Profiling the Membrane and Glycosaminoglycan-Binding Proteomes of Moraxella catarrhalis.

Moraxella catarrhalis, a Gram-negative bacterium, is an important respiratory pathogen causing acute otitis media and exacerbations of chronic obstructive pulmonary disease. Adhesion of the pathogen to human epithelial cells is mediated via bacterial membrane adhesin proteins. To identify the surface proteome of Moraxella catarrhalis, we applied different membrane protein extraction methods in combination with different proteomic technologies. Proteins from preparations of outer membrane vesicles and from carbonate extractions were analyzed using either a gel-based nano-HPLC-MS/MS technique or 2D-LC-MS/MS. Furthermore, because glycosaminoglycans (GAGs) play an important role for microbial entry into human cells, the GAG-binding membranome of Moraxella catarrhalis was investigated using a glycan-based pull-down approach. By these means, potential vaccine protein candidates that were previously selected by the ANTIGENome technology were confirmed, but importantly also novel proteins were identified as candidates.

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence.

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.

Species Identification in Malaise Trap Samples by DNA Barcoding Based on NGS Technologies and a Scoring Matrix.

The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection. Aliquots of each ordinal-level DNA extract were combined to roughly simulate a DNA extract from a non-sorted malaise sample. Each DNA extract was amplified using four primer sets targeting the CO1-5' fragment. The resulting PCR products (150-400bp) were sequenced separately on an Illumina Mi-SEQ platform, resulting in 1.5 million sequences and 5,500 clusters (coverage ≥10; CD-HIT-EST, 98%). Using a total of 120,000 DNA barcodes of identified, Central European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded from BOLD we established a reference sequence database for a local CUSTOM BLAST. This allowed us to identify 529 Barcode Index Numbers (BINs) from our sequence clusters derived from pooled Malaise trap samples. We introduce a scoring matrix based on the sequence match percentages of each amplicon in order to gain plausibility for each detected BIN, leading to 390 high score BINs in the sorted samples; whereas 268 of these high score BINs (69%) could be identified in the combined sample. The results indicate that a time consuming presorting process will yield approximately 30% more high score BINs compared to the non-sorted sample in our case. These promising results indicate that a fast, efficient and reliable analysis of next generation data from malaise trap samples can be achieved using this pipeline.

Complete genome sequence of DSM 30083(T), the type strain (U5/41(T)) of Escherichia coli, and a proposal for delineating subspecies in microbial taxonomy.

Although Escherichia coli is the most widely studied bacterial model organism and often considered to be the model bacterium per se, its type strain was until now forgotten from microbial genomics. As a part of the G enomic E ncyclopedia of B acteria and A rchaea project, we here describe the features of E. coli DSM 30083(T) together with its genome sequence and annotation as well as novel aspects of its phenotype. The 5,038,133 bp containing genome sequence includes 4,762 protein-coding genes and 175 RNA genes as well as a single plasmid. Affiliation of a set of 250 genome-sequenced E. coli strains, Shigella and outgroup strains to the type strain of E. coli was investigated using digital DNA:DNA-hybridization (dDDH) similarities and differences in genomic G+C content. As in the majority of previous studies, results show Shigella spp. embedded within E. coli and in most cases forming a single subgroup of it. Phylogenomic trees also recover the proposed E. coli phylotypes as monophyla with minor exceptions and place DSM 30083(T) in phylotype B2 with E. coli S88 as its closest neighbor. The widely used lab strain K-12 is not only genomically but also physiologically strongly different from the type strain. The phylotypes do not express a uniform level of character divergence as measured using dDDH, however, thus an alternative arrangement is proposed and discussed in the context of bacterial subspecies. Analyses of the genome sequences of a large number of E. coli strains and of strains from > 100 other bacterial genera indicate a value of 79-80% dDDH as the most promising threshold for delineating subspecies, which in turn suggests the presence of five subspecies within E. coli.

Integrated mapping, chromosomal sequencing and sequence analysis of Cryptosporidium parvum.

The apicomplexan Cryptosporidium parvum is one of the most prevalent protozoan parasites of humans. We report the physical mapping of the genome of the Iowa isolate, sequencing and analysis of chromosome 6, and approximately 0.9 Mbp of sequence sampled from the remainder of the genome. To construct a robust physical map, we devised a novel and general strategy, enabling accurate placement of clones regardless of clone artefacts. Analysis reveals a compact genome, unusually rich in membrane proteins. As in Plasmodium falciparum, the mean size of the predicted proteins is larger than that in other sequenced eukaryotes. We find several predicted proteins of interest as potential therapeutic targets, including one exhibiting similarity to the chloroquine resistance protein of Plasmodium. Coding sequence analysis argues against the conventional phylogenetic position of Cryptosporidium and supports an earlier suggestion that this genus arose from an early branching within the Apicomplexa. In agreement with this, we find no significant synteny and surprisingly little protein similarity with Plasmodium. Finally, we find two unusual and abundant repeats throughout the genome. Among sequenced genomes, one motif is abundant only in C. parvum, whereas the other is shared with (but has previously gone unnoticed in) all known genomes of the Coccidia and Haemosporida. These motifs appear to be unique in their structure, distribution and sequences.

Illuminating the evolutionary history of chlamydiae.

Chlamydiae are the major cause of preventable blindness and sexually transmitted disease. Genome analysis of a chlamydia-related symbiont of free-living amoebae revealed that it is twice as large as any of the pathogenic chlamydiae and had few signs of recent lateral gene acquisition. We showed that about 700 million years ago the last common ancestor of pathogenic and symbiotic chlamydiae was already adapted to intracellular survival in early eukaryotes and contained many virulence factors found in modern pathogenic chlamydiae, including a type III secretion system. Ancient chlamydiae appear to be the originators of mechanisms for the exploitation of eukaryotic cells.

The complete genome sequence of the carcinogenic bacterium Helicobacter hepaticus.

Helicobacter hepaticus causes chronic hepatitis and liver cancer in mice. It is the prototype enterohepatic Helicobacter species and a close relative of Helicobacter pylori, also a recognized carcinogen. Here we report the complete genome sequence of H. hepaticus ATCC51449. H. hepaticus has a circular chromosome of 1,799,146 base pairs, predicted to encode 1,875 proteins. A total of 938, 953, and 821 proteins have orthologs in H. pylori, Campylobacter jejuni, and both pathogens, respectively. H. hepaticus lacks orthologs of most known H. pylori virulence factors, including adhesins, the VacA cytotoxin, and almost all cag pathogenicity island proteins, but has orthologs of the C. jejuni adhesin PEB1 and the cytolethal distending toxin (CDT). The genome contains a 71-kb genomic island (HHGI1) and several genomic islets whose G+C content differs from the rest of the genome. HHGI1 encodes three basic components of a type IV secretion system and other virulence protein homologs, suggesting a role of HHGI1 in pathogenicity. The genomic variability of H. hepaticus was assessed by comparing the genomes of 12 H. hepaticus strains with the sequenced genome by microarray hybridization. Although five strains, including all those known to have caused liver disease, were indistinguishable from ATCC51449, other strains lacked between 85 and 229 genes, including large parts of HHGI1, demonstrating extensive variation of genome content within the species.