The complete genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum)

This paper describes the genome sequence of Moorella thermoacetica (f. Clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the Wood-Ljungdahl pathway of CO and CO(2) fixation. This pathway, which is also known as the reductive acetyl-CoA pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into 3 mol of acetate and to grow autotrophically using H(2) and CO as electron donors and CO(2) as an electron acceptor. Methanogenic archaea use this pathway in reverse to grow by converting acetate into methane and CO(2). Acetogenic bacteria also couple the Wood-Ljungdahl pathway to a variety of other pathways to allow the metabolism of a wide variety of carbon sources and electron donors (sugars, carboxylic acids, alcohols and aromatic compounds) and electron acceptors (CO(2), nitrate, nitrite, thiosulfate, dimethylsulfoxide and aromatic carboxyl groups). The genome consists of a single circular 2 628 784 bp chromosome encoding 2615 open reading frames (ORFs), which includes 2523 predicted protein-encoding genes. Of these, 1834 genes (70.13%) have been assigned tentative functions, 665 (25.43%) matched genes of unknown function, and the remaining 24 (0.92%) had no database match. A total of 2384 (91.17%) of the ORFs in the M. thermoacetica genome can be grouped in orthologue clusters. This first genome sequence of an acetogenic bacterium provides important information related to how acetogens engage their extreme metabolic diversity by switching among different carbon substrates and electron donors/acceptors and how they conserve energy by anaerobic respiration. Our genome analysis indicates that the key genetic trait for homoacetogenesis is the core acs gene cluster of the Wood-Ljungdahl pathway.     

A novel genomics approach for the identification of drug targets in pathogens, with special reference to Pseudomonas aeruginosa

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We have used three-way genome comparisons to identify essential genes from Pseudomonas aeruginosa. Our approach identified 306 essential genes that may be considered as potential drug targets. The resultant analyses are in good agreement with the results of systematic gene deletion experiments. This approach enables rapid potential drug target identification, thereby greatly facilitating the search for new antibiotics. These results underscore the utility of large genomic databases for in silico systematic drug target identification in the post-genomic era.     

Metal-dependent anaerobic methane oxidation in marine sediment: Insights from marine settings and other systems

Anaerobic oxidation of methane(AOM) plays a crucial role in controlling global methane emission. This is a microbial process that relies on the reduction of external electron acceptors such as sulfate, nitrate/nitrite, and transient metal ions. In marine settings, the dominant electron acceptor for AOM is sulfate, while other known electron acceptors are transient metal ions such as iron and manganese oxides. Despite the AOM process coupled with sulfate reduction being relatively well characterized,researches on metal-dependent AOM process are few, and no microorganism has to date been identified as being responsible for this reaction in natural marine environments. In this review, geochemical evidences of metal-dependent AOM from sediment cores in various marine environments are summarized. Studies have showed that iron and manganese are reduced in accordance with methane oxidation in seeps or diffusive profiles below the methanogenesis zone. The potential biochemical basis and mechanisms for metal-dependent AOM processes are here presented and discussed. Future research will shed light on the microbes involved in this process and also on the molecular basis of the electron transfer between these microbes and metals in natural marine environments.     

Complete genome sequence of the dehalorespiring bacterium Desulfitobacterium hafniense Y51 and comparison with Dehalococcoides ethenogenes 195

Desulfitobacterium strains have the ability to dechlorinate halogenated compounds under anaerobic conditions by dehalorespiration. The complete genome of the tetrachloroethene (PCE)-dechlorinating strain Desulfitobacterium hafniense Y51 is a 5,727,534-bp circular chromosome harboring 5,060 predicted protein coding sequences. This genome contains only two reductive dehalogenase genes, a lower number than reported in most other dehalorespiring strains. More than 50 members of the dimethyl sulfoxide reductase superfamily and 30 paralogs of the flavoprotein subunit of the fumarate reductase are encoded as well. A remarkable feature of the genome is the large number of O-demethylase paralogs, which allow utilization of lignin-derived phenyl methyl ethers as electron donors. The large genome reveals a more versatile microorganism that can utilize a larger set of specialized electron donors and acceptors than previously thought. This is in sharp contrast to the PCE-dechlorinating strain Dehalococcoides ethenogenes 195, which has a relatively small genome with a narrow metabolic repertoire. A genomic comparison of these two very different strains allowed us to narrow down the potential candidates implicated in the dechlorination process. Our results provide further impetus to the use of desulfitobacteria as tools for bioremediation.     

Genomics of the evolutionary process

Comparative analysis of genome sequences has become the primary means by which functional elements are first identified, often preceding even the identification of their function. Although this approach capitalizes on the conservation of homologous functions, it has also been successful in identifying evolutionary novelties, including new genes and pathways. As I discuss here, the analysis of multiple alignments of sequences from species on a known phylogeny has provided rich detail about the heterogeneities in the process of genome changes. Inferences of positive selection acting on protein-encoding genes have provided clues about the role of adaptive evolution in the past. These methods also identify negatively selected genes, providing some clue to genes that are most likely to be mutable to a disease-causing state.     

The physics of DNA and the annotation of the Plasmodium falciparum genome

A gene identification procedure is formulated, based on large-scale structural analyses of genomic sequences. The structural property is the physical - thermal - stability of the DNA double-helix, as described by the classical helix-coil model. The analyses are detailed for the Plasmodium falciparum genome, which represents one of the most difficult cases for the gene identification problem (notably because of the extreme AT-richness of the genome). In this genome, the coding domains (either uninterrupted genes or exons in split genes) are accurately identified as regions of high thermal stability. The conclusion is based on the study of the available cloned genes, of which 17 examples are described in detail. These examples demonstrate that the physical criterion is valid for the detection of coding regions whose lengths extend from a few base pairs up to several thousand base pairs. Accordingly, the structural analyses can provide a powerful and convenient tool for the identification of complex genes in the P. falciparum genome. The limits of such a scheme are discussed. The gene identification procedure is applied to the completely sequenced chromosomes (2 and 3), and the results are compared with the database annotations. The structural analyses suggest more or less extensive revision to the annotations, and also allow new putative genes to be identified in the chromosome sequences. Several examples of such new genes are described in detail.     

Comparative genomics identifies genes shared by distantly related insect-transmitted plant pathogenic mollicutes

Phytoplasmas and spiroplasmas are distantly related insect-transmitted plant pathogens within the class Mollicutes. Genome sequencing projects of phytoplasma strain Aster Yellows-Witches' Broom (AY-WB) and Spiroplasma kunkelii are near completion. Complete genome sequences of seven obligate animal and human pathogenic mollicutes (Mycoplasma and Ureaplasma spp.), and OY phytoplasma have been reported. Putative ORFs predicted from the genome sequences of AY-WB and S. kunkelii were compared to those of the completed genomes. This resulted in identification of at least three ORFs present in AY-WB, OY and S. kunkelii but not in the obligate animal and human pathogenic mollicutes. Moreover, we identified ORFs that seemed more closely related between AY-WB and S. kunkelii than to their mycoplasma counterparts. Phylogenetic analyses using parsimony were employed to study the origin of these genes, resulting in identification of one gene that may have undergone horizontal gene transfer. The possible involvement of these genes in plant pathogenicity is discussed.     

Finding Protein-Coding Genes through Human Polymorphisms

Human gene catalogs are fundamental to the study of human biology and medicine. But they are all based on open reading frames (ORFs) in a reference genome sequence (with allowance for introns). Individual genomes, however, are polymorphic: their sequences are not identical. There has been much research on how polymorphism affects previously-identified genes, but no research has been done on how it affects gene identification itself. We computationally predict protein-coding genes in a straightforward manner, by finding long ORFs in mRNA sequences aligned to the reference genome. We systematically test the effect of known polymorphisms with this procedure. Polymorphisms can not only disrupt ORFs, they can also create long ORFs that do not exist in the reference sequence. We found 5,737 putative protein-coding genes that do not exist in the reference, whose protein-coding status is supported by homology to known proteins. On average 10% of these genes are located in the genomic regions devoid of annotated genes in 12 other catalogs. Our statistical analysis showed that these ORFs are unlikely to occur by chance.     

Gene identification using rice genome sequences

Identifying useful gene(s) is one of the most important objectives of plant geneticists. Various strategies can be used, which are based on the characteristics of plant reproduction and available technology. Rice is the first model crop whose whole genome sequence has been reported. In addition, information on the whole genome sequences of two important rice subspecies (japonica and indica rice) is also available. Rice is a self-pollinating crop and methods of artificial crossing are relatively easy to perform; such methods enable the production of numerous seeds for genetic analyses. Based on these features, a map-based cloning (i.e., positional cloning) strategy has been successfully applied over the last decade to identify rice genes. Recently, advanced next-generation sequencing (NGS) technology was used to ascertain the genome sequences of individual plants, opening up a new strategy for gene identification. This strategy has been used successfully to identify the genes responsible for certain qualitative traits in rice. However, to identify the gene(s) involved in a quantitative trait, a map-based cloning strategy is still required after quantitative trait loci analysis using NGS technology. In this review, we discuss both map-based cloning (which is still the primary strategy used to identify rice genes) and NGS-based strategies.     

In silico identification of potential therapeutic targets in the human pathogen Helicobacter pylori

Availability of genome sequences of pathogens has provided a tremendous amount of information that can be useful in drug target and vaccine target identification. One of the recently adopted strategies is based on a subtractive genomics approach, in which the subtraction dataset between the host and pathogen genome provides information for a set of genes that are likely to be essential to the pathogen but absent in the host. This approach has been used successfully in recent times to identify essential genes in Pseudomonas aeruginosa. We have used the same methodology to analyse the whole genome sequence of the human gastric pathogen Helicobacter pylori. Our analysis revealed that out of the 1590 coding sequences of the pathogen, 40 represent essential genes that have no human homolog. We have further analysed these 40 genes by the protein sequence databases to list some 10 genes whose products are possibly exposed on the pathogen surface. This preliminary work reported here identifies a small subset of the Helicobacter proteome that might be investigated further for identifying potential drug and vaccine targets in this pathogen.     

Functional features of a single chromosome arm in wheat (1AL) determined from its structure

Bread wheat (Triticum aestivum L.) is one of the most important crops globally and a high priority for genetic improvement, but its large and complex genome has been seen as intractable to whole genome sequencing. Isolation of individual wheat chromosome arms has facilitated large-scale sequence analyses. However, so far there is no such survey of sequences from the A genome of wheat. Greater understanding of an A chromosome could facilitate wheat improvement and future sequencing of the entire genome. We have constructed BAC library from the long arm of T. aestivum chromosome 1A (1AL) and obtained BAC end sequences from 7,470 clones encompassing the arm. We obtained 13,445 (89.99%) useful sequences with a cumulative length of 7.57 Mb, representing 1.43% of 1AL and about 0.14% of the entire A genome. The GC content of the sequences was 44.7%, and 90% of the chromosome was estimated to comprise repeat sequences, while just over 1% encoded expressed genes. From the sequence data, we identified a large number of sites suitable for development of molecular markers (362 SSR and 6,948 ISBP) which will have utility for mapping this chromosome and for marker assisted breeding. From 44 putative ISBP markers tested 23 (52.3%) were found to be useful. The BAC end sequence data also enabled the identification of genes and syntenic blocks specific to chromosome 1AL, suggesting regions of particular functional interest and targets for future research.     

Analysis of fragment homology among DNA sequences from cytochrome P450 families 4 and 6

Cytochrome P450s comprise a diverse superfamily of proteins that often share as little as 12% amino acid identity. Accordingly, the identification of novel gene families, subfamilies and alleles has been based primarily on ‘benchmark’ levels of global amino acid identity and, more recently, phylogenetic relatedness has been used to resolve ambiguous relationships. However, PCR-based cloning strategies have resulted in a large increase in the number of short DNA sequences, particularly among insects. Many of these fragments remain unnamed and even their gene family membership remains unknown due to the uncertainty as to whether these fragments accurately reflect the levels of sequence identity or patterns of evolutionary divergence exhibited by groups of full-length P450 sequences. As a result, the nature of P450 diversity among insects remains obscure. In this paper, P450 sequences belonging to the two major gene families, that is, families CYP4 and CYP6, are analyzed by comparing segments to full-length sequences. A parameter called the Segmental Divergence Index is used to characterize segments of P450s with respect to the degree to which they mirror the divergence of full-length sequences. The reliability of these fragments in phylogenetic analyses is also tested. The results of these analyses support the use of some commonly amplified P450 gene fragments for naming new P450 genes and for studies of P450 diversity and evolution. This revised version was published online in July 2006 with corrections to the Cover Date.