The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

Wang, Jieping; Han, Mei Lan K.; Cao, Zheng; Qian, Hongliang; Cui, Cui; Fu, Yang; Su, Jianmei; Liu, Ziduo; Zhou, Yu; He, Jin

doi:10.1074/mcp.m112.023986

Cited by 85 publications

(110 citation statements)

References 73 publications

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“…Natural PHA producers are capable of metabolizing PHA by inherent PHA depolymerases (Tseng et al 2006); thus, PHA depolymerases would be mainly responsible for this molecular weight decrease. The metabolic regulation of sporulation and PHA mobilization in Bacillus is of great interest (Slepecky and Law 1961;Chen et al 2010;Wang et al 2013). Since low-molecular-weight PHA seems to be favored as a storage material for bacteria that can be quickly metabolized, the decreasing molecular weight might be associated with providing a rapid energy supply for endospore development.…”

Section: Pha-producing Bacillusmentioning

confidence: 99%

Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus

Tsuge

Hyakutake

Mizuno

2015

Appl Microbiol Biotechnol

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This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40 kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20 kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials.

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Section: Pha-producing Bacillusmentioning

confidence: 99%

Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus

Tsuge

Hyakutake

Mizuno

2015

Appl Microbiol Biotechnol

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“…The cells were disrupted with disposable tissue-grinding pestles for 5 min and then treated by ultra sonication (Sonics & Materials) for 10 min (38). DNAse I and RNAse A were added, and the mixture was incubated on ice for 20 min.…”

Section: Sample Preparation and Isobaric Labelingmentioning

confidence: 99%

“…Total eukaryotic initiation factor-4E (eIF4E), S6 kinase (S6K), phospho-S6K, mammalian target of rapamycin signaling pathway (mTOR), phospho-mTOR, eukaryotic initiation factor-4E binding protein-1 (4EBP1), phospho-4EBP1, and PCNA protein abundances in epithelial cells along the crypt-villus axis of jejunum were determined by Western blotting, as previously reported (34,38). Total protein were extracted using ice-cold radioimmunoprecipitation assay lysis buffer (Biyuntian, Shanghai) containing phenylmethylsulfonyl fluoride.…”

Section: Western Blot Analysismentioning

confidence: 99%

Differential expression of proteins involved in energy production along the crypt-villus axis in early-weaning pig small intestine

Xiong

Yang

Tan

et al. 2015

American Journal of Physiology-Gastrointestinal and Liver Physi

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Weaning of piglets reflects intestinal dysfunction and atrophy and affected the physiological state of enterocytes. However, few studies have defined physiological state of enterocytes along the crypt-villus axis in early-weaning piglets. A total of 16 piglets from 8 litters were used in the experiment. One group of piglets was nursed by sows until age 21 days, and another group was weaned at age 14 days and then fed creep feed instead of breast milk for 7 days. Piglets were killed at 21 days, and the jejunum segments were dissected. After sequential isolation of jejunum epithelial cells along the crypt-villus axis, their proteins were analyzed through the isobaric tags for relative and absolute quantification, and proteins involved in the mammalian target of rapamycin signaling pathway and proliferating cell nuclear antigen abundances in jejunal epithelial cells of weaning or suckling group were determined by Western blotting. The differential proteins in three cell fractions were identified and analyzed. The results showed that proteins involved in the tricarboxylic acid cycle, ␤-oxidation, and the glycolysis pathway were significantly downregulated in the upper and middle villus of the early-weaned group. However, proteins involved in glycolysis were significantly upregulated in crypt cells. In addition, Western blot analysis showed that the expression of mammalian target of rapamycin pathway-related proteins was decreased (P ＜ 0.05) in the earlyweaned group. The present results showed that early-weaning differentially affect the expression of proteins involved in energy production of enterocytes along the jejunal crypt-villus axis.weaning; enterocyte; crypt-villus axis; piglet EARLY WEANING (14-to 21-day weaning age) of piglets is stressful and is characterized by poor growth, decreased feed intake, and diarrhea, because they are abruptly forced to combined stressors, including maternal and littermate separations, changes in diet, and establishment of a new social hierarchy (30, 31). Early weaning stress results in increased intestinal dysfunction and atrophy, impaired mucosal barrier function, and an imbalance of intestinal microbiota (13, 30). Inadequate food intake immediately after weaning and insufficient energy intake may contribute to the changes in intestinal morphology (25,35).The intestinal epithelium is characterized by rapidly proliferating cells organized into structures called crypts, which invaginate into the underlying mesenchyme, and villi, which project into the lumen (6). Furthermore, epithelial cells undergo functional and morphological differentiation during migration along the crypt-villus axis from the crypt to the tip of the villus (22, 43). Intestinal epithelial cell proliferation, differentiation, and apoptosis play important roles in intestinal development, maintenance, and recovery from tissue damage (44). In addition, epithelial cells in the pig intestine have notably high energy demands due to the rapid renewal of the epithelium (within few days) (2, 36). Previous studies showed...

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“…This list includes two ATP synthase subunits, AtpF and AtpB; elongation factor 4 (LepA); and the transcription termination factor Rho. It was found previously that in Bacillus thuringiensis, the translation of most ATP synthase subunits was upregulated during late sporulation to meet energy demands (52). We believe that the ATP synthase subunits were present at high levels during sporulation after being carried over from mother cells to the spores.…”

Section: Discussionmentioning

confidence: 61%

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly

et al. 2016

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Heat-resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of L-arginine-poly(L-aspartic acid), to ␤-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase. IMPORTANCEClostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria. C lostridium perfringens is a Gram-positive, anaerobic, sporeforming bacterium that causes myonecrosis (gas gangrene) as well as acute food poisoning and nonfoodborne gastrointestinal diseases (1). Due to their general resistance properties (2), spores are believed to be the major means of transmission of this pathogen (3). After spore germination, C. perfringens has the...

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The Metabolic Regulation of Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis Revealed by Transcriptomics and Proteomics

Cited by 85 publications

References 73 publications

Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus

Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus

Differential expression of proteins involved in energy production along the crypt-villus axis in early-weaning pig small intestine

Analysis of the Spore Membrane Proteome in Clostridium perfringens Implicates Cyanophycin in Spore Assembly

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