Bacterial Genome Sizes Determined by DNA Renaturation Studies

Al, Bak; Christiansen, Claus; Stenderup, A

doi:10.1099/00221287-64-3-377

Cited by 120 publications

(30 citation statements)

References 17 publications

(4 reference statements)

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“…This is at the upper end of reported bacterial genomic weights ( 1.7-11.5 fg . cell-') (Kingsbury 1969;Bak et al 1970) and is larger than those reported from the 0.2-0.6-pm fractions of waters of the California Bight (Fuhrman and Azam 1982). Differences may be inherent in DNA methods (Hoechst 3 3258 vs. diaminobenzoic acid) or due to inclusion of larger bacteria or some microeucaryotes in our 0.2-l -pm fractions.…”

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confidence: 70%

Genetic material in the marine environment: Implication for bacterial DNA

Paul

Carlson

1984

Limnology & Oceanography

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Particulate DNA content (DNAp) in water samples from freshwater, estuarine, and marine environments was measured with the DNA‐specific fluorochrome Hoechst 33258. A good correlation (r = 0.94) was found between DNA content and bacterial number for all samples. Size fractionation with 12‐, 3‐, and 1‐µm Nuclepore filters indicated that the 0.2‐–‐µm fraction contained 40–54% of the total DNAp of fresh/brackish and 71–91% of that of oceanic waters. This fraction also contained most of the bacterioplankton, while the phytoplankton (as indicated by Chl a) was found primarily in the 12–110‐µm fraction. The results suggest that most of the DNAp in the marine environment is associated with bacterioplankton and not with phytoplankton.

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confidence: 70%

Genetic material in the marine environment: Implication for bacterial DNA

Paul

Carlson

1984

Limnology & Oceanography

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“…Of the total phage, a proportion of 20% were particles carrying recombinant genomes. Assuming that the average insert size was about 10 kb (see below) and the size of the Streptococcus genome is 2000 kb (24), the number of recombinant phage particles required to have any target DNA sequence represented at a probability of 0.99 is approximately 900 (25). The yield of recombinant phages resulting from ligation and in vitro packaging exceeded this figure by a factor of about 20.…”

Section: Resultsmentioning

confidence: 99%

Streptokinase: cloning, expression, and excretion by Escherichia coli.

Malke¹,

Ferretti²

1984

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

121

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Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage X replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene (skc). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized. One recombinant clone was used to subclone skc in E. coli plasmid vectors. Plasmid pMF2 [10.4 kilobases (kb)] consisting of pACYC184 with a 6.4-kb H46A DNA fragment in the EcoRI site and pMF5 (6.9 kb) carrying a 2.5-kb fragment in the Pst I site of pBR322 were among the recombinant plasmids determining streptokinase production in three different E. coli host strains. Expression of skc was independent of its orientation in either vector, indicating that its own promoter was present and functional in E. coli. However, expression in pBR322 was more efficient in one orientation than in the other, suggesting that one or both of the bla gene promoters contributed to skc expression. Several lines of evidence, including proof obtained by the immunodiffusion technique, established the identity of E. coli streptokinase. Testing cell-free culture supernatant fluids, osmotic shock fluids, and sonicates of osmotically shocked cells for streptokinase activity revealed the substance to be present in all three principal locations, indicating that E. coli cells were capable of releasing substantial amounts of streptokinase into the culture medium.Streptokinases are a well-defined group of proteins exported by many strains of hemolytic streptococci to the growth medium. They interact stoichiometrically with the enzymatically inert plasma plasminogen to yield the active enzyme plasmin. The plasmin so formed then degrades, by limited proteolysis, the fibrin network to form soluble products (1, 2). Although, unlike other plasminogen activators, streptokinases are not proteases, the recently determined amino acid sequence of one streptokinase species revealed homology to the sequences of bovine trypsin and Streptomyces griseus proteases, suggesting that it evolved from a seine protease (3).The role of streptokinases in the pathogenicity of streptococci is unclear. Potentially, these substances may be determinants of virulence that contribute to the invasiveness of the organisms by preventing the formation of fibrin barriers around infectious lesions. Physical and immunological differences, paralleled by differences in substrate specificity, testify to the molecular heterogeneity of streptokinases from different sources (4, 5). Although these proteins are closely related in function, the genetic basis of their heterogeneity is unknown. To achieve a better understanding of the genetic aspects of this important streptococcal product, we have undertaken to clone a streptokinase gene from a group C Streptococcus and report here its expression in Escherichia coli. Besides providing approach...

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“…1). The genome complexities of all prokaryotes so far tested varies only from 2000 to 12,000kb (23), suggesting that the difference in Nif hybridization intensities between K. pneumoniae and the other species is not due simply to lower complexity of the K. pneumoniae genome. We estimate that the DNA sequence divergence between K. pneumoniae and R. meliloti Nif genes is from 8 to 20%, assuming a 10C decrease in tm per 1.5% mismatch (24).…”

Section: Discussionmentioning

confidence: 99%

Interspecies homology of nitrogenase genes.

Ruvkun¹,

Ausubel²

1980

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

334

127

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Cloned nitrogen fixation (nifl) genes from Klebsiella pneumoniae hybridize to DNA from 19 out of 19 widely divergent nitrogen-fixing bacterial strains but do not hybridize to DNA from 10 different non-nitrogen-fixing species.K pneumoniae nif DNA fragments that hybridize to DNA from other species contain part of the three structural genes that code for nitrogenase polypeptides. We have utilized this homology to clone an EcoRI restriction endonuclease fragment from Rhizobium meliloti that hybridizes to the K. pneumoniae nif structural genes. Some of the species whose DNA hybridizes with K. pneumoniae nif DNA have been postulated to have diverged from K pneumoniae 3 X 109 years ago. Nitrogenase genes are the only known example of such highly conserved prokaryotic translated genes. Nitrogenase genes are either extraordinarily conserved in evolution or have been exchanged between different nitrogen-fixing species relatively recently in evolutionary time. The ability to fix atmospheric nitrogen is widely distributed among divergent prokaryotic taxonomic groups, including Azotobacteraceae, Enterobacteriaceae, Rhodospirillaceae, Bacillaceae, Rhizobiaceae, Actinomycetaceae, and Cyanobacteria (1). Although the physiological conditions under which prokaryotes fix nitrogen vary considerably, the enzymatic apparatus involved in nitrogen fixation is remarkably similar. Each nitrogen-fixing species that has been studied produces an enzyme complex called nitrogenase composed of two characteristic components: a molybdenum-iron protein (MoFe protein), which reduces substrate, and an iron protein (Fe protein), which binds MgATP and transfers electrons to the MoFe protein (2). Neither purified component in the absence of the other reduces nitrogen, but it is possible to reconstitute an active nitrogenase complex in vitro from purified MoFe and Fe proteins (2).In some cases, purified MoFe protein from one bacterial species reconstitutes an enzymatically active hybrid nitrogenase with Fe protein from another species (3). In addition, amino acid compositions of nitrogenases from several species are very similar (4). These observations suggest that the structure of nitrogenase has been conserved between evolutionarily distant organisms. We hypothesized, therefore, that the DNA sequences coding for nitrogenase proteins may also have been conserved and that DNA that codes for nitrogenase from one nitrogen-fixing species would hybridize specifically to DNA from other nitrogen-fixing species. (9), and the DNA was electrophoresed in a 13 X 24 X 0.5 cm 0.8% agarose horizontal gel (Dankar plastics) at 2 V/cm for 12-16 hr. The DNA in the gel was transferred to nitrocellulose sheets (Schleicher and Schuell type BA85) by the method of Southern (7), using modifications from Botchan et al. (10).Supercoiled plasmids were prepared by the cleared lysate technique (11). Purified restriction fragments were obtained by gel electrophoresis and eluted by inserting a gel slice containing the desired fragment into a dialysis bag and placing the ba...

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Bacterial Genome Sizes Determined by DNA Renaturation Studies

Cited by 120 publications

References 17 publications

Genetic material in the marine environment: Implication for bacterial DNA

Genetic material in the marine environment: Implication for bacterial DNA

Streptokinase: cloning, expression, and excretion by Escherichia coli.

Interspecies homology of nitrogenase genes.

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