Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.

Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of β-lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized.

Klebsiella pneumoniae is one of the most clinically relevant species in immunocompromised individuals responsible for community-acquired and nosocomial infections, including pneumonias, urinary tract infections, bacteremias, and liver abscesses. Since the mid-1980s, hypervirulent K. pneumoniae, generally associated with the hypermucoviscosity phenotype, has emerged as a clinically significant pathogen responsible for serious disseminated infections, such as pyogenic liver abscesses, osteomyelitis, and endophthalmitis, in a generally younger and healthier population. Hypervirulent K. pneumoniae infections were primarily found in East Asia and now are increasingly being reported worldwide. Although most hypervirulent K. pneumoniae isolates are antibiotic-susceptible, some isolates with combined virulence and resistance, such as the carbapenem-resistant hypervirulent K. pneumoniae isolates, are increasingly being detected. The combination of multidrug resistance and enhanced virulence has the potential to cause the next clinical crisis. To better understand the basic biology of hypervirulent K. pneumoniae, this review will provide a summarization and discussion focused on epidemiology, hypervirulence-associated factors, and antibiotic resistance mechanisms of such hypervirulent strains. Epidemiological analysis of recent clinical isolates in China warns the global dissemination of hypervirulent K. pneumoniae strains with extensive antibiotic resistance in the near future. Therefore, an immediate response to recognize the global dissemination of this hypervirulent strain with resistance determinants is an urgent priority.

SummaryThe differentiating bacterium Streptomyces coelicolor harbours some 66 sigma factors, which support its complex life cycle.

The protein kinase mammalian target of rapamycin (mTOR) plays an important role in the coordinate regulation of cellular responses to nutritional and growth factor conditions. mTOR achieves these roles through interacting with raptor and rictor to form two distinct protein complexes, mTORC1 and mTORC2. Previous studies have been focused on mTORC1 to elucidate the central roles of the complex in mediating nutritional and growth factor signals to the protein synthesis machinery. Cell growth relies on coordinated regulation of signaling pathways that integrate cellular physiological status in response to nutrient levels, growth factor signals, and environmental stress. Impairment of the coordinated regulation can lead to disastrous effects on cell physiology, resulting in cell death or uncontrolled growth. mTOR, 2 a member of the phosphatidylinositol kinase-related kinase family, has been known as a central player in the signaling pathway that regulates cell growth in response to a variety of cellular signals derived from nutrient levels, growth factors, and environmental stress (2-4). mTOR plays a central role in the signaling network that regulates a variety of cellular processes including ribosome biogenesis, protein synthesis, autophagy, and actin cytoskeleton organization; human diseases such as cancer, diabetes, obesity, and harmatoma syndrome are associated with defects in mTOR signaling (5-9).Recent years have seen discoveries of several mTOR effectors and binding proteins. mTOR exists in two multiprotein complexes, mTORC1 and mTORC2. mTORC1 consists of mTOR, raptor, G␤L, and PRAS40, and it functions to regulate protein synthesis and cell growth in response to nutrient levels and growth factor signals (10 -14). mTORC1 regulates phosphorylations of at least two regulators of protein synthesis, S6K1 and 4E-BP1, and mediates nutrient and insulin signals to the cell growth machinery (2, 15). mTORC1 is regulated by TSC-Rheb (tuberous sclerosis complex-Ras homolog-enriched in brain) signaling (16 -19). mTORC2 consists of mTOR, rictor, G␤L, and Sin1, and it does not likely bind rapamycin-FK506-binding protein 12 complex, which makes mTORC2 distinctive from mTORC1 (13,20,21). Saccharomyces cerevisiae TORC2 consists of TOR2, LST8, AVO1 (Sin1 ortholog), and AVO3 (rictor ortholog) and two other components, AVO2 and BIT61, whose homologues have not been identified in higher eukaryotes (13,22,23). Functions and regulatory mechanisms of mTORC2 remain largely unknown. Recent studies showed that mTORC2 regulates protein kinase C ␣ phosphorylation, actin cytoskeleton organization, and Akt phosphorylation at 21,24,25). Recognizing the complex relationship between mTOR, S6K1, and * This study was supported by the Tuberous Sclerosis Alliance, the Minnesota Medical Foundation, American Heart Association Grant 0655706Z, and National Institutes of Health Grant DK072004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in acc...

Cellular nutritional and energy status regulates a wide range of nuclear processes important for cell growth, survival, and metabolic homeostasis. Mammalian target of rapamycin (mTOR) plays a key role in the cellular responses to nutrients. However, the nuclear processes governed by mTOR have not been clearly defined. Using isobaric peptide tagging coupled with linear ion trap mass spectrometry, we performed quantitative proteomics analysis to identify nuclear processes in human cells under control of mTOR. Within 3 h of inhibiting mTOR with rapamycin in HeLa cells, we observed downregulation of nuclear abundance of many proteins involved in translation and RNA modification. Unexpectedly, mTOR inhibition also down-regulated several proteins functioning in chromosomal integrity and upregulated those involved in DNA damage responses (DDRs) such as 53BP1. Consistent with these proteomic changes and DDR activation, mTOR inhibition enhanced interaction between 53BP1 and p53 and increased phosphorylation of ataxia telangiectasia mutated (ATM) kinase substrates. ATM substrate phosphorylation was also induced by inhibiting protein synthesis and suppressed by inhibiting proteasomal activity, suggesting that mTOR inhibition reduces steady-state (abundance) levels of proteins that function in cellular pathways of DDR activation. Finally, rapamycin-induced changes led to increased survival after radiation exposure in HeLa cells. These findings reveal a novel functional link between mTOR and DDR pathways in the nucleus potentially operating as a survival mechanism against unfavorable growth conditions. Molecular & Cellular Proteomics 9:403-414, 2010.Eukaryotic cells coordinately regulate molecular processes in distinct subcellular compartments for growth and survival in response to nutritional status and environmental stress. A crucial integrator/coordinator for these cellular responses is mTOR, 1 a nutrient-responsive protein kinase belonging to the phosphatidylinositol kinase-related kinase family (1). mTOR, as a downstream element of the insulin/IGF-1-phosphoinositide 3-kinase-Akt pathway, plays an important role in the regulation of a variety of cellular processes in response to nutrient and growth factor signals (1, 2). mTOR is mainly known for its regulation of translation and protein synthesis, and it is also involved in the regulation of diverse cellular and biological processes such as cell cycle progression, actin cytoskeleton rearrangement, transcription, autophagy, and development (1, 2). Despite the pervasive role of mTOR in different cellular functions, its ability to coordinately regulate diverse processes in distinct cellular compartments, particularly those occurring in the nucleus of mammalian cells, remains poorly defined.There has been growing evidence that TOR regulates diverse processes in the nucleus. In Saccharomyces cerevisiae, TOR regulates the nucleocytoplasmic shuttling of several transcription factors (1, 3). TOR complex 1, TORC1, itself undergoes translocation to the nucleus and interacts with c...

We investigated the effect of consuming probiotic fermented milk (PFM) on the microbial community structure in the human intestinal tract by using high-throughput barcoded pyrosequencing. Six healthy adults ingested 2 servings of PFM daily for 3 wk, and their fecal microbiota were analyzed before and after 3 wk of PFM ingestion period and for another 3 wk following the termination of PFM ingestion (the noningestion period). Fecal microbial communities were characterized by sequencing of the V1-V3 hypervariable regions of the 16S rRNA gene. All subjects showed a similar pattern of microbiota at the phylum level, where the relative abundance of Bacteriodetes species increased during the PFM ingestion period and decreased during the noningestion period. The increase in Bacteroidetes was found to be due to an increase in members of the families Bacteroidaceae or Prevotellaceae. In contrast to PFM-induced adaptation at the phylum level, the taxonomic composition at the genus level showed a considerable alteration in fecal microbiota induced by PFM ingestion. As revealed by analysis of operational taxonomic units (OTU), the numbers of shared OTU were low among the 3 different treatments (before, during, and after PFM ingestion), but the abundance of the shared OTU was relatively high, indicating that the majority (>77.8%) of total microbiota was maintained by shared OTU during PFM ingestion and after its termination. Our results suggest that PFM consumption could alter microbial community structure in the gastrointestinal tract of adult humans while maintaining the stability of microbiota.

An endoglucanase that is able to degrade both crystalline and amorphous cellulose was purified from the culture filtrates of the brown-rot fungus Fomitopsis pinicola grown on cellulose. An apparent molecular weight of the purified enzyme was approximately 32 kDa by SDS-PAGE analysis. The enzyme was purified 11-fold with a specific activity of 944 U/mg protein against CMC. The partial amino acid sequences of the purified endoglucanase had high homology with endo-beta-1,4-glucanase of glycosyl hydrolase family 5 from other fungi. The K(m) and K(cat)values for CMC were 12 mg CMC/ml and 670/s, respectively. The purified EG hydrolyzed both cellotetraose (G4) and cellopentaose (G5), but did not degrade either cellobiose (G2) or cellotriose (G3).