Background Mycobacterium tuberculosis (Mtb) becomes dormant and phenotypically drug resistant when it encounters multiple stresses within the host. Inability of currently available drugs to kill latent Mtb is a major impediment to curing and possibly eradicating tuberculosis (TB). Most in vitro dormancy models, using single stress factors, fail to generate a truly dormant Mtb population. An in vitro model that generates truly dormant Mtb cells is needed to elucidate the metabolic requirements that allow Mtb to successfully go through dormancy, identify new drug targets, and to screen drug candidates to discover novel drugs that can kill dormant pathogen.Methodology/Principal FindingsWe developed a novel in vitro multiple-stress dormancy model for Mtb by applying combined stresses of low oxygen (5%), high CO2 (10%), low nutrient (10% Dubos medium) and acidic pH (5.0), conditions Mtb is thought to encounter in the host. Under this condition, Mtb stopped replicating, lost acid-fastness, accumulated triacylglycerol (TG) and wax ester (WE), and concomitantly acquired phenotypic antibiotic-resistance. Putative neutral lipid biosynthetic genes were up-regulated. These genes may serve as potential targets for new antilatency drugs. The triacylglycerol synthase1 (tgs1) deletion mutant, with impaired ability to accumulate TG, exhibited a lesser degree of antibiotic tolerance and complementation restored antibiotic tolerance. Transcriptome analysis with microarray revealed the achievement of dormant state showing repression of energy generation, transcription and translation machineries and induction of stress-responsive genes. We adapted this model for drug screening using the Alamar Blue dye to quantify the antibiotic tolerant dormant cells.Conclusions/SignificanceThe new in vitro multiple stress dormancy model efficiently generates Mtb cells meeting all criteria of dormancy, and this method is adaptable to high-throughput screening for drugs that can kill dormant Mtb. A critical link between storage-lipid accumulation and development of phenotypic drug-resistance in Mtb was established. Storage lipid biosynthetic genes may be appropriate targets for novel drugs that can kill latent Mtb.

SummarySIT4 encodes the multifunctional catalytic subunit of a type 2A-related protein phosphatase of Saccharomyces cerevisiae and has been implicated in cell cycle regulation and nitrogen sensing. We have identified the Candida albicans homologue of SIT4 , and we show that its disruption caused a significant reduction in general growth rate, in hyphal outgrowth and in virulence in a mouse infection model. These phenotypes were reversed by the reintroduction of the wild-type SIT4 gene. We used glass DNA microarrays to measure the transcriptional profiles of 6287 open reading frames in sit4 cells undergoing the yeast-to-hypha transition induced by serum. Although differential expression of many of the hyphae-specific genes was not affected by the SIT4 deletion, the transcription of two new hyphae-induced genes, XOG1 and YNR67 , was entirely reliant upon Sit4p. Both genes represent glucanases, indicating that SIT4 may play a role in controlling cell wall biogenesis. Furthermore, sit4 cells exhibited a reduced heat shock response to treatment with serum/37 ∞ ∞ ∞ ∞ C, suggesting that SIT4 acts to co-ordinate the stress response signals during morphological switching. Finally, sit4 cells displayed reduced transcript levels for the genes encoding the Hog1p MAP kinase and several modulators of protein biosynthesis. Sit4p thus plays important roles during hyphal growth in Candida albicans through the regulation of cell wall biogenesis, osmosensing and protein translation.

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we reveal the genome sequence of S. tora at the chromosome level with 526 Mb (96%) assembled into 13 chromosomes. Comparison among related plant species shows that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identify a CHS-L gene contributing to the biosynthesis of anthraquinones. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants.

To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase.

The Mycobacterium tuberculosis acyl-coenzyme A (CoA) carboxylases provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase I (FAS I) and for the elongation of FAS I end products by the FAS II complex to produce meromycolic acids. The M. tuberculosis genome contains three biotin carboxylase subunits (AccA1 to -3) and six carboxyltransferase subunits (AccD1 to -6), with accD6 located in a genetic locus that contains members of the FAS II complex. We found by quantitative real-time PCR analysis that the transcripts of accA3, accD4, accD5, and accD6 are expressed at high levels during the exponential growth phases of M. tuberculosis in vitro. Microarray analysis of M. tuberculosis transcripts indicated that the transcripts for accA3, accD4, accD5, accD6, and accE were repressed during later growth stages. AccD4 and AccD5 have been previously studied, but there are no reports on the function of AccD6. We expressed AccA3 (␣ 3 ) and AccD6 (␤ 6 ) in E. coli and purified them by affinity chromatography. We report here that reconstitution of the ␣ 3 -␤ 6 complex yielded an active acyl-CoA carboxylase. Kinetic characterization of this carboxylase showed that it preferentially carboxylated acetyl-CoA (1.1 nmol/mg/min) over propionyl-CoA (0.36 nmol/mg/min). The activity of the ␣ 3 -␤ 6 complex was inhibited by the subunit. The ␣ 3 -␤ 6 carboxylase was inhibited significantly by dimethyl itaconate, C75, haloxyfop, cerulenin, and 1,2-cyclohexanedione. Our results suggest that the ␤ 6 subunit could play an important role in mycolic acid biosynthesis by providing malonyl-CoA to the FAS II complex.Tuberculosis causes 2 million deaths each year, according to the World Health Organization. Mycobacterium tuberculosis, the pathogen that causes the disease, infects 8 million people each year and is one of the world's deadliest pathogens (9). The ongoing AIDS pandemic has developed a deadly synergy with tuberculosis, which is the leading cause of death among AIDS patients (2). Multidrug-resistant M. tuberculosis strains have been emerging rapidly (9), and the need for identifying novel drug targets in this pathogen has become urgent. The cell wall of M. tuberculosis is lipid enriched and acts as an impermeable barrier to many common broad-spectrum antibiotics (14).The first committed step of fatty-acid biosynthesis, which is the biotin-dependent carboxylation of acyl-coenzyme A (CoA) to produce malonyl-CoA and methylmalonyl-CoA, is catalyzed by the acyl-CoA carboxylase. The reaction consists of two catalytic steps, which involve the biotin carboxylase and the carboxyltransferase (8). In M. tuberculosis, the biotin carboxylation step is catalyzed by the ␣ subunit; there are three open reading frames (ORFs) that can encode the ␣ subunit (accA1 to -A3) in the genome. Carboxyl transfer is catalyzed by the ␤ subunit, and there are six ␤ subunits (accD1 to -D6) in the genome of the pathogen (6).Previously, the catalytic activities of the ␣ 3 , ␤ 4 , and ␤ 5 subunits were studied (10,11,22,24). However, the level...

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β-lactam antibiotics. This result explains the weak β-lactamase activity of EstU1 compared with class C β-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum.

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A Gram-negative, short rod-shaped bacterium, strain GA2-M15 T , was isolated from a sea-sand sample at Homi Cape, Pohang city, Republic of Korea. 16S rRNA gene sequence analysis demonstrated that this isolate was unique, showing 95.9 % sequence similarity to the type strain of Thalassobacter stenotrophicus and similarities of 94.0-95.2 % to the type strains of species of the genera Octadecabacter (94.4-95.2 %), Jannaschia (94.0-94.4 %) and Thalassobius (94.0-94.7 %). Chemotaxonomic characteristics (diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine as the major polar lipids and C 18 : 1 v7c as the predominant fatty acid) and DNA G+C content (56 mol%) were also similar to those of Thalassobacter stenotrophicus. 16S rRNA gene sequence similarity, physiological properties and some fatty acid components showed that strain GA2-M15T could be differentiated fromThalassobacter stenotrophicus. On the basis of these results, strain GA2-M15 T is considered to represent a novel species of the genus Thalassobacter, for which the name Thalassobacter arenae sp. nov. is proposed. The type strain is GA2-M15 T (5KACC 12675The genus Thalassobacter was described to accommodate a single bacterial strain isolated from Mediterranean seawater (Macián et al., 2005). A subsequent polyphasic taxonomic approach revealed that the type strains of Jannaschia cystaugens (Adachi et al., 2004) and Thalassobacter stenotrophicus (Macián et al., 2005) were highly similar and should be considered to be members of the same species. Pujalte et al. (2005) thus proposed the unification of the two species as Thalassobacter stenotrophicus and provided an emended description of the genus Thalassobacter. Phylogenetically, this genus is a member of the Alphaproteobacteria, and is closely related to the genera Jannaschia, Octadecabacter and Thalassobius. Thalassobacter was characterized as comprising Gramnegative, strictly aerobic, chemo-organotrophic, slightly halophilic bacteria, with C 18 : 1 v7c as the major fatty acid and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine as the major polar lipids.Strain GA2-M15 T was isolated from a sea-sand sample collected from the coast of Homi Cape, Pohang city, Republic of Korea. Isolation of the strain was performed by using the standard dilution-plating method on marine agar 2216 (MA; Difco) kept at 30 u C for 10 days. The culture was maintained routinely on MA at 30 uC and was preserved as glycerol suspensions (15 %, v/v) at 280 u C. Strain GA2-M15 T grew weakly on nutrient agar (NA), but did not grow on R2A, trypticase soy agar (TSA) or MacConkey agar (all from Difco). Thalassobacter stenotrophicus DSM 16310 T was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Braunschweig, Germany, and was used as a reference strain.The methods described by Smibert & Krieg (1994) were used for Gram staining and to detect accumulation of polyhydroxybutyrate, the presence of catalase and oxidase, and hydrolysis of casein, DNA and sta...