Bacterial tubulin distinct loop sequences and primitive assembly properties support its origin from a eukaryotic tubulin ancestor.

The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic αß-tubulin but, strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either α- or ß-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide-binding loops are more conserved, and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic cytosolic chaperonin, which is absent from the Prosthecobacter dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than αß-tubulin, forming pairs of protofilaments that coalesce into bundles instead of microtubules, and it lacks the ability to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like ß-tubulin. BtubA/B chimera bearing the ß-tubulin loops M, H1-S2, and S9-S10 in BtubB fold, assemble, and have reduced GTPase activity. However, introduction of the α-tubulin loop S9-S10 with its unique eight-residue insertion impaired folding. From the sequence analyses, its primitive assembly features, and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding α- and ß-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution.

Genome sequence of the fish pathogen Renibacterium salmoninarum suggests reductive evolution away from an environmental Arthrobacter ancestor.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of R. salmoninarum ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The R. salmoninarum genome has extended regions of synteny to the Arthrobacter sp. strain FB24 and Arthrobacter aurescens TC1 genomes, but it is approximately 1.9 Mb smaller than both Arthrobacter genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding beta-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into R. salmoninarum evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.

The Wolbachia genome of Brugia malayi: endosymbiont evolution within a human pathogenic nematode.

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.

Monitoring of transcriptional regulation in Pichia pastoris under protein production conditions.

BACKGROUND: It has become evident that host cells react to recombinant protein production with a variety of metabolic and intrinsic stresses such as the unfolded protein response (UPR) pathway. Additionally, environmental conditions such as growth temperature may have a strong impact on cell physiology and specific productivity. However, there is little information about the molecular reactions of the host cells on a genomic level, especially in context to recombinant protein secretion. For the first time, we monitored transcriptional regulation of a subset of marker genes in the common production host Pichia pastoris to gain insights into the general physiological status of the cells under protein production conditions, with the main focus on secretion stress related genes. RESULTS: Overexpression of the UPR activating transcription factor Hac1p was employed to identify UPR target genes in P. pastoris and the responses were compared to those known for Saccharomyces cerevisiae. Most of the folding/secretion related genes showed similar regulation patterns in both yeasts, whereas genes associated with the general stress response were differentially regulated. Secretion of an antibody Fab fragment led to induction of UPR target genes in P. pastoris, however not to the same magnitude as Hac1p overproduction. Overexpression of S. cerevisiae protein disulfide isomerase (PDI1) enhances Fab secretion rates 1.9 fold, but did not relief UPR stress. Reduction of cultivation temperature from 25 degrees C to 20 degrees C led to a 1.4-fold increase of specific product secretion rate in chemostat cultivations, although the transcriptional levels of the product genes (Fab light and heavy chain) were significantly reduced at the lower temperature. A subset of folding related genes appeared to be down-regulated at the reduced temperature, whereas transcription of components of the ER associated degradation and the secretory transport was enhanced. CONCLUSION: Monitoring of genomic regulation of marker genes with the transcriptional profiling method TRAC in P. pastoris revealed similarities and discrepancies of the responses compared to S. cerevisiae. Thus our results emphasize the importance to analyse the individual hosts under real production conditions instead of drawing conclusions from model organisms. Cultivation temperature has a significant influence on specific productivity that cannot be related just to thermodynamic effects, but strongly impacts the regulation of specific genes.

The ERGO genome analysis and discovery system.

The ERGO (http://ergo.integratedgenomics.com/ERGO/) genome analysis and discovery suite is an integration of biological data from genomics, biochemistry, high-throughput expression profiling, genetics and peer-reviewed journals to achieve a comprehensive analysis of genes and genomes. Far beyond any conventional systems that facilitate functional assignments, ERGO combines pattern-based analysis with comparative genomics by visualizing genes within the context of regulation, expression profiling, phylogenetic clusters, fusion events, networked cellular pathways and chromosomal neighborhoods of other functionally related genes. The result of this multifaceted approach is to provide an extensively curated database of the largest available integration of genomes, with a vast collection of reconstructed cellular pathways spanning all domains of life. Although access to ERGO is provided only under subscription, it is already widely used by the academic community. The current version of the system integrates 500 genomes from all domains of life in various levels of completion, 403 of which are available for subscription.

The genome of Syntrophus aciditrophicus: life at the thermodynamic limit of microbial growth.

Biochemically, the syntrophic bacteria constitute the missing link in our understanding of anaerobic flow of carbon in the biosphere. The completed genome sequence of Syntrophus aciditrophicus SB, a model fatty acid- and aromatic acid-degrading syntrophic bacterium, provides a glimpse of the composition and architecture of the electron transfer and energy-transducing systems needed to exist on marginal energy economies of a syntrophic lifestyle. The genome contains 3,179,300 base pairs and 3,169 genes where 1,618 genes were assigned putative functions. Metabolic reconstruction of the gene inventory revealed that most biosynthetic pathways of a typical Gram-negative microbe were present. A distinctive feature of syntrophic metabolism is the need for reverse electron transport; the presence of a unique Rnf-type ion-translocating electron transfer complex, menaquinone, and membrane-bound Fe-S proteins with associated heterodisulfide reductase domains suggests mechanisms to accomplish this task. Previously undescribed approaches to degrade fatty and aromatic acids, including multiple AMP-forming CoA ligases and acyl-CoA synthetases seem to be present as ways to form and dissipate ion gradients by using a sodium-based energy strategy. Thus, S. aciditrophicus, although nutritionally self-sufficient, seems to be a syntrophic specialist with limited fermentative and respiratory metabolism. Genomic analysis confirms the S. aciditrophicus metabolic and regulatory commitment to a nonconventional mode of life compared with our prevailing understanding of microbiology.

Genome analysis of F. nucleatum sub spp vincentii and its comparison with the genome of F. nucleatum ATCC 25586.

We present the draft genome sequence and its analysis for Fusobacterium nucleatum sub spp. vincentii (FNV), and compare that genome with F. nucleatum ATCC 25586 (FN). A total of 441 FNV open reading frames (ORFs) with no orthologs in FN have been identified. Of these, 118 ORFs have no known function and are unique to FNV, whereas 323 ORFs have functional orthologs in other organisms. In addition to the excretion of butyrate, H2S and ammonia-like FN, FNV has the additional capability to excrete lactate and aminobutyrate. Unlike FN, FNV is likely to incorporate galactopyranose, galacturonate, and sialic acid into its O-antigen. It appears to transport ferrous iron by an anaerobic ferrous transporter. Genes for eukaryotic type serine/threonine kinase and phosphatase, transpeptidase E-transglycosylase Pbp1A are found in FNV but not in FN. Unique ABC transporters, cryptic phages, and three types of restriction-modification systems have been identified in FNV. ORFs for ethanolamine utilization, thermostable carboxypeptidase, gamma glutamyl-transpeptidase, and deblocking aminopeptidases are absent from FNV. FNV, like FN, lacks the classical catalase-peroxidase system, but thioredoxin/glutaredoxin enzymes might alleviate oxidative stress. Genes for resistance to antibiotics such as acriflavin, bacitracin, bleomycin, daunorubicin, florfenicol, and other general multidrug resistance are present. These capabilities allow Fusobacteria to survive in a mixed culture in the mouth.

Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586.

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H(2)S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth.

Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis.

Bacillus cereus is an opportunistic pathogen causing food poisoning manifested by diarrhoeal or emetic syndromes. It is closely related to the animal and human pathogen Bacillus anthracis and the insect pathogen Bacillus thuringiensis, the former being used as a biological weapon and the latter as a pesticide. B. anthracis and B. thuringiensis are readily distinguished from B. cereus by the presence of plasmid-borne specific toxins (B. anthracis and B. thuringiensis) and capsule (B. anthracis). But phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B. cereus, B. anthracis and B. thuringiensis are varieties of the same species or different species. Here we report the sequencing and analysis of the type strain B. cereus ATCC 14579. The complete genome sequence of B. cereus ATCC 14579 together with the gapped genome of B. anthracis A2012 enables us to perform comparative analysis, and hence to identify the genes that are conserved between B. cereus and B. anthracis, and the genes that are unique for each species. We use the former to clarify the phylogeny of the cereus group, and the latter to determine plasmid-independent species-specific markers.

Draft sequencing and comparative genomics of Xylella fastidiosa strains reveal novel biological insights.

Draft sequencing is a rapid and efficient method for determining the near-complete sequence of microbial genomes. Here we report a comparative analysis of one complete and two draft genome sequences of the phytopathogenic bacterium, Xylella fastidiosa, which causes serious disease in plants, including citrus, almond, and oleander. We present highlights of an in silico analysis based on a comparison of reconstructions of core biological subsystems. Cellular pathway reconstructions have been used to identify a small number of genes, which are likely to reside within the draft genomes but are not captured in the draft assembly. These represented only a small fraction of all genes and were predominantly large and small ribosomal subunit protein components. By using this approach, some of the inherent limitations of draft sequence can be significantly reduced. Despite the incomplete nature of the draft genomes, it is possible to identify several phage-related genes, which appear to be absent from the draft genomes and not the result of insufficient sequence sampling. This region may therefore identify potential host-specific functions. Based on this first functional reconstruction of a phytopathogenic microbe, we spotlight an unusual respiration machinery as a potential target for biological control. We also predicted and developed a new defined growth medium for Xylella.

The genome sequence of the facultative intracellular pathogen Brucella melitensis.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.

Whole-genome comparative analysis of three phytopathogenic Xylella fastidiosa strains.

Xylella fastidiosa (Xf) causes wilt disease in plants and is responsible for major economic and crop losses globally. Owing to the public importance of this phytopathogen we embarked on a comparative analysis of the complete genome of Xf pv citrus and the partial genomes of two recently sequenced strains of this species: Xf pv almond and Xf pv oleander, which cause leaf scorch in almond and oleander plants, respectively. We report a reanalysis of the previously sequenced Xf 9a5c (CVC, citrus) strain and the two "gapped" Xf genomes revealing ORFs encoding critical functions in pathogenicity and conjugative transfer. Second, a detailed whole-genome functional comparison was based on the three sequenced Xf strains, identifying the unique genes present in each strain, in addition to those shared between strains. Third, an "in silico" cellular reconstruction of these organisms was made, based on a comparison of their core functional subsystems that led to a characterization of their conjugative transfer machinery, identification of potential differences in their adhesion mechanisms, and highlighting of the absence of a classical quorum-sensing mechanism. This study demonstrates the effectiveness of comparative analysis strategies in the interpretation of genomes that are closely related.

The genome of Methanosarcina mazei: evidence for lateral gene transfer between bacteria and archaea.

The Archaeon Methanosarcina mazei and related species are of great ecological importance as they are the only organisms fermenting acetate, methylamines and methanol to methane, carbon dioxide and ammonia (in case of methylamines). Since acetate is the precursor of 60% of the methane produced on earth these organisms contribute significantly to the production of this greenhouse gas, e.g. in rice paddies. The 4,096,345 base pairs circular chromosome of M. mazei is more than twice as large as the genomes of the methanogenic Archaea currently completely sequenced (Bult et al., 1996; Smith et al., 1997). 3,371 open reading frames (ORFs) were identified. Based on currently available sequence data 376 of these ORFs are Methanosarcina-specific and 1,043 ORFs find their closest homologue in the bacterial domain. 544 of these ORFs reach significant similarity values only in the bacterial domain. They include 56 of the 102 transposases, and proteins involved in gluconeogenesis, proline biosynthesis, transport processes, DNA-repair, environmental sensing, gene regulation, and stress response. Striking examples are the occurrence of the bacterial GroEL/GroES chaperone system and the presence of tetrahydrofolate-dependent enzymes. These findings might indicate that lateral gene transfer has played an important evolutionary role in forging the physiology of this metabolically versatile methanogen.