Bacterial genomics pdf

Further, when the clones from the smaller hybridization contigs were mapped to the FPC database, their positions were located within the larger contigs rather than at the ends of the physical maps. The sizes of the small hybridization contigs based on fingerprints of clones in the minimum tile were estimated to be and kb for contigs 3 and 4, respectively. In one strain of 0. Gene survey by BAC end sequencing Sequencing of BAC ends using both forward and reverse primers was performed on the 69 clones from the reduced tiles, generating a total of sequences.

The results were sorted using scripts developed at the CUGI. Overall, I 10 of BES resulted in hits during the database search. A summary of these hits describing best match accession number, gene type, organism, amino acid identity, and amino acid similarity is shown in Table 3. All of the BES identified and listed in Table 3 are sorted by function of their putative protein product.

Unidentified and unclassified sequences may represent a subset of those proteins that are specific to 0.

The E. The library has an average insert size of kb and provides an estimated 70 genome equivalents. The library is an ideal substrate for a number of genomics- based applications, including physical mapping, DNA sequencing, and the cloning of important genes and pathways involved in biodegradation. Using this new BAC library, we successfully constructed a physical map of the 0. This approach was accomplished by using the hybridization-based data as the foundation and verifying the resulting contigs by comparison to the FPC data.

The hybridization-based data were considered to be quite robust because bacterial genomes are relatively void of repetitive sequences. Repetitive DNA in eukaryotic genomes can confound a hybridization without replacement strategy by giving false physical associations among unrelated clones.

Furthermore, it is difficult to develop extensive contigs from fingerprint data without the use of molecular markers. In newly characterized bacterial genomes, there is a paucity of molecular markers available for aligning contigs. In the case of 0. It is noteworthy that although it was difficult to completely reconstruct the replicons using the fingerprint data, the physical associations accurately verified the data obtained by hybridization.

Using the 69 clones from reduced tiles, BAC end sequencing was performed to provide a gene survey. Based on an estimated genome size of 4.

Some of the strongest hits were from closely related proteobacterial genomes, such as Agrobacterium tumefaciens and various species of Rhizobium.

Although virtually nothing was previously known about gene structure in 0. However, no members of the Rhizobiaceae group, which includes 0. We have effectively demonstrated the utility of using BAC clones in bacterial genomics research. These techniques have allowed the construction of a detailed physical map combined with a gene survey of a bacterial genome for which little information currently existed. This framework approach can provide essential information related to genome structure and organization that can serve as a guide prior to a full-scale sequencing effort.

Furthermore, if resources for a full-scale genome sequencing effort were not available, this system would still provide the necessary tools needed for the cloning and analysis of important genes and whole operons or genetic pathways. This is technical contribution No. The Oligonucteotide Probe Data- base. Appl Environ Microbiol 62, Infections with the unusual human pathogens Agrobacterium species and Ochrobactrum anthropi. Clin Infect Dis 18, Nucleic Acids Res 25, Genomic mapping by anchoring ran- dom clones: A mathematical analysis.

Genomics 11, Still, even where from 1 to 4, genes per cluster that are present in radical species-founding evolutionary novelties would some strains but absent from others O. Zhaxybayeva, originate as mutations occurring within the ancestral bacter- C. D, unpublished work. From a similar ial population. In some species []. Best studied, not surprisingly, are bacteria that have such as Bacillus anthracis the depth of the pangenome become pathogens by the acquisition of novel plasmids, may have been plumbed after only a few genomes have been chromosomal genes or mobile pathogenicity islands [19], but sequenced.

For others, such as the ecologically versatile non-pathogens also evolve in this saltatory fashion. From a Streptococcus agalactiae, Tettelin et al. Island genes ognizing species as coherent natural units in the environ- appear to have been acquired in part by phage-mediated ment, namely as tight clusters of strains with very similar lateral gene transfer, and some are differentially expressed sequences for certain marker genes sometimes 16S rRNA, under light and nutrient stress.

What clearly cannot be sup- reports ported, however, is the notion that species qua ecotypes are genomically coherent. Homologous recombination in bacteria Another surprise of the past decade is that bacteria are not all deposited research asexuals lacking recombination, but that in some homolo- gous recombination is so frequent that it easily outperforms mutation as a source of strain-to-strain sequence differences [26]. The evidence for this comes from multi-locus sequence analysis MLSA based on sequences from five to seven unlinked core housekeeping genes amplified from scores or hundreds of strains of a species and, more recently, from the use of recombination detection algorithms [27] with aligned Figure 1 long segments or entire genomes from fewer strains.

As refereed research Microdiversity and diversity in gene content. Environmental surveys, using PCR amplification and sequencing of marker genes such as 16S rRNA or Dykhuizen and Green presciently observed some 15 years ago more rapidly evolving protein-coding genes and intergenic spacers, often [12], we might apply to such recombining groups something reveal microdiverse clusters of strains with closely related sequences.

In this diagram shows a hypothetical phylogenetic tree compiled from such sequences, with each cluster indicated by a set of circles of the same context the BSC would require that a bacterial species main- color.

Such a pattern of clustering by sequence might be expected if there tains genomic coherence because its members share an exclu- were process other than random divergence and extinction of lineages at sive common gene pool see Figure 2.

Different species play see Figure 2 , and has been attributed [11,23,24] to an ecotype would have separate gene pools, and diverge and adapt speciation process see text. In this context, a microdiverse cluster might generally be a species.

Comparisons of sequenced genomes for multiple through the separate fixation within them of favorable muta- interactions strains of many designated species, and of genome sizes from isolates of tions or laterally acquired genetic novelties. The different sizes of the circles represent on an exaggerated scale the diversity in genome size in closely related strains traditional model we must know first, whether biological found by such studies.

Both are in question. The minor variations in marker gene sequences frequency of homologous recombination as sequences within a microdiverse cluster of isolates from a given site diverge.

Black arrowheads indicate organisms or isolates. The crosses in a indicate the clones eliminated in the process, while the red arrows in b indicate recombination between genomes. Blue lines indicate speciation. In the ecotype periodic selection model in a , which is applicable to organisms without significant genetic recombination, favorable mutations sweep to fixation, carrying the genome in which they first occurred along, so that diversity is reduced to zero at all loci.

Accumulation of neutral mutations, prior to the next sweep, generates the sort of microdiversity illustrated in Figure 1. Gray bars are niche boundaries. In the biological species model, it is individual favorable mutations that are fixed, because recombination indicated by red arrows separates them from alleles at other loci in the genome in which they first occurred. Still, recombination at all loci will in time promote genomic coherence within populations and divergence between populations, because with time all alleles at all loci will be traceable to mutations that occurred within the population.

The gray block indicates a barrier to recombination. But some agents of machinery. More interestingly, it should also vary between bacterial gene transfer plasmids and conjugation machin- genes because of their different rates of sequence diver- ery are highly promiscuous, mobilizing DNA transfer gence.

And it does vary within species, thanks to mutations between phyla or even across domain boundaries: in the mismatch repair system, which can increase homolo- Escherichia coli can in fact conjugate with yeast [29]! This book provides an in-depth analysis of the mechanisms and biological consequences of genome rearrangements in bacteria. Science Posted on Author : Charles J. Medical Posted on Due to the shorter read length of the pyrosequencing platform, a specific assembler optimized on shorter read lengths is needed, because many existing assembly algorithms have been implemented and optimized for mate-paired Sanger-based reads, and thus do not perform well on short single reads.

Due to the shorter read length, a higher coverage of about 20—30 fold is needed to get a proper genome assembly with pyrosequencing reads. Anyhow, repetitive elements, which are longer than the average read length still constitute a major challenge during the genome assembly and finishing process.

As a consequence of this currently most sequencing centers use a high coverage of pyrosequencing reads in combination with low-coverage Sanger sequencing reads. They first assemble only reads using Newbler and later transform large Newbler contigs longer than bp into overlapping pseudo-reads modeling typical Sanger reads Chaisson and Pevzner, As examples for a whole genome sequencing strategy combining next- generation sequencing with traditional Sanger sequencing the genome projects of lipid- requiring, urealytic bacterium Corynebacterium urealyticum DSM Tauch et al.

The 2,, bp genome sequence of C. Altogether the Genome Sequencer GS 20 system Roche, Penzberg, Germany delivered , pyrosequence reads with 68,, detected bases that were used for de novo genome assembly with the Newbler Assembler Roche, Penzberg, Germany. By applying a contig length cut-off of bp, a total of 2,, bp were assembled into 69 contigs Tauch et al.

For the manually driven sequencing and linking phase, a large insert fosmid library was constructed and terminal DNA sequences of large genomic fosmid inserts were determined with an ABI xl DNA analyzer applying dye terminator chemistry Tauch et al. Both the terminal fosmid insert sequences and the pseudo phd files of the Newbler derived contigs were combined using the Phred Phrap programs included in the Consed finishing tool Gordon et al.

The remaining gaps in the genome sequence were closed by primer walking on selected fosmid templates. In total, walking reads on fosmid DNA templates were necessary to close all gaps in the C. Assembly of the pyrosequencing reads revealed one small contig bp and six large contigs, of which the smallest one bp represented the consensus sequence of the ribosomal RNA rrn operons of C.

The genomic sequence contigs could be ordered into a circular chromosome by performing synteny analyses at the protein level with the genome of Corynebacterium glutamicum ATCC Kalinowski et al.

The assembled genomic contigs were linked by long-range PCR assays, thereby indicating a tandem duplication of the small contig and the presence of three rrn copies in the genome of C. This low number of genomic contigs demonstrates the ideal case of a genome sequencing project where all of the gaps could be explained by one duplicated sequence and the presence of multiple copies of three rrn operons only.

This ideal sequencing result is a very rare event, because many genomes carry many more repetitive or short duplicated sequence elements. Usually, this process consists of two steps. Based on the finished high-quality genome sequence, potentially functional regions are predicted before — in the second step — the assignment of biological functions to every potential gene is performed.

In this section, both the steps are described in detail. Because of their coding capacity, the protein coding regions in bacterial genomes typically exhibit certain characteristic sequence properties that distinguish them from noncoding parts of the sequence. Additionally, sequence homology of a potential coding region to genes of other organisms is a useful property for gene identification.

Ab initio or intrinsic gene-finders exclusively use the statistical analysis of sequence properties e. Other systems, like Critica Coding Region Identification Tool Invoking Comparative Analysis Badger and Olsen, , for instance, use additionally homology-based information for gene prediction. There- fore, this combined approach is called extrinsic gene-finding. Modern genome annotation platforms like the GenDB system Meyer et al.

Using a combined interpretation of the different tool results, a very accurate and reliable gene prediction is possible that builds up the basis for the functional gene annotation. The large amounts of data, which have to be evaluated in a whole-genome annotation project, cause the need for an automatic functional annotation system that assists the human annotator by suggesting most probable annotation features.

Current genome annotation systems include assistance for computation, storage, retrieval, and analysis of relevant data, which is essential for the success of any genome project. This leads to consistent automatic gene annotations assigning gene names, gene products, EC numbers, descriptions, functional categories, numbers, and other attributes.

Based on this automatic annotation, human experts can check manually each gene annotation by confirming, correct- ing, or extending the automatic predictions. This integrates biological expert knowledge in manually curated gene annotations. Anyhow, most genome annotations are based on in-silico gene and function prediction. To demonstrate the real biological function of a gene of interest, still wetlab experiments need to be performed.

In the following para- graph, a detailed description of the sequencing using an optimized shotgun sequencing approach Kaiser et al.