RESUMEN

Clostridium cellulovorans, an anaerobic and mesophilic bacterium, degrades native substrates in soft biomass such as corn fibre and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Recently, we have reported the whole-genome sequence of C. cellulovorans comprising 4220 predicted genes in 5.10 Mbp [Y. Tamaru et al., (2010) J. Bacteriol., 192: 901­902]. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, and they coded for not only carbohydrate-degrading enzymes but also a lipase, peptidases and proteinase inhibitors. Interestingly, two novel genes encoding scaffolding proteins were found in the genome. According to KEGG metabolic pathways and their comparison with 11 Clostridial genomes, gene expansion in the C. cellulovorans genome indicated mainly non-cellulosomal genes encoding hemicellulases and pectin-degrading enzymes. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced cellulosome-producing Clostridium strains for industrial applications such as biofuel production.

Asunto(s)

Proteínas Bacterianas/genética , Celulosomas/enzimología , Clostridium cellulovorans/genética , Clostridium/genética , Genoma Bacteriano , Proteínas Bacterianas/metabolismo , Celulosomas/genética , Clostridium/enzimología , Clostridium cellulovorans/enzimología , Tamaño del Genoma , Datos de Secuencia Molecular

RESUMEN

Clostridium cellulovorans is an anaerobic, mesophilic bacterium that efficiently degrades native substrates in soft biomass such as corn fibre and rice straw by producing an extracellular enzyme complex called the cellulosomes. By examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Recently, we reported the whole genome sequence of C. cellulovorans. A total of 57 cellulosomal genes were found in the C. cellulovorans genome and coded for not only carbohydrate-active enzymes but also lipase, peptidase and proteinase inhibitors, in addition to two novel genes encoding scaffolding proteins CbpB and CbpC. Interestingly, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome-producing clostridia: mesophilic C. cellulolyticum and thermophilic C. thermocellum. Since the C. cellulovorans genome included not only cellulosomal genes but also a large number of genes encoding non-cellulosomal enzymes, the genome expansion of C. cellulovorans included genes more related to degradation of polysaccharides, such as hemicelluloses and pectins, than to cellulose. In this review, we propose a strategy for industrial applications such as biofuel production using enhanced mesophilic cellulosome- and solvent-producing clostridia.

Asunto(s)

Biocombustibles , Celulasa/metabolismo , Celulosa/metabolismo , Clostridium cellulovorans/metabolismo , Genómica/métodos , Microbiología Industrial/métodos , Complejos Multienzimáticos/metabolismo , Clostridium cellulovorans/genética

RESUMEN

Clostridium cellulovorans produces large extracellular enzyme complex, called cellulosomes. The diversity of the cellulosomal enzymes, which are secreted by C. cellulovorans that has been cultured on different carbon sources, such as Avicel, xylan, AXP (Avicel-xylan-pectin, 3:1:1) and cellobiose, was explored by two-dimensional gel electrophoresis. To identify the cellulosomal enzymes, we constructed a biomarker using cohesin 6, one of the CbpA cohesins, that was labeled with fluorescence. The major apparent spots were isolated and identified by ESI MS/MS protein sequencing. Fluorescently labeled cohesin clearly showed that the amount of the cellulosomal enzymes was influenced by the available carbon source. EngE, ExgS, EngK, XynB and ManA were most frequently expressed under all conditions. However, EngY was only observed on the AXP culture. We found two novel putative cellulosomal proteins, NC1[GH9] and NC2[GH26], and five unknown proteins, NU1, NU2, NU3, NU4 and NU5. The cohesin biomarker clearly showed different production patterns of the cellulosomal subunits under different culture conditions and revealed novel cellulosomal subunits.

Asunto(s)

Biotecnología/métodos , Carbono/farmacología , Proteínas de Ciclo Celular/metabolismo , Celulosomas/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Clostridium cellulovorans/crecimiento & desarrollo , Clostridium cellulovorans/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biomarcadores/metabolismo , Celulosomas/efectos de los fármacos , Celulosomas/enzimología , Clostridium cellulovorans/efectos de los fármacos , Clostridium cellulovorans/enzimología , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Péptidos/química , Proteínas Recombinantes/aislamiento & purificación , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Cohesinas

RESUMEN

Clostridium cellulovorans 743B was isolated from a wood chip pile and is an anaerobic and mesophilic spore-forming bacterium. This organism degrades native substrates in soft biomass such as corn fiber and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Here we report the genome sequence of C. cellulovorans 743B.

Asunto(s)

Celulosomas/metabolismo , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Genoma Bacteriano/genética , Genoma Bacteriano/inmunología , Sistemas de Lectura Abierta/genética , Análisis de Secuencia de ADN

RESUMEN

We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3' half of alpha-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of beta-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus beta-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed beta-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.

Asunto(s)

Enzimas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Celulasa/genética , Celulasa/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Glucosa/metabolismo , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , beta-Glucanos/metabolismo , Cohesinas

RESUMEN

The cellulolytic activity of mesophilic bacteria and fungi is described, with special emphasis on the large extracellular enzyme complex called the cellulosome. The cellulosome is composed of a scaffolding protein, which is attached to various cellulolytic and hemicellulolytic enzymes, and this complex allows the organisms to degrade plant cell walls very efficently. The enzymes include a variety of cellulases, hemicellulases, and pectinases that work synergistically to degrade complex cell-wall molecules.

Asunto(s)

Bacterias/enzimología , Celulasa/metabolismo , Celulosa/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Plantas/metabolismo

RESUMEN

Cellulosomes in Clostridium cellulovorans are assembled by the interaction between the repeated cohesin domains of a scaffolding protein (CbpA) and the dockerin domain of enzyme components. In this study, we determined the synergistic effects on cellulosic and hemicellulosic substrates by three different recombinant mini-cellulosomes containing either endoglucanase EngB or endoxylanase XynA bound to mini-CbpA with one cohesin domain (mini-CbpA1), two cohesins (mini-CbpA12), or four cohesins (mini-CbpA1234). The assembly of EngB or XynA with mini-CbpA increased the activity against carboxymethyl cellulose, acid-swollen cellulose, Avicel, xylan, and corn fiber 1.1-1.8-fold compared with that for the corresponding enzyme alone. A most distinct improvement was shown with corn fiber, a natural substrate containing xylan, arabinan, and cellulose. However, there was little difference in activity between the three different mini-cellulosomes when the cellulosomal enzyme concentration was held constant regardless of the copy number of cohesins in the cellulosome. A synergistic effect was observed when the enzyme concentration was increased to be proportional to the number of cohesins in the mini-cellulosome. The highest degree of synergy was observed with mini-CbpA1234 (1.8-fold) and then mini-CbpA12 (1.3-fold), and the lowest synergy was observed with mini-CbpA1 (1.2-fold) when Avicel was used as the substrate. As the copy number of cohesin was increased, there was more synergy. These results indicate that the clustering effect (physical enzyme proximity) of the enzyme within the mini-cellulosome is one of the important factors for efficient degradation of plant cell walls.

Asunto(s)

Proteínas de Ciclo Celular/fisiología , Celulasa/metabolismo , Celulosomas/enzimología , Proteínas Cromosómicas no Histona/fisiología , Clostridium cellulovorans/enzimología , Glicósido Hidrolasas/metabolismo , Proteínas Nucleares/fisiología , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Cohesinas

RESUMEN

Bacterial cells contain many large, spatially extended assemblies of ions, molecules, and macromolecules, called hyperstructures, that are implicated in functions that range from DNA replication and cell division to chemotaxis and secretion. Interactions between these hyperstructures would create a level of organization intermediate between macromolecules and the cell itself. To explore this level, a taxonomy is needed. Here, we describe classification criteria based on the form of the hyperstructure and on the processes responsible for this form. These processes include those dependent on coupled transcription-translation, protein-protein affinities, chromosome site-binding by protein, and membrane structures. Various combinations of processes determine the formation, maturation, and demise of many hyperstructures that therefore follow a trajectory within the space of classification by form/process. Hence a taxonomy by trajectory may be desirable. Finally, we suggest that working toward a taxonomy based on speculative interactions between hyperstructures promises most insight into life at this level.

Asunto(s)

Bacterias/clasificación , Bacterias/citología , Bacterias/genética , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Metabolismo Energético , Biosíntesis de Proteínas , Transcripción Genética

RESUMEN

Clostridium cellulovorans, an anaerobic bacterium, produces a small nonenzymatic protein called HbpA, which has a surface layer homology domain and a type I cohesin domain similar to those found in the cellulosomal scaffolding protein CbpA. In this study, we demonstrated that HbpA could bind to cell wall fragments from C. cellulovorans and insoluble polysaccharides and form a complex with cellulosomal cellulases endoglucanase B (EngB) and endoglucanase L (EngL). Synergistic degradative action of the cellulosomal cellulase and HbpA complexes was demonstrated on acid-swollen cellulose, Avicel, and corn fiber. We propose that HbpA functions to bind dockerin-containing cellulosomal enzymes to the cell surface and complements the activity of cellulosomes.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Celulasas/metabolismo , Clostridium cellulovorans/enzimología , Celulosa/metabolismo , Unión Proteica , Zea mays/metabolismo

RESUMEN

The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures. Here, we review a variety of spatially extended structures, complexes, and assemblies that might be termed hyperstructures. These include ribosomal or "nucleolar" hyperstructures; transertion hyperstructures; putative phosphotransferase system and glycolytic hyperstructures; chemosignaling and flagellar hyperstructures; DNA repair hyperstructures; cytoskeletal hyperstructures based on EF-Tu, FtsZ, and MreB; and cell cycle hyperstructures responsible for DNA replication, sequestration of newly replicated origins, segregation, compaction, and division. We propose principles for classifying these hyperstructures and finally illustrate how thinking in terms of hyperstructures may lead to a different vision of the bacterial cell.

Asunto(s)

Bacterias/genética , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Bacterias/citología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/metabolismo , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas

RESUMEN

The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon "intercellular complementation." Interesting implications concerning bacterial cooperation are suggested from these results.

Asunto(s)

Celulosomas/química , Celulosomas/metabolismo , Clostridium cellulovorans/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Bioquímica/métodos , Proteínas Portadoras/genética , Dominio Catalítico , Celulasa/genética , Clonación Molecular , Técnicas de Cocultivo , Endo-1,4-beta Xilanasas/genética , Complejos Multienzimáticos/química , Plásmidos/metabolismo , Unión Proteica , Especificidad de la Especie , beta-Glucosidasa/genética

RESUMEN

Clostridium cellulovorans degrades cellulose efficiently to small oligosaccharides, which are used as an energy source. To characterize enzymes related to degrading small oligosaccharides, a gene was cloned for an extracellular non-cellulosomal beta-glucan glucohydrolase (BglA) classified as a family-1 glycosyl hydrolase in C. cellulovorans. Recombinant BglA (rBglA) had higher activity on long glucooligomers than on cellobiose. When cellulosomes and rBglA were incubated with cellulose, the oligosaccharides produced were degraded more effectively to cellobiose and glucose, than with cellulosomes alone, indicating that BglA facilitated the degradation of accessible cello-oligosaccharides produced from cellulose by C. cellulovorans cellulosomes. Thus, this is an example of an extracellular non-cellulosomal enzyme working in a cooperative manner with cellulosomes to degrade cellulose to sugars.

Asunto(s)

Celulasa/química , Celulosomas/química , Clostridium cellulovorans/enzimología , Glucano 1,4-beta-Glucosidasa/química , Oligosacáridos/química , beta-Glucanos/química , Carbohidratos/química , Carbono/química , Proteínas Recombinantes/química

RESUMEN

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHDeltaCBM and EngYDeltaCBM devoid of the CBM lost activity toward all substrates including CMC. EngKDeltaCBM and EngMDeltaCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.

Asunto(s)

Celulasa/metabolismo , Celulosa/metabolismo , Clostridium cellulovorans/enzimología , Complejos Multienzimáticos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dominio Catalítico/genética , Celulasa/química , Celulasa/genética , Celulasa/aislamiento & purificación , Clostridium cellulovorans/química , Clostridium cellulovorans/genética , Clostridium cellulovorans/crecimiento & desarrollo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Especificidad por Sustrato

RESUMEN

The planar and anchoring residues of the family IIIa cellulose binding domain (CBD) from the cellulosomal scaffolding protein of Clostridium cellulovorans were investigated by site-directed mutagenesis and cellulose binding studies. By fusion with maltose binding protein, the family IIIa recombinant wild-type and mutant CBDs from C. cellulovorans were expressed as soluble forms. Cellulose binding tests of the mutant CBDs indicated that the planar strip residues played a major role in cellulose binding and that the anchoring residues played only a minor role.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Celulosa/metabolismo , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Proteínas Portadoras/química , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad

RESUMEN

Clostridium cellulovorans produces a major noncellulosomal family 9 endoglucanase EngO. A genomic DNA fragment (40 kb) containing engO and neighboring genes was cloned. The nucleotide sequence contained reading frames for endoglucanase EngO, a putative response regulator, and a putative sensor histidine kinase protein. The engO gene consists of 2,172 bp and encodes a protein of 724 amino acids with a molecular weight of 79,474. Northern hybridizations revealed that the engO gene is transcribed as a monocistronic 2.6-kb mRNA. 5' RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) PCR analysis indicated that the single transcriptional start site of engO was located 264 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the engO promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the sigma(A) consensus promoter sequences of gram-positive bacteria. EngO contains a typical N-terminal signal peptide of 28 amino acid residues, followed by a 149-amino-acid sequence which is homologous to the family 4-9 carbohydrate-binding domain. Downstream of this domain was an immunoglobulin-like domain of 89 amino acids. The C terminus contains a family 9 catalytic domain of glycosyl hydrolase. Mass spectrometry analysis of EngO was in agreement with that deduced from the nucleotide sequence. Expression of engO mRNA increased from early to middle exponential phase and decreased during the early stationary phase. EngO was highly active toward carboxymethyl cellulose but showed no activity towards xylan. It was optimally active at 40 to 50 degrees C and pH 5 to 6. The analysis of the products from the cellulose hydrolysis through thin-layer chromatography indicated its endoglucanase activity.

Asunto(s)

Celulasa/genética , Clostridium cellulolyticum/enzimología , Clostridium cellulolyticum/genética , Enzimas/genética , Regiones Promotoras Genéticas , Transcripción Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Enzimas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa

RESUMEN

Clostridium cellulovorans, an anaerobic bacterium, degrades native substrates efficiently by producing an extracellular enzyme complex called the cellulosome. All cellulosomal enzyme subunits contain dockerin domains that can bind to hydrophobic domains termed cohesins which are repeated nine times in CbpA, the nonenzymatic scaffolding protein of C. cellulovorans cellulosomes. In this study, the synergistic interactions of cellulases (endoglucanase E, EngE; endoglucanase L, EngL) and hemicellulases (arabinofuranosidase A, ArfA; xylanase A, XynA) were determined on the degradation of corn fiber, a natural substrate containing mainly xylan, arabinan, and cellulose. The degradation by XynA and ArfA of cellulose/arabinoxylan was greater than that of corn fiber and resulted in 2.6-fold and 1.4-fold increases in synergy, respectively. Synergistic effects were observed in increments in both simultaneous and sequential reactions with ArfA and XynA. These synergistic enzymes appear to represent potential rate-limiting enzymes for efficient hemicellulose degradation. When mini-cellulosomes were constructed from the cellulosomal enzymes (XynA and EngL) and mini-CbpA with cohesins 1 and 2 (mini-CbpA1&2) and mini-CbpA with cohesins 5 and 6 (mini-CbpA5&6), higher activity was observed than that for the corresponding enzymes alone. Based on the degradation of different types of celluloses and hemicelluloses, the interaction between cellulosomal enzymes (XynA and EngL) and mini-CbpA displayed a diversity that suggests that dockerin-cohesin interaction from C. cellulovorans may be more selective than random.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Celulasas/metabolismo , Clostridium cellulovorans/enzimología , Glicósido Hidrolasas/metabolismo , Complejos Multienzimáticos/metabolismo , Zea mays/metabolismo , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Celulasas/genética , Clostridium cellulovorans/genética , Clostridium cellulovorans/crecimiento & desarrollo , Medios de Cultivo , Glicósido Hidrolasas/genética , Complejos Multienzimáticos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Zea mays/química

RESUMEN

Clostridium cellulovorans produces a cellulase enzyme complex called the cellulosome. When cells were grown on different carbon substrates such as Avicel, pectin, xylan, or a mixture of all three, the subunit composition of the cellulosomal subpopulations and their enzymic activities varied significantly. Fractionation of the cellulosomes (7-11 fractions) indicated that the cellulosome population was heterogeneous, although the composition of the scaffolding protein CbpA, endoglucanase EngE and cellobiohydrolase ExgS was relatively constant. One of the cellulosomal fractions with the greatest endoglucanase activity also showed the highest or second highest cellulase activity under all growth conditions tested. The cellulosomal fractions produced from cells grown on a mixture of carbon substrates showed the greatest cellulase activity and contained CbpA, EngE/EngK, ExgS/EngH and EngL. High xylanase activity in cellulose, pectin and mixed carbon-grown cells was detected with a specific cellulosomal fraction which had relatively larger amounts of XynB, XynA and unknown proteins (35-45 kDa). These results in toto indicate that the assembly of cellulosomes occurs in a non-random fashion.

Asunto(s)

Carbono/metabolismo , Celulosomas/metabolismo , Clostridium cellulovorans/enzimología , Clostridium cellulovorans/crecimiento & desarrollo , Western Blotting , Carboximetilcelulosa de Sodio/metabolismo , Celulasa/metabolismo , Celulosa/metabolismo , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Pectinas/metabolismo , Xilanos/metabolismo

RESUMEN

This review is a concise description of the study undertaken to examine the modulation by opioids of simian acquired immunodeficiency syndrome (SAIDS) induced by inoculation of rhesus monkeys with simian AIDS virus SIVmac239. The study showed that the replication rate of the virus was several times greater in monkeys made dependent on morphine than in those of non-morphine treated monkeys. Further, a significant change in the mutation rate of the infecting virus in morphine-treated monkeys resulted in the production of mutants that were silent to conventional serological screening tests as well as resistant to AZT. In addition, opioid and chemokine receptors involved were identified in immune cells and a full comparative spectrum of the compromise of the immune system was examined allowing subsequent studies to evaluate wherein the modulation of the development of the syndrome could be better characterized. The data gathered to date are unique and germane to furthering our understanding of AIDS in humans and its subsequent treatment thereof.

Asunto(s)

Sistema Inmunológico/efectos de los fármacos , Morfina/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Animales , Progresión de la Enfermedad , Humanos , Macaca mulatta , Mutación , Narcóticos/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Replicación Viral/efectos de los fármacos , Zidovudina/farmacología

RESUMEN

The nucleotide sequence of the Clostridium cellulovorans xynB gene, which encodes the XynB xylanase, consists of 1,821 bp and encodes a protein of 607 amino acids with a molecular weight of 65,976. XynB contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 147-amino-acid sequence that is homologous to the family 4-9 (subfamily 9 in family 4) carbohydrate-binding domain. Downstream of this domain is a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The XynB sequence from mass spectrometry and N-terminal amino acid sequence analyses agreed with that deduced from the nucleotide sequence. XynB was highly active toward xylan, but not active toward carboxymethyl cellulose. The enzyme was optimally active at 40 degrees C and pH 5.0. Northern hybridizations revealed that xynB is transcribed as a monocistronic 1.9-kb mRNA. RNA ligase-mediated rapid amplification of 5' cDNA ends by PCR (RLM-5'RACE PCR) analysis of C. cellulovorans RNA identified a single transcriptional start site of xynB located 47 bp upstream from the first nucleotide of the translation initiation codon. Alignment of the xynB promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the sigmaA consensus promoter sequences of gram-positive bacteria. Expression of xynB mRNA increased from early to middle exponential phase and decreased during the early stationary phase when the cells were grown on cellobiose. No alternative promoter was observed by RLM-5'RACE PCR and reverse transcriptase PCR analyses during expression. The analysis of the products from xylan hydrolysis by thin-layer chromatography indicated its endoxylanase activity. The results suggest that XynB is a consistent and major cellulosomal enzyme during growth on cellulose or xylan.

Asunto(s)

Celulasa/metabolismo , Clostridium cellulovorans/enzimología , Clostridium cellulovorans/crecimiento & desarrollo , Endo-1,4-beta Xilanasas , Regulación Bacteriana de la Expresión Génica , Complejos Multienzimáticos/metabolismo , beta-Glucosidasa , Secuencia de Aminoácidos , Secuencia de Bases , Celulasa/genética , Celulosa/metabolismo , Clostridium cellulovorans/genética , Clostridium cellulovorans/metabolismo , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/aislamiento & purificación , Endo-1,4-beta Xilanasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Xilanos/metabolismo , beta-Glucosidasa/química , beta-Glucosidasa/genética , beta-Glucosidasa/aislamiento & purificación , beta-Glucosidasa/metabolismo

RESUMEN

CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs). Among the three types of domains, the function of the HLDs is still unknown. We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE. Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C. cellulovorans cell wall fraction. Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed. EngE was shown to have a higher affinity for cell walls than rHLDs have. C. cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have. When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C. cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface. During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose. The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose. These results suggest that the CbpA HLDs play a major role and a minor role in C. cellulovorans cellulosomes. The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C. cellulovorans cell surface.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Portadoras/química , Celulosa/metabolismo , Celulosomas/metabolismo , Clostridium/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Pared Celular/metabolismo , Polisacáridos Bacterianos/metabolismo , Homología de Secuencia