Microbial genescapes: phyletic and functional patterns of ORF distribution among prokaryotes

We have implemented a statistically based approach to comparative genomics that allows us to define and characterize distributional patterns of conceptually translated open reading frames (ORFs) at different confidence levels based on pairwise FASTA matches. In this report, we apply this methodology to nine microbial genomes, focusing particularly on phyletic and functional patterns of ORF distribution within and between the two prokaryotic domains of life, Bacteria and Archaea. We examine patterns of presence and absence of matches, determine the universal ORF set, analyze features of genome specialization between closely related organisms, and present genomic evidence for the monophyly of Archaea. These analyses illustrate how a quantitative approach to comparative genomics can illuminate questions of fundamental biological significance.     

PGAAS: a prokaryotic genome assembly assistant system

MOTIVATION: In order to accelerate the finishing phase of genome assembly, especially for the whole genome shotgun approach of prokaryotic species, we have developed a software package designated prokaryotic genome assembly assistant system (PGAAS). The approach upon which PGAAS is based is to confirm the order of contigs and fill gaps between contigs through peptide links obtained by searching each contig end with BLASTX against protein databases. RESULTS: We used the contig dataset of the cyanobacterium Synechococcus sp. strain PCC7002 (PCC7002), which was sequenced with six-fold coverage and assembled using the Phrap package. The subject database is the protein database of the cyanobacterium, Synechocystis sp. strain PCC6803 (PCC6803). We found more than 100 non-redundant peptide segments which can link at least 2 contigs. We tested one pair of linked contigs by sequencing and obtained satisfactory result. PGAAS provides a graphic user interface to show the bridge peptides and pier contigs. We integrated Primer3 into our package to design PCR primers at the adjacent ends of the pier contigs. AVAILABILITY: We tested PGAAS on a Linux (Redhat 6.2) PC machine. It is developed with free software (MySQL, PHP and Apache). The whole package is distributed freely and can be downloaded as UNIX compress file: ftp://ftp.cbi.pku.edu.cn/pub/software/unix/pgaas1.0.tar.gz. The package is being continually updated.     

Decoding the genome: a modified view

Transfer RNA’s role in decoding the genome is critical to the accuracy and efficiency of protein synthesis. Though modified nucleosides were identified in RNA 50 years ago, only recently has their importance to tRNA’s ability to decode cognate and wobble codons become apparent. RNA modifications are ubiquitous. To date, some 100 different posttranslational modifications have been identified. Modifications of tRNA are the most extensively investigated; however, many other RNAs have modified nucleosides. The modifications that occur at the first, or wobble position, of tRNA’s anticodon and those 3′-adjacent to the anticodon are of particular interest. The tRNAs most affected by individual and combinations of modifications respond to codons in mixed codon boxes where distinction of the third codon base is important for discriminating between the correct cognate or wobble codons and the incorrect near-cognate codons (e.g. AAA/G for lysine versus AAU/C asparagine). In contrast, other modifications expand wobble codon recognition, such as U·U base pairing, for tRNAs that respond to multiple codons of a 4-fold degenerate codon box (e.g. GUU/A/C/G for valine). Whether restricting codon recognition, expanding wobble, enabling translocation, or maintaining the messenger RNA, reading frame modifications appear to reduce anticodon loop dynamics to that accepted by the ribosome. Therefore, we suggest that anticodon stem and loop domain nucleoside modifications allow a limited number of tRNAs to accurately and efficiently decode the 61 amino acid codons by selectively restricting some anticodon–codon interactions and expanding others.     

Corynebacterium diphtheriae: a PTS view to the genome

We have surveyed the publicly available genome sequence of Corynebacterium diphtheriae (www.sanger.ac.uk) to identify components of the phosphotransferase system (PTS), which plays a central role in carbon metabolism in many bacteria. Three gene loci were found to contain putative pts genes. These comprise: (i) the genes of the general phosphotransferases enzyme I (ptsI) and HPr (ptsH), a fructose-specific enzyme IIABC permease (fruA), and a fructose 1-phosphate kinase (fruK); (ii) a gene that encodes an enzyme IIAB of the fructose/mannitol family, and a novel HPr-like gene, ptsF, that encodes an HPr domain fused to a domain of unknown function; (iii) and a gene for a glucose-specific enzyme IIBCA (ptsG). A search for genes that may be putative PTS-targets or that may operate in general carbon regulation revealed a possible regulatory gene encoding an antiterminator protein downstream from ptsG. Furthermore, genes were detected encoding glycerol kinase, glucose kinase, and a homologue of the global activator of carbon catabolite repression in Escherichia coli, CAP. The possible significance of these observations in carbon metabolism and the novel features of the detected genes are discussed.     

Selection and gene duplication: a view from the genome

Immediately after a gene duplication event, the duplicate genes have redundant functions. Is natural selection therefore completely relaxed after duplication? Does one gene evolve more rapidly than the other? Several recent genome-wide studies have suggested that duplicate genes are always under purifying selection and do not always evolve at the same rate.     

Trichomonas vaginalis surface proteins: a view from the genome

Surface proteins of mucosal microbial pathogens play multiple and essential roles in initiating and sustaining the colonization of the heavily defended mucosa. The protist Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa. However, little is known about its surface proteins. The recently completed draft genome sequence of T. vaginalis provides an invaluable resource to guide molecular and cellular characterization of surface proteins and to investigate their role in pathogenicity. Here, we review the existing data on T. vaginalis surface proteins and summarize some of the main findings from the recent in silico characterization of its candidate surface proteins.     

Principles of prokaryotic genome organization]

The peculiarities of bacterial chromosome organization are discussed, based mainly on the data on Escherichia coli. Highly important for bacterial genome organization is its division into two approx. equal half-genomes undergoing periodically "exchanges" of some kind displayed as continuous inversions including the oriC region of replication initiation. It is believed that short oligonucleotides are comprised in either of half-genomes. The former are predominantly oriented as direct repeats, which ensures the possibility of formation of tandem duplications consisting of identical genes--under conditions when selection for enhancing functions of corresponding genes takes place. Multiple tandem duplications capable of excision of plasmatic gene copies seem to initiate horizontal gene transfer in bacteria. Tandem gene duplications are probably being formed in the process of bacterial genetic recombination as well, when, as a result of non-equal crossing over, gene alleles derived from different strains are united into a tandem.     

Concept of a bacterial species and the evolution of the prokaryotic genome

The present concepts of evolution and species delineation in prokaryotes are considered. Recently a considerable extension of knowledge on the processes of microevolution of medically significant bacteria was noted alongside with the importance of horizontal and lateral transfer of genes. The phylophenetic concept of species was considered in detail. The inclusion of the ecological criterion into a phylophenetic concept of a species is supposed to facilitate the development of more adequate notion on the evolution of bacteria, the improvement of species delineation in prokaryotes, their classification and nomenclature.     

Building a genome engineering toolbox in nonmodel prokaryotic microbes

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO₂, and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop “design rules” for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems.     

The human genome: a view from above.

The genome of vertebrates (and of eukaryotes in general) is not simply formed by genes that are randomly scattered over vast expanses of "junk DNA", but is organized in a system which obeys precise rules, that amount to a genomic code. Moreover, genes are concentrated in the chromosomal regions which are the richest in G (guanine) and C (cytosine) and seem to correspond to the telomeric regions of certain chromosome arms (T-bands). The study of the genome organization in different vertebrate classes allowed us to approach in a novel way a number of fundamental problems of genome evolution.     

Prophage Finder: a prophage loci prediction tool for prokaryotic genome sequences

Prophage loci often remain under-annotated or even unrecognized in prokaryotic genome sequencing projects. A PHP application, Prophage Finder, has been developed and implemented to predict prophage loci, based upon clusters of phage-related gene products encoded within DNA sequences. This application provides results detailing several facets of these clusters to facilitate rapid prediction and analysis of prophage sequences. Prophage Finder was tested using previously annotated prokaryotic genomic sequences with manually curated prophage loci as benchmarks. Additional analyses from Prophage Finder searches of several draft prokaryotic genome sequences are available through the Web site (http://bioinformatics.uwp.edu/~phage/DOEResults.php) to illustrate the potential of this application.     

Microbial genome evolution: sources of variability

Comparative genome analyses of close relatives have yielded exciting insight into the sources of microbial genome variability with respect to gene content, gene order and evolution of genes with unknown functions. The genomes of free-living bacteria often carry phages and repetitive sequences that mediate genomic rearrangements in contrast to the small genomes of obligate host-associated bacteria. This suggests that genomic stability correlates with the genomic content of repeated sequences and movable genetic elements, and thereby with bacterial lifestyle. Genes with unknown functions present in a single species tend to be shorter than conserved, functional genes, indicating that the fraction of unique genes in microbial genomes has been overestimated.     

Complex prokaryotic genome structure: rapid evolution of chromosome II

Although many bacteria with two chromosomes have been sequenced, the roles of such complex genome structuring are still unclear. To uncover levels of chromosome I (CI) and chromosome II (CII) sequence divergence, Mauve 2.2.0 was used to align the CI- and CII-specific sequences of bacteria with complex genome structuring in two sets of comparisons: the first set was conducted among the CI and CII of bacterial strains of the same species, while the second set was conducted among the CI and CII of species in Alphaproteobacteria that possess two chromosomes. The analyses revealed a rapid evolution of CII-specific DNA sequences compared with CI-specific sequences in a majority of organisms. In addition, levels of protein divergence between CI-specific and CII-specific genes were determined using phylogenetic analyses and confirmed the DNA alignment findings. Analysis of synonymous and nonsynonymous substitutions revealed that the structural and functional constraints on CI and CII genes are not significantly different. Also, horizontal gene transfer estimates in selected organisms demonstrated that CII in many species has acquired higher levels of horizontally transferred segments than CI. In summary, rapid evolution of CII may perform particular roles for organisms such as aiding in adapting to specialized niches.     

Microbial macroecology: highly structured prokaryotic soil assemblages in a tropical deciduous forest

Aim To assess the hypothesis that free‐living prokaryotes show a pattern of ‘no biogeography’ by examining the scaling of soil prokaryotic diversity and by comparing it with other groups’ biogeographical patterns. Location Two sites in the tropical deciduous forest of Chamela, Jalisco, on the western coast of Mexico. Methods We examined the diversity and distribution of soil prokaryotes in two 8 × 8 m quadrats divided in such manner that we could sample at four spatial scales. Restriction fragment length polymorphisms of 16S rRNA genes were used to define operational taxonomic units (OTUs) that we used in lieu of species to assess diversity. Results We found highly structured species assemblages that allowed us to reject multiple predictions of the hypothesis that soil bacteria show ‘no biogeography’. The frequency distribution of range size (measured as the occupancy of quadrats) of OTUs followed a hollow curve similar to that of vertebrates on continents. Assemblages showed high levels of beta diversity and a non‐random nested pattern of diversity. OTU diversity scaled with area followed a power function with slopes z = 0.42 and 0.47. Main conclusions We demonstrate a non‐ubiquitous dispersal for soil prokaryotes, which suggests a complex biogeography similar to that found for terrestrial vertebrates.