The recent plethora of sequenced genomes has just ushered in a new era of genetics-based research. Although an impressive number of species have been, or are planned to be sequenced, the full value of such efforts will be fully accrued when patterns of variation can be discerned and annotated for many strains or isolates within a given species. As such, bacteria are an ideal place to start investigations aimed at the discernment of genome rearrangements, chromosome deletions, and horizontal transfer of foreign DNA, since these events help drive bacterial evolution. For example, genome remodeling events may cause irreversible gene loss, or add novel functionalities to an organism. Unfortunately, current approaches do not adequately identify and characterize such large-scale genomic rearrangements. The Optical Mapping System, developed in our laboratory creates high resolution maps of entire genomes, using DNA directly extracted from cells—this approach obviates the need for libraries, PCR, and probe technologies. The system uses a complex blend of single molecule technologies to enable high throughput and the construction of reliable maps. This capability has been proven by the construction and sequence comparison of 13 bacterial optical maps, and 3 parasites. Recent advances in both throughput and resolution of the Optical Mapping System has enabled genomic comparisons amongst different strains of the same species or closely related species, allowing for the pinpoint discernment of insertions, deletions and rearrangements. Comparisons of optical maps vs. in silico maps, and in silico vs. in silico maps constructed for two strains of Yersinia pestis (CO-92 biovar Orientalis and KIM), E. coli K12 and Shigella flexneri 2a, two strains of S. flexneri (2a and Y), and two strains of Rhodobacter sphaeroides (2.4.1 and ATCC 17029) have revealed regions of homology, insertion sites and a panoply of rearrangements. These results portend the wide use of Optical Mapping to uniquely provide genome structural details for a large number of strains, isolates or even closely related species, in ways that would complement direct sequence analysis.

Rhodopseudomonas palustris is a photosynthetic bacterium that can use many forms of carbon, nitrogen, and electron donors. Under anaerobic conditions it can generate energy from light and convert nitrogen gas to ammonia and hydrogen (a biofuel) by nitrogen fixation. A striking feature of the genome sequence of R. palustris CGA009 is genes encoding three different nitrogenases and the accessory proteins needed for nitrogenase assembly. AnfHDGK, nifHDK, and vnfHDGK genes encode iron (Fe)-containing, molybdenum (Mo)-containing, and vanadium (V)-containing nitrogenases. To address the question of how R. palustris differentially regulates nitrogenase gene expression, we constructed anfHnifH, anfHvnfH, and nifHvnfH double mutants and analyzed the whole genome gene expression profiles of each mutant and wild-type cells grown under nitrogen-fixing conditions. Each mutant expressed a single functional nitrogenase (Mo, V or Fe) in a minimal medium that contained molybdenum and other trace elements. Wild-type and the Mo-nitrogenase active mutant cells expressed over 150 genes at levels of 2-fold or higher when grown under nitrogen-fixing conditions as compared to when grown with ammonia. Among these were the 30 genes in the nif gene cluster, which were expressed at 5to 200-fold higher (depending on the gene) levels in cells grown under nitrogen-fixing conditions. Genes in the anf and vnf clusters were not expressed. By contrast, cells with an active Fe-nitrogenase only or an active V-nitrogenase only expressed all of the nif genes (except nifH which was deleted), all of the anf genes, and all of the vnf genes. It makes sense that nif genes would be expressed because many of them are needed for the assembly of the Feand V-nitrogenases. These results indicate that R. palustris synthesizes both its Feand V-nitrogenases in situations where it is unable to synthesize an active Mo-nitrogenase. The mechanism by which Feand V-nitrogenase gene expression is activated is not known, but does not involve relief of Mo repression.

Leishmania is a group of protozoan parasites which causes a broad spectrum of diseases resulting in widespread human suffering and death, as well as economic loss from the infection of some domestic animals and wildlife. To further understand the fundamental genomic architecture of this parasite, and to accelerate the on-going sequencing project, a whole-genome XbaI restriction map was constructed using the optical mapping system. This map supplemented traditional physical maps that were generated by fingerprinting and hybridization of cosmid and P1 clone libraries. Thirty-six optical map contigs were constructed for the corresponding known 36 chromosomes of the Leishmania major Friedlin genome. The chromosome sizes ranged from 326.9 to 2821.3 kb, with a total genome size of 34.7 Mb; the average XbaI restriction fragment was 25.3 kb, and ranged from 15.7 to 77.8 kb on a per chromosomes basis. Comparison between the optical maps and the in silico maps of sequence drawn from completed, nearly finished, or large sequence contigs showed that optical maps served several useful functions within the path to create finished sequence by: guiding aspects of the sequence assembly, identifying misassemblies, detection of cosmid or PAC clones misplacements to chromosomes, and validation of sequence stemming from varying degrees of finishing. Our results also showed the potential use of optical maps as a means to detect and characterize map segmental duplication within genomes.

ABSTRACT Modern comparative genomics has been established, in part, by the sequencing and annotation of a broad range of microbial species. To gain further insights, new sequencing efforts are now dealing with the variety of strains or isolates that gives a species definition and range; however, this number vastly outstrips our ability to sequence them. Given the availability of a large number of microbial species, new whole genome approaches must be developed to fully leverage this information at the level of strain diversity that maximize discovery. Here, we describe how optical mapping, a single-molecule system, was used to identify and annotate chromosomal alterations between bacterial strains represented by several species. Since whole-genome optical maps are ordered restriction maps, sequenced strains of Shigella flexneri serotype 2a (2457T and 301), Yersinia pestis (CO 92 and KIM), and Escherichia coli were aligned as maps to identify regions of homology and to further characterize them as possible insertions, deletions, inversions, or translocations. Importantly, an unsequenced Shigella flexneri strain (serotype Y strain AMC[328Y]) was optically mapped and aligned with two sequenced ones to reveal one novel locus implicated in serotype conversion and several other loci containing insertion sequence elements or phage-related gene insertions. Our results suggest that genomic rearrangements and chromosomal breakpoints are readily identified and annotated against a prototypic sequenced strain by using the tools of optical mapping.

Rhodobacter sphaeroides 2.4.1 is a facultative photoheterotrophic bacterium with tremendous metabolic diversity, which has significantly contributed to our understanding of the molecular genetics of photosynthesis, photoheterotrophy, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, taxis, and tetrapyrrole biosynthesis. To further understand this remarkable bacterium, and to accelerate an ongoing sequencing project, two whole-genome restriction maps (EcoRI and HindIII) of R. sphaeroides strain 2.4.1 were constructed using shotgun optical mapping. The approach directly mapped genomic DNA by the random mapping of single molecules. The two maps were used to facilitate sequence assembly by providing an optical scaffold for high-resolution alignment and verification of sequence contigs. Our results show that such maps facilitated the closure of sequence gaps by the early detection of nascent sequence contigs during the course of the whole-genome shotgun sequencing process.

ABSTRACT Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps ( Xho I and Pvu II) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the Xho I map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the Pvu II map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.