Small Bacterial Genomes Research Articles

Computational Structural Genomics of a Complete Minimal Organism

Structural genomics aims to provide an experimental structure or computational model of every tractable protein in a complete genome. A considerable fraction of the genes in all sequenced genomes have no known function, and have diverged sufficiently from functionally characterized homologues that the evolutionary relationship cannot be detected from sequence alone. Determining the structure of these proteins may reveal distant homology, which can be used to infer cellular and molecular functions. The structure is also important for acquiring a detailed understanding of enzymatic catalysis and interaction with small molecule ligands and other proteins. More generally, knowledge of an increasingly complete repertoire of protein structures will aid structure prediction methods, improve understanding of protein structure, and ultimately lend insight into molecular interactions and pathways. Mycoplasma genitalium has the smallest bacterial genome, with only 480 proteins. Its close relative, Mycoplasma pneumoniae, has 677. The small size of these genomes should allow us to obtain structures or models for all tractable proteins in these genomes within 5 years. This is expected to yield important insight into the minimal set of genes necessary for life; many genes in more complex organisms may be variations on genes in the minimal set.

Paradigms of plasmid organization.

Plasmids are extrachromosomal elements built from a selection of generally quite well understood survival and propagation functions, including replication, partitioning, multimer resolution, post-segregational killing and conjugative transfer. Evolution has favoured clustering of these modules to form plasmid cores or backbones. Co-regulation of these core genes can also provide advantages that favour retention of the backbone organization. Tumour-inducing and symbiosis-determining plasmids appear to co-regulate replication and transfer in response to cell density, both being stimulated at high density. Broad-host-range plasmids of the IncP-1 group, on the other hand, have autogenous control circuits, which allow a burst of expression during establishment in a new host, but a minimum of expression during maintenance. The lessons that plasmids have for clustering and co-regulation may explain the logic and organization of many small bacterial genomes currently being investigated.

Structure-based assignment of the biochemical function of a hypothetical protein: a test case of structural genomics.

Many small bacterial, archaebacterial, and eukaryotic genomes have been sequenced, and the larger eukaryotic genomes are predicted to be completely sequenced within the next decade. In all genomes sequenced to date, a large portion of these organisms' predicted protein coding regions encode polypeptides of unknown biochemical, biophysical, and/or cellular functions. Three-dimensional structures of these proteins may suggest biochemical or biophysical functions. Here we report the crystal structure of one such protein, MJ0577, from a hyperthermophile, Methanococcus jannaschii, at 1.7-A resolution. The structure contains a bound ATP, suggesting MJ0577 is an ATPase or an ATP-mediated molecular switch, which we confirm by biochemical experiments. Furthermore, the structure reveals different ATP binding motifs that are shared among many homologous hypothetical proteins in this family. This result indicates that structure-based assignment of molecular function is a viable approach for the large-scale biochemical assignment of proteins and for discovering new motifs, a basic premise of structural genomics.

Sequencing and analysis of bacterial genomes

The complete sequences of two small bacterial genomes have recently become available, and those of several more species should follow within the next two years. Sequence comparisons show that the most bacterial proteins are highly conserved in evolution, allowing predictions to be made about the functions of most products of an uncharacterized genome. Bacterial genomes differ vastly in their gene repertoires. Although genes for components of the translation and transcription machinery, and for molecular chaperones, are typically maintained, many regulatory and metabolic systems are absent in bacteria with small genomes. Mycoplasma genitalium, with the smallest known genome of any cellular life form, lacks virtually all known regulatory genes, and its gene expression may be regulated differently than in other bacteria. Genome organization is evolutionarily labile: extensive gene shuffling leaves only very few conserved gene arrays in distantly related bacteria.

Genome analysis of Theileria parva

Recent technological developments in the field of genome analyses have advanced our knowledge of the structures of prokaryotic and eukoryotic genomes. Examples of these range from small bacterial genomes, such as Escherichia coli, to the more complex genomes of Caenorhabditis elegans, Drosophila, humans and mouse. Here, Subhash Morzona and John Young review developments in mapping the genome of on economically important protozoan parasite o f cattle, Theileria parva. This map provides a framework for more detailed analysis of the genome structure o f this organism. The methodologies developed in constructing the map also have application to the mapping of other protozoan genomes.

DNA amplification fingerprinting of bacteria.

We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng.microliters-1 and high MgCl2 levels was especially important for reproducibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.