Genomics and future biological weapons: the need for preventive action by the biomedical community.

There is an increasing concern within both the scientific and security communities that the ongoing revolution in biology has great potential to be misused in offensive biological weapons programs. In light of the 11 September tragedy, we can no longer afford to be complacent about the possibility of biological terrorism. Here we review the major relevant trends in genomics research and development, and discuss how these capabilities might be misused in the design of new bioweapons. We also discuss how the breakthroughs that have come from the genomics revolution may be used to enhance detection, protection and treatment so that biological warfare agents are never used.

Autoantibodies to beta 2-adrenergic receptors: a possible cause of adrenergic hyporesponsiveness in allergic rhinitis and asthma.

Autoantibodies to beta 2-adrenergic receptors have been identified in the serum of one patient with allergic rhinitis ("hay fever") and two patients with asthma. The antibodies precipitate solubilized dog lung beta receptors in an indirect immunoprecipitation assay and inhibit the specific binding of iodine-125-labeled iodohydroxybenzylpindolol to membrane-associated receptors from dog lung, calf lung, and human placenta. Ligand binding to canine heart beta 1 receptors is not affected by the antibodies.

Global analysis of the genetic network controlling a bacterial cell cycle.

This report presents full-genome evidence that bacterial cells use discrete transcription patterns to control cell cycle progression. Global transcription analysis of synchronized Caulobacter crescentus cells was used to identify 553 genes (19% of the genome) whose messenger RNA levels varied as a function of the cell cycle. We conclude that in bacteria, as in yeast, (i) genes involved in a given cell function are activated at the time of execution of that function, (ii) genes encoding proteins that function in complexes are coexpressed, and (iii) temporal cascades of gene expression control multiprotein structure biogenesis. A single regulatory factor, the CtrA member of the two-component signal transduction family, is directly or indirectly involved in the control of 26% of the cell cycle-regulated genes.

Global transposon mutagenesis and a minimal Mycoplasma genome.

Mycoplasma genitalium with 517 genes has the smallest gene complement of any independently replicating cell so far identified. Global transposon mutagenesis was used to identify nonessential genes in an effort to learn whether the naturally occurring gene complement is a true minimal genome under laboratory growth conditions. The positions of 2209 transposon insertions in the completely sequenced genomes of M. genitalium and its close relative M. pneumoniae were determined by sequencing across the junction of the transposon and the genomic DNA. These junctions defined 1354 distinct sites of insertion that were not lethal. The analysis suggests that 265 to 350 of the 480 protein-coding genes of M. genitalium are essential under laboratory growth conditions, including about 100 genes of unknown function.

Mutation of a mutL homolog in hereditary colon cancer.

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Chromosome 2 sequence of the human malaria parasite Plasmodium falciparum.

Chromosome 2 of Plasmodium falciparum was sequenced; this sequence contains 947,103 base pairs and encodes 210 predicted genes. In comparison with the Saccharomyces cerevisiae genome, chromosome 2 has a lower gene density, introns are more frequent, and proteins are markedly enriched in nonglobular domains. A family of surface proteins, rifins, that may play a role in antigenic variation was identified. The complete sequencing of chromosome 2 has shown that sequencing of the A+T-rich P. falciparum genome is technically feasible.

Complete genome sequence of Treponema pallidum, the syphilis spirochete.

The complete genome sequence of Treponema pallidum was determined and shown to be 1,138,006 base pairs containing 1041 predicted coding sequences (open reading frames). Systems for DNA replication, transcription, translation, and repair are intact, but catabolic and biosynthetic activities are minimized. The number of identifiable transporters is small, and no phosphoenolpyruvate:phosphotransferase carbohydrate transporters were found. Potential virulence factors include a family of 12 potential membrane proteins and several putative hemolysins. Comparison of the T. pallidum genome sequence with that of another pathogenic spirochete, Borrelia burgdorferi, the agent of Lyme disease, identified unique and common genes and substantiates the considerable diversity observed among pathogenic spirochetes.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

Urban blue: A global analysis of the factors shaping people's perceptions of the marine environment and ecological engineering in harbours.

Marine harbours are the focus of a diverse range of activities and subject to multiple anthropogenically induced pressures. Support for environmental management options aimed at improving degraded harbours depends on understanding the factors which influence people's perceptions of harbour environments. We used an online survey, across 12 harbours, to assess sources of variation people's perceptions of harbour health and ecological engineering. We tested the hypotheses: 1) people living near impacted harbours would consider their environment to be more unhealthy and degraded, be more concerned about the environment and supportive of and willing to pay for ecological engineering relative to those living by less impacted harbours, and 2) people with greater connectedness to the harbour would be more concerned about and have greater perceived knowledge of the environment, and be more supportive of, knowledgeable about and willing to pay for ecological engineering, than those with less connectedness. Across twelve locations, the levels of degradation and modification by artificial structures were lower and the concern and knowledge about the environment and ecological engineering were greater in the six Australasian and American than the six European and Asian harbours surveyed. We found that people's perception of harbours as healthy or degraded, but not their concern for the environment, reflected the degree to which harbours were impacted. There was a positive relationship between the percentage of shoreline modified and the extent of support for and people's willingness to pay indirect costs for ecological engineering. At the individual level, measures of connectedness to the harbour environment were good predictors of concern for and perceived knowledge about the environment but not support for and perceived knowledge about ecological engineering. To make informed decisions, it is important that people are empowered with sufficient knowledge of the environmental issues facing their harbour and ecological engineering options.

Cloning, localization, and permanent expression of a Drosophila octopamine receptor.

A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human beta 2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor.

Systematic review of economic evaluations of laparoscopic surgery for colorectal cancer.

OBJECTIVE: Colorectal cancer is one of the most common cancers and the standard surgical treatment for this cancer is open resection (OS), but laparoscopic surgery (LS) may be an alternative treatment. In 2000, a Health Technology Assessment (HTA) review found little evidence on costs and cost-effectiveness in comparing the two methods. The evidence base has since expanded and this study systematically reviews the economic evaluations on the subject published since 2000. METHOD: Systematic review of studies reporting costs and outcomes of LS vs OS for colorectal cancer. National Health Service Economic Evaluation Database (NHS EED) methods for abstract writing were followed. Studies were summarized and incremental cost-effectiveness ratios (ICER) for common outcomes were calculated. RESULTS: Five studies met the inclusion criteria. LS generally had higher healthcare costs. Most studies reported longer operational time and shorter length of stay and similar long-term outcomes with LS vs OS. Only one outcome, complications, was common across all studies but results lacked consistency (e.g. in two studies, OS was less costly but more effective; in another study, LS was less costly but more effective; and in the further two studies, LS could potentially be cost effective depending on the decision-makers' willingness to pay for the health gain). CONCLUSION: The evidence on cost-effectiveness is not consistent. LS was generally more costly than OS. However, the effectiveness data used in individual economic evaluation were imprecise and unreliable when compared with data from systematic reviews of effectiveness. Nevertheless, short-term benefits of LS (e.g. shorter recovery) may make LS appear less costly when productivity gains are considered.

Salmonella Typhi Colonization Provokes Extensive Transcriptional Changes Aimed at Evading Host Mucosal Immune Defense During Early Infection of Human Intestinal Tissue.

Commensal microorganisms influence a variety of host functions in the gut, including immune response, glucose homeostasis, metabolic pathways and oxidative stress, among others. This study describes how Salmonella Typhi, the pathogen responsible for typhoid fever, uses similar strategies to escape immune defense responses and survive within its human host. To elucidate the early mechanisms of typhoid fever, we performed studies using healthy human intestinal tissue samples and "mini-guts," organoids grown from intestinal tissue taken from biopsy specimens. We analyzed gene expression changes in human intestinal specimens and bacterial cells both separately and after colonization. Our results showed mechanistic strategies that S. Typhi uses to rearrange the cellular machinery of the host cytoskeleton to successfully invade the intestinal epithelium, promote polarized cytokine release and evade immune system activation by downregulating genes involved in antigen sampling and presentation during infection. This work adds novel information regarding S. Typhi infection pathogenesis in humans, by replicating work shown in traditional cell models, and providing new data that can be applied to future vaccine development strategies.

Genome data: what do we learn?

Genome sequence information has continued to accumulate at a spectacular pace during the past year. Details of the sequence and gene content of human chromosome 22 were published. The sequencing and annotation of the first two Arabidopsis thaliana chromosomes was completed. The sequence of chromosome 3 from Plasmodium falciparum, the second sequenced malaria chromosome, was reported, as was that of chromosome 1 from Leishmania major. The complete genomic sequences of five microbes were reported. Approaches to using data from completely sequenced microbial genomes in phylogenetic studies are being explored, as is the application of microarrays to whole genome expression analysis.

Status of genome projects for nonpathogenic bacteria and archaea.

Since the first microbial genome was sequenced in 1995, 30 others have been completed and an additional 99 are known to be in progress. Although the early emphasis of microbial genomics was on human pathogens for obvious reasons, a significant number of sequencing projects have focused on nonpathogenic organisms, beginning with the release of the complete genome sequence of the archaeon Methanococcus jannaschii in 1996. The past 18 months have seen the completion of the genomes of several unusual organisms, including Thermotoga maritima, whose genome reveals extensive potential lateral transfer with archaea; Deinococcus radiodurans, the most radiation-resistant microorganism known; and Aeropyrum pernix, the first Crenarchaeota to be completely sequenced. Although the functional characterization of genomic data is still in its initial stages, it is likely that microbial genomics will have a significant impact on environmental, food, and industrial biotechnology as well as on genomic medicine.

Findings emerging from complete microbial genome sequences.

Sixteen microorganisms, including one eukaryote, four archaeons, and 11 eubacteria, have been completely sequenced and published. More than 50 genomes are scheduled to be completed by the year 2000. This explosive growth of information is forcing change in many scientific disciplines (e.g. bioinformatics and molecular genetics), spawning new fields, and even changing the way scientific information is used and shared. Novel, global genome sequence comparisons seem slow to appear but the infrastructure for these projects is being built, and we expect exciting developments in the near future.

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39.

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.