Timothy D. Read

The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria

Bacillus anthracis is an endospore-forming bacterium that causes inhalational anthrax. Key virulence genes are found on plasmids (extra-chromosomal, circular, double-stranded DNA molecules) pXO1 (ref. 2) and pXO2 (ref. 3). To identify additional genes that might contribute to virulence, we analysed the complete sequence of the chromosome of B. anthracis Ames (about 5.23 megabases). We found several chromosomally encoded proteins that may contribute to pathogenicity—including haemolysins, phospholipases and iron acquisition functions—and identified numerous surface proteins that might be important targets for vaccines and drugs. Almost all these putative chromosomal virulence and surface proteins have homologues in Bacillus cereus, highlighting the similarity of B. anthracis to near-neighbours that are not associated with anthrax. By performing a comparative genome hybridization of 19 B. cereus and Bacillus thuringiensis strains against a B. anthracis DNA microarray, we confirmed the general similarity of chromosomal genes among this group of close relatives. However, we found that the gene sequences of pXO1 and pXO2 were more variable between strains, suggesting plasmid mobility in the group. The complete sequence of B. anthracis is a step towards a better understanding of anthrax pathogenesis.

Genome sequence of the dissimilatory metal ion-reducing bacterium Shewanella oneidensis

Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities, conferred in part by multicomponent, branched electron transport systems. Here we report the sequencing of the S. oneidensis genome, which consists of a 4,969,803–base pair circular chromosome with 4,758 predicted protein-encoding open reading frames (CDS) and a 161,613–base pair plasmid with 173 CDSs. We identified the first Shewanella lambda-like phage, providing a potential tool for further genome engineering. Genome analysis revealed 39 c-type cytochromes, including 32 previously unidentified in S. oneidensis, and a novel periplasmic [Fe] hydrogenase, which are integral members of the electron transport system. This genome sequence represents a critical step in the elucidation of the pathways for reduction (and bioremediation) of pollutants such as uranium (U) and chromium (Cr), and offers a starting point for defining this organisms complex electron transport systems and metal ion–reducing capabilities.

Complete genome sequence and comparative genomic analysis of an emerging human pathogen, serotype V Streptococcus agalactiae

The 2,160,267 bp genome sequence of Streptococcus agalactiae, the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe, is predicted to encode 2,175 genes. Genome comparisons among S. agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and the other completely sequenced genomes identified genes specific to the streptococci and to S. agalactiae. These in silico analyses, combined with comparative genome hybridization experiments between the sequenced serotype V strain 2603 V/R and 19 S. agalactiae strains from several serotypes using whole-genome microarrays, revealed the genetic heterogeneity among S. agalactiae strains, even of the same serotype, and provided insights into the evolution of virulence mechanisms.

The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts

The 3.31-Mb genome sequence of the intracellular pathogen and potential bioterrorism agent, Brucella suis, was determined. Comparison of B. suis with Brucella melitensis has defined a finite set of differences that could be responsible for the differences in virulence and host preference between these organisms, and indicates that phage have played a significant role in their divergence. Analysis of the B. suis genome reveals transport and metabolic capabilities akin to soil/plant-associated bacteria. Extensive gene synteny between B. suis chromosome 1 and the genome of the plant symbiont Mesorhizobium loti emphasizes the similarity between this animal pathogen and plant pathogens and symbionts. A limited repertoire of genes homologous to known bacterial virulence factors were identified.

Complete genome sequence of the Q-fever pathogen Coxiella burnetii.

The 1,995,275-bp genome of Coxiella burnetii, Nine Mile phase I RSA493, a highly virulent zoonotic pathogen and category B bioterrorism agent, was sequenced by the random shotgun method. This bacterium is an obligate intracellular acidophile that is highly adapted for life within the eukaryotic phagolysosome. Genome analysis revealed many genes with potential roles in adhesion, invasion, intracellular trafficking, host-cell modulation, and detoxification. A previously uncharacterized 13-member family of ankyrin repeat-containing proteins is implicated in the pathogenesis of this organism. Although the lifestyle and parasitic strategies of C. burnetii resemble that of Rickettsiae and Chlamydiae, their genome architectures differ considerably in terms of presence of mobile elements, extent of genome reduction, metabolic capabilities, and transporter profiles. The presence of 83 pseudogenes displays an ongoing process of gene degradation. Unlike other obligate intracellular bacteria, 32 insertion sequences are found dispersed in the chromosome, indicating some plasticity in the C. burnetii genome. These analyses suggest that the obligate intracellular lifestyle of C. burnetii may be a relatively recent innovation.

Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus capsulatus (Bath)

Methanotrophs are ubiquitous bacteria that can use the greenhouse gas methane as a sole carbon and energy source for growth, thus playing major roles in global carbon cycles, and in particular, substantially reducing emissions of biologically generated methane to the atmosphere. Despite their importance, and in contrast to organisms that play roles in other major parts of the carbon cycle such as photosynthesis, no genome-level studies have been published on the biology of methanotrophs. We report the first complete genome sequence to our knowledge from an obligate methanotroph, Methylococcus capsulatus (Bath), obtained by the shotgun sequencing approach. Analysis revealed a 3.3-Mb genome highly specialized for a methanotrophic lifestyle, including redundant pathways predicted to be involved in methanotrophy and duplicated genes for essential enzymes such as the methane monooxygenases. We used phylogenomic analysis, gene order information, and comparative analysis with the partially sequenced methylotroph Methylobacterium extorquens to detect genes of unknown function likely to be involved in methanotrophy and methylotrophy. Genome analysis suggests the ability of M. capsulatus to scavenge copper (including a previously unreported nonribosomal peptide synthetase) and to use copper in regulation of methanotrophy, but the exact regulatory mechanisms remain unclear. One of the most surprising outcomes of the project is evidence suggesting the existence of previously unsuspected metabolic flexibility in M. capsulatus, including an ability to grow on sugars, oxidize chemolithotrophic hydrogen and sulfur, and live under reduced oxygen tension, all of which have implications for methanotroph ecology. The availability of the complete genome of M. capsulatus (Bath) deepens our understanding of methanotroph biology and its relationship to global carbon cycles. We have gained evidence for greater metabolic flexibility than was previously known, and for genetic components that may have biotechnological potential.

Formation and Composition of the Bacillus anthracis Endospore

The endospores of Bacillus anthracis are the infectious particles of anthrax. Spores are dormant bacterial morphotypes able to withstand harsh environments for decades, which contributes to their ability to be formulated and dispersed as a biological weapon. We monitored gene expression in B. anthracis during growth and sporulation using full genome DNA microarrays and matched the results against a comprehensive analysis of the mature anthrax spore proteome. A large portion (approximately 36%) of the B. anthracis genome is regulated in a growth phase-dependent manner, and this regulation is marked by five distinct waves of gene expression as cells proceed from exponential growth through sporulation. The identities of more than 750 proteins present in the spore were determined by multidimensional chromatography and tandem mass spectrometry. Comparison of data sets revealed that while the genes responsible for assembly and maturation of the spore are tightly regulated in discrete stages, many of the components ultimately found in the spore are expressed throughout and even before sporulation, suggesting that gene expression during sporulation may be mainly related to the physical construction of the spore, rather than synthesis of eventual spore content. The spore also contains an assortment of specialized, but not obviously related, metabolic and protective proteins. These findings contribute to our understanding of spore formation and function and will be useful in the detection, prevention, and early treatment of anthrax. This study also highlights the complementary nature of genomic and proteomic analyses and the benefits of combining these approaches in a single study.

Strain-Specific Single-Nucleotide Polymorphism Assays for the Bacillus anthracis Ames Strain

ABSTRACT Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.

Scaffolding and validation of bacterial genome assemblies using optical restriction maps

Motivation: New, high-throughput sequencing technologies have made it feasible to cheaply generate vast amounts of sequence information from a genome of interest. The computational reconstruction of the complete sequence of a genome is complicated by specific features of these new sequencing technologies, such as the short length of the sequencing reads and absence of mate-pair information. In this article we propose methods to overcome such limitations by incorporating information from optical restriction maps. Results: We demonstrate the robustness of our methods to sequencing and assembly errors using extensive experiments on simulated datasets. We then present the results obtained by applying our algorithms to data generated from two bacterial genomes Yersinia aldovae and Yersinia kristensenii. The resulting assemblies contain a single scaffold covering a large fraction of the respective genomes, suggesting that the careful use of optical maps can provide a cost-effective framework for the assembly of genomes. Availability: The tools described here are available as an open-source package at ftp://ftp.cbcb.umd.edu/pub/software/soma Contact: mpop@umiacs.umd.edu

Identification and characterization of the gerH operon of Bacillus anthracis endospores: a differential role for purine nucleosides in germination

We identified a tri-cistronic operon, gerH, in Bacillus anthracis that is important for endospore germination triggered by two distinct germination response pathways termed inosine-His and purine-Ala. Together, the two pathways allow B. anthracis endospores a broader recognition of purines and amino acids that may be important for host-mediated germination.