Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria.

Stress is a powerful modulator of neuroendocrine, behavioral, and immunological functions. After 4.5-d repeated combined acoustic and restraint stress as a murine model of chronic psychological stress, severe metabolic dysregulations became detectable in female BALB/c mice. Stress-induced alterations of metabolic processes that were found in a hepatic mRNA expression profiling were verified by in vivo analyses. Repeatedly stressed mice developed a hypermetabolic syndrome with the severe loss of lean body mass, hyperglycemia, dyslipidemia, increased amino acid turnover, and acidosis. This was associated with hypercortisolism, hyperleptinemia, insulin resistance, and hypothyroidism. In contrast, after a single acute stress exposure, changes in expression of metabolic genes were much less pronounced and predominantly confined to gluconeogenesis, probably indicating that metabolic disturbances might be initiated already early but will only manifest in repeatedly stressed mice. Thus, in our murine model, repeated stress caused severe metabolic dysregulations, leading to a drastic reduction of the individual's energy reserves. Under such circumstances stress may further reduce the ability to cope with new stressors such as infection or cancer.

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B gene (BCL11B) encodes a Kru¨ppel-like zincfinger protein, which plays a crucial role in thymopoiesis and has been associated with hematopoietic malignancies. It was hypothesized that BCL11B may act as a tumorsuppressor gene, but its precise function has not yet been elucidated. Here, we demonstrate that the survival of human T-cell leukemia and lymphoma cell lines is critically dependent on Bcl11b. Suppression of Bcl11b by RNA interference selectively induced apoptosis in transformed T cells whereas normal mature T cells remained unaffected. The apoptosis was effected by simultaneous activation of death receptor-mediated and intrinsic apoptotic pathways, most likely as a result of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) upregulation and suppression of the Bcl-xL antiapoptotic protein. Our data indicate an antiapoptotic function of Bcl11b. The resistance of normal mature T lymphocytes to Bcl11b suppression-induced apoptosis and restricted expression pattern make it an attractive therapeutic target in T-cell malignancies.

The structure of the SigB-dependent general stress regulon of Bacillus subtilis has previously been characterized by proteomics approaches as well as DNA array-based expression studies. However, comparing the SigB targets published in three previous major transcriptional profiling studies it is obvious that although each of them identified well above 100 target genes, only 67 were identified in all three studies. These substantial differences can likely be attributed to the different strains, growth conditions, microarray platforms and experimental setups used in the studies. In order to gain a better understanding of the structure of this important regulon, a targeted DNA microarray analysis covering most of the known SigB-inducing conditions was performed, and the changes in expression kinetics of 252 potential members of the SigB regulon and appropriate control genes were recorded. Transcriptional data for the B. subtilis wild-type strain 168 and its isogenic sigB mutant BSM29 were analysed using random forest, a machine learning algorithm, by incorporating the knowledge from previous studies. This analysis revealed a strictly SigB-dependent expression pattern for 166 genes following ethanol, butanol, osmotic and oxidative stress, low-temperature growth and heat shock, as well as limitation of oxygen or glucose. Kinetic analysis of the data for the wild-type strain identified 30 additional members of the SigB regulon, which were also subject to control by additional transcriptional regulators, thus displaying atypical SigB-independent induction patterns in the mutant strain under some of the conditions tested. For 19 of these 30 SigB regulon members, published reports support control by secondary regulators along with SigB. Thus, this microarray-based study assigns a total of 196 genes to the SigB-dependent general stress regulon of B. subtilis. INTRODUCTIONAs an organism living in the upper layer of soil, Bacillus subtilis is exposed to a wide range of transitory stress and starvation conditions, and many of these activate the alternative sigma factor SigB. The collection of SigBactivating stress conditions includes sudden environmental stresses such as heat, acid, ethanol, butanol and osmotic stress, Mn 2+ , nitric oxide (NO), sodium nitroprusside (SNP) and blue light; energy stresses such as starvation for glucose, phosphate and oxygen; inhibitors that collectively trigger a decrease in the ATP pool [azide, carbonyl cyanide m-chlorophenylhydrazone (CCCP), mycophenolic acid], addition of antibiotics such as vancomycin and bacitracin; as well as growth at extreme temperatures, both high and low (Hecker et al., 2007;Price, 2000Price, , 2002Price, , 2011. The increased expression of the SigB regulon provides B. subtilis cells with a non-specific, multiple stress resistance against a wide range of stresses (Gaidenko & Price, 1998; Höper et al., 2005; Völker et al., 1999). SigB-dependent gene Abbreviations: ANOVA, analysis of variance; OOB, out-of-bag; RF, random forest.The complete microarray dataset discussed...

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 105  S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.