Size of DNA determined by viscoelastic measurements: Results on bacteriophages, Bacillus subtilis and Escherichia coti

Klotz, Lynn C.; Zimm, Bruno H.

doi:10.1016/0022-2836(72)90191-x

Cited by 176 publications

(54 citation statements)

References 24 publications

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“…The size of the S. pneumoniae genome (2,240 to 2,270 kbp) is in reasonable agreement with the size estimated by viscoelastic measurements (1,700 + 500 kbp) (18,21). The fact that the S. pneumoniae chromosome is smaller than that of E. coli (22,44) may be related to its many requirements for growth, i.e., 12 amino acids and six vitamins (40).…”

Section: Resultssupporting

confidence: 73%

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae

et al. 1991

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A physical map of the Streptococcus (Diplococcus ) pneumoniae chromosome, which is circular and 2,270 kbp in circumference, has been constructed. The restriction enzymes ApaI, SmaI, and SacII were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). The digests produced 22, 20, and 29 fragments, respectively. The order of the fragments was deduced from Southern blot hybridization of isolated labeled fragments to separated fragments of the various restriction digests. Genetic markers were correlated with the physical map by transformation of recipient cells with FIGE-isolated DNA fragments derived from genetically marked S. pneumoniae strains. In addition, markers were mapped by the hybridization of cloned genes to FIGE-separated restriction fragments. Six rRNA gene (rrn) clusters were mapped by hybridization to rrn-containing fragments of Haemophilus influenzae.Transformation of Streptococcus (Diplococcus) pneumoniae was the first method found to transfer genetic information in bacteria (1, 14), and it has had a monumental impact on the development of molecular biology. Although the pneumococcal transformation system has been extensively investigated for the past 45 years (6,17,40,41), understanding of its genetics and biochemistry, compared with that of Escherichia coli (2, 44) and/or Bacillus subtilis (16), is still relatively primitive. The absence of a genetic map has been a major impediment to the investigation of the molecular genetics of S. pneumoniae.The ability to separate large fragments of DNA by pulsedfield gel electrophoresis has provided the technology to map the chromosomes of bacteria (10, 19, 20, 25-27, 43, 44), and this technology has gained wide acceptance for analyzing the chromosomes of more complex organisms as well. The map of the S. pneumoniae chromosome should provide a useful framework for further molecular and genetic investigations. MATERIALS AND METHODSBacterial strains and growth conditions. All bacteria used in this study are derived from pneumococcus strain R6. A subculture of S. pneumoniae 800 or 801 was used in this study (28), along with a multiply marked strain (strain 119) resistant to 200 ,ug of streptomycin (str41) per ml, 75 ,ug of fusidic acid (fus-rA) per ml, 4 ,ug of novobiocin (nov-rl) per ml, 2 ,ug of optochin (opt-r2) per ml, 1 ,ug of rifampin (rif-rF) per ml, and 5 ,ug of streptolydigin (stg-rF) per ml (47). The uvr resistance gene (48) was located with the use of the cloned gene (42). To map the amiA gene, strain 801 bearing amiA3 was used (12). The methotrexate-sensitive bacteria in the resistant population were selected in synthetic medium containing an excess of isoleucine (40). The strain resistant to 0.1 ,ug of cefotaxime per ml was obtained by transforma- tion of strain 801 with DNA extracted from strain C506, which is resistant to a higher level of cefotaxime (1 ,ug/ml) (23). A second level of resistance (0.4 ,ug/ml) was obtained by transformation of a strain resistant to 0.1 ,ug of cefotaxime per ...

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Section: Resultssupporting

confidence: 73%

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae

et al. 1991

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“…The size of the T. ferrooxidans genome (2.8 x 106 bp) is about two-thirds that of E. coli (4 x 106 bp) but is larger than those of two members of the archaebacterial group, Thermoplasma acidophilum (1.2 x 106 bp) and Methanobacterium thermoautotrophicum (1.7 x 10' bp) (16,21,28 The reiteration frequency of family 1 has also been determined by kinetic analysis. This was done by determining the CoT112 of a radioactively labeled fragment of a repeated DNA sequence derived from family 1 reassociated with an excess of unlabeled total DNA (Fig.…”

Section: Discussionmentioning

confidence: 99%

Two families of repeated DNA sequences in Thiobacillus ferrooxidans

Yates¹,

Holmes²

1987

J Bacteriol

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The genome of Thiobacillus ferrooxidans ATCC 19859 is about 2.8 X 10(6) base pairs as determined by analysis of reassociation kinetics of sheared DNA. This is 70% of the size of the genome of Escherichia coli. About 6% of the genome of T. ferrooxidans consists of moderately repetitive DNA sequences that are repeated an average of 20 times per genome. Two distinct repeated sequences, designated family 1 and family 2, have been analyzed in more detail. Both families are approximately 1 kilobase in length and are repeated 20 to 30 times per genome. Preliminary evidence from restriction enzyme analysis, Southern blotting experiments, and thermal melting analysis indicates that members of both families are conserved and are interspersed with single-copy DNA. Six copies of one family are present on the 45-kilobase-pair plasmid of strain ATCC 19859.

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“…DNA manipulations. E. chrysanthemi was transformed by electroporation as described by Ausubel et al (1), and chromosomal DNA was extracted by the method of Klotz and Zimm (22). The following procedures were carried out by the standard methods described by Sambrook et al (37): preparation of plasmids and chromosomal DNA from E. coli, DNA ligation, bacterial transformation, agarose gel electrophoresis, and Southern blotting.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA

et al. 1996

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Growth of Erwinia chrysanthemi in media of elevated osmolarity can be achieved by the uptake and accumulation of various osmoprotectants. This study deals with the cloning and sequencing of the ousA geneencoded osmoprotectant uptake system A from E. chrysanthemi 3937. OusA belongs to the superfamily of solute ion cotransporters. This osmotically inducible system allows the uptake of glycine betaine, proline, ectoine, and pipecolic acid and presents strong similarities in nucleotide sequence and protein function with the proline/ betaine porter of Escherichia coli encoded by proP. The control of ousA expression is clearly different from that of proP. It is induced by osmotic strength and repressed by osmoprotectants. Its expression in E. coli is controlled by H-NS and is rpoS dependent in the exponential phase but unaffected by the stationary phase.Microbial pathogens encounter extremely diverse environments both inside and outside their hosts. In response to these adverse conditions, they undergo striking adaptations in order to survive and retain virulence. The growth of Erwinia chrysanthemi, which is involved in a systemic soft rot disease on a variety of higher plants, is influenced by desiccation (18, 21). We have recently analyzed the influence of osmotic strength on E. chrysanthemi growth and pathogenicity in the absence and presence of osmoprotectants (10). The consequences for pathogenicity were estimated by the effect of osmotic pressure on transcription of pel genes and pectate lyase activity. The transcription of the pelE gene, encoding the major extracellular pectate lyase enzyme, is induced in medium of high osmolarity, whereas the cellular growth rate was reduced. Osmoprotectants such as glycine betaine, proline, ectoine, and pipecolic acid were shown to be accumulated in the cells through an osmoinducible mechanism and stimulated growth. However, pelE transcription was reduced to basal levels.Although uptake and accumulation of osmoprotectants have been observed in many bacteria, transporter structural genes have been characterized in only a few microorganisms. Considering the regulation of their transcription, a knowledge of which is essential to understanding osmoregulation, only the proP and proU operons of Escherichia coli and Salmonella typhimurium have been analyzed in depth (6-8, 13, 14, 20, 27, 28, 31, 32). The identification of structural domains involved in osmosensing and substrate binding could be improved by comparison of ProP and ProU with other osmoprotectant transporters in other bacteria. In this report, we characterize one of the osmoprotectant transporters of E. chrysanthemi, analogous to ProP of E. coli. MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. The E. coli and E. chrysanthemi strains and plasmids used are described in Table 1. Cells were grown in LB or M63 glucose (0.2%) medium (33). E. chrysanthemi cells were usually incubated at 30ЊC, and E. coli cells were incubated at 37ЊC. The osmoprotectants choline, glycine betaine, proline, ectoin...

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Size of DNA determined by viscoelastic measurements: Results on bacteriophages, Bacillus subtilis and Escherichia coti

Cited by 176 publications

References 24 publications

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae

Gene localization, size, and physical map of the chromosome of Streptococcus pneumoniae

Two families of repeated DNA sequences in Thiobacillus ferrooxidans

Characterization of the Erwinia chrysanthemi osmoprotectant transporter gene ousA

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