Characterization of exoglucanase and synergistic hydrolysis of cellulose in Clostridium stercorarium

Abstract A cellobiohydrolase component was isolated from an anaerobic thermophilic cellulolytic bacterium, Clostridium stercorarium . When acting alone, the enzyme showed minimal activity towards ordered substrates such as cellulose and filter paper but it has been shown to attack phosphoric-acid swollen cellulose giving cellobiose as principal product. When recombined with endoglucanase it did allow an extensive hydrolysis demonstrating a marked synergism in the action of those two components; the addition of β-glucosidase resulted in a further increase in activity.     

Distinctive ligand-binding specificities of tandem PA14 biomass-sensory elements from Clostridium thermocellum and Clostridium clariflavum

Cellulolytic clostridia use a highly efficient cellulosome system to degrade polysaccharides. To regulate genes encoding enzymes of the multi-enzyme cellulosome complex, certain clostridia contain alternative sigma I (σ^I) factors that have cognate membrane-associated anti-σ^I factors (RsgIs) which act as polysaccharide sensors. In this work, we analyzed the structure-function relationship of the extracellular sensory elements of Clostridium (Ruminiclostridium) thermocellum and Clostridium clariflavum (RsgI3 and RsgI4, respectively). These elements were selected for comparison, as each comprised two tandem PA14-superfamily motifs. The X-ray structures of the PA14 modular dyads from the two bacterial species were determined, both of which showed a high degree of structural and sequence similarity, although their binding preferences differed. Bioinformatic approaches indicated that the DNA sequence of promoter of sigI/rsgI operons represents a strong signature, which helps to differentiate binding specificity of the structurally similar modules. The σ^I4-dependent C. clariflavum promoter sequence correlates with binding of RsgI4_PA14 to xylan and was identified in genes encoding xylanases, whereas the σ^I3-dependent C. thermocellum promoter sequence correlates with RsgI3_PA14 binding to pectin and regulates pectin degradation-related genes. Structural similarity between clostridial PA14 dyads to PA14-containing proteins in yeast helped identify another crucial signature element: the calcium-binding loop 2 (CBL2), which governs binding specificity. Variations in the five amino acids that constitute this loop distinguish the pectin vs xylan specificities. We propose that the first module (PA14^A) is dominant in directing the binding to the ligand in both bacteria. The two X-ray structures of the different PA14 dyads represent the first reported structures of tandem PA14 modules.     

Complete Genome Sequence of Mycobacterium fortuitum subsp. fortuitum Type Strain DSM46621

Mycobacterium fortuitum is a member of the rapidly growing nontuberculous mycobacteria (NTM). It is ubiquitous in water and soil habitats, including hospital environments. M. fortuitum is increasingly recognized as an opportunistic nosocomial pathogen causing disseminated infection. Here we report the genome sequence of M. fortuitum subsp. fortuitum type strain DSM46621.     

Genetic engineering of grass cell wall polysaccharides for biorefining

Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass‐derived lignocellulose processing industry.     

Draft Genome Sequence of Lactobacillus rossiae DSM 15814T

The draft genome sequence of Lactobacillus rossiae DSM 15814(T) (CS1, ATCC BAA-88) was determined by a whole-genome shotgun approach. Reads were assembled to a 2.9-Mb draft version. RAST genome annotation evidenced 2,723 predicted coding sequences. Many carbohydrate, amino acid, and amino acid derivative subsystem features were found.     

科学家完成金小蜂基因组测序

<正>近日,美国罗切斯特大学生物学教授John H.Werren和贝勒医学院基因组测序中心的Stephen Richards领导完成了3种寄生性金小蜂(Nasonia vitripennis,N.giraulti和N.longicornis)的基因组测序。这一成果揭示     

Complete Genome Sequence of the B12-Producing Shimwellia blattae Strain DSM 4481, Isolated from a Cockroach

Here we announce the complete genome sequence of the coenzyme B(12)-producing enteric bacterium Shimwellia blattae (formerly Escherichia blattae). The genome consists of a single chromosome (4,158,636 bp). The genome size is smaller than that of most other enteric bacteria. Genome comparison revealed significant differences from the Escherichia coli genome.     

Complete Genome Sequence of Vibrio parahaemolyticus Bacteriophage vB_VpaM_MAR

Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global food safety issue. Our objective was to isolate and completely sequence a specific phage against this bacterium. Phage vB_VpaM_MAR is able to lyse 76% of the V. parahaemolyticus strains tested. MAR belongs to the Myoviridae family and has a genome comprised of double-stranded DNA with a size of 41,351 bp, a G+C content of 51.3%, and 62 open reading frames (ORFs). Bioinformatic analysis showed that phage MAR is closely related to Vibrio phages VHML, VP58.5, and VP882 and Halomonas aquamarina phage ΦHAP-1.     

Complete Genome Sequence of Haemophilus parasuis SH0165

Haemophilus parasuis is the causative agent of Glässer's disease, which produces big losses in swine populations worldwide. H. parasuis SH0165, belonging to the dominant serovar 5 in China, is a clinically isolated strain with high-level virulence. Here, we report the first completed genome sequence of this species.     

Complete Genome Sequence of Canine Papillomavirus Type 10

Papillomaviruses are epitheliotropic, nonenveloped, circular, double-stranded DNA viruses within the family Papillomaviridae that are associated with benign and malignant tumors in humans and animals. We report the complete genome sequence of canine papillomavirus type 10 identified from a pigmented plaque located on the head of a mixed-breed bloodhound.     

Complete Genome Sequence of Equine Herpesvirus Type 9

Equine herpesvirus type 9 (EHV-9), which we isolated from a case of epizootic encephalitis in a herd of Thomson''s gazelles (Gazella thomsoni) in 1993, has been known to cause fatal encephalitis in Thomson''s gazelle, giraffe, and polar bear in natural infections. Our previous report indicated that EHV-9 was similar to the equine pathogen equine herpesvirus type 1 (EHV-1), which mainly causes abortion, respiratory infection, and equine herpesvirus myeloencephalopathy. We determined the genome sequence of EHV-9. The genome has a length of 148,371 bp and all 80 of the open reading frames (ORFs) found in the genome of EHV-1. The nucleotide sequences of the ORFs in EHV-9 were 86 to 95% identical to those in EHV-1. The whole genome sequence should help to reveal the neuropathogenicity of EHV-9.     

Genome Sequence of the Antarctic Psychrophile Bacterium Planococcus antarcticus DSM 14505

Planococcus antarcticus DSM 14505 is a psychrophile bacterium that was isolated from cyanobacterial mat samples, originally collected from ponds in McMurdo, Antarctica. This orange-pigmented bacterium grows at 4°C and may possess interesting enzymatic activities at low temperatures. Here we report the first genomic sequence of P. antarcticus DSM 14505.     

Complete Genome Sequence of Borrelia afzelii K78 and Comparative Genome Analysis

The main Borrelia species causing Lyme borreliosis in Europe and Asia are Borrelia afzelii, B. garinii, B. burgdorferi and B. bavariensis. This is in contrast to the United States, where infections are exclusively caused by B. burgdorferi. Until to date the genome sequences of four B. afzelii strains, of which only two include the numerous plasmids, are available. In order to further assess the genetic diversity of B. afzelii, the most common species in Europe, responsible for the large variety of clinical manifestations of Lyme borreliosis, we have determined the full genome sequence of the B. afzelii strain K78, a clinical isolate from Austria. The K78 genome contains a linear chromosome (905,949 bp) and 13 plasmids (8 linear and 5 circular) together presenting 1,309 open reading frames of which 496 are located on plasmids. With the exception of lp28-8, all linear replicons in their full length including their telomeres have been sequenced. The comparison with the genomes of the four other B. afzelii strains, ACA-1, PKo, HLJ01 and Tom3107, as well as the one of B. burgdorferi strain B31, confirmed a high degree of conservation within the linear chromosome of B. afzelii, whereas plasmid encoded genes showed a much larger diversity. Since some plasmids present in B. burgdorferi are missing in the B. afzelii genomes, the corresponding virulence factors of B. burgdorferi are found in B. afzelii on other unrelated plasmids. In addition, we have identified a species specific region in the circular plasmid, cp26, which could be used for species determination. Different non-coding RNAs have been located on the B. afzelii K78 genome, which have not previously been annotated in any of the published Borrelia genomes.     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

鼠尾草叶绿体基因组序列分析

鼠尾草（Salvia japonica）是唇形科（Labiatae）鼠尾草属（Salvia）的一种多年生草本植物,具有十分重要的药用和经济价值。本文采用第二代测序技术Illumina Hiseq平台对鼠尾草的叶绿体基因组进行测序,同时以鼠尾草近缘物种丹参叶绿体基因组作为参考,组装得到完整叶绿体基因组序列。结果表明,鼠尾草叶绿体基因组序列全长153 995 bp,呈典型的四段式结构,其中LSC区长84 573 bp,SSC区长19 874 bp,两个IR区分别长24 774 bp;鼠尾草叶绿体基因组成功注释13组叶绿体基因,基因的种类、数目及GC含量等与唇形科中其它物种较为类似。这些研究结果丰富了鼠尾草属的叶绿体基因组数据,为今后鼠尾草属植物系统发育关系重建积累了基础性数据。     

Complete Genome Sequence of Clostridium sp. Strain BNL1100, a Cellulolytic Mesophile Isolated from Corn Stover

We present the full genome sequence of Clostridium sp. strain BNL1100, a Gram-positive, endospore-forming, lignocellulolytic bacterium isolated from a corn stover enrichment culture. The 4,613,747-bp genome of strain BNL1100 contains 4,025 putative protein-coding genes, of which 103 are glycoside hydrolases, the highest detected number in cluster III clostridia.     

Complete Genome Sequence of Anaplasma marginale subsp. centrale

Anaplasma marginale subsp. centrale is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent senso stricto A. marginale strains. The comparison markedly narrows the number of outer membrane protein candidates for development of a safer inactivated vaccine and provides insight into the diversity among strains of senso lato A. marginale.Sir Arnold Theiler described Anaplasma marginale as the “cause of a specific tick-borne disease of cattle” in 1908 (14), providing the first identification of a rickettsial pathogen. Two years later, Theiler isolated a less virulent organism, which he designated A. marginale subtype centrale (15). This naturally attenuated strain has been used as a live vaccine to prevent severe disease due to A. marginale senso stricto strains for 100 years. Understanding the genetic similarities and differences between the vaccine strain and wild-type A. marginale strains will provide clues as to how the vaccine provides protection. To that end, we have sequenced the A. marginale subsp. centrale vaccine strain using a whole-genome shotgun sequencing strategy.Genomic DNA, obtained from Kimron Veterinary institute, was fragmented by hydroshearing and ligated into pSmartLCKan (Lucigen). A total of 10,752 paired-end sequence reads (∼6.5× coverage) were generated. Assembly with Phrap (www.phrap.org) resulted in 148 contigs. Closure was achieved by applying the genome walking method across gap-spanning subclones and genomic DNA amplicons. For polymorphic loci, the most frequently observed subclone sequence was selected.Coding sequences (CDSs) in the single, circular, 1,206,806-bp chromosome were predicted using Glimmer2 and Glimmer3 (4, 5, 12). Annotation was as described previously for A. marginale senso stricto genomes (2, 3). There are 925 predicted CDSs, 19 pseudogenes, 37 tRNA genes, and a single set of rRNA genes in the genome. A. marginale subsp. centrale contains 10 putative genes not found in the closed-core genomes of senso stricto strains (3). Similarly, 18 genes found in senso stricto strains are absent from A. marginale subsp. centrale. This divergence is consistent with the subspecies nomenclature (15), but the findings do not resolve whether these genetic differences warrant classification of the vaccine strain as a distinct species within the genus Anaplasma (6).The ability of live A. marginale subsp. centrale to protect against a diversity of A. marginale strains indicates that epitopes critical for protective immunity are broadly conserved (11). As immunity against A. marginale can be induced by immunization with purified outer membrane protein (OMP) complexes (8-10, 13), identification of OMPs conserved between A. marginale subsp. centrale and senso stricto A. marginale may narrow the vaccine candidate list. A. marginale OMPs cluster predominately into two protein superfamilies, major surface protein 1 (Msp1) and Pfam01617/Msp2 (2). Members of the Msp1 superfamily from senso stricto strains (1, 2) are not well conserved (e.g., Msp1a, Msp1b-1, Msp1b-2, and Mlp2 to Mlp4; 13 to 48% amino acid identity) or are nonexistent (e.g., the products of Msp1b partial genes 1 to 3) in A. marginale subsp. centrale, suggesting that immunity induced by the live vaccine strain is unlikely to be associated with the Msp1 superfamily.Comparative analysis of the Pfam01617/Msp2 superfamily (2, 8) reveals both conservation and diversity. OpAG1 to OpAG3 and Msp4 are generally well conserved, while the family comprising Omp1 to Omp15 found in senso stricto strains (2, 3, 8) is reduced in A. marginale subsp. centrale: genes for the closely related proteins Omp7 to Omp9 are collapsed into a single CDS, and genes for homologs of Omp2, Omp3, Omp6, and Omp15 are missing. The OMP complex capable of inducing protective immunity contains 11 proteins (7, 8). By excluding those without homologs in the vaccine strain and the highly variable Msp2 and Msp3, the number of candidates is narrowed to six: four Msp2 superfamily members (Msp4, Omp1, Omp7, and OpAG2) and two non-superfamily members (AM779/ACIS557 and AM854/ACIS486). The degree of identity among these candidates from the vaccine strain and senso stricto A. marginale strains ranges from 63% (for OpAG2 proteins) to 88% (for Msp4 homologs). While the next steps in vaccine development will require strain analysis for epitope conservation in these candidates and immunization trials to test in vivo efficacy, progress will be accelerated using the minimal candidate list defined by the comparative genomics approach.     

Complete Genome Sequence of Beijerinckia indica subsp. indica

Beijerinckia indica subsp. indica is an aerobic, acidophilic, exopolysaccharide-producing, N₂-fixing soil bacterium. It is a generalist chemoorganotroph that is phylogenetically closely related to facultative and obligate methanotrophs of the genera Methylocella and Methylocapsa. Here we report the full genome sequence of this bacterium.Beijerinckia indica subsp. indica ATCC 9039 is the type strain of the genus Beijerinckia (6), a member of the Rhizobiales order of the Alphaproteobacteria. Beijerinckia spp. are commonly found as free-living bacteria in acidic soils and also in plant rhizosphere and phyllosphere environments (4). Research on Beijerinckia has suffered from chronic taxonomic confusion, with some strains of Sphingomonas and Azotobacter being misidentified in the literature: e.g., a “Beijerinckia” reported to degrade PAH has been reclassified (3). However, some Beijerinckia spp. have received research attention due to their plant growth-promoting properties (7) and for their abundant production of exoheteropolysaccharide with potential biotechnological uses (5).Genomic DNA from Beijerinckia indica subsp. indica was used to create 3-kb, 8-kb, and 40-kb DNA libraries. Sequencing, assembly, and automated annotation were performed at the Joint Genome Institute using standard procedures (U.S. Department of Energy; http://www.jgi.doe.gov/sequencing/strategy.html). The total number of paired-end shotgun Sanger reads in the assembly was 33,870. In addition, Roche 454 sequence data were included into the final assembly. Large Newbler contigs of 454 reads were chopped into 4,975 overlapping fragments of 1,000 bp and entered into the assembly as pseudoreads.The genome of B. indica subsp. indica was 4,170,153 bp. In addition, two plasmids of 181,736 and 66,727 bp were present. There are a total of 3,982 open reading frames (ORFs) predicted using Glimmer, of which 3,784 are predicted protein-coding genes and 2,695 (70%) have been assigned a predicted function. There are 134 pseudogenes, 52 tRNA genes, and three operons each containing 16S, 23S, and 5S rRNA genes. The G+C content is 57.0% (56% and 54% in the plasmids).The bacterium lacks phosphofructokinase, the key enzyme of the Embden-Meyerhof pathway. Instead, it uses the Entner-Doudoroff or pentose phosphate pathway to catabolize sugars, which is typical of free-living Rhizobiales. The majority of the genes involved in N₂ fixation are clustered in two genomic islands (10 kb and 51 kb), with the notable exception of the nifS gene encoding cysteine desulfurase.Beijerinckia indica is a metabolically versatile bacterium capable of growth on a variety of organic acids, sugars, and alcohols (4). In contrast, its close phylogenetic cousins Methylocella and Methylocapsa are highly specialized methanotrophs capable of growth on very few substrates (2). However, the genome size of Beijerinckia indica compared to that of Methylocella silvestris (4.17 versus 4.30 Mbp) and the numbers of predicted protein-encoding genes (3,788 versus 3,917) are remarkably similar. A BLAST analysis indicated that the 57% of the genes in the genome of B. indica have homologues in M. silvestris (stringency threshold expectation value [E] of 1e−50). Some key pathways of one-carbon metabolism (such as the tetrahydromethanopterin and serine pathways of formaldehyde metabolism) that are present in M. silvestris appear to be absent or incomplete in B. indica, which confirms previous experiments showing that the organism is incapable of methylotrophic growth (1). However, an operon encoding a putative propane monooxygenase homologous to soluble propane/methane monooxygenases of Methylocella silvestris BL2 was identified. More in-depth comparison of these genomes will help elucidate what defines their distinct lifestyles.