Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

ABSTRACTThe genome ofNeurospora crassaencodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome ofN. crassaand preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced inPichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochromec, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (kcatandKmvalues) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the hemebcofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) fromN. crassawas expressed inP. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

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Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

Applied and Environmental Microbiology ◽

10.1128/aem.02942-12 ◽

2012 ◽

Vol 79 (2) ◽

pp. 488-496 ◽

Cited By ~ 111

Author(s):

Mathieu Bey ◽

Simeng Zhou ◽

Laetitia Poidevin ◽

Bernard Henrissat ◽

Pedro M. Coutinho ◽

...

Keyword(s):

Cellulose Hydrolysis ◽

Podospora Anserina ◽

Striking Difference ◽

Glycoside Hydrolase Family ◽

Carbohydrate Binding ◽

Pycnoporus Cinnabarinus ◽

Content Type ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

ABSTRACTThe genome of the coprophilic ascomycetePodospora anserinaencodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserinaGH61A [PaGH61A] andPaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced inPichia pastoris. Synergistic cooperation betweenPaGH61A orPaGH61B with the cellobiose dehydrogenase (CDH) ofPycnoporus cinnabarinuson cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference betweenPaGH61A andPaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination ofPaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties ofPaGH61A andPaGH61B and their effect on the interaction with CDH are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

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Degradation of Granular Starch by the Bacterium Microbacterium aurum Strain B8.A Involves a Modular α-Amylase Enzyme System with FNIII and CBM25 Domains

Applied and Environmental Microbiology ◽

10.1128/aem.01029-15 ◽

2015 ◽

Vol 81 (19) ◽

pp. 6610-6620 ◽

Cited By ~ 18

Author(s):

Vincent Valk ◽

Wieger Eeuwema ◽

Fean D. Sarian ◽

Rachel M. van der Kaaij ◽

Lubbert Dijkhuizen

Keyword(s):

Enzyme System ◽

Recombinant Expression ◽

Potato Starch ◽

Carbohydrate Binding ◽

Starch Granules ◽

Catalytic Core ◽

Content Type ◽

C Terminus ◽

Carbohydrate Binding Modules

ABSTRACTThe bacteriumMicrobacterium aurumstrain B8.A, originally isolated from a potato plant wastewater facility, is able to degrade different types of starch granules. Here we report the characterization of an unusually large, multidomainM. aurumB8.A α-amylase enzyme (MaAmyA). MaAmyA is a 1,417-amino-acid (aa) protein with a predicted molecular mass of 148 kDa. Sequence analysis of MaAmyA showed that its catalytic core is a family GH13_32 α-amylase with the typical ABC domain structure, followed by a fibronectin (FNIII) domain, two carbohydrate binding modules (CBM25), and another three FNIII domains. Recombinant expression and purification yielded an enzyme with the ability to degrade wheat and potato starch granules by introducing pores. Characterization of various truncated mutants of MaAmyA revealed a direct relationship between the presence of CBM25 domains and the ability of MaAmyA to form pores in starch granules, while the FNIII domains most likely function as stable linkers. At the C terminus, MaAmyA carries a 300-aa domain which is uniquely associated with large multidomain amylases; its function remains to be elucidated. We concluded thatM. aurumB8.A employs a multidomain enzyme system to initiate degradation of starch granules via pore formation.

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Genome Sequence of the Polysaccharide-Degrading, Thermophilic Anaerobe Spirochaeta thermophila DSM 6192

Journal of Bacteriology ◽

10.1128/jb.01023-10 ◽

2010 ◽

Vol 192 (24) ◽

pp. 6492-6493 ◽

Cited By ~ 13

Author(s):

Angel Angelov ◽

Susanne Liebl ◽

Meike Ballschmiter ◽

Mechthild Bömeke ◽

Rüdiger Lehmann ◽

...

Keyword(s):

Genome Sequence ◽

Complete Genome Sequence ◽

Glycoside Hydrolase ◽

Enzyme System ◽

Cellulose Degradation ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Free Living ◽

Genome Data ◽

Genes Encoding

ABSTRACT Spirochaeta thermophila is a thermophilic, free-living anaerobe that is able to degrade various α- and β-linked sugar polymers, including cellulose. We report here the complete genome sequence of S. thermophila DSM 6192, which is the first genome sequence of a thermophilic, free-living member of the Spirochaetes phylum. The genome data reveal a high density of genes encoding enzymes from more than 30 glycoside hydrolase families, a noncellulosomal enzyme system for (hemi)cellulose degradation, and indicate the presence of a novel carbohydrate-binding module.

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The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00778-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4446-4452 ◽

Cited By ~ 37

Author(s):

Vadim Makarov ◽

João Neres ◽

Ruben C. Hartkoorn ◽

Olga B. Ryabova ◽

Elena Kazakova ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nitro Group ◽

Covalent Bond ◽

Antimycobacterial Activity ◽

Content Type ◽

Sar Studies ◽

Covalent Bond Formation

ABSTRACT8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity againstMycobacterium tuberculosisin vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml againstM. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorablein vitroabsorption-distribution-metabolism-excretion/toxicity (ADME/T) andin vivopharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

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Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04694-14 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4669-4679 ◽

Cited By ~ 23

Author(s):

Nilmar Silvio Moretti ◽

Leonardo da Silva Augusto ◽

Tatiana Mordente Clemente ◽

Raysa Paes Pinto Antunes ◽

Nobuko Yoshida ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Drug Targets ◽

Wild Type ◽

Content Type ◽

Damage Repair ◽

Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

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Characterization of New Virulence Factors Involved in the Intracellular Growth and Survival of Burkholderia pseudomallei

Infection and Immunity ◽

10.1128/iai.01102-15 ◽

2015 ◽

Vol 84 (3) ◽

pp. 701-710 ◽

Cited By ~ 23

Author(s):

Madeleine G. Moule ◽

Natasha Spink ◽

Sam Willcocks ◽

Jiali Lim ◽

José Afonso Guerra-Assunção ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Insertion Site ◽

Wild Type ◽

Content Type ◽

Growth And Survival ◽

New Genes ◽

Insertion Sites

Burkholderia pseudomallei, the causative agent of melioidosis, has complex and poorly understood extracellular and intracellular lifestyles. We used transposon-directed insertion site sequencing (TraDIS) to retrospectively analyze a transposon library that had previously been screened through a BALB/c mouse model to identify genes important for growth and survivalin vivo. This allowed us to identify the insertion sites and phenotypes of negatively selected mutants that were previously overlooked due to technical constraints. All 23 unique genes identified in the original screen were confirmed by TraDIS, and an additional 105 mutants with various degrees of attenuationin vivowere identified. Five of the newly identified genes were chosen for further characterization, and clean, unmarkedbpsl2248,tex,rpiR,bpsl1728, andbpss1528deletion mutants were constructed from the wild-type strain K96243. Each of these mutants was testedin vitroandin vivoto confirm their attenuated phenotypes and investigate the nature of the attenuation. Our results confirm that we have identified new genes important toin vivovirulence with roles in different stages ofB. pseudomalleipathogenesis, including extracellular and intracellular survival. Of particular interest, deletion of the transcription accessory protein Tex was shown to be highly attenuating, and thetexmutant was capable of providing protective immunity against challenge with wild-typeB. pseudomallei, suggesting that the genes identified in our TraDIS screen have the potential to be investigated as live vaccine candidates.

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Characterization of Intracellular Growth RegulatoricgRby Utilizing Transcriptomics To Identify Mediators of Pathogenesis in Shigella flexneri

Infection and Immunity ◽

10.1128/iai.00537-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3068-3076 ◽

Cited By ~ 8

Author(s):

Carolyn R. Morris ◽

Christen L. Grassel ◽

Julia C. Redman ◽

Jason W. Sahl ◽

Eileen M. Barry ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Diarrheal Disease ◽

Intracellular Growth ◽

Bioinformatics Analyses ◽

Content Type ◽

Regulatory Pathways

ABSTRACTShigellaspecies Gram-negative bacteria which cause a diarrheal disease, known as shigellosis, by invading and destroying the colonic mucosa and inducing a robust inflammatory response. With no vaccine available, shigellosis annually kills over 600,000 children in developing countries. This study demonstrates the utility of combining high-throughput bioinformatic methods within vitroandin vivoassays to provide new insights into pathogenesis. Comparisons ofin vivoandin vitrogene expression identified genes associated with intracellular growth. Additional bioinformatics analyses identified genes that are present inS. flexneriisolates but not in the three otherShigellaspecies. Comparison of these two analyses revealed nine genes that are differentially expressed during invasion and that are specific toS. flexneri. One gene, a DeoR family transcriptional regulator with decreased expression during invasion, was further characterized and is now designatedicgR, forintracellulargrowthregulator. Deletion oficgRcaused no difference in growthin vitrobut resulted in increased intracellular replication in HCT-8 cells. Furtherin vitroandin vivostudies using high-throughput sequencing of RNA transcripts (RNA-seq) of an isogenic ΔicgRmutant identified 34 genes that were upregulated under both growth conditions. This combined informatics and functional approach has allowed the characterization of a gene and pathway previously unknown inShigellapathogenesis and provides a framework for further identification of novel virulence factors and regulatory pathways.

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Multifunctional Nutrient-Binding Proteins Adapt Human Symbiotic Bacteria for Glycan Competition in the Gut by Separately Promoting Enhanced Sensing and Catalysis

mBio ◽

10.1128/mbio.01441-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 35

Author(s):

Elizabeth A. Cameron ◽

Kurt J. Kwiatkowski ◽

Byung-Hoo Lee ◽

Bruce R. Hamaker ◽

Nicole M. Koropatkin ◽

...

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Human Microbiome ◽

Gut Bacteria ◽

Nutrient Acquisition ◽

Carbohydrate Binding ◽

Content Type ◽

Carbohydrate Binding Proteins ◽

Gnotobiotic Mice

ABSTRACT To compete for the dynamic stream of nutrients flowing into their ecosystem, colonic bacteria must respond rapidly to new resources and then catabolize them efficiently once they are detected. The Bacteroides thetaiotaomicron starch utilization system (Sus) is a model for nutrient acquisition by symbiotic gut bacteria, which harbor thousands of related Sus-like systems. Structural investigation of the four Sus outer membrane proteins (SusD, -E, -F, and -G) revealed that they contain a total of eight starch-binding sites that we demonstrated, using genetic and biochemical approaches, to play distinct roles in starch metabolism in vitro and in vivo in gnotobiotic mice. SusD, whose homologs are abundant in the human microbiome, is critical for the initial sensing of available starch, allowing sus transcriptional activation at much lower concentrations than without this function. In contrast, seven additional binding sites across SusE, -F, and -G are dispensable for sus activation. However, they optimize the rate of growth on starch in a manner dependent on the expression of the bacterial polysaccharide capsule, suggesting that they have evolved to offset the diffusion barrier created by this structure. These findings demonstrate how proteins with similar biochemical behavior can serve orthogonal functions during different stages of cellular adaptation to nutrients. Finally, we demonstrated in gnotobiotic mice fed a starch-rich diet that the Sus binding sites confer a competitive advantage to B. thetaiotaomicron in vivo in a manner that is dependent on other colonizing microbes. This study reveals how numerically dominant families of carbohydrate-binding proteins in the human microbiome fulfill separate and sometimes cooperative roles to optimize gut commensal bacteria for nutrient acquisition. IMPORTANCE Our intestinal tract harbors trillions of symbiotic microbes. A critical function contributed by this microbial community is the ability to degrade most of the complex carbohydrates in our diet, which not only change from meal to meal but also cannot be digested by our own bodies. A numerically abundant group of gut bacteria called the Bacteroidetes plays a prominent role in carbohydrate digestion in humans and other animals. Currently, the mechanisms that allow this bacterial group to rapidly respond to available carbohydrates and then digest them efficiently are unclear. Here, we present novel functions for four carbohydrate-binding proteins present in one member of the Bacteroidetes, revealing that these proteins serve unique and separable roles in either initial nutrient sensing or subsequent digestion. Because the protein families investigated are numerous in other gut bacteria colonizing nearly all humans and animals, our findings are fundamentally important to understanding how symbiotic microbes assist human digestion.

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Molecular and Biochemical Analyses of the GH44 Module of CbMan5B/Cel44A, a Bifunctional Enzyme from the Hyperthermophilic Bacterium Caldicellulosiruptor bescii

Applied and Environmental Microbiology ◽

10.1128/aem.02009-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 7048-7059 ◽

Cited By ~ 26

Author(s):

Libin Ye ◽

Xiaoyun Su ◽

George E. Schmitz ◽

Young Hwan Moon ◽

Jing Zhang ◽

...

Keyword(s):

Specific Activity ◽

Cellulose Hydrolysis ◽

Thermophilic Bacterium ◽

Carbohydrate Binding ◽

Content Type ◽

Caldicellulosiruptor Bescii ◽

C Terminus ◽

Carbohydrate Binding Modules ◽

Β Sheets ◽

Biochemical Analyses

ABSTRACTA large polypeptide encoded in the genome of the thermophilic bacteriumCaldicellulosiruptor besciiwas determined to consist of two glycoside hydrolase (GH) modules separated by two carbohydrate-binding modules (CBMs). Based on the detection of mannanase and endoglucanase activities in the N-terminal GH5 and the C-terminal GH44 module, respectively, the protein was designated CbMan5B/Cel44A. A GH5 module with >99% identity from the same organism was characterized previously (X. Su, R. I. Mackie, and I. K. Cann, Appl. Environ. Microbiol.78:2230-2240, 2012); therefore, attention was focused on CbMan5A/Cel44A-TM2 (or TM2), which harbors the GH44 module and the two CBMs. On cellulosic substrates, TM2 had an optimal temperature and pH of 85°C and 5.0, respectively. Although the amino acid sequence of the GH44 module of TM2 was similar to those of other GH44 modules that hydrolyzed cello-oligosaccharides, cellulose, lichenan, and xyloglucan, it was unique that TM2 also displayed modest activity on mannose-configured substrates and xylan. The TM2 protein also degraded Avicel with higher specific activity than activities reported for its homologs. The GH44 catalytic module is composed of a TIM-like domain and a β-sandwich domain, which consists of one β-sheet at the N terminus and nine β-sheets at the C terminus. Deletion of one or more β-sheets from the β-sandwich domain resulted in insoluble proteins, suggesting that the β-sandwich domain is essential for proper folding of the polypeptide. Combining TM2 with three other endoglucanases fromC. besciiled to modest synergistic activities during degradation of cellulose, and based on our results, we propose a model for cellulose hydrolysis and utilization byC. bescii.

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Two Zinc Uptake Systems Contribute to the Full Virulence of Listeria monocytogenes during GrowthIn VitroandIn Vivo

Infection and Immunity ◽

10.1128/iai.05904-11 ◽

2011 ◽

Vol 80 (1) ◽

pp. 14-21 ◽

Cited By ~ 39

Author(s):

David Corbett ◽

Jiahui Wang ◽

Stephanie Schuler ◽

Gloria Lopez-Castejon ◽

Sarah Glenn ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Zinc Uptake ◽

Detectable Effect ◽

Intracellular Growth ◽

Content Type ◽

Zinc Sensing ◽

Identification And Characterization ◽

Cell Deletion

ABSTRACTWe report here the identification and characterization of two zinc uptake systems, ZurAM and ZinABC, in the intracellular pathogenListeria monocytogenes. Transcription of both operons was zinc responsive and regulated by the zinc-sensing repressor Zur. Deletion of eitherzurAMorzinAhad no detectable effect on growth in defined media, but a doublezurAM zinAmutant was unable to grow in the absence of zinc supplementation. Deletion ofzinAhad no detectable effect on intracellular growth in HeLa epithelial cells. In contrast, growth of thezurAMmutant was significantly impaired in these cells, indicating the importance of the ZurAM system during intracellular growth. Notably, the deletion of bothzinAandzurAMseverely attenuated intracellular growth, with the double mutant being defective in actin-based motility and unable to spread from cell to cell. Deletion of eitherzurAMorzinAhad a significant effect on virulence in an oral mouse model, indicating that both zinc uptake systems are importantin vivoand establishing the importance of zinc acquisition during infection byL. monocytogenes. The presence of two zinc uptake systems may offer a mechanism by whichL. monocytogenescan respond to zinc deficiency within a variety of environments and during different stages of infection, with each system making distinct contributions under different stress conditions.

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