We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300 -400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.hole-genome sequences provide the foundation for the creation of relatively complete collections of strains carrying defined mutations in individual genes. Such libraries should facilitate the comprehensive identification of genes required for a wide range of biological processes. A nearly complete library of single-gene deletions of Saccharomyces cerevisiae has been assembled by an international consortium using a PCR-based mutagenesis approach (1). Other projects, also following a strategy of gene-by-gene disruption, are underway for Escherichia coli (E. coli genome project, www. genome.wisc.edu͞functional͞tnmutagenesis.htm), and have recently been completed for Bacillus subtilis (2).An alternative strategy for generating mutant libraries consists of ''random'' whole-genome transposon-insertion mutagenesis followed by sequence-based identification of insertion sites. The approach is cost-effective and applicable to a wide variety of microbes (3, 4). Studies with yeast, in which a collection of mutants corresponding to about one-third of the genes were represented, have illustrated that the generation of large, arrayed collections of insertion mutants is feasible (5). Other studies with bacteria have analyzed large numbers of transposon insertion mutants to identify genes essential for growth, although the mutants were analyzed within populations rather than being archived in a format allowing additional phenotypes to be examined (6)(7)(8). In this report, we describe the generation and initial phenotypic analysis of a near-saturation library of transposon insertion mutants of the opportunistic pathogen Pseudomonas aeruginos...

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Pathogenicity in Francisella tularensis subspecies

.Sequencing of the non-pathogenic Francisella tularensis sub-species novicida U112, and comparison with two pathogenic sub-species, provides insights into the evolution of pathogenicity in these species.

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. The methanogenic Archaea (methanogens) occupy a unique metabolic niche, as they produce methane, which is a useful energy source and a powerful greenhouse gas. These organisms are found in diverse anaerobic habitats, ranging from aquatic and marine sediments to sewage digesters and the rumens and large intestines of herbivores and other mammals (127). In these habitats, the degradation of organic matter results in the production of H 2 and other intermediates by fermentative organisms. By maintaining an extremely low partial pressure of H 2 , the methanogens keep fermentative pathways energetically favorable. In addition, some methanogens may occupy niches where hydrogen is produced predominately by geothermal reactions.Metabolically, methanogens are divided into those that specialize in CO 2 reduction and those that also use acetate and/or methyl compounds. The former group, the hydrogenotrophs, use H 2 as an electron donor to reduce CO 2 to methane. Many hydrogenotrophic species can substitute formate or certain low-molecular-weight alcohols and ketones for H 2 . Complete genome sequences have been published for three hydrogenotrophic methanogens, Methanocaldococcus jannaschii (13), Methanothermobacter thermautotrophicus (105), and Methanopyrus kandleri (104), all of which are thermophiles or hyperthermophiles. Of the methanogens that utilize acetate and methyl compounds, complete genome sequences have been published for two species, Methanosarcina acetivorans (26) and Methanosarcina mazei (19), both of which are mesophiles. In addition, partial sequences have been published for two psychrophiles, the hydrogenotroph Methanogenium frigidum and the methylotroph Methanolobus burtonii (97).Genome sequences of methanogens have answered many questions, but they have inspired many others. More than half of the genes in Methanocaldococcus jannaschii lack a predicted function (13), and this proportion has not declined significantly as other methanogen sequences have been determined. The proportions of genes of unknown functions, which are either homologous to other genes of unknown function...

There has been much speculation as to what role balancing selection has played in evolution. In an attempt to identify regions, such as HLA, at which polymorphism has been maintained in the human population for millions of years, we scanned the human genome for regions of high SNP density. We found 16 regions that, outside of HLA and ABO, are the most highly polymorphic regions yet described; however, evidence for balancing selection at these sites is notably lacking-indeed, whole-genome simulations indicate that our findings are expected under neutrality. We propose that (i) because it is rarely stable, longterm balancing selection is an evolutionary oddity, and (ii) when a balanced polymorphism is ancient in origin, the requirements for detection by means of SNP data alone will rarely be met.