Physical map of the genome of Rhodobacter capsulatus SB 1003

Fonstein, Michael; Zheng, Su; Haselkorn, Robert

doi:10.1128/jb.174.12.4070-4077.1992

Cited by 46 publications

(46 citation statements)

References 28 publications

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“…Genomic libraries of Rhodobacter species were constructed by using cosmid vector Lorist 6 (11). Chromosomal DNA was partially digested with Sau3A, size selected by using sucrose gradients, and cloned in cosmid arms as described previously (10). A device made it possible to print 96 E. coli cultures from each of nine microtiter plates on one 150-mm-diameter petri dish in an ordered way and to prepare six such plates at once (10).…”

Section: Methodsmentioning

confidence: 99%

“…Alkaline plasmid extraction for mapping and labeling and preparation of chromosomal DNA were done as described elsewhere (10). Restriction endonucleases and Klenow fragment of DNA polymerase were purchased from New England Biolabs and used as instructed by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

“…Enzymes for SP6 or T7 in vitro transcription were obtained from Promega Corp. and used according to the Promega manual. DNA extraction, digestions, and PFGE separation were performed as described previously (10).…”

Section: Methodsmentioning

confidence: 99%

“…Chromosomal DNA was partially digested with Sau3A, size selected by using sucrose gradients, and cloned in cosmid arms as described previously (10). A device made it possible to print 96 E. coli cultures from each of nine microtiter plates on one 150-mm-diameter petri dish in an ordered way and to prepare six such plates at once (10). After overnight growth on DuPont Colony Screen membranes, colony replicas were picked, processed as instructed by the membrane manufacturer, and hybridized with different DNA probes.…”

Section: Methodsmentioning

confidence: 99%

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

1995

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A detailed restriction map of the genome of Rhodobacter capsulatus SB1003 was constructed recently by using an ordered set of overlapping cosmids. Pulsed-field gel electrophoresis-generated restriction patterns of the chromosomes of 14 other R. capsulatus strains were compared. Two of them, St. Louis and 2.3.1, were chosen for high-resolution alignment of their genomes with that of SB1003. A 1-Mb segment of the R. capsulatus SB1003 cosmid set was used as a source of ordered probes to group cosmids from the other strains. Selected cosmids were linked into one 800-kb contig and two smaller contigs of 100 kb each. EcoRV and BamHI restriction maps of the newly ordered cosmids were constructed by using terminase. Long-range gene order in the new strains was mainly conserved for the regions studied. However, one large genome rearrangement inverted a 470-kb DNA fragment of the St. Louis strain between the rrnA and rrnB operons. A 50-kb deletion covering three SB1003 probes was found in strain 2.3.1 near rrnB. Conservation of about 50% of the positions of restriction sites in all these strains and nearly 80% for the pair 2.3.1-St. Louis made it possible to produce high-resolution alignment of the contiguous 800-kb genome segment. Ten deletions of 2 to 27 kb, one 30-kb inversion, and three translocations were found in this region. Strong clustering of the positions of polymorphic restriction sites was observed. For a 50-kb size interval, two patterns of the distribution of restriction sites were found, one with about 90% and the other with 5 to 30% conservation of sites. This mosaic structure may be explained by independent acquisition of these divergent regions from other Rhodobacter strains.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

1995

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“…1 a ; Shaw et al., 1991). The dctP gene has previously been mapped at a distance of more than 1000 kb from the nifHDK genes (Fonstein et al, 1992).…”

Section: A M G T D G --R R R Y R a S Y L R L L K G W L F D S N N A mentioning

confidence: 99%

The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase

Masepohl

Krey

Klipp

1993

Journal of General Microbiology

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Synthetic oligonucleotides, which were designed according to amino acid sequences conserved between Rhodospirillum rubrum and Azospirillum brasileizse DraT and DraG, respectively, were used to identify the corresponding genes of Rhodobacter capsulatus. Sequence analysis of a 1904bp DNA fragment proved the existence of R. capsulatus draT and draG. These two genes were separated by 11 bp only, suggesting that R. capsulatus draT and draG were part of one transcriptional unit. In contrast to R. rubrum, A. brasilense and AzospirUum lipoferum, the R. capsulatus draTG genes were not located upstream of the structural genes of nitrogenase nzmDK but close to the dctP gene at a distance of about lo00 kb from the nzjHDK genes. Deletion mutations in the draTG gene region were constructed and introduced into R. capsulatus wild-type and a nijHDK deletion strain. The resulting mutant strains were examined for post-translational regulation of the molybdenum and the alternative nitrogenase in response to ammonia and darkness. Under 'switch-off' conditions the modified (ADP-ribosylated) and the non-modified forms of component I1 of both the molybdenum and the alternative nitrogenase were detected in a ,draTG wild-type background by immunoblot analysis, whereas only the nonmodified forms were present in the draTG deletion strains. Nitrogenase activity in these strains was followed by the acetylene reduction assay. In contrast to the wild-type, draTG mutants were not affected in nitrogenase activity in response to ammonia or darkness. These results demonstrated that the draTG genes are required for posttranslational regulation of both the molybdenum and the heterometal-free nitrogenase in R. capsulatus.

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Physical genome analysis of bacteria

et al. 1992

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Pulsed-field gel electrophoresis (PFGE) is a general analytical tool to separate large DNA molecules and may therefore be applied to problems from all areas of bacteriology. The genome size of bacteria covers the range of 0.6 to 10 megabase pairs. For genome fingerprinting, the bacterial chromosome is cleaved with a restriction endonuclease that gives a resolvable and informative number of five to one hundred fragments on the PFGE gel. Restriction enzymes are chosen according to GC content, degree of methylation, and codon usage of the respective bacterial genus. Macrorestriction fingerprinting allows the identification of bacterial strains and the distinction between related and unrelated strains. If fragment patterns of several restriction digestions are quantitatively evaluated, strains can be classified according to genetic relatedness at the level of genus, species, and biovar. In particular, members of a clonal lineage can be uncovered. Hence, any problem from applied, environmental, and clinical microbiology may be addressed by PFGE restriction analysis where the spatiotemporal spread of a bacterial clone is of interest. In bacterial genomics, PFGE is employed for the top-down construction of macrorestriction maps of the chromosome which yields data about genome organization, mobile genetic elements, and the arrangement of gene loci and gene families. The genomic diversity of a bacterial species is elucidated by comparative chromosome mapping. Map positions of restriction sites and gene loci of interest serve as landmarks to assess the extent of gross chromosomal modification, namely insertions, deletions and inversions. Intra- and interspecies comparisons of genome organization provide insights into the structure and diversity of bacterial populations and the phylogeny of bacterial taxa.

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Physical map of the genome of Rhodobacter capsulatus SB 1003

Cited by 46 publications

References 28 publications

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

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

The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase

Physical genome analysis of bacteria

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