An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Genome sequence of the ethanol-producing Zymomonas mobilis subsp. pomaceae lectotype strain ATCC 29192

Zymomonas mobilis is an alphaproteobacterium studied for bioethanol production. Different strains of this organism have been hitherto sequenced; they all belong to the Z. mobilis subsp. mobilis taxon. Here we report the finished and annotated genome sequence of strain ATCC 29192, a cider-spoiling agent isolated in the United Kingdom. ATCC 29192 is the lectotype of the second-best-characterized subspecies of Z. mobilis, Z. mobilis subsp. pomaceae. The nucleotide sequence of ATCC 29192 deviates from that of Z. mobilis subsp. mobilis representatives, which justifies its distinct taxonomic positioning and proves particularly useful for comparative and functional genomic analyses.     

Complete Genome Sequence of the Ethanol Producer Zymomonas mobilis NCIMB 11163

Zymomonas mobilis is an ethanol-producing alphaproteobacterium currently considered a major candidate organism for bioethanol production. Here we report the finished and annotated genome sequence of Z. mobilis subsp. mobilis strain NCIMB 11163, a British ale-infecting isolate. This is the first Z. mobilis strain whose genome, chromosomal and plasmid, is presented in its entirety.Zymomonas mobilis is a bacterium vigorously studied as a platform organism for bioethanol production in North America and other parts of the world. Z. mobilis converts sugars such as glucose or sucrose into ethanol and carbon dioxide to almost theoretical yields and to rates higher than those of yeasts (17). Genetically engineered strains that ferment pentoses in addition to naturally utilized hexoses also hold great promise for use in lignocellulosic biomass degradations (5, 22). Besides ethanol, Z. mobilis can produce other high-value chemicals such as sorbitol, levan, or phenylacetylcarbinol and has attracted interest for its unusual membrane steroid content (11). Lastly, Zymomonas is regarded as a safe organism and is even used for medicinal purposes (12, 20), which further facilitates its employment in large-scale biotechnological endeavors.The chromosomal sequence of the Z. mobilis subsp. mobilis industrial strain ATCC 31821 (ZM4) was recently published (19). Here we announce the first entire genome sequence of a Z. mobilis subsp. mobilis strain, that of the United Kingdom-originating strain NCIMB 11163 (B70) (20). Total DNA from NCIMB 11163 (16) was used for whole-genome shotgun sequencing at the U.S. DOE Joint Genome Institute. For this, an 8.7-kb DNA library and 454 and Solexa reads were used (http://www.jgi.doe.gov). Draft assemblies were based on 8,551 Sanger reads and 454 pyrosequencing to 20× coverage, whereas the Phred/Phrap/Consed software package was used for sequence assembly and quality assessment (6, 7, 9; http://www.phrap.com). After the shotgun stage, reads were assembled with parallel Phrap (High Performance Software, LLC), and misassemblies were corrected with Dupfinisher (10) or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). A total of 144 primer walk reactions, five transposon bomb libraries, 53 PCR end reads, and two PCR shatter libraries were necessary to close gaps, resolve repetitive regions, and raise the quality of the finished sequence. The completed genome sequence of NCIMB 11163 was based on 11,048 reads, with an error rate of less than 6 bp out of 100,000 bp.Open reading frame prediction and annotation were performed using Prodigal (http://compbio.ornl.gov/prodigal/) and BLAST (1); tRNAscan-SE and RNAmmer (14, 15) were used for tRNA and rRNA recognition, respectively. Functional assignment of genes was performed by searching translated open reading frames against sequences in the SPTR (TrEMBL) (2), Pfam (8), TIGRFAMs (18), COG (21), and KEGG (13) databases.Z. mobilis NCIMB 11163 contains a single, circular chromosome of 2,124,771 bp and three plasmids, p11163_1, p11163_2, and p11163_3 of 53,380 bp, 40,818 bp, and 4,551 bp, respectively. The overall GC content of the chromosome is 46.83%, whereas those of the plasmids are 42.32%, 43.80%, and 36.37%, respectively. The entire genome of NCIMB 11163 contains 1,884 protein-encoding genes and 51 tRNA and nine rRNA genes, which are chromosomally located.The chromosome of NCIMB 11163 is 68,355 bp larger than that of ZM4 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_006526","term_id":"283856168","term_text":"NC_006526"}}NC_006526) (19) and colinear at its largest part with that of ZM4 (genome structure comparisons were performed using ACT) (3). It bears several unique regions, among which are two genomic islands of ca. 25 and 79 kb, with no detectable nucleotide homology to same-species sequences and high regional similarity to chromosomal stretches of Paracoccus denitrificans PD1222 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000489.1","term_id":"119372524","term_text":"CP000489.1"}}CP000489.1), Xanthobacter autotrophicus Py2 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000781.1","term_id":"154158043","term_text":"CP000781.1"}}CP000781.1), and Gluconacetobacter diazotrophicus PAl 5 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP001189.1","term_id":"209529865","term_text":"CP001189.1"}}CP001189.1). Genome plasticity in NCIMB 11163 is further indicated by the presence of a type IV secretion system on the 79-kb island, syntenous to the Agrobacterium tumefaciens Ti (IncRh1) conjugal trb system (4), and also by multiple transposase and phage-related genes.In plasmids, housekeeping genes implicated in replication, active partitioning, and plasmid addiction are recognized, as well as genes involved in metabolism, transport, regulation, transposition, and DNA modification. Most notably, p11163_1 bears an arsenical resistance operon inserted in a type II secretion locus, whereas p11163_2, otherwise homologous to the 41-kb ZM4 plasmid (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY057845","term_id":"26983909","term_text":"AY057845"}}AY057845), harbors a unique ca. 12-kb CRISPR insertion that interrupts nucleotide colinearity with the aforementioned replicon.     

Mitochondrial gene sequences alone or combined with ITS region sequences provide firm molecular criteria for the classification of Lecanicillium species

The recent revision of Verticillium sect. Prostrata led to the introduction of the genus Lecanicillium, which comprises the majority of the entomopathogenic strains. Sixty-five strains previously classified as Verticillium lecanii or Verticillium sp. from different geographical regions and hosts were examined and their phylogenetic relationships were determined using sequences from three mitochondrial (mt) genes [the small rRNA subunit (rns), the NADH dehydrogenase subunits 1 (nad1) and 3 (nad3)] and the ITS region. In general, single gene phylogenetic trees differentiated and placed the strains examined in well-supported (by BS analysis) groups of L. lecanii, L. longisporum, L. muscarium, and L. nodulosum, although in some cases a few uncertainties still remained. nad1 was the most informative single gene in phylogenetic analyses and was also found to contain group I introns with putative open reading frames (ORFs) encoding for GIY–YIG endonucleases. The combined use of mt gene sequences resolved taxonomic uncertainties arisen from ITS analysis and, alone or in combination with ITS sequences, helped in placing uncharacterised Verticillium lecanii and Verticillium sp. firmly into Lecanicillium species. Combined gene data from all the mt genes and all the mt genes and the ITS region together, were very similar. Furthermore, a relaxed correlation with host specificity—at least for Homoptera—was indicated for the rns and the combined mt gene sequences. Thus, the usefulness of mt gene sequences as a convenient molecular tool in phylogenetic studies of entomopathogenic fungi was demonstrated.     

Differentiation of mitochondrial subgroups in the Verticillium lecanii species complex

Presumptive mitochondrial DNA (mtDNA) restriction fragment length polymorphisms (RFLPs) were obtained from 54 fungal isolates identified as Verticillium lecanii, V. psalliotae or ' V. lecanii -like'. Analysis of the mtDNA RFLPs showed 20 different patterns, indicating considerable genetic variation within the V. lecanii species complex. There was no direct correlation between host and mtDNA pattern, or between mtDNA patterns and previously described isoenzyme-defined specific groups. Isolates from tropical and subtropical areas showed considerable variation in genotypes, while isolates from temperate regions appeared less variable.     

Phylogenetic and biogeographic implications inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2 of the entomopathogenic fungi <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">B. brongniartii</Emphasis>

Background  

The entomopathogenic fungi of the genus Beauveria are cosmopolitan with a variety of different insect hosts. The two most important species, B. bassiana and B. brongniartii, have already been used as biological control agents of pests in agriculture and as models for the study of insect host - pathogen interactions. Mitochondrial (mt) genomes, due to their properties to evolve faster than the nuclear DNA, to contain introns and mobile elements and to exhibit extended polymorphisms, are ideal tools to examine genetic diversity within fungal populations and genetically identify a species or a particular isolate. Moreover, mt intergenic region can provide valuable phylogenetic information to study the biogeography of the fungus.     

The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation

The mitochondrial genome (mtDNA) of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, with a total size of 24,673 bp, was one of the smallest known mtDNAs of Pezizomycotina. It contained the 14 typical genes coding for proteins related to oxidative phosphorylation, the two rRNA genes, a single intron that harbored an intronic ORF coding for a putative ribosomal protein (rps) within the large rRNA gene (rnl), and a set of 24 tRNA genes which recognized codons for all amino acids, except proline and valine. Gene order comparison with all known mtDNAs of Sordariomycetes illustrated a highly conserved genome organization for all the protein- and rRNA-coding genes, as well as three clusters of tRNA genes. By considering all mitochondrial essential protein-coding genes as one unit a phylogenetic study of these small genomes strongly supported the common evolutionary course of Sordariomycetes (100% bootstrap support) and highlighted the advantages of analyzing small genomes (mtDNA) over single genes. In addition, comparative analysis of three intergenic regions demonstrated sequence variability that can be exploited for intra- and inter-specific identification of Metarhizium. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Assessing the cytotoxic and mutagenic effects of secondary metabolites produced by several fungal biological control agents with the Ames assay and the VITOTOX(®) test

The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites.