High-resolution alignment of a 1-megabase-long genome region of three strains of Rhodobacter capsulatus

Fonstein, Michael; Nikolskaya, Tatiana; Haselkorn, Robert

doi:10.1128/jb.177.9.2368-2372.1995

Cited by 8 publications

(10 citation statements)

References 25 publications

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“…The second pattern associated the conservation of gene order with a high restriction polymorphism (4,25,32). In a third pattern, comparison of genome mapping revealed a mosaic structure showing the presence of conserved and modified regions (3,11,23). In the most extreme case, the restriction site or gene order differed markedly (19,43).…”

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confidence: 99%

Genome structure and phylogeny in the genus Brucella

et al. 1997

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PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb SpeI fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus.Brucella is a small gram-negative bacterium pathogenic for animals and occasionally for humans. The genus is divided into six species on the basis of cultural, metabolic, and antigenic characteristics: B. melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae (6). However, it was shown by DNA-DNA hybridization that the genus is more probably monospecific (42). In a previous work (1), we found that each of the socalled species exhibited a specific macrorestriction pattern by digestion of genomic DNA with XbaI, with only minor variations between biovars within a species. Recently, a physical map was proposed for the genus type strain, B. melitensis 16M, by a cross-hybridization method (30). We showed that the Brucella genome was constituted by two circular chromosomes with sizes of about 2.05 and 1.15 Mb.In this study, we established the PacI and SpeI restriction maps of the two chromosomes of the other reference strains in the genus Brucella: B. abortus 544, B. suis 1330, B. canis 62/290, B. ovis 63/290, and B. neotomae 5K33. Three different but complementary techniques were employed: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping strategy using I-SceI. I-SceI is an endonuclease encoded by a group I intron of the Saccharomyces cerevisiae mitochondrial 21S rRNA gene (31). Recently, by using artificially inserted I-SceI sites, a physical map of chromosome XI of S. cerevisiae (40) and a physical map of YAC inserts (5) were made. Since bacterial genomes lack natural I-SceI sites, we showed that the introduction of a unique restriction site recognized by I-SceI could be a useful tool for the study of the genome organization (20). In this study, we used an indirect end-labeling method to localize the SpeI sites, derived from a previously described procedure involving partial digestion of linearized DNA, followed by Southern blotting and h...

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confidence: 99%

Genome structure and phylogeny in the genus Brucella

et al. 1997

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“…Recently, cosmid encyclopedias of genomes of three different strains of R. capsulatus were assembled and mapped with high resolution (11). These cosmids were used in global expression studies (12) and for the comparison of chromosome organization in different strains (13).…”

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“…We chose the first strategy for the following reasons: (i) R. capsulatus has a genome size of 3.8 Mb, which we considered to be too large to be assembled by shotgun sequencing without great complications; (ii) the high GC content of this genome (68%) results in many sequencing stops; it is easier to link contigs interrupted by these stops if the genome already is represented by an ordered clone encyclopedia; and (iii) a cosmid encyclopedia of R. capsulatus already had been constructed and mapped with high resolution (11,13). The current report describes the results of a pilot project (5% of the genome) aimed at testing the sequencing tactics and computer tools to be applied to the remainder of the Rhodobacter genome.…”

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Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003

Vlček

Pačes

Maltsev

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Cosmids from the 1A3-1A10 region of the complete miniset were individually subcloned by using the vector M13 mp18. Sequences of each cosmid were assembled from about 400 DNA fragments generated from the ends of these phage subclones and merged into one 189-kb contig. About 160 ORFs identified by the CodonUse program were subjected to similarity searches. The biological functions of 80 ORFs could be assigned reliably by using the WIT and Magpie genome investigation tools. Eighty percent of these recognizable ORFs were organized in functional clusters, which simplified assignment decisions and increased the strength of the predictions. A set of 26 genes for cobalamin biosynthesis, genes for polyhydroxyalkanoic acid metabolism, DNA replication and recombination, and DNA gyrase were among those identified. Most of the ORFs lacking significant similarity with reference databases also were grouped. There are two large clusters of these ORFs, one located between 45 and 67 kb of the map, and the other between 150 and 183 kb. Nine of the loosely identified ORFs (of 15) of the first of these clusters match ORFs from phages or transposons. The other cluster also has four ORFs of possible phage origin.

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“…The small number of common loci used in these comparisons often makes the degree of similarity present easy to determine simply by looking at the data. Examples of comparisons in which many loci were used, such as those with E. coli and S. typhimurium (40) and Rhodobacter capsulatus (16), showed clear-cut results. Such easily interpreted results are unlikely to make up the majority of future comparisons.…”

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“…(4,5), Rhodobacter spp. (16), and Leptospira interrogans (48). The criteria for deciding in which group a comparison will be included have not been rigorously established, however.…”

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Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement

Jean

Charlebois

1996

J Bacteriol

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Anonymous probes from the genome of Halobacterium salinarium GRB and 12 gene probes were hybridized to the cosmid clones representing the chromosome and plasmids of Halobacterium salinarium GRB and Haloferax volcanii DS2. The order of and pairwise distances between 35 loci uniquely cross-hybridizing to both chromosomes were analyzed in a search for conservation. No conservation between the genomes could be detected at the 15-kbp resolution used in this study. We found distinct sets of low-copy-number repeated sequences in the chromosome and plasmids of Halobacterium salinarium GRB, indicating some degree of partitioning between these replicons. We propose alternative courses for the evolution of the haloarchaeal genome: (i) that the majority of genomic differences that exist between genera came about at the inception of this group or (ii) that the differences have accumulated over the lifetime of the lineage. The strengths and limitations of investigating these models through comparative genomic studies are discussed.In recent years much effort has been devoted to genomelevel analysis among prokaryotes (11,15). Most of this effort involved the use of pulsed-field gel electrophoresis (PFGE), either in the construction of physical maps or in the direct comparison of restriction fragment patterns. The latter method allows for a large number of genomes to be quickly and relatively easily compared but suffers from poor resolution, although it has been useful in the typing of strains (18,22,32). A large number of studies have been conducted by using PFGE to construct physical maps (for a review, see reference 11). Most often, genetic markers are then localized to specific regions of the map through hybridization, allowing the investigation of gross rearrangements at the genomic level. Genetic loci occurring on the same restriction fragment, however, cannot be ordered on the map, masking any differences in their arrangement. This limitation restricts such comparisons to closely related genomes.The majority of genomic comparisons performed to date have used PFGE-derived maps to compare genomes at the strain or species level. The results of these comparisons have prompted their division into two groups (15), organisms with highly conserved genetic maps and those with divergent maps. (16), and Leptospira interrogans (48). The criteria for deciding in which group a comparison will be included have not been rigorously established, however. Typically, no objective measure is used to determine the degree of similarity between genomes, which makes relating the results of one comparison to those of another problematic. Indeed, genomes showing a moderate number of differences could be considered either conserved or divergent, depending on the context of the study.Of the more than 100 chromosomal maps available, only 10 are from members of the domain Archaea. To date, three archaeal genomic comparisons have been performed, one between two methanogens (46) and two between different members of the haloarchaea (19, 31). The most de...

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High-resolution alignment of a 1-megabase-long genome region of three strains of Rhodobacter capsulatus

Cited by 8 publications

References 25 publications

Genome structure and phylogeny in the genus Brucella

Genome structure and phylogeny in the genus Brucella

Sequence of a 189-kb segment of the chromosome of Rhodobacter capsulatus SB1003

Comparative genomic analysis of the Haloferax volcanii DS2 and Halobacterium salinarium GRB contig maps reveals extensive rearrangement

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