with coronary heart disease., Conclusions: We identified 18 loci that were associated with CRP levels. Our study highlights immune response and metabolic regulatory pathways involved in the regulation of chronic inflammation.

Rationale: Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium)., Objectives: To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample., Methods: We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/-10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations., Results: The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV(1) or FEV(1)/FVC traits using a carefully defined significance threshold of 1.3×10(-5). The most significant loci associated with FEV(1) include SNPs tagging MACROD2 (P = 6.81×10(-5)), CNTN5 (P = 4.37×10(-4)), and TRPV4 (P = 1.58×10(-3)). Among ever-smokers, SERPINA1 showed the most significant association with FEV(1) (P = 8.41×10(-5)), followed by PDE4D (P = 1.22×10(-4)). The strongest association with FEV(1)/FVC ratio was observed with ABCC1 (P = 4.38×10(-4)), and ESR1 (P = 5.42×10(-4)) among ever-smokers., Conclusions: Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV(1) among smokers in the general population.

Objective: Glycated hemoglobin (HbA₁(c)), used to monitor and diagnose diabetes, is influenced by average glycemia over a 2- to 3-month period. Genetic factors affecting expression, turnover, and abnormal glycation of hemoglobin could also be associated with increased levels of HbA₁(c). We aimed to identify such genetic factors and investigate the extent to which they influence diabetes classification based on HbA₁(c) levels., Research Design and Methods: We studied associations with HbA₁(c) in up to 46,368 nondiabetic adults of European descent from 23 genome-wide association studies (GWAS) and 8 cohorts with de novo genotyped single nucleotide polymorphisms (SNPs). We combined studies using inverse-variance meta-analysis and tested mediation by glycemia using conditional analyses. We estimated the global effect of HbA₁(c) loci using a multilocus risk score, and used net reclassification to estimate genetic effects on diabetes screening., Results: Ten loci reached genome-wide significant association with HbA(1c), including six new loci near FN3K (lead SNP/P value, rs1046896/P = 1.6 × 10⁻²⁶), HFE (rs1800562/P = 2.6 × 10⁻²⁰), TMPRSS6 (rs855791/P = 2.7 × 10⁻¹⁴), ANK1 (rs4737009/P = 6.1 × 10⁻¹²), SPTA1 (rs2779116/P = 2.8 × 10⁻⁹) and ATP11A/TUBGCP3 (rs7998202/P = 5.2 × 10⁻⁹), and four known HbA₁(c) loci: HK1 (rs16926246/P = 3.1 × 10⁻⁵⁴), MTNR1B (rs1387153/P = 4.0 × 10⁻¹¹), GCK (rs1799884/P = 1.5 × 10⁻²⁰) and G6PC2/ABCB11 (rs552976/P = 8.2 × 10⁻¹⁸). We show that associations with HbA₁(c) are partly a function of hyperglycemia associated with 3 of the 10 loci (GCK, G6PC2 and MTNR1B). The seven nonglycemic loci accounted for a 0.19 (% HbA₁(c)) difference between the extreme 10% tails of the risk score, and would reclassify ∼2% of a general white population screened for diabetes with HbA₁(c)., Conclusions: GWAS identified 10 genetic loci reproducibly associated with HbA₁(c). Six are novel and seven map to loci where rarer variants cause hereditary anemias and iron storage disorders. Common variants at these loci likely influence HbA₁(c) levels via erythrocyte biology, and confer a small but detectable reclassification of diabetes diagnosis by HbA₁(c).

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation.

The Lia system, a cell envelope stress response module of Bacillus subtilis, is comprised of the LiaRS two-component system and a membrane-anchored inhibitor protein, LiaF. It is highly conserved in the Firmicutes bacteria, and all orthologs investigated so far are activated by cell wall antibiotics. In response to envelope stress, the systems in Firmicutes cocci induce the expression of a number of genes that are involved in conferring resistance against its inducers. In contrast, a complete picture of the LiaR regulon of B. subtilis is still missing and no phenotypes could be associated with mutants lacking LiaRS. Here, we performed genome-wide transcriptomic, proteomic, and in-depth phenotypic profiling of constitutive "Lia ON" and "Lia OFF" mutants to obtain a comprehensive picture of the Lia response of Bacillus subtilis. In addition to the known targets liaIH and yhcYZ-yhdA, we identified ydhE as a novel gene affected by LiaR-dependent regulation. The results of detailed follow-up gene expression studies, together with proteomic analysis, demonstrate that the liaIH operon represents the only relevant LiaR target locus in vivo. It encodes a small membrane protein (LiaI) and a phage shock protein homolog (LiaH). LiaH forms large oligomeric rings reminiscent of those described for Escherichia coli PspA or Arabidopsis thaliana Vipp1. The results of comprehensive phenotype studies demonstrated that the gene products of the liaIH operon are involved in protecting the cell against oxidative stress and some cell wall antibiotics. Our data suggest that the LiaFSR system of B. subtilis and, presumably, other Firmicutes bacilli coordinates a phage shock protein-like response.

Magnesium, potassium, and sodium, cations commonly measured in serum, are involved in many physiological processes including energy metabolism, nerve and muscle function, signal transduction, and fluid and blood pressure regulation. To evaluate the contribution of common genetic variation to normal physiologic variation in serum concentrations of these cations, we conducted genome-wide association studies of serum magnesium, potassium, and sodium concentrations using approximately 2.5 million genotyped and imputed common single nucleotide polymorphisms (SNPs) in 15,366 participants of European descent from the international CHARGE Consortium. Study-specific results were combined using fixed-effects inverse-variance weighted meta-analysis. SNPs demonstrating genome-wide significant (p<5 x 10(-8)) or suggestive associations (p<4 x 10(-7)) were evaluated for replication in an additional 8,463 subjects of European descent. The association of common variants at six genomic regions (in or near MUC1, ATP2B1, DCDC5, TRPM6, SHROOM3, and MDS1) with serum magnesium levels was genome-wide significant when meta-analyzed with the replication dataset. All initially significant SNPs from the CHARGE Consortium showed nominal association with clinically defined hypomagnesemia, two showed association with kidney function, two with bone mineral density, and one of these also associated with fasting glucose levels. Common variants in CNNM2, a magnesium transporter studied only in model systems to date, as well as in CNNM3 and CNNM4, were also associated with magnesium concentrations in this study. We observed no associations with serum sodium or potassium levels exceeding p<4 x 10(-7). Follow-up studies of newly implicated genomic loci may provide additional insights into the regulation and homeostasis of human serum magnesium levels., Competing Interests: Aravinda Chakravarti is a paid member of the Scientific Advisory Board of Affymetrix, a role that is managed by the Committee on Conflict of Interest of the Johns Hopkins University School of Medicine.

The acoABCL and acuABC operons of Bacillus licheniformis DSM13 are strongly induced at the transcriptional level during glucose starvation conditions. Primer extension analyses of this study indicate that the acoABCL operon is controlled by a sigmaL-dependent promoter and the acuABC operon by a sigmaA-dependent promoter. Transcription at the acoA promoter is repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The acuA promoter shows a similar induction pattern, but its activity is independent from the presence of acetoin. It is demonstrated that the acoABCL operon is mainly responsible for acetoin and 2,3-butanediol degradation in B. licheniformis.

Pulmonary function measures are heritable traits that predict morbidity and mortality and define chronic obstructive pulmonary disease (COPD). We tested genome-wide association with forced expiratory volume in 1 s (FEV(1)) and the ratio of FEV(1) to forced vital capacity (FVC) in the SpiroMeta consortium (n = 20,288 individuals of European ancestry). We conducted a meta-analysis of top signals with data from direct genotyping (n < or = 32,184 additional individuals) and in silico summary association data from the CHARGE Consortium (n = 21,209) and the Health 2000 survey (n < or = 883). We confirmed the reported locus at 4q31 and identified associations with FEV(1) or FEV(1)/FVC and common variants at five additional loci: 2q35 in TNS1 (P = 1.11 x 10(-12)), 4q24 in GSTCD (2.18 x 10(-23)), 5q33 in HTR4 (P = 4.29 x 10(-9)), 6p21 in AGER (P = 3.07 x 10(-15)) and 15q23 in THSD4 (P = 7.24 x 10(-15)). mRNA analyses showed expression of TNS1, GSTCD, AGER, HTR4 and THSD4 in human lung tissue. These associations offer mechanistic insight into pulmonary function regulation and indicate potential targets for interventions to alleviate respiratory disease.

Elevated blood pressure is a common, heritable cause of cardiovascular disease worldwide. To date, identification of common genetic variants influencing blood pressure has proven challenging. We tested 2.5 million genotyped and imputed SNPs for association with systolic and diastolic blood pressure in 34,433 subjects of European ancestry from the Global BPgen consortium and followed up findings with direct genotyping (N ≤ 71,225 European ancestry, N ≤ 12,889 Indian Asian ancestry) and in silico comparison (CHARGE consortium, N = 29,136). We identified association between systolic or diastolic blood pressure and common variants in eight regions near the CYP17A1 (P = 7 × 10(-24)), CYP1A2 (P = 1 × 10(-23)), FGF5 (P = 1 × 10(-21)), SH2B3 (P = 3 × 10(-18)), MTHFR (P = 2 × 10(-13)), c10orf107 (P = 1 × 10(-9)), ZNF652 (P = 5 × 10(-9)) and PLCD3 (P = 1 × 10(-8)) genes. All variants associated with continuous blood pressure were associated with dichotomous hypertension. These associations between common variants and blood pressure and hypertension offer mechanistic insights into the regulation of blood pressure and may point to novel targets for interventions to prevent cardiovascular disease.

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p = 5.2x10(-201)), ABCG2 (p = 3.1x10(-26)), SLC17A1 (p = 3.0x10(-14)), SLC22A11 (p = 6.7x10(-14)), SLC22A12 (p = 2.0x10(-9)), SLC16A9 (p = 1.1x10(-8)), GCKR (p = 1.4x10(-9)), LRRC16A (p = 8.5x10(-9)), and near PDZK1 (p = 2.7x10(-9)). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p = 4.0x10(-26)) and propionyl-L-carnitine (p = 5.0x10(-8)) concentrations, which in turn were associated with serum UA levels (p = 1.4x10(-57) and p = 8.1x10(-54), respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels., Competing Interests: Vincent Mooser and Dawn Waterworth are full-time employees of GlaxoSmithKline, a pharmaceutical company. Peter Vollenweider and Gerard Waeber received financial support from GlaxoSmithKline to build the CoLaus study.

The related lipo(depsi)peptide antibiotics daptomycin and friulimicin B show great potential in the treatment of multiply resistant gram-positive pathogens. Applying genome-wide in-depth expression profiling, we compared the respective stress responses of Bacillus subtilis. Both antibiotics target envelope integrity, based on the strong induction of extracytoplasmic function sigma factor-dependent gene expression. The cell envelope stress-sensing two-component system LiaRS is exclusively and strongly induced by daptomycin, indicative of different mechanisms of action in the two compounds.

Bacteria secrete numerous proteins into their environment for growth and survival under complex and ever-changing conditions. The highly different characteristics of secreted proteins pose major challenges to the cellular protein export machinery and, accordingly, different pathways have evolved. While the main secretion (Sec) pathway transports proteins in an unfolded state, the twin-arginine translocation (Tat) pathway transports folded proteins. To date, these pathways were believed to act in strictly independent ways. Here, we have employed proteogenomics to investigate the secretion mechanism of the esterase LipA of Bacillus subtilis, using a serendipitously obtained hyper-producing strain. While LipA is secreted Sec-dependently under standard conditions, hyper-produced LipA is secreted predominantly Tat-dependently via an unprecedented overflow mechanism. Two previously identified B. subtilis Tat substrates, PhoD and YwbN, require each a distinct Tat translocase for secretion. In contrast, hyper-produced LipA is transported by both Tat translocases of B. subtilis, showing that they have distinct but overlapping specificities. The identified overflow secretion mechanism for LipA focuses interest on the possibility that secretion pathway choice can be determined by environmental and intracellular conditions. This may provide an explanation for the previous observation that many Sec-dependently transported proteins have potential twin-arginine signal peptides for export via the Tat pathway.

Ribonucleases J1 and J2 of Bacillus subtilis are evolutionarily conserved enzymes combining an endoribonucleolytic and a 5'-3' exoribonucleolytic activity in a single polypeptide. Their endoribonucleolytic cleavage specificity resembles that of RNase E, a key player in the processing and degradation of RNA in Escherichia coli. The biological significance of the paralogous RNases J1 and J2 in Bacillus subtilis is still unknown. Based on the premise that cleavage of an mRNA might alter its stability and hence its abundance, we have analysed the transcriptomes and proteomes of single and double mutant strains. The absence or decrease of both RNases J1 and J2 together profoundly alters the expression level of hundreds of genes. By contrast, the effect on global gene expression is minimal in single mutant strains, suggesting that the two nucleases have largely overlapping substrate specificities. Half-life measurements of individual mRNAs show that RNases J1/J2 can alter gene expression by modulating transcript stability. The absence/decrease of RNases J1 and J2 results in similar numbers of transcripts whose abundance is either increased or decreased, suggesting a complex role of these ribonucleases in both degradative and regulatory processing events that have an important impact on gene expression.

One of the clinical characteristics associated with septic shock is heart failure. Several lines of evidence indicate that functional consequences of heart failure in septic shock are linked to the activated NO-cyclic guanosine monophosphate (NO-cGMP) pathway. We have previously shown that the high-affinity cGMP export transporter, multidrug resistance protein 5 (MRP5), is expressed in the heart, which modulates intracellular concentrations and, hence, the effects of cGMP. Thus, modified expression of cardiac MRP5 in septic shock can alter cGMP concentrations and contribute to the development of heart failure. We therefore investigated MRP5 expression in the heart using two established murine models of septic shock (intraperitoneal LPS injection and surgical implantation of a stent into the ascending colon, resulting in a multibacterial peritonitis [CASP, colon ascendens stent peritonitis] in C57BL/6N mice, respectively; n = 38). Cardiac MRP5 was assessed by quantitative polymerase chain reaction and immunofluorescence. The protein was localized in the endothelial wall, smooth muscle, and cardiac myocytes. MRP5 mRNA expression was significantly reduced compared with controls both in the LPS (31.9 +/- 16.8 x 10(-4) vs. 54.1 +/- 14.8 x 10(-4), P = 0.025) and CASP model (18.3 +/- 9.4 x 10(-4) vs. 42.8 +/- 12.1 x 10(-4), P = 0.009; MRP5/glyceraldehyde 3-phosphate dehydrogenase copy numbers, respectively). In parallel, IL-6 plasma levels were significantly increased in both models. Incubation of cultured murine cardiomyocytes (HL1) with 5 ng/mL IL-6 resulted in decreased expression of MRP5 (54% of control), as did incubation of the cells with serum from septic mice (LPS serum, 22% of control; CASP serum, 11% of control). In conclusion, cardiac expression of the cGMP export transporter MRP5 is decreased in two murine models of septic shock, most likely by a transcriptional mechanism. Reduced cGMP export as a consequence of decreased MRP5 expression can attenuate heart failure in sepsis.

Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript.

In this study global changes in gene expression were monitored in Bacillus subtilis cells entering stationary growth phase owing to starvation for glucose. Gene expression was analysed in growing and starving cells at different time points by full-genome mRNA profiling using DNA macroarrays. During the transition to stationary phase we observed extensive reprogramming of gene expression, with approximately 1,000 genes being strongly repressed and approximately 900 strongly up-regulated in a time-dependent manner. The genes involved in the response to glucose starvation can be assigned to two main classes: (i) general stress/starvation genes which respond to various stress or starvation stimuli, and (ii) genes that respond specifically to starvation for glucose. The first class includes members of the sigma(B)-dependent general stress regulon, as well as 90 vegetative genes, which are strongly down regulated in the course of the stringent response. Among the genes in the second class, we observed a decrease in the expression of genes encoding proteins required for glucose uptake, glycolysis and the tricarboxylic acid cycle. Conversely, many carbohydrate utilisation systems that depend on phosphotransferase systems (PTS) or ABC transporters were activated. The expression of genes required for utilisation or generation of acetate indicates that acetate constitutes an important energy source for B. subtilis during periods of glucose starvation. Finally, genome wide mRNA profiling data can be used to predict new metabolic pathways in B. subtilis. Thus, our data suggest that glucose-starved cells are able to degrade branched-chain fatty acids to pyruvate and succinate via propionyl-CoA using the methylcitrate pathway. This pathway appears to link lipid degradation to gluconeogenesis in glucose-starved cells.

Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogen Helicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of the H. pylori iron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of the sodB gene was iron induced in H. pylori wild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of the fur gene resulted in constitutive, iron-independent expression of SodB. Recombinant H. pylori Fur protein bound with low affinity to the sodB promoter region, but addition of the iron substitute Mn2+ abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of the sodB gene at positions -5 to -47 from the transcription start site. The direct role of Fur in regulation of the H. pylori sodB gene contrasts with the small-RNA-mediated sodB regulation observed in Escherichia coli. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.

Intracellular iron homeostasis is a necessity for almost all living organisms, since both iron restriction and iron overload can result in cell death. The ferric uptake regulator protein, Fur, controls iron homeostasis in most Gram-negative bacteria. In the human gastric pathogen Helicobacter pylori, Fur is thought to have acquired extra functions to compensate for the relative paucity of regulatory genes. To identify H. pylori genes regulated by iron and Fur, we used DNA array-based transcriptional profiling with RNA isolated from H. pylori 26695 wild-type and fur mutant cells grown in iron-restricted and iron-replete conditions. Sixteen genes encoding proteins involved in metal metabolism, nitrogen metabolism, motility, cell wall synthesis and cofactor synthesis displayed iron-dependent Fur-repressed expression. Conversely, 16 genes encoding proteins involved in iron storage, respiration, energy metabolism, chemotaxis, and oxygen scavenging displayed iron-induced Fur-dependent expression. Several Fur-regulated genes have been previously shown to be essential for acid resistance or gastric colonization in animal models, such as those encoding the hydrogenase and superoxide dismutase enzymes. Overall, there was a partial overlap between the sets of genes regulated by Fur and those previously identified as growth-phase, iron or acid regulated. Regulatory patterns were confirmed for five selected genes using Northern hybridization. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization. These findings further delineate the central role of Fur in regulating the unique capacity of H. pylori to colonize the human stomach.

In Bacillus subtilis, the genes of the branched-chain amino acids biosynthetic pathway are organized in three genetic loci: the ilvBHC-leuABCD (ilv-leu) operon, ilvA, and ilvD. These genes, as well as ybgE, encoding a branched-chain amino acid aminotransferase, were recently demonstrated to represent direct targets of the global transcriptional regulator CodY. In the present study, the transcriptional organization and posttranscriptional regulation of these genes were analyzed. Whereas ybgE and ilvD are transcribed monocistronically, the ilvA gene forms a bicistronic operon with the downstream located ypmP gene, encoding a protein of unknown function. The ypmP gene is also directly preceded by a promoter sharing the regulatory pattern of the ilvA promoter. The ilv-leu operon revealed complex posttranscriptional regulation: three mRNA species of 8.5, 5.8, and 1.2 kb were detected. Among them, the 8.5-kb full-length primary transcript exhibits the shortest half-life (1.2 min). Endoribonucleolytic cleavage of this transcript generates the 5.8-kb mRNA, which lacks the coding sequences of the first two genes of the operon and is predicted to carry a stem-loop structure at its 5' end. This processing product has a significantly longer half-life (3 min) than the full-length precursor. The most stable transcript (half-life, 7.6 min) is the 1.2-kb mRNA generated by the processing event and exonucleolytic degradation of the large transcripts or partial transcriptional termination. This mRNA, which encompasses exclusively the ilvC coding sequence, is predicted to carry a further stable stem-loop structure at its 3' end. The very different steady-state amounts of mRNA resulting from their different stabilities are also reflected at the protein level: proteome studies revealed that the cellular amount of IlvC protein is 10-fold greater than that of the other proteins encoded by the ilv-leu operon. Therefore, differential segmental stability resulting from mRNA processing ensures the fine-tuning of the expression of the individual genes of the operon.

The Gram-positive soil bacterium Bacillus subtilis responds to oxidative stress by the activation of different cellular defence mechanisms. These are composed of scavenging enzymes as well as protection and repair systems organized in highly sophisticated networks. In this study, the peroxide and the superoxide stress stimulons of B. subtilis were characterized by means of transcriptomics and proteomics. The results demonstrate that oxidative-stress-responsive genes can be classified into two groups. One group encompasses genes which show similar expression patterns in the presence of both reactive oxygen species. Examples are members of the PerR and the Fur regulon which were induced by peroxide and superoxide stress. Similarly, both kinds of stress stimulated the activation of the stringent response. The second group is composed of genes primarily responding to one stimulus, like the members of the SOS regulon which were particularly upregulated in the presence of peroxide, and many genes involved in sulfate assimilation and methionine biosynthesis which were only induced by superoxide. Several genes encoding proteins of unknown function could be assigned to one of these groups.

Chemoheterotrophic bacteria use a few central metabolic pathways for carbon catabolism and energy production as well as for the generation of the main precursors for anabolic reactions. All sources of carbon and energy are converted to intermediates of these central pathways and then further metabolized. While the regulation of genes encoding enzymes used to introduce specific substrates into the central metabolism has already been studied to some detail, much less is known about the regulation of the central metabolic pathways. In this study, we investigated the responses of the Bacillus subtilis transcriptome to the presence of glucose and analyzed the role of the pleiotropic transcriptional regulator CcpA in these responses. We found that CcpA directly represses genes involved in the utilization of secondary carbon sources. In contrast, induction by glucose seems to be mediated by a variety of different mechanisms. In the presence of glucose, the genes encoding glycolytic enzymes are induced. Moreover, the genes responsible for the production of acetate from pyruvate with a concomitant substrate-level phosphorylation are induced by glucose. In contrast, the genes required for the complete oxidation of the sugar (Krebs cycle, respiration) are repressed if excess glucose is available for the bacteria. In the absence of glucose, the genes of the Krebs cycle as well as gluconeogenic genes are derepressed. The genes encoding enzymes of the pentose phosphate pathway are expressed both in the presence and the absence of glucose, as suggested by the central role of this pathway in generating anabolic precursors.

The production of high levels of ammonia allows the human gastric pathogen Helicobacter pylori to survive the acidic conditions in the human stomach. H. pylori produces ammonia through urease-mediated degradation of urea, but it is also able to convert a range of amide substrates into ammonia via its AmiE amidase and AmiF formamidase enzymes. Here data are provided that demonstrate that the iron-responsive regulatory protein Fur directly and indirectly regulates the activity of the two H. pylori amidases. In contrast to other amidase-positive bacteria, amidase and formamidase enzyme activities were not induced by medium supplementation with their respective substrates, acrylamide and formamide. AmiE protein expression and amidase enzyme activity were iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the transcriptional level via the binding of Fur to the amiE promoter region. In contrast, formamidase enzyme activity was not iron-repressed but was significantly higher in the fur mutant. This effect was not mediated at the transcriptional level, and Fur did not bind to the amiF promoter region. These roles of Fur in regulation of the H. pylori amidases suggest that the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems.

The transcriptome of Bacillus subtilis was analysed at different time points (30, 60 and 90 min) after a temperature downshift from 37 to 18 degrees C using DNA macroarrays. This approach allowed the identification of around 50 genes exhibiting an increased mRNA level and around 50 genes exhibiting a decreased mRNA level under cold-shock conditions. Many of the repressed genes encode enzymes involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating metabolic adaptation of the cells to the decreased growth rate at the lower temperature. The strongest cold-inducible gene encodes fatty acid desaturase, which forms unsaturated fatty acids from saturated phospholipid precursors, thereby increasing membrane fluidity. The cold-shock-induced increase of mRNA levels of the classical cold-shock genes cspB, cspC and cspD could be verified. Furthermore, besides many genes encoding proteins of unknown function, some genes encoding ribosomal proteins were transcriptionally up-regulated, which points to an adaptive reprogramming of the ribosomes under cold-shock conditions. Interestingly, the amount of mRNA specified by the operon ptb-bcd-buk-lpd-bkdA1-bkdA2-bkdB, which encodes enzymes involved in degradation of branched-chain amino acids, also increases after a temperature downshift. As cells utilize the isoleucine and valine degradation intermediates alpha-methylbutyryl-CoA and isobutyryl-CoA for synthesis of branched-chain fatty acids, this finding reflects the adaptation of membrane lipid composition, ensuring the maintenance of appropriate membrane fluidity at low temperatures. The results of the DNA array analyses were verified for several selected genes by RNA slot-blot analysis and compared with two-dimensional PAGE analyses.

A comprehensive study of Bacillus subtilis gene expression patterns in response to amino acid availability was performed by means of proteomics and transcriptomics. The methods of two-dimensional protein gel electrophoresis and DNA macroarray technology were combined to analyze cells exponentially grown in minimal medium with and without 0.2% Casamino Acids (CAA). This approach revealed about 120 genes predominantly involved in amino acid biosynthesis, sporulation, and competence, which were downregulated in CAA-containing medium. Determination of sporulation frequencies confirmed the physiological relevance of the expression data.

The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to DL-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

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.

When confronted with a stress factor, bacteria react with a specific stress response, a genetically encoded programme resulting in the transiently enhanced expression of a subset of genes. One of these stress factors is a sudden increase in the external pH. As a first step to understand the response of Bacillus subtilis cells towards an alkali shock at the transcriptional level, we attempted to identify alkali-inducible genes using the DNA macroarray technique. To define the appropriate challenging conditions, we used the ydjF gene, the orthologue of the Escherichia coli pspA, as a model gene for an alkali-inducible gene. Hybridization of 33P-labelled cDNA to a DNA macroarray revealed induction of more than 80 genes by a sudden increase in the external pH value from 6.3 to 8.9. It was discovered that a large subset of these genes belong to the recently described sigmaW regulon, which was confirmed by the analysis of a sigW knockout. A comparison of B. subtilis wild type with the congenic sigW knockout also led to the discovery of new members of the sigmaW regulon. In addition, we found several genes clearly not belonging to that regulon. This analysis represents the first report of an extracellular stimulus inducing the sigmaW regulon.

The Bacillus subtilis lacA gene, coding for beta-galactosidase, has been explored as a new site able to accept DNA sequences from nonreplicating delivery vectors. Two such delivery expression vectors have been constructed and shown to be useful in obtaining regulated expression from the chromosomal location. In another experiment, it was shown that the integration of a regulatory gene at the lacA locus was able to control the expression of a transcriptional fusion at the amyE locus. These experiments demonstrate that both integration sites can be used simultaneously to obtain regulated expression of desired genes.

In silico analysis of the complete Bacillus subtilis genome revealed the presence of three genes whose deduced amino acid sequences exhibit an alpha-crystallin domain characteristic for the family of small heat shock proteins: cotM (which has already been identified [Henriques et al. (1997) J. Bacteriol 179, 1887-1897]), yocM, and cotP (formerly ydfT). Analysis of the expression of all three genes by slot-blot experiments and by transcriptional fusions revealed that none of them was heat-inducible. Transcription of cotP was induced late during sporulation by the sporulation-specific sigma factor sigma(K) and negatively controlled by the GerE repressor. No expression of the yocM gene was found under all standard laboratory conditions tested. Both a cotP knockout mutant as well as a cotM cotP double knockout turned out to be viable and form spores and exhibited no germination defect.

The transcriptional organization and heat inducibility of the major heat shock genes hrcA, dnaK, dnaJ, groEL, and htpG were analyzed on the transcriptional level in Helicobacter pylori strain 69A. The strongly heat-induced dnaK operon was found to be tricistronic, consisting of the genes hrcA, grpE, and dnaK. The dnaJ gene specified one monocistronic mRNA which was also heat inducible. The genes groES and groEL were transcribed as one strongly heat-inducible bicistronic mRNA which exhibited exactly the same induction kinetic as the dnaK operon. Surprisingly, transcription of the monocistronic htpG gene was switched off after heat shock. The data presented are discussed with regard to the different mechanisms regulating expression of heat shock genes in H. pylori

Previous characterization of Bacillus subtilis hemN, encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation, indicated the presence of a second hemN-like gene (B. Hippler, G. Homuth, T. Hoffmann, C. Hungerer, W. Schumann, and D. Jahn, J. Bacteriol. 179:7181-7185, 1997). The corresponding hemZ gene was found to be split into the two potential open reading frames yhaV and yhaW by a sequencing error of the genome sequencing project. The hemZ gene, encoding a 501-amino-acid protein with a calculated molecular mass of 57,533 Da, complemented a Salmonella typhimurium hemF hemN double mutant under aerobic and anaerobic growth conditions. A B. subtilis hemZ mutant accumulated coproporphyrinogen III under anaerobic growth conditions. A hemN hemZ double mutant exhibited normal aerobic and anaerobic growth, indicating the presence of a third alternative oxygen-independent enzymatic system for coproporphyrinogen III oxidation. The hemY gene, encoding oxygen-dependent protoporphyrinogen IX oxidase with coproporphyrinogen III oxidase side activity, did not significantly contribute to this newly identified system. Growth behavior of hemY mutants revealed the presence of an oxygen-independent protoporphyrinogen IX oxidase in B. subtilis. A monocistronic hemZ mRNA, starting 31 bp upstream of the translational start codon, was detected. Reporter gene fusions of hemZ and hemN demonstrated a fivefold anaerobic induction of both genes under nitrate ammonifying growth conditions. No anaerobic induction was observed for fermentatively growing B. subtilis. The B. subtilis redox regulatory systems encoded by resDE, fnr, and ywiD were indispensable for the observed transcriptional induction. A redox regulation cascade proceeding from an unknown sensor via resDE, through fnr and ywiD to hemN/hemZ, is suggested for the observed coregulation of heme biosynthesis and the anaerobic respiratory energy metabolism. Finally, only hemZ was found to be fivefold induced by the presence of H(2)O(2), indicating further coregulation of heme biosynthesis with the formation of the tetrapyrrole enzyme catalase.

The heptacistronic dnaK heat shock operon of Bacillus subtilis consists of the genes hrcA, grpE, dnaK, dnaJ, orf35, orf28 and orf50. It is controlled by the CIRCE/HrcA operator/repressor system and specifies three primary transcripts, two of which are processed into three different products. We have analysed the regulatory consequences of this complex transcriptional organization in detail. First, the seven genes were heat induced to different extents at the mRNA level and can be classified into three groups by their induction factors. This differential induction was also reflected at the protein level. Secondly, the cellular amounts of the proteins HrcA, DnaK and DnaJ in B. subtilis differed drastically both under non-heat shock conditions and after thermal upshock. Thirdly, Northern blot analyses demonstrated that an mRNA-processing reaction generating products of differential stabilities plays an essential role during the regulation of gene expression. A crucial factor determining the low stability of two transcripts is the presence of the CIRCE element at their 5' ends. We demonstrate that CIRCE leads to the destabilization of mRNAs, but only if it is located in the immediate vicinity of a Shine-Dalgarno sequence. These results show that B. subtilis is using various, especially post-transcriptional, regulatory mechanisms to fine tune the expression of the individual genes of the heptacistronic dnaK operon.

A recently cloned Bacillus subtilis open reading frame (hemN) upstream of the dnaK operon was identified as encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation. B. subtilis hemN functionally complemented two Salmonella typhimurium hemF hemN double mutants under aerobic and anaerobic conditions. A B. subtilis hemN mutant accumulated coproporphyrinogen III only under anaerobic conditions. Interestingly, growth experiments using the B. subtilis hemN mutant revealed normal aerobic and anaerobic growth, indicating the presence of an alternative oxygen-independent enzymatic system. Northern blot experiments identified hemN mRNA as part of an approximately 7-kb pentacistronic transcript consisting of lepA, hemN, hrcA, grpE, and dnaK. One potential start site for aerobic and anaerobic transcription was located 37 bp upstream of the translational start codon of lepA. Comparable amounts of hemN transcript were observed under aerobic and anaerobic growth conditions. No experimental evidence for the presence of hemF in B. subtilis was obtained. Moreover, B. subtilis hemY did not substitute for hemF hemN deficiency in S. typhimurium. These results indicate the absence of hemF and suggest the presence of a second hemN-like gene in B. subtilis.

Class I heat-inducible genes in Bacillus subtilis consist of the heptacistronic dnaK and the bicistronic groE operon and form the CIRCE regulon. Both operons are negatively regulated at the level of transcription by the HrcA repressor interacting with its operator, the CIRCE element. Here, we demonstrate that the DnaK chaperone machine is not involved in the regulation of HrcA and that the GroE chaperonin exerts a negative effect in the post-transcriptional control of HrcA. When expression of the groE operon was turned off, the dnaK operon was significantly activated and large amounts of apparently inactive HrcA repressor were produced. Overproduction of GroEL, on the other hand, resulted in decreased expression of the dnaK operon. Introduction of the hrcA gene and its operator into Escherichia coli was sufficient to elicit a transient heat shock response, indicating that no additional Bacillus-specific gene(s) was needed. As in B. subtilis, the groEL gene of E. coli negatively influenced the activity of HrcA. HrcA could be overproduced in E. coli, but formed inclusion bodies which could be dissolved in 8 M urea. Upon removal of urea, HrcA had a strong tendency to aggregate, but aggregation could be suppressed significantly by the addition of GroEL. Purified HrcA repressor was able specifically to retard a DNA fragment containing the CIRCE element, and the amount of retarded DNA was increased significantly in the presence of GroEL. These results suggest that the GroE chaperonin machine modulates the activity of the HrcA repressor and therefore point to a novel function of GroE as a modulator of the heat shock response.

We show that the htpG gene of Bacillus subtilis is induced by heat, as has been reported for the Escherichia coli homolog. Analysis of different mutants revealed that the htpG gene belongs to class III heat shock genes in B. subtilis. An about 10-fold induction after thermal upshock was found at the levels of both transcription and translation, and this induction resulted from enhanced synthesis of mRNA. By primer extension, we identified one potential transcription start site immediately downstream of a putative sigmaA-dependent promoter which became activated after thermal upshift. Northern blot analysis revealed that htpG is part of a monocistronic transcriptional unit. An operon fusion where the complete region between htpG and its upstream gene was fused to the bgaB reporter gene accurately reflected htpG expression. Analysis of this fusion revealed that, in contrast to other class III heat shock genes, htpG was not induced by osmotic upshock, by ethanol, or by oxygen limitation, suggesting that it belongs to a subgroup within class III. Deletion of the region upstream of the putative promoter resulted in an enhanced basal level of htpG expression, but the 10-fold induction was retained, suggesting that the upstream sequences are involved in the regulation of expression in the absence of heat shock.

In 1992, we described the cloning and sequencing of the dnaK locus of Bacillus subtilis which, together with transcriptional studies, implied a tetracistronic structure of the operon consisting of the genes hrcA, grpE, dnaK, and dnaJ. We have repeated the Northern blot analysis, this time using riboprobes instead of oligonucleotides, and have detected a heat-inducible 8-kb transcript, suggesting the existence of additional heat shock genes downstream of dnaJ. Cloning and sequencing of that region revealed the existence of three novel heat shock genes named orf35, orf28, and orf50, extending the tetra- into a heptacistronic operon. This is now the largest dnaK operon to be described to date. The three new genes are transcribed as a part of the entire dnaK operon (8.0-kb heptacistronic heat-inducible transcript) and as part of a suboperon starting at an internal vegetative promoter immediately upstream of dnaJ (4.3-kb tetracistronic non-heat-inducible transcript). In addition, the Northern blot analysis detected several processing products of these two primary transcripts. To demonstrate the existence of the internal promoter, a DNA fragment containing this putative promoter structure was inserted upstream of a promoterless bgaB gene, resulting in the synthesis of beta-galactosidase. Challenging this transcriptional fusion with various stress factors did not result in the activation of this promoter. To assign a biological function to the three novel genes, they have each been inactivated by the insertion of a cat cassette. All of the mutants were viable, and furthermore, these genes are (i) not essential for growth at high temperatures, (ii) not involved in the regulation of the heat shock response, and (iii) sporulation proficient. Blocking transcription of the suboperon from the upstream heat-inducible promoter did not impair growth and viability at high temperatures.

The IepA operon of Bacillus subtilis was found to be bicistronic and to consist of the two genes IepA and hemN, which encode a putative GTP-binding protein and an oxygen-independent coproporhyrinogen III oxidase, respectively. The IepA operon is located immediately upstream of the dnaK operon. Both operons are transcribed in the same direction and are not separated by an obvious transcription-terminator-like structure. The IepA operon is preceded by a potential vegetative promoter, and there is a putative strong intergenic terminator between IepA and hemN. Northern blot experiments revealed only a transcript corresponding to IepA, but expression of hemN was demonstrated in slot-blot and immunoblot experiments using antibodies raised against His-tagged HemN. The data suggest that most of the transcripts originating at the potential vegetative promoter are terminated at the intergenic terminator. Readthrough transcription into the downstream dnaK operon was not found.